PD-1/PD-L1 Blockade Can Enhance HIV-1 Gag-specific T Cell Immunity Elicited by Dendritic Cell-Directed Lentiviral Vaccines

Exhaustion of CD8⁺ T cells and upregulation of programmed death 1 (PD-1), a negative regulator of T cell activation, are characteristic features of individuals chronically infected with human immunodeficiency virus type 1. In a previous study, we showed in mice that a dendritic cell-directed lentiviral vector (DCLV) system encoding the human immunodeficiency virus (HIV)-1 Gag protein was an efficient vaccine modality to induce a durable Gag-specific T cell immune response. In this study, we demonstrate that blocking of the PD-1/PD-1 ligand (PD-L) inhibitory signal via an anti-PD-L1 antibody generated an enhanced HIV-1 Gag-specific CD8⁺ immune response following both a single round of DCLV immunization and a homologous prime/boost regimen. The prime/boost regimen combined with PD-L1 blockade generated very high levels of Gag-specific CD8⁺ T cells comprising several valuable features: improved ability to produce multiple cytokines, responding to a broader range of Gag-derived epitopes, and long-lasting memory. This enhanced cellular immune response generated by DCLV immunization combined with anti-PD-L1 blockade correlated with improved viral control following challenge with Gag-expressing vaccinia virus. Taken together, our studies offer evidence to support the use of PD-1/PD-L1 blockade as an adjuvant modality to enhance antigen-specific immune responses elicited by T cell-based immunizations such as DCLV.     

Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells

Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4⁺ and CD8⁺ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8⁺ T cells is qualitatively different from that in CD4⁺ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C β4 (PLCβ4). TCR-mediated responses were severely impaired in PLCβ4-deficient CD8⁺ T cells, whereas those in CD4⁺ T cells were intact. PLCβ4-deficient CD8⁺ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP₃ generation. Binding of PLCβ4 to the cytoplasmic tail of CD8α was important for CD8⁺ T cell activation. Furthermore, GNAQ interacted with PLCβ4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCβ4, and activated CD8⁺ T cells in a PLCβ4-dependent fashion. PLCβ4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCβ4 differentiates TCR signaling in CD4⁺ and CD8⁺ T cells and selectively promotes CD8⁺ T cell–dependent adaptive immunity.     

Resting Memory CD8+ T Cells are Hyperreactive to Antigenic Challenge In Vitro

The characteristics of CD8⁺ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8⁺ T cell populations. In most experimental systems, heterogeneous populations of primed CD8⁺ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8⁺ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8⁺ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8⁺ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8⁺ T cells were compared in vitro. We found that CD8⁺ T cells from primed mice are activated by peptide concentrations 10–50-fold lower than naive mice. In addition, the kinetics of interleukin 2Rα chain upregulation by primed CD8⁺ T cells differ from naive CD8⁺ T cells. These primed hyperresponsive CD8⁺ T cells might play an important role in the memory response.     

Fusion of HCV Nonstructural Antigen to MHC Class II–associated Invariant Chain Enhances T-cell Responses Induced by Vectored Vaccines in Nonhuman Primates

Despite viral vectors being potent inducers of antigen-specific T cells, strategies to further improve their immunogenicity are actively pursued. Of the numerous approaches investigated, fusion of the encoded antigen to major histocompatibility complex class II–associated invariant chain (Ii) has been reported to enhance CD8⁺ T-cell responses. We have previously shown that adenovirus vaccine encoding nonstructural (NS) hepatitis C virus (HCV) proteins induces potent T-cell responses in humans. However, even higher T-cell responses might be required to achieve efficacy against different HCV genotypes or therapeutic effect in chronically infected HCV patients. In this study, we assessed fusion of the HCV NS antigen to murine and human Ii expressed by the chimpanzee adenovirus vector ChAd3 or recombinant modified vaccinia Ankara in mice and nonhuman primates (NHPs). A dramatic increase was observed in outbred mice in which vaccination with ChAd3 expressing the fusion antigen resulted in a 10-fold increase in interferon-γ⁺ CD8⁺ T cells. In NHPs, CD8⁺ T-cell responses were enhanced and accelerated with vectors encoding the Ii-fused antigen. These data show for the first time that the enhancement induced by vector vaccines encoding li-fused antigen was not species specific and can be translated from mice to NHPs, opening the way for testing in humans.     

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.     

