JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease.
Science
PUBLISHED: 10-18-2014
Show Abstract
Hide Abstract
T cells that mediate autoimmune diseases such as rheumatoid arthritis (RA) are difficult to characterize because they are likely to be deleted or inactivated in the thymus if the self antigens they recognize are ubiquitously expressed. One way to obtain and analyze these autoimmune T cells is to alter T cell receptor (TCR) signaling in developing T cells to change their sensitivity to thymic negative selection, thereby allowing their thymic production. From mice thus engineered to generate T cells mediating autoimmune arthritis, we isolated arthritogenic TCRs and characterized the self antigens they recognized. One of them was the ubiquitously expressed 60S ribosomal protein L23a (RPL23A), with which T cells and autoantibodies from RA patients reacted. This strategy may improve our understanding of the underlying drivers of autoimmunity.
Related JoVE Video
Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-29-2014
Show Abstract
Hide Abstract
Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA(g7) in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.
Related JoVE Video
Regulatory T cells modulate granulomatous inflammation in an HLA-DP2 transgenic murine model of beryllium-induced disease.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-27-2014
Show Abstract
Hide Abstract
Susceptibility to chronic beryllium disease (CBD) is linked to certain HLA-DP molecules, including HLA-DP2. To elucidate the molecular basis of this association, we exposed mice transgenic (Tg) for HLA-DP2 to beryllium oxide (BeO) via oropharyngeal aspiration. As opposed to WT mice, BeO-exposed HLA-DP2 Tg mice developed mononuclear infiltrates in a peribronchovascular distribution that were composed of CD4(+) T cells and included regulatory T (Treg) cells. Beryllium-responsive, HLA-DP2-restricted CD4(+) T cells expressing IFN-? and IL-2 were present in BeO-exposed HLA-DP2 Tg mice and not in WT mice. Using Be-loaded HLA-DP2-peptide tetramers, we identified Be-specific CD4(+) T cells in the mouse lung that recognize identical ligands as CD4(+) T cells derived from the human lung. Importantly, a subset of HLA-DP2 tetramer-binding CD4(+) T cells expressed forkhead box P3, consistent with the expansion of antigen-specific Treg cells. Depletion of Treg cells in BeO-exposed HLA-DP2 Tg mice exacerbated lung inflammation and enhanced granuloma formation. These findings document, for the first time to our knowledge, the development of a Be-specific adaptive immune response in mice expressing HLA-DP2 and the ability of Treg cells to modulate the beryllium-induced granulomatous immune response.
Related JoVE Video
Tolerance induction in memory CD4 T cells requires two rounds of antigen-specific activation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-12-2014
Show Abstract
Hide Abstract
A major goal for immunotherapy is to tolerize the immune cells that coordinate tissue damage in autoimmune and alloantigen responses. CD4 T cells play a central role in many of these conditions and improved antigen-specific regulation or removal of these cells could revolutionize current treatments. A confounding factor is that little is known about whether and how tolerance is induced in memory CD4 T cells. We used MHC class II tetramers to track and analyze a population of endogenous antigen-specific memory CD4 T cells exposed to soluble peptide in the absence of adjuvant. We found that such memory T cells proliferated and reentered the memory pool apparently unperturbed by the incomplete activation signals provided by the peptide. Upon further restimulation in vivo, CD4 memory T cells that had been previously exposed to peptide proliferated, provided help to primary responding B cells, and migrated to inflamed sites. However, these reactivated memory cells failed to survive. The reduction in T-cell number was marked by low expression of the antiapoptotic molecule B cell lymphoma 2 (Bcl2) and increased expression of activated caspase molecules. Consequently, these cells failed to sustain a delayed-type hypersensitivity response. Moreover, following two separate exposures to soluble antigen, no T-cell recall response and no helper activity for B cells could be detected. These results suggest that the induction of tolerance in memory CD4 T cells is possible but that deletion and permanent removal of the antigen-specific T cells requires reactivation following exposure to the tolerogenic antigen.
Related JoVE Video
Identification of multiple public TCR repertoires in chronic beryllium disease.
J. Immunol.
PUBLISHED: 04-09-2014
Show Abstract
Hide Abstract
Chronic beryllium disease (CBD) is a granulomatous lung disease characterized by the accumulation of beryllium (Be)-specific CD4(+) T cells in bronchoalveolar lavage. These expanded CD4(+) T cells are composed of oligoclonal T cell subsets, suggesting their recruitment to the lung in response to conventional Ag. In the current study, we noted that all bronchoalveolar lavage-derived T cell lines from HLA-DP2-expressing CBD patients contained an expansion of Be-responsive V?5.1(+) CD4(+) T cells. Using Be-loaded HLA-DP2-peptide tetramers, the majority of tetramer-binding T cells also expressed V?5.1 with a highly conserved CDR3? motif. Interestingly, Be-specific, V?5.1-expressing CD4(+) T cells displayed differential HLA-DP2-peptide tetramer staining intensity, and sequence analysis of the distinct tetramer-binding subsets showed that the two populations differed by a single conserved amino acid in the CDR3? motif. TCR V?-chain analysis of purified V?5.1(+) CD4(+) T cells based on differential tetramer-binding intensity showed differing TCR V?-chain pairing requirements, with the high-affinity population having promiscuous V?-chain pairing and the low-affinity subset requiring restricted V?-chain usage. Importantly, disease severity, as measured by loss of lung function, was inversely correlated with the frequency of tetramer-binding CD4(+) T cells in the lung. Our findings suggest the presence of a dominant Be-specific, V?5.1-expressing public T cell repertoire in the lungs of HLA-DP2-expressing CBD patients using promiscuous V?-chain pairing to recognize an identical HLA-DP2-peptide/Be complex. Importantly, the inverse relationship between expansion of CD4(+) T cells expressing these public TCRs and disease severity suggests a pathogenic role for these T cells in CBD.
