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.     

Evidence of premature immune aging in patients thymectomized during early childhood

While the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgery in newborns, the thymus is completely resected to enable better access to the heart to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Here, we show that young adults thymectomized during early childhood exhibit an altered T cell compartment. Specifically, absolute CD4⁺ and CD8⁺ T cell counts were decreased, and these T cell populations showed substantial loss of naive cells and accumulation of oligoclonal memory cells. A subgroup of these young patients (22 years old) exhibited a particularly altered T cell profile that is usually seen in elderly individuals (more than 75 years old). This condition was directly related to CMV infection and the induction of strong CMV-specific T cell responses, which may exhaust the naive T cell pool in the absence of adequate T cell renewal from the thymus. Together, these marked immunological alterations are reminiscent of the immune risk phenotype, which is defined by a cluster of immune markers predictive of increased mortality in the elderly. Overall, our data highlight the importance of the thymus in maintaining the integrity of T cell immunity during adult life.     

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.     

Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus

A diverse T cell repertoire is essential for a vigorous immune response to new infections, and decreasing repertoire diversity has been implicated in the age-associated decline in CD8 T cell immunity. In this study, using the well-characterized mouse influenza virus model, we show that although comparable numbers of CD8 T cells are elicited in the lung and lung airways of young and aged mice after de novo infection, a majority of aged mice exhibit profound shifts in epitope immunodominance and restricted diversity in the TCR repertoire of responding cells. A preferential decline in reactivity to viral epitopes with a low naive precursor frequency was observed, in some cases leading to "holes" in the T cell repertoire. These effects were also seen in young thymectomized mice, consistent with the role of the thymus in maintaining naive repertoire diversity. Furthermore, a decline in repertoire diversity generally correlated with impaired responses to heterosubtypic challenge. This study formally demonstrates in a mouse infection model that naturally occurring contraction of the naive T cell repertoire can result in impaired CD8 T cell responses to known immunodominant epitopes and decline in heterosubtypic immunity. These observations have important implications for the design of vaccine strategies for the elderly.     

An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions.

It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.     

Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4⁺ and CD8⁺ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4⁺ and CD8⁺ T cells isolated from the majority of participants exhibited human influenza–specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4⁺ and CD8⁺ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4⁺ and CD8⁺ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell–mediated immunity may confer broad protection against avian and human influenza A viruses.     

Resistance to and Recovery from Lethal Influenza Virus Infection in B Lymphocyte–deficient Mice

In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4⁺ and CD8⁺ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte–deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell–deficient mice have a 50– 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell–deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4⁺ and CD8⁺ effector T cells in this process, defined clonal populations of influenza-specific CD4⁺ and CD8⁺ effector T cells were adoptively transferred into lethally infected B cell–deficient mice. Cloned CD8⁺ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4⁺ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.     

Contribution of ?Virus-specific CD8+ Cytotoxic T Cells to Virus Clearance or Pathologic Manifestations of Influenza Virus Infection in a T Cell Receptor Transgenic Mouse Model

The ability of influenza virus to evade immune surveillance by neutralizing antibodies (Abs) directed against its variable surface antigens provides a challenge to the development of effective vaccines. CD8⁺ cytotoxic T lymphocytes (CTLs) restricted by class I major histocompatibility complex molecules are important in establishing immunity to influenza virus because they recognize internal viral proteins which are conserved between multiple viral strains. In contrast, protective Abs are strain-specific. However, the precise role of effector CD8⁺ CTLs in protection from influenza virus infection, critical for understanding disease pathogenesis, has not been well defined. In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8⁺ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose. The data suggest a dual role for CD8⁺ CTLs against influenza, which may present a challenge to the development of effective CTL vaccines. Effector mechanisms used by CD8⁺ CTLs in orchestrating clearance of virus and recovery from experimental influenza infection, or potentiation of lethal pathology, are discussed.     

