Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

Cutting edge: IFN-gamma regulates the induction and expansion of IL-17-producing CD4 T cells during mycobacterial infection

T cell responses are important to the control of infection but are deleterious if not regulated. IFN-gamma-deficient mice infected with mycobacteria exhibit enhanced accumulation of activated effector T cells and neutrophils within granulomatous lesions. These cells do not control bacterial growth and compromise the integrity of the infected tissue. We show that IFN-gamma-deficient mice have increased numbers of IL-17-producing T cells following infection with Mycobacterium bovis bacille Calmette Guérin. Furthermore, exogenous IFN-gamma increases IL-12 and decreases IL-23 production by bacille Calmette Guérin-infected bone marrow-derived dendritic cells and reduces the frequency of IL-17-producing T cells induced by these bone marrow-derived dendritic cells. These data support the hypothesis that, during mycobacterial infection, both IFN-gamma- and IL-17-producing T cells are induced, but that IFN-gamma serves to limit the IL-17-producing T cell population. This counterregulation pathway may be an important factor in limiting mycobacterially associated immune-mediated pathology.     

Cutting edge: rapid in vivo killing by memory CD8 T cells

In this study, we examined the cytotoxic activity of effector and memory CD8 T cells in vivo. At the peak of the CTL response following an acute lymphocytic choriomeningitis virus infection, effector CD8 T cells exhibited extremely rapid killing and started to eliminate adoptively transferred target cells within 15 min by a perforin-dependent mechanism. Although resting memory CD8 T cells are poorly cytolytic by in vitro (51)Cr release assays, there was rapid elimination (within 1-4 h) of target cells after transfer into immune mice, and both CD62L(high) and CD62L(low) memory CD8 T cells were able to kill rapidly in vivo. Strikingly, when directly compared on a per cell basis, memory CD8 T cells were only slightly slower than effector cells in eliminating target cells. These data indicate that virus specific memory CD8 T cells can rapidly acquire cytotoxic function upon re-exposure to Ag and are much more efficient killers in vivo than previously appreciated.     

Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.     

Cutting edge: soluble IL-6R is produced by IL-6R ectodomain shedding in activated CD4 T cells

IL-6 trans-signaling via the soluble IL-6R (sIL-6R) plays an important role in the progression of several autoimmune diseases and cancer by providing IL-6-responsiveness to cells lacking IL-6R. However, the potential sources of sIL-6R are less understood. In this study we show that sIL-6R is produced by both naive and memory CD4 T cells upon TCR activation. The production of sIL-6R by activated CD4 T cells is mediated by shedding of the membrane-bound IL-6R, and this process correlates with the expression of the metalloproteinase ADAM17 in these cells. In contrast to CD4 T cells, CD8 T cells do not express ADAM17 and their production of sIL-6R is negligible. Thus, during an immune response CD4 T cells are an important source of sIL-6R. Production of sIL-6R by autoreactive CD4 T cells may contribute to their role in the development of autoimmune disease by conferring IL-6-responsiveness to cells lacking IL-6R such as synoviocytes.     

Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses

In this study, we compared the proliferation and differentiation of Ag-specific CD4 and CD8 T cells following Listeria infection. Our results show that CD4 T cells responding to infection divide a limited number of times, with progeny exhibiting proliferative arrest in early divisions. Even with increased infectious doses, CD4 T cells display this restricted proliferative pattern and are not driven to undergo extensive clonal expansion. This is in striking contrast to CD8 T cells, which undergo extensive proliferation in response to infection. These differences are also evident when CD4 and CD8 T cells receive uniform anti-CD3 stimulation in vitro. Together, these results suggest that CD4 and CD8 T cells are programmed to undergo limited and extensive proliferation, respectively, to suit their function as regulator and effector cells.     

Activation and IL-10 production of specific CD4+ T cells are regulated by IL-27 during chronic infection with Plasmodium chabaudi

IL-27, a regulatory cytokine, plays critical roles in the prevention of immunopathology during Plasmodium infection. We examined these roles in the immune responses against Plasmodium chabaudi infection using the Il-27ra^−/− mice. While IL-27 was expressed at high levels during the early phase of the infection, enhanced CD4⁺ T cell function and reduction in parasitemia were observed mainly during the chronic phase in the mutant mice. In mice infected with P. chabaudi and cured with drug, CD4⁺ T cells in the Il-27ra^−/− mice exhibited enhanced CD4⁺ T-cell responses, indicating the inhibitory role of IL-27 on the protective immune responses. To determine the role of IL-27 in detail, we performed CD4⁺ T-cell transfer experiments. The Il-27ra^−/− and Il27p28^−/− mice were first infected with P. chabaudi and then cured using drug treatment. Plasmodium-antigen primed CD4⁺ T cells were prepared from these mice and transferred into the recipient mice, followed by infection with the heterologous parasite P. berghei ANKA. Il-27ra^−/− CD4⁺ T cells in the infected recipient mice did not produce IL-10, indicating that IL-10 production by primed CD4⁺ T cells is IL-27 dependent. Il27p28^−/− CD4⁺ T cells that were primed in the absence of IL-27 exhibited enhanced recall responses during the challenge infection with P. berghei ANKA, implying that IL-27 receptor signaling during the primary infection affects recall responses in the long-term via the regulation of the memory CD4⁺ T cell generation. These features highlighted direct and time-transcending roles of IL-27 in the regulation of immune responses against chronic infection with Plasmodium parasites.     