Related JoVE Video
Monoclonal antibody blocking the recognition of an insulin peptide-MHC complex modulates type 1 diabetes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-03-2014
Show Abstract
Hide Abstract
The primary autoantigen triggering spontaneous type 1 diabetes mellitus in nonobese diabetic (NOD) mice is insulin. The major T-cell insulin epitope lies within the amino acid 9-23 peptide of the ?-chain (B:9-23). This peptide can bind within the peptide binding groove of the NOD MHC class II molecule (MHCII), IA(g7), in multiple positions or "registers." However, the majority of pathogenic CD4 T cells recognize this complex only when the insulin peptide is bound in register 3 (R3). We hypothesized that antibodies reacting specifically with R3 insulin-IA(g7) complexes would inhibit autoimmune diabetes specifically without interfering with recognition of other IA(g7)-presented antigens. To test this hypothesis, we generated a monoclonal antibody (mAb287), which selectively binds to B:9-23 and related variants when presented by IA(g7) in R3, but not other registers. The monoclonal antibody blocks binding of IA(g7)-B:10-23 R3 tetramers to cognate T cells and inhibits T-cell responses to soluble B:9-23 peptides and NOD islets. However, mAb287 has no effect on recognition of other peptides bound to IA(g7) or other MHCII molecules. Intervention with mAb287, but not irrelevant isotype matched antibody, at either early or late stages of disease development, significantly delayed diabetes onset by inhibiting infiltration by not only insulin-specific CD4 T cells, but also by CD4 and CD8 T cells of other specificities. We propose that peptide-MHC-specific monoclonal antibodies can modulate autoimmune disease without the pleiotropic effects of nonselective reagents and, thus, could be applicable to the treatment of multiple T-cell mediated autoimmune disorders.
Related JoVE Video
Structural basis of chronic beryllium disease: linking allergic hypersensitivity and autoimmunity.
Cell
PUBLISHED: 02-02-2014
Show Abstract
Hide Abstract
T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.
Related JoVE Video
Molecules in medicine mini review: the ?? T cell receptor.
J. Mol. Med.
PUBLISHED: 01-21-2014
Show Abstract
Hide Abstract
As an integral part of the mammalian immune system, a distributed network of tissues, cells, and extracellular factors, T lymphocytes perform and control a multitude of activities that collectively contribute to the effective establishment, maintenance, and restoration of tissue and organismal integrity. Development and function of T cells is controlled by the T cell receptor (TCR), a heterodimeric cell surface protein uniquely expressed on T cells. During T cell development, the TCR undergoes extensive somatic diversification that generates a diverse T cell repertoire capable of recognizing an extraordinary range of protein and nonprotein antigens presented in the context of major histocompatibility complex molecules (MHC). In this review, we provide an introduction to the TCR, describing underlying principles that position this molecule as a central regulator of the adaptive immune system involved in responses ranging from tissue protection and preservation to pathology and autoimmunity.
Related JoVE Video
Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-16-2013
Show Abstract
Hide Abstract
The self-reactivity of their T-cell antigen receptor (TCR) is thought to contribute to the development of immune regulatory cells, such as invariant NK T cells (iNKT). In the mouse, iNKT cells express TCRs composed of a unique V?14-J?18 rearrangement and recognize lipid antigens presented by CD1d molecules. We created mice expressing a transgenic TCR-? chain that confers high affinity for self-lipid/CD1d complexes when randomly paired with the mouse iNKT V?14-J?18 rearrangement to study their development. We show that although iNKT cells undergo agonist selection, their development is also shaped by negative selection in vivo. In addition, iNKT cells that avoid negative selection in these mice express natural sequence variants of the canonical TCR-? and decreased affinity for self/CD1d. However, limiting the affinity of the iNKT TCRs for "self" leads to inefficient Egr2 induction, poor expression of the iNKT lineage-specific zinc-finger transcription factor PLZF, inadequate proliferation of iNKT cell precursors, defects in trafficking, and impaired effector functions. Thus, proper development of fully functional iNKT cells is constrained by a limited range of TCR affinity that plays a key role in triggering the iNKT cell-differentiation pathway. These results provide a direct link between the affinity of the TCR expressed by T-cell precursors for self-antigens and the proper development of a unique population of lymphocytes essential to immune responses.
Related JoVE Video
Improving antigenic peptide vaccines for cancer immunotherapy using a dominant tumor-specific T cell receptor.
J. Biol. Chem.
PUBLISHED: 10-08-2013
Show Abstract
Hide Abstract
Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react with or that have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.
Related JoVE Video
T-box transcription factor T-bet, a key player in a unique type of B-cell activation essential for effective viral clearance.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-06-2013
Show Abstract
Hide Abstract
IgG2a is known to be the most efficient antibody isotype for viral clearance. Here, we demonstrate a unique pathway of B-cell activation, leading to IgG2a production, and involving synergistic stimulation via B-cell antigen receptors, toll-like receptor 7 (TLR7), and IFN? receptors on B cells. This synergistic stimulation leads to induction of T-box transcription factor T-bet expression in B cells, which, in turn, drives expression of CD11b and CD11c on B cells. T-bet/CD11b/CD11c positive B cells appear during antiviral responses and produce high titers of antiviral IgG2a antibodies that are critical for efficient viral clearance. The results thus demonstrate a previously unknown role for T-bet expression in B cells during viral infections. Moreover, the appearance of T-bet(+) B cells during antiviral responses and during autoimmunity suggests a possible link between these two processes.
