CD8+ T cells are a major source of IFN-γ, a key effector cytokine in immune responses against many viruses and protozoa. Although the transcription factor T-bet is required for IFN-γ expression in CD4+ T cells, it is reportedly dispensable in CD8+ T cells, where the transcription factor Eomesodermin is thought to be sufficient. The diverse functions of IFN-γ are mediated through the IFN-γR and STAT1. In CD4+ T cells, STAT1 appears to be critical for the activation of T-bet and IFN-γ, suggesting an IFN-γ-dependent positive feedback loop. However, STAT1 can also be activated by other cytokines, including IL-27. In the present study we show that, in contrast to in vitro conditions and the prevailing paradigm, T-bet is critical for the in vivo IFN-γ production by CD8+ T cells upon infection of mice with diverse pathogens. Whereas IFN-γR signals are dispensable for the T-bet-dependent IFN-γ production, direct IL-27Rα signals are critical.

The CD8+ T cell subset is a major source of IFN-γ, a key effector cytokine in immune responses against diverse pathogens including viruses and protozoa (1, 2, 3, 4). Although the transcription factor T-bet is critical for Th1 polarization and IFN-γ expression in CD4+ T cells, in vitro it is reportedly dispensable in CD8+ T cells (5, 6, 7, 8). Consistent with this, it has been demonstrated that the transcription factor Eomesodermin is sufficient for in vitro IFN-γ production by CD8+ T cells (7, 8). The diverse effects of IFN-γ on numerous immune cells are mediated through the widely expressed IFN-γR and the activation of STAT1 (3). In CD4+ T cells, STAT1 appears to be critical for the activation of T-bet and IFN-γ, suggesting an IFN-γ-dependent positive feedback loop (9, 10). In addition to type I IFNs and IFN-γ, STAT1 phosphorylation can also be induced by other cytokine receptors including the receptor for IL-27, an IL-12 family cytokine that is generally discussed for its effects on CD4+ T cells (11, 12, 13, 14, 15). However, our current understanding of the role of IFN-γ, IL-27, and T-bet in Th1 development and IFN-γ expression is based extensively on in vitro culture systems. Because the molecular mechanisms regulating the production of IFN-γ by CD8+ T cells in vitro are significantly dependent on the method of restimulation (16), we wished to examine the expression of IFN-γ by CD8+ T cells directly in vivo. We therefore used Yeti IFN-γ reporter mice (17, 18) to bypass the need for in vitro restimulation. In Yeti mice the IFN-γ-YFP reporter is faithfully expressed only under conditions known to induce IFN-γ, and the reporter fluorescence intensity correlates directly with both the abundance of IFN-γ transcripts and the production of IFN-γ upon restimulation (17, 18). In the present study we revisited the CD8+ T cell-intrinsic roles of IFN-γR, IL-27R, and T-bet for IFN-γ expression in response to viral and protozoan infection by using Yeti IFN-γ reporter mice, direct in vivo cytokine staining, MHC class I multimer reagents, and the generation of mixed bone marrow (BM)4chimeras.

IFN-γ reporter (Yeti) (17), T-bet−/− (B6.129S6-Tbx21tm1Glm/J) (6), IFN-γR1−/− (B6.129S7-Ifngr1tm1Agt/J) (1), IL-27Rα−/− (19), IFN-γ−/− (B6.129S7-Ifngtm1Ts/J) (2), and CD45.1 congenic mice (B6.SJL-PtprcaPep3b/BoyJ) mice on a C57BL/6 background (n > 10) were kept at the animal facility of Trudeau Institute. All Yeti mice were heterozygous for the IFN-γ-enhanced yellow fluorescent protein (YFP) reporter. Infections with influenza A virus (300 EID50 (50% egg infective doses) of A/HK/x31 (H3N2)) and 10 cysts of Toxoplasma gondii (ME49) were initiated as described (18). Experimental animals were analyzed on day 9 after intranasal infection with influenza A virus or day 7 after oral infection with T. gondii. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the Trudeau Institute (Saranac Lake, NY).

