CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis

CD4 T cell activation during peripheral infections not only is essential in inducing protective CD8 T cell memory but also promotes CD8 T cell function and survival. However, the contributions of CD4 T cell help to antiviral CD8 T cell immunity during central nervous system (CNS) infection are not well established. Encephalitis induced by the sublethal coronavirus JHMV was used to identify when CD4 T cells regulate CD8 T cell responses following CNS infection. Peripheral expansion of virus-specific CD8 T cells was impaired when CD4 T cells were ablated prior to infection but not at 4 days postinfection. Delayed CD4 T cell depletion abrogated CD4 T cell recruitment to the CNS but only slightly diminished CD8 T cell recruitment. Nevertheless, the absence of CNS CD4 T cells was associated with reduced gamma interferon (IFN-γ) and granzyme B expression by infiltrating CD8 T cells, increased CD8 T cell apoptosis, and impaired control of infectious virus. CD4 T cell depletion subsequent to CD4 T cell CNS migration restored CD8 T cell activity and virus control. Analysis of γc-dependent cytokine expression indicated interleukin-21 (IL-21) as a primary candidate optimizing CD8 T cell activity within the CNS. These results demonstrate that CD4 T cells play critical roles in both enhancing peripheral activation of CD8 T cells and prolonging their antiviral function within the CNS. The data highlight the necessity for temporally and spatially distinct CD4 T cell helper functions in sustaining CD8 T cell activity during CNS infection.     

Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases.

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-gamma, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-gamma-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.     

Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4⁺ and CD8⁺ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4⁺ and CD8⁺ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.     

IL-12 signaling drives CD8+ T cell IFN-gamma production and differentiation of KLRG1+ effector subpopulations during Toxoplasma gondii Infection

IFN-gamma-producing CD8(+) T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8(+) T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8(+) T cell-intrinsic IL-12 signaling was required for the generation of IFN-gamma-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1(+) effector subpopulations, but not the memory precursor-containing KLRG1(-) effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1(+) effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-gamma expression but also in the development of heterogeneous subpopulations of effector CD8(+) T cells generated in response to the intracellular parasite T. gondii.     

Targeting an anti-viral CD8+ T cell response to a growing tumor facilitates its rejection

 We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional ‘spontaneous’ rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8⁺ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA–immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70–80% of the tumor-bearing animals. All rejecting animals showed a CD8⁺ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral (‘strong’) CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against (‘weak’) TAA in this system. Received: 18 December 1996 / Accepted: 6 February 1997     

Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector function and induces herpes simplex virus type 1 reactivation

Clinical research suggests hormonal contraceptive use is associated with increased frequencies of HSV reactivation and shedding. We examined the effects of medroxyprogesterone acetate (MPA), the compound most commonly used for injectable hormonal contraception, on HSV type 1 (HSV-1) reactivation and CD8(+) T cell function in murine trigeminal ganglia (TG). In ex vivo TG cultures, MPA dramatically inhibited canonical CD8(+) T cell effector functions, including IFN-gamma production and lytic granule release, and increased HSV-1 reactivation from latency. In vivo, MPA treatment of latently infected ovariectomized mice inhibited IFN-gamma production and lytic granule release by TG resident CD8(+) T cells stimulated directly ex vivo. RNA specific for the essential immediate early viral gene ICP4 as well as viral genome DNA copy number were increased in mice that received MPA during latency, suggesting that treatment increased in vivo reactivation. The increase in HSV-1 copy number appeared to be the result of a two-tine effect, as MPA induced higher reactivation frequencies from latently infected explanted TG neurons in the presence or absence of CD45(+) cells. Our data suggest hormonal contraceptives that contain MPA may promote increased frequency of HSV reactivation from latency through the combinatory effects of inhibiting protective CD8(+) T cell responses and by a leukocyte-independent effect on infected neurons.     

Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation

In response to infection, CD8(+) T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127(low)KLRG1(high)) and memory precursor effector cells (CD127(high)KLRG1(low)) from an early effector cell that is CD127(low)KLRG1(low) in phenotype. CD8(+) T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12-dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8(+) T cell functionality and memory subset distribution, but in an IL-12-independent manner. Population dynamics were dramatically different during secondary CD8(+) T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127(high)KLRG1(high) memory cells, both of which were intrinsic to the memory CD8(+) T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8(+) T cell differentiation.     

Regulatory T cells selectively control CD8+ T cell effector pool size via IL-2 restriction

Regulatory T cells (Treg) are key players in maintaining immune homeostasis but have also been shown to regulate immune responses against infectious pathogens. Therefore, Treg are a promising target for modulating immune responses to vaccines to improve their efficacy. Using a viral vector system, we found that Treg act on the developing immune response early postinfection by reducing the extent of dendritic cell costimulatory molecule expression. Due to this change and the lower IL-2 production that results, a substantial fraction of CD8(+) effector T cells lose CD25 expression several days after activation. Surprisingly, such Treg-dependent limitations in IL-2 signaling by Ag-activated CD8(+) T cells prevent effector differentiation without interfering with memory cell formation. In this way, Treg fine-tune the numbers of effector T cells generated while preserving the capacity for a rapid recall response upon pathogen re-exposure. This selective effect of Treg on a subpopulation of CD8(+) T cells indicates that although manipulation of the Treg compartment might not be optimal for prophylactic vaccinations, it can be potentially exploited to optimize vaccine efficacy for therapeutic interventions.     