Related JoVE Video
Identification of beryllium-dependent peptides recognized by CD4+ T cells in chronic beryllium disease.
J. Exp. Med.
PUBLISHED: 06-26-2013
Show Abstract
Hide Abstract
Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of beryllium (Be)-specific CD4? T cells into the lung. The vast majority of these T cells recognize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition. However, the peptides that stimulate Be-specific T cells are unknown. Using positional scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP molecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to MHCII and Be, forming a complex recognized by pathogenic CD4? T cells in CBD. These peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that surround the putative Be-binding site and cooperate with HLA-DP2 in Be coordination. Endogenous plexin A peptides and proteins, which share the core motif and are expressed in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4 tetramers detected high frequencies of CD4? T cells specific for these ligands in all HLADP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4? T cell involved in metal-induced hypersensitivity and suggest a unique role of these peptides in metal ion coordination and the generation of a common antigen specificity in CBD.
Related JoVE Video
TLR7 drives accumulation of ABCs and autoantibody production in autoimmune-prone mice.
Immunol. Res.
PUBLISHED: 06-14-2013
Show Abstract
Hide Abstract
Although autoantibodies are the hallmarks of most autoimmune diseases, the mechanisms by which autoreactive B cells are generated and accumulate are still poorly understood. Overexpression of Toll-like receptor 7 (TLR7) that recognizes single-stranded RNAs has been implicated in systemic lupus erythematosus (SLE), although the cellular mechanism by which this receptor drives the disease is unknown. We recently identified a population of CD11c(+) age-associated B cells (ABCs) which is driven by TLR7 signaling, secretes autoantibodies and appears in autoimmune-prone mice by the time of onset of autoimmunity. Mice lacking the Mer receptor develop autoantibodies and splenomegaly similar to other mouse models of SLE. Here, we show that Mer(-/-) mice that lack TLR7 fail to develop anti-chromatin IgG antibodies, perhaps because they also fail to develop ABCs. Moreover, depletion of CD11c(+) ABCs from Mer(-/-) mice leads to rapid reduction in autoantibodies. Together, these data strongly suggest that ABCs and/or their descendants are the primary source of autoantibodies in Mer(-/-) mice and that TLR7 signaling is crucial for accumulation of ABCs and development of autoantibodies. These data demonstrate for the first time that TLR7, and not TLR9, is responsible for generation of anti-chromatin IgG antibodies in Mer(-/-) mice.
Related JoVE Video
Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-27-2013
Show Abstract
Hide Abstract
Many vaccines include aluminum salts (alum) as adjuvants despite little knowledge of alums functions. Host DNA rapidly coats injected alum. Here, we further investigated the mechanism of alum and DNAs adjuvant function. Our data show that DNase coinjection reduces CD4 T-cell priming by i.m. injected antigen + alum. This effect is partially replicated in mice lacking stimulator of IFN genes, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNase treatment impairs dendritic cell (DC) migration from the peritoneal cavity to the draining lymph node in mice immunized i.p. with alum. However, our data show that DNase does not affect accumulation of, or expression of costimulatory proteins on, antigen-loaded DCs in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNase does inhibit prolonged T-cell-DC conjugate formation and antigen presentation between antigen-positive DCs and antigen-specific CD4 T cells following i.m. injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DCs, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC class II presentation and better DC-T-cell interactions.
Related JoVE Video
The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.
PLoS ONE
PUBLISHED: 01-21-2013
Show Abstract
Hide Abstract
The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.
Related JoVE Video
MAIT cell recognition of MR1 on bacterially infected and uninfected cells.
PLoS ONE
PUBLISHED: 01-14-2013
Show Abstract
Hide Abstract
Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant ?? TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.
Related JoVE Video
Recognition of self and altered self by T cells in autoimmunity and allergy.
Protein Cell
PUBLISHED: 01-11-2013
Show Abstract
Hide Abstract
T cell recognition of foreign peptide antigen and tolerance to self peptides is key to the proper function of the immune system. Usually, in the thymus T cells that recognize self MHC + self peptides are deleted and those with the potential to recognize self MHC + foreign peptides are selected to mature. However there are exceptions to these rules. Autoimmunity and allergy are two of the most common immune diseases that can be related to recognition of self. Many genes work together to lead to autoimmunity. Of those, particular MHC alleles are the most strongly associated, reflecting the key importance of MHC presentation of self peptides in autoimmunity. T cells specific for combinations of self MHC and self peptides may escape thymus deletion, and thus be able to drive autoimmunity, for several reasons: the relevant self peptide may be presented at low abundance in the thymus but at high level in particular peripheral tissues; the relevant self peptide may bind to MHC in an unusual register, not present in the thymus but apparent elsewhere; finally the relevant self peptide may be post translationally modified in a tissue specific fashion. In some types of allergy, the peptide + MHC combination may also be fully derived from self. However the combination in question may be modified by the presence of other ligands, such as small drug molecules or metal ions. Thus these types of allergies may act like the post translationally modified peptides involved some types of autoimmunity.
Related JoVE Video
Influenza nucleoprotein delivered with aluminium salts protects mice from an influenza A virus that expresses an altered nucleoprotein sequence.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.
Related JoVE Video
Immunization with an insulin peptide-MHC complex to prevent type 1 diabetes of NOD mice.
Diabetes Metab. Res. Rev.
PUBLISHED: 11-10-2011
Show Abstract
Hide Abstract
Mutating the insulin B:9-23 peptide prevents diabetes in NOD mice. Thus, the trimolecular complex of I-Ag7-insulin B:9-23 peptide-TCR may be essential for the development of spontaneous diabetes. Pathogenic T cells recognize the B:9-23 peptide presented by I-Ag7 in what is termed register 3, with the B22 basic amino acid (arginine) of the peptide bound in pocket 9 of I-Ag7. Our hypothesis is that immunization with an insulin B:12-22 peptide linked to I-Ag7 in register 3 (I-Ag7-B:RE#3 complex) can induce specific antibodies to the complex, block pathogenic TCRs, and thus prevent diabetes.