Naive (CD44lowYFP) CD8α+ T cells were sorted from the lymph nodes and spleens of naive mice and cultured (1 × 106/ml) in the presence of anti-CD3ε (clone 145-2C11; 2 μg/ml) and anti-CD28 (clone 37.51; 5 μg/ml), rIL-2 (5 ng/ml), and irradiated IFN-γ−/− splenocytes as APCs (5 × 106/ml) for 3 days as described (18). The following cytokines and mAb were added as indicated: rIL-4 (50 ng/ml), rIL-12 (5 ng/ml), anti-IL-4 (clone 11B11; 20 μg/ml), and anti-IFN-γ (clone XMG1.2; 20 μg/ml).

The following mAbs against mouse Ags were used as PE- or allophycocyanin conjugates: CD8α (clone CT-CD8a), CD44 (clone IM7), and CD45.1 (clone A20). Surface staining with mAb and MHC class I peptide tetramers specific for the influenza virus nucleoprotein NP366–374/Db (FluNP) and flow cytometry were performed as described (18). Samples were acquired on a FACSCalibur (BD Biosciences) flow cytometer and analyzed using FlowJo (Tree Star) software.

C57BL/6 mice (CD45.2 or CD45.1) were lethally irradiated (950 rad) and reconstituted with a total of 1 × 107 donor BM cells from CD45.1 wild-type (WT) Yeti mice mixed at equal parts with either CD45.2 T-bet−/− Yeti, IFN-γR1−/− Yeti, or IL-27Rα−/− BM cells. Mice were infected 6–8 wks later.

T. gondii-infected mice were injected i.v. with 250 μg brefeldin A and the mesenteric lymph nodes were harvested 5 h later. Intracellular staining was performed as described (20).

Data from mixed BM chimeras were analyzed by two-way ANOVA with two-tailed post tests performed by the Bonferroni method (Prism; GraphPad Software). Significance levels refer to comparisons between wilt-type (WT) and gene-deficient cells for each tissue within the individual mice (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). Data were not significantly different unless indicated.

Activation of naive WT, T-bet−/− (Fig. 1,A), and IFN-γR−/− (Fig. 1,B) Yeti CD8+ T cells with mAb to TCR/CD28 in vitro induced IFN-γ-YFP reporter expression. However, the frequency of IFN-γ-YFP+ cells and the abundance of IFN-γ transcripts were extensively modulated by a polarizing cytokine environment (18). Under neutral conditions the expression of IFN-γ-YFP was strikingly and similarly impaired in both T-bet−/− and IFN-γR−/− CD8+ T cells as compared with WT controls, indicating an autocrine, IFN-γ-dependent, positive feedback loop on T-bet as suggested for the Th1 development of CD4+ T cells (9, 10). However, when IL-12 was added, IFN-γ expression was substantially enhanced in T-bet−/− and IFN-γR−/− CD8+ T cells and, in both cases, was identical with that of WT controls (Fig. 1, A and B). These in vitro data suggest that CD8+ T cells require both T-bet and IFN-γR for optimal IFN-γ expression under neutral conditions, whereas both molecules are dispensable when IL-12 is added.

FIGURE 1.

IFN-γR and T-bet are required for normal IFN-γ expression by CD8+ T cells in vitro. Naive (CD44low/YFP) CD8+ T cells were sorted from T-bet−/− Yeti (A), IFN-γR−/− Yeti (B), and WT Yeti mice, stimulated in the presence of the indicated cytokines and cytokine-neutralizing mAb, and analyzed on day 3 for YFP fluorescence. The frequency (%) of the enhanced YFP+ cells is noted. α-IFN-γ, anti-IFN-γ; α-IL-4, anti-IL-4.

FIGURE 1.

