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. 2009 Dec;128(4):589–599. doi: 10.1111/j.1365-2567.2009.03169.x

T-bet-dependent regulation of CD8+ T-cell expansion during experimental Trypanosoma cruzi infection

Dustin Cobb 1, Siqi Guo 1, Ana M Lara 1, Patricio Manque 1, Gregory Buck 1, Ronald B Smeltz 1
PMCID: PMC2792143  PMID: 19824916

Abstract

The transcription factor T-bet (T-box, expressed in T cells), promotes type I immunity to pathogens through effects involving T cells and dendritic cells. In CD8+ T cells, many of the functions of T-bet are redundant with those of eomesodermin (Eomes), a paralogue of T-bet. We therefore investigated the role of T-bet in immunity to Trypanosoma cruzi, an intracellular pathogen that causes Chagas disease, and which requires CD8+ T cells for resistance. T-bet-deficient mice (tbx21−/−) were highly susceptible to T. cruzi infection, marked by severe liver pathology. CD8+ T cells from infected tbx21−/− mice expressed typical markers of activation, including CD44 and CD25. In striking contrast, there was a 10-fold reduction in the number of antigen-specific CD8+ T cells in tbx21−/− mice. This reduction was not a consequence of increased apoptosis or altered tissue-specific migration. Further, antigen-presenting cell (APC) functions in tbx21−/− mice were normal as we observed comparable levels of B7-1, B7-2 and CD40 expression as well as normal antigen-driven proliferation of wild-type CD8+ T cells in infected tbx21−/− mice. However, adoptive transfer of naïve T cells from tbx21−/− donors into infected Rag-2-deficient mice (tbx21+/+) demonstrated a similar quantitative defect in CD8+ T-cell expansion. These data demonstrate that T-bet facilitates immunity to T. cruzi by promoting the expansion of T. cruzi-specific CD8+ T cells in a T cell-intrinsic manner. They also serve to further illustrate the multifaceted functions of T-box proteins in regulating quantitative aspects of T-cell immunity, in addition to qualitative components such as cytokine production.

Keywords: infection, T cells, transcription factors/gene regulation

Introduction

CD8+ T cells play a critical role in type 1/interferon (IFN)-γ immunity to intracellular pathogens, including the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease in humans.1–3 Although the factors that promote IFN-γ production and subsequent resistance to T. cruzi infection have been well characterized,4,5 much less is known about the molecular factors that regulate CD8+ T-cell responses to T. cruzi. Two primary candidates, however, are the T-box transcription factors T-bet (T-box, expressed in T cells) and eomesodermin (Eomes).6,7 T-bet is expressed in CD4+ T helper type 1 (Th1) cells, CD8+ T cells, dendritic cells (DCs) and natural killer (NK) T cells.8–12 Expression of T-bet in T cells is induced by a combination of T-cell receptor (TCR) and IFN-γ signalling.13,14 Not surprisingly, mice genetically deficient in T-bet (tbx21−/−) have increased susceptibility to infections caused by various pathogens, and susceptibility is typically a consequence of altered CD4+ T-cell cytokine production and/or reduced Th1 differentiation.15–19 However, there is evidence that T-bet can regulate CD8+ T-cell proliferation, including CD8+ T-cell responses to vaccinia and leukotriene cytomegalovirus (LCMV) viruses.17–20 Eomes, which is expressed in naïve CD8+ T cells and whose expression is increased upon activation, performs functions similar to those of T-bet, including regulation of IFN-γ and cytotoxicity.7,21 Studies with mutant mice suggest that Eomes can compensate for loss of T-bet function in CD8+ T cells,22 and therefore the extent to which T-bet regulates quantitative aspects of CD8+ T-cell responses to pathogens, particularly parasites, has not been thoroughly investigated. Infection with the parasite T. cruzi generates a vigorous T-cell response, including activation of CD8+ T cells that respond to the immunodominant epitope trans-sialidase, H-2Kb-bound (Tskb20).23 Although CD4+ and CD8+ T cells are both required for acute resistance,24 CD8+ T cells are critical for long-term immunity. Because of the known compensatory functions of Eomes in CD8+ T cells, we hypothesized that T-bet may be dispensable for immunity to T. cruzi. Contrary to our predictions, we observed that infection of T-bet-deficient (tbx21−/−) mice with T. cruzi resulted in increased morbidity and mortality. Importantly, we observed a significant reduction in the number of T. cruzi-specific CD8+ T cells in tbx21−/− mice. This quantitative defect was not attributable to increased apoptosis or T-cell priming, but to a CD8+ T cell-intrinsic function of T-bet. These data illustrate the multifaceted role of T-box transcription factors in regulating T-cell responses to infection, in addition to their roles in regulating cytokine production.