A spontaneously arising pancreatic tumor does not promote the differentiation of naive CD8+ T lymphocytes into effector CTL

In this report, we address whether a growing tumor provides sufficient inflammatory signals to promote activation, clonal expansion, and acquisition of effector functions by naive tumor-specific CD8(+) T lymphocytes. CD8(+) T lymphocytes obtained from hemagglutinin (HA)-specific clone 4 TCR-transgenic mice were injected into recipient mice that spontaneously develop pancreatic tumors expressing HA as a tumor-associated Ag (RIP-Tag2-HA mice). When 3 x 10(6) clone 4 CD8(+) T cells were transferred into tumor-bearing mice, the cells became activated in the pancreatic lymph nodes where they proliferated and acquired effector functions such as cytolytic activity and IFN-gamma production. Surprisingly, reducing the number of adoptively transferred CD8(+) T cells led to a parallel reduction in the proportion of the activated cells that exhibited effector functions, suggesting that CTL differentiation was induced by the large numbers of activated CD8(+) T cells and not the tumor environment. Provision of tumor-specific CD4(+) helper cells provided the signals required to promote both the development of CTL effector functions and increased clonal expansion, resulting in tumor eradication. Considering that only small numbers of tumor-specific CD8(+) T cells would be present in a conventional T cell repertoire, these data suggest that tumor growth alone may not provide the inflammatory signals necessary to support the development of CD8(+) T cell effector functions.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.     

Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor's growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.     

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.     

Cutting edge: regulation of CD8+ T cell effector population size

Naive CD8(+) T cells are activated on encounter with Ag presented on dendritic cells and proliferate rapidly. To investigate the regulation of naive CD8(+) T cells proliferation, we adoptively transferred TCR-transgenic CD8(+) T cells into intact mice together with Ag-pulsed dendritic cells. Regardless of the number of cells initially transferred, the expansion of activated Ag-specific CD8(+) T cells was limited to a ceiling of effector cells. This limit was reached from a wide range of T cell doses, including a physiological number of precursor cells, and was not altered by changing the amount of Ag or APCs. The total Ag-specific response was composed of similar numbers of host and donor transgenic cells regardless of donor cell input, suggesting that these populations were independently regulated. Regulation of the transgenic donor cell population was TCR specific. We hypothesize that a clone-specific regulatory mechanism controls the extent of CD8(+) T cell responses to Ag.     

Radiotherapy promotes tumor-specific effector CD8+ T cells via dendritic cell activation

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity.     

Regulation of effector and memory CD8+ T cell function by inflammatory cytokines

Cells communicate with each other through the production and secretion of cytokines, which are integral to the host response to infection. Once recognized by specific cytokine receptors expressed on the cell surface, these exogenous signals direct the biological function of a cell in order to adapt to their microenvironment. CD8⁺ T cells are critical immune cells that play an important role in the control and elimination of intracellular pathogens. Current findings have demonstrated that cytokines influence all aspects of the CD8⁺ T cell response to infection or immunization. The cytokine milieu induced at the time of activation impacts the overall magnitude and function of the effector CD8⁺ T cell response and the generation of functional memory CD8⁺ T cells. This review will focus on the impact of inflammatory cytokines on different aspects of CD8⁺ T cell biology.     

Evolution of CD8+ T cell immunity and viral escape following acute HIV-1 infection

Induction of HIV-1-specific CD8(+) T cells during acute infection is associated with a decline in viremia. The role CD8(+) effectors play in subsequently establishing viral set point remains unclear. To address this, we focused on two acutely infected patients with the same initial Tat-specific CD8(+) response, analyzing their CD8(+) T cell responses longitudinally in conjunction with viral load and sequence evolution. In one patient initiating treatment during acute infection, the frequencies of Tat-specific CD8(+) T cells gradually diminished but persisted, and the Tat epitope sequence was unaltered. By contrast, in the second patient who declined treatment, the Tat-specific CD8(+) T cells disappeared below detection, in conjunction with Gag-specific CD4(+) T cell loss, as plasma viremia reached a set point. This coincided with the emergence of an escape variant within the Tat epitope and an additional Vpr epitope. New CD8(+) T cell responses emerged but with no further associated decline in viremia. These findings indicate that, in the absence of treatment, the initial CD8(+) T cell responses have the greatest impact on reducing viremia, and that later, continuously evolving responses are less efficient in further reducing viral load. The results also suggest that T cell help may contribute to the antiviral efficiency of the acute CD8(+) T cell response.