IFN-γR and T-bet are required for normal IFN-γ expression by CD8+ T cells in vitro. Naive (CD44low/YFP) CD8+ T cells were sorted from T-bet−/− Yeti (A), IFN-γR−/− Yeti (B), and WT Yeti mice, stimulated in the presence of the indicated cytokines and cytokine-neutralizing mAb, and analyzed on day 3 for YFP fluorescence. The frequency (%) of the enhanced YFP+ cells is noted. α-IFN-γ, anti-IFN-γ; α-IL-4, anti-IL-4.

Close modal

However, it is apparent that Ag-nonspecific stimulation of CD8+ T cells in vitro does not mimic their in vivo activation by pathogen-derived Ags presented in response to infection (16, 21). Therefore, we wished to analyze the role of T-bet and IFN-γR in vivo in response to infection with diverse pathogens. Because T-bet and IFN-γR are expressed by various cellular subsets and the defects in T-bet−/− and IFN-γR−/− mice are not limited to CD8+ T cells, T-bet−/− and IFN-γR−/− animals are not suitable for revealing their CD8+ T cell-intrinsic roles (1, 3, 5, 6, 22, 23). To overcome this problem, we generated mixed BM chimeras in which lethally irradiated C57BL/6 mice (CD45.1 or CD45.2) were reconstituted with BM from WT Yeti mice (CD45.1) mixed 1:1 with either T-bet−/− Yeti (T-bet−/−/WT; Fig. 2, A–C) or IFN-γR−/− Yeti (IFN-γR−/−/WT; Fig. 2, D–F) CD45.2 donors. This approach allowed us to directly compare T-bet−/− and IFN-γR−/− T cells with WT T cells in the same animal in a T-bet- and IFN-γR-sufficient environment. Mixed T-bet−/−/WT BM chimeras were infected with influenza virus (Fig. 2, A–C) or Sendai virus (data not shown) and various organs were analyzed 9 days later. To assess the IFN-γ response within the population of Ag-specific cells generated by the respective donor BM, we used MHC class I tetramers specific for the influenza virus nucleoprotein NP366–374/Db (FluNP) or the Sendai virus nucleoprotein NP324–332/Kb (SenNP) (18) (Fig. 2,C and data not shown). Surprisingly, in striking contrast to our in vitro studies (Fig. 1,A) and the current literature (6, 7, 8), very few Ag-specific T-bet−/− CD8+ T cells in influenza virus-infected (Fig. 2,A) or Sendai virus-infected (data not shown) mixed BM chimeras expressed IFN-γ, while the vast majority of Ag-specific WT cells were YFP+ (18) (Fig. 2,B). Importantly, the defective IFN-γ response was not due to a failure of the T-bet−/− compartment to prime, expand. or disseminate a population of Ag-specific CD8+ T cells, because the frequency of Ag-specific cells was comparable to that of the internal WT control in all analyzed tissues. Because T-bet−/− and IFN-γR−/− CD8+ T cells primed in vitro under neutral conditions displayed a similar defect in IFN-γ expression (Fig. 1) and T-bet is reportedly induced by the IFN-γR/STAT1 pathway (9, 10), we reasoned that IFN-γR−/− CD8+ T cells would have a similar in vivo defect in IFN-γ expression as that of T-bet−/− T cells. However, IFN-γ expression in IFN-γR−/− CD8+ T cells was not impaired in mixed IFN-γR−/−/WT BM chimeras infected with influenza virus (Fig. 2, D and E) or Sendai virus (data not shown) as compared with the internal WT control cells. Moreover, the frequency of Ag-specific CD8+ T cells within the IFN-γR−/− population was comparable to that of the internal WT control, demonstrating that direct IFN-γR signals are dispensable for the priming, expansion, or dissemination of Ag-specific CD8+ T cells (Fig. 1 F). The latter observations in mixed BM chimeras infected with two different viruses or a protozoan parasite (see below) are in striking contrast to a recent adoptive transfer study suggesting that direct IFN-γR signals are critical for the expansion of a CD8+ T cell response (24).

FIGURE 2.