Materials and methods

Mice and infections

Female C57BL/6 and tbx21−/− mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and used at between 6 and 8 weeks of age. Mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility under pathogen-free conditions and procedures were conducted according to an Institutional Animal Care and Use Committee (IACUC)-approved protocol and United States Department of Agriculture (USDA) guidelines. For infections, 5–10 × 105T. cruzi trypomastigotes (CL strain) were injected intraperitoneally (i.p.) and infected mice observed weekly for weight loss and evidence of blood parasitaemia. In most instances mice were killed on day 9 post-infection (p.i.) for analysis of T cells.

Fluorescence-activated cell sorting (FACS)/tetramer analysis

Peripheral blood leucocytes and splenocytes were harvested from infected mice and preincubated with purified anti-CD16/32 (Fc block), and then labelled with fluorescein isothiocyanate (FITC) anti-CD8a (5H10 clone; Biolegend, San Diego, CA), phycoerythrin (PE) anti-CD11b, PE anti-CD4, and PE anti-B220, followed by allophycocyanin-coupled major histocompatibility complex (MHC) class I tetramers consisting of Tskb20 or Tskb18 peptides bound to H-2Kb molecules. Tetramers were synthesized by the National Institutes of Health (NIH) Tetramer Core Facility (Atlanta, GA, USA) and used according to instructions. For detection of tetramer-positive cells, PE-positive cells were excluded from analysis and a gate was established for CD8+ T cells. The absolute number of Tskb20+/Tskb18+ T cells was calculated by multiplying the frequency of tetramer-positive CD8+ T cells by the total number of CD8+ T cells per 50 000 events. Alternatively, T cells were labelled with FITC anti-CD62L, allophycocyanin anti-CD44, and PE anti-CD25 fluorescent antibodies and analysed by FACS.

Detection of apoptosis

Cells were harvested from the blood and spleens of infected mice on day 9 p.i., and labelled with FITC anti-CD4 and Cy7 anti-CD8 antibodies as described above. Cells were subsequently re-suspended in Annexin V binding buffer, and then stained with PE anti-Annexin V according to the manufacturer’s instructions and analysed by FACS.

Dendritic cell activation

On days 2, 4 and 6 p.i., C57BL/6 and tbx21−/− mice were killed and spleens removed. Splenocytes were subsequently treated with purified anti-mouse CD16/CD32 to block Fc receptor binding, followed by incubation with allophycocyanin anti-CD11c, Cy7 anti-CD8 (clone 53-6·7), FITC anti-B7-1, PE anti-B7-2, PE anti-CD40, FITC anti-H-2Kb, and PE anti-I-Ab (all from Biolegend). Expression of these markers in CD11c+ DCs was determined by FACS.

In vivo priming assay

Naïve CD8+ T cells were purified from pooled spleen/lymph nodes of OT-1 TCR transgenic mice (The Jackson Laboratories) using CD8+ T-cell isolation kits and negative selection (Miltenyi Biotec, Auburn, CA). CD8+ T cells were labelled with carboxyfluorescein succinimidyl ester (CFSE), and 5 million T cells injected intravenously (i.v.) into C57BL/6 and tbx21−/− mice previously infected with T. cruzi (day 2 p.i.). Twenty-four hours post-transfer (day 3 p.i.), infected mice were immunized i.p. with 200 μg of ovalbumin protein emulsified in incomplete Freund’s adjuvant. Infected mice were killed on day 6 p.i., and the number of CFSEdim CD8+ T cells was determined by FACS.

Adoptive transfer into rag-2−/− recipients

Thy1·2+ T cells were purified from pooled spleens/lymph nodes of naïve C57BL/6 or tbx21−/− mice using Thy1·2+ microbeads and positive selection (Miltenyi Biotec). Approximately 20 million T cells were injected i.v. into naïve Rag-2-deficient (rag-2−/−) mouse recipients, which were subsequently infected 24 hr later i.p. with 5–10 × 105T. cruzi parasites. On day 9 p.i., recipient mice were killed and blood/spleens removed for analysis of Tskb20-tetramer binding.