Gut epithelial IL-27 confers intestinal immunity through the induction of intraepithelial lymphocytes

IL-27 controls a diverse range of immune responses in many disease settings. Here, we identify intestinal epithelial cells (IECs) as one of the major IL-27 cellular sources in the gut-associated tissue. Unlike IL-27 secreted by innate immune cells, gut epithelial IL-27 is dispensable for T-bet⁺ regulatory T cell (T reg cell) differentiation or IL-10 induction. Rather, IEC-derived IL-27 specifically promotes a distinct CD8αα⁺CD4⁺ intraepithelial lymphocyte (IEL) population that acquires their functional differentiation at the intestinal epithelium. Loss of IL-27 in IECs leads to a selective defect in CD8αα⁺CD4⁺ IELs over time. Consequently, mice with IEC-specific IL-27 ablation exhibited elevated pathogen burden during parasitic infection, and this could be rescued by transfer of exogenous CD8αα⁺CD4⁺ IELs. Collectively, our data reveal that in addition to its known regulatory properties in preventing immune hyperactivity, gut epithelial IL-27 confers barrier immunity by inducing a specific IEL subset and further suggest that IL-27 produced by different cell types plays distinct roles in maintaining intestinal homeostasis.     

NKTR-255 is a polymer-conjugated IL-15 with unique mechanisms of action on T and natural killer cells

NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8⁺ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8⁺ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα^−/− mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα^−/− OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα^−/− OT-I cell responses were partially impaired, suggesting that naive CD8⁺ T cells are more dependent on cis presentation of NKTR-255 than memory CD8⁺ T cells. In bone marrow chimera studies, IL-15Rα^−/− and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rβ agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαβ agonist on CD8⁺ T cells.     

Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus

DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime–modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase 3 (Flt3)–ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8⁺ interferon (IFN) γ⁺ T cells and polyfunctional CD8⁺ T cells producing IFN-γ, tumor necrosis factor α, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac₂₅₁ and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL.     

The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion

Memory T cells exhibit superior responses to pathogens and tumors compared with their naive counterparts. Memory is typically generated via an immune response to a foreign antigen, but functional memory T cells can also be produced from naive cells by homeostatic mechanisms. Using a recently developed method, we studied CD8 T cells, which are specific for model (ovalbumin) and viral (HSV, vaccinia) antigens, in unimmunized mice and found a subpopulation bearing markers of memory cells. Based on their phenotypic markers and by their presence in germ-free mice, these preexisting memory-like CD44^hi CD8 T cells are likely to arise via physiological homeostatic proliferation rather than a response to environmental microbes. These antigen-inexperienced memory phenotype CD8 T cells display several functions that distinguish them from their CD44^lo counterparts, including a rapid initiation of proliferation after T cell stimulation and rapid IFN-γ production after exposure to proinflammatory cytokines. Collectively, these data indicate that the unprimed antigen-specific CD8 T cell repertoire contains antigen-inexperienced cells that display phenotypic and functional traits of memory cells.     

Virus-specific CD8+ T Lymphocytes Downregulate T Helper Cell Type 2 Cytokine Secretion and Pulmonary Eosinophilia during Experimental Murine Respiratory Syncytial Virus Infection