T-bet, but not IFN-γR, is critical for IFN-γ expression by CD8+ T cells in response to influenza virus infection. T-bet−/−/WT (A–C) and IFN-γR−/−/WT (D–F) mixed BM chimeras were infected with influenza virus and FluNP-specific CD8+ T cells in the indicated organs were analyzed for IFN-γ-YFP reporter expression. A and D, Representative FACS plots of the bronchoalveolar lavage (BAL). B and E, Frequency of YFP+ cells within the FluNP-specific population. C and F, Frequency of FluNP+ cells within the respective CD8+ T cell population. Bar graphs depict mean and + SD (n = 4–5). mesLN, mesenteric lymph node.

FIGURE 2.

T-bet, but not IFN-γR, is critical for IFN-γ expression by CD8+ T cells in response to influenza virus infection. T-bet−/−/WT (A–C) and IFN-γR−/−/WT (D–F) mixed BM chimeras were infected with influenza virus and FluNP-specific CD8+ T cells in the indicated organs were analyzed for IFN-γ-YFP reporter expression. A and D, Representative FACS plots of the bronchoalveolar lavage (BAL). B and E, Frequency of YFP+ cells within the FluNP-specific population. C and F, Frequency of FluNP+ cells within the respective CD8+ T cell population. Bar graphs depict mean and + SD (n = 4–5). mesLN, mesenteric lymph node.

Close modal

Because the addition of IL-12 overcame the defect in IFN-γ expression by T-bet−/− CD8+ T cells activated under neutral conditions in vitro (Fig. 1,A), we infected mixed T-bet−/−/WT BM chimeras with T. gondii, a paradigm disease model for high IL-12 production and IFN-γ-dependent immunity (Fig. 3, A–C) (4, 25). However, even T. gondii infection did not overcome the CD8+ T cell intrinsic T-bet requirement for IFN-γ expression, while the frequency of activated CD8+ T cells was identical with that of the WT control (Fig. 3,C). As expected (Fig. 2, D–F), IFN-γ expression in IFN-γR−/− CD8+ T cells of T. gondii-infected IFN-γR−/−/WT BM chimeras was unimpaired (Fig. 3, D–F). Thus, IFN-γ expression by CD8+ T cells in response to various infections is dependent upon T-bet, whereas IFN-γR-mediated signals are dispensable.

FIGURE 3.

T-bet, but not IFN-γR, is critical for in vivo IFN-γ production by CD8+ T cells in response to infection with T. gondii. T-bet−/−/WT (A–C) and IFN-γR−/−/WT (D–F) mixed BM chimeras were infected with T. gondii and CD44high CD8+ T cells in the indicated organs were analyzed for IFN-γ-YFP reporter expression. A and D, Representative FACS plots of the lung. B and E, Frequency of YFP+ cells within the CD44highCD8+ T cell population. C and F, Frequency of CD44high cells within the respective CD8+ T cell population. Bar graphs depict mean and + SD (n = 4–5). G, T-bet−/−/WT and IFN-γR−/−/WT mixed BM chimeras and IFN-γ−/− mice were infected with T. gondii, and CD8+CD44high cells in the mesenteric lymph node were analyzed for IFN-γ production using direct in vivo intracellular cytokine staining. Numbers indicate the frequencies of IFN-γ+ cells within the respective CD45 population.

FIGURE 3.

T-bet, but not IFN-γR, is critical for in vivo IFN-γ production by CD8+ T cells in response to infection with T. gondii. T-bet−/−/WT (A–C) and IFN-γR−/−/WT (D–F) mixed BM chimeras were infected with T. gondii and CD44high CD8+ T cells in the indicated organs were analyzed for IFN-γ-YFP reporter expression. A and D, Representative FACS plots of the lung. B and E, Frequency of YFP+ cells within the CD44highCD8+ T cell population. C and F, Frequency of CD44high cells within the respective CD8+ T cell population. Bar graphs depict mean and + SD (n = 4–5). G, T-bet−/−/WT and IFN-γR−/−/WT mixed BM chimeras and IFN-γ−/− mice were infected with T. gondii, and CD8+CD44high cells in the mesenteric lymph node were analyzed for IFN-γ production using direct in vivo intracellular cytokine staining. Numbers indicate the frequencies of IFN-γ+ cells within the respective CD45 population.

