Selection influences naive CD8+ TCR-ß repertoire sharing.

Within each individual, the adaptive immune system generates a repertoire of cells expressing receptors capable of recognizing diverse potential pathogens. The theoretical diversity of the T-cell receptor (TCR) repertoire exceeds the actual size of the T-cell population in an individual by several orders of magnitude - making the observation of identical TCRs in different individuals extremely improbable if all receptors were equally likely. Despite this disparity between the theoretical and the realized diversity of the repertoire, these 'public' receptor sequences have been identified in autoimmune, cancer and pathogen interaction contexts. Biased generation processes explain the presence of public TCRs in the naive repertoire, but do not adequately explain the different abundances of these public TCRs. We investigate and characterize the distribution of genomic TCR-ß sequences of naive CD8+ T cells from three genetically identical mice, comparing non-productive (non-functional sequences) and productive sequences. We find public TCR-ß sequences at higher abundances compared with unshared sequences in the productive, but not in the non-productive, repertoire. We show that neutral processes such as recombination biases, codon degeneracy and generation probability do not fully account for these differences, and conclude that thymic or peripheral selection plays an important role in increasing the abundances of public TCR-ß sequences.

TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes.

Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8(+) T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development.

Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes.

BACKGROUND: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems. METHODS: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe. RESULTS: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA. CONCLUSIONS: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Enhanced immunity in a mouse model of malignant glioma is mediated by a therapeutic ketogenic diet.

BACKGROUND: Glioblastoma multiforme is a highly aggressive brain tumor with a poor prognosis, and advances in treatment have led to only marginal increases in overall survival. We and others have shown previously that the therapeutic ketogenic diet (KD) prolongs survival in mouse models of glioma, explained by both direct tumor growth inhibition and suppression of pro-inflammatory microenvironment conditions. The aim of this study is to assess the effects of the KD on the glioma reactive immune response. METHODS: The GL261-Luc2 intracranial mouse model of glioma was used to investigate the effects of the KD on the tumor-specific immune response. Tumor-infiltrating CD8+ T cells, CD4+ T cells and natural killer (NK) cells were analyzed by flow cytometry. The expression of immune inhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) on CD8+ T cells were also analyzed by flow cytometry. Analysis of intracellular cytokine production was used to determine production of IFN, IL-2 and IFN- in tumor-infiltrating CD8+ T and natural killer (NK) cells and IL-10 production by T regulatory cells. RESULTS: We demonstrate that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells. CONCLUSIONS: The KD may work in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression. This evidence suggests that the KD increases tumor-reactive immune responses, and may have implications in combinational treatment approaches.

Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.

Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo.

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.

Viral antigen and extensive division maintain virus-specific CD8 T cells during chronic infection.

Efficient maintenance of memory CD8 T cells is central to long-term protective immunity. IL-7- and IL-15-driven homeostatic proliferation is essential for long-term memory CD8 T cell persistence after acute infections. During chronic infections, however, virus-specific CD8 T cells respond poorly to these cytokines. Yet, virus-specific CD8 T cells often persist for long periods of time during chronic infections. We have addressed this apparent paradox by examining the mechanism for maintaining virus-specific CD8 T cells during chronic infection. We find that homeostatic cytokines (e.g., IL-7/15), inflammatory signals, and priming of recent thymic emigrants are not sufficient to maintain virus-specific CD8 T cells over time during chronic infection. Rather, our results demonstrate that viral peptide is required for virus-specific CD8 T cell persistence during chronic infection. Moreover, this viral antigen-dependent maintenance results in a dramatically different type of T cell division than is normally observed during memory T cell homeostasis. Rather than undergoing slow, steady homeostatic turnover during chronic viral infection, CD8 T cells undergo extensive peptide-dependent division, yet cell numbers remain relatively stable. These results indicate that antigen-specific CD8 T cell responses during persisting infection are maintained by a mechanism distinct from that after acute infection.

Flow Cytometry Analysis of Immune Cell Responses.

Flow cytometry is a fluorescence-based technology that allows for the identification and characterization of immune cell subsets within a heterogenous population. Briefly, isolated immune cells are stained in suspension with fluorescently tagged antibodies to identify cells of interest prior to being run through a flow cytometer. Here we describe how to isolate murine immune cells from various body regions, including the inguinal lymph nodes (ILNs), spleen, thymus, and peripheral blood, and tag them with primary fluorescent antibodies for flow cytometric analysis of CD4+ and CD8+ T cell populations. This chapter also details how to use flow cytometry to measure T cell expression of chemokine receptor 7 (CCR7), the major chemokine receptor lymphocytes use to enter lymph nodes. The methods described in this chapter can be used for characterizing other proteins of interest, as well as other immune cell subsets.

The impact of microbiome dysbiosis on T cell function within the tumor microenvironment (TME).

