CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1

It has become evident that naturally occurring CD25(+) regulatory T cells (T(reg) cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if T(reg) cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25(+) T(reg) cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8(+) T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8(+) T-cell reactivity in T(reg) cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25(+) T(reg) cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8(+) T-cell memory pool. Moreover, in the challenge experiments, memory CD8(+) T cells generated with plasmid DNA in the absence of T(reg) cells cleared the virus more effectively compared with control groups. We conclude that CD25(+) T(reg) cells quantitatively as well as qualitatively affect the memory CD8(+) T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25(+) T(reg) cells and if it is possible to devise means of limiting T(reg) cell activity to enhance vaccine efficacy.     

CD8(+) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection

During an initial encounter with herpes simplex virus type 1 (HSV-1) it takes several days for an adaptive immune response to develop and for herpes-specific CD8(+) T cells to infiltrate sites of infection. By this time the virus has firmly established itself within the innervating sensory nervous system where it then persists indefinitely. Preventing the establishment of viral latency would require blocking the skin to nervous system transmission of the virus. We wished to examine if CD8(+) T cells present early during acute HSV-1 infection could block this transmission. We show that effector CD8(+) T cells failed to prevent the establishment of HSV latency even when present prior to infection. This lack of blocking likely reflects the delayed infiltration of the CD8(+) T cells into the infected skin. Examination of the kinetics of HSV-1 infection highlighted the rapidity at which the virus infects the sensory ganglia and singled out early viral replication within the skin as an important factor in determining the magnitude of the ensuing latent infection. Though unable to prevent the establishment of latency, CD8(+) T cells could reduce the average viral copy number of the residual latent infection by dampening the skin infection and thus limiting the skin-to-nerve transmission of virus.     

Rates of reactivation of latent herpes simplex virus from mouse trigeminal ganglia ex vivo correlate directly with viral load and inversely with number of infiltrating CD8+ T cells

Herpes simplex viruses (HSV) reactivate at rates proportional to the viral loads in latently infected ganglia. However, these rates vary substantially among infected animals. We assessed whether the numbers of HSV-specific CD8(+) T cells infiltrating latently infected ganglia also affect reactivation rates and contribute to their variability. Following corneal infection of mice with HSV type 2 (HSV-2), we quantified the latent viral loads in dissociated trigeminal ganglia by real-time PCR, the numbers of infiltrating CD8(+) T cells by flow cytometry, and the rates of reactivation by the detection of cell-free virus released from ganglion cells cultured in 96-well plates. The reactivation rates correlated directly with the latent viral loads (P = 0.001) but did so more strongly (P = 10(-7)) when cultures were depleted of CD8(+) T cells. Reactivation rates were reduced in a dose-dependent fashion by adding back ganglion CD8(+) T cells to the cultures (P = 0.003). We related the latent viral loads, numbers of CD8(+) T cells, and reactivation rates by mathematical equations. The rates of reactivation predicted from latent viral loads and numbers of infiltrating CD8(+) T cells in dissociated ganglia correlated with the observed rates of reactivation (P = 0.04). The reactivation of HSV-2 from ganglia ex vivo is determined both by the latent viral load and the number of infiltrating CD8(+) T cells.     

Herpetic stromal keratitis in the absence of viral antigen recognition

Herpetic stromal keratitis (HSK), resulting from ocular infection with herpes simplex virus (HSV), is thought to represent a T cell mediated immunopathologic lesion. Antigens recognized by the inflammatory T cells remain unresolved and non-TCR mediated activation of T cells (bystander activation) is considered as also involved. This report documents further evidence for the bystander activation mechanisms using three T cell transgenic RAG-/- mouse strains. Accordingly HSK occurred in PCC RAG-/-, P14 RAG-/-, and OT-1 RAG-/- mice. In none of the models could HSV specific T cell reactivity be demonstrated and animals were unprotected from lesion development by immunization prior to HSV ocular infection. The results support the role of bystander activation as a mechanism of T cell mediated immunopathology and show that CD8(+) as well as CD4(+) T cells can participate in HSK lesion development.     

T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4(+)Foxp3(+) Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8(+) T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8(+) and CD4(+) T cell IFN-gamma responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-beta in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.     

