Molecular Anatomy and Number of Antigen Specific CD8 T Cells Required to Cause Type 1 Diabetes

We quantified CD8 T cells needed to cause type 1 diabetes and studied the anatomy of the CD8 T cell/beta (β) cell interaction at the immunologic synapse. We used a transgenic model, in situ tetramer staining to distinguish antigen specific CD8 T cells from total T cells infiltrating islets and a variety of viral mutants selected for functional deletion(s) of various CD8 T cell epitopes. Twenty percent of CD8 T cells in the spleen were specific for all immunodominant and subdominant viral glycoprotein (GP) epitopes. CTLs to the immunodominant LCMV GP33-41 epitope accounted for 63% of the total (12.5% of tetramers). In situ hybridization analysis demonstrated only 1 to 2% of total infiltrating CD8 T cells were specific for GP33 CD8 T cell epitope, yet diabetes occurred in 94% of mice. The immunologic synapse between GP33 CD8 CTL and β cell contained LFA-1 and perforin. Silencing both immunodominant epitopes (GP33, GP276–286) in the infecting virus led to a four-fold reduction in viral specific CD8 CTL responses, negligible lymphocyte infiltration into islets and absence of diabetes.     

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.     

Dominant clonotypes within HIV-specific T cell responses are programmed death-1high and CD127low and display reduced variant cross-reactivity

HIV epitope-specific T cell responses are often comprised of clonotypic expansions with distinct functional properties. In HIV(+) individuals, we measured programmed death-1 (PD-1) and IL-7Rα expression, MHC class I tetramer binding, cytokine production, and proliferation profiles of dominant and subdominant TCR clonotypes to evaluate the relationship between the composition of the HIV-specific T cell repertoire and clonotypic phenotype and function. Dominant clonotypes are characterized by higher PD-1 expression and lower C127 expression compared with subdominant clonotypes, and TCR avidity positively correlates with PD-1 expression. At low peptide concentrations, dominant clonotypes fail to survive in culture. In response to stimulation with peptides representing variant epitopes, subdominant clonotypes produce higher relative levels of cytokines and display greater capacity for cross-recognition compared with dominant clonotypes. These data indicate that dominant clonotypes within HIV-specific T cell responses display a phenotype consistent with ongoing exposure to cognate viral epitopes and suggest that cross-reactive, subdominant clonotypes may retain greater capacity to suppress replication of viral variants as well as to survive in the absence of strong antigenic signaling.     

Hierarchy of alpha fetoprotein (AFP)-specific T cell responses in subjects with AFP-positive hepatocellular cancer

We identified a series of immunodominant and subdominant epitopes from alpha fetoprotein (AFP), restricted by HLA-A*0201, which are recognized by the human T cell repertoire. The four immunodominant epitopes have been tested for immunogenicity in vivo, in HLA-A*0201+AFP+ advanced stage hepatocellular cancer (HCC) patients, and have activated and expanded AFP-specific IFN-gamma-producing T cells in these patients, despite high serum levels of this self Ag. Here, we have examined the frequency, function, and avidity of the T cells specific for subdominant epitopes from AFP. We find that T cells specific for several of these epitopes are of similar or higher avidity than those specific for immunodominant epitopes. We then tested the peripheral blood of subjects ex vivo with different levels of serum AFP for the hierarchy of response to epitopes from this Ag and find that HCC patients have detectable frequencies of circulating IFN-gamma-producing AFP-specific CD8+ T cells to both immunodominant and subdominant epitopes. We find the immunodominant and subdominant peptide-specific T cells to be differentially expanded with different modes of Ag presentation. Whereas spontaneous and AFP protein-stimulated responses show evidence for immunodominance, AdVhAFP-transduced dendritic cell-stimulated responses were broader and not skewed. Importantly, these data identify subdominant epitopes from AFP that can activate high-avidity T cells, and that can be detected and expanded in HCC subjects. These subdominant epitope-specific T cells can also recognize tumor cells and may be important therapeutically.     

Clonally diverse CTL response to a dominant viral epitope recognizes potential epitope variants.

