Identification of Type II Collagen Peptide 261-273-specific T Cell Clones in a Patient with Relapsing Polychondritis

Arthritis and Rheumatism. Jan, 2002  |  Pubmed ID: 11817597

To characterize and clone T cells specific for type II collagen (CII) in a patient with relapsing polychondritis (RP) and to establish whether the immunodominant epitope of CII determined in HLA transgenic mice is used in the human autoimmune response to CII.

HLA Class II Tetramers: Tools for Direct Analysis of Antigen-specific CD4+ T Cells

Arthritis and Rheumatism. Jan, 2002  |  Pubmed ID: 11817608

Immunotherapies for human autoimmune and immune-mediated diseases are proliferating rapidly, and with these changes comes the opportunity to monitor patients for immune responses to therapy based on early surrogate markers for clinical responses. Class II tetramers have the potential to serve as these sorts of markers for immune monitoring, and thereby assist with patient management, therapy selection, and improved outcomes. However, important issues of TCR avidity require resolution, because much is still unknown regarding location, quantitation, and characterization of the human T cell response. Opportunities for application of tetramer technologies in the near future will enable both clinical progress and the development of new insights into human CD4+ T cell biology in vivo.

Mutational Analysis of Critical Residues Determining Antigen Presentation and Activation of HLA-DQ0602 Restricted T-cell Clones

Human Immunology. Mar, 2002  |  Pubmed ID: 11872236

Three different HLA-DQ0602 restricted T-lymphocyte clones (clones 5, 44, and 48) specific for two different Herpes simplex virus type 2 (HSV-2) VP16 peptides were used in a series of proliferation assays with BLS-1 cell lines expressing mutated HLA-DQ0604 molecules as APC. Up to four residues in the peptide-binding region of DQ0604 were replaced by the respective DQ0602 residue. For all three clones, residue beta70 played a crucial role in TCR recognition; beta30 and beta57 were important, although beta86 was less significant. Clone 5 and 48, specific to the HSV-2 VP16 369--379 peptide, responded to the same mutated DQ0604 molecules. Both clones could be stimulated only when the antigen presenting DQ molecule contained the DQ0602-like Gly at position beta70. Stimulation of clone 44, which recognized a different HSV-2 VP16 epitope (VP16 40-50), was less restricted. Molecular homology modeling showed that the beta70Arg of DQ0604 partially covered the peptide around P5/P6. Interactions of beta70 with residues from the antigen-peptide and polymorphic residues at positions beta30 and beta57 can modulate this effect. Supported by molecular modeling data, we conclude that the distinct molecular topography of DQ0602 is not contributed by a single residue, but rather the interactions of various polymorphic DQ residues with particular antigenic peptides.

Detection of GAD65-specific T-cells by Major Histocompatibility Complex Class II Tetramers in Type 1 Diabetic Patients and At-risk Subjects

Diabetes. May, 2002  |  Pubmed ID: 11978633

Soluble HLA-DR401 or -DR404 tetramers containing a peptide corresponding to an immunodominant epitope from human GAD65 were used to analyze peripheral blood T-cells of newly diagnosed type 1 diabetic patients and at-risk subjects. Peripheral blood mononuclear cells were expanded on antigen-presenting cells presenting GAD65 peptide and subsequently activated with specific plate-bound class II-peptide monomers. T-cell activation defined in flow cytometry by CD4(high) and/or CD25 markers were observed in all type 1 diabetic patients and some at-risk subjects, but not in normal control subjects. The activated T-cells stained positive with tetramers containing the GAD65 epitope 555-567. Tetramer-positive cells were CD4(high) T-cells with high avidity for an immunodominant GAD65 T-cell epitope. Phenotyping of T-cells utilizing HLA class II tetramers provides a new tool to characterize the autoimmune response in type 1 diabetes.

Linear B-cell Epitopes in Lambert-Eaton Myasthenic Syndrome Defined by Cell-free Synthetic Peptide Binding

Journal of Neuroimmunology. May, 2002  |  Pubmed ID: 12020970

Lambert-Eaton Myasthenic Syndrome (LEMS) is a rare autoimmune disorder characterized by autoantibodies that bind to P/Q voltage-gated calcium channels at the neuromuscular junction. Using cell-free direct peptide binding in 12 LEMS patients, we find linear B-cell epitopes that reside on the extracellular peptide linkers between the S5-S6 transmembrane peptides of Domains II and IV. A major epitope is located within the S5-S6 linker peptide of Domain IV defined by the acidic amino acid sequence EDEDSDEDE.

HLA Class II Peptide-binding and Autoimmunity

Tissue Antigens. Feb, 2002  |  Pubmed ID: 12028533

The HLA class II locus is located in the 6p21.3 region on the short arm of chromosome 6 and encompasses approximately 700 kb. It consists of over 30 gene loci including the major class II structural genes DP, DQ and DR. While autoimmune disease correlates to specific DP, DQ or DR alleles have been documented, due to the strong linkage disequilibrium between the different HLA alleles, especially between the DR and DQ, the precise identification of susceptible MHC alleles for a number of autoimmune diseases remains elusive.

Defining Antigen-specific Responses with Human MHC Class II Tetramers

The Journal of Allergy and Clinical Immunology. Aug, 2002  |  Pubmed ID: 12170258

Antigen-specific CD4(+) T lymphocytes play key roles in the orchestration of immune responses, including the initiation and amplification of allergic and immune-mediated disorders. Direct detection and quantitation of these cells is made possible by the use of HLA class II tetramers, soluble recombinant forms of a complex of HLA molecule and antigenic peptide that bind the antigen-specific T-cell receptor used for T-cell recognition. By using flow cytometry with fluorescent tetramers, specific T cells can be identified, recovered, and analyzed for functional markers and transcripts, helping to characterize the lineage and commitment program for individual lymphocytes. Several newly emerging uses for tetramers in clinical immunology are under development for patient management or prognosis when the number or nature of antigen-specific T cells can be clinically important. Tetramers might be useful as patient-monitoring tools for evaluating response to immunotherapy, providing a surrogate marker for the immunologic response foreshadowing a clinical response to either immune stimulation, suppression, or deviation therapeutic modalities.

Disease-associated Bias in T Helper Type 1 (Th1)/Th2 CD4(+) T Cell Responses Against MAGE-6 in HLA-DRB10401(+) Patients with Renal Cell Carcinoma or Melanoma

The Journal of Experimental Medicine. Sep, 2002  |  Pubmed ID: 12208877

T helper type 1 (Th1)-type CD4(+) antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4(+) T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4(+) T cells from human histocompatibility leukocyte antigens (HLA)-DRbeta1*0401(+) patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-gamma and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6-derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus- or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4(+) T cell secretion of IL-10 and transforming growth factor (TGF)-beta1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4(+) subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4(+) T cell responses to provide optimal clinical benefit.

Use of Class II Tetramers for Identification of CD4+ T Cells

Journal of Immunological Methods. Oct, 2002  |  Pubmed ID: 12213344

Multivalent MHC class II molecules containing peptide antigens are useful tools for the detection of antigen specific human CD4+ T cells. Tetramers produced by exogenous peptide loading onto empty class II molecules are comparable to tetramers with peptide tethered to the class II chain covalently, but have many practical advantages. Conditions for optimal peptide loading to generate tetramers are discussed and optimal conditions of using tetramers for staining T cells are examined. As the frequency of antigen specific CD4+ T cells in peripheral blood is low, we demonstrate that an in vitro expansion step is effective in detecting low frequency T cells. Two new applications with tetramers, their uses for mapping T cell epitopes and for the detection of low affinity T cells are described. In a clinical setting, potential applications include using these reagents for monitoring disease progression during clinical intervention.

Differential Antigen Sensitivity and Costimulatory Requirements in Human Th1 and Th2 Antigen-specific CD4+ Cells with Similar TCR Avidity

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2003  |  Pubmed ID: 12538679

The differentiation of naive CD4(+) Th cells into Th1 and Th2 phenotypes is influenced by cytokines, concentration of Ag, accessory molecules, and the affinity of the MHC-TCR interaction. To study these factors in human memory T cells, T cell lines with Th1 or Th2 phenotypes specific for the peptide hemagglutinin (HA)(307-319) in the context of DRB1*0401 were established from the peripheral blood of an individual previously vaccinated for influenza virus. Flow cytometric analysis with fluorescent-labeled MHC class II tetramers was used to analyze TCR avidity: the Th2 line bound the HLA-DR*0401-HA(307-319) tetramers with higher mean avidity, although the range of binding avidity largely overlapped with the Th1 line. High-affinity Th1 and Th2 lines were established for further study by FACS sorting. When activated with plate-bound HLA-DR*0401-HA(307-319) monomers, the Th1 line proliferated and produced IFN-gamma without additional costimulation whereas the Th2 line required the addition of soluble anti-CD28 Ab to induce proliferation and IL-5 production, but this requirement could be overcome with high concentrations of plate-bound monomer alone. IL-2 production was dependent on costimulation in both cell lines. These findings demonstrate that upon antigenic rechallenge, Th1 and Th2 cells differ in their response to Ag-specific stimulation. Th2 cells were sensitive to the strength of signal to a greater degree than Th1 cells and required costimulation through CD28 for maximal proliferation. These distinctions between Th1 and Th2 activation are not consistent with a simple avidity model of Ag recognition and indicate both qualitative and quantitative differences in determining cell lineage commitment.

HLA Tetramer-based Artificial Antigen-presenting Cells for Stimulation of CD4+ T Cells

Clinical Immunology (Orlando, Fla.). Jan, 2003  |  Pubmed ID: 12584046

Current evidence suggests that immunotherapy for cancer or infectious diseases will require the activation of CD4(+) T cells in addition to the activation of cytotoxic CD8(+) T cells. To complement and overcome some of the limitations of dendritic-cell-based vaccines and ex vivo expansion of human T cells, we sought to engineer artificial antigen-presenting cells (aAPCs) for the stimulation of antigen-specific human CD4(+) T cells. We have designed a variety of aAPCs using magnetic beads as a scaffold on which to coat HLA-peptide tetrameric complexes along with costimulatory molecules such as anti-CD28. Here, we tested various forms of conjugation of the tetramers onto beads, characterized the relative concentration of antigen available on the surface of the beads, and evaluated the ability of different types of beads to promote activation of antigen-specific CD4(+) T cells. We find that an indirect coating of HLA-peptide tetramers on beads via an anti-Class II antibody provides specific stimulation of antigen-specific CD4(+) T cells.

