IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomesodermin expression

Exposure of naive Th cell precursors (Thp) to IL-21 inhibits IFN-gamma production from developing Th1 cells. The inhibition of IFN-gamma seen in IL-21-treated Thp cells is specific as the expression of other Th1 cytokines is unaffected. Recently, it has been reported that Eomesodermin (Eomes), a member of the T-box gene family, is expressed in developing CD8+ T cells and plays an important role in regulating IFN-gamma production and cytolytic effector function. In this study, we show that Eomes mRNA and protein are also expressed in developing Th1 cells, and exposure of naive Thp cells to IL-21 results in a decrease in Eomes expression. Moreover, the repression of Eomes expression by IL-21 is not due to an indirect effect of IL-21 on the expression of IFN-gamma or STAT4 and is independent of STAT1 and T-bet expression. Finally, we show that ectopic expression of Eomes prevents the inhibition of IFN-gamma production from IL-21-treated Thp cells. Taken together, these results demonstrate that Eomes plays a role in regulating IFN-gamma production in CD4+ T cells and IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomes expression.     

Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation

In this study, we show that IFN-alpha beta can have a direct role in linking innate and adaptive responses by providing the "third signal" needed by naive CD8 T cells responding to Ag and costimulatory ligands. Stimulation of CD8 T cells in the absence of a third signal leads to proliferation, but clonal expansion is limited by poor survival and effector functions do not develop. We show that IFN-alpha beta can provide the third signal directly to CD8 T cells via a STAT4-dependent pathway to stimulate survival, development of cytolytic function, and production of IFN-gamma. Provision of the third signal by either IFN-alpha beta or IL-12 results in regulation of the expression of a number of genes, including several that encode proteins critical for effector function.     

Increasing the CD4+ T cell precursor frequency leads to competition for IFN-gamma thereby degrading memory cell quantity and quality

The precursor frequency of naive CD4(+) T cells shows an inverse relationship with the number of memory cells generated after exposure to cognate Ag. Using the lymphocytic choriomeningitis virus (LCMV) model, we show here that only when the initial number of naive virus-specific CD4(+) T cell precursors is low (< or =10(4) per spleen) do they give rise to abundant and homogeneous memory cells that are CD62L(low), IL-7R(high), and imbued with an enhanced capacity to produce cytokine, proliferate, and survive over time. Furthermore, memory cells derived from a high naive precursor number show functional deficits upon secondary exposure to virus. The negative effect of higher naive precursor frequency was not attributable to competition for limiting amounts of Ag, because LCMV-naive CD4(+) TCR-transgenic CD4 T cells were recruited into the LCMV-induced response even when their initial number was high. Instead, the T cells appear to compete for direct IFN-gamma signals as they differentiate into memory cells. These results are consistent with a model of T cell development in which the most fit effector T cells that receive sufficient direct IFN-gamma signals are selected to differentiate further into memory cells.     

Profiling the CD8low phenotype, an alternative career choice for CD8 T cells during primary differentiation

A CD8+ T cell of naive phenotype has multiple career choices during its primary differentiation into an effector cell population. One of these career options is becoming a CD8low T cell. We have previously shown by in vitro studies that CD8low T cells have lost expression of CD8 surface protein and mRNA and are poorly cytolytic. In line with poor cytolytic function, CD8low T cells express low levels of perforin and granzyme B and C, mediators of the granule-exocytosis machinery. However, CD8low T cells express IFN-gamma and substantial amounts of IL-4, the signature cytokines of type 1 and type 2 T-cell polarization, respectively. Here, we argue that the CD8low phenotype is an alternative career choice for any naive CD8+ T cell during primary activation but that the probability of choosing this option is greatly enhanced by both IL-4 and strong activation conditions. CD8low T cells have downregulated CD8 alpha/beta heterodimers and no preferential CD8 alpha/alpha homodimer expression. As shown by anti-CD8 Ab blocking experiments, surface CD8 substantially contributes to the CD8 T cell's effector function (i.e. cytokine expression and cytolytic activity). The distinct effector profile of CD8low T cells gives an example of the complexity of different CD8 T cell careers during primary effector differentiation.     