Statistic analysis and comparisons

One-way analysis of variance (anova), two-tailed tests, and Tukey–Kramer multiple comparison post-tests were performed using graphpad instat version 3·0a for Macintosh (GraphPad Software, San Diego, CA; http://www.graphpad.com). A P-value of less than 0·05 was considered significant.

Results

T-bet is required for resistance to T. cruzi infection

To initially determine if T-bet was required for resistance to T. cruzi infection, C57BL/6 and tbx21−/− mice were infected with T. cruzi parasites and observed for clinical signs of disease. tbx21−/− mice developed significant weight loss (Fig. 1a; P<0·0001) and increased signs of morbidity. Eventually, 100% of tbx21−/− mice succumbed to T. cruzi infection or were killed because of excessive morbidity (Fig. 1b; P<0·0001). Haematoxylin and eosin (H&E) staining of liver sections demonstrated multiple inflammatory foci in infected tbx21−/− mice, with features strikingly similar to those reported for visceral leishmaniasis18 (Figs 1c–d, arrows). Thus, T-bet is required for resistance to T. cruzi infection.

Figure 1.

Figure 1

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T-bet (T-box, expressed in T cells) is required for resistance to Trypanosoma cruzi infection. (a, b) C57BL/6 (dark diamonds) and T-bet-deficient (tbx21−/−) mice (open circles) were infected with T. cruzi trypomastigotes and observed for morbidity (i.e. weight loss) (a) and mortality (b). (c) Livers from infected C57BL/6 and tbx21−/− mice [day 16 post-infection (p.i.)] were subjected to haematoxylin and eosin (H&E) staining and analysed microscopically. Arrows indicate the presence of inflammatory infiltrates (magnification ×100). Data are representative of five independent experiments, with 4–5 mice per experiment. ***P<0·0001.

Normal signs of T-cell activation in tbx21−/− mice infected with T. cruzi

To determine if the increased susceptibility of tbx21−/− mice was secondary to defects in T-cell activation, we analysed CD4+ and CD8+ T cells from spleens of infected C57BL/6 and tbx21−/− mice for expression of CD62L, CD44 and CD25. Phenotypic analysis was performed on day 9 p.i. because this represented the peak of Tskb20-specific CD8+ T-cell responses in wild-type C57BL/6 mice. We observed a significant increase in the frequency of CD25+ cells in the spleens of infected tbx21−/− mice (Fig. 2a; 27% versus 6·5% for CD4+; 26% versus 5% for CD8+), including a sharp increase in the mean fluorescence intensity (MFI) of CD25 expression. This observation is strikingly similar to that reported for CD8+ T cells from tbx21−/− × eomes−/− mice infected with LCMV.22 However, we did not detect any increase in forkhead box P3 (FoxP3) expression among the CD4+ CD25+ T cells, indicating that increased CD25 expression was the result of T-cell activation and not an increase in the numbers of regulatory T cells (Treg) (not shown). Next, we analysed splenic T cells for expression of CD62Llo CD44hi to identify T cells with an activated phenotype. FACS analysis revealed a similar increase in the total number of CD62Llo CD44hi cells per spleen in infected tbx21−/− mice, again indicative of normal T-cell activation (Fig. 2b; 63% versus 55% CD4+; Fig. 2c; 54% versus 44% CD8+). Because of the systemic nature of T. cruzi infection, we wanted to determine if a parallel increase in CD62Llo CD44hi cells could be detected in the blood. As expected for an acute parasitic infection, there was a sharp increase in the number of CD8+ CD62Llo CD44hi cells in the blood of infected C57BL/6 mice (Fig. 2e; 221 per 50 000 cells for naïve mice versus 4165 per 50 000 cells for infected C57BL/6 mice). In striking contrast, the number of circulating CD8+ CD62Llo CD44hi cells was significantly reduced in infected tbx21−/− mice relative to infected C57BL/6 mice (Fig. 2e; 761 per 50 000 cells; P<0·0001). Paradoxically, there was no evidence of a similar decrease in CD4+ T cells, as the number of CD62Llo CD44hi cells in tbx21−/− mice was slightly elevated relative to infected C57BL/6 mice, although this was not statistically significant (Fig. 2d). Thus, tbx21−/− mice have increased numbers of activated T cells in the spleen without a concomitant increase in peripheral blood.

Figure 2.

