Rapid Progressing Allele HLA-B35 Px Restricted Anti-HIV-1 CD8+ T Cells Recognize Vestigial CTL Epitopes

BackgroundThe HLA-B*35-Px allele has been associated with rapid disease progression in HIV-1 infection, in contrast to the HLA-B*35-Py allele.Conclusions/SignificanceThis demonstrates a novel phenomenon that distinguishes individuals with the HLA-B*35-Px rapid progressing allele and those with the HLA-B*35-Py slower progressing allele.     

Reevaluating the CD8 T-Cell Response to Herpes Simplex Virus Type 1: Involvement of CD8 T Cells Reactive to Subdominant Epitopes

In C57BL/6 (B6) mice, most herpes simplex virus (HSV)-specific CD8 T cells recognize a strongly immunodominant epitope on glycoprotein B (gB₄₉₈) and can inhibit HSV type 1 (HSV-1) reactivation from latency in trigeminal ganglia (TG). However, half of the CD8 T cells retained in latently infected TG of B6 mice are not gB₄₉₈ specific and have been largely ignored. The following observations from our current study indicate that these gB₄₉₈-nonspecific CD8 T cells are HSV specific and may contribute to the control of HSV-1 latency. First, following corneal infection, OVA₂₅₇-specific OT-1 CD8 T cells do not infiltrate the infected TG unless mice are simultaneously immunized with OVA₂₅₇ peptide, and then they are not retained. Second, 30% of CD8 T cells in acutely infected TG that produce gamma interferon in response to HSV-1 stimulation directly ex vivo are gB₄₉₈ nonspecific, and these cells maintain an activation phenotype during viral latency. Finally, gB₄₉₈-nonspecific CD8 T cells are expanded in ex vivo cultures of latently infected TG and inhibit HSV-1 reactivation from latency in the absence of gB₄₉₈-specific CD8 T cells. We conclude that many of the CD8 T cells that infiltrate and are retained in infected TG are HSV specific and potentially contribute to maintenance of HSV-1 latency. Identification of the viral proteins recognized by these cells will contribute to a better understanding of the dynamics of HSV-1 latency.The generation and maintenance of a CD8 T-cell response represent an important line of defense against many viral pathogens. Such responses are typically initiated when host antigen-presenting cells at the site of infection capture and process viral proteins and transport them to local draining lymph nodes (DLN). There the antigen-presenting cells either directly present viral antigens to naïve CD8 T cells or pass them to a distinct LN-resident dendritic cell (DC) subset for antigen presentation in the context of major histocompatibility complex class I (1). Antigen-specific CD8 T cells then undergo robust division and differentiation into effector populations armed to infiltrate infected tissue and eliminate the invading pathogen. The magnitude of the CD8 T-cell response against different viral epitopes is typically aligned within a defined hierarchy. Those epitopes recognized by the largest portion of the pathogen-specific CD8 T-cell population are referred to as immunodominant, while those inciting lesser responses are referred to as subdominant (17). Manipulation of this hierarchal system by the elimination of an immunodominant epitope often results in the expansion of a normally silent or “cryptic” determinant (2, 17, 21).Although the HSV-1 genome contains at least 84 open reading frames (13), it is estimated that 70 to 95% of the acute CD8 T-cell response in lymphoid organs of B6 mice is directed against the single immunodominant gB₄₉₈ epitope (11, 21, 24, 26, 27). The remaining HSV-specific CD8 T cells are thought to be directed against a subdominant epitope on the viral ribonucleotide reductase (RR1₈₂₂) (16). These conclusions are derived from studies characterizing the specificity of CD8 T cells at the peak of the effector response in lymphoid tissue. Interestingly, a recombinant HSV-1 lacking the immunodominant gB₄₉₈ epitope induced an HSV-specific CD8 T-cell response of normal magnitude, while the RR1₈₂₂ epitope remained subdominant (21), suggesting the emergence of previously unrecognized or cryptic epitopes.Following HSV-1 corneal infection of B6 mice, virus is transmitted to the trigeminal ganglia (TG), where it replicates briefly (up to 6 days postinfection [dpi]) and then establishes a latent infection. CD8 effector T cells accumulate to peak levels in the TG by 8 dpi and then undergo contraction, and then a memory population of constant size is maintained for the life of the animal. While 50% of both the effector and memory CD8 T-cell populations are specific for the immunodominant gB₄₉₈ epitope (11, 18), the remaining TG-resident CD8 T cells are specific for neither the dominant gB₄₉₈ nor the subdominant RR1₈₂₂ epitope. Although the phenotype and function of the gB₄₉₈-specific CD8 T cells in sensory ganglia and their role in maintaining HSV-1 latency have been well characterized (3, 5, 9, 11, 12, 14, 18, 19, 22, 24, 25, 27), the properties of the gB₄₉₈-nonspecific TG-resident CD8 T-cell population and their role in maintaining viral latency remain unexplored. Here we demonstrate that many of the gB₄₉₈-nonspecific CD8 T cells in latently infected TG proliferate and some produce gamma interferon (IFN-γ) when stimulated with HSV-1 antigens directly ex vivo. These cells also persistently exhibit an activation phenotype within latently infected TG, are expanded in ex vivo cultures of latently infected TG, and can block HSV-1 reactivation in TG neurons in the absence of gB₄₉₈-specific CD8 T cells.     

