HIV-1 Variation Diminishes CD4 T Lymphocyte Recognition

Effective long-term antiviral immunity requires specific cytotoxic T lymphocytes and CD4⁺ T lymphocyte help. Failure of these helper responses can be a principle cause of viral persistence. We sought evidence that variation in HIV-1 CD4⁺ T helper epitopes might contribute to this phenomenon. To determine this, we assayed fresh peripheral blood mononuclear cells from 43 asymptomatic HIV-1⁺ patients for proliferative responses to HIV-1 antigens. 12 (28%) showed a positive response, and we went on to map dominant epitopes in two individuals, to p24 Gag restricted by human histocompatibility leukocyte antigen (HLA)-DR1 and to p17 Gag restricted by HLA-DRB52c. Nine naturally occurring variants of the p24 Gag epitope were found in the proviral DNA of the individual in whom this response was detected. All variants bound to HLA-DR1, but three of these peptides failed to stimulate a CD4⁺ T lymphocyte line which recognized the index sequence. Antigenic variation was also detected in the p17 Gag epitope; a dominant viral variant present in the patient was well recognized by a specific CD4⁺ T lymphocyte line, whereas several natural mutants were not. Importantly, variants detected at both epitopes also failed to stimulate fresh uncultured cells while index peptide stimulated successfully. These results demonstrate that variant antigens arise in HIV-1⁺ patients which fail to stimulate the T cell antigen receptor of HLA class II–restricted lymphocytes, although the peptide epitopes are capable of being presented on the cell surface. In HIV-1 infection, naturally occurring HLA class II–restricted altered peptide ligands that fail to stimulate the circulating T lymphocyte repertoire may curtail helper responses at sites where variant viruses predominate.     

HLA-E–restricted HIV-1–specific CD8+ T cell responses in natural infection

CD8⁺ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E–restricted CD8⁺ T cell responses in HIV infection, however, remains unknown. In this study, CD8⁺ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E–restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8⁺ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8⁺ T cells. Importantly, bulk CD8⁺ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E–restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E–restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8⁺ T cell responses in HIV infection.     

Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27–restricted human immune response

There is an association between expression of the MHC class I molecule HLA-B27 and protection following human infection with either HIV or HCV. In both cases, protection has been linked to HLA-B27 presentation of a single immunodominant viral peptide epitope to CD8⁺ T cells. If HIV mutates the HLA-B27–binding anchor of this epitope to escape the protective immune response, the result is a less-fit virus that requires additional compensatory clustered mutations. Here, we sought to determine whether the immunodominant HLA-B27–restricted HCV epitope was similarly constrained by analyzing the replication competence and immunogenicity of different escape mutants. Interestingly, in most HLA-B27–positive patients chronically infected with HCV, the escape mutations spared the HLA-B27–binding anchor. Instead, the escape mutations were clustered at other sites within the epitope and had only a modest impact on replication competence. Further analysis revealed that the cluster of mutations is required for efficient escape because a combination of mutations is needed to impair T cell recognition of the epitope. Artificially introduced mutations at the HLA-B27–binding anchors were found to be either completely cross-reactive or to lead to substantial loss of fitness. These results suggest that protection by HLA-B27 in HCV infection can be explained by the requirement to accumulate a cluster of mutations within the immunodominant epitope to escape T cell recognition.     