Signal 3 availability limits the CD8 T cell response to a solid tumor

CD8 T cells need a third signal, along with Ag and costimulation, for effective survival and development of effector functions, and this can be provided by IL-12 or type I IFN. Adoptively transferred OT-I T cells, specific for H-2K(b) and OVA, encounter Ag in the draining lymph nodes of mice with the OVA-expressing E.G7 tumor growing at a s.c. site. The OT-I cells respond by undergoing limited clonal expansion and development of effector functions (granzyme B expression and IFN-gamma production), and they migrate to the tumor where they persist but fail to control tumor growth. In contrast, OT-I T cells deficient for both the IL-12 and type I IFN receptors expand only transiently and rapidly disappear. These results suggested that some signal 3 cytokine is available, but that it is insufficient to support a CTL response that can control tumor growth. Consistent with this, administration of IL-12 at day 10 of tumor growth resulted in a large and sustained expansion of wild-type OT-I cells with enhanced effector functions, and tumor growth was controlled. This did not occur when the OT-I cells lacked the IL-12 and type I IFN receptors, demonstrating that the therapeutic effect of IL-12 results from direct delivery of signal 3 to the CD8 T cells responding to tumor Ag in the signal 3-deficient environment of the tumor.     

PD-1 regulates self-reactive CD8+ T cell responses to antigen in lymph nodes and tissues

PD-1, an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. We tested the role of PD-1:PD-1 ligand (PD-L) interactions in cross-presentation and the generation and control of CD8(+) responses against self-Ag. Ag-naive PD-1(-/-) OVA-specific OT-I CD8(+) T cells exhibited exacerbated responses to cross-presented Ag in mice expressing soluble OVA under the control of the rat insulin promoter (RIP-ova(high)). Following adoptive transfer into RIP-ova(high) recipients, PD-1(-/-) OT-I T cells expanded in the pancreatic lymph node. In contrast to wild-type OT-I cells, PD-1(-/-) OT-I T cells secreted IFN-gamma and migrated into the pancreas, ultimately causing diabetes. Loss of PD-1 affected CD8(+) cells intrinsically, and did not significantly alter the responses of wild-type OT-I T cells adoptively transferred into the same RIP-ova(high) recipient mouse. PD-1:PD-L interactions also limited CD8(+) effector cells, and PD-L1 expression on parenchymal tissues protected against effector OT-I T cell attack. Finally, we found that the loss of PD-1 on effector OT-I cells lowers the threshold for Ag recognition in peripheral tissues. These findings indicate two checkpoints where PD-1 attenuates self-reactive T cell responses: presentation of self-Ag to naive self-reactive T cells by dendritic cells in the draining lymph node and reactivation of pathogenic self-reactive T cells in the target organ.     

A clonal culture system demonstrates that IL-4 induces a subpopulation of noncytolytic T cells with low CD8, perforin, and granzyme expression

Immune deviation of cytolytic T cell function, induced by type 2 cytokines like IL-4, is an attractive concept to explain failure of the immune system in some diseases. However, this concept is challenged by previous conflicting results on whether type 2 cytokine-producing CD8(+) T cells are cytolytic. Therefore, we have analyzed the relationship between cytolytic activity and cytokine production among large numbers of primary CD8(+) T cell clones. Single murine CD8(+) T cells of naive phenotype were activated at high efficiency with immobilized Abs to CD3, CD8, and CD11a in the presence of IL-2 (neutral conditions) or IL-2, IL-4, and anti-IFN-gamma Ab (type 2-polarizing conditions) for 8-9 days. Under neutral conditions, most clones produced IFN-gamma without IL-4 and were cytolytic. Under type 2-polarizing conditions, most clones produced IFN-gamma and IL-4 but displayed variable cytolytic activity and CD8 expression. Separation on the basis of surface CD8 levels revealed that, compared with CD8(high) cells from the same cultures, CD8(low) cells were poorly cytolytic and expressed low levels of perforin mRNA and protein and granzyme A, B, and C mRNA. A similar, smaller population of noncytolytic CD8(low) cells was identified among CD8(+) T cells activated in mixed lymphocyte reaction with IL-4. Variable efficiency of generation of the noncytolytic cells may account for the differing results of earlier studies. We conclude that IL-4 promotes the development of a noncytolytic CD8(low) T cell phenotype that might be important in tumor- or pathogen-induced immune deviation.     