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.     

Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.     

Dominant CD8+ T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8⁺ T-lymphocyte responses in Mamu-A*01⁺ rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01⁺ rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8⁺ T-lymphocyte response in the absence of secondary Gag p11C-specific CD8⁺ T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8⁺ T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8⁺ T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.CD8⁺ T lymphocyte responses play a central role in controlling human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) infections in nonhuman primates (18, 20, 29, 41). Naturally occurring virus-specific CD8⁺ T-lymphocyte responses typically focus on a limited number of dominant epitopes (52). However, accumulating data indicate that a broad cellular immune response, in which multiple viral epitopes are recognized by CD8⁺ T lymphocytes, confers better protection against viral replication than a restricted cellular immune response (26, 33). Therefore, it has been suggested that increasing the magnitude of subdominant epitope-specific responses may increase the breadth of a cellular immune response and provide enhanced protection against HIV/SIV replication.An understanding of the factors that influence the immunodominance hierarchy of viral epitopes will be needed to develop vaccination strategies that can generate the greatest breadth of virus-specific CD8⁺ T-lymphocyte responses. Differences in antigen processing, competition between epitope peptides for major histocompatibility complex (MHC) class I molecules, T-cell receptor (TCR) repertoire, TCR affinity for peptide class I complexes, and immunodomination have been shown to contribute to the dominance of an epitope-specific response (6, 10, 24, 32, 45, 52). In addition, studies have shown that immunodominance patterns for T-lymphocyte epitopes may differ following a primary and secondary exposure to the same viral antigen (4, 5, 43).In the present study, we observed that Mamu-A*01⁺ rhesus monkeys primed with plasmid DNA and boosted with recombinant adenovirus (rAd) vaccines encoding SIVmac239 Gag-Pol-Nef and HIV-1 Env proteins generated Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses of comparable magnitude. However, while there was a significant expansion of Gag p11C-specific CD8⁺ T-lymphocyte populations following challenge with pathogenic simian-human immunodeficiency virus 89.6P (SHIV-89.6P), there was no significant expansion of the Env p41A-specific CD8⁺ T-lymphocyte populations. We hypothesized that factors influencing the relative immunodominance of the primed Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses after viral challenge may have contributed to the observed differences in their secondary expansion. In the present study, we sought to identify the potential factors contributing to this immunodominance.     

Immune Surveillance by Rhinovirus-Specific Circulating CD4+ and CD8+ T Lymphocytes

Background

It is difficult to experimentally infect volunteers with RV strains to which the subject demonstrates serological immunity. However, in RV challenges, viral clearance begins before de novo adaptive immune responses would develop. We speculated that adaptive immunity to RV reflects heterologous immunity by effector memory cells.