Specific T Helper Cell Requirement for Optimal Induction of Cytotoxic T Lymphocytes against Major Histocompatibility Complex Class II Negative Tumors

This study shows that induction of tumor-specific CD4⁺ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4⁺ T cells were of the T helper cell 1 type. The peptide-specific CD4⁺ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8⁺ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.Adequate T helper cell activation is essential in the initiation of an immune reaction. The inability to control tumor outgrowth can be due to inadequate T helper responses underlying poor tumor-specific immunity. In the cellular immune response, specialized APCs process protein and present antigenic peptide fragments in MHC class II molecules to CD4⁺ T helper lymphocytes. These provide “help” to effector cells via the production of cytokines. Although tumor cells can directly present endogenously processed antigenic peptide in surface MHC class I molecules to CD8⁺ CTL precursors, initiation of tumor-specific CTL responses is likely to involve indirect presentation of tumor antigens by specialized APCs.Evidence for a role of T helper cell–mediated immunity comes from studies with genetically modified tumor cells. CD4⁺ cells can be directly activated by transfection of MHC class II α and β chain genes in mouse tumor cells (1–4). These cells become immunogenic, lose their tumorigenicity, and even induce protection against wild-type MHC class II negative tumors, indicating that direct MHC class II presentation of tumor expressed antigens can induce efficient anti–tumor responses.A central role of CD4⁺ T cells emerged from studies of immunity against FMR (Friend, Moloney, Rauscher)¹ murine leukemia virus (MuLV) type tumors by Greenberg (5). Transfer of purified polyclonal T cells from FBL (Friend MuLV-induced erythroleukemia cell line) vaccinated mice in naive animals can protect these mice against subsequent tumor challenge. Both purified CD4⁺ and CD8⁺ T cells transfer protection to FBL tumors (6). FBL cells do not express MHC class II molecules, but CD4⁺ T cells can protect mice even in the absence of CD8⁺ T cells. In this case, macrophages seem to play an important effector role. CD8⁺ T cells can only be effective if CD4⁺ T cells are present or if exogenous IL-2 is administered. Neither B cells nor NK cells seem to exert a significant role in the FBL sytem. These data suggest involvement of APCs, presenting tumor antigens, and a crucial regulatory role of Ths, which was strongly supported by experiments performed in Friend MuLV env-transgenic mice (7). These mice were rendered tolerant for env-specific Th responses and it was not possible to protect these mice against FBL tumors by vaccination.Immune responsiveness to MuLV is classically regulated by the genes of the H-2 (MHC) complex (8). In particular, the H-2^b haplotype confers resistance, and studies using H-2 recombinant and H-2 mutant mouse strains have mapped the protective effects to the class II I-A^b locus (9, 10). This MHC class II association indicates an important role of T helper cells influencing both CTL activity as well as class switching of antiviral antibodies from IgM to IgG. The H-2 I-A^b phenotype protects against early lymphomagenesis. The identification of two Friend MuLV env-derived epitopes, presented by MHC class II, I-A^b and I-E^b/d, respectively, indicated that tumor-directed T helper immunity is virus specific (11). The few lymphomas that arise in H-2^b mice have abrogated viral antigen or (more rarely) MHC class I expression (12), indicating that CTLs also play a crucial role. CTLs have been proven to recognize viral antigens, both gag and env proteins encode CTL epitopes (13). We have identified a K^b-presented, env-derived Moloney and Rauscher CTL epitope that is subdominant in C57BL/6 mice making use of the D^b mutant BM13 mouse strain (14). The D^b-presented gag-leader (gag-L) derived immunodominant CTL epitope for the FMR type of MuLV has been identified only recently (15).Vaccination with a synthetic peptide comprising a relevant T cell epitope is a powerful method to induce highly specific T cells. Protective vaccination using CTL peptide epitopes has been achieved in pathogenic viral models (16, 17) and tumor models (18–20). Peptide vaccination in IFA led to measurable specific CTL induction and protective immunity against virulent virus or tumor cells. Importantly, peptide vaccination can also be applied succesfully for therapy of established tumors by presenting the peptide in IFA, on RMA-S cells, or on activated dendritic cells (21, 22).We now report the induction of tumor-protective immunity by a single vaccination with a tumor-specific MuLV env-encoded T helper peptide. Strong protection can be achieved against highly aggressive tumor cells that lack MHC class II expression. This indicates the requirement of cross-priming of tumor antigens by local APCs. We show that CD8⁺ T cells, recognizing the gag-L–encoded CTL epitope, are crucial effector cells that are efficiently activated with help from peptide-primed tumor-specific CD4⁺ T cells. Vaccination with a mixture of the T helper peptide and the immunodominant CTL epitope resulted in synergistic, long-term tumor protection.     