Memory CD8+ T cells require CD28 costimulation

CD8(+) T cells are a critical component of the adaptive immune response against infections and tumors. A current paradigm in immunology is that naive CD8(+) T cells require CD28 costimulation, whereas memory CD8(+) T cells do not. We show here, however, that during viral infections of mice, costimulation is required in vivo for the reactivation of memory CD8(+) T cells. In the absence of CD28 costimulation, secondary CD8(+) T cell responses are greatly reduced and this impairs viral clearance. The failure of CD8(+) T cells to expand in the absence of CD28 costimulation is CD4(+) T cell help independent and is accompanied by a failure to down-regulate Bcl-2 and by cell cycle arrest. This requirement for CD28 costimulation was shown in both influenza A and HSV infections. Thus, contrary to current dogma, memory CD8(+) T cells require CD28 costimulation to generate maximal secondary responses against pathogens. Importantly, this CD28 requirement was shown in the context of real infections were multiple other cytokines and costimulators may be up-regulated. Our findings have important implications for pathogens, such as HIV and measles virus, and tumors that evade the immune response by failing to provide CD28 costimulation. These findings also raise questions about the efficacy of CD8(+) T cell-based vaccines against such pathogens and tumors.     

Selective delivery of augmented IL-2 receptor signals to responding CD8+ T cells increases the size of the acute antiviral response and of the resulting memory T cell pool

CD8(+) T cells respond to IL-2 produced both endogenously and by CD4(+) Th during an antiviral response. However, IL-2R signals can potentially promote CD8(+) T cell death as well as proliferation, making it unclear whether IL-2R signals provide a predominantly positive or negative effect upon CD8(+) T cell responses to viral infection. To more precisely define the direct role of IL-2R signaling on CD8(+) T cells during the response to a virus, we examined the effect of delivering augmented IL-2R signals selectively to CD8(+) T cells responding to lymphocytic choriomeningitis virus infection. Although naive CD8(+) T cells are competent to produce IL-2, CD8(+) T cells lose this capacity upon differentiation into effector CD8(+) T cells. However, effector CD8(+) T cells do retain the capacity to produce GM-CSF upon Ag stimulation. Thus, to deliver enhanced autocrine IL-2R signals to CD8(+) T cells, we established a transgenic mouse strain expressing a chimeric GM-CSF/IL-2R (GMIL2R). As GM-CSF production is Ag dependent, the GMIL2R delivers an augmented IL-2R signal exclusively to CD8(+) T cells responding to Ag. Following lymphocytic choriomeningitis virus infection, GMIL2R transgenic mice exhibited an increase in both the peak CD8(+) T cell response achieved and the size of the resulting memory pool established. Upon secondary viral challenge, the GMIL2R also enhanced the proliferative response of memory CD8(+) T cells. Thus, our findings indicate that IL-2 delivery to responding CD8(+) T cells is a limiting factor in both the acute and memory antiviral responses.     

Granzyme A, a noncytolytic component of CD8(+) cell granules, restricts the spread of herpes simplex virus in the peripheral nervous systems of experimentally infected mice

Control of ganglionic herpes simplex virus (HSV) infection depends on CD8(+) cells but not on the death of infected neurons. Primarily, perforin and granzyme B mediate CD8(+) cell cytotoxicity, whereas the in vivo functions of granzyme A, a third granule protein, are unknown. Here, it is shown that granzyme A restricts the interneuronal spread of HSV and significantly influences ganglionic virus load.     

Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.     

CD137 costimulation of CD8+ T cells confers resistance to suppression by virus-induced regulatory T cells

Chronic viral infections cause high levels of morbidity and mortality worldwide, making the development of effective therapies a high priority for improving human health. We have used mice infected with Friend virus as a model to study immunotherapeutic approaches to the cure of chronic retroviral infections. In chronic Friend virus infections CD4(+) T regulatory (Treg) cells suppress CD8(+) T cell effector functions critical for virus clearance. In this study, we demonstrate that immunotherapy with a combination of agonistic anti-CD137 Ab and virus-specific, TCR-transgenic CD8(+) T cells produced greater than 99% reductions of virus levels within 2 wk. In vitro studies indicated that the CD137-specific Ab rendered the CD8(+) T cells resistant to Treg cell-mediated suppression with no direct effect on the suppressive function of the Treg cells. By 2 weeks after transfer, the adoptively transferred CD8(+) T cells were lost, likely due to activation-induced cell death. The highly focused immunological pressure placed on the virus by the single specificity CD8(+) T cells led to the appearance of escape variants, indicating that broader epitope specificity will be required for long-term virus control. However, the results demonstrate a potent strategy to potentiate the function of CD8(+) T cells in the context of immunosuppressive Treg cells.     

CD8alpha+ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.     