Challenges in Staining T Cells Using HLA Class II Tetramers

Clinical Immunology (Orlando, Fla.). Jan, 2003  |  Pubmed ID: 12584047

Use of HLA Class II Tetramers in Tracking Antigen-specific T Cells and Mapping T-cell Epitopes

Methods (San Diego, Calif.). Mar, 2003  |  Pubmed ID: 12725793

The highly specific interaction between major histocompatibility complex (MHC)/peptide and its cognate T-cell receptor (TCR) directs to the specificity of the T-cell response. Although the interaction affinity of individual MHC/peptide and TCR molecules is low, multimerization of the MHC/peptide complexes increases the avidity of their interaction with TCRs, and enables the complexes to be used as staining reagents for antigen-specific T cells. Different approaches are used to generate class I and class II tetramer reagents, and these reagents have been demonstrated to be useful in identifying CD8+ and CD4+ T cells, respectively. This article focuses on the uses of human lymphocyte antigen (HLA) class II tetramers in tracking antigen-specific T cells. Class II tetramers can also be used to map antigenic epitopes of known antigens through a novel peptide screening procedure known as tetramer-guided epitope mapping (TGEM). In this article, we provide a description of the methodological approach of using HLA class II tetramers in tracking and isolating T cells, and the use of class II tetramers in mapping T-cell epitopes.

Low-avidity Recognition by CD4+ T Cells Directed to Self-antigens

European Journal of Immunology. May, 2003  |  Pubmed ID: 12731068

Self-reactive T cells populate the peripheral immune system, and likely form the reservoir from which autoreactive cells are derived. We analyzed a panel of self and non-self peptides presented by HLA-DR4, a class II molecule associated with autoimmunity, by immunization of mice transgenic for HLA-DR4. Significant structural avidity for T cell recognition, as measured by MHC class II tetramer binding to CD4(+) T cells was only observed in mice immunized with the non-self antigens. T cell hybridomas were generated from mice immunized with the naturally processed self-peptide hGAD65 (552-572) and also from mice immunized with an influenza-derived non-self epitope (HA 306-318). T cells specific for the self peptide failed to bind tetramers and exhibited low functional avidity as measured by the peptide concentration required to reach half-maximum proliferation values. In contrast, T cells specific for the non-self HA (306-318) peptide exhibited high structural and functional avidity profiles. As recently described in studies of murine CD8(+) T cell function, the predominance of low avidity recognition of self-peptide epitopes may be a characteristic feature of CD4(+) T cells responding to autoantigens.

P53(110-124)-specific Human CD4+ T-helper Cells Enhance in Vitro Generation and Antitumor Function of Tumor-reactive CD8+ T Cells

Cancer Research. Jul, 2003  |  Pubmed ID: 12839958

Current evidence suggests that the optimal vaccines for cancer should incorporate tumor-specific cytotoxic as well as helper epitopes. Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, which could combine multiple tumor epitopes defined by CD8(+) CTLs, as well as CD4(+) T-helper cells. To test this possibility, we generated anti-p53 CD4(+) T cells from peripheral blood obtained from an HLA-DRB1*0401(+) donor by in vitro stimulation with dendritic cells and recombinant human p53 protein. We identified the wt p53(110-124) peptide as a naturally presented epitope. In a series of ex vivo experiments, performed in an autologous human system, we then demonstrated the ability of anti-wt p53(110-124) CD4(+) T cells to enhance the generation and antitumor functions of CD8(+) effector cells. The results demonstrate the crucial role of T helper-defined epitopes in shaping the immune response to multiepitope cancer vaccines targeting p53. This model of tumor-specific CD8(+) and CD4(+) T-cell interactions suggests that future vaccination strategies targeting tumor cells should incorporate helper and cytotoxic T cell-defined epitopes.

HLA Class II-restricted CD4+ T Cell Responses Directed Against Influenza Viral Antigens Postinfluenza Vaccination

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2003  |  Pubmed ID: 12960344

The memory T cell response is polyclonal, with the magnitude and specificity of the response controlled in part by the burst size of T cells expanded from effector/memory precursors. Sensitive assays using HLA class II multimers were used to detect low-frequency Ag-specific T cells directed against influenza viral Ags in subjects immunized with the influenza vaccine. Direct ex vivo tetramer staining of PBMC from five individuals identified frequencies of hemagglutinin (HA) 306-318 tetramer binding CD4(+) T cells in the peripheral blood ranging from 1 in 600 to 1 in 30,000 CD4(+) T cells. These frequencies were validated by counting CFSE(low), tetramer-positive T cells after in vitro expansion. Low frequency of T cells directed to other influenza epitopes, including DRA1*0101/DRB1*0401-restricted matrix protein 60-73, DRA1*0101/DRB1*0101-restricted matrix protein 18-29, DRA1*0101/DRB1*0701-restricted HA 232-244 and DRA1*0101/DRB1*0101-restricted nucleoprotein 206-217 were also determined. T cells which occurred at a frequency as low as 1 in 350,000 could be ascertained by in vitro expansion of precursors. Peripheral HA(306-318)-responsive T cells expanded 2- to 5-fold following influenza vaccination. Examination of phenotypic markers of the HA(306-318)-responsive T cells in the peripheral blood indicated that the majority were CD45RA(-), CD27(+), CD25(-), CD28(+), and CD62L(-), while T cell clones derived from this population were CD45RA(-), CD27(-), CD25(+), CD28(+), and CD62L(-).

Diminished Proliferation of Human Immunodeficiency Virus-specific CD4+ T Cells is Associated with Diminished Interleukin-2 (IL-2) Production and is Recovered by Exogenous IL-2

Journal of Virology. Oct, 2003  |  Pubmed ID: 14512540

Virus-specific CD4(+) T-cell function is thought to play a central role in induction and maintenance of effective CD8(+) T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4(+) T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4(+) T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-gamma in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer(+) or total-Gag-specific CD4(+) T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4(+) T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4(+) T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.

Detection of CD4+ Autoreactive T Cells in T1D Using HLA Class II Tetramers

Annals of the New York Academy of Sciences. Nov, 2003  |  Pubmed ID: 14679042

The use of soluble class II MHC tetramers has enabled the identification of autoantigen-specific T cells in the peripheral blood of type 1 diabetes patients. Our approach takes advantage of the appearance of highly activated T cells expressing a CD25(+)/CD4(high+) phenotype induced by immobilized class II MHC monomer containing the GAD65 peptide. Almost all T cells that stain with the specific tetramer reside in this population and, since this activation profile is not present in normal subjects, it may provide a useful tool for analysis of the T cell response in autoimmune diabetes. The utilization of tetramer techniques in the detection of autoreactive T cells is a powerful tool to gain insight into mechanisms of the molecular basis of autoimmunity. The phenotyping of T cells should provide useful markers for progression of immune-mediated beta cell reactivity and can be utilized in clinical trials to evaluate the efficacy of the immunomodulatory therapies targeting intervention/prevention of autoimmune diseases.

HLA Class II Polymorphisms Determine Responses to Bacterial Superantigens

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2004  |  Pubmed ID: 14734754

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ alpha-chain polymorphisms such that HLA-DQA1*01 alpha-chains show greater binding than DQA1*03/05 alpha-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vbeta8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vbeta specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

Differential Recognition and Activation Thresholds in Human Autoreactive GAD-specific T-cells

Diabetes. Apr, 2004  |  Pubmed ID: 15047612

The activation requirements of autoreactive CD4(+) T-cells were investigated in GAD65-specific HLA-DR0401-restricted clones derived from a diabetic patient using major histocompatibility complex (MHC) class II tetramers (TMrs) as stimulating agents. Despite the fact that TMrs loaded with an immunodominant-altered GAD peptide (TMr-GAD) bound a limited number of T-cell receptors, they were capable of efficiently delivering activation signals. These signals ranged from the early steps of phospholipase C (PLC)-gamma(1) phosphorylation and Ca(2+) mobilization to more complex events, such as CD69 upregulation, cytokine mRNA transcription and secretion, and proliferation. All the effects triggered by TMr-GAD were dose dependent. On the contrary, [(3)H]-thymidine incorporation decreased at high TMr-GAD concentrations because of activation-induced cell death (AICD) after initial proliferation. Lower-avidity clones (as defined by TMr-GAD binding) were less sensitive to activation as well as less susceptible to AICD compared with higher-avidity clones. Induction of apoptosis is a potential immunomodulatory target for therapeutic applications of MHC class II multimers, but the relative resistance of low-avidity T-cells may limit its benefits.

Autoreactive T Cells in Healthy Individuals

Journal of Immunology (Baltimore, Md. : 1950). May, 2004  |  Pubmed ID: 15128778

The presence of autoreactive CD4(+) T cells in the peripheral blood of healthy human subjects was investigated after removal of CD4(+)CD25(+) regulatory T cells (Treg). CD4(+) T cells that were directed against the type 1 diabetes-associated autoantigen glutamic acid decarboxylase 65, the melanocyte differentiation Ag tyrosinase, and the cancer/testis tumor Ag NY-ESO-1 were readily derived from PBMC of healthy individuals. These autoreactive T cells could be visualized, using Ag-specific class II tetramer reagents, in the peripheral blood of most individuals examined. Addition of CD4(+)CD25(+) Treg back to the CD4(+)CD25(-) population suppressed the expansion of the autoreactive T cells. Autoreactive T cells were cloned based on tetramer binding, and expressed characteristic activation markers upon self-Ag stimulation. These results show that autoreactive T cells are present in most healthy individuals and that Treg likely play an important role of keeping these autoreactive T cells in check.

Extensive Replicative Capacity of Human Central Memory T Cells

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2004  |  Pubmed ID: 15153483

To characterize the replicative capacity of human central memory (T(CM)) CD4 T cells, we have developed a defined culture system optimized for the ex vivo expansion of Ag-specific CD4(+) T cells. Artificial APCs (aAPCs) consisting of magnetic beads coated with Abs to HLA class II and a costimulatory Ab to CD28 were prepared; peptide-charged HLA class II tetramers were then loaded on the beads to provide Ag specificity. Influenza-specific DR*0401 CD4 T(CM) were isolated from the peripheral blood of normal donors by flow cytometry. Peptide-loaded aAPC were not sufficient to induce resting CD4 T(CM) to proliferate. In contrast, we found that the beads efficiently promoted the growth of previously activated CD4 T(CM) cells, yielding cultures with >80% Ag-specific CD4 cells after two stimulations. Further stimulation with peptide-loaded aAPC increased purity to >99% Ag-specific T cells. After in vitro culture for 3-12 wk, the flu-specific CD4 T(CM) had surface markers that were generally consistent with an effector phenotype described for CD8 T cells, except for the maintenance of CD28 expression. The T(CM) were capable of 20-40 mean population doublings in vitro, and the expanded cells produced IFN-gamma, IL-2, and TNF-alpha in response to Ag, and a subset of cells also secreted IL-4 with PMA/ionomycin treatment. In conclusion, aAPCs expand T(CM) that have extensive replicative capacity, and have potential applications in adoptive immunotherapy as well as for studying the biology of human MHC class II-restricted T cells.