CD8⁺ T cells with an intraepithelial phenotype upregulate cytotoxic function upon influenza infection in human lung

The human lung T cell compartment contains many CD8⁺ T cells specific for respiratory viruses, suggesting that the lung is protected from recurring respiratory infections by a resident T cell pool. The entry site for respiratory viruses is the epithelium, in which a subset of lung CD8⁺ T cells expressing CD103 (αE integrin) resides. Here, we determined the specificity and function of CD103⁺CD8⁺ T cells in protecting human lung against viral infection. Mononuclear cells were isolated from human blood and lung resection samples. Variable numbers of CD103⁺CD8⁺ T cells were retrieved from the lung tissue. Interestingly, expression of CD103 was seen only in lung CD8⁺ T cells specific for influenza but not in those specific for EBV or CMV. CD103⁺ and influenza-reactive cells preferentially expressed NKG2A, an inhibitor of CD8⁺ T cell cytotoxic function. In contrast to CD103^–CD8⁺ T cells, most CD103⁺CD8⁺ cells did not contain perforin or granzyme B. However, they could quickly upregulate these cytotoxic mediators when exposed to a type I IFN milieu or via contact with their specific antigen. This mechanism may provide a rapid and efficient response to influenza infection, without inducing cytotoxic damage to the delicate epithelial barrier.     

Characterization of pandemic influenza immune memory signature after vaccination or infection

The magnitude, quality, and maintenance of immunological memory after infection or vaccination must be considered for future design of effective influenza vaccines. In 2009, the influenza pandemic produced disease that ranged from mild to severe, even fatal, illness in infected healthy adults and led to vaccination of a portion of the population with the adjuvanted, inactivated influenza A(H1N1)pdm09 vaccine. Here, we have proposed a multiparameter quantitative and qualitative approach to comparing adaptive immune memory to influenza 1 year after mild or severe infection or vaccination. One year after antigen encounter, severely ill subjects maintained high levels of humoral and polyfunctional effector/memory CD4⁺ T cells responses, while mildly ill and vaccinated subjects retained strong cellular immunity, as indicated by high levels of mucosal homing and degranulation markers on IFN-γ⁺ antigen-specific T cells. A principal component analysis distinguished 3 distinct clusters of individuals. The first group comprised vaccinated and mildly ill subjects, while clusters 2 and 3 included mainly infected individuals. Each cluster had immune memory profiles that differed in magnitude and quality. These data provide evidence that there are substantial similarities between the antiinfluenza response that mildly ill and vaccinated individuals develop and that this immune memory signature is different from that seen in severely ill individuals.     

Memory CD4+ T cells protect against influenza through multiple synergizing mechanisms

Memory CD4+ T cells combat viral infection and contribute to protective immune responses through multiple mechanisms, but how these pathways interact is unclear. We found that several pathways involving memory CD4+ T cells act together to effectively clear influenza A virus (IAV) in otherwise unprimed mice. Memory CD4+ T cell protection was enhanced through synergy with naive B cells or CD8+ T cells and maximized when both were present. However, memory CD4+ T cells protected against lower viral doses independently of other lymphocytes through production of IFN-γ. Moreover, memory CD4+ T cells selected for epitope-specific viral escape mutants via a perforin-dependent pathway. By deconstructing protective immunity mediated by memory CD4+ T cells, we demonstrated that this population simultaneously acts through multiple pathways to provide a high level of protection that ensures eradication of rapidly mutating pathogens such as IAV. This redundancy indicates the need for reductionist approaches for delineating the individual mechanisms of protection mediated by memory CD4+ T cells responding to pathogens.     

IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking

Memory and naive CD8⁺ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8⁺ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8⁺ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8⁺ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8⁺ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8⁺ T cells. After unrelated infection or inflammatory challenge, memory CD8⁺ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8⁺ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8⁺ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15–dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8⁺ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8⁺ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy.     

Protective Heterologous Antiviral Immunity and Enhanced Immunopathogenesis Mediated by Memory T Cell Populations

A basic principle of immunology is that prior immunity results in complete protection against a homologous agent. In this study, we show that memory T cells specific to unrelated viruses may alter the host''s primary immune response to a second virus. Studies with a panel of heterologous viruses, including lymphocytic choriomeningitis (LCMV), Pichinde (PV), vaccinia (VV), and murine cytomegalo (MCMV) viruses showed that prior immunity with one of these viruses in many cases enhanced clearance of a second unrelated virus early in infection. Such protective immunity was common, but it depended on the virus sequence and was not necessarily reciprocal. Cell transfer studies showed that both CD4 and CD8 T cell populations from LCMV-immune mice were required to transfer protective immunity to naive hosts challenged with PV or VV. In the case of LCMV-immune versus naive mice challenged with VV, there was an enhanced early recruitment of memory phenotype interferon (IFN) γ–secreting CD4⁺ and CD8⁺ cells into the peritoneal cavity and increased IFN-γ levels in this initial site of virus replication. Studies with IFN-γ receptor knockout mice confirmed a role for IFN-γ in mediating the protective effect by LCMV-immune T cell populations when mice were challenged with VV but not PV. In some virus sequences memory cell populations, although clearing the challenge virus more rapidly, elicited enhanced IFN-γ–dependent immunopathogenesis in the form of acute fatty necrosis. These results indicate that how a host responds to an infectious agent is a function of its history of previous infections and their influence on the memory T cell pool.     