Methods

DCs were generated from monocytes using GM-CSF and IL-4 and RV39 loading accomplished with a dose of ∼350 TCID₅₀/10⁵ cells. RV-induced maturation was established as modulation of MHC class II, CD80, CD83, and CD86. Circulating RV targeting CD4 and CD8 T cells were investigated as induction of RV-specific proliferation (CFSE-dilution).

Results

Maturation of DC by RV was confirmed as upregulation of MHC Class II (83.3±5.0% to 87.8±4.1%), CD80 (39.4±7.2% to 47.6±7.7%) and CD86 (78.4±4.7% to 84.1±3.4%). Both CD4 and CD8 memory T cells were recognized in the circulation of healthy subjects.

Conclusions

RV drives DC maturation and results in their ability to present RV antigens to both T helper and cytotoxic lymphocytes. Both CD4 and CD8 cells capable of recognizing RV-associated antigens are present in the circulation of healthy subjects where they are presumably involved in immune surveillance and explain the rapid recruitment of an adaptive immune response during RV infection.     

Negative Immune Regulator Tim-3 Is Overexpressed on T Cells in Hepatitis C Virus Infection and Its Blockade Rescues Dysfunctional CD4+ and CD8+ T Cells

A number of emerging molecules and pathways have been implicated in mediating the T-cell exhaustion characteristic of chronic viral infection. Not all dysfunctional T cells express PD-1, nor are they all rescued by blockade of the PD-1/PD-1 ligand pathway. In this study, we characterize the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in chronic hepatitis C infection. For the first time, we found that Tim-3 expression is increased on CD4⁺ and CD8⁺ T cells in chronic hepatitis C virus (HCV) infection. The proportion of dually PD-1/Tim-3-expressing cells is greatest in liver-resident T cells, significantly more so in HCV-specific than in cytomegalovirus-specific cytotoxic T lymphocytes. Tim-3 expression correlates with a dysfunctional and senescent phenotype (CD127^low CD57^high), a central rather than effector memory profile (CD45RA^negative CCR7^high), and reduced Th1/Tc1 cytokine production. We also demonstrate the ability to enhance T-cell proliferation and gamma interferon production in response to HCV-specific antigens by blocking the Tim-3-Tim-3 ligand interaction. These findings have implications for the development of novel immunotherapeutic approaches to this common viral infection.Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, affecting approximately 200 million people throughout the world; the majority of individuals exposed to HCV become persistently infected (19). A broad array of functional impairments of virus-specific T cells from early to chronic stages of infection, including exhaustion (decreased antiviral cytokine production, cytotoxicity, and proliferative capacity) (8, 24) and arrested stages of differentiation (1, 13), is supported by considerable evidence. Recently, upregulation of programmed death 1 (PD-1) and downmodulation of CD127 (interleukin-7 [IL-7] receptor) have been linked to functional exhaustion of T cells in chronic HCV infection (5-7, 15, 21, 22). However, not all exhausted T cells express these phenotypic changes, and blockade of the PD-1/PD-L1 signaling pathway does not always reconstitute Th1/Tc1 cytokine production (4, 5), indicating that other molecules may contribute to the exhaustion typically associated with chronic viral infections (9). One such molecule is Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule 3), a membrane protein initially identified on terminally differentiated Th1 but not Th2 cells in mice (9). A recent analysis of human immunodeficiency virus (HIV) infection demonstrates that Tim-3 is upregulated on both CD4⁺ and CD8⁺ T cells from patients with chronic infection relative to uninfected individuals and that virus-specific cells expressing high levels of Tim-3 secrete less IFN-γ than do Tim-3-negative cells (10). In light of these findings, for the first time, this study assessed the expression of Tim-3 in chronic HCV infection. We found a higher frequency of Tim3-expressing CD4⁺ and CD8⁺ T cells in chronic HCV infection, with the highest on HCV-specific cytotoxic T lymphocytes (CTLs). Tim-3 expression correlates with a dysfunctional phenotype and reduced Th1/Tc1 cytokine production but not viral load. We also demonstrated the ability to enhance T-cell proliferation in response to HCV-specific antigens by blocking the Tim-3-Tim-3 ligand interaction. These findings have implications for the development of novel immunotherapeutic approaches to this common disease.     

CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⁺ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⁺ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⁺ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⁺ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⁺ T cell exhaustion can be rescued by CD40-mediated mDC-activation.     

Dynamics of Cytokine/Chemokine Responses in Intestinal CD4+ and CD8+ T Cells during Acute Simian Immunodeficiency Virus Infection

Loss of intestinal CD4⁺ T cells was associated with decreased production of several T-helper 1 (T_H1) and T_H2 cytokines and increased production of interleukin 17 (IL-17), gamma interferon (IFN-γ), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by CD8⁺ T cells 21 days after simian immunodeficiency virus (SIV) infection in rhesus macaques. Shifting of mucosal T_H1 to T_H2 or T-cytotoxic 1 (T_C1) to T_C2 cytokine profiles was not evident. Additionally, both CD4⁺ and CD8⁺ T cells showed upregulation of macrophage migration inhibition factor (MIF) and basic fibroblast growth factor (FGF-basic) cytokines that have been linked to HIV disease progression.     

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

The role of CD4⁺ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4⁺ T cells are needed for the generation of the protective HSV-1-specific CD8⁺-T-cell response. This study examined the contribution of CD4⁺ T cells in the generation of the primary CD8⁺-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8⁺-T-cell response generated in the draining lymph nodes of CD4⁺-T-cell-depleted C57BL/6 mice and B6-MHC-II^−/− mice is quantitatively and qualitatively distinct from the CD8⁺ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8⁺ T cells express comparable levels of the activation marker CD44 in mice lacking CD4⁺ T cells and normal mice. In contrast, CD8⁺ T cells generated in the absence of CD4⁺ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8⁺ T cells in the CD4⁺-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8⁺ T cells is diminished in the absence of CD4⁺-T-cell help. These results suggest that CD4⁺-T-cell help is essential for the generation of fully functional CD8⁺ T cells during the primary response to HSV-1 infection.Infection due to herpes simplex virus type 1 (HSV-1) results in a wide spectrum of clinical presentations depending on the host''s age, the host''s immune status, and the route of inoculation (47). HSV-1 typically causes mild and self-limited lesions on the orofacial areas or genital sites. However, the disease can be life-threatening, as in the case of neonatal and central nervous system infections (18). The host''s immune responses, particularly CD8⁺ T cells, play an important role in determining the outcome of HSV infections in both the natural human host (18, 19, 28) and experimental murine models (11, 43). Immunodepletion and adoptive transfer studies have demonstrated the role of CD8⁺ T cells in reducing viral replication, resolving cutaneous disease, and providing overall protection upon rechallenge (6, 25, 26). CD8⁺ T cells play a particularly important role in preventing infection of the peripheral nervous system (PNS) and the reactivation of latent virus from neurons in the sensory ganglia of infected mice (21, 24, 36). The mechanisms that CD8⁺ T cells employ include gamma interferon (IFN-γ) production and functions associated with cytolytic granule content at the sites of primary infection (23, 31, 38). In the PNS of infected mice, the mechanisms primarily involve IFN-γ secretion (16, 20, 29), particularly against infected neurons expressing surface Qa-1 (41). Histopathological evidence from HSV-1-infected human ganglion sections show a large CD8⁺-T-cell infiltrate and the presence of inflammatory cytokines, suggesting that the presence of activated, effector memory cells within the PNS is important for maintaining HSV-1 latency in the natural human host (10, 42).The generation of a robust CD8⁺-T-cell response is essential for the control of various infectious pathogens. Some studies suggest that a brief interaction with antigen-presenting cells (APCs) is sufficient for CD8⁺-T-cell activation and expansion into functional effectors (44). However, the magnitude and quality of the overall CD8⁺-T-cell response generated may be dependent on additional factors (49). Recent