Portable flanking sequences modulate CTL epitope processing

Peptide presentation is critical for immune recognition of pathogen-infected cells by CD8+ T lymphocytes. Although a limited number of immunodominant peptide epitopes are consistently observed in diseases such as HIV-1 infection, the relationship between immunodominance and antigen processing in humans is largely unknown. Here, we have demonstrated that endogenous processing and presentation of a human immunodominant HIV-1 epitope is more efficient than that of a subdominant epitope. Furthermore, we have shown that the regions flanking the immunodominant epitope constitute a portable motif that increases the production and antigenicity of otherwise subdominant epitopes. We used a novel in vitro degradation assay involving cytosolic extracts as well as endogenous intracellular processing assays to examine 2 well-characterized HIV-1 Gag overlapping epitopes presented by the same HLA class I allele, one of which is consistently immunodominant and the other subdominant in infected persons. The kinetics and products of degradation of HIV-1 Gag favored the production of peptides encompassing the immunodominant epitope and destruction of the subdominant one. Notably, cytosolic digestion experiments revealed flanking residues proximal to the immunodominant epitope that increased the production and antigenicity of otherwise subdominant epitopes. Furthermore, specific point mutations in these portable flanking sequences modulated the production and antigenicity of epitopes. Such portable epitope processing determinants provide what we believe is a novel approach to optimizing CTL responses elicited by vaccine vectors.     

CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

The final pathway of β cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill β cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8⁺ T cells from HLA-A2⁺ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide–specific CD8⁺ T cells killed human β cells in vitro. Critically, at high glucose concentration, β cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human β cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing β cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining β cells.     

Identification of High Potency Microbial and Self Ligands for a Human Autoreactive Class II–restricted T Cell Clone