Related JoVE Video
Specificity and detection of insulin-reactive CD4+ T cells in type 1 diabetes in the nonobese diabetic (NOD) mouse.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-26-2011
Show Abstract
Hide Abstract
In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9-23) is a major target for pathogenic CD4(+) T cells. However, there is no consensus on the relative importance of the various positions or "registers" this peptide can take when bound in the groove of the NOD MHCII molecule, IA(g7). This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9-23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4(+) T cells react to B:9-23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IA(g7) tetramers that bind to most insulin-specific T-cell clones tested. A mixture of these tetramers detected a high frequency of B:9-23-reactive CD4(+) T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9-23 binding to IA(g7) in register 3 may explain the escape of insulin-specific CD4(+) T cells from the mechanisms that usually eliminate self-reactive T cells.
Related JoVE Video
Mutagenesis of beryllium-specific TCRs suggests an unusual binding topology for antigen recognition.
J. Immunol.
PUBLISHED: 08-26-2011
Show Abstract
Hide Abstract
Unconventional Ags, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of Be-specific TCRs derived from the lung of a chronic beryllium disease patient. These TCRs are HLA-DP2-restricted and express nearly identical TCR V?5.1 chains coupled with different TCR ?-chains. Site-specific mutagenesis of all amino acids comprising the CDRs of the TCRA and TCRB genes showed a dominant role for V?5.1 residues in Be recognition, with little contribution from the TCR ?-chain. Solvent-exposed residues along the ?-helices of the HLA-DP2 ?- and ?-chains were also mutated to alanine. Two ?-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition with no contribution from the HLA-DP2 ?-chain. These findings suggest that Be-specific T cells recognize Ag using an unconventional binding topology, with the majority of interactions contributed by TCR V?5.1 residues and the HLA-DP2 ?1-chain. Thus, unusual docking topologies are not exclusively used by autoreactive T cells, but also for the recognition of unconventional metal Ags, such as Be.
Related JoVE Video
IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity.
J. Immunol.
PUBLISHED: 08-03-2011
Show Abstract
Hide Abstract
Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. In this study, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild-type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, Mer(KD), MRL(lpr), and New Zealand Black/WF1 did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum, and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by Abs against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.
Related JoVE Video
Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c? B-cell population is important for the development of autoimmunity.
Blood
PUBLISHED: 05-04-2011
Show Abstract
Hide Abstract
Females are more susceptible than males to many autoimmune diseases. The processes causing this phenomenon are incompletely understood. Here, we demonstrate that aged female mice acquire a previously uncharacterized population of B cells that we call age-associated B cells (ABCs) and that these cells express integrin ?(X) chain (CD11c). This unexpected population also appears in young lupus-prone mice. On stimulation, CD11c(+) B cells, both from autoimmune-prone and healthy strains of mice, secrete autoantibodies, and depletion of these cells in vivo leads to reduction of autoreactive antibodies, suggesting that the cells might have a direct role in the development of autoimmunity. We have explored factors that contribute to appearance of ABCs and demonstrated that signaling through Toll-like receptor 7 is crucial for development of this B cell population. We were able to detect a similar population of B cells in the peripheral blood of some elderly women with autoimmune disease, suggesting that there may be parallels between the creation of ABC-like cells between mice and humans.
Related JoVE Video
Evolutionarily conserved features contribute to ?? T cell receptor specificity.
Immunity
PUBLISHED: 04-28-2011
Show Abstract
Hide Abstract
?? T cell receptors (TCRs) bind specifically to foreign antigens presented by major histocompatibility complex proteins (MHC) or MHC-like molecules. Accumulating evidence indicates that the germline-encoded TCR segments have features that promote binding to MHC and MHC-like molecules, suggesting coevolution between TCR and MHC molecules. Here, we assess directly the evolutionary conservation of ?? TCR specificity for MHC. Sequence comparisons showed that some V?s from distantly related jawed vertebrates share amino acids in their complementarity determining region 2 (CDR2). Chimeric TCRs containing amphibian, bony fish, or cartilaginous fish V?s can recognize antigens presented by mouse MHC class II and CD1d (an MHC-like protein), and this recognition is dependent upon the shared CDR2 amino acids. These results indicate that features of the TCR that control specificity for MHC and MHC-like molecules were selected early in evolution and maintained between species that last shared a common ancestor more than 400 million years ago.
Related JoVE Video
Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-25-2011
Show Abstract
Hide Abstract
Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells.
Related JoVE Video
A single T cell receptor bound to major histocompatibility complex class I and class II glycoproteins reveals switchable TCR conformers.
Immunity
PUBLISHED: 03-01-2011
Show Abstract
Hide Abstract
Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, V? disengages from the J? ? strand, switching V?s position relative to V?. In several other structures, either V? or V? undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands.
Related JoVE Video
Memory CD4 T cells that express CXCR5 provide accelerated help to B cells.
J. Immunol.
PUBLISHED: 01-26-2011
Show Abstract
Hide Abstract
CD4 T cell help for B cells is critical for effective Ab responses. Although many of the molecules involved in helper functions of naive CD4 T cells have been characterized, much less is known about the helper capabilities of memory CD4 T cells, an important consideration for the design of vaccines that aim to prime protective memory CD4 T cells. In this study, we demonstrate that memory CD4 T cells enable B cells to expand more rapidly and class switch earlier than do primary responding CD4 T cells. This accelerated response does not require large numbers of memory cells, and similar numbers of primary responding cells provide less effective help than do memory cells. However, only memory CD4 T cells that express the B cell follicle homing molecule, CXCR5, are able to accelerate the response, suggesting that the rapidity of the Ab response depends on the ability of CD4 memory T cells to migrate quickly toward B cells.