Cutting edge: IL-15-independent maintenance of mucosally generated memory CD8 T cells

Effective vaccines against intracellular pathogens rely on the generation and maintenance of memory CD8 T cells (T(mem)). Hitherto, evidence has indicated that CD8 T(mem) use the common γ-chain cytokine IL-15 for their steady-state maintenance in the absence of Ag. This evidence, however, has been amassed predominantly from models of acute, systemic infections. Given that the route of infection can have significant impact on the quantity and quality of the resultant T(mem), reliance on limited models of infection may restrict our understanding of long-term CD8 T(mem) survival. In this article, we show IL-15-independent generation, maintenance, and function of CD8 T(mem) after respiratory infection with influenza virus. Importantly, we demonstrate that alternating between mucosal and systemic deliveries of the identical virus prompts this change in IL-15 dependence, necessitating a re-evaluation of the current model of CD8 T(mem) maintenance.     

Cutting edge: dysfunctional CD8+ T cells reside in nonlymphoid tissues during chronic Trypanosoma cruzi infection

Chagas disease is caused by persistent Trypanosoma cruzi infection in muscle tissue that ultimately results in chronic inflammation and tissue destruction. It is unclear why T. cruzi is cleared from some tissues but persists in others, despite an active inflammatory response. In this study, we show that the majority of CD8(+) T cells present in muscle tissue express memory and effector cell surface markers but have sharply attenuated effector function compared with their splenic counterparts. The dysfunction of CD8(+) T cells in the muscle tissue suggests a mechanism by which T. cruzi can persist in that location and cause inflammatory damage.     

IL-27 promotes IL-10 production by effector Th1 CD4+ T cells: a critical mechanism for protection from severe immunopathology during malaria infection

Infection with the malaria parasite, Plasmodium, is characterized by excessive inflammation. The establishment of a precise balance between the pro- and anti-inflammatory responses is critical to guarantee control of the parasite and survival of the host. IL-10, a key regulatory cytokine produced by many cells of the immune system, has been shown to protect mice against pathology during acute Plasmodium0 chabaudi chabaudi AS model of malaria. However, the critical cellular source of IL-10 is still unknown. In this article, we demonstrate that T cell-derived IL-10 is necessary for the control of pathology during acute malaria, as mice bearing specific deletion of Il10 in T cells fully reproduce the phenotype observed in Il10(-)(/)(-) mice, with significant weight loss, decline in temperature, and increased mortality. Furthermore, we show that IFN-γ(+) Th1 cells are the main producers of IL-10 throughout acute infection, expressing high levels of CD44 and ICOS, and low levels of CD127. Although Foxp3(+) regulatory CD4(+) T cells produce IL-10 during infection, highly activated IFN-γ(+) Th1 cells were shown to be the essential and sufficient source of IL-10 to guarantee protection against severe immune-mediated pathology. Finally, in this model of malaria, we demonstrate that the generation of protective IL10(+)IFN-γ(+) Th1 cells is dependent on IL-27 signaling and independent of IL-21.     

Cutting edge: a critical role for CD70 in CD8 T cell priming by CD40-licensed APCs

The CD154/CD40 interaction is an important pathway of CD4 T cell help for CD8 T cell responses. In this study, we address the role of CD70, a member of the TNF superfamily and the ligand for the T cell costimulatory receptor CD27, in CD40-mediated priming of CD8 T cells. Using an agonistic anti-CD40 mAb to mimic the CD154/CD40 interaction we demonstrate that the priming of OT-I TCR transgenic or endogenous mouse OVA-specific CD8 T cells is critically dependent on CD70/CD27 interaction. CD70 blockade inhibited CD40-mediated clonal expansion of CD8 T cells and reduced the number of memory CD8 T cells generated. Furthermore, CD70 blockade during the initial priming of CD8 T cells inhibited the ability of memory CD8 T cells to expand in response to a second encounter with Ag. Our data indicate that CD70 expression on APCs plays a key role in CD40-dependent CD8 T cell responses.     