Figure 2

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T cells from tbx21−/− mice infected with Trypanosoma cruzi display phenotypic markers of activation. Spleens (a–c) and blood (d–e) from naïve and infected C57BL/6 and tbx21−/− mice were harvested on day 9 post-infection (p.i.) and the frequency of CD25+ (a) and the number of CD44hi CD62Llo (b–e) cells were determined by fluorescence-activated cell sorting (FACS). In (d) and (e), the absolute numbers of CD44hi CD62Llo cells were determined by collecting 50 000 total peripheral blood cells, and multiplying by the fraction of CD4+ CD44hi CD62Llo (d) or CD8+ CD44hi CD62Llo (e) cells. Numbers in (a) reflect the mean percentages among three independent experiments (n = 3 mice per group), as well as the average of the mean fluorescence intensity of CD25 expression.

T-bet deficiency results in a profound reduction in the number of T. cruzi-specific CD8+ T cells in vivo

We were intrigued by the dichotomy between the numbers of activated CD8+ T cells in the spleen and in the blood of infected tbx21−/− mice (Fig. 2c,e). As polyclonal T-cell activation can be elicited in an antigen non-specific manner, particularly by parasite-derived proteins (including those encoded by T. cruzi),25,26 we reasoned that local activation in the spleen may mask a more general defect in antigen-specific T-cell responses. Further, we reasoned that CD44hi CD62Llo cells in the blood would probably reflect true antigen-specific CD8+ T cells due to their ability to traffic to peripheral tissues infected by T. cruzi. Because of the availability of MHC class I tetramers to track T. cruzi-specific CD8+ T cells, and the established role of CD8+ T cells in immunity to T. cruzi, we used tetramers to quantify the number of Tskb20-specific CD8+ T cells in infected mice. As expected, there was a significant increase in the frequency and absolute number of Tskb20-specific CD8+ T cells in the spleen (Fig. 3a,b) and blood (Fig. 3c) of infected C57BL/6 mice. Strikingly, and in contrast to increased numbers of CD44hi CD62Llo CD8+ T cells in the spleen (Fig. 2c), there was a 10-fold reduction in the number of Tskb20+ CD8+ T cells in infected tbx21−/− mice (Fig. 3a–c; P<0·0001). The low number of Tskb20+ cells was evident throughout the course of infection, indicating that the kinetics of the Tskb20 response were not delayed in tbx21−/− mice (data not shown). Furthermore, there was no evidence of a shift in the pattern of immunodominance, as CD8+ T-cell responses to Tskb18 (a response that typically peaks later in infection) were similarly impaired (data not shown). These data suggest that tbx21−/− mice exhibit a quantitative defect in the generation of antigen-specific CD8+ T cells during T. cruzi infection.

Figure 3.

Figure 3

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Reduced numbers of Trypanosoma cruzi-specific CD8+ T cells in infected tbx21−/− mice. Spleens (a–b) and blood (c) were harvested from infected mice on day 9 post-infection (p.i.). CD8+ T cells were analysed for staining with trans-sialidase, H-2Kb-bound (Tskb20)-positive tetramers as described in the text, and the absolute number of Tskb20+ T cells per 50 000 cells (a, c) or the total number of Tskb20+ cells per spleen (b) was determined by fluorescence-activated cell sorting (FACS). Dot plots shown to the right (arrows) are representative of the tetramer staining pattern as well as the frequency of Tskb20+ cells. Results shown represent multiple (n > 5) experiments (n = 3 mice per group). ***P<0·0001.

Although we consistently detected low numbers of Tskb20-specific CD8+ T cells in the spleens and blood of infected tbx21−/− mice, it remained possible that our inability to detect T. cruzi-specific T cells was secondary to altered trafficking and tissue-specific migration. This possibility was strengthened by our observation (unpublished data), and the reports of others, that CD8+ T cells from tbx21−/− mice do not express the chemokine (C-X-C motif) receptor 3 (CXCR3), which is important for migration of T cells into inflamed tissues.27–29 Therefore we analysed mononuclear cells obtained from the livers of infected mice for the presence of CD8+ T cells, as the strain of T. cruzi used in these studies (CL) has a particular tropism for liver. We limited our FACS analyses to total CD8+ T cells as the antigen-specific defect observed in tbx21−/− mice (Fig. 3a,c) was reflected in total effector CD8+ T cells (Fig. 2e), which would include CD8+ T cells responding to T. cruzi epitopes other than Tskb20. As shown in Fig. 4a–c, there was a substantial increase in the number of infiltrating CD8+ T cells in the liver of infected C57BL/6 mice (Fig. 4b–c; two representative donors shown), consistent with the blood data (Fig. 4b–c). Despite an increase in CD8+ T cells in livers of infected tbx21−/− mice relative to naïve mice (Fig. 4a, d–e; two representative donors shown), there were significantly fewer CD8+ T cells compared with infected C57BL/6 mice. Thus, the failure to detect significant numbers of T. cruzi-specific CD8+ T cells in the spleen or blood was not attributable to altered T-cell trafficking.