Related JoVE Video
Aluminum adjuvants elicit fibrin-dependent extracellular traps in vivo.
Blood
PUBLISHED: 09-27-2010
Show Abstract
Hide Abstract
It has been recognized for nearly 80 years that insoluble aluminum salts are good immunologic adjuvants and that they form long-lived nodules in vivo. Nodule formation has long been presumed to be central for adjuvant activity by providing an antigen depot, but the composition and function of these nodules is poorly understood. We show here that aluminum salt nodules formed within hours of injection and contained the clotting protein fibrinogen. Fibrinogen was critical for nodule formation and required processing to insoluble fibrin by thrombin. DNase treatment partially disrupted the nodules, and the nodules contained histone H3 and citrullinated H3, features consistent with extracellular traps. Although neutrophils were not essential for nodule formation, CD11b(+) cells were implicated. Vaccination of fibrinogen-deficient mice resulted in normal CD4 T-cell and antibody responses and enhanced CD8 T-cell responses, indicating that nodules are not required for aluminums adjuvant effect. Moreover, the ability of aluminum salts to retain antigen in the body, the well-known depot effect, was unaffected by the absence of nodules. We conclude that aluminum adjuvants form fibrin-dependent nodules in vivo, that these nodules have properties of extracellular traps, and the nodules are not required for aluminum salts to act as adjuvants.
Related JoVE Video
Interaction of JMJD6 with single-stranded RNA.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-02-2010
Show Abstract
Hide Abstract
JMJD6 is a Jumonji C domain-containing hydroxylase. JMJD6 binds alpha-ketoglutarate and iron and has been characterized as either a histone arginine demethylase or U2AF65 lysyl hydroxylase. Here, we describe the structures of JMJD6 with and without alpha-ketoglutarate, which revealed a novel substrate binding groove and two positively charged surfaces. The structures also contain a stack of aromatic residues located near the active center. The side chain of one residue within this stack assumed different conformations in the two structures. Interestingly, JMJD6 bound efficiently to single-stranded RNA, but not to single-stranded DNA, double-stranded RNA, or double-stranded DNA. These structural features and truncation analysis of JMJD6 suggest that JMJD6 may bind and modify single-stand RNA rather than the previously reported peptide substrates.
Related JoVE Video
Diabetogenic T cells recognize insulin bound to IAg7 in an unexpected, weakly binding register.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-01-2010
Show Abstract
Hide Abstract
A peptide derived from the insulin B chain contains a major epitope for diabetogenic CD4(+) T cells in the NOD mouse model of type 1 diabetes (T1D). This peptide can fill the binding groove of the NOD MHCII molecule, IA(g7), in a number of ways or "registers." We show here that a diverse set of NOD anti-insulin T cells all recognize this peptide bound in the same register. Surprisingly, this register results in the poorest binding of peptide to IA(g7). The poor binding is due to an incompatibility between the p9 amino acid of the peptide and the unique IA(g7) p9 pocket polymorphisms that are strongly associated with susceptibility to T1D. Our findings suggest that the association of autoimmunity with particular MHCII alleles may be do to poorer, rather than more favorable, binding of the critical self-epitopes, allowing T-cell escape from thymic deletion.
Related JoVE Video
Linking genetic susceptibility and T cell activation in beryllium-induced disease.
Proc Am Thorac Soc
PUBLISHED: 04-30-2010
Show Abstract
Hide Abstract
Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium (Be) exposure in the workplace. It is characterized by the accumulation of Be-specific CD4(+) T cells in the lung as well as persistent lung inflammation, culminating in the development of lung fibrosis. CBD occurs in 2 to 16% of Be-exposed workers depending on the individuals genetic susceptibility and the characteristics of the exposure. Genetic susceptibility to Be-induced disease has been linked to major histocompatibility complex class II molecules. In particular, HLA-DP alleles possessing a glutamic acid at the 69th position of the beta-chain (betaGlu69) are most strongly linked to disease susceptibility. The HLA-DP alleles that present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. However, the structural features of betaGlu69-containing HLA-DP molecules that explain the disease association remain unknown. We have recently crystallized HLA-DP2, which is the most prevalent of the betaGlu69-containing HLA-DP molecules. Its unique structure, which includes surface exposure of betaGlu69, provides an explanation of the genetic linkage between betaGlu69-containing HLA-DP alleles and Be-induced disease.
Related JoVE Video
Immune mechanisms of protection: can adjuvants rise to the challenge?
BMC Biol.
PUBLISHED: 04-08-2010
Show Abstract
Hide Abstract
For many diseases vaccines are lacking or only partly effective. Research on protective immunity and adjuvants that generate vigorous immune responses may help generate effective vaccines against such pathogens.
Related JoVE Video
Crystal structure of HLA-DP2 and implications for chronic beryllium disease.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-31-2010
Show Abstract
Hide Abstract
Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.
Related JoVE Video
Memory CD4 T cells: generation, reactivation and re-assignment.
Immunology
PUBLISHED: 03-16-2010
Show Abstract
Hide Abstract
Immunological memory is one of the features that define the adaptive immune response: by generating specific memory cells after infection or vaccination, the host provides itself with a set of cells and molecules that can prevent future infections and disease. Despite the obvious importance of memory cells, memory CD4 T cells are incompletely understood. Here we discuss recent progress in understanding which activated T cells surmount the barrier to enter into the memory pool and, once generated, what signals are important for memory cell survival. There is still, however, little understanding of how (or even whether) memory CD4 T cells are useful once they have been created; a surprising thought considering the critical role CD4 T cells play in all adaptive primary immune responses. In light of this, we will discuss how CD4 T memory T cells respond to reactivation in vivo and whether they are malleable to a re-assignment of their effector response.