CD4⁺ class II–restricted T cells specific for self antigens are thought to be involved in the pathogenesis of most human autoimmune diseases and molecular mimicry between foreign and self ligands has been implicated as a possible mechanism for their activation. In this report we introduce combinatorial peptide libraries as a powerful tool to identify cross-reactive ligands for these T cells. The antigen recognition of a CD4⁺ T cell clone (TCC) specific for myelin basic protein peptide (MBP) (86-96) was dissected by the response to a set of 220 11-mer peptide sublibraries. Based on the results obtained with the libraries for each position of the antigen, artificial peptides were found that induced proliferative responses at much lower concentrations than MBP(86-96). In addition stimulatory ligands derived from protein sequences of self and microbial proteins were identified, some of them even more potent agonists than MBP(86-96). These results indicate that: (a) for at least some autoreactive CD4⁺ T cells antigen recognition is highly degenerate; (b) the autoantigen used to establish the TCC represents only a suboptimal ligand for the TCC; (c) a completely random and unbiased approach such as combinatorial peptide libraries can decrypt the spectrum of stimulatory ligands for a T cell receptor (TCR).CD4⁺ T cells recognize via their TCR 11-14-mer  peptides derived from exogenous proteins after processing and presentation by a MHC class II–expressing APC (1). CD4⁺ MHC class II–restricted T cells are not only essential for the specific immune response to foreign pathogens but also are thought to be involved in the pathogenesis of many autoimmune diseases. Based on sequence homologies, cross-recognition by autoreactive T cells of foreign antigenic peptides has been demonstrated, and it was hypothesized that self-reactive T cells expanded by foreign antigens may be involved in the pathogenesis of autoimmune diseases (2–4).In recent years, peptide binding motifs of class II molecules have been identified by several experimental approaches. These motifs allow predictions about the binding of peptides to the MHC molecule and have helped to understand which ligands are available for recognition by CD4⁺ T cells. In parallel, determinants derived from foreign and self antigens that are important for T cell recognition have been mapped in detail using modified (truncated, single amino acid [aa]¹–substituted) peptides. Based on the concept of conserved TCR contact positions (5) it is well established that CD4⁺ T cells can recognize different peptides, but the number of possible ligands for a single T cell receptor is thought to be limited. However, it was also demonstrated that peptides sharing only a few amino acids are able to stimulate the same T cell clone (6) as long as they have conserved TCR contact residues (7). Using this information, a novel strategy to identify molecular mimicry peptides has been described, and stimulatory ligands derived from microbial proteins have been identified for autoreactive T cells, which do not share sequence homologies with the autoantigen in positions other than TCR contacts (7).However, approaches using single amino acid exchanges of the peptide antigen in order to identify the anatomy of antigen recognition and to search for cross-reactive ligands are impaired by the large number of individual peptides necessary to define a single T cell epitope. Furthermore, little is known about how multiple alterations of a ligand influence T cell recognition. Therefore, it has been impossible to define by formerly used approaches, how many ligands are recognized by a single T cell receptor and the full extent to which molecular mimicry can occur.In contrast to the experiments mentioned above, combinatorial peptide libraries were introduced recently as an approach to allow epitope identification that is unbiased by sequence homologies or binding motifs. Peptide libraries have been successfully applied to define unknown antibody specificities (8), peptide recognition by SH2-domains (9) or enzyme substrate specificity (10). Peptide libraries have also allowed a more detailed dissection of MHC class I and II binding motifs (11, 12). Finally, 9-mer peptide libraries were used to identify stimulatory ligands for tumor-specific (13) or alloreactive CD8⁺ T cell clones (14–16).In this report, we used combinatorial peptide libraries to study antigen recognition of a CD4⁺ T cell clone and to search for cross-reactive high potency ligands. The response of a MBP(86-96)-specific DR2b-restricted TCC to an undecapeptide library in the positional scan format (17, 18) was used to dissect its recognition pattern. Artificial peptide ligands based on the results derived from the library were shown to be more potent ligands than MBP(86-96). Furthermore, screening of protein databases with the library information revealed not only MBP as potential ligand for the TCC but also several peptides derived from self and foreign antigens. These ligands induced proliferation of the TCC, some of them even at lower concentration than MBP(86-96).These data indicate: (a) for at least some class II–restricted T cells antigen recognition is highly degenerate; (b) for these TCC a high number of ligands with different stimulatory potency exist; (c) the autoantigen that was used to establish the TCC represents a suboptimal ligand; (d) peptide ligands from self and microbial antigens can be identified which are more potent than MBP(86-96).These results not only have important implications for the understanding of how cross-reactivity is related to autoimmunity, but may also provide a model as to how degeneracy of antigen recognition might contribute to thymic selection.     

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation

Discovering dominant epitopes for T cells, particularly CD4⁺ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2–expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4⁺ T cells in the lung. We discovered lung-resident CD4⁺ T cells that expressed a disease-specific public CDR3β T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLA-DP2–CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4⁺ T cells specific for the HLA-DP2–CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation.     

Vertical T cell immunodominance and epitope entropy determine HIV-1 escape

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8⁺ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell–mediated in vivo control of HIV-1. Primary HIV-1–specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or “vertical” immunodominance as the primary determinant of in vivo CD8⁺ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8⁺ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.     