Figure 4.

Figure 4

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CD8+ T cells in infected tbx21−/− mice do not exhibit altered T-cell trafficking to non-lymphoid tissues. Livers and blood were harvested from naïve (a) and infected C57BL/6 (b) and tbx21−/− (c) mice on day 9 post-infection (p.i.), and the frequency of CD8+ T cells was determined by fluorescence-activated cell sorting (FACS). Results shown are representative of two experiments, with two to three mice per group.

CD8+ T cells from infected tbx21−/− mice do not exhibit increased apoptosis

The results shown in Fig. 3 clearly show that infected tbx21−/− mice are impaired in their ability to generate sufficient numbers of T. cruzi-specific CD8+ T cells. To determine if this quantitative defect was secondary to an increase in apoptosis, CD8+ T cells obtained from infected mice on day 9 p.i. were analysed for Annexin V expression. We observed a marginal increase in CD8+ T-cell apoptosis in the spleens of infected mice relative to those of naïve mice (50% versus 35%; Fig. 5). However, there was no significant difference in apoptosis between infected C57BL/6 and tbx21−/− mice. Furthermore, the degree of apoptosis in the blood was similar among all groups (Fig. 5). Thus, increased apoptosis does not appear to contribute to the numeric reduction in T. cruzi-specific CD8+ T cells in infected tbx21−/− mice.

Figure 5.

Figure 5

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CD8+ T cells from infected tbx21−/− mice do not exhibit increased apoptosis. Spleens and blood were collected from naïve and Trypanosoma cruzi-infected mice [on day 9 post-infection (p.i.)], and CD8+ T cells were analysed for Annexin V expression by fluorescence-activated cell sorting (FACS). Results shown are representative of three independent experiments (n = 2–3 mice per group). WT, wild type.

Normal activation of dendritic cells in infected tbx21−/− mice

As the low numbers of Tskb20+ CD8+ T cells in infected tbx21−/− mice did not appear to be attributable to increased cell death (Fig. 5), we reasoned that the defect could be the result of impaired CD8+ T-cell expansion. In light of recent reports demonstrating a critical role for T-bet in DC functions,10,11,30 we hypothesized that DCs from T. cruzi-infected tbx21−/− mice may be defective in their ability to programme CD8+ T-cell responses to T. cruzi. To characterize the state of DC activation, C57BL/6 and tbx21−/− mice were infected with T. cruzi and splenic CD11c+ DCs were subsequently analysed by FACS. Phenotypic evidence of DC activation could be detected as early as day 2 p.i. in infected C57BL/6 and tbx21−/− mice (data not shown). On day 6 p.i., DCs from infected C57BL/6 mice expressed increased levels of CD80, CD86 and CD40 (Fig. 6a,b), with marked up-regulation of CD86 and CD40. Importantly, CD11c+ DCs from infected tbx21−/− mice expressed levels of B7-1, B7-2 and CD40 that were comparable to those of infected wild-type mice (Fig. 6b,c). Furthermore, expression of H-2Kb was comparable in infected C57BL/6 and tbx21−/− mice (Fig. 6b,c), arguing against a possible defect in antigen presentation. These data suggest that failure of tbx21−/− mice to generate sufficient numbers of Tskb20-specific CD8+ T cells is not attributable to changes in DC activation/maturation.

Figure 6.

Figure 6

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Dendritic cells from infected tbx21−/− mice display normal signs of activation. Spleen cells were harvested from naïve and infected mice on day 6 post-infection (p.i.), and labelled with anti-CD11c, anti-CD80 (B7-1), CD86 (B7-2), CD40 and H-2Kb [major histocompatibility complex (MHC) class I]. A total of 5000 CD11c+ dendritic cells were collected and analysed by fluorescence-activated cell sorting (FACS). Numbers reflect the relative proportion of CD11c+ dendritic cells expressing the indicated surface marker. Results shown are representative of four independent experiments (n = 2 mice per group).