IL-6, in synergy with IL-7 or IL-15, stimulates TCR-independent proliferation and functional differentiation of CD8+ T lymphocytes

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8(+) T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8(+) T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8(+) T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8(+) T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8(+) T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8(+) T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.     

Cutting Edge: Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens

Constitutive presentation of tissue Ags by dendritic cells results in tolerance of autoreactive CD8+ T cells; however, the underlying molecular mechanisms are not well understood. In this study we show that programmed death (PD)-1, an inhibitory receptor of the CD28 family, is required for tolerance induction of autoreactive CD8+ T cells. An antagonistic Ab against PD-1 provoked destructive autoimmune diabetes in RIP-mOVA mice expressing chicken OVA in the pancreatic islet cells, which received naive OVA-specific CD8+ OT-I cells. This effect was mediated by the PD ligand (PD-L) PD-L1 but not by PD-L2. An increased number of effector OT-I cells recovered from the pancreatic lymph nodes of anti-PD-L1-treated mice showed down-regulation of PD-1. Furthermore, the blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect OT-I cell division but enhanced its granzyme B, IFN-gamma, and IL-2 production. Thus, during the presentation of tissue Ags to CD8+ T cells, PD-1/PD-L1 interaction crucially controls the effector differentiation of autoreactive T cells to maintain self-tolerance.     

Autocrine IFN-γ promotes naive CD8 T cell differentiation and synergizes with IFN-α to stimulate strong function

Autocrine IFN-γ signaling is important for CD4 differentiation to Th1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show in this paper that naive murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at ～8 h and declining by 24 h. The autocrine IFN-γ signals for upregulation of expression of T-bet and granzyme B and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, whereas IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or type I IFN.     

Progressive differentiation and commitment of CD8+ T cells to a poorly cytolytic CD8low phenotype in the presence of IL-4

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4-CD8- cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.     

Differential regulation of primary and secondary CD8+ T cells in the central nervous system

T cell accumulation and effector function following CNS infection is limited by a paucity of Ag presentation and inhibitory factors characteristic of the CNS environment. Differential susceptibilities of primary and recall CD8+ T cell responses to the inhibitory CNS environment were monitored in naive and CD8+ T cell-immune mice challenged with a neurotropic coronavirus. Accelerated virus clearance and limited spread in immunized mice was associated with a rapid and increased CNS influx of virus-specific secondary CD8+ T cells. CNS-derived secondary CD8+ T cells exhibited increased cytolytic activity and IFN-gamma expression per cell compared with primary CD8+ T cells. However, both Ag-specific primary and secondary CD8+ T cells demonstrated similar contraction rates. Thus, CNS persistence of increased numbers of secondary CD8+ T cells reflected differences in the initial pool size during peak inflammation rather than enhanced survival. Unlike primary CD8+ T cells, persisting secondary CD8+ T cells retained ex vivo cytolytic activity and expressed high levels of IFN-gamma following Ag stimulation. However, both primary and secondary CD8+ T cells exhibited reduced capacity to produce TNF-alpha, differentiating them from effector memory T cells. Activation of primary and secondary CD8+ T cells in the same host using adoptive transfers confirmed similar survival, but enhanced and prolonged effector function of secondary CD8+ T cells in the CNS. These data suggest that an instructional program intrinsic to T cell differentiation, rather than Ag load or factors in the inflamed CNS, prominently regulate CD8+ T cell function.     

Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed APC

In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.     

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.     

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.