Infection with Trypanosoma cruzi restricts the repertoire of parasite-specific CD8+ T cells leading to immunodominance

Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.     

Immunodominance in virus-induced CD8(+) T-cell responses is dramatically modified by DNA immunization and is regulated by gamma interferon

The phenomenon whereby the host immune system responds to only a few of the many possible epitopes in a foreign protein is termed immunodominance. Immunodominance occurs not only during microbial infection but also following vaccination, and clarification of the underlying mechanism may permit the rational design of vaccines which can circumvent immunodominance, thereby inducing responses to all epitopes, dominant and subdominant. Here, we show that immunodominance affects DNA vaccines and that the effects can be avoided by the simple expedient of epitope separation. DNA vaccines encoding isolated dominant and subdominant epitopes induce equivalent responses, confirming a previous demonstration that coexpression of dominant and subdominant epitopes on the same antigen-presenting cell (APC) is central to immunodominance. We conclude that multiepitope DNA vaccines should comprise a cocktail of plasmids, each with its own epitope, to allow maximal epitope dispersal among APCs. In addition, we demonstrate that subdominant responses are actively suppressed by dominant CD8(+) T-cell responses and that gamma interferon (IFN-gamma) is required for this suppression. Furthermore, priming of CD8(+) T cells to a single dominant epitope results in strong suppression of responses to other normally dominant epitopes in immunocompetent mice, in effect rendering these epitopes subdominant; however, responses to these epitopes are increased 6- to 20-fold in mice lacking IFN-gamma. We suggest that, in agreement with our previous observations, IFN-gamma secretion by CD8(+) T cells is highly localized, and we propose that its immunosuppressive effect is focused on the APC with which the dominant CD8(+) T cell is in contact.     

Mycobacterium tuberculosis directs immunofocusing of CD8+ T cell responses despite vaccination

Vaccines that elicit T cell responses try to mimic protective memory T cell immunity after infection by increasing the frequency of Ag-specific T cells in the immune repertoire. However, the factors that determine immunodominance during infection and after vaccination and the relation between immunodominance and protection are incompletely understood. We previously identified TB10.4(20-28) as an immunodominant epitope recognized by H2-K(d)-restricted CD8(+) T cells after M. tuberculosis infection. Here we report a second epitope, EspA(150-158), that is recognized by a substantial number of pulmonary CD8(+) T cells. The relative abundance of these T cells in the naive repertoire only partially predicts their relative frequency after M. tuberculosis infection. Furthermore, although vaccination with recombinant vaccinia virus expressing these epitopes changes their relative immunodominance in the preinfection T cell repertoire, this change is transient after challenge with M. tuberculosis. We speculate that factors intrinsic to the chronic nature of M. tuberculosis infection establishes the hierarchy of immunodominance and may explain the failure of some vaccines to provide protection.     

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.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy

Negative selection is designed to purge the immune system of high-avidity, self-reactive T cells and thereby protect the host from overt autoimmunity. In this in vivo viral infection model, we show that there is a previously unappreciated dichotomy involved in negative selection in which high-avidity CD8(+) T cells specific for a dominant epitope are eliminated, whereas T cells specific for a subdominant epitope on the same protein preferentially escape deletion. Although this resulted in significant skewing of immunodominance and a substantial depletion of the most promiscuous T cells, thymic and/or peripheral deletion of high-avidity CD8(+) T cells was not accompanied by any major change in the TCR V beta gene family usage or an absolute deletion of a single preferred complementarity-determining region 3 length polymorphism. This suggests that negative selection allows high-avidity CD8(+) T cells specific for subdominant or cryptic epitopes to persist while effectively deleting high-avidity T cells specific for dominant epitopes. By allowing the escape of subdominant T cells, this process still preserves a relatively broad peripheral TCR repertoire that can actively participate in antiviral and/or autoreactive immune responses.     

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ～84 HSV-1 proteins are recognized by CD8(+) T cells, and most (～80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.     

Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies

Generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Virus-specific CD8(+) T-lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8(+) T-lymphocyte responses may prove to be critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Here we show that epitope modification strategies can alter CD8(+) T-lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing simian immunodeficiency virus Gag lacking the dominant D(b)-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D(b)-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes.     