Normal antigen presentation functions of dendritic cells in infected tbx21−/− mice

As a corollary to DC activation, we next determined if APC functions were intact in tbx21−/− mice infected with T. cruzi. Specifically, we wanted to determine if APCs could capture, process and present a model protein [i.e. ovalbumin (OVA) protein] to CD8+ T cells. To do this, we utilized an adoptive transfer strategy in which OVA-specific CD8+ T cells from naïve OT-1 TCR transgenic mice were labelled with CFSE and subsequently injected into mice infected with T. cruzi (day 2 p.i.). Twenty-four hours post-transfer, mice were immunized with OVA protein. Three days after immunization (day 6 p.i.), mice were killed and spleens and lymph nodes were removed. To assess the degree of CD8+ T-cell proliferation, CFSE dilution was analysed among transferred cells by FACS. As shown in Fig. 7, up to six cell divisions could be detected in OT-1 T cells after transfer into infected C57BL/6 mice (Fig. 7), with a majority of OT-1 cells accumulating in the six cell division peak. Importantly, CFSE dilution among OT-1 T cells transferred into infected tbx21−/− mice was virtually identical, with no apparent defect in the ability of OT-1 CD8+ T cells to undergo antigen-specific proliferation. Previous experiments confirmed that OT-1 cell division did not exceed six divisions during the 3-day immunization period (not shown). In conclusion, these data (along with those presented in Fig. 6) show that APC functions in infected tbx21−/− mice are intact, and point to the possibility that defective expansion of T. cruzi-specific CD8+ T cells occurs in a T cell-intrinsic manner.

Figure 7.

Figure 7

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Dendritic cells from infected tbx21−/− mice exhibit normal antigen presentation to CD8+ T cells in vivo. CD8+ T cells from naïve OT-1 T-cell receptor (TCR) transgenic mice were purified and labelled with carboxyfluorescein succinimidyl ester (CFSE), and transferred into C57BL/6 or tbx21−/− mice previously infected with Trypanosoma cruzi [on day 2 post-infection (p.i.)]. Twenty-four hours post-transfer, infected mice were immunized with ovalbumin (OVA) protein emulsified in oil and subsequently killed on day 6 p.i. Spleens and lymph nodes were removed and CFSE+ cells were analysed for CFSE dilution to determine the number of cell divisions induced by OVA. Results from three independent experiments (n = 2 mice per group) are shown.

T-bet regulates CD8+ T-cell expansion in response to infection in a T cell-intrinsic manner

The data presented in Figs 5–7 suggested that the expansion of Tskb20+ T cells in tbx21−/− mice was regulated by T-bet in a T cell-intrinsic manner. To test this possibility, we utilized an adoptive transfer strategy in which Thy1·2+ T cells from naïve C57BL/6 or tbx21−/− donors were transferred into rag-2−/− recipient mice, which possess wild-type (tbx21+/+) APCs. Recipient mice were subsequently infected with T. cruzi and killed on day 9 p.i. Blood and spleens were collected from recipient mice on day 9 p.i. and analysed for the relative proportions of CD8+ T cells. As shown in Fig. 8a, there were significantly fewer CD8+ T cells in the spleens of rag-2−/− mice that received Thy1·2+ T cells from tbx21−/− mice (P=0·01). Because of donor-to-donor variability between experiments, a scatter plot is shown. Importantly, and similarly to the results obtained with intact tbx21−/− mice, there was a marked reduction in the number of CD8+ T cells when T cells from tbx21−/− donors were used. Furthermore, the dichotomy between blood and spleen was recapitulated (Fig. 8b; P=0·0045). In conclusion, these data confirm that T-bet regulates the expansion of CD8+ T cells during T. cruzi infection in a T cell-intrinsic manner.

Figure 8.

Figure 8

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T-bet (T-box, expressed in T cells) regulates the expansion of CD8+ T cells during Trypanosoma cruzi infection in a T cell-intrinsic manner. Thy1·2+ T cells were purified from spleens and lymph nodes of naïve C57BL/6 or tbx21−/− mice, and approximately 20 million T cells transferred intravenously (i.v.) into naïve rag-2−/− recipients. rag-2−/− mice were subsequently infected 24 hr post-transfer with T. cruzi trypomastigotes, and recipient mice were killed on day 9 post-infection (p.i.). Spleens and blood were collected, and analysed for the presence of CD8+ T cells. Results shown are from five independent experiments (n = 2 mice per group), with each data point representing a single mouse.