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8⁺ T cytolytic T cells on the differentiation and activation of functionally distinct CD4⁺ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8⁺ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8⁺ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8⁺ T cells in this process was further supported by the results in CD8⁺ T cell deficient, β₂ microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8⁺ T cell compartment. These results indicate that CD8⁺ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4⁺ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.Multiple arms of the immune system are activated in response to infection by foreign organisms. The outcomes of the infection, such as the clearance of the organisms and the generation of tissue injury, depend on these immune effector mechanisms that are mobilized against the pathogen. The role of T cells in the immune response to viral infections has been clearly established (1, 2). Mature T cells that express α/β T cell receptors can be divided into cells expressing either CD4 or CD8 surface antigen. CD8⁺ T cells recognize processed antigen in the context of MHC class I molecules, whereas CD4⁺ T cells recognize peptides in the context of MHC class II molecules (3, 4). The conventional view of CD8⁺ T cells has been primarily the killing of virally infected cells by direct cytolysis or by cytokines secreted by these T cells such as IFN-γ and TNF (1, 2). Functional subsets of CD4⁺ T cells have been described based on the cytokines produced by these cells, Th1 CD4⁺ T cells that secrete IL-2 and IFN-γ and Th2 T cells that secrete IL-4 and IL-5 (5, 6). The physiological relevance of these subsets of T cells with diverse functions have been shown in several models of viral infection including respiratory syncytial virus (RSV)¹.In the murine model of RSV infection, the roles of T cells and their products in the eradication of the virus as well as the pathogenesis of lung inflammation have been well documented (7–12). Recent studies have shown that mice sensitized to either of the two major glycoproteins of this virus developed distinct lung pathology when subsequently infected with RSV (12–15). Mice sensitized to the attachment glycoprotein (G) of RSV developed pulmonary eosinophilia that correlated with a strong induction of Th2 type response, whereas mice primed to the fusion (F) glycoprotein mounted a weak Th2 response and developed lung inflammation characterized by mononuclear cell infiltration. The underlying mechanisms that lead to these apparent distinct patterns of cytokine production and lung injury still remain unclear.The process by which CD4⁺ T cells mature and differentiate has been the subject of intense investigation (16). CD4⁺ T cells can be induced to differentiate into effector cells of either the Th1 or Th2 subsets, depending on the milieu in which these cells are activated. Cytokines such as IL-4 and IL-12 have been shown to play a crucial part in the regulation of CD4⁺ T cell differentiation (17–19). The presence of certain immune effector functions during the differentiation and expansion of CD4⁺ T cells may potentially be a key factor in determining the functional phenotypes acquired by these cells. In the murine model of experimental RSV infection, one important difference in the immune response to F and G glycoprotein of RSV is the lack of MHC class I–restricted cytolytic response to the G glycoprotein (13, 20). In the following studies, we began to investigate the impact of CD8⁺ T cells and MHC class I–restricted CTL responses on cytokine secretion and the subsequent development of pulmonary eosinophilia during experimental murine RSV infection. We have found that the induction of CD8⁺ T cell response to RSV proteins resulted in dramatically decreased levels of Th2 type cytokines and eosinophil recruitment into the lungs of RSV-infected mice previously sensitized to the RSV-G glycoprotein. The possible mechanisms underlying these observations and their potential significance are discussed.     

IL-1 enhances expansion,effector function,tissue localization,and memory response of antigen-specific CD8 T cells

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1^−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B⁺, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.A major goal in developing protective immune responses is to obtain robust CD8 T cell expansion, effector differentiation, and memory generation using simple protein antigens as immunogens. We have previously reported that IL-1 strikingly enhances CD4 T cell responses when administered to mice during the period immediately after priming (Ben-Sasson et al., 2009), and thus wished to determine whether it would have a comparable effect on CD8 cells.IL-1’s effect on CD4 T cells was observed in vivo, was direct, and largely reflected enhanced survival rather than increased proliferative rate. Furthermore, when wild-type and IL1R1^−/− CD4 TCR transgenic T cells specific for an OVA peptide were jointly transferred to IL1R1^−/− recipients, only the wild-type cells responded to IL-1 with enhanced antigen-driven expansion (Ben-Sasson et al., 2009). This result indicates that IL-1 acts directly on the antigen-responding CD4 cell. Of a wide range of cytokines, including IL-2, -4, -6, -7, -9, -15, -18, -21, and -33, as well as TNF, only IL-1α and IL-1β showed such profound enhancement activity (Ben-Sasson et al., 2009). The IL-1 effect was observed in both IL-6– and in CD28-deficient recipients. Neutralizing IL-1 diminished responses to protein plus LPS by ∼60%, implying that endogenous IL-1 enhanced antigen-specific CD4 T cells responses.IL-1 strikingly enhanced antigen-driven expansion in vivo and enhances in vitro expansion of Th17 cells, which express large amounts of IL-1R1 (Guo et al., 2009; Lee et al., 2010), but it had no detectable effect on in vitro expansion of Th1 or Th2 cells. However, administering IL-1 in vivo during CD4 T cell priming, while increasing the proportion of Th17 cells among responders, also causes striking expansion of both IFN-γ and IL-4–producing cells (Ben-Sasson et al., 2009).The role of IL-1 in regulating CD8 T cell responses has not been clear. Some have reported that IL-1 enhances in vitro expansion of CD8 cells responding to polyclonal stimulants (Mizuochi et al., 1988; Hope et al., 2001). Where studied, it appears that the in vitro effects of IL-1 have been limited to cells expressing large amounts of IL-1R1 (Klarnet et al., 1989; Nagoya et al., 1994). In one instance, enhanced capacity to produce IFN-γ was observed (Fischer et al., 1990). However, others have failed to observe IL-1–mediated enhancement of in vitro TCR-driven CD8 T cell expansion (Halvorsen et al., 1987; Panzer et al., 1990; Curtsinger et al., 1999).IL1R1^−/− mice have been reported to have diminished CD8 responses to infection with LCMV (Joeckel et al., 2012), influenza (Ichinohe et al., 2009), Mycobacterium tuberculosis (Fremond et al., 2007), vaccinia (Staib et al., 2005), and certain tumors (Elkabets et al., 2009; Ghiringhelli et al., 2009). In addition, Myd88^−/− and/or IRAK-4^−/− mice, both of which have defective IL-1–mediated signaling, have impaired responses to LCMV (Lye et al., 2008), vaccinia (Zhao et al., 2009), and malaria (Gowda et al., 2012). CD8 T cells specific for LCMV appearing in infected IL1R1^−/− mice were reported not to express granzyme B (Joeckel et al., 2012). Furthermore, vaccinia that fail to display a virally encoded soluble IL-1β receptor elicit greater protection and improved CD8 memory responses (Staib et al., 2005) implying that neutralizing endogenous IL-1 normally limits immunity to vaccinia. However, in these infection models, the cell target of IL-1 was not established.We sought to determine the importance of IL-1 in in vivo priming and differentiation of antigen-specific CD8 T cells. To that end, we transferred WT OT-I cells to WT or IL1R1^−/− C57BL/6 recipients that were then immunized with OVA plus LPS. IL-1R1^−/− recipients showed increases of WT OT-I T cells comparable to WT recipients in response to IL-1 in lymph nodes and spleen, but not in liver and lung. IL-1 administration also resulted in a striking enhancement in the frequency of granzyme B⁺ cells, in cytotoxic activity, and in cells that produced IFN-γ in response to PMA and ionomycin. Mice primed in the presence of IL-1 developed secondary CD8 T cells responses marked by enhanced expression of granzyme B and augmented capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhanced the protective value of weak immunogens. Thus, IL-1 has a striking effect on enhancing antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.     