GAD65-specific CD4+ T-cells with High Antigen Avidity Are Prevalent in Peripheral Blood of Patients with Type 1 Diabetes

Diabetes. Aug, 2004  |  Pubmed ID: 15277377

Negative selection of self-reactive T-cells during thymic development, along with activation-induced cell death in peripheral lymphocytes, is designed to limit the expansion and persistence of autoreactive T-cells. Autoreactive T-cells are nevertheless present, both in patients with type 1 diabetes and in at-risk subjects. By using MHC class II tetramers to probe the T-cell receptor (TcR) specificity and avidity of GAD65 reactive T-cell clones isolated from patients with type 1 diabetes, we identified high-avidity CD4+ T-cells in peripheral blood, coexisting with low-avidity cells directed to the same GAD65 epitope specificity. A variety of cytokine patterns was observed, even among T-cells with high MHC-peptide avidity, and the clones utilize a biased set of TcR genes that favor two combinations, Valpha12-beta5.1 and Valpha17-Vbeta4. Presence of these high-avidity TcRs indicates a failure to delete autoreactive T-cells that likely arise from oligoclonal expansion in response to autoantigen exposure during the progression of type 1 diabetes.

In Vivo Biotinylation of the Major Histocompatibility Complex (MHC) Class II/peptide Complex by Coexpression of BirA Enzyme for the Generation of MHC Class II/tetramers

Human Immunology. Jul, 2004  |  Pubmed ID: 15301857

Success in generation of major histocompatibility complex (MHC) tetramer relies on application of a key technique, biotinylation of MHC molecule specifically on a single lysine residue using the BirA enzyme. However, in vitro biotinylation of MHC-BSP (BirA enzyme substrate peptide) fusion protein using BirA enzyme is laborious and is prone to losses of target proteins to unacceptable levels. To circumvent this problem, an in vivo biotinylation strategy was developed where the BirA enzyme was coexpressed with target protein, HLA-DR2BSP/MBP, in an insect cell expression system. Bacterial BirA enzyme expressed in Drosophila melanogaster 2 (D. Mel-2) cell lines was biologically functional and was able to biotinylate secretary target protein (on specific lysine residue present on the BSP tag). Biotinylation efficiency was maximized by providing exogenous d-biotin in the culture medium and optimization of the expression vector ratios for cotransfection. By limiting dilution cloning, a clone was identified where the expressed DR2BSP/MBP protein was completely biotinylated. DR2BSP/MBP protein expressed and purified from such a clone was ready to be tetramerized with streptavidin to be used for staining antigen-specific T cells.

Direct Killing of Epstein-Barr Virus (EBV)-infected B Cells by CD4 T Cells Directed Against the EBV Lytic Protein BHRF1

Blood. Feb, 2004  |  Pubmed ID: 14563644

Due to their low frequency, CD4 T-cell responses to Epstein-Barr virus (EBV) lytic antigens are, so far, poorly characterized. Human peptide major histocompatibility complex (MHC) class II multimers provide a means to detect and characterize such rare T cells. Along a screening of T-cell responses to lytic or latent EBV antigens within peripheral blood leukocyte (PBL)- or synovial-derived CD4 T-cell lines, we identified an human leukocyte antigen-DR*0401 (HLA-DR*0401)-restricted epitope derived from BHRF1 (BamHI fragment H rightward open reading frame 1), a viral protein produced during the early stages of the lytic cycle. We show here that T-cell responses to this particular BHRF1 epitope are shared by most EBV-infected DR*0401(+) individuals, as BHRF1-specific CD4 T cells could be sorted out from all the DRB*0401 T-cell lines analyzed, using magnetic beads coated with recombinant BHRF1/DR*0401 complexes. Sorting with these peptide MHC class II multimers was very efficient, as the yield of recovery of BHRF1-specific T cells was nearly 100%. Functional analysis of a large number of clones responding to BHRF1/DR*0401 demonstrated their cytolytic action against autologous and allogeneic DR*0401(+) EBV-transformed B-lymphoblastoid cell lines (B-LCLs), with 40% to 80% killing efficiency and potent interferon gamma production, thus suggesting that this CD4 T-cell population contributes to the control of EBV replication. B-LCL lysis by these T-cell clones was DR*0401 dependent, EBV dependent, and was not merely due to bystander killing. Taken together, these data provide the first demonstration that a lytic antigen can induce a direct cytolytic response against EBV-infected cells.

Expression of Cutaneous Lymphocyte-associated Antigen and E-selectin Ligand by Circulating Human Memory CD4+ T Lymphocytes Specific for Herpes Simplex Virus Type 2

The Journal of Infectious Diseases. Jan, 2005  |  Pubmed ID: 15609235

Virus-specific memory T lymphocytes traffic to sites of viral infection. Herpes simplex virus (HSV) type 2-specific CD4(+) and CD8(+) T lymphocytes differ with regard to their homing kinetics to infected tissues. We studied the expression of cutaneous lymphocyte-associated antigen (CLA) and E-selectin ligand (ESL) by HSV-2-specific CD4(+) T lymphocytes. Virus-reactive T lymphocytes were identified ex vivo by CD154 or interferon-gamma up-regulation. We detected selective expression of CLA by HSV-2-reactive CD4(+) T lymphocytes, but at levels lower than those we previously observed for CD8(+) T lymphocytes. Short-term HSV-2-reactive CD4(+) lines generated from peripheral-blood mononuclear cells preferentially express CLA, compared with cytomegalovirus- or influenza-specific cells. CLA is expressed by HSV-2-reactive cells that are initially CLA negative before restimulation. Short-term culture-expanded HSV-2-specific CD4(+) T lymphocytes also selectively express ESL. These findings have implications for the optimization of vaccines for HSV and other cutaneous pathogens.

Clustering of T Cell Ligands on Artificial APC Membranes Influences T Cell Activation and Protein Kinase C Theta Translocation to the T Cell Plasma Membrane

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2005  |  Pubmed ID: 15749850

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

Functional Avidity Directs T-cell Fate in Autoreactive CD4+ T Cells

Blood. Oct, 2005  |  Pubmed ID: 16030184

Major histocompatibility complex class II tetramer staining and activation analysis identified 2 distinct types of antigen-specific CD4+ T cells in the peripheral blood of humans with type 1 (autoimmune) diabetes. T cells with low-avidity recognition of peptide-MHC ligands had low sensitivity to activation and inefficient activation-induced apoptosis. In contrast, high-avidity T cells were highly sensitive to antigen-induced cell death through apoptotic mechanisms, and both apoptosis-resistant high- and low-avidity T cells that survived prolonged tetramer treatment were rendered anergic to restimulation by antigen. In addition, however, apoptosis-resistant high-avidity T cells acquired regulatory features, being able to suppress both antigen-specific and nonspecific CD4+ T-cell responses. This suppression was contact-dependent and correlated with the down-regulation of HLA class II and costimulatory molecules on antigen-presenting cells, including B cells and dendritic cells. T cells face a variety of fates following antigen exposure, including the paradoxic maintenance of high-avidity autoreactive T cells in the peripheral circulation, perhaps due to this capability of acquiring anergic and suppressive properties. Regulation via down-modulation of antigen-presenting cell function, a form of cell-to-cell licensing for suppression, also offers possibilities for the application of peptide-MHC therapeutics.

Persistence of Herpes Simplex Virus Type 2 VP16-specific CD4+ T Cells

Human Immunology. Jul, 2005  |  Pubmed ID: 16112025

Patients with genital herpes have frequent viral reactivations. The repeated antigenic rechallenges can modulate the CD4+ memory T-cell repertoires during the course of infection. In this study, the CD4+ T-cell responses against the herpes simplex virus type 2 (HSV-2) tegument protein VP16 were studied in two HSV-2-infected subjects at two different time points that spanned a 5-year period. Although the VP16-specific T cells did exhibit variation of T-cell receptor Vbeta usages at the two time points, T cells that used identical Vbeta and CDR3 junction sequences were also observed at the two time points. These experiments demonstrate that the CD4+ T cells that are directed against HSV-2 VP16 protein in chronically infected individuals are oligoclonal and that T cells of specific clonotypes can be maintained throughout the course of the disease.

Expression of HLA-DP0401 Molecules for Identification of DP0401 Restricted Antigen Specific T Cells

Journal of Clinical Immunology. Sep, 2005  |  Pubmed ID: 16160911

Human histocompatibility leukocyte antigen (HLA)-DPA1*0103/DPB1*0401 (DP0401) is the most common HLA class II molecule and is present in approximately 45% of the Caucasian population. In this study, soluble HLA-DP0401 molecules were expressed as "empty'' class II molecules in insect cells. Utilizing these soluble DP molecules and the Tetramer Guided Epitope Mapping (TGEM) approach, the influenza A Puerto Rico/8/34 matrix protein (MP) derived peptide MP(41-60) VLMEWLKTRPILSPLTKGIL and the Clostridium tetani Tetanus Toxin (TT) derived peptide TT(634-653) DKISDVSTIVPYIGPALNIV were identified as the DP0401 restricted MP and TT epitopes, respectively. In addition, T cells specific for the cancer testis antigen NY-ESO-1 and the breast/ovarian cancer over-expressing antigen Her-2/neu were detected in DP0401 subjects by DP0401 tetramers. The availability of HLA-DP0401 tetramers should facilitate the study of DP restricted T cell responses.

