CD8(+) T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.
Signals from the TCR that specifically contribute to effector versus memory CD8? T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras-deficient CD8? T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)-AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras-deficient CD8? T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8? T cell memory fate.
Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) in long-term nonprogressor-elite controller patients (LTNP-EC) has been described only in sporadic cases and then, consequences in disease progression are not clearly established. To fill-up this limited knowledge, we analyzed, for the first time, the prevalence, host genetic polymorphisms, and clinical consequences of HIV-1 DI in a group of LTNP-EC.
The generation of peptides presented by MHC class I molecules requires the proteolytic activity of the proteasome and/or other peptidases. The processing of a short vaccinia virus-encoded antigen can take place by a proteasome-independent pathway involving initiator caspase-5 and -10, which generate antigenic peptides recognized by CD8(+) T lymphocytes. In the present study, comparing single versus double enzyme digestions by mass spectrometry analysis, both qualitative and quantitative differences in the products obtained were identified. These in vitro data suggest that each enzyme can use the degradation products of the other as substrate for new cleavages, indicating concerted endoproteolytic activity of caspase-5 and -10.
CD8(+) T lymphocytes screen the surface of all cells in the body to detect pathogen infection or oncogenic transformation. They recognize peptides derived from cellular proteins displayed at the plasma membrane by major histocompatibility complex (MHC) class I molecules. Peptides are mostly by-products of cytosolic proteolytic enzymes. Peptidic ligands of MHC class I molecules are also generated in the secretory and vesicular pathways. Features of protein substrates, of proteases and of available MHC class I molecules for loading peptides in these compartments shape a singular collection of ligands that also contain different, longer, and lower affinity peptides than ligands produced in the cytosol. Especially in individuals who lack the transporters associated with antigen processing, TAP, and in infected and tumor cells where TAP is blocked, which thus have no supply of peptides derived from the cytosol, MHC class I ligands generated in the secretory and vesicular pathways contribute to shaping the CD8(+) T lymphocyte response.
Short viral antigens bound to human major histocompatibility complex (HLA) class I molecules are presented on infected cells. Vaccine development frequently relies on synthetic peptides to identify optimal HLA class I ligands. However, when natural peptides are analyzed, more complex mixtures are found. By immunoproteomics analysis, we identify in this study a physiologically processed HLA ligand derived from the human respiratory syncytial virus matrix protein that is very different from what was expected from studies with synthetic peptides. This natural HLA-Cw4 class I ligand uses alternative interactions to the anchor motifs previously described for its presenting HLA-Cw4 class I molecule. Finally, this octameric peptide shares its C-terminal core with the H-2D(b) nonamer ligand previously identified in the mouse model. These data have implications for the identification of antiviral cytotoxic T lymphocyte responses and for vaccine development.
In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitts lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.
CD8(+) cytotoxic T lymphocytes recognize infected cells in which MHC class I molecules present pathogen-derived peptides that have been processed mainly by proteasomes. Many infections induce a set of proteases, the caspases involved in apoptosis or inflammation. In this study, we report that processing and presentation of a short vaccinia virus-encoded Ag can take place also by a nonproteasomal pathway, which was blocked in infected cells with chemical inhibitors of caspases. By cleaving at noncanonical sites, at least two caspases generated antigenic peptides recognized by T lymphocytes. The sites and the peptidic products were partially overlapping but different to those used and produced by proteasomes in vitro. Antigenic natural peptides produced in infected cells by either pathway were quantitatively and qualitatively similar. Finally, coexpression of the natural vaccinia virus protein B13, which is an inhibitor of caspases and apoptosis, impaired Ag presentation by the caspase pathway in infected cells. These data support the hypothesis that numerous cellular proteolytic systems, including those induced during infection, such as caspases involved in apoptosis or in inflammation, contribute to the repertoire of presented peptides, thereby facilitating immunosurveillance.
In the classical MHC class I Ag presentation pathway, antigenic peptides derived from viral proteins by multiple proteolytic cleavages are transported to the endoplasmic reticulum lumen and are then exposed to ami-nopeptidase activity. In the current study, a long MHC class I natural ligand recognized by cytotoxic T lymphocytes was used to study the kinetics of degradation by aminopeptidase. The in vitro data indicate that this N-extended peptide is efficiently trimmed to a 9-mer, unless its binding to the MHC molecules protects the full-length peptide.
Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. In addition, HRSV poses a serious health risk in immunocompromised individuals and the elderly. It has been reported that this virus can infect mouse antigen-presenting cells, including B lymphocytes. In these B cells, HRSV infection upregulates the expression of activation markers, including MHC class II and CD86, but not MHC class I molecules. Here, we report that HRSV infection of spleen B lymphocytes downregulated TLR4. Either blocking with anti-TLR4 antibody or genetic deletion, but not functional deficiency of TLR4, moderately reduced the infectivity of HRSV in B lymphocytes. HRSV-infected B lymphocytes with deleted TLR4 upregulated MHC class II and CD86 molecules to the same levels as TLR4(+) wild type B cells. Since the activation of monocytes and macrophages by HRSV was previously reported to depend on TLR4, the current study indicates that these cells and B lymphocytes respond to HRSV infection with different activation pathways.
Most pathogen-derived peptides recognized by CD8+ CTL are produced by proteasomes and delivered to the endoplasmic reticulum by the TAP transporters associated with Ag processing. Alternative proteases also produce antigenic peptides, but their actual relevance is unclear. There is a need to quantify the contribution of these supplementary pathways in vitro and in vivo. A well-defined TAP-independent secretory route of Ag processing involves the trans-Golgi network protease furin. Quantitation of this route by using OVA constructs encoded by vaccinia viruses indicates that it provides approximately one-third of all surface complexes of peptide and MHC class I molecules. Generation of the epitope carboxyl terminus is a dramatic rate-limiting step, since bypassing it increased efficiency by at least 1000-fold. Notably, the secretory construct activated a similar percentage of Ag-specific CD8+ T cells in wild type as in TAP1-deficient mice, which allow only secretory routes but which have a 10- to 20-fold smaller CD8 compartment. Moreover, these TAP1(-/-) OVA-specific CD8+ T lymphocytes accomplished elimination of epitope-bearing cells in vivo. The results obtained with this experimental system underscore the potential of secretory pathways of MHC class I Ag presentation to elicit functional CD8+ T lymphocytes in vivo and support the hypothesis that noncytosolic processing mechanisms may compensate in vivo for the lack of proteasome participation in Ag processing in persons genetically deficient in TAP and thus contribute to pathogen control.
Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. Although human airway epithelial cells are the main target of HRSV, it has been reported that this virus can also infect professional antigen-presenting cells such as macrophages and dendritic cells, promoting upregulation of maturation markers. Here, we report that mouse spleen B220(+) B lymphocytes were susceptible to HRSV infection in vitro, probably involving a glycosaminoglycan-dependent mechanism. In contrast, neither CD4(+) nor CD8(+) T lymphocytes were infected. In B lymphocytes, HRSV infection upregulated major histocompatibility complex (MHC) class II but not MHC class I molecules and induced the expression of the activation marker CD86.
Recognition of infected or altered cells by CD8(+) cytotoxic T lymphocytes is mediated by direct interaction of their T-cell receptor with peptides presented by MHC class I molecules. Peptides are transferred for assembly with newly synthesized MHC molecules by the transporters associated with antigen processing (TAP). Yet, a fraction of described epitopes are presented independently of TAP. Current belief is that most of them derive from membrane proteins, mostly from their signal sequences, and are processed by vesicular proteases. A thorough review of the published data may challenge some of these views.
Respiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell-mediated immune responses. CD8(+) cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249-258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule K(d). In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249-258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249-258 can be generated from non-infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus-specific CTLs.
Susceptibility to HIV infection and disease progression are complex traits modulated by environmental and genetic factors, affecting innate and adaptive immune responses, among other cellular processes. A single nucleotide polymorphism (SNP) 35 kb upstream of the HLA-C gene locus (-35C/T) was previously shown to correlate with increased HLA-C expression and improved control of HIV-1. Here, we genotyped the -35C/T SNP in 639 subjects (180 uninfected patients, 304 HIV progressors and 155 LTNP) and confirmed the association of the -35C/T variant with the LTNP phenotype. The genotype frequencies in the general population subjects did not differ significantly from those seen in HIV progressors (p-value=0.472). However, a significant higher frequency of the protective CC genotype was identified when LTNP were compared either with HIV progressors alone (p-value<0.0001) or progressors and uninfected subjects together (p-value<0.0001). When considering aviremic LTNP alone (elite controllers; viral load below 50 copies/ml), the -35 CC genotype was not overrepresented compared to HIV progressors. Conversely, a significant association was found with the viremic LTNP groups (viral loads below 10,000 copies/ml). These results suggest that other factors alone or in combination with the -35 CC genotype may play an important role in differentiating the elite controller status from LTNP. Combination of different genetic variants may have additive or epistatic effects determining the HIV course of infection.
Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of ?B kinase (IKK) ? activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.
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