Self-antigen–specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response

A primary goal of cancer immunotherapy is to improve the naturally occurring, but weak, immune response to tumors. Ineffective responses to cancer vaccines may be caused, in part, by low numbers of self-reactive lymphocytes surviving negative selection. Here, we estimated the frequency of CD8⁺ T cells recognizing a self-antigen to be <0.0001% (∼1 in 1 million CD8⁺ T cells), which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity, but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit, most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more, acquire polyfunctionality, and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8⁺ T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells, followed by in vivo activation, is a new approach that can be applied to human cancer immunotherapy. Further, precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8⁺ T cells.Over a century ago, Ehrlich realized that animals must avoid “horror autotoxicus” and prevent immune attack on self-tissues. This is accomplished, in part, by deletion of highly self-reactive clones. The current T cell development paradigm reasons that thymocytes recognizing self-peptide–MHC complexes with high affinity are programmed to die. Yet, T cells are positively selected because of their ability to recognize a diverse set of self-peptides bound to MHC complexes (1, 2) and weak-to-moderate affinity self-reactive T cells do escape negative selection (3). Some of these CD8⁺ and CD4⁺ T cells are capable of recognizing self-antigens expressed by tumors, including cancer-testes antigens, and differentiation antigens (4). Melanosomal membrane glycoproteins are a well-characterized family of differentiation antigens that are recognized by low-frequency CD8⁺ T cells of melanoma patients (5). In the mouse, this family includes gp100/pmel-17, a type I melanosomal membrane protein involved in the polymerization of melanin (6).To generate a robust immune response against a self-protein, mechanisms of tolerance must be circumvented. Vaccination with altered forms of the antigen is an effective, widely studied method of overcoming peripheral tolerance (7–9). We have reported preclinical and clinical use of a plasmid delivery system that utilizes high pressure to drive cutaneous particle bombardment of DNA, leading to presentation of antigen by dendritic cells in skin draining LNs (DLNs) (10, 11). Active immunization of mice with plasmid encoding human gp100 (hgp100) generates a response composed of CD8⁺ T cells that cross-react with an immunodominant epitope (amino acids 25–33) from the wild-type self-protein presented by H-2D^b (10, 12). Although this strategy allows for the generation of low-level, detectable responses, the growth of established tumors is barely impeded. Indeed, active vaccination strategies alone, although considered safe, have yielded poor responses in clinical trials (13).Recent work has shown that delivering antitumor vaccines while cells are undergoing lymphopenia-induced homeostatic expansion enhances CD8⁺ T cell responses (14, 15). This augmentation is caused by the availability of homeostatic cytokines, removal of regulatory populations, increased self-peptide–MHC reactivity of homeostatically proliferating cells, and irradiation-induced release of danger signals (16–18). We designed a strategy to induce more potent tumor immunity that combines sublethal irradiation, adoptive transfer of naive splenocytes, and vaccination against gp100. This treatment is moderately effective at preventing melanoma growth in a prophylactic setting, but is much less potent when administered to a tumor-bearing mouse (unpublished data). Fortunately, the literature suggests many ways to improve this treatment, including but not limited to, addition of adjuvants, multi-epitope vaccination, and targeted removal of suppressor populations. Here, we chose to focus on a previously unexplored parameter: the frequency of self-antigen–specific CD8⁺ T cells present at the time of in vivo priming.How many of the ∼20 million naive CD8⁺ T cells in a mouse are specific for a particular epitope? The theoretical diversity of TCRs on mature T cells has been estimated at ∼10¹³ possibilities (19), but this may be an overestimate if documented constraints and biases on the generation of functional TCRs are considered. The study of individual clonotypes in the preimmune repertoire is made difficult because it requires the detection of small numbers of cells among millions. Nevertheless, immunologists have used indirect and direct methods to assess the preimmune repertoire for several well-studied foreign epitopes. Estimates range from 10 to 3,000 antigen-specific cells/antigen/mouse (20–26). To date, the lowest reported estimates for epitope-specific frequency is ∼16 CD4⁺ cells recognizing OVA and the highest is ∼3,000 CD8⁺ T cells that recognize gp₃₃ from LCMV (21, 24).Two recent reports demonstrate that natural differences in the size of three CD4⁺ (21) and six CD8⁺ foreign antigen–specific T cell populations (22) are important determinants of the magnitude of the effector and memory response. Further, it has been suggested that low CD8⁺ T cell precursor frequency may partially account for patterns of immunodominance and explain why certain well-processed and presented foreign epitopes fail to elicit strong immune responses in the context of infection (27, 28). Mechanisms of central and peripheral deletion might be expected to leave the mouse with even fewer precursors specific for self-antigens (29), thus limiting the generation of robust antitumor responses. We hypothesized that the strength of the antitumor immune response was in part determined by the size of the tumor-specific precursor pool. Therefore, we sought to determine the endogenous frequency of CD8⁺ T cells specific for a self-antigen, and to characterize the antitumor immune response generated from different T cell precursor frequencies.Here, we present our studies combining vaccination with adoptively transferred, naive, gp100-specific CD8⁺ T cells activated in vivo. We estimate the frequency of endogenous CD8⁺ cells recognizing gp100_25-33 to be extremely low, <0.0001% of CD8⁺ T cells, an implication of which is that some genetically identical animals do not mount strong, protective responses to vaccination. We demonstrate that supplementing the naive repertoire with gp100_25-33-specific cells increases vaccine-elicited tumor immunity. However, at very high input numbers, this benefit is negated because of intraclonal competition. These data show that CD8⁺ T cell precursor frequency has a striking effect on clinically relevant disease. Our results demonstrate that precursor frequency can be manipulated to advantage, and must at least be considered in therapeutic strategies of adoptive immunotherapy.     