Aerosol-induced Immunoglobulin (Ig)-E Unresponsiveness to Ovalbumin Does Not Require CD8+ or T Cell Receptor (TCR)-γ/δ+ T Cells or Interferon (IFN)-γ in a Murine Model of Allergen Sensitization

Mice exposed for 20 min daily to aerosolized ovalbumin (OVA) for 10 d at concentrations from 1 to 0.01% OVA made greatly reduced immunoglobulin (Ig)-E responses to subsequent immunogenic OVA challenges, given either intraperitoneally or by aerosol. This IgE-specific unresponsiveness lasted for at least four months. However, these aerosol-treated mice were primed for larger OVA-specific IgG1 and IgG2a responses. The specific reduction in IgE responses was not due to preferential induction of a T helper (Th)-1 response as aerosol OVA– primed mice made greatly reduced Th2 and no detectable Th1 response after rechallenge in vitro. Consistent with this, the increase in circulating eosinophils observed in control Th2-primed mice was absent in aerosol OVA–treated animals. Interferon (IFN)-γ was not required for this unresponsiveness, as IFN-γ knockout mice and anti–IFN-γ antibody-treated wild-type mice had greatly reduced levels of IgE similar to wild-type controls. CD8⁺ T cells played a relatively small role as IgE responses were reduced to about the same extent in β₂ microglobulin-deficient, or in anti-CD8–treated wild-type mice as in normal mice after aerosol OVA treatment. Similarly, T cell receptor (TCR)-γ/δ T cells were not required for maximal inhibition of the IgE response. These results demonstrate that exposure to inhaled protein antigens can induce a state of unresponsiveness of CD4⁺ T cells that results in a prolonged loss of IgE and eosinophil responses to subsequent challenges. This T cell unresponsiveness was shown not to require CD8⁺ or TCR-γ/δ⁺ T cells or IFN-γ.     