Immunomodulatory Potential of Heteroclitic Analogs of the Dominant T-cell Epitope of Lipocalin Allergen Bos D 2 on Specific T Cells

International Immunology. Dec, 2005  |  Pubmed ID: 16221722

Peptide-based allergen immunotherapy is a novel alternative for conventional allergen immunotherapy. Here, we have characterized the immunomodulatory potential of heteroclitic peptide analogs of the immunodominant epitope of lipocalin allergen Bos d 2 on specific human T-cell clones. The TCR affinity of Bos d 2-specific T-cell clones for the natural peptide ligand and its heteroclitic analogs was assessed with fluorescent-labeled MHC class II tetramers. The activation and cytokine production of the clones were analyzed upon stimulation with the different ligands. Moreover, the capacity of the heteroclitic analogs to induce hyporesponsiveness and cell death was examined. The T-cell clones F1-9 and K3-2 bound MHC class II tetramers loaded with the heteroclitic peptide analogs of the immunodominant epitope of Bos d 2 with increased affinity. At similar peptide concentrations, stimulation of the clones with the heteroclitic analogs favored increased IFN-gamma/IL-4 and IFN-gamma/IL-5 ratios in comparison with stimulation with the natural peptide ligand. Moreover, the T-cell clones stimulated with the heteroclitic analogs exhibited an increased susceptibility to cell death or hyporesponsiveness upon re-stimulation. Our results suggest that heteroclitic analogs of a T-cell epitope of an allergen may enhance the efficacy of peptide-based allergen immunotherapy.

The Heat Shock Protein Hsp70 Enhances Antigen-specific Proliferation of Human CD4+ Memory T Cells

European Journal of Immunology. Nov, 2005  |  Pubmed ID: 16245362

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress-inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen-specific CD4+ T cells as confirmed by HLA-DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen-specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen-specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.

Comparative Study of GAD65-specific CD4+ T Cells in Healthy and Type 1 Diabetic Subjects

Journal of Autoimmunity. Dec, 2005  |  Pubmed ID: 16249070

Glutamic acid decarboxylase 65 (GAD65) is a putative autoantigen associated with the pathogenesis of type 1 diabetes (T1D). The prevalence of autoreactive CD4+ T cells towards the immunodominant GAD65(555-567) epitope in DR4 healthy and T1D subjects was investigated with class II tetramers. A slightly higher percentage of diabetic subjects had GAD65(555-567) tetramer-positive T cells upon GAD65(555-567) peptide stimulation on the total CD4+ T-cell populations compared to healthy subjects. In contrast, three quarters of subjects in both groups had tetramer-positive T cells resulting from stimulation of the CD4+CD25+ regulatory T-cell depleted CD4+ T cells. The frequencies and TCR Vbeta gene usages of GAD65(555-567) T cells were also similar in both groups. Experiments demonstrated that GAD65(555-567)-reactive T cells in healthy and diabetic subjects had different CD45RA phenotypes. For the healthy group, GAD65(555-567)-reactive T cells were generally found in the CD45RA+ naïve T-cell pool while GAD65(555-567)-reactive T cells from T1D subjects were present in both CD45RA+ naïve and CD45RA- memory T-cell pools. These findings suggested that there is no difference in thymic selection of DR4 restricted GAD-reactive T cells amongst healthy and T1D individuals but GAD65(555-567)-reactive T cells have been preferentially activated in diabetic patients.

GAD65- and Proinsulin-specific CD4+ T-cells Detected by MHC Class II Tetramers in Peripheral Blood of Type 1 Diabetes Patients and At-risk Subjects

Journal of Autoimmunity. Nov, 2005  |  Pubmed ID: 16263242

In type 1 diabetes the major loss of insulin producing beta-cells is caused by autoreactive T-cells specific for antigens expressed by the pancreatic islets. In this study we have analyzed the prevalence of glutamate decarboxylase 65 (GAD65)- and proinsulin-specific CD4(+) T-cells in type 1 diabetes patients, at-risk subjects and in HLA-matched control children. Peripheral blood mononuclear cells were cultured in the presence of two different GAD65 peptides (555-567, 557I and 274-286) or with a proinsulin (B24-C36) peptide for 10-11days. The autoreactive T-cells were detected using antigen specific-MHC class II tetramers by flow cytometry. Our results show that 11 of 18 (61%) type 1 diabetes patients and 7 of the 20 (35%) at-risk subjects were positive for one of the three GAD65 or proinsulin-containing tetramers, whereas only 2 of 21 (9.5%) controls had tetramer binding cells (p = 0.0007 type 1 diabetes vs. controls and p = 0.0488 at-risk subjects vs. controls, Chi-square test). Type 1 diabetes patients responded to all three peptides. At-risk subjects recognized also the GAD65 555-567 557I peptide, while none of the controls responded to it. In conclusion, type 1 diabetes patients and at-risk subjects have a significantly higher prevalence of GAD65- and proinsulin-specific CD4(+) T-cells than the control subjects.

Allelic Variation in Key Peptide-binding Pockets Discriminates Between Closely Related Diabetes-protective and Diabetes-susceptible HLA-DQB1*06 Alleles

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2006  |  Pubmed ID: 16424231

HLA-DQA1*0102-DQB1*0602 is associated with protection against type 1 diabetes (T1D). A similar allele, HLA-DQA1*0102-DQB1*0604, contributes to T1D susceptibility in certain populations but differs only at seven amino acids from HLA-DQA1*0102-DQB1*0602. Five of these polymorphisms are found within the peptide-binding groove, suggesting that differences in peptide binding contribute to the mechanism of their association with T1D. In this study, we determine the peptide-binding motif for HLA-DQA1*0102-DQB1*0604 allelic protein (DQ0604) in comparison to the established HLA-DQA1*0102-DQB1*0602 (DQ0602) motif using binding assays with model peptides from T1D autoantigens and homology modeling using the coordinates of the DQ0602-hypocretin 1-13 crystal structure. The peptide binding preferences were deduced with a peptide from insulin that bound both with a 2- to 3-fold difference in avidity using the same amino acids in the peptide as anchors. Peptide binding differences directly influenced by the polymorphisms in or nearby pockets 1, 6, and 9 were observed. In pocket 1, DQ0604 was better able to accommodate aromatic residues due to the beta86 and beta87 polymorphisms. A negatively charged amino acid was preferred by DQ0604 in pocket 6 due to the positively charged beta30His. In pocket 9, DQ0604 preferred aromatic amino acids due to the beta9 and beta30 polymorphisms and had low tolerance of acidic residues. beta57Val in DQ0604 functions differently than beta57Ala, in that it pushes alpha76Arg outside of the pocket, preventing the formation of a salt bridge with an acidic amino acid in the peptide. This study furthers our understanding of the structure-function relationships of MHC class II polymorphisms.

Cytotoxic Herpes Simplex Type 2-specific, DQ0602-restricted CD4 T+-cell Clones Show Alloreactivity to DQ0601

Immunology. Mar, 2006  |  Pubmed ID: 16476054

Alloreactivity is one of the most serious problems in organ transplantation. It has been hypothesized that pre-existing alloreactive T cells are actually cross-reacting cells that have been primed by the autologous major histocompatibility complex (MHC) and a specific peptide. CD8+ cytotoxic T lymphocytes that are alloreactive and recognize a virus-peptide that is presented by the autologous MHC have been reported. Here we demonstrate a cross-reactivity that exists between DQ0602 restricted, herpes simplex type 2 VP16 40-50 specific CD4+ T-cell clones, which can be alloreactive to DQ0601. Though most of the DQ0602 restricted T-cell clones we isolated from two different donors were not alloreactive, weakly cross-reacting T-cell clones could be isolated from both donors. Two strongly cross-reacting T-cell clones with high affinity interaction of their T-cell receptor (TCR) with both DQ0602/VP16 40-50 and DQ0601 could be isolated from one donor. DNA sequencing of the a fragment of the Vbeta gene used in their TCR confirmed that these two T cells indeed are two independent clones. These clones are cytotoxic and produce cytokines of a T helper 2-like pattern. Possible implications in a DR-matched transplantation setting are discussed.

Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein-reactive CD4+ T Cells in Human Subjects

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2006  |  Pubmed ID: 16493034

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4+ T cell responses toward IGRP in human subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4+ T cell epitopes. IGRP(23-35) and IGRP(247-259) were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP(13-25) and IGRP(226-238) were identified as DRA1*0101/DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4+ T cell responses for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both gamma-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP(247-259)-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general population.

Antibiotic-refractory Lyme Arthritis is Associated with HLA-DR Molecules That Bind a Borrelia Burgdorferi Peptide

The Journal of Experimental Medicine. Apr, 2006  |  Pubmed ID: 16585267

An association has previously been shown between antibiotic-refractory Lyme arthritis, the human histocompatibility leukocyte antigen (HLA)-DR4 molecule, and T cell recognition of an epitope of Borrelia burgdorferi outer-surface protein A (OspA163-175). We studied the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes in 121 patients with antibiotic-refractory or antibiotic-responsive Lyme arthritis and correlated these frequencies with in vitro binding of the OspA163-175 peptide to 14 DRB molecules. Among the 121 patients, the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes were similar to those in control subjects. However, when stratified by antibiotic response, the frequencies of DRB1 alleles in the 71 patients with antibiotic-refractory arthritis differed significantly from those in the 50 antibiotic-responsive patients (log likelihood test, P = 0.006; exact test, P = 0.008; effect size, Wn = 0.38). 7 of the 14 DRB molecules (DRB1*0401, 0101, 0404, 0405, DRB5*0101, DRB1*0402, and 0102) showed strong to weak binding of OspA163-175, whereas the other seven showed negligible or no binding of the peptide. Altogether, 79% of the antibiotic-refractory patients had at least one of the seven known OspA peptide-binding DR molecules compared with 46% of the antibiotic-responsive patients (odds ratio = 4.4; P < 0.001). We conclude that binding of a single spirochetal peptide to certain DRB molecules is a marker for antibiotic-refractory Lyme arthritis and might play a role in the pathogenesis of the disease.

Allergen-specific MHC Class II Tetramer+ Cells Are Detectable in Allergic, but Not in Nonallergic, Individuals

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2006  |  Pubmed ID: 16585604

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-gamma. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

Multiplex Mapping of CD4 T Cell Epitopes Using Class II Tetramers

Clinical Immunology (Orlando, Fla.). Jul, 2006  |  Pubmed ID: 16677863

With the advent of class II tetramer technology, a tetramer-guided epitope mapping (TGEM) technique was developed for the identification of CD4+ T cell epitopes. This allowed the direct identification of epitopes recognized by the responding T cells, which were restricted to the single MHC allele of interest. However, as each individual carries multiple class II alleles, it would be advantageous to design an approach to identify CD4+ epitopes presented by different class II alleles at the same time. In the present study, a multiplex TGEM approach was developed to identify antigenic epitopes presented by multiple HLA class II alleles simultaneously. In this new approach, CD4+ T cells were stained with multiple sets of MHC class II tetramers-each labeled with a unique fluorescent label. Using this multiplex approach, novel epitopes from influenza antigens hemagglutinin and matrix protein presented by multiple class II alleles were identified in a single experimental setting.