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.     

Inflammatory IL-15 is required for optimal memory T cell responses

Due to their ability to rapidly proliferate and produce effector cytokines, memory CD8⁺ T cells increase protection following reexposure to a pathogen. However, low inflammatory immunizations do not provide memory CD8⁺ T cells with a proliferation advantage over naive CD8⁺ T cells, suggesting that cell-extrinsic factors enhance memory CD8⁺ T cell proliferation in vivo. Herein, we demonstrate that inflammatory signals are critical for the rapid proliferation of memory CD8⁺ T cells following infection. Using murine models of viral infection and antigen exposure, we found that type I IFN–driven expression of IL-15 in response to viral infection prepares memory CD8⁺ T cells for rapid division independently of antigen reexposure by transiently inducing cell-cycle progression via a pathway dependent on mTOR complex-1 (mTORC1). Moreover, exposure to IL-15 allowed more rapid division of memory CD8⁺ T cells following antigen encounter and enhanced their protective capacity against viral infection. Together, these data reveal that inflammatory IL-15 promotes optimal responses by memory CD8⁺ T cells.     

Absence of mouse 2B4 promotes NK cell–mediated killing of activated CD8+ T cells,leading to prolonged viral persistence and altered pathogenesis

Persistent viral infections are often associated with inefficient T cell responses and sustained high-level expression of inhibitory receptors, such as the NK cell receptor 2B4 (also known as CD244), on virus-specific T cells. However, the role of 2B4 in T cell dysfunction is undefined, and it is unknown whether NK cells contribute to regulation of these processes. We show here that persistent lymphocytic choriomeningitis virus (LCMV) infection of mice lacking 2B4 resulted in diminished LCMV-specific CD8⁺ T cell responses, prolonged viral persistence, and spleen and thymic pathologies that differed from those observed in infected wild-type mice. Surprisingly, these altered phenotypes were not caused by 2B4 deficiency in T cells. Rather, the entire and long-lasting pathology and viral persistence were regulated by 2B4-deficient NK cells acting early in infection. In the absence of 2B4, NK cells lysed activated (defined as CD44^hi) but not naive (defined as CD44^lo) CD8⁺ T cells in a perforin-dependent manner in vitro and in vivo. These results illustrate the importance of NK cell self-tolerance to activated CD8⁺ T cells and demonstrate how an apparent T cell–associated persistent infection can actually be regulated by NK cells.     