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

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

CD8 T cells utilize TRAIL to control influenza virus infection

Elimination of influenza virus-infected cells during primary influenza virus infections is thought to be mediated by CD8(+) T cells though perforin- and FasL-mediated mechanisms. However, recent studies suggest that CD8(+) T cells can also utilize TRAIL to kill virally infected cells. Therefore, we herein examined the importance of TRAIL to influenza-specific CD8(+) T cell immunity and to the control of influenza virus infections. Our results show that TRAIL deficiency increases influenza-associated morbidity and influenza virus titers, and that these changes in disease severity are coupled to decreased influenza-specific CD8(+) T cell cytotoxicity in TRAIL(-/-) mice, a decrease that occurs despite equivalent numbers of pulmonary influenza-specific CD8(+) T cells. Furthermore, TRAIL expression occurs selectively on influenza-specific CD8(+) T cells, and high TRAIL receptor (DR5) expression occurs selectively on influenza virus-infected pulmonary epithelial cells. Finally, we show that adoptive transfer of TRAIL(+/+) but not TRAIL(-/-) CD8(+) effector T cells alters the mortality associated with lethal dose influenza virus infections. Collectively, our results suggest that TRAIL is an important component of immunity to influenza infections and that TRAIL deficiency decreases CD8(+) T cell-mediated cytotoxicity, leading to more severe influenza infections.     

The NK cell response to mouse cytomegalovirus infection affects the level and kinetics of the early CD8(+) T-cell response

Natural killer (NK) cells and CD8(+) T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8(+) T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8(+) T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8(+) T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8(+) T cells has only a minor effect on the early control of wild-type MCMV, CD8(+) T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8(+) T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8(+) T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H-m157 interaction.     

Cutting edge: conventional CD8 alpha+ dendritic cells are generally involved in priming CTL immunity to viruses

Dendritic cells (DCs) play a central role in initiating immune responses. Despite this, there is little understanding how different DC subsets contribute to immunity to different pathogens. CD8alpha(+) DC have been shown to prime immunity to HSV. Whether this very limited capacity of a single DC subset priming CTL immunity is restricted to HSV infection or is a more general property of anti-viral immunity was examined. Here, we show that the CD8alpha(+) DCs are the principal DC subset that initiates CTL immunity to s.c. infection by influenza virus, HSV, and vaccinia virus. This same subset also dominated immunity after i.v. infection with all three viruses, suggesting a similar involvement in other routes of infection. These data highlight the general role played by CD8alpha(+) DCs in CTL priming to viral infection and raises the possibility that this DC subset is specialized for viral immunity.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

A novel functional CTL avidity/activity compartmentalization to the site of mucosal immunization contributes to protection of macaques against simian/human immunodeficiency viral depletion of mucosal CD4+ T cells

The presence of high-avidity CTLs in the right compartment can greatly affect clearance of a virus infection (for example, AIDS viral infection of and dissemination from mucosa). Comparing mucosal vs systemic immunization, we observed a novel compartmentalization of CTL avidity and proportion of functionally active Ag-specific CD8(+) T cells to tissues proximal to sites of immunization. Whereas both s.c. and intrarectal routes of immunization induced tetramer(+) cells in the spleen and gut, the mucosal vaccine induced a higher percentage of functioning IFN-gamma(+) Ag-specific CD8(+) T cells in the gut mucosa in mice. Translating to the CD8(+) CTL avidity distribution in rhesus macaques, intrarectal vaccination induced more high-avidity mucosal CTL than s.c. vaccination and protection of mucosal CD4(+) T cells from AIDS viral depletion, whereas systemic immunization induced higher avidity IFN-gamma-secreting cells in the draining lymph nodes but no protection of mucosal CD4(+) T cells, after mucosal challenge with pathogenic simian/human immunodeficiency virus. Mucosal CD4(+) T cell loss is an early critical step in AIDS pathogenesis. The preservation of CD4(+) T cells in colonic lamina propria and the reduction of virus in the intestine correlated better with high-avidity mucosal CTL induced by the mucosal AIDS vaccine. This preferential localization of high-avidity CTL may explain previous differences in vaccination results and may guide future vaccination strategy.     

Cell surface expression of H2 antigens on primary sensory neurons in response to acute but not latent herpes simplex virus infection in vivo.

CD8(+) T lymphocytes and class I major histocompatibility complex (MHC-I) molecules profoundly influence the severity of neuronal herpes simplex virus (HSV) infection in experimentally infected mice. Paradoxically, neurons are classically regarded as MHC-I deficient. However, it is shown here that H2-encoded heavy chains (alphaCs) and their associated light chain, beta2 microglobulin, are present on the surfaces of primary sensory neurons recovered from sensory ganglia within 1 to 2 weeks of HSV infection. During this time, some neurons are found to be tightly associated with T cells in vivo. Prior data showed that termination of productive HSV infection in the peripheral nervous system is not dependent on cell-mediated lysis of infected neurons. Consistent with these data, immunogold electron microscopy showed that the density of cell surface H2 on neurons is an order of magnitude lower than on satellite glia, which is predicted to favor a noncytolytic CD8 cell response.