HLA-DQ2 and -DQ8 Signatures of Gluten T Cell Epitopes in Celiac Disease

The Journal of Clinical Investigation. Aug, 2006  |  Pubmed ID: 16878175

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

Melan-A/MART-1-specific CD4 T Cells in Melanoma Patients: Identification of New Epitopes and Ex Vivo Visualization of Specific T Cells by MHC Class II Tetramers

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2006  |  Pubmed ID: 17082590

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Detection of Low Avidity Desmoglein 3-reactive T Cells in Pemphigus Vulgaris Using HLA-DR Beta 1*0402 Tetramers

Clinical Immunology (Orlando, Fla.). Mar, 2007  |  Pubmed ID: 17113829

In the present study, we developed a HLA class II tetramer-based detection system utilizing DRB1*0402 tetramers loaded with recently identified immunodominant peptides of desmoglein 3 (Dsg3), the major autoantigen of pemphigus vulgaris (PV). Initial experiments demonstrated staining of a Dsg3-reactive T cell hybridoma which was derived from HLA-DR0402-transgenic mice with loaded PE-labeled DRbeta1*0402 tetramers. However, staining of autoreactive T cell clones (TCC) derived from PV patients resulted only in positive staining by addition of exogenous peptides to the staining reactions. There was a dose-dependent specific binding of TCC to the tetramers with the agonistic Dsg3 peptide which was not altered by exogenous unrelated Dsg3 peptide. Noteworthy, the TCC did not stain with HLA-DR4 tetramers complexed with unrelated Dsg3 peptides. The findings of this study suggest that HLA class II tetramers may provide a highly specific approach to monitor ex vivo the T cellular autoimmune response against Dsg3 in patients with PV.

CD8+ Suppressor-mediated Regulation of Human CD4+ T Cell Responses to Glutamic Acid Decarboxylase 65

European Journal of Immunology. Jan, 2007  |  Pubmed ID: 17163450

Although potentially autoreactive T cells are present even in healthy subjects, most individuals do not develop autoimmune disease. It has been well demonstrated that CD4+ CD25+ regulatory T cells play a significant role in controlling the expansion of autoreactive T cells in the periphery. However, some healthy individuals exhibit measurable responses to self peptide even in the presence of CD4+ CD25+ regulatory cells. This article describes the regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65 (GAD65), an autoantigen implicated in type-1 diabetes, by autologous CD8+ suppressor T cells. In cells cultured from healthy individuals, the inclusion of autologous CD8+ T cells at physiological levels resulted in a dramatic decrease in the magnitude of in vitro CD4+ T cell responses to GAD65 peptide. Based on transwell experiments, the observed suppression was cell contact-dependent. However, antibody blocking studies indicated that suppression was mediated by IL-10. Cell fractionation studies suggested that CD8+ suppressor T cells originate from the CD45RA+ CD27- population. The suppression of CD4+ T cell responses to GAD65 in healthy individuals raises the possibility that CD8+ suppressor T cells play an important role in controlling potentially autoreactive T cells in the general population.

Antigen-specific CD4+ T Cells Recognize Epitopes of Protective Antigen Following Vaccination with an Anthrax Vaccine

Infection and Immunity. Apr, 2007  |  Pubmed ID: 17283103

Detection of antigen-specific CD4+ T cells is facilitated by the use of fluorescently labeled soluble peptide-major histocompatibility complex (MHC) multimers which mirror the antigen specificity of T-cell receptor recognition. We have used soluble peptide-MHC class II tetramers containing peptides from the protective antigen (PA) of Bacillus anthracis to detect circulating T cells in peripheral blood of subjects vaccinated with an anthrax vaccine. PA-specific HLA class II-restricted T lymphocytes were isolated which displayed both TH1- and TH2-like characteristics, indicating heterogeneity of the lymphocyte lineage within the CD4+ response. Presentation of antigen to these T-cell clones by HLA-matched antigen-presenting cells exposed to the intact PA protein confirmed that the identified epitopes are indeed naturally processed by the human immune system. Specific tetramer-derived T-cell profiling may be useful for monitoring helper CD4+ T-cell responses to anthrax vaccination.

Potential of an Altered Peptide Ligand of Lipocalin Allergen Bos D 2 for Peptide Immunotherapy

The Journal of Allergy and Clinical Immunology. Apr, 2007  |  Pubmed ID: 17335888

Peptide immunotherapy is a promising alternative for treating allergic diseases. One way to enhance the efficacy of peptide immunotherapy is to use altered peptide ligands (APLs) that contain amino acid substitutions compared with the natural peptide.

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-competent Vaccinia Virus

Journal of Immunology (Baltimore, Md. : 1950). May, 2007  |  Pubmed ID: 17475867

Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication-incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relationship between Ab and CD4 T cell specificity for complex pathogens.

Tetramer-guided Epitope Mapping Reveals Broad, Individualized Repertoires of Tetanus Toxin-specific CD4+ T Cells and Suggests HLA-based Differences in Epitope Recognition

International Immunology. Nov, 2007  |  Pubmed ID: 17906339

Tetanus toxoid is a routine positive control antigen for cellular assays. Previous studies identified multiple tetanus toxin (TT) epitopes, including some 'universal' epitopes. However, rigorous HLA-restricted study of tetanus toxoid responses is still lacking. In this study, the tetramer-guided epitope mapping approach was used to identify CD4+ T-cell epitopes within the TT heavy chain restricted by 10 different class II alleles. Of 106 peptides tested, 52 contained epitopes. Response frequencies toward specific epitopes varied, indicating prevalent and rare specificities. Most antigenic peptides (85%) were presented by one or two class II alleles. For peptides presented by three or more alleles, truncation studies revealed that some contained multiple epitopes. These results contrast with the perceived notion that tetanus toxoid responses are dominated by universal CD4+ T-cell epitopes. Rather these results illustrate heterogeneous T-cell responses for different class II alleles and individual-specific variation of the T-cell repertoire.

Decline in the Frequencies of Borrelia Burgdorferi OspA161 175-specific T Cells After Antibiotic Therapy in HLA-DRB1*0401-positive Patients with Antibiotic-responsive or Antibiotic-refractory Lyme Arthritis

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2007  |  Pubmed ID: 17947711

Synovitis in patients with antibiotic-refractory Lyme arthritis persists for months to several years after antibiotic therapy. This course, which may result from infection-induced autoimmunity, is associated with T cell recognition of Borrelia burgdorferi outer surface protein A (OspA(161-175)) and with HLA-DR molecules that bind this epitope, including the DRB1*0401 molecule. In this study, we used tetramer reagents to determine the frequencies of OspA(161-175)-specific T cells in samples of PBMC and synovial fluid mononuclear cells (SFMC) from 13 DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory arthritis. Initially, three of the six patients (50%) with antibiotic-responsive arthritis and four of the seven patients (57%) with antibiotic-refractory arthritis had frequencies of OspA(161-175)-specific CD4(+) T cells in peripheral blood above the cutoff value of 4 per 10(5) cells. Among the five patients with concomitant PBMC and SFMC, four (80%) had OspA tetramer-positive cells at both sites, but the mean frequency of such cells was 16 times higher in SFMC, reaching levels as high as 1,177 per 10(5) cells. In the two patients in each patient group in whom serial samples were available, the frequencies of OspA(161-175)-specific T cells declined to low or undetectable levels during or soon after antibiotic therapy, months before the resolution of synovitis in the two patients with antibiotic-refractory arthritis. Thus, the majority of patients with Lyme arthritis initially have increased frequencies of OspA(161-175)-specific T cells. However, the marked decline in the frequency of such cells with antibiotic therapy suggests that persistent synovitis in the refractory group is not perpetuated by these cells.

Tracking Antigen Specific CD4+ T-cells with Soluble MHC Molecules

Methods in Molecular Medicine. 2007  |  Pubmed ID: 17983139

The advent of soluble MHC multimer technology has allowed for the flow-cytometric direct identification of specific-MHC restricted antigen-specific T cells in mixed cell populations and also enabled the direct phenotyping and cloning of these cells at the same time. To date, MHC multimers have been used in characterizing the adaptive T cell repertoire under infectious, cancerous, and autoimmune states and has increased our understanding of the dynamics of T-cell immunity. Recombinant MHC multimers have been produced where MHC-binding peptide antigens are either covalently or noncovalently bound to the MHC, with the latter having the advantage of the ability to use a single recombinant MHC to investigate multiple MHC-binding peptides and their interacting T cells. In this method we describe how to generate recombinant non-covalently bound peptide MHC-multimers in insect cells. MHC multimers are generated as tetravalent complexes using a streptavidin scaffold.

High Level of PD-1 Expression on Hepatitis C Virus (HCV)-specific CD8+ and CD4+ T Cells During Acute HCV Infection, Irrespective of Clinical Outcome

Journal of Virology. Mar, 2008  |  Pubmed ID: 18160439

We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8(+) and CD4(+) T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.

Searching for Borrelial T Cell Epitopes Associated with Antibiotic-refractory Lyme Arthritis

Molecular Immunology. Apr, 2008  |  Pubmed ID: 18191206

Antibiotic-refractory Lyme arthritis is believed to result from an infection-induced autoimmune response triggered by the spirochete Borrelia burgdorferi (Bb). Disease susceptibility is associated with the HLA alleles DRB1*0101, 0401, 0402, 0404, 0405 and DRB5*0101, and all these MHC molecules bind the Bb epitope OspA(163-175.) However, not all patients have a proliferative response to this epitope. To identify other possible Bb epitopes involved in this disease process, the algorithm TEPITOPE was used to scan 17 immunogenic Bb proteins for potential T cell epitopes with a refractory arthritis-associated MHC binding profile, and the Bb proteome was searched for peptides with sequence homology to OspA(165-173). Sixteen promising T epitopes were identified and their MHC binding profiles to 13 MHC molecules were verified using in vitro MHC/peptide binding assays. One peptide, BBK32(392-404), had a strong refractory arthritis-associated MHC binding profile, and another GK(297-306) shared sequence homology to OspA(165-173). However, patient cells did not proliferate in response to either peptide making it highly unlikely they were involved in a refractory course. A comparison of the in silico and in vitro results revealed that TEPITOPE correctly predicted 74% of the in vitro binding peptides, but it incorrectly predicted that 44% of the in vitrononbinding peptides would bind. For a particular MHC molecule, concordance between the in silico and in vitro results varied anywhere between 33% and 100%. Therefore, while additional Bb epitopes may be involved in the development of antibiotic-refractory Lyme arthritis, recognition of OspA(163-175) remains the only known Bb epitope associated with this disease course.