evidence suggests that CD4⁺ T cells facilitate the activation and development of CD8⁺-T-cell responses either directly through the provision of cytokines or indirectly by the conditioning of dendritic cells (DC) (8, 48, 51). Those studies suggested that the latter mechanism is the dominant pathway, wherein CD4⁺ T cells assist CD8⁺-T-cell priming via the engagement of CD40 ligand (CD154) on CD4⁺ T cells and CD40 expressed on DC (4, 30, 33). This interaction results in the activation and maturation of DC, making them competent to stimulate antigen-specific CD8⁺-T-cell responses (35, 37).The requirement for CD4⁺-T-cell help in the generation of primary and secondary CD8⁺-T-cell responses to antigen varies. Primary CD8⁺-T-cell responses to infectious pathogens, such as Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), influenza virus, and vaccinia virus, can be mounted effectively independently of CD4⁺-T-cell help (3, 12, 22, 34). In contrast, primary CD8⁺-T-cell responses to nonmicrobial antigens display an absolute dependence on CD4⁺-T-cell help (4, 5, 30, 33, 46). This observed difference in the requirement for CD4⁺-T-cell help may ultimately be a product of the initial inflammatory stimulus generated following immunization (49). Microbial antigens trigger an inflammatory response that can lead to the direct activation and priming of APCs, such as DC, thereby bypassing the need for CD4⁺-T-cell help. Nonmicrobial antigens, however, trigger an attenuated inflammatory response that does not directly activate and prime DCs. In the absence of this inflammation, CD4⁺ T cells are thought to condition and license DC functions through CD154/CD40 interactions, which leads to the subsequent activation of antigen-specific CD8⁺-T-cell responses (5, 49). Even in the case of pathogens where primary CD8⁺-T-cell responses were independent of CD4⁺-T-cell help, the secondary responses to these pathogens were found to be defective in the absence of CD4⁺-T-cell help (3, 12, 34, 40).The requirement for CD4⁺-T-cell help in priming CD8⁺-T-cell responses against HSV-1 infection is not well defined. Earlier studies with HSV-1 suggested that CD4⁺ T cells play an important role in the generation of primary CD8⁺-T-cell responses, detected in vitro, to acute infection with HSV-1 (14), principally through the provision of interleukin 2 (IL-2) for optimal CD8⁺-T-cell differentiation and proliferation. Subsequent studies, utilizing an in vivo approach, indicated that CD4⁺ T cells were not required for CD8⁺-T-cell-mediated cytolytic function (23). CD4⁺ T cells are thought to provide help by conditioning DC in a cognate, antigen-specific manner, thereby making them competent to stimulate HSV-1-specific CD8⁺-T-cell responses (37). By contrast, findings from other studies show that CD4⁺-T-cell-depleted mice were able to fully recover from acute infection with HSV-1 (38). These studies imply that the absence of CD4⁺ T cells does not prevent priming of CD8⁺ T cells in vivo.Studies from this laboratory have identified two distinct HSV-1-specific CD8⁺-T-cell subpopulations generated during the primary response, based upon the ability to synthesize IFN-γ following antigenic stimulation in vitro (1). To better understand the need for CD4⁺-T-cell help, we examined the functional characteristics and phenotypes of these CD8⁺-T-cell populations generated during a primary response to acute infection with HSV-1 in mice lacking CD4⁺ T cells. Our findings show that primary CD8⁺-T-cell responses to HSV-1 are compromised in the absence of CD4⁺-T-cell help. Specifically, the HSV-1 gB-specific CD8⁺ T cells produced in the absence of CD4⁺ T cells were found to be active with regard to cytolysis in vivo but were functionally impaired in the production of IFN-γ and TNF-α compared with intact C57BL/6 mice. Virus-specific CD8⁺ T cells were also reduced in number in CD4-depleted mice and in B6 mice lacking major histocompatibility complex (MHC) class II expression (B6-MHC-II^−/−) compared to wild-type (WT) mice. In addition, our data showed higher virus burdens in the infectious tissues obtained from mice lacking CD4⁺ T cells than in those from intact mice. Collectively, these findings demonstrate that CD4⁺-T-cell help is essential for the generation of primary CD8⁺-T-cell responses following acute cutaneous infection with HSV-1.     