Mobilizing monocytes to cross-present circulating viral antigen in chronic infection

Selection of antigens for therapeutic vaccination against chronic viral infections is complicated by pathogen genetic variations. We tested whether antigens present during persistent viral infections could provide a personalized antigenic reservoir for therapeutic T cell expansion in humans. We focused our study on the HBV surface antigen (HBsAg), which is present in microgram quantities in the serum of chronic HBV patients. We demonstrated by quantitative fluorescent microscopy that, out of 6 professional APC populations in the circulation, only CD14 monocytes (MNs) retained an HBsAg depot. Using TCR-redirected CD8⁺ T cells specific for MHC-I–restricted HBV epitopes, we showed that, despite being constantly exposed to antigen, ex vivo–isolated APCs did not constitutively activate HBV-specific CD8⁺ T cells. However, differentiation of HBsAg⁺ CD14 MNs from chronic patients to MN-derived DCs (moDCs) induced cross-presentation of the intracellular reservoir of viral antigen. We exploited this mechanism to cross-present circulating viral antigen and showed that moDCs from chronically infected patients stimulated expansion of autologous HBV-specific T cells. Thus, these data demonstrate that circulating viral antigen produced during chronic infection can serve as a personalized antigenic reservoir to activate virus-specific T cells.     

SARS-CoV-2–specific CD8+ T cell responses in convalescent COVID-19 individuals

Characterization of the T cell response in individuals who recover from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8⁺ T cell recognition in a cross-sectional sample of 30 coronavirus disease 2019 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis and from humoral and inflammatory responses. There were 132 SARS-CoV-2–specific CD8⁺ T cell responses detected across 6 different HLAs, corresponding to 52 unique epitope reactivities. CD8⁺ T cell responses were detected in almost all convalescent individuals and were directed against several structural and nonstructural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2–specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8⁺ T cell response. Overall, T cells exhibited distinct differentiation into stem cell and transitional memory states (subsets), which may be key to developing durable protection.     

Cellular immune selection with hepatitis C virus persistence in humans

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8+ T cell responses to epitopes covering the entire HCV polyprotein in subjects who were followed prospectively from before infection to beyond the first year. There were no substitutions in T cell epitopes for a year after infection in a subject who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (P < 0.001, range 5-38). T lymphocyte recognition of 8 of 10 mutant peptides was markedly reduced compared with the initial sequence, indicating viral escape. Of 16 nonenvelope substitutions that occurred outside of known T cell epitopes, 8 represented conversion to consensus (P = 0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8+ T cell responses and optimization of replicative capacity.     

SENP7 senses oxidative stress to sustain metabolic fitness and antitumor functions of CD8+ T cells

The functional integrity of CD8⁺ T cells is tightly coupled to metabolic reprogramming, but how oxidative stress directs CD8⁺ T cell metabolic fitness in the tumor microenvironment (TME) remains elusive. Here, we report that SUMO-specific protease 7 (SENP7) senses oxidative stress to maintain the CD8⁺ T cell metabolic state and antitumor functions. SENP7-deficient CD8⁺ T cells exhibited decreased glycolysis and oxidative phosphorylation, resulting in attenuated proliferation in vitro and dampened antitumor functions in vivo. Mechanistically, CD8⁺ T cell–derived ROS triggered cytosolic SENP7–mediated PTEN deSUMOylation, thereby promoting PTEN degradation and preventing PTEN-dependent metabolic defects. Importantly, lowering T cell–intrinsic ROS restricted SENP7 cytosolic translocation and repressed CD8⁺ T cell metabolic and functional activity in human colorectal cancer samples. Our findings reveal that SENP7, as an oxidative stress sensor, sustains CD8⁺ T cell metabolic fitness and effector functions and unveil an oxidative stress–sensing machinery in tumor-infiltrating CD8⁺ T cells.     