Related JoVE Video
Molecular targeting of islet autoantigens.
Immunity
PUBLISHED: 02-19-2010
Show Abstract
Hide Abstract
Type 1 diabetes of man and animal models results from immune-mediated specific beta cell destruction. Multiple islet antigens are targets of autoimmunity and most of these are not beta cell specific. Immune responses to insulin appear to be essential for the development of diabetes of the NOD mouse. In this review, we will emphasize the unusual manner in which selected autoantigenic peptides (particularly the recently discovered target of BDC2.5 T cells [chromagranin A]) are presented and recognized by autoreactive CD4(+) T cell receptors. We hypothesize that "unusual" structural interactions of specific trimolecular complexes (MHC class II, peptide, and T cell receptors) are fundamental to the escape from the thymus of autoreactive T cells able to cause type 1 diabetes.
Related JoVE Video
Genetic and hormonal factors in female-biased autoimmunity.
Autoimmun Rev
PUBLISHED: 02-06-2010
Show Abstract
Hide Abstract
Autoimmunity is controlled both by the environment and by genetic factors. One of the most well defined genetic factors is polymorphisms, with some alleles of particular genes promoting autoimmune diseases, whereas other alleles either not affecting susceptibility to disease or, in some cases actually inhibiting the appearance of such illnesses. Another genetically controlled factor, gender, also plays a profound role in the incidence of autoimmune diseases. For example, Systemic Lupus Erythematosus (SLE) occurs much more frequently in females than in males in both mice and man. The genetic differences that make some individuals susceptible to autoimmunity and protect others could act in many ways and affect many tissues. In this review we will discuss how gender may act on the cells of the immune system and thereby influence the predisposition of the host to autoimmune diseases.
Related JoVE Video
Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-26-2010
Show Abstract
Hide Abstract
Peptide vaccines enhance the response of T cells toward tumor antigens and represent a strategy to augment antigen-independent immunotherapies of cancer. However, peptide vaccines that include native tumor antigens rarely prevent tumor growth. We have assembled a set of peptide variants for a mouse-colon tumor model to determine how to improve T-cell responses. These peptides have similar affinity for MHC molecules, but differ in the affinity of the peptide-MHC/T-cell receptor interaction with a tumor-specific T-cell clone. We systematically demonstrated that effective antitumor responses are generated after vaccination with variant peptides that stimulate the largest proportion of endogenous T cells specific for the native tumor antigen. Importantly, we found some variant peptides that strongly stimulated a specific T-cell clone in vitro, but elicited fewer tumor-specific T cells in vivo, and were not protective. The T cells expanded by the effective vaccines responded to the wild-type antigen by making cytokines and killing target cells, whereas most of the T cells expanded by the ineffective vaccines only responded to the peptide variants. We conclude that peptide-variant vaccines are most effective when the peptides react with a large responsive part of the tumor-specific T-cell repertoire.
Related JoVE Video
Chromogranin A is an autoantigen in type 1 diabetes.
Nat. Immunol.
PUBLISHED: 01-15-2010
Show Abstract
Hide Abstract
Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.
Related JoVE Video
Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity.
J. Immunol.
PUBLISHED: 09-04-2009
Show Abstract
Hide Abstract
To understand more about how the body recognizes alum we characterized the early innate and adaptive responses in mice injected with the adjuvant. Within hours of exposure, alum induces a type 2 innate response characterized by an influx of eosinophils, monocytes, neutrophils, DCs, NK cells and NKT cells. In addition, at least 13 cytokines and chemokines are produced within 4 h of injection including IL-1beta and IL-5. Optimal production of some of these, including IL-1beta, depends upon both macrophages and mast cells, whereas production of others, such as IL-5, depends on mast cells only, suggesting that both of these cell types can detect alum. Alum induces eosinophil accumulation partly through the production of mast cell derived IL-5 and histamine. Alum greatly enhances priming of endogenous CD4 and CD8 T cells independently of mast cells, macrophages, and of eosinophils. In addition, Ab levels and Th2 bias was similar in the absence of these cells. We found that the inflammation induced by alum was unchanged in caspase-1-deficient mice, which cannot produce IL-1beta. Furthermore, endogenous CD4 and CD8 T cell responses, Ab responses and the Th2 bias were also not impacted by the absence of caspase-1 or NLRP3. These data suggest that activation of the inflammasome and the type 2 innate response orchestrated by macrophages and mast cells in vivo are not required for adjuvant effect of alum on endogenous T and B cell responses.
Related JoVE Video
T cell allorecognition via molecular mimicry.
Immunity
PUBLISHED: 06-10-2009
Show Abstract
Hide Abstract
T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.
Related JoVE Video
Many different Vbeta CDR3s can reveal the inherent MHC reactivity of germline-encoded TCR V regions.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-28-2009
Show Abstract
Hide Abstract
We have hypothesized that in the prenegative selection TCR repertoire, many somatically generated complementary-determining region (CDR) 3 loops combine with evolutionarily selected germline Valpha/Vbeta CDR1/CDR2 loops to create highly MHC/peptide cross-reactive T cells that are subsequently deleted by negative selection. Here, we present a mutational analysis of the Vbeta CDR3 of such a cross-reactive T-cell receptor (TCR), YAe62. Most YAe62 TCRs with the mutant CDR3s became less MHC promiscuous. However, others with CDR3s unrelated in sequence to the original recognized even more MHC alleles than the original TCR. Most importantly, this recognition was still dependent on the conserved CDR1/CDR2 residues. These results bolster the idea that germline TCR V elements are inherently reactive to MHC but that this reactivity is fine-tuned by the somatically generated CDR3 loops.