Insights into the effect of the microbiome's composition on immune cell function have recently been discerned and further characterized. Microbiome dysbiosis can result in functional alterations across immune cells, including those required for innate and adaptive immune responses to malignancies and immunotherapy treatment. Dysbiosis can yield changes in or elimination of metabolite secretions, such as short-chain fatty acids (SCFAs), from certain bacterial species that are believed to impact proper immune cell function. Such alterations within the tumor microenvironment (TME) can significantly affect T cell function and survival necessary for eliminating cancerous cells. Understanding these effects is essential to improve the immune system's ability to fight malignancies and the subsequent efficacy of immunotherapies that rely on T cells. In this review, we assess typical T cell response to malignancies, classify the known impact of the microbiome and particular metabolites on T cells, discuss how dysbiosis can affect their function in the TME then further describe the impact of the microbiome on T cell-based immunotherapy treatment, with an emphasis on recent developments in the field. Understanding the impact of dysbiosis on T cell function within the TME can carry substantial implications for the design of immunotherapy treatments and further our understanding of factors that could impact how the immune system combats malignancies.

Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge.

Vaccine-induced memory T cells localized at mucosal sites can provide rapid protection from viral infection. All-trans-retinoic acid (ATRA) has been shown to act physiologically to induce the expression of gut-homing receptors on lymphocytes. We tested whether the administration of exogenous ATRA during a systemic vaccination of mice could enhance the generation of mucosal CD8(+) T cell immunity, which might represent a strategy for establishing better protection from viral infection via mucosal routes. ATRA induced the expression of CCR9 and α4ß7 on both mouse and human CD8(+) T cells activated in vitro. The administration of ATRA to mice during in vivo priming with a replication-defective recombinant adenovirus vector expressing the lymphocytic choriomeningitis virus glycoprotein (LCMVgp) (Ad5gp) increased numbers of both effector and memory T cells in intestinal mucosal tissues and showed higher frequencies of systemic central memory-like T cells that exhibited enhanced proliferation during boosting immunization with recombinant modified vaccinia virus Ankara expressing LCMVgp (MVAgp). Mice that received ATRA during Ad5gp vaccination were more resistant to intravaginal challenge by recombinant vaccinia virus expressing LCMVgp (VVgp), reflecting in part stronger T cell recall responses in situ. Thus, ATRA appears to be useful as an adjuvant during vaccination to increase memory T cell responses and protection from viral infection at mucosal sites and may facilitate the development of more effective vaccines against mucosally transmitted pathogens such as HIV.

Vaccination alters the balance between protective immunity, exhaustion, escape, and death in chronic infections.

While T cell-based vaccines have the potential to provide protection against chronic virus infections, they also have the potential to generate immunopathology following subsequent virus infection. We develop a mathematical model to investigate the conditions under which T cells lead to protection versus adverse pathology. The model illustrates how the balance between virus clearance and immune exhaustion may be disrupted when vaccination generates intermediate numbers of specific CD8 T cells. Surprisingly, our model suggests that this adverse effect of vaccination is largely unaffected by the generation of mutant viruses that evade T cell recognition and cannot be avoided by simply increasing the quality (affinity) or diversity of the T cell response. These findings should be taken into account when developing vaccines against persistent infections.

Multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors.

The innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggered by vaccine vectors have largely focused on in vitro studies involving antigen-presenting cells and on early in vivo inflammatory responses. Here, we systematically explore the Toll-like receptor (TLR) signaling requirements for the generation of cellular immune responses by intramuscular immunization with common and alternative serotype rAd vectors in mice. Antigen-specific CD8(+) T-lymphocyte responses elicited by these rAd vectors were significantly diminished in MyD88(-/-) mice but not in TRIF(-/-) or TLR3(-/-) mice, suggesting the importance of MyD88-dependent TLR signaling. However, the absence of each individual TLR resulted in minimal to no effect on vaccine-elicited cellular immune responses. Moreover, responses were not diminished in IL-1R(-/-) or IL-18R(-/-) mice. These data suggest that rAd vectors engage multiple MyD88-dependent signaling pathways, none of which are individually critical; rather, they are integrated to contribute to the potent immunogenicity of rAd vectors. Stimulation of multiple innate immune mechanisms may prove a generalizable property of potent vaccines, and this strategy could be harnessed in the development of next-generation vaccine vectors and adjuvants.

SHP-1 in T cells limits the production of CD8 effector cells without impacting the formation of long-lived central memory cells.

During responses against viruses and malignancies, naive CD8 T lymphocytes expand to form both short-lived effector cells and a population containing cells with the potential to be long-lived and participate in memory responses (memory precursor effector cells). The strength of antigenic, costimulatory, and cytokine signals during responses impacts the magnitude and type of CD8 populations formed. In vitro studies have revealed that the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) regulates signal transduction from receptors on T cells including the TCR, helping set the activation threshold, and therefore may shape responses of mature CD8 T cells in vivo. Analysis of CD8 T cells from motheaten mice, which are globally deficient in SHP-1, proved problematic due to cell-extrinsic effects of SHP-1 deficiency in non-T cells on CD8 T cells. Therefore, a conditional knockout of SHP-1 in mature single-positive T cells was developed to analyze cell-intrinsic consequences of complete and partial SHP-1 deficiency on CD8 T cell responses to acute viral infection. The results demonstrated that SHP-1 has disparate effects on subpopulations of responding cells, limiting the magnitude and quality of primary and secondary responses by reducing the number of short-lived effector cells generated without affecting the size of the memory precursor effector cell pool that leads to formation of long-term memory.

Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction.

Adoptive T cell immunotherapy is an evolving technology with the potential of providing a means to safely and effectively target tumor cells for destruction.

Rexinoids Modulate Effector T Cell Expression of Mucosal Homing Markers CCR9 and α4ß7 Integrin and Direct Their Migration In Vitro.

Altering T cell trafficking to mucosal regions can enhance immune responses towards pathogenic infections and cancers at these sites, leading to better outcomes. All-trans-retinoic acid (ATRA) promotes T cell migration to mucosal surfaces by inducing transcription of the mucosal-homing receptors CCR9 and α4ß7 via binding to retinoic acid receptors (RARs), which heterodimerize with retinoid X receptors (RXRs) to function. However, the unstable nature and toxicity of ATRA limit its use as a widespread treatment modality for mucosal diseases. Therefore, identifying alternatives that could reduce or eliminate the use of ATRA are needed. Rexinoids are synthetically derived compounds structurally similar to ATRA. Originally named for their ability to bind RXRs, rexinoids can enhance RAR-mediated gene transcription. Furthermore, rexinoids are more stable than ATRA and possess an improved safety profile, making them attractive candidates for use in clinical settings. Here we show that select novel rexinoids act as ATRA mimics, as they cause increased CCR9 and α4ß7 expression and enhanced migration to the CCR9 ligand, CCL25 in vitro, even in the absence of ATRA. Conversely, other rexinoids act synergistically with ATRA, as culturing cells with suboptimal doses of both compounds resulted in CCR9 expression and migration to CCL25. Overall, our findings show that rexinoids can be used independently or synergistically with ATRA to promote mucosal homing of T cells in vitro, and lends support for the prospective clinical use of these compounds in immunotherapeutic approaches for pathogenic infections or cancers at mucosal surfaces.

Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma.

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo.

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)-infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.

Restoration of CD28 expression in CD28- CD8+ memory effector T cells reconstitutes antigen-induced IL-2 production.

The control of many persistent viral infections by Ag-specific cytolytic CD8+ T cells requires a concurrent virus-specific CD4+ Th cell response. This reflects in part a requirement of activated effector CD8+ T cells for paracrine IL-2 production as a growth and survival factor. In human CMV and HIV infection, the majority of differentiated virus-specific CD8+ T cells notably lose the ability to produce IL-2 but also lose expression of CD28, a costimulatory molecule. Analysis of the fraction of memory CD8+ T cells that continue to express CD28 revealed these cells retain the ability to produce IL-2. Therefore, we examined if IL-2 production by CD28- CD8+ T cells could be restored by introduction of a constitutively expressed CD28 gene. Expression of CD28 in CD28- CD8+ CMV- and HIV-specific CD8+ T cells reconstituted the ability to produce IL-2, which could sustain an autocrine proliferative response after Ag recognition. These results suggest that the loss of CD28 expression during differentiation of memory/effector CD8+ T cells represents a decisive step in establishing regulation of responding CD8+ T cells, increasing the dependence on CD4+ Th for proliferation after target recognition, and has implications for the treatment of viral disease with adoptively transferred CD8+ T cells.

Estimating the precursor frequency of naive antigen-specific CD8 T cells.

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.

Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.

During some persistent viral infections, virus-specific T-cell responses wane due to the antigen-specific deletion or functional inactivation (i.e., exhaustion) of responding CD8 T cells. T-cell exhaustion often correlates with high viral load and is associated with the expression of the inhibitory receptor PD-1. In other infections, functional T cells are observed despite high levels of pathogen persistence. The reasons for these different T-cell fates during chronic viral infections are not clear. Here, we tracked the fate of virus-specific CD8 T cells in lymphocytic choriomeningitis virus (LCMV)-infected mice during viral clearance, the persistence of wild-type virus, or the selection and persistence of a viral variant that abrogates the presentation of a single epitope. Viral clearance results in PD-1(lo) functional virus-specific CD8 T cells, while the persistence of wild-type LCMV results in high PD-1 levels and T-cell exhaustion. However, following the emergence of a GP35V-->A variant virus that abrogates the presentation of the GP33 epitope, GP33-specific CD8 T cells remained functional, continued to show low levels of PD-1, and reexpressed CD127, a marker of memory T-cell differentiation. In the same animals and under identical environmental conditions, CD8 T cells recognizing nonmutated viral epitopes became physically deleted or were PD-1(hi) and nonfunctional. Thus, the upregulation of PD-1 and the functional inactivation of virus-specific T cells during chronic viral infection is dependent upon continued epitope recognition. These data suggest that optimal strategies for vaccination should induce high-magnitude broadly specific T-cell responses that prevent cytotoxic T-lymphocyte escape and highlight the need to evaluate the function of vaccine-induced T cells in the context of antigens presented during virus persistence.