HLA-E–restricted regulatory CD8+ T cells are involved in development and control of human autoimmune type 1 diabetes

A key feature of the immune system is its ability to discriminate self from nonself. Breakdown in any of the mechanisms that maintain unresponsiveness to self (a state known as self-tolerance) contributes to the development of autoimmune conditions. Recent studies in mice show that CD8⁺ T cells specific for the unconventional MHC class I molecule Qa-1 bound to peptides derived from the signal sequence of Hsp60 (Hsp60sp) contribute to self/nonself discrimination. However, it is unclear whether they exist in humans and play a role in human autoimmune diseases. Here we have shown that CD8⁺ T cells specific for Hsp60sp bound to HLA-E (the human homolog of Qa-1) exist and play an important role in maintaining peripheral self-tolerance by discriminating self from nonself in humans. Furthermore, in the majority of type 1 diabetes (T1D) patients tested, there was a specific defect in CD8⁺ T cell recognition of HLA-E/Hsp60sp, which was associated with failure of self/nonself discrimination. However, the defect in the CD8⁺ T cells from most of the T1D patients tested could be corrected in vitro by exposure to autologous immature DCs loaded with the Hsp60sp peptide. These data suggest that HLA-E–restricted CD8⁺ T cells may play an important role in keeping self-reactive T cells in check. Thus, correction of this defect could be a potentially effective and safe approach in the therapy of T1D.     

Analysis of Gag-specific Cytotoxic T Lymphocytes in Simian Immunodeficiency Virus–infected Rhesus Monkeys by Cell Staining with a Tetrameric Major Histocompatibility Complex Class I–Peptide Complex

A tetrameric recombinant major histocompatibility complex (MHC) class I–peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and β₂-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C–M) representing the optimal nine–amino acid peptide of Mamu-A*01–restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C–M complex. Tetrameric Mamu-A*01/p11C, C–M complex bound to T cells of SIVmac-infected, Mamu-A*01⁺, but not uninfected, Mamu-A*01⁺, or infected, Mamu-A*01⁻ rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01⁺ rhesus monkeys was only found in the cluster of differentiation (CD)8α/β⁺ T lymphocyte subset and the percentage of CD8α/β⁺ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01⁺ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C–M complex–binding, but not nonbinding, CD8α/β⁺ T cells. Furthermore, the percentage of CD8α/β⁺ T cells binding this tetrameric Mamu-A*01/p11C, C–M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.Cytotoxic T lymphocytes (CTLs) play an important role in containing virus spread in many viral infections. However, the activity of this cell population in vivo has proven difficult to study because its evaluation has relied on cumbersome, functional assays that require extensive cell manipulation and lengthy in vitro periods of cell cultivation. Altman et al. have recently reported that fluorescence dye-coupled tetrameric MHC class I–peptide complexes can specifically bind to subpopulations of epitope-specific cluster of differentiation (CD)¹8⁺ T cells, raising the possibility that CTLs might be studied using flow cytometric technology (1).There is accumulating evidence for the importance of CTLs in controlling HIV-1 and simian immunodeficiency virus replication in both primary and chronic infections (2– 6). We have been studying the role of this cellular immune response in AIDS immunopathogenesis in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Much of this work has focused on the evaluation of SIVmac Gag recognition by CTL in rhesus monkeys expressing the HLA-A homologue molecule Mamu-A*01. In fact, we have shown that CTL recognition of Gag in SIVmac-infected or vaccinated Mamu-A*01⁺ rhesus monkeys is restricted to a single epitope, 12–amino acid fragment of SIVmac 251 Gag (amino acid 179–190) (p11C), bound to Mamu-A*01 (7). Through studying the monkeys'' response to this dominant CTL epitope, we have been able to evaluate efficiently a variety of novel vaccine strategies for eliciting SIVmac-specific CTL responses and assess the role of CTLs in containing the replication of SIVmac during primary and chronic infections (8–11).In these studies, we have generated tetrameric Mamu-A*01/p11C, C–M complex using the optimal nine–amino acid fragment of SIVmac (amino acids 181–189) p11C, C-M (12) and evaluated its binding specificity in PBMCs of SIVmac-infected, Mamu-A*01⁺ rhesus monkeys. We demonstrate that the enumeration of CD8⁺ T cells that bind this complex in flow cytometric analyses correlates quantitatively with functional CTL activity and that this cell population is phenotypically heterogeneous.     