Immunoproteasomes shape immunodominance hierarchies of antiviral CD8(+) T cells at the levels of T cell repertoire and presentation of viral antigens

Vertebrates express three cytokine-inducible proteasome subunits that are incorporated in the place of their constitutively synthesized counterparts. There is increasing evidence that the set of peptides generated by proteasomes containing these subunits (immunoproteasomes) differs from that produced by standard proteasomes. In this study, we use mice lacking one of the immunoproteasome subunits (LMP2) to show that immunoproteasomes play an important role in establishing the immunodominance hierarchy of CD8(+) T cells (T(CD8+)) responding to seven defined determinants in influenza virus. In LMP2(-/)- mice, responses to the two most dominant determinants drop precipitously, whereas responses to two subdominant determinants are greatly enhanced. Adoptive transfer experiments with naive normal and transgenic T(CD8+) reveal that the reduced immunogenicity of one determinant (PA(224-233)) can be attributed to decreased generation by antigen presenting cells (APCs), whereas the other determinant (NP(366-374)) is less immunogenic due to alterations in the T(CD8+) repertoire, and not, as reported previously, to the decreased capacity of LMP2(-/)- APCs to generate the determinant. The enhanced response to one of the subdominant determinants (PB1F2(62-70)) correlates with increased generation by LMP2(-)(/)- virus-infected cells. These findings indicate that in addition to their effects on the presentation of foreign antigens, immunoproteasomes influence T(CD8+) responses by modifying the repertoire of responding T(CD8+).     

Memory CD8+ T cells exhibit increased antigen threshold requirements for recall proliferation

