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. 2008 Oct;57(10):2684-92.
doi: 10.2337/db08-0609. Epub 2008 Aug 8.

Transforming growth factor-beta suppresses the activation of CD8+ T-cells when naive but promotes their survival and function once antigen experienced: a two-faced impact on autoimmunity

Christophe M Filippi  1 , Amy E Juedes, Janine E Oldham, Ellie Ling, Lisa Togher, Yufeng Peng, Richard A Flavell, Matthias G von Herrath
Affiliations

Transforming growth factor-beta suppresses the activation of CD8+ T-cells when naive but promotes their survival and function once antigen experienced: a two-faced impact on autoimmunity

Christophe M Filippi et al. Diabetes. 2008 Oct.

Abstract

Objective: Transforming growth factor-beta (TGF-beta) can exhibit strong immune suppression but has also been shown to promote T-cell growth. We investigated the differential effect of this cytokine on CD8(+) T-cells in autoimmunity and antiviral immunity.

Research design and methods: We used mouse models for virally induced type 1 diabetes in conjunction with transgenic systems enabling manipulation of TGF-beta expression or signaling in vivo.

Results: Surprisingly, when expressed selectively in the pancreas, TGF-beta reduced apoptosis of differentiated autoreactive CD8(+) T-cells, favoring their expansion and infiltration of the islets. These results pointed to drastically opposite roles of TGF-beta on naïve compared with antigen-experienced/memory CD8(+) T-cells. Indeed, in the absence of functional TGF-beta signaling in T-cells, fast-onset type 1 diabetes caused by activation of naïve CD8(+) T-cells occurred faster, whereas slow-onset disease depending on accumulation and activation of antigen-experienced/memory CD8(+) T-cells was decreased. TGF-beta receptor-deficient CD8(+) T-cells showed enhanced activation and expansion after lymphocytic choriomeningitis virus infection in vivo but were more prone to apoptosis once antigen experienced and failed to survive as functional memory cells. In vitro, TGF-beta suppressed naïve CD8(+) T-cell activation and gamma-interferon production, whereas memory CD8(+) T-cells stimulated in the presence of TGF-beta showed enhanced survival and increased production of interleukin-17 in conjunction with gamma-interferon.

Conclusions: The effect of TGF-beta on CD8(+) T-cells is dependent on their differentiation status and activation history. These results highlight a novel aspect of the pleiotropic nature of TGF-beta and have implications for the design of immune therapies involving this cytokine.