Related JoVE Video
Vaccination of mice with baculovirus-infected insect cells expressing antigenic proteins.
Curr Protoc Immunol
PUBLISHED: 04-07-2009
Show Abstract
Hide Abstract
Methods to induce antigen-specific immune responses in mice using insect cells infected with recombinant baculoviruses are described in this unit. Although this vaccine strategy has been used to generate both antibody and T cell responses, it has been more thoroughly characterized for the peptide-specific cytotoxic T cell responses. Nonspecific responses to the vaccine vehicle are controlled for by vaccinating with insect cells infected with baculoviruses encoding irrelevant antigens or no antigen. The baculovirus-infected insect cells alone are an effective immune adjuvant to elicit antigen-specific T cells. Overall, immune responses generated using this approach are similar to those generated by more conventional vaccine strategies.
Related JoVE Video
The Epstein-Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a limited amount of Bim.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-17-2009
Show Abstract
Hide Abstract
Current knowledge suggests that the balance between life and death within a cell can be controlled by the stable engagement of Bcl-2-related proapoptotic proteins such as Bak, Bax, and Bim by survival proteins such as Bcl-2. BHRF1 is a prosurvival molecule from Epstein-Barr virus that has a high degree of homology to Bcl-2. To understand how BHRF1 blocks apoptosis, BHRF1 and mutants of BHRF1 were expressed in primary cells and an IL-2-dependent T cell line. BHRF1 bound the Executioner Bak and, when cells were cultured without cytokines, BHRF1 associated with Bim. A point mutation that lost the ability to bind Bak retained its ability to bind Bim and to protect cells. This result demonstrated that it was the capacity of BHRF1 to bind Bim, not Bak, that provided protection. Interestingly, the amount of Bim bound by BHRF1 was minimal when compared with the amount of Bim induced by apoptosis. Thus, BHRF1 does not act by simply absorbing the excess Bim produced while cells prepare for death. Rather, BHRF1 may act either by binding preferentially the most lethal form of Bim or by acting catalytically on Bim to block apoptosis.
Related JoVE Video
Transgenic Bcl-3 slows T cell proliferation.
Int. Immunol.
PUBLISHED: 02-10-2009
Show Abstract
Hide Abstract
Immunological adjuvants, such as bacterial LPS, increase the mRNA levels of the IkB-related NF-kappaB transcriptional transactivator, Bcl-3, in activated T cells. Adjuvants also increase the life expectancy of activated T cells, as does over-expression of Bcl-3, suggesting that Bcl-3 is part of the pathway whereby adjuvants affect T cell lifespans. However, previous reports, confirmed here, show that adjuvants also increase the life expectancies of Bcl-3-deficient T cells, making Bcl-3s role and effects in adjuvant-induced survival uncertain. To investigate the functions of Bcl-3 further, here we confirm the adjuvant-induced expression of Bcl-3 mRNA and show Bcl-3 induction at the protein level. Bcl-3 was expressed in mice via a transgene driven by the human CD2 promoter. Like other protective events, over-expression of Bcl-3 slows T cell activation very early in T cell responses to antigen, both in vitro and in vivo. This property was intrinsic to the T cells over-expressing the Bcl-3 and did not require Bcl-3 expression by other cells such as antigen-presenting cells.
Related JoVE Video
CD4 memory T cells: what are they and what can they do?
Semin. Immunol.
PUBLISHED: 02-04-2009
Show Abstract
Hide Abstract
Immunological memory provides the basis for successful vaccines. It is important to understand the properties of memory cells. There is much known about the phenotype and functions of memory CD8 T cells, less about memory B cells, while CD4 memory T cells have proved difficult to study. Differences in the types of memory CD4 cells studied and the difficulties of tracking the small number of cells have led to conflicting and unclear results. Here we discuss the different systems used to study CD4 memory cells and ask whether, and in what circumstances, memory CD4 cells could provide protection against infections.
Related JoVE Video
Germline-encoded amino acids in the alphabeta T-cell receptor control thymic selection.
Nature
PUBLISHED: 01-20-2009
Show Abstract
Hide Abstract
An alphabeta T-cell response depends on the recognition of antigen plus major histocompatibility complex (MHC) proteins by its antigen receptor (TCR). The ability of peripheral alphabeta T cells to recognize MHC is at least partly determined by MHC-dependent thymic selection, by which an immature T cell survives only if its TCR can recognize self MHC. This process may allow MHC-reactive TCRs to be selected from a repertoire with completely random and unbiased specificities. However, analysis of thymocytes before positive selection indicated that TCR proteins might have a predetermined ability to bind MHC. Here we show that specific germline-encoded amino acids in the TCR promote generic MHC recognition and control thymic selection. In mice expressing single, rearranged TCR beta-chains, individual mutation of amino acids in the complementarity-determining region (CDR) 2beta to Ala reduced development of the entire TCR repertoire. Altogether, these results show that thymic selection is controlled by germline-encoded MHC contact points in the alphabeta TCR and indicate that the diversity of the peripheral T-cell repertoire is enhanced by this built-in specificity.
Related JoVE Video
A conserved CXXC motif in CD3epsilon is critical for T cell development and TCR signaling.
PLoS Biol.