Identification of conserved SARS-CoV-2 spike epitopes that expand public cTfh clonotypes in mild COVID-19 patients

Adaptive immunity is a fundamental component in controlling COVID-19. In this process, follicular helper T (Tfh) cells are a subset of CD4⁺ T cells that mediate the production of protective antibodies; however, the SARS-CoV-2 epitopes activating Tfh cells are not well characterized. Here, we identified and crystallized TCRs of public circulating Tfh (cTfh) clonotypes that are expanded in patients who have recovered from mild symptoms. These public clonotypes recognized the SARS-CoV-2 spike (S) epitopes conserved across emerging variants. The epitope of the most prevalent cTfh clonotype, S_864–882, was presented by multiple HLAs and activated T cells in most healthy donors, suggesting that this S region is a universal T cell epitope useful for booster antigen. SARS-CoV-2–specific public cTfh clonotypes also cross-reacted with specific commensal bacteria. In this study, we identified conserved SARS-CoV-2 S epitopes that activate public cTfh clonotypes associated with mild symptoms.     

Increased Valency of Conserved-mosaic Vaccines Enhances the Breadth and Depth of Epitope Recognition

The biggest roadblock in development of effective vaccines against human immunodeficiency virus type 1 (HIV-1) is the virus genetic diversity. For T-cell vaccine, this can be tackled by focusing the vaccine-elicited T-cells on the highly functionally conserved regions of HIV-1 proteins, mutations in which typically cause a replicative fitness loss, and by computing multivalent mosaic proteins, which maximize the coverage of potential 9-mer T-cell epitopes of the input viral sequences. Our first conserved region vaccines HIVconsv employed clade alternating consensus sequences and showed promise in the initial clinical trials in terms of magnitude and breadth of elicited CD8⁺ T-cells. Here, monitoring T-cells restricted by HLA-A*02:01 in transgenic mice, we assessed whether or not the tHIVconsv design (HIVconsv with a tissue plasminogen activator leader sequence) benefits from combining with a complementing conserved mosaic immunogen tHIVcmo, and compared the bivalent immunization to that with trivalent conserved mosaic vaccines. A hierarchy of tHIVconsv ≤ tHIVconsv+tHIVcmo < tCmo1+tCmo2+tCmo3 vaccinations for induction of CD8⁺ T-cell responses was observed in terms of recognition of tested peptide variants. Thus, our HLA-A*02:01-restricted epitope data concur with previously published mouse and macaque observations and suggest that even conserved region vaccines benefit from oligovalent mosaic design.     

Proteome-wide analysis of HIV-specific naive and memory CD4+ T cells in unexposed blood donors