Healthy Human Subjects Have CD4+ T Cells Directed Against H5N1 Influenza Virus

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2008  |  Pubmed ID: 18209073

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4(+) T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4(+) T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4(+) T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-gamma, TNF-alpha, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.

Definition of the Peptide Binding Motif Within DRB1*1401 Restricted Epitopes by Peptide Competition and Structural Modeling

Molecular Immunology. May, 2008  |  Pubmed ID: 18276010

This study identified the peptide-binding motif of HLA-DRA/DRB1*1401 (DR1401). First, peptides containing DR1401 restricted epitopes were identified using tetramer-guided epitope mapping. Among these, an influenza B peptide was selected for the motif study. After confirming the binding register for this peptide using a set of arginine substitutions, binding affinities were determined for 33 peptides derived from this influenza B sequence with single amino acid substitutions. The DR1401 peptide-binding motif was deduced from the relative binding affinities of these peptides and confirmed by structural modeling. Pocket 1 demonstrated a preference for aliphatic anchor residues and methionine. Pocket 4 accommodated methionine and aliphatic residues, but also allowed some polar and charged amino acids. Pocket 6 preferred basic residues but also allowed some polar and aliphatic amino acids. Pocket 9 preferred aliphatic and aromatic amino acids and tolerated some polar residues but excluded all charged residues. Together these preferences define a distinct set of peptides that can be presented by DR1401. The resulting motif was used to verify T cell epitopes within the novel antigenic peptides identified by tetramer-guided epitope mapping and within peptides from published reports that contain putative DR1401 epitopes.

Identification and in Vitro Expansion of Functional Antigen-specific CD25+ FoxP3+ Regulatory T Cells in Hepatitis C Virus Infection

Journal of Virology. May, 2008  |  Pubmed ID: 18337568

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction, we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.

CD4+ T Cells from Type 1 Diabetic and Healthy Subjects Exhibit Different Thresholds of Activation to a Naturally Processed Proinsulin Epitope

Journal of Autoimmunity. Aug, 2008  |  Pubmed ID: 18385016

Recent studies suggest that insulin is a primary autoantigen for type 1 diabetes. Several studies have identified preproinsulin (PPI) 76-90 as an immunodominant CD4+ T cell epitope. We developed a class II tetramer reagent using a modified PPI peptide with a lysine to serine substitution at position 88 (PPI 78-90(88S)) that has high binding affinity to DRA1*0101/DRB1*0401 (DR0401). Using this tetramer, positive responses were observed from both DR0401 healthy and type 1 diabetic subjects when T cells were stimulated with the PPI 78-90(88S) peptide. Seventy percent of these T cells proliferated in response to both the wild type PPI 76-90 and PPI 78-90(88S) peptides. However, when T cells were stimulated with wild type peptide and assayed with DR0401/PPI 78-90(88S), positive responses were only detected in the diabetic group but not in healthy subjects. When highly purified CD4+CD25-CD45RA+ T cells were stimulated with PPI 78-90(88S) peptide in the absence of antigen-presenting cells, T cells from the diabetic group were able to respond to peptide stimulation, while T cells from healthy subjects were not. These data suggest that T cells from type 1 diabetic subjects have a lower threshold of activation in response to PPI peptide stimulation as compared to healthy subjects.

Low-affinity Major Histocompatibility Complex-binding Peptides in Type 1 Diabetes

Diabetes. Jul, 2008  |  Pubmed ID: 18586912

The Anthrax Vaccine Adsorbed Vaccine Generates Protective Antigen (PA)-Specific CD4+ T Cells with a Phenotype Distinct from That of Naive PA T Cells

Infection and Immunity. Oct, 2008  |  Pubmed ID: 18678674

Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4(+) T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4(+) T cells with a CD45RA(-) phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4(+) T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA(+) and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4(+) T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4(+) T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4(+) cell responses and lineage development after vaccination.

Human Homologues of a Borrelia T Cell Epitope Associated with Antibiotic-refractory Lyme Arthritis

Molecular Immunology. Jan, 2008  |  Pubmed ID: 17555819

Antibiotic-refractory Lyme arthritis, which may result from infection-induced autoimmunity, is associated with HLA-DR molecules that bind an epitope of Borrelia burgdorferi (Bb) outer-surface protein A (OspA(165-173)) and with T cell reactivity with this epitope. One potential mechanism to explain these associations is molecular mimicry between OspA(165-173) and a self-peptide. Here, we searched the published human genome for peptides with sequence homology with OspA(165-173). The two peptides identified with the greatest sequence homology with the OspA epitope were MAWD-BP(276-288), which had identity at eight of the nine core amino acid residues, and T-span7(58-70), which had identity at six residues. MAWD-BP mRNA was expressed by synoviocytes, while T-span7 mRNA was not. However, neither peptide bound all of the HLA-DR molecules associated with antibiotic-refractory Lyme arthritis. Among 11 patients, 9 had T cell reactivity with OspA(161-170), 6 had responses to MAWD-BP(276-288), and 3 had reactivity with T-span7(58-70), but reactivity with the self-peptides was lower than that induced by the spirochetal epitope. Thus, there remains an association between OspA(165-173) and antibiotic-refractory Lyme arthritis, and infection-induced autoimmunity is an attractive hypothesis to explain this outcome. However, molecular mimicry due to sequence homology between OspA(165-173) and a human peptide seems unlikely to be the critical mechanism.

Searching Immunodominant Epitopes Prior to Epidemic: HLA Class II-restricted SARS-CoV Spike Protein Epitopes in Unexposed Individuals

International Immunology. Jan, 2009  |  Pubmed ID: 19050106

Identification of dominant T cell epitopes within newly emerging and re-emerging infectious organisms is valuable in understanding pathogenic immune responses and potential vaccine designs. However, difficulties in obtaining samples from patients or convalescent subjects have hampered research in this direction. We demonstrated a strategy, tetramer-guided epitope mapping, that specific CD4+ T cell epitopes can be identified by using PBMC from subjects that have not been exposed to the infectious organism. Sixteen HLA-DR0401- and 14 HLA-DR0701-restricted epitopes within spike protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) were identified. Among these, spike protein residues 159-171, 166-178, 449-461 and 1083-1097 were identified to contain naturally processed immunodominant epitopes based on strong in vitro T cell responses of PBMC (as assayed by tetramer staining) to intact spike protein stimulation. These immunodominant epitopes were confirmed in vivo in HLA-DR0401 transgenic mice by immunizing with spike protein. Furthermore, the epitope-specific T cells from naive donors secreted IFN-gamma and IL-13 upon re-stimulation with corresponding tetramers. Our study demonstrates a strategy to determine potential immunodominant epitopes for emerging infectious pathogens prior to their epidemic circulation.

Functional Islet-specific Treg Can Be Generated from CD4+CD25- T Cells of Healthy and Type 1 Diabetic Subjects

European Journal of Immunology. Feb, 2009  |  Pubmed ID: 19180473

CD4(+)CD25(+)FOXP3(+) Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self-antigen-specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen-specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen-specific adaptive Treg were induced in vitro by activation of CD4(+)FOXP3(-) T cells with glutamic acid decarboxylase and islet-specific glucose-6-phosphate catalytic subunit-related protein peptides in the context of T1D-associated HLA-DRbeta alleles. Antigen-specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza-specific Treg from the same study population. The function of tetramer(+) cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of islet-specific Treg may potentially be used as a targeted immunotherapy in T1D.

H5N1 Strain-specific Hemagglutinin CD4+ T Cell Epitopes Restricted by HLA DR4

Vaccine. Jun, 2009  |  Pubmed ID: 19446935

CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain-specific HLA class II restricted epitopes is essential for monitoring cellular strain-specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA(441-460) and H5HA(57-76) were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA(57-76) epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA(57-76) response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA(57-76) immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.

Lineages of Human T-cell Clones, Including T Helper 17/T Helper 1 Cells, Isolated at Different Stages of Anti-factor VIII Immune Responses

Blood. Aug, 2009  |  Pubmed ID: 19549986

The development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject (the proband) 19 weeks and 21 months after his development of a high-titer inhibitor. Clones responding to the same epitope were also isolated from his multiply infused brother, who has not developed a clinically significant inhibitor. The 19-week proband clones were T helper (T(H))17/T(H)1- or T(H)1/T(H)2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were T(H)2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were T(H)1-polarized, indicating that tolerance to FVIII can be maintained even with circulating T(H)1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that T(H)17/T(H)1-polarized cells play a role in hemophilic immune responses to FVIII. Furthermore, this is the first report of successful isolation and expansion of antigen-specific human T(H)17/T(H)1 clones using standard culture conditions.

The Binding of Antigenic Peptides to HLA-DR is Influenced by Interactions Between Pocket 6 and Pocket 9

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2009  |  Pubmed ID: 19648278

Peptide binding to class II MHC protein is commonly viewed as a combination of discrete anchor residue preferences for pockets 1, 4, 6/7, and 9. However, previous studies have suggested cooperative effects during the peptide binding process. Investigation of the DRB1*0901 binding motif demonstrated a clear interaction between peptide binding pockets 6 and 9. In agreement with prior studies, pockets 1 and 4 exhibited clear binding preferences. Previously uncharacterized pockets 6 and 7 accommodated a wide variety of residues. However, although it was previously reported that pocket 9 is completely permissive, several substitutions at this position were unable to bind. Structural modeling revealed a probable interaction between pockets 6 and 9 through beta9Lys. Additional binding studies with doubly substituted peptides confirmed that the amino acid bound within pocket 6 profoundly influences the binding preferences for pocket 9 of DRB1*0901, causing complete permissiveness of pocket 9 when a small polar residue is anchored in pocket 6 but accepting relatively few residues when a basic residue is anchored in pocket 6. The beta9Lys residue is unique to DR9 alleles. However, similar studies with doubly substituted peptides confirmed an analogous interaction effect for DRA1/B1*0301, a beta9Glu allele. Accounting for this interaction resulted in improved epitope prediction. These findings provide a structural explanation for observations that an amino acid in one pocket can influence binding elsewhere in the MHC class II peptide binding groove.