Close modal

Although Yeti mice are a faithful and sensitive reporter system for directly analyzing IFN-γ expression (17, 18), we wished to corroborate our observations at the IFN-γ protein level. Due to the potential artifact introduced by the in vitro restimulation required for standard intracellular cytokine staining, we used a recently developed direct in vivo staining technique (20). T-bet−/−/WT and IFN-γR−/−/WT mixed BM chimeras and IFN-γ−/− negative control mice were infected with T. gondii and 1 wk later injected i.v. with brefeldin A. The mesenteric lymph nodes (Fig. 3,G), peripheral blood, and spleen (data not shown) were harvested 5 h later, stained directly ex vivo for IFN-γ, and analyzed within the CD8+CD44high population. Very little IFN-γ staining was observed in T-bet−/− CD8+ T cells as compared with that in WT CD8+ T cell controls in the same animal, whereas robust IFN-γ production was observed in IFN-γR−/− CD8+ T cells. No IFN-γ staining was observed in T. gondii-infected IFN-γ−/− mice, confirming the specificity of this method (Fig. 3 G). Similar results were observed in the spleen and blood (data not shown). These data are identical with our results obtained in Yeti mixed BM chimeras and demonstrate that in vivo both ifng gene expression and IFN-γ protein production by CD8+ T cells during infection is critically dependent on T-bet but not on the IFN-γR.

Because T-bet reportedly functions downstream of STAT1 (9, 10, 15, 21) and, as we show here, the IFN-γR is dispensable for T-bet-dependent IFN-γ expression (Figs. 2 and 3), we explored whether the STAT1-activating IL-27R regulates T-bet-dependent IFN-γ expression by CD8+ T cells in vivo (11, 15). Mixed BM chimeras were generated from IL-27Rα−/− Yeti (CD45.2) and WT Yeti (CD45.1) donors (IL-27Rα−/−/WT) and infected with influenza virus (Fig. 4, A–C) (19). Similar to the T-bet−/−/WT and IFN-γR−/−/WT mixed BM chimeras (Fig. 3, C, and F), a robust FluNP-specific response was generated within the IL-27Rα−/− CD8+ T cell population that was not significantly different from that in the internal WT controls in all analyzed tissues (Fig. 4,C). However, the frequency of IFN-γ-expressing cells was profoundly reduced within Ag-specific IL-27Rα−/− CD8+ T cells as compared with the internal WT control (Fig. 4, A and B). The IFN-γ response of activated CD8+ T cells was similarly impaired in the IL-27R−/− population of IL-27Rα−/−/WT mixed BM chimeras infected with T. gondii (Fig. 4, D and E) while the frequency of activated cells was not reduced (Fig. 4 F). Thus, IL-27R signals, but not IFN-γR signals, are critical for IFN-γ expression by CD8+ T cells in response to infection.

FIGURE 4.

IL-27R is critical for IFN-γ expression by CD8+ T cells in response to infection. IL-27R−/−/WT mixed BM chimeras were infected with influenza virus (A–C) or T. gondii (D–F), and FluNP-specific (A–C) or activated (CD44high) (D–F) CD8+ T cells in the indicated organs were analyzed for IFN-γ-YFP reporter expression. A and D, Representative FACS plots of the indicated organ. B and E, Frequency of YFP+ cells within the FluNP-specific (B) or CD44high (E) population. C and F, Frequency of FluNP+ (C) or CD44high (F) cells within the respective CD8+ T cell population. Bar graphs depict mean + SD (n = 3); ns, Not significant.

FIGURE 4.