Discussion

The T-box family of transcription factors plays an important role in normal growth and development.31 In particular, the T-box members Tbx21 (T-bet) and Eomes regulate the differentiation and effector functions of Th1 cells and cytotoxic T lymphocytes (CTLs),6,21,32 and thus are important for regulating type I/IFN-γ immunity to intracellular pathogens. For example, T-bet-deficient mice (tbx21−/−) have an increased susceptibility to infection, and in many instances susceptibility is attributed to decreased Th1 and/or increased Th2/Th17 cytokine responses.15,16,19 While Eomes deficiency is embryonic lethal, Eomes and T-bet have been shown to have redundant functions in CD8+ T cells.7,21 Because of this redundancy, we were interested in the role of T-bet in immunity to infection with T. cruzi, particularly as CD8+ T cells play a critical role in resistance and long-term immunity to the parasite.2,33,34 We hypothesized that T-bet may be dispensable for T. cruzi-specific CD8+ T-cell responses in vivo. However, we report here that T-bet is indeed required for resistance, as tbx21−/− mice exhibited increased morbidity and mortality subsequent to infection. CD8+ T cells from infected tbx21−/− mice showed normal signs of activation in the spleen (i.e. CD62Llow, CD44hi), which is in contrast to results obtained with T-bet-deficient CD8+ T cells stimulated in vitro.35 Despite this apparent activation, infected tbx21−/− mice exhibited a marked reduction in the number of Tskb20 antigen-specific CD8+ T cells. We observed a similar reduction in the number of circulating Tskb20-specific CD8+ T cells in the blood, including total numbers of effector CD8+ cells that were induced as a consequence of T. cruzi infection. While CD8+ T cells from infected tbx21−/− mice failed to up-regulate the chemokine receptor CXCR3 (unpublished data), we failed to detect an increase in Tskb20-specific or effector CD8+ T cells in non-lymphoid tissues such as the liver, suggesting that the distribution of lymphocytes was not altered in tbx21−/− mice. Despite a significant number of CD25-expressing cells and an increase in the MFI of CD25 expression, we failed to detect any increase in the number of CD4+ CD25+ FoxP3+ regulatory T cells in infected tbx21−/− mice, ruling out the possibility that increased suppression accounted for low numbers of Tskb20-specific T cells. Importantly, the observed defect in CD8+ T-cell expansion in tbx21−/− mice was recapitulated in rag-2−/− mice, as CD8+ T cells from tbx21−/− mice failed to undergo expansion in vivo despite antigen presentation by wild-type, T-bet-expressing APCs. Despite limited reports, defects in CD8+ T-cell proliferation have been observed in tbx21−/− mice after infection with vaccinia virus,17 and in the rat insulin promoter (RIP)-LCMV model of type 1 diabetes.20 Conversely, infection of tbx21−/− mice with influenza or Sendai virus revealed no defects in CD8+ T-cell expansion.36 Further, infection of tbx21−/− mice with herpes simplex virus (HSV)-2 led to increased numbers of antigen-specific CD8+ T cells.19 In the latter case, however, primary CD8+ T-cell responses were generated using an attenuated strain of HSV. One possibility is that persistent antigen stimulation and/or increased virulence, particularly after infection of tbx21−/− mice with live parasites, leads to a greater defect in CD8+ T-cell expansion in vivo. Alternatively, intrinsic differences may exist between viral and parasitic infections. The data presented here for T. cruzi infection are most consistent with vaccinia and LCMV studies.17–20 However, in the RIP-LCMV model of type 1 diabetes (T1D), the development of diabetogenic CD8+ T cells does not require CD4+ T-cell help. In contrast, the development of T. cruzi-specific CD8+ T cells requires CD4+ T-cell help,37 although this is controversial.38 To determine if the defect in CD8+ T-cell expansion in infected tbx21−/− mice was attributable to T-bet-dependent CD4+ T-cell help, we performed additional experiments in which wild-type CD4+ T cells were co-transferred with T-bet-deficient CD8+ T cells into rag-2−/− mice. We observed that provision of wild-type CD4+ T cells was not sufficient to restore CD8+ T-cell expansion subsequent to T. cruzi infection (D. A. Cobb, S. Guo, R. B. Smeltz, unpublished data). Interestingly, it also suggests that the development of CD4+ Th17 responses in infected tbx21−/− mice is independent of the defect in CD8+ T-cell expansion in tbx21−/− mice.38 This conclusion is supported by a recent study that demonstrated that CD8+ T-cell responses to Listeria can develop normally and concomitant with Th17 responses.40 A second major difference is that LCMV-specific CD8+ T-cell numbers were comparable between wild-type and