A hallmark of immunological memory is the ability of previously primed T cells to undergo rapid recall responses upon antigen reencounter. Classic work has suggested that memory T cells proliferate in response to lower doses of antigen than naive T cells and with reduced requirements for co-stimulation. In contrast to this premise, we observed that naive but not memory T cells proliferate in vivo in response to limited antigen presentation. To reconcile these observations, we tested the antigen threshold requirement for cell cycle entry in naive and central memory CD8⁺ T cells. Although both naive and memory T cells detect low dose antigen, only naive T cells activate cell cycle effectors. Direct comparison of TCR signaling on a single cell basis indicated that central memory T cells do not activate Zap70, induce cMyc expression, or degrade p27 in response to antigen levels that activate these functions in naive T cells. The reduced sensitivity of memory T cells may result from both decreased surface TCR expression and increased expression of protein tyrosine phosphatases as compared with naive T cells. Our data describe a novel aspect of memory T cell antigen threshold sensitivity that may critically regulate recall expansion.The ability of the adaptive immune system to respond more rapidly and effectively to pathogens that have been previously encountered is the basis of immunological memory. This attribute of CD8⁺ T cell memory is primarily due to an estimated 5–100-fold increase in the frequency of antigen-specific cells after memory formation over that found in naive individuals (Ahmed and Gray, 1996). Additionally, evidence suggests that clonal competition during the expansion phase of T cell priming may increase the affinity of the resulting antigen-specific effector and memory CD8⁺ T cell pool compared with the naive pool (Busch and Pamer, 1999; Zehn et al., 2009). Indeed, based on functionality, memory CD8⁺ T cells appear to be more sensitive to TCR-mediated stimulation than naive cells. Multiple studies have observed that resting memory but not naive CD8⁺ T cells can secrete cytokines and produce cytolytic effectors more rapidly than naive cells upon antigen encounter (Zimmermann et al., 1999; Veiga-Fernandes et al., 2000; Slifka and Whitton, 2001). Consistent with this ability, memory CD8⁺ T cells show epigenetic changes at cytokine gene loci that are consistent with more rapid gene expression (Kersh et al., 2006; Northrop et al., 2006). In addition, memory T cells redistribute their TCR into higher order oligomers that may increase antigen sensitivity (Kumar et al., 2011). Multiple phenotypic differences between naive and memory CD8⁺ T cells have also been described that may influence TCR reactivity including up-regulation of adhesion molecules and increased surface expression of the IL-2Rβ chain CD122 (Berard and Tough, 2002).However, the characteristics ascribed to naive and memory T cells may have been influenced by the experimental systems used to test them. For example, although memory CD8⁺ T cells reportedly proliferate in response to lower doses of antigen than naive T cells (Pihlgren et al., 1996; Curtsinger et al., 1998; London et al., 2000), little difference in peptide sensitivity was observed in the absence of exogenous IL-2 (Curtsinger et al., 1998; Zimmermann et al., 1999). Thus, the increased sensitivity of memory T cells to cytokine may be responsible for their superior response. Additionally, although some in vitro studies have found that memory CD8⁺ T cells do not require CD28-mediated co-stimulation to initiate recall expansion (Flynn and Müllbacher, 1996; Bachmann et al., 1999), B-7 expression appears to be necessary for recall expansion in vivo (Borowski et al., 2007; Boesteanu and Katsikis, 2009). These inconsistent data may be attributable to comparison of in vitro and in vivo results or inadequate analysis of the contribution of distinct memory CD8⁺ T cell subsets. Extensive phenotyping of antigen-specific T cell responses has suggested that multiple markers may co-segregate with proliferative capacity. CD8⁺ central memory T cells expressing CD44^hi, CD62L^hi, CD27^hi, CXCR3^hi, CD43^lo, KLRG1^lo, and CD127^hi exhibit the most robust recall proliferation, whereas CD44^hi, CD62L^lo, CD27^hi, CXCR3^hi, CD43^hi, KLRG1^lo, and CD127^hi effector memory T cells exhibit sustained cytotoxicity but poorer recall expansion (Wherry et al., 2003; Sallusto et al., 2004; Hikono et al., 2007; Olson et al., 2013).Intriguingly, it has been reported that after clearance of acute influenza infection, residual viral antigen presentation can drive proliferation and expansion of naive but not memory CD8⁺ T cells of the same specificity (Belz et al., 2007; Khanna et al., 2008). This observation is in contrast to the expectation that memory T cells exhibit greater responsiveness than naive cells. It has been suggested that naive and memory T cells may respond to antigen presentation by distinct DC subsets or migrate to different areas of the lymph node (Belz et al., 2007; Kastenmüller et al., 2013). Currently, it remains unclear why residual, long-lived antigen does not stimulate memory T recall proliferation. To better understand the requirements for efficient proliferative recall expansion, we have compared the activation and proliferation of TCR transgenic CD62L^hiCD44^lo naive and CD62L^hiCD44^hi central memory CD8⁺ T cells in multiple models of noninfectious antigen presentation at limiting levels in vitro and in vivo. Although we found that both naive and central memory cells received a TCR stimulus and were activated by limiting levels of antigen presentation, only naive CD8⁺ T cells entered cell cycle and expanded. This effect appeared T cell intrinsic, as naive T cells preferentially proliferated in response to limiting levels of peptide presented by multiple DC subsets. Direct comparison of naive and memory T cells indicated that resting memory CD8⁺ T cells express more cyclin-dependent kinase inhibitor p27 and do not activate effectors of cell cycle progression or Zap70 upon low dose peptide stimulation, although no defect was observed at saturating concentrations. Additionally, we found that memory T cells expressed lower levels of surface TCR and higher levels of non-receptor tyrosine phosphatases involved in negative regulation of TCR signaling. Our data clearly indicates that, surprisingly, memory CD8⁺ T cells actually exhibit a higher antigen threshold than naive CD8⁺ T cells to stimulate cell cycle entry in vitro and in vivo.     

Epitope-specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection

Primary virus infection often elicits a large CD8(+) T cell response which subsequently contracts to a smaller memory T cell pool; the relationship between these two virus-specific populations is not well understood. Here we follow the human CD8(+) T cell response to Epstein-Barr virus (EBV) from its primary phase in infectious mononucleosis (IM) through to the persistent carrier state. Using HLA-A2.1 or B8 tetramers specific for four lytic cycle and three latent cycle epitopes, we find marked differences in the epitope-specific composition of the T cell populations between the two phases of infection. The primary response is dominated by lytic epitope specificities which are severely culled (and in one case extinguished) with resolution of the acute infection; in contrast latent epitope specificities are less abundant, if present at all, in acute IM but often then increase their percentage representation in the CD8 pool. Even comparing epitopes of the same type, the relative size of responses seen in primary infection does not necessarily correlate with that seen in the longer term. We also follow the evolution of phenotypic change in these populations and show that, from a uniform CD45RA(-)RO(+)CCR7(-) phenotype in IM, lytic epitope responses show greater reversion to a CD45RA(+)RO(-) phenotype whereas latent epitope responses remain CD45RA(-)RO(+) with a greater tendency to acquire CCR7. Interestingly these phenotypic distinctions reflect the source of the epitope as lytic or latent, and not the extent to which the response has been amplified in vivo.