PUBLISHED: 01-14-2009
Show Abstract
Hide Abstract
Virtually all T cell development and functions depend on its antigen receptor. The T cell receptor (TCR) is a multi-protein complex, comprised of a ligand binding module and a signal transmission module. The signal transmission module includes proteins from CD3 family (CD3epsilon, CD3delta, CD3gamma) as well as the zeta chain protein. The CD3 proteins have a short extracellular stalk connecting their Ig-like domains to their transmembrane regions. These stalks contain a highly evolutionarily conserved CXXC motif, whose function is unknown. To understand the function of these two conserved cysteines, we generated mice that lacked endogenous CD3epsilon but expressed a transgenic CD3epsilon molecule in which these cysteines were mutated to serines. Our results show that the mutated CD3epsilon could incorporate into the TCR complex and rescue surface TCR expression in CD3epsilon null mice. In the CD3epsilon mutant mice, all stages of T cell development and activation that are TCR-dependent were impaired, but not eliminated, including activation of mature naïve T cells with the MHCII presented superantigen, staphylococcal enterotoxin B, or with a strong TCR cross-linking antibody specific for either TCR-Cbeta or CD3epsilon. These results argue against a simple aggregation model for TCR signaling and suggest that the stalks of the CD3 proteins may be critical in transmitting part of the activation signal directly through the membrane.
Related JoVE Video
Ikaros promotes rearrangement of TCR ? genes in an Ikaros null thymoma cell line.
Eur. J. Immunol.
Show Abstract
Hide Abstract
Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR ? gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR ? loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the ? locus and TCR V?/J? recombination resulting in the appearance of many new ??TCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR ? enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR ? rearrangement. Our data suggest that Ikaros controls V?/J? recombination in T cells by controlling access of the transcription and recombination machinery to the TCR ? loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to ??TCR(+) CD4(+) CD8(+) thymocyte transition.
Related JoVE Video
T-cell receptor (TCR) interaction with peptides that mimic nickel offers insight into nickel contact allergy.
Proc. Natl. Acad. Sci. U.S.A.
Show Abstract
Hide Abstract
T cell-mediated allergy to Ni(++) is one of the most common forms of allergic contact dermatitis, but how the T-cell receptor (TCR) recognizes Ni(++) is unknown. We studied a TCR from an allergic patient that recognizes Ni(++) bound to the MHCII molecule DR52c containing an unknown self-peptide. We identified mimotope peptides that can replace both the self-peptide and Ni(++) in this ligand. They share a p7 lysine whose ?NH(2) group is surface-exposed when bound to DR52c. Whereas the TCR uses germ-line complementary-determining region (CDR)1/2 amino acids to dock in the conventional diagonal mode on the mimotope-DR52c complex, the interface is dominated by the TCR V? CDR3 interaction with the p7 lysine. Mutations in the TCR CDR loops have similar effects on the T-cell response to either the mimotope or Ni(++) ligand. We suggest that the mimotope p7 lysine mimics Ni(++) in the natural TCR ligand and that MHCII ?-chain flexibility in the area around the peptide p7 position forms a common site for cation binding in metal allergies.
Related JoVE Video
T cells and their eons-old obsession with MHC.
Immunol. Rev.
Show Abstract
Hide Abstract
T cells bearing receptors made up of ? and ? chains (TCRs) usually react with peptides bound to major histocompatibility complex proteins (MHC). This bias could be imposed by positive selection, the phenomenon that selects thymocytes to mature into T cells only if the TCRs they bear react with low but appreciable affinity with MHC + peptide combinations in the thymus cortex. However, it is also possible that the polypeptides of TCRs themselves do not have random specificities but rather are biased toward reaction with MHC. Evolution would therefore have selected for a collection of TCR variable elements that are prone to react with MHC. If this were to be so, positive selection would act on thymocytes bearing a pre biased collection of TCRs to pick out those that react to some extent, but not too well, with self MHC + self-peptides. A problem with studies of this evolutionary idea is the fact that there are many TCR variable elements and that these differ considerably in the amino acids with which they contact MHC. However, recent experiments by our group and others suggest that one group of TCR variable elements, those related to the mouse V?8 family, has amino acids in their CDR2 regions that consistently bind a particular site on an MHC ?-helix. Other groups of variable elements may use different patterns of amino acids to achieve the same goal. Mutation of these amino acids reduces the ability of T cells and thymocytes to react with MHC. These amino acids are present in the variable regions of distantly related species such as sharks and human. Overall the data indicate that TCR elements have indeed been selected by evolution to react with MHC proteins. Many mysteries about TCRs remain to be solved, including the nature of auto-recognition, the basis of MHC allele specificity, and the very nature and complexity of TCRs on mature T cells.
Related JoVE Video
Do MHCII-presented neoantigens drive type 1 diabetes and other autoimmune diseases?
Cold Spring Harb Perspect Med
Show Abstract
Hide Abstract
The strong association between particular MHCII alleles and type 1 diabetes is not fully understood. Two ideas that have been considered for many years are that autoimmunity is driven by (1) low-affinity CD4(+) T cells that escape thymic negative selection and respond to certain autoantigen peptides that are particularly well presented by particular MHCII molecules, or (2) CD4(+) T cells responding to neoantigens that are absent in the thymus, but uniquely created in the target tissue in the periphery and presented by particular MHCII alleles. Here we discuss the recent structural data in favor of the second idea. We review studies suggesting that peptide antigens recognized by autoimmune T cells are uniquely proteolytically processed and/or posttranslationally modified in the target tissue, thus allowing these T cells to escape deletion in the thymus during T-cell development. We postulate that an encounter with these tissue-specific neoantigenic peptides presented by the particular susceptible MHCII alleles in the peripheral tissues when accompanied by the appropriate inflammatory milieu activates these T-cell escapees leading to the onset of autoimmune disease.
Related JoVE Video
TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines.
Cancer Immunol. Immunother.
Show Abstract
Hide Abstract
A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3? motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.