tbx21−/− mice early during LCMV infection (day 8 p.i.), but differences became evident 2 weeks p.i. In contrast, we failed to detect a significant increase in antigen-specific CD8+ T cells at any time during infection, including those specific for other T. cruzi epitopes such as Tskb18.23 Therefore, these data demonstrate that, during the normal course of infection with T. cruzi, T-bet promotes CD8+ T-cell expansion in a CD8+ T cell-intrinsic manner. It is interesting that, despite redundant functions between T-bet and Eomes in CD8+ T cells, the ability to promote CD8+ T-cell expansion may be a unique function of T-bet. We did not detect any differences in Eomes expression between CD8+ T cells from wild-type and tbx21−/− mice infected with T. cruzi (data not shown), in contrast to a previous report.39 Although we do not fully understand this apparent discrepancy, we hypothesize that it reflects chronic interleukin (IL)-12 production as a result of persistent T. cruzi infection in tbx21−/− mice. Consistent with this, in the aforementioned study the increase in Eomes expression was observed after IL-12 signalling had been extinguished, concomitant with the clearance of Listeria.41 In addition to Eomes, real-time polymerase chain reaction (PCR) analysis of CD8+ T cells from infected tbx21−/− mice revealed that runt-related transcription factor 3 (Runx3) expression was normally up-regulated, indicating that defective expansion was not attributable to a lack of Runx3 expression (data not shown). Runt-related transcription factor 3 (Runx3) is a transcription factor that is normally involved in silencing of CD4 expression during the appearance of single-positive CD8+ thymocytes, and has recently been shown to regulate peripheral CD8+ T-cell functions.42,43 We were surprised that Runx3 levels in CD8+ T cells from infected tbx21−/− mice were equivalent to those in wild-type CD8+ T cells, as T-bet is required for induction of Runx3 in CD4+ T cells.44 With respect to the function(s) of T-bet that regulate CD8+ T-cell expansion in vivo, we have not yet identified these functions but speculate that cytokine signalling may be involved. Because of the importance of IL-2 in T-cell growth, we performed preliminary FACS experiments to determine if IL-2 production was affected. However, consistent with previous reports that identified T-bet as a repressor of IL-2,45 we observed increased IL-2 expression in T cells from tbx21−/− mice, suggesting that decreased CD8+ T-cell expansion was not attributable to a lack of IL-2 (data not shown). With respect to interferons (including the type I interferons IFN-α/β), the situation is less clear. IFN-γ/signal transducer and activator of transcription (Stat)-1 signalling can negatively regulate CD8+ T-cell proliferation.46–48 For example, antigen-specific CD8+ T cells induced by Listeria infection quickly lose IFN-γ responsiveness during the time corresponding to CD8+ T-cell expansion.48 Furthermore, stimulation of Stat-1-deficient T cells in vitro led to hyperproliferation that was not evident in ifn-γ−/− cells.49 Conversely, both type I IFN and IFN-γ have been shown to promote CD8+ T-cell expansion in vivo.50,51 If IFN-γ/Stat-1 are inhibitory to CD8+ T-cell expansion during T. cruzi infection, and because of the positive feedback loop between T-bet and IFN-γ/Stat-1 signalling,13,14 the prediction in this scenario is that CD8+ T cells in tbx21−/− would undergo increased expansion, which is clearly not the case in our studies. This raises the possibility that IFN-γ/Stat-1 and T-bet may have divergent effects on CD8+ T-cell expansion during parasitic infection. Indeed, preliminary experiments show that, in contrast to tbx21−/− T cells, stat-1−/− CD8+ T cells expand normally during T. cruzi infection (unpublished data). Experiments are underway in our laboratory to address these issues. In conclusion, the data reported here demonstrate a critical role for T-bet in promoting the expansion of parasite-specific CD8+ T cells during T. cruzi infection, and shed new light on the role of T-bet in regulating quantitative aspects of type 1 immunity to intracellular pathogens.

Acknowledgments

RBS would like to thank Dr William C. Hooper for his mentoring, advice and support. The study was supported by funds provided by Virginia Commonwealth University and the Department of Microbiology and Immunology, as well as a grant from the Thomas F. and Kate Miller Jeffress Memorial Trust. This work is dedicated to the memory of Dr Karen Cozad, University of Akron.

Glossary

Abbreviations:

T-bet

T-box, expressed in T cells

CTL

cytotoxic T lymphocyte

Tbx21

T-box gene 21

Disclosure

The authors declare no financial interests.

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