RNA viruses undergo rapid sequence variation as the result of error-prone RNA replication mechanisms. When viable mutations arise in RNA regions encoding B or T cell epitopes, mutant viruses that can evade immune detection may be selected. In the carefully studied CTL response to the Gag p11C(C-M) epitope in SIVmac-infected Mamu-A*01(+) rhesus monkeys, it has been shown that CTL recognition of that epitope can occur even in the face of accruing mutations. To explore the underlying mechanism for this breadth of recognition, we have constructed Mamu-A*01 tetramers which discriminate T cells specific for epitope variants. Using these reagents we have defined discrete subsets of p11C(C-M)-specific T cells that cross-react with cells presenting variant peptides. We have found that individual Mamu-A*01(+) monkeys differ functionally in their ability to recognize epitope variants despite consistently strong recognition of the p11C(C-M) epitope. This functional difference is accounted for by the relative number of variant-specific T cells and by differences in the functionally relevant TCR repertoire of the infected monkeys. We have also found that monkeys immunized with DNA vaccine constructs encoding only the wild-type epitope sequence develop p11C(C-M)-specific CTL cross-reactive with variant peptides. Thus, cross-reactive CTL do not merely arise secondary to the emergence and immune presentation of viral CTL escape mutants but rather arise de novo following priming with a dominant epitope peptide sequence. Taken together, our results support the concept that the CTL response to a dominant viral epitope, although highly focused, can be clonally diverse and recognize potential epitope variants.     

Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8(+) T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-specific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer(+) CD8(+) T cells allowed the isolation of tetramer(bright) and tetramer(dull) populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1(157-165)) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.     

Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein

CD8⁺ T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8⁺ T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein–derived S^269–277 peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2^S269–277 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2^S269–277-specific CD8⁺ T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12⁺ TCRs which bound the HLA-A2^S269–277 complex with low micromolar affinity and determined the crystal structure of the HLA-A2^S269–277 binary complex, and subsequently a ternary structure of the TRAV12⁺ TCR complexed to HLA-A2^S269–277. We found that the TCR made extensive contacts along the entire length of the S^269–277 peptide, suggesting that the TRAV12⁺ TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12⁺ T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8⁺ T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S^269–277 peptide.     

Broadly Reactive Human CD8 T Cells that Recognize an Epitope Conserved between VZV,HSV and EBV

Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV), we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1) along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a “pan-herpesvirus” vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.     

Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.     

Loss of Anti-Viral Immunity by Infection with a Virus Encoding a Cross-Reactive Pathogenic Epitope

T cell cross-reactivity between different strains of the same virus, between different members of the same virus group, and even between unrelated viruses is a common occurrence. We questioned here how an intervening infection with a virus containing a sub-dominant cross-reactive T cell epitope would affect protective immunity to a previously encountered virus. Pichinde virus (PV) and lymphocytic choriomeningitis virus (LCMV) encode subdominant cross-reactive NP_205–212 CD8 T cell epitopes sharing 6 of 8 amino acids, differing only in the MHC anchoring regions. These pMHC epitopes induce cross-reactive but non-identical T cell receptor (TCR) repertoires, and structural studies showed that the differing anchoring amino acids altered the conformation of the MHC landscape presented to the TCR. PV-immune mice receiving an intervening infection with wild type but not NP205-mutant LCMV developed severe immunopathology in the form of acute fatty necrosis on re-challenge with PV, and this pathology could be predicted by the ratio of NP205-specific to the normally immunodominant PV NP_38–45 -specific T cells. Thus, cross-reactive epitopes can exert pathogenic properties that compromise protective immunity by impairing more protective T cell responses.     

Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent.

Tetrameric peptide-MHC class I complexes ("tetramers") are proving invaluable as reagents for characterizing immune responses involving CTLs. However, because the TCR can exhibit a degree of promiscuity for binding peptide-MHC class I ligands, there is potential for cross-reactivity. Recent reports showing that the TCR/peptide-MHC interaction is dramatically dependent upon temperature led us to investigate the effects of incubation temperature on tetramer staining. We find that tetramers rapidly stain CTLs with high intensity at 37 degrees C. We examine the fine specificity of tetramer staining using a well-characterized set of natural epitope variants. Peptide variants that elicit little or no functional cellular response from CTLs can stain these cells at 4 degrees C but not at 37 degrees C when incorporated into tetramers. These results suggest that some studies reporting tetramer incubations at 4 degrees C could detect cross-reactive populations of CTLs with minimal avidity for the tetramer peptide, especially in the tetramer-low population. For identifying specific CTLs among polyclonal cell populations such as PBLs, incubation with tetramers at 37 degrees C improves the staining intensity of specific CTLs, resulting in improved separation of tetramer-high CD8+ cells. Confocal microscopy reveals that tetramers incubated at 37 degrees C can be rapidly internalized by specific CTLs into vesicles that overlap with the early endocytic compartment. This TCR-specific internalization suggests that coupling of tetramers or analogues with toxins, which are activated only after receptor internalization, may create immunotoxins capable of killing CTLs of single specificities.     