Effective Simian Immunodeficiency Virus-Specific CD8+ T Cells Lack an Easily Detectable,Shared Characteristic

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8⁺ T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8⁺ T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8⁺ T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8⁺ T cells to suppress viral replication from SIVmac239-infected CD4⁺ T cells. Using this assay, we established an antiviral hierarchy when we compared CD8⁺ T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8⁺ T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8⁺ T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8⁺ T-cell response may be comprised of several factors.CD8⁺ T cells may play a critical role in blunting peak viremia and controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. The transient depletion of CD8⁺ cells in SIV-infected macaques results in increased viral replication (26, 31, 51, 70). The emergence of virus-specific CD8⁺ T cells coincides with the reduction of peak viremia (12, 39, 42, 63), and CD8⁺ T-cell pressure selects for escape mutants (6, 9, 13, 28, 29, 38, 60, 61, 85). Furthermore, particular major histocompatibility complex (MHC) class I alleles are overrepresented in SIV- and HIV-infected elite controllers (15, 29, 33, 34, 46, 56, 88).Because it has been difficult to induce broadly neutralizing antibodies (Abs), the AIDS vaccine field is currently focused on developing a vaccine designed to elicit HIV-specific CD8⁺ T cells (8, 52, 53, 82). Investigators have tried to define the immune correlates of HIV control. Neither the magnitude nor the breadth of epitopes recognized by virus-specific CD8⁺ T-cell responses correlates with the control of viral replication (1). The quality of the immune response may, however, contribute to the antiviral efficacy of the effector cells. It has been suggested that the number of cytokines that virus-specific CD8⁺ T cells secrete may correlate with viral control, since HIV-infected nonprogressors appear to maintain CD8⁺ T cells that secrete several cytokines, compared to HIV-infected progressors (11, 27). An increased amount of perforin secretion may also be related to the proliferation of HIV-specific CD8⁺ T cells in HIV-infected nonprogressors (55). While those studies offer insight into the different immune systems of progressors and nonprogressors, they did not address the mechanism of viral control. Previously, we found no association between the ability of SIV-specific CD8⁺ T-cell clones to suppress viral replication in vitro and their ability to secrete gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-2 (IL-2) (18).Evidence suggests that some HIV/SIV proteins may be better vaccine targets than others. CD8⁺ T cells recognize epitopes derived from Gag as early as 2 h postinfection, whereas CD8⁺ T cells specific for epitopes in Env recognize infected cells only at 18 h postinfection (68). Additionally, a previously reported study of HIV-infected individuals showed that an increased breadth of Gag-specific responses was associated with lower viral loads (35, 59, 65, 66). CD8⁺ T-cell responses specific for Env, Rev, Tat, Vif, Vpr, Vpu, and Nef were associated with higher viral loads, with increased breadth of Env in particular being significantly associated with a higher chronic-phase viral set point.None of the many sophisticated methods employed for analyzing the characteristics of HIV- or SIV-specific immune responses clearly demarcate the critical qualities of an effective antiviral response. In an attempt to address these questions, we developed a new assay to measure the antiviral efficacy of individual SIV-specific CD8⁺ T-cell responses sorted directly from fresh peripheral blood mononuclear cells (PBMC). Using MHC class I tetramers specific for the epitope of interest, we sorted freshly isolated virus-specific CD8⁺ T cells and determined their ability to suppress virus production from SIV-infected CD4⁺ T cells. We then looked for a common characteristic of efficacious epitope-specific CD8⁺ T cells using traditional methods.     

TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01 + Rhesus Monkeys

Many of the factors that contribute to CD8⁺ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8⁺ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8⁺ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8⁺ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8⁺ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8⁺ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.     

CD4+ and CD8+ T Cell Activation Are Associated with HIV DNA in Resting CD4+ T Cells

The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4⁺ and CD8⁺ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.     

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8⁺ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8⁺ T cells require IL-2 provided by activated CD4⁺ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4⁺ cells is mediated through the IL-2Rβγ of CD8⁺ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8⁺ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8⁺ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8⁺ T-cell response is elicited by a critical temporal function of IL-2 released from CD4⁺ T cells, providing mechanistic insights into the regulation of CD8⁺ T cell activation and differentiation.