IL-27R is critical for IFN-γ expression by CD8+ T cells in response to infection. IL-27R−/−/WT mixed BM chimeras were infected with influenza virus (A–C) or T. gondii (D–F), and FluNP-specific (A–C) or activated (CD44high) (D–F) CD8+ T cells in the indicated organs were analyzed for IFN-γ-YFP reporter expression. A and D, Representative FACS plots of the indicated organ. B and E, Frequency of YFP+ cells within the FluNP-specific (B) or CD44high (E) population. C and F, Frequency of FluNP+ (C) or CD44high (F) cells within the respective CD8+ T cell population. Bar graphs depict mean + SD (n = 3); ns, Not significant.

Close modal

It is well established that T-bet is critical for the expression of IFN-γ in CD4+ T cells (5, 6). In contrast, its role in regulating IFN-γ expression in CD8+ T cells is controversial, with some studies showing T-bet dependence (16, 26) while others demonstrate T-bet independence (6, 7, 8, 27). Moreover, while T-bet is reportedly regulated by STAT1 (9, 10), none of these studies have investigated which cytokine receptor regulates IFN-γ expression in CD8+ T cells. In this study we demonstrate that T-bet is a nonredundant activator of ifng gene expression (Fig. 2) and IFN-γ protein production (Fig. 3) in CD8+ T cells in vivo. Moreover, we show that direct IL-27R signals, but not IFN-γR signals, are critical for the IFN-γ response in CD8+ T cells. Consistent with these data, it has been shown that IL-27 activates STAT1 and augments T-bet expression in activated CD8+ T cells in vitro (15, 28).

We can exclude that the defect in IFN-γ expression is due to an impaired pathogen-specific response or differential homing, because the frequencies of Ag-specific T-bet−/− and IL-27R−/− CD8+ T cells in mixed BM chimeras are, in all organs, comparable to those in the internal WT controls. Moreover, in contrast with another study (16), we can exclude that critical T-bet functions in non-T cells, such as dendritic cells or B cells, obscure the CD8+ T cell-intrinsic requirement for T-bet, even in disease models where CD4+ T cells appear to be dispensable (22, 23). Similarly, while Hunter and colleagues found an unabated CD8+ IFN-γ response upon polyclonal in vitro restimulation of splenocytes from T. gondii-infected IL-27R−/− mice, this study cannot exclude indirect effects (12). The nonredundant functions of T-bet and IL-27R for IFN-γ expression in CD8+ T cells are not limited to viral pathogens but are also critical in response to infection with a protozoan parasite, are required in both localized and systemic infections, and are apparent in lymphoid and nonlymphoid tissues. In contrast with some studies (7, 8), the CD8+ T cell-intrinsic requirements for T-bet apparently cannot be by-passed in vivo by its paralogue Eomesodermin. Similarly, other STAT1-activating receptors, such as the IFN-γR, the type 1 IFNR, and others, cannot compensate for direct IL-27R signals. Although we cannot exclude the possibility that IL-27 acts through other STAT pathways (11, 28, 29) or that IL-27-induced IFN-γ expression (Fig. 4) is T-bet-independent, it seems likely that STAT1 is critical because IFN-γ expression is T-bet dependent (Figs. 2 and 3) and T-bet is induced by STAT1 (9, 10, 15). Although it is possible that IL-27 regulates the expression of other cytokine receptors, direct IL-27 signals are nonetheless indispensable for normal IFN-γ expression. In summary, we demonstrate that T-bet and direct IL-27R signals are critical for IFN-γ expression by CD8+ T cells in response to diverse infections.

We thank Nico Ghilardi for providing us with IL-27Rα−/− mice and critical comments on the manuscript, Laurie Glimcher for T-bet−/− mice, and Scottie Adams and Jessica Hoffman of the molecular biology core facility for producing tetramer reagents.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by funds from Trudeau Institute and by National Institutes of Health Grants AI46530 and AI067723 (to A.M.C.), AI61587 (to L.L.J.), AI067967 and AI49823 (to D.L.W.), and AI046530 (to M.M.).

4

Abbreviations used in this paper: BM, bone marrow; FluNP, MHC class I peptide tetramer specific for the influenza virus nucleoprotein NP366–374/Db; WT, wild type; YFP, yellow fluorescent protein.

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