Cutting edge: dendritic cells are essential for in vivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice

A powerful IFN-gamma response is triggered upon infection with the protozoan parasite, Toxoplasma gondii. Several cell populations, including dendritic cells (DCs), macrophages, and neutrophils, produce IL-12, a key cytokine for IFN-gamma induction. However, it is still unclear which of the above cell populations is its main source. Diphtheria toxin (DT) injection causes transient DC depletion in a transgenic mouse expressing Simian DT receptors under the control of the CD11c promoter, allowing us to investigate the role of DCs in IL-12 production. T. gondii-inoculated DT-treated and control groups were monitored for IL-12 levels and survival. We show in this study that DC depletion abolished IL-12 production and led to mortality. Furthermore, replenishment with wild-type, but not MyD88- or IL-12p35-deficient, DCs rescued IL-12 production, IFN-gamma-induction, and resistance to infection in DC-depleted mice. Taken together, the results presented in this study indicate that DCs constitute the major IL-12-producing cell population in vivo during T. gondii infection.     

Cutting edge: latecomer CD8 T cells are imprinted with a unique differentiation program

Factors that influence T cell responses, such as Ag load, APCs, costimulatory molecules, and cytokines, dramatically change during the course of an immune response. We observed that antiviral CD8 T cells were not recruited from circulation simultaneously, but over a period of 3-4 days. Consequently, locally resident T cells and those that entered secondary lymphoid tissue later were primed in very different environments. The cells recruited later in the response were imprinted with a unique differentiation program, such that their magnitude of proliferation was reduced and their kinetics of expansion was delayed. In addition, we found that the "latecomer" CD8 T cells displayed a unique surface phenotype indicative of reduced stimulation but were not preferentially recruited into the surviving pool of memory cells. This finding demonstrates that the timing of recruitment of individual T cell clones determines the population dynamics of the subsequent immune response.     

Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells

IL-15 and IL-15Ralpha are required for generation of memory-phenotype CD8 T cells in unimmunized mice. However, the role of IL-15 in primary expansion and generation of Ag-specific memory CD8 T cells in vivo has not been investigated. We characterized the CD8 T cell response against vesicular stomatitis virus (VSV) in IL-15(-/-) and IL-15Ralpha(-/-) mice. Surprisingly, IL-15 was required for primary expansion of VSV-specific CD8 T cells. The generation of VSV-specific memory CD8 T cells was also impaired without IL-15 signaling, and this defect correlated with a decrease in memory CD8 T cell turnover. Despite minimal proliferation without IL-15, a subset of memory cells survived long-term. IL-15Ralpha expression was low on naive CD8 T cells, up-regulated on Ag-specific effector cells, and sustained on memory cells. Thus, IL-15 was important for the generation and the subsequent maintenance of antiviral memory CD8 T cells.     

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.     

Cutting edge: selective IL-18 requirements for induction of compartmental IFN-gamma responses during viral infection

Optimal protective effects for defense against infection require orchestration of immune responses spanning multiple host compartments and divergent local regulation at particular sites. During murine cytomegalovirus infections known to target spleen and liver, IL-12-induced IFN-gamma from NK cells is crucial for resistance. However, the roles for IL-18 and/or IL-12 in regulating hepatic IFN-gamma responses, as compared with systemic or splenic responses, have not been defined. In this report, mice genetically deficient in either IL-18 or IL-12p35 exhibited up to 95% reductions in systemic and splenic IFN-gamma responses. Surprisingly, IFN-gamma responses were preserved in the livers of IL-18-deficient, but not IL-12p35-deficient, mice. Cytokine requirements for host survival also differed. Under conditions where mice lacking IL-12p35 exhibited 100% mortality, those lacking IL-18 survived. Taken together, our results delineate contrasting compartmental requirements for IL-18 and suggest that preservation of local, hepatic IFN-gamma production is critical for host defense during murine cytomegalovirus challenge.     

Cutting edge: CD40-CD40 ligand pathway plays a critical CD8-intrinsic and -extrinsic role during rescue of exhausted CD8 T cells

CD8 exhaustion mediated by an inhibitory programmed death-1-programmed death ligand-1 (PD-L1) pathway occurs in several chronic infections, including toxoplasmosis. Although blockade of the programmed death-1-PD-L1 pathway revives this response, the role of costimulatory receptors involved in this rescue has not been ascertained in any model of CD8 exhaustion. This report demonstrates that one such costimulatory pathway, CD40-CD40L, plays a critical role during rescue of exhausted CD8 T cells. Blockade of this pathway abrogates the ameliorative effects of anti-PD-L1 treatment on CD8 T cells. Additionally, we demonstrate in an infectious disease model that CD8-intrinsic CD40 signaling is important for optimal CD8 polyfunctionality, proliferation, T-bet upregulation, and IL-21 signaling, albeit in the context of CD8 rescue. The critical role of CD40 during the rescue of exhausted CD8 T cells may provide a rational basis for designing novel therapeutic vaccination approaches.