Tumor Antigen-specific FOXP3+ CD4 T Cells Identified in Human Metastatic Melanoma: Peptide Vaccination Results in Selective Expansion of Th1-like Counterparts

Cancer Research. Oct, 2009  |  Pubmed ID: 19808957

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Reassessing the Role of HLA-DRB3 T-cell Responses: Evidence for Significant Expression and Complementary Antigen Presentation

European Journal of Immunology. Jan, 2010  |  Pubmed ID: 19830726

In humans, several HLA-DRB loci (DRB1/3/4/5) encode diverse beta-chains that pair with alpha-chains to form DR molecules on the surface of APC. While DRB1 and DRB5 have been extensively studied, the role of DRB3/4 products of DR52/DR53 haplotypes has been largely neglected. To clarify the relative expression of DRB3, we quantified DRB3 mRNA levels in comparison with DRB1 mRNA from the same haplotype in both B cells and monocytes, observing quantitatively significant DRB3 synthesis. In CD19+ cells, DRB1*03/11/13 was 3.5-fold more abundant than DRB3, but in CD14+ this difference was only two-fold. Monocytes also had lower overall levels of DR mRNA compared with B cells, which was confirmed by cell surface staining of DRB1 and DRB3. To evaluate the functional role of DRB3, tetramer-guided epitope mapping was used to detect T cells against tetanus toxin and several influenza antigens presented by DRB3*0101/0202 or DRB1*03/11/13. None of the epitopes discovered were shared among any of the DR molecules. Quantitative assessment of DRB3-tetanus toxin specific T cells revealed that they are present at similar frequencies as those observed for DRB1. These results suggest that DRB3 plays a significant role in antigen presentation with different epitopic preferences to DRB1. Therefore, DRB3, like DRB5, serves to extend and complement the peptide repertoire of DRB1 in antigen presentation.

Assessment of Seasonal Influenza A Virus-specific CD4 T-cell Responses to 2009 Pandemic H1N1 Swine-origin Influenza A Virus

Journal of Virology. Apr, 2010  |  Pubmed ID: 20071564

Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.

Involvement of CD91 and Scavenger Receptors in Hsp70-facilitated Activation of Human Antigen-specific CD4+ Memory T Cells

European Journal of Immunology. Apr, 2010  |  Pubmed ID: 20101615

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress-inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen-specific CD4(+) T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen-specific cells were detected combining HLA-DRB1*0401 or HLA-DRB1*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen-specific CD4(+) T cell responses.

HIV Controller CD4+ T Cells Respond to Minimal Amounts of Gag Antigen Due to High TCR Avidity

PLoS Pathogens. Feb, 2010  |  Pubmed ID: 20195518

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vbeta repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-gamma responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vbeta repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia.

Direct Ex Vivo Analysis of Allergen-specific CD4+ T Cells

The Journal of Allergy and Clinical Immunology. Jun, 2010  |  Pubmed ID: 20513526

HLA-DR1001 Presents "altered-self" Peptides Derived from Joint-associated Proteins by Accepting Citrulline in Three of Its Binding Pockets

Arthritis and Rheumatism. Oct, 2010  |  Pubmed ID: 20533291

HLA-DRB1*1001 (DR1001) is a shared epitope allele associated with rheumatoid arthritis (RA). The present study was undertaken to assess the capacity of DR1001 to accommodate citrulline in its binding pockets and to identify citrullinated T cell epitopes derived from joint-associated proteins.

Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-cell Responses

PloS One. 2010  |  Pubmed ID: 20634998

Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.

Allergen-specific Naïve and Memory CD4+ T Cells Exhibit Functional and Phenotypic Differences Between Individuals with or Without Allergy

European Journal of Immunology. Sep, 2010  |  Pubmed ID: 20690179

Although allergen-specific CD4(+) T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401-restricted responses of peripheral blood-derived memory (CD4(+)CD45RO(+)) and naïve (CD4(+)CD45RA(+)) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2(127-142)-specific memory T cells in the peripheral blood-derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2(127-142)-specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2(127-142)-specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long-term Bos d 2(127-142)-specific T-cell lines generated from both memory and naïve T-cell pools from individuals with allergy proliferated more strongly, produced more IL-4 and IL-10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T-cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen-specific T-cell repertoires differ between individuals with or without allergy.

Autoantigen-specific Regulatory T Cells, a Potential Tool for Immune-tolerance Reconstitution in Type-2 Autoimmune Hepatitis

Hepatology (Baltimore, Md.). Oct, 2010  |  Pubmed ID: 21190158

Effector CD4 and CD8 T cell immune responses to cytochrome P450IID6 (CYP2D6), the autoantigen of autoimmune hepatitis type 2 (AIH-2), are permitted by a numerical and functional impairment of CD4(pos)CD25(high) regulatory T cells (T-regs). We aimed to investigate whether T-regs specific for CYP2D6 immunodominant regions and restricted by the appropriate human leukocyte antigen (HLA)-DR molecule can be generated in patients with AIH-2 and can control CD4 and CD8 T cell effectors targeting identical or overlapping CYP2D6 regions. CYP2D6-specific regulatory T cells (CYP2D6 T-regs) were obtained from peptide-pulsed monocyte-depleted peripheral blood mononuclear cells of 17 patients with AIH-2, who were positive for the predisposing HLA-DR7 and/or HLA-DR3 alleles. Their antigen specificity was assessed by cytofluorimetry using HLA class II tetramers and their cytokine profile by intracellular staining. T-reg ability to suppress was ascertained by measuring reduction of CD4(pos)CD25(neg) cell proliferation/effector cytokine secretion and of CD8 T cell cytotoxicity. The most efficient suppression of effector T cell proliferation, inflammatory cytokine release, and cytotoxicity was obtained by coculturing T-regs with CYP2D6-peptide-loaded semimature dendritic cells (smDCs), and smDC-CYP2D6 T-regs also expressed high levels of FOXP3 (forkhead box P3). Possession of the appropriate HLA-DR molecule and recognition of the CYP2D6 autoantigenic sequence were critical to the synergistic smDC-CYP2D6 T-reg immunoregulatory functions, and lack of either element led to poor control of responder cell proliferation and cytokine secretion. Moreover, interferon-γ neutralization significantly boosted the suppressive ability of CYP2D6 T-regs. Conclusion: T-regs generated under CYP2D6-specific conditions and cocultured with smDCs are highly effective at controlling autoreactive T cells, thus providing the basis for a powerful and tailored form of immunotherapy for AIH-2. (HEPATOLOGY 2010;).

Differences in Self-peptide Binding Between T1D-related Susceptible and Protective DR4 Subtypes

Journal of Autoimmunity. Mar, 2011  |  Pubmed ID: 21257290

HLA-DR0401, 0403 and 0405 are associated with variable T1D susceptibilities when linked with a common HLA-DQ8 (DQA1∗0301/DQB1∗0302). It is unknown how the modest differences within the peptide binding regions of DR4 subtypes lead to distinct autoimmune risks. Since all Class II HLA molecules share the same intracellular compartments during biosynthesis, it is possible that DQ and DR compete with one another to bind and present antigenic peptides. As such, it is reasonable to hypothesize that a strong DR4 self-peptide binder down-modulates DQ8 epitope presentation more than a weak one. In this study, we first examined the binding of the peptides derived from two putative beta-cell autoantigens - GAD65 and insulin. Protective DR0403 bound similar number of self-peptides as susceptible DR0401 while highly susceptible DR0405 bound substantially less self-peptides than rest two molecules. Kinetic assays were used to further compare the stability of peptide:DR complexes formed between DR0401, 0403 and selected GAD65 peptides, which also bound DQ8. Two peptides with naturally processed DQ8 epitopes bound protective DR0403 with longer half-life and lower dissociation rate than susceptible DR0401, confirming DR0403 as a better peptide competitor than DR0401. The distinguishing peptide binding features of DR0401, DR0403, and DR0405 highlighted in this study help to explain the hierarchy of genetic associations between T1D and these DR4 subtypes. The enhanced peptide competition of DR0403 leads to a down-modulation of DQ8 epitope presentation, as compared to weak competitors such as DR0401 and DR0405, and therefore contributes to disease protection.

Autoantigen-specific Regulatory T Cells, a Potential Tool for Immune-tolerance Reconstitution in Type-2 Autoimmune Hepatitis

Hepatology (Baltimore, Md.). Feb, 2011  |  Pubmed ID: 21274874

Effector CD4 and CD8 T cell immune responses to cytochrome P450IID6 (CYP2D6), the autoantigen of autoimmune hepatitis type 2 (AIH-2), are permitted by a numerical and functional impairment of CD4(pos) CD25(high) regulatory T cells (T-regs). We aimed to investigate whether T-regs specific for CYP2D6 immunodominant regions and restricted by the appropriate human leukocyte antigen (HLA)-DR molecule can be generated in patients with AIH-2 and can control CD4 and CD8 T cell effectors targeting identical or overlapping CYP2D6 regions. CYP2D6-specific regulatory T cells (CYP2D6 T-regs) were obtained from peptide-pulsed monocyte-depleted peripheral blood mononuclear cells of 17 patients with AIH-2, who were positive for the predisposing HLA-DR7 and/or HLA-DR3 alleles. Their antigen specificity was assessed by cytofluorimetry using HLA class II tetramers and their cytokine profile by intracellular staining. T-reg ability to suppress was ascertained by measuring reduction of CD4(pos) CD25(neg) cell proliferation/effector cytokine secretion and of CD8 T cell cytotoxicity. The most efficient suppression of effector T cell proliferation, inflammatory cytokine release, and cytotoxicity was obtained by coculturing T-regs with CYP2D6-peptide-loaded semimature dendritic cells (smDCs), and smDC-CYP2D6 T-regs also expressed high levels of FOXP3 (forkhead box P3). Possession of the appropriate HLA-DR molecule and recognition of the CYP2D6 autoantigenic sequence were critical to the synergistic smDC-CYP2D6 T-reg immunoregulatory functions, and lack of either element led to poor control of responder cell proliferation and cytokine secretion. Moreover, interferon-γ neutralization significantly boosted the suppressive ability of CYP2D6 T-regs. CONCLUSION: T-regs generated under CYP2D6-specific conditions and cocultured with smDCs are highly effective at controlling autoreactive T cells, thus providing the basis for a powerful and tailored form of immunotherapy for AIH-2.

Ara H 1-reactive T Cells in Individuals with Peanut Allergy

The Journal of Allergy and Clinical Immunology. May, 2011  |  Pubmed ID: 21459424

Effective immunotherapy for peanut allergy is hampered by a lack of understanding of peanut-reactive CD4(+) T cells.

Papillomavirus-specific CD4+ T Cells Exhibit Reduced STAT-5 Signaling and Altered Cytokine Profiles in Patients with Recurrent Respiratory Papillomatosis

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2011  |  Pubmed ID: 21531896

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.