Effect of chronic viral infection on epitope selection, cytokine production, and surface phenotype of CD8 T cells and the role of IFN-gamma receptor in immune regulation

Regulation of CD8 T cell responses in chronic viral infections is not well understood. In this study, we have compared the CD8 T cell responses to immunodominant and subdominant epitopes during an acute and a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The epitope hierarchy of the primary CD8 T cell response was similar in acute and chronic LCMV infections. However, strikingly, the epitope hierarchy of the primary CD8 T cell response was conserved in the T cell memory only in an acute but not in a chronic LCMV infection. Interestingly, in an acute infection, increasing the viral dose caused significant changes in the epitope hierarchy of the LCMV-specific memory CD8 T cell pool, with no effect on the primary CD8 T cell response. Functional and phenotypic analyses revealed that exposure of CD8 T cells to extended periods of antigenic stimulation could lead to long-term defects in cytokine production and alteration in expression of cell surface L-selectin (CD62L). Whereas expression of CD44 was minimally altered, a greater proportion of LCMV-specific memory CD8 T cells were CD62L(low) in mice that have recovered from a chronic LCMV infection, compared with acutely infected mice. Mechanistic studies showed that IFN-gammaR deficiency altered the epitope hierarchy of the pool of LCMV-specific memory CD8 T cells without significantly affecting the immunodominance of the primary CD8 T cell response in an acute infection. Taken together, these findings should further our understanding about the regulation of T cell responses in human chronic viral infections.     

Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.     

Immunodominant tuberculosis CD8 antigens preferentially restricted by HLA-B

CD8(+) T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-gamma ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8(+) T cells recognizing known immunodominant CD4(+) T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8(+) T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8(+) T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics.     

Two Overlapping Subdominant Epitopes Identified by DNA Immunization Induce Protective CD8+ T-Cell Populations with Differing Cytolytic Activities

Subdominant CD8(+) T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by L(d) and the other presented by K(d). This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8(+) T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8(+) response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the "dominant" vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the "subdominant" vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8(+) T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysis(low)-cytokine(competent) phenotype associated with most memory CD8(+) T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8(+) T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8(+) T cells with distinct functional attributes.     

Variable Processing and Cross-presentation of HIV by Dendritic Cells and Macrophages Shapes CTL Immunodominance and Immune Escape

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.     

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.     

CD8+ T cell immunodominance in lymphocytic choriomeningitis virus infection is modified in the presence of toll-like receptor agonists

Currently, we have limited understanding of how Toll-like receptor (TLR) engagement by microbial products influences the immune response during a concurrent virus infection. In this study, we established that dual TLR2 plus TLR3 (designated TLR2+3) stimulation alters the immunodominance hierarchies of lymphocytic choriomeningitis virus (LCMV) epitopes by reducing NP396-specific CD8+ T cell responses and shifting it to a subdominant position. The shift in immunodominance occurred due to a reduction in antigen uptake and the reduced cross-presentation of NP396, a major LCMV immunodominant epitope that is efficiently cross-presented. Moreover, the altered immunodominance was dependent on TLR stimulation occurring at the site of infection. Finally, as lipopolysaccharide failed to induce the same phenomenon, the data suggest that these findings are dependent not only on the dual engagement of the TRIF/MyD88 pathways but also on how TLR agonists activate antigen-presenting cells. Taken together, our data demonstrate a novel role for TLR ligands in regulating antiviral CD8+ T cell responses due to the regulation of the cross-presentation of cell-associated antigens.     

Elimination of an immunodominant CD4+ T cell epitope in human IFN-beta does not result in an in vivo response directed at the subdominant epitope

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4(+) T cell epitope peptide region in human IFN-beta, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-beta molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-beta responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-beta molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.     

Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.     

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.     

The immunodominant CD8 T cell response to the human cytomegalovirus tegument phosphoprotein pp65(495-503) epitope critically depends on CD4 T cell help in vaccinated HLA-A*0201 transgenic mice

Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-A(b+)/A2-HHD-II and HLA-DR1(+)/A2-DR1 mice the HLA-A*0201-restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ(+) CD8 T cell frequencies to the pp65(495-503)/(e6) epitope and low responses to the pp65(320-328)/(e3) and pp65(522-530)/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65(Δ501-503) DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65(487-503) domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.     

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.