The preexisting HIV-1–specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4⁺ T cell repertoire in healthy HIV-1–negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4⁺ T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1–unexposed, seronegative donors. HIV-1–specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4⁺ T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8–12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1–specific helper cell responses by vaccination.Only one candidate HIV vaccine, a canarypox vectored gp120 with a protein boost, has shown any efficacy (Rerks-Ngarm et al., 2009). The limited protection correlated with induction of nonneutralizing antibodies to the VI/V2 region of the virus Envelope protein (Env; Rerks-Ngarm et al., 2009; Haynes et al., 2012). This modest success has stimulated efforts to design vaccines that generate more efficient neutralizing antibodies, together with potent CD4⁺ T cell responses capable of providing help to B cells and cytotoxic T cells (Burton et al., 2012). Understanding how the magnitude and specificity of these helper T cells can be optimized will be critical to the design of an effective vaccine.Primary immune responses are probably influenced strongly by the preexisting repertoire of B and T cells. However, characterization and quantification of these repertoires is difficult due to the extremely low number of circulating naive precursor cells (Jenkins et al., 2001; Su et al., 2013). Previous studies of naive CD4⁺ T cell repertoires in humans and mice have relied on magnetic beads to enrich MHC tetramer binding cells (Moon et al., 2007; Kwok et al., 2012; Su et al., 2013). However, although this approach gives precise information on responses to particular MHC-peptide epitopes, it does not measure the total repertoire and misses previously unknown epitopes. An alternative T cell library technique requires no prior knowledge of donor HLA type or epitope specificity (Geiger et al., 2009). The method presorts circulating T cells into naive and memory subsets which are seeded at limiting dilution before polyclonal expansion in the presence of PHA, allogeneic feeder cells, and IL-2. Individual cultures are then screened for proliferative responses to a protein or series of peptides representing the pathogen of interest (Geiger et al., 2009). Combined with epitope mapping and the Poisson distribution, the T cell library technique can provide quantitative data on the specificity of the entire preexisting naive and memory repertoire.The existence of HIV-1–specific memory cells in seronegative donors was originally suggested by studies of highly exposed HIV-1 seronegative (HESN) donors. It has been shown that 25–61% of HESNs have demonstrable HIV-1–specific memory cells, probably primed by exposure to the virus. Surprisingly, HIV-1–specific CD4⁺ T cells were also detected in 24–44% of unexposed donors (Ritchie et al., 2011), although it was not clear whether the latter came from cross-reactive memory T cells or naive T cells primed in vitro. More recently, the existence of low frequency (1–10/million) memory CD4⁺ T cells, specific for a known HIV-1 Gag epitope, was demonstrated by HLA DR4 tetramers in 50% of HIV-1 unexposed HLA DR4⁺ adults (Su et al., 2013), but it was not clear how generalizable the HIV-1 result was beyond the single epitope–HLA DR4 combination.The present study first validates the library technique by direct comparison with the tetramer enrichment method for measuring precursor T cell frequencies. We then use the T cell library technique to provide the first proteome-wide analysis of the frequencies and specificities of preexposure HIV-1–specific naive and memory CD4⁺ T cells in a HLA diverse population of HIV-1 unexposed donors.     

gp100/pmel 17 Is a Murine Tumor Rejection Antigen: Induction of “Self”-reactive,Tumoricidal T Cells Using High-affinity,Altered Peptide Ligand

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to “self”-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8⁺ T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2D^b, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I–restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp100_25–33) and mouse (mgp100_25–33) epitopes were homologous, differences in the three NH₂-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize “empty” D^b on RMA-S cells and a 3-log increase in its ability to trigger interferon γ release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.     

Processing of a multiple membrane spanning Epstein-Barr virus protein for CD8(+) T cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway

Epstein-Barr virus (EBV) latent membrane protein (LMP)2 is a multiple membrane spanning molecule which lacks ectodomains projecting into the lumen of the endoplasmic reticulum (ER). Human CD8(+) cytotoxic T lymphocytes (CTL)s recognize a number of epitopes within LMP2. Assays with epitope-specific CTLs in two different cell backgrounds lacking the transporter associated with antigen processing (TAP) consistently show that some, but not all, LMP2 epitopes are presented in a TAP-independent manner. However, unlike published examples of TAP-independent processing from endogenously expressed antigens, presentation of TAP-independent LMP2 epitopes was abrogated by inhibition of proteasomal activity. We found a clear correlation between hydrophobicity of the LMP2 epitope sequence and TAP independence, and experiments with vaccinia minigene constructs expressing cytosolic epitope peptides confirmed that these more hydrophobic peptides were selectively able to access the HLA class I pathway in TAP-negative cells. Furthermore, the TAP-independent phenotype of particular epitope sequences did not require membrane location of the source antigen since (i) TAP-independent LMP2 epitopes inserted into an EBV nuclear antigen and (ii) hydrophobic epitope sequences native to EBV nuclear antigens were both presented in TAP-negative cells. We infer that there is a proteasome-dependent, TAP-independent pathway of antigen presentation which hydrophobic epitopes can selectively access.