Autoreactive CD4(+) T Cells in Patients with Atopic Dermatitis

The Journal of Allergy and Clinical Immunology. Jul, 2011  |  Pubmed ID: 21620451

Increased Frequencies of Myelin Oligodendrocyte Glycoprotein/MHC Class II-binding CD4 Cells in Patients with Multiple Sclerosis

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2011  |  Pubmed ID: 21653833

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG(97-109(107E-S)) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG(97-109)-reactive CD4(+) T cells, isolated with DRB1*0401/MOG(97-109) tetramers, and after a short-term culture of PMBCs with MOG(97-109) peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG(97-109(107E-S)) tetramer(+) cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG(97-109)-reactive T cells is greater in patients with MS as compared with healthy controls.

Uveitis-associated Epitopes of Retinal Antigens Are Pathogenic in the Humanized Mouse Model of Uveitis and Identify Autoaggressive T Cells

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2011  |  Pubmed ID: 21765017

Noninfectious uveitis is a leading cause of blindness and thought to involve autoimmune T cell responses to retinal proteins (e.g., retinal arrestin [soluble-Ag (S-Ag)]). There are no known biomarkers for the disease. Susceptibility is associated with HLA, but little is known about susceptible class II alleles or the potentially pathogenic epitopes that they present. Using a humanized HLA-transgenic mouse model of S-Ag-induced autoimmune uveitis, we identified several susceptible and resistant alleles of HLA-DR and -DQ genes and defined pathogenic epitopes of S-Ag presented by the susceptible alleles. The sequences of these epitopes overlap with some previously identified peptides of S-Ag ("M" and "N"), known to elicit memory responses in lymphocytes of uveitis patients. HLA-DR-restricted, S-Ag-specific CD4(+) T cells could be detected in blood and draining lymph nodes of uveitic mice with HLA class II tetramers and transferred the disease to healthy mice. Importantly, tetramer-positive cells were detected in peripheral blood of a uveitis patient. To our knowledge, these findings provide the first tangible evidence that an autoimmune response to retina is causally involved in pathogenesis of human uveitis, demonstrating the feasibility of identifying and isolating retinal Ag-specific T cells from uveitis patients and may facilitate their development as biomarkers for the disease.

Human CD8⁺ and CD4⁺ T Cell Memory to Lymphocytic Choriomeningitis Virus Infection

Journal of Virology. Nov, 2011  |  Pubmed ID: 21900169

Although cellular immunity to acute lymphocytic choriomeningitis virus (LCMV) infection has been well characterized in experimental studies in mice, the T cell response to this virus in humans is incompletely understood. Thus, we analyzed the breadths, magnitudes, and differentiation phenotypes of memory LCMV-specific CD8(+) and CD4(+) T cells in three human donors displaying a variety of disease outcomes after accidental needle stick injury or exposure to LCMV. Although only a small cohort of donors was analyzed at a single time point postinfection, several interesting observations were made. First, we were able to detect LCMV-specific CD8(+) and CD4(+) T cell responses directly ex vivo at 4 to 8 years after exposure, demonstrating the longevity of T cell memory in humans. Second, unlike in murine models of LCMV infection, we found that the breadths of memory CD8(+) and CD4(+) T cell responses were not significantly different from one another. Third, it seemed that the overall CD8(+) T cell response was augmented with increasing severity of disease, while the LCMV-specific CD4(+) T cell response magnitude was highly variable between the three different donors. Next, we found that LCMV-specific CD8(+) T cells in the three donors analyzed seemed to undergo an effector memory differentiation program distinct from that of CD4(+) T cells. Finally, the levels of expression of memory, costimulatory, and inhibitory receptors on CD8(+) and CD4(+) T cell subsets, in some instances, correlated with disease outcome. These data demonstrate for the first time LCMV-specific CD8(+) and CD4(+) T cells in infected humans and begin to provide new insights into memory T cell responses following an acute virus infection.

Acquisition of Pneumococci Specific Effector and Regulatory Cd4+ T Cells Localising Within Human Upper Respiratory-tract Mucosal Lymphoid Tissue

PLoS Pathogens. Dec, 2011  |  Pubmed ID: 22144893

The upper respiratory tract mucosa is the location for commensal Streptococcus (S.) pneumoniae colonization and therefore represents a major site of contact between host and bacteria. The CD4(+) T cell response to pneumococcus is increasingly recognised as an important mediator of immunity that protects against invasive disease, with data suggesting a critical role for Th17 cells in mucosal clearance. By assessing CD4 T cell proliferative responses we demonstrate age-related sequestration of Th1 and Th17 CD4(+) T cells reactive to pneumococcal protein antigens within mucosal lymphoid tissue. CD25(hi) T cell depletion and utilisation of pneumococcal specific MHCII tetramers revealed the presence of antigen specific Tregs that utilised CTLA-4 and PDL-1 surface molecules to suppress these responses. The balance between mucosal effector and regulatory CD4(+) T cell immunity is likely to be critical to pneumococcal commensalism and the prevention of unwanted pathology associated with carriage. However, if dysregulated, such responses may render the host more susceptible to invasive pneumococcal infection and adversely affect the successful implementation of both polysaccharide-conjugate and novel protein-based pneumococcal vaccines.

DRB1*12:01 Presents a Unique Subset of Epitopes by Preferring Aromatics in Pocket 9

Molecular Immunology. Feb, 2012  |  Pubmed ID: 22196942

This study characterized the unique peptide-binding characteristics of HLA-DRB1*12:01 (DR1201), an allele studied in the context of various autoimmune diseases, using a peptide competition assay and structural modeling. After defining Influenza A/Puerto Rico/8/34 Matrix Protein M1 (H1MP) 40-54 as a DR1201 restricted epitope, the critical anchor residues within this sequence were confirmed by measuring the relative binding of peptides with non-conservative substitutions in competition with biotin labeled H1MP(40-54) peptide. Based on this information, a set of peptides was designed with single amino acid substitutions at these anchor positions. The overall peptide binding preferences for the DR1201 allele were deduced by incubating these peptides in competition with the reference H1MP(40-54) to determine the relative binding affinities of each to recombinant DR1201 protein. As expected, pocket 1 preferred methionine and aliphatic residues, and tolerated phenylalanine. Pocket 4 was mostly composed of hydrophobic residues, thereby preferentially accommodating aliphatic residues, but could also weakly accommodate lysine due to its slightly acidic environment. Pocket 6 accepted a wide range of amino acids because of the diverse residues that comprise this pocket. Pocket 9 accepted aliphatic and negatively charged amino acids, but showed a remarkable preference for aromatic residues due to the conformation of the pocket, which lacks the typical salt bridge between β57Asp and α76Arg. These binding characteristics contrast with the closely related DR1104 allele, distinguishing DR1201 among the alleles of the HLA-DR5 group. These empirical results were used to develop an algorithm to predict peptide binding to DR1201. This algorithm was used to verify T cell epitopes within novel antigenic peptides identified by tetramer staining and within peptides from published reports that contain putative DR1201 epitopes.

Broadly Directed Virus-specific CD4+ T Cell Responses Are Primed During Acute Hepatitis C Infection, but Rapidly Disappear from Human Blood with Viral Persistence

The Journal of Experimental Medicine. Jan, 2012  |  Pubmed ID: 22213804

Vigorous proliferative CD4(+) T cell responses are the hallmark of spontaneous clearance of acute hepatitis C virus (HCV) infection, whereas comparable responses are absent in chronically evolving infection. Here, we comprehensively characterized the breadth, specificity, and quality of the HCV-specific CD4(+) T cell response in 31 patients with acute HCV infection and varying clinical outcomes. We analyzed in vitro T cell expansion in the presence of interleukin-2, and ex vivo staining with HCV peptide-loaded MHC class II tetramers. Surprisingly, broadly directed HCV-specific CD4(+) T cell responses were universally detectable at early stages of infection, regardless of the clinical outcome. However, persistent viremia was associated with early proliferative defects of the HCV-specific CD4(+) T cells, followed by rapid deletion of the HCV-specific response. Only early initiation of antiviral therapy was able to preserve CD4(+) T cell responses in acute, chronically evolving infection. Our results challenge the paradigm that HCV persistence is the result of a failure to prime HCV-specific CD4(+) T cells. Instead, broadly directed HCV-specific CD4(+) T cell responses are usually generated, but rapid exhaustion and deletion of these cells occurs in the majority of patients. The data further suggest a short window of opportunity to prevent the loss of CD4(+) T cell responses through antiviral therapy.

Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2012  |  Pubmed ID: 22327072

The frequency of epitope-specific naive CD4(+) T cells in humans has not been extensively examined. In this study, a systematic approach was used to examine the frequency of CD4(+) T cells that recognize the protective Ag of Bacillus anthracis in both anthrax vaccine-adsorbed vaccinees and nonvaccinees with HLA-DRB1*01:01 haplotypes. Three epitopes were identified that had distinct degrees of immunodominance in subjects that had received the vaccine. Average naive precursor frequencies of T cells specific for these different epitopes in the human repertoire ranged from 0.2 to 10 per million naive CD4(+) T cells, which is comparable to precursor frequencies observed in the murine repertoire. Frequencies of protective Ag-specific T cells were two orders of magnitude higher in immunized subjects than in nonvaccinees. The frequencies of epitope-specific memory CD4(+) T cells in vaccinees were directly correlated with the frequencies of precursors in the naive repertoire. At the level of TCR usage, at least one preferred Vβ in the naive repertoire was present in the memory repertoire. These findings implicate naive frequencies as a crucial factor in shaping the epitope specificity of memory CD4(+) T cell responses.

Differentiation Stage Determines Pathologic and Protective Allergen-specific CD4(+) T-cell Outcomes During Specific Immunotherapy

The Journal of Allergy and Clinical Immunology. Feb, 2012  |  Pubmed ID: 21975176

The main obstacle to elucidating the role of CD4(+) T cells in allergen-specific immunotherapy (SIT) has been the absence of an adequately sensitive approach to directly characterize rare allergen-specific T cells without introducing substantial phenotypic modifications by means of in vitro amplification.

Induction and Maintenance of Allergen-specific FOXP3(+) Treg Cells in Human Tonsils As Potential First-line Organs of Oral Tolerance

The Journal of Allergy and Clinical Immunology. Feb, 2012  |  Pubmed ID: 22051696

Tonsils are strategically located in the gateway of both alimentary and respiratory tracts representing the first contact point of food and aeroallergens with the immune system. Tonsillectomy removes only the palatine tonsils and sometimes adenoids. Lingual tonsil is anatomically big and remains lifelong intact.