A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized after vaccinia virus infection

Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8(+) T cell responses demonstrated that approximately one-fortieth of all possible 9- to 10-mer peptides were high-affinity HLA-A*0201 binders. Peptide immunization and T cell recognition data generated from 90 peptides indicated that about one-half of the binders were capable of eliciting T cell responses, and that one-seventh of immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes approximately 150 dominant and subdominant epitopes restricted in by HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide-binding affinity, nor complex stability, nor TCR avidity, nor amount of processed epitope appeared to strictly correlate with immunodominance status. Additional experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that additional factors, including immunoregulatory mechanisms, restrict the repertoire of T cell specificities after vaccinia infection by a factor of at least 10.     

Very diverse CD8 T cell clonotypic responses after virus infections

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-gamma capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vbeta chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1-2 orders of magnitude than predicted previously.     

A similarity in peptide cross-reactivity between alloantigen- and nominal antigen-induced CD8+ T cell responses in vitro

Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (FcγRI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8⁺ T cells after co-culturing. The CD8⁺ T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-γ) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8⁺ T cell responses. Interestingly, the allo- and self-HLA-restricted CD8⁺ T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.     

Multiple Distinct Forms of CD8+ T Cell Cross-Reactivity and Specificities Revealed after 2009 H1N1 Influenza A Virus Infection in Mice

Influenza primed mice are protected against lethal infection with H1N1 A/CA/04/E3/09 virus, and T depletion and serum transfer studies suggest a T-dependent mechanism. We therefore set out to investigate the quality of the cross-reactive T cell response to CA/E3/09 in mice primed with H3N2 influenza A/Hong Kong/X31 virus. Sequences of the immunodominant nucleoprotein (NP) NP366–374 and acid polymerase (PA) PA224–233 CD8 epitopes from X31 each differ from the CA/E3/09 virus by one amino acid: an M371V substitution at position 6 of the NP peptide, and an S224P substitution at position 1 of the PA peptide, raising questions about the role of these epitopes in protection. PA224–233 peptides from either virus could elicit IFN-γ spot forming cells from mice infected with X31, indicating cross-reactivity of these two peptides. However, no T cell responses to either PA224–233 peptide were detectable after primary CA/E3/09 infection, suggesting it is cryptic in this virus. In contrast, primary responses to the NP366 peptides were detectable after infection with either virus, but did not cross-react in vitro. Similarly, H2-D^b tetramers of each NP epitope stained CD8+ T cells from each respective virus infection, but did not obviously cross-react. Early after lethal CA/E3/09 challenge, X31 primed mice had enhanced IFN-γ responses toward both NP366 peptides, as well as recall responses to a set of subdominant NP and PA peptides not detectable after primary X31 infection alone. Furthermore, dual-tetramer staining revealed an expanded population of CD8 T cells reactive to both NP366 variant peptides also not seen after the priming infection alone. These observations demonstrate unusual CD8+ T cell cross-reactivity and specificity are elicited after primary and secondary CA/E3/09 influenza virus infections.     

Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis

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.     

Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers

Antigen-specific CD4⁺ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4⁺ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4⁺ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4⁺ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4⁺ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4⁺ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4⁺ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.     

Identification of seventeen new simian immunodeficiency virus-derived CD8+ T cell epitopes restricted by the high frequency molecule, Mamu-A*02, and potential escape from CTL recognition

MHC class I-restricted CD8+ T cells play an important role in controlling HIV and SIV replication. In SIV-infected Indian rhesus macaques (Macaca mulatta), comprehensive CD8+ T cell epitope identification has only been undertaken for two alleles, Mamu-A*01 and Mamu-B*17. As a result, these two molecules account for virtually all known MHC class I-restricted SIV-derived CD8+ T cell epitopes. SIV pathogenesis research and vaccine testing have intensified the demand for epitopes restricted by additional MHC class I alleles due to the shortage of Mamu-A*01+ animals. Mamu-A*02 is a high frequency allele present in over 20% of macaques. In this study, we characterized the peptide binding of Mamu-A*02 using a panel of single amino acid substitution analogues and a library of 497 unrelated peptides. Of 230 SIVmac239 peptides that fit the Mamu-A*02 peptide-binding motif, 75 peptides bound Mamu-A*02 with IC50 values of < or = 500 nM. We assessed the antigenicity of these 75 peptides using an IFN-gamma ELISPOT assay with freshly isolated PBMC from eight Mamu-A*02+ SIV-infected macaques and identified 17 new epitopes for Mamu-A*02. The synthesis of five Mamu-A*02 tetramers demonstrated the discrepancy between tetramer binding and IFN-gamma secretion by SIV-specific CD8+ T cells during chronic SIV infection. Bulk sequencing determined that 2 of the 17 epitopes accumulated amino acid replacements in SIV-infected macaques by the chronic phase of infection, suggestive of CD8+ T cell escape in vivo. This work enhances the use of the SIV-infected macaque model for HIV and increases our understanding of the breadth of CD8+ T cell responses in SIV infection.     

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.