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Figures

FIG. 1.
FIG. 1.
Mice induced to express TGF-β in the islets show increased islet infiltration by autoreactive CD8+ T-cells activated in the periphery. TTA/TGF-β BALB/c mice were crossed with BALB/c RIP-NP mice to generate double-transgenic RIP-NP.TGFβ offspring. RIP-NP.TGFβ mice and their single-transgenic RIP-NP littermates were fed a diet containing doxycycline (dox) during gestation and after birth. A: Doxycycline was either maintained or removed from the diet 7 days before infection with LCMV, and pancreata were harvested 7 days later (day 14 after doxycycline removal). Tissue sections were analyzed for TGF-β production and CD8+ T-cell infiltration after staining with anti–TGF-β (top panels) or anti-CD8 (bottom panels) and counterstaining with hematoxylin. B: Single-cell suspensions were prepared from the pancreas, spleen, or pancreatic draining lymph nodes (PDLN) of mice fed a doxycycline-free diet. The frequency of NP118-specific/responsive CD8+ T-cells was evaluated in pooled pancreata (three to four) by tetramer staining and in the spleen and pancreatic draining lymph nodes by flow cytometry after stimulation with NP118 ex vivo. (Please see http://dx.doi.org/10.2337/db08-0609 for a high-quality digital representation of this figure.)
FIG. 2.
FIG. 2.
TGF-β promotes the survival of previously activated autoreactive CD8+ T-cells by decreasing their apoptosis. A: Apoptosis was evaluated by TUNEL in pancreatic tissue sections obtained from mice fed a doxycycline (dox)-free diet (top panels). Cell suspensions were prepared from the pooled pancreatic draining lymph nodes of three to four mice and stained with H2-ld/NP118 tetramers and annexin V (bottom panels). Histograms show the frequency of tetramer-positive CD8+ T-cells stained with annexin V. B: CD8+ T-cells were purified from the spleen of LCMV-immune BALB/c mice, labeled with CFSE, and cultured with LCMV-infected, irradiated, antigen-presenting cells in the presence or absence of 3 ng/ml rhTGF-β1. Cell proliferation was assessed by measuring CFSE dilution by flow cytometry after 6 days in culture (top panel) or cell cycle protein RNA was quantitated by RNase protection assay after 24 h (bottom panel). C: CD8+ T-cells were isolated from the spleen of LCMV-immune BALB/c mice and cultured with LCMV-infected, irradiated, antigen-presenting cells in the presence or absence of 3 ng/ml rhTGF-β1. After 1 and 3 days in culture, T-cells were analyzed for apoptosis by flow cytometry after staining with annexin V. Data represents the average frequency of CD8+ T-cells stained with annexin V ± SD from three independent experiments. (Please see http://dx.doi.org/10.2337/db08-0609 for a high-quality digital representation of this figure.)
FIG. 3.
FIG. 3.
Absence of TGF-β receptor signaling in T-cells has opposite effects on virally induced autoimmune diabetes depending on the effector mechanism. dnTGFβR mice were crossed onto fast-onset diabetes RIP-GP or slow-onset diabetes RIP-NP lines. Groups of RIP-GP.dnTGFβR (A) mice, RIP-NP.dnTGFβR (B) mice, and their single transgenic RIP-GP or RIP-NP littermates were infected with LCMV, and diabetes incidence was monitored by measuring blood glucose. Mice were considered diabetic when blood glucose exceeded 300 mg/dl.
FIG. 4.
FIG. 4.
Absence of TGF-β receptor signaling in T-cells enhances antiviral CD8+ T-cell responses at the acute phase but reduces memory responses. dnTGFβR mice and their wild-type (WT) C57BL/6 littermates were infected with LCMV. A and B: Mice were killed and spleens harvested on day 7 (peak) (A) or 28 (memory) (B) postinfection. The frequency of IFN-γ–producing cells (measured by flow cytometry after stimulation with GP33) was multiplied by the total number of splenocytes to obtain the number of GP33-responsive CD8+ T-cells per spleen. Data represents the average number of GP33-responsive CD8+ T-cells per spleen ± SD from three individual mice per group. ▪, wild-type; □, dnTGFβR. C: Splenocytes were harvested from dnTGFβR mice 28 days postinfection, labeled with CFSE, and cultured with LCMV-infected, irradiated, antigen-presenting cells. Cells were harvested 2 or 3 days later and stained for expression of CD8 and annexin V. Data represents the average frequency of CD8+ T-cells stained with annexin V ± SD from three mice per group. D: Alternatively, cells were harvested 6 days later and analyzed for IFN-γ production by flow cytometry after stimulation with GP33. Representative fluorescence-activated cell sorting plots are shown.
FIG. 5.
FIG. 5.
TGF-β suppresses naïve CD8+ T-cell activation and enhances the survival and effector function of antigen-experienced/memory CD8+ T-cells. CD8+ T-cells were purified from the spleen of naïve P14 or LCMV-immune C57BL/6 mice, labeled with CFSE, cultured with GP33-loaded dendritic cells in the presence or absence of 3 ng/ml rhTGF-β1, and harvested 2 days later. A and B: Cells were analyzed for IFN-γ and IL-17 production by flow cytometry after stimulation with GP33. Data represents the average frequency of naïve (A) and memory (B) CD8+ T-cells producing IFN-γ or IL-17 ± SD from three individual mice per group. C: Cell proliferation was assessed by measuring CFSE dilution by flow cytometry. Representative fluorescence-activated cell sorting plots are shown. Values show the percentage of naïve (left panel) or memory (right panel) CD8+ T-cells that have undergone at least one division on stimulation in the presence or absence of TGF-β ± SD from three individual mice per group. D: Naïve GP33-specific CD8+ T-cells were analyzed for cell cycle protein RNA by RNase protection assay as described in Fig. 2B. E: Cells were analyzed by flow cytometry for apoptosis after staining with annexin V or for expression of PD-1. Data represents the average frequency of CD8+ T-cells stained with annexin V (left panel) or expressing PD-1 (right panel) ± SD from three individual mice per group.
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