Rapid Effector Function in CD8+ Memory T Cells

The nature of the CD8⁺ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8⁺ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon γ (IFN-γ) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8⁺ T cells displayed IFN-γ release within 6 h of antigen contact, identifying a population of memory CD8⁺ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8⁺ effector T cells specific for six different MHC class I–restricted influenza virus epitopes. Enumeration of these CD8⁺ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8⁺ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.After recovery from an acute viral infection, the frequency of antigen-specific CD8⁺ T cells is too low to permit direct analysis. Instead, assays have used antigen- experienced T cells that have been expanded by in vitro restimulation with cognate antigen, a process that may introduce quantitative and qualitative biases, particularly with respect to the activation state of the cell. It has therefore been difficult to establish, for viral infections in humans, the phenotype of antigen-specific memory T cells in their natural state. In particular, it remains uncertain whether antiviral protective immunological memory is subserved by long-lived quiescent T cells (1–3) that must divide and differentiate over several days to become effectors, or by circulating effector T cells continuously activated either by persisting antigen or by cross-reactive environmental antigens (4, 5).We have studied individuals previously exposed to influenza virus but without acute clinical influenza, that is, individuals in the memory state with respect to influenza virus infection. Influenza infection is well suited for the study of CD8⁺ T cell memory. First, CD8⁺ CTLs are crucial for host defense against influenza virus; they are important in the clearance of intranasal virus (6, 7) and, since CTLs recognize the relatively conserved internal viral proteins (8, 9), they cross-react between viruses of different strains which evade neutralizing antibody through variation in surface glycoproteins (10, 11). Second, influenza virus–specific CTLs secrete IFN-γ which has direct effects on virus replication in infected cells (12) and may be more important in vivo for protection against influenza virus than perforin- or fas-mediated lysis (13). Examination of IFN-γ secretion by antigen-specific CD8⁺ T cells is therefore expected to be of at least as much relevance to protection as conventional measurements of lytic activity.We have applied a sensitive enzyme-linked immunospot (ELISPOT)¹ assay for single cell IFN-γ secretion in a novel way to detect low frequencies of uncultured influenza peptide–specific CD8⁺ T lymphocytes freshly isolated from peripheral blood. The ELISPOT assay detects secreted cytokine molecules in the immediate vicinity of the cell from which they are derived, while still at a relatively high concentration; each spot in the read-out represents a ‘footprint'' of the original cytokine-producing cell. Quantitation of these IFN-γ spot-forming cells (SFCs) by this technique is highly sensitive; for influenza virus–specific CD8⁺ CTL lines and bulk cultures, the ELISPOT assay is an order of magnitude more sensitive than the ⁵¹Cr-release cytotoxicity assay for detecting low numbers of peptide-specific CTLs (data not shown). We have exploited this enhanced sensitivity to demonstrate the presence of circulating influenza virus–specific CD8⁺ memory T cells capable of rapid effector function, long after exposure to the virus.     

Concurrent Generation of Effector and Central Memory CD8 T Cells during Vaccinia Virus Infection

It is generally thought that during the contraction phase of an acute anti-viral T cell reponse, the effector T cells that escape activation-induced cell death eventually differentiate into central memory T cells over the next several weeks. Here we report that antigen-specific CD8T cells with the phenotype and function of central memory cells develop concomitantly with effector T cells during vaccinia virus (vv) infection. As soon as 5 days after an intraperitoneal infection with vv, we could identify a subset of CD44^hi and CD62L⁺ vv-specific CD8 T cells in the peritoneal exudate lymphocytes. This population constituted approximately 10% of all antigen-specific T cells and like central memory T cells, they also expressed high levels of CCR7 and IL-7R but expressed little granzyme B. Importantly, upon adoptive transfer into naïve congenic hosts, CD62L⁺, but not CD62L⁻ CD8 T cells were able to expand and mediate a rapid recall response to a new vv challenge initiated 6 weeks after transfer, confirming that the CD62L⁺ vv-specific CD8 T cells are bonafide memory cells. Our results are thus consistent with the branched differentiation model, where effector and memory cells develop simultaneously. These results are likely to have implications in the context of vaccine design, particularly those based on vaccinia virus recombinants.     

An Alternate Pathway for T Cell Development Supported by the Bone Marrow Microenvironment: Recapitulation of Thymic Maturation

In the principal pathway of α/β T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4⁺ and CD8⁺ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4⁺ and CD8⁺ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4⁺CD8⁺ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.     

CD8+ T cells fail to limit SIV reactivation following ART withdrawal until after viral amplification

To define the contribution of CD8⁺ T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8⁺ T cell depletion using a rhesusized anti-CD8β monoclonal antibody on barcoded SIVmac239 dynamics on stable ART and after ART cessation in rhesus macaques (RMs). Among the RMs with full CD8⁺ T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA⁺ cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RMs during ART. Upon ART cessation, both CD8⁺ T cell–depleted and control RMs rebounded in fewer than 12 days, with no difference in the time to viral rebound or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8⁺ T cell–depleted RMs showed a stable, approximately 2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent antiviral CD8⁺ T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.     

Immunoregulatory mechanisms triggered by viral infections protect from type 1 diabetes in mice

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing β cells. Viral infections induce immune responses that can damage β cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on β cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death–1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8⁺ T cells expressing programmed cell death–1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4⁺CD25⁺ Tregs, which produced TGF-β and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4⁺CD25⁺ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.