TGFpMediated Immunoregulation

Y. Peng, L. Gorelik, Y. Laouar, M.O. Li, R.A. Flavell

9.1 Introduction 156

9.2 Materials and Methods 156

9.2.1 Transgenic Mice 156

9.2.2 Tumor Model 156

9.2.3 Infection Model 157

9.2.4 Diabetes Model 157

9.3 Results 157

9.3.1 TGFP and Effector T Cells 157

9.3.2 TGFP and Regulatory T Cells 158

9.4 Conclusions 159

References 160

Abstract. T cell homeostasis is required for normal immune responses and prevention of pathological responses. Transforming growth factor | (TGF|) plays an essential role in that regulation. Owing to its broad expression and inhibitory effects on multiple immune cell types, TGF| regulation is complex. Through recent advances in cell-specific targeting of TGF| signaling in vivo, the role of TGF| in T cell regulation is emerging. We demonstrated here a critical role for TGF| in regulating effector vs regulatory T cell homeostasis.

9.1 Introduction

Understanding immune regulation and tolerance remains a major challenge in immunology. As an immunosuppressive cytokine, TGF^ plays a key role in attenuating excessive pathological immune responses. Mice deficient in TGF^, developed severe multifocal inflammatory disease and died shortly after birth (Shull et al. 1992). Since TGFg. can be produced by and act on virtually all cell types, the regulatory network invoked by TGF^ remains incompletely understood. To investigate the role of TGFP signaling in T cells, we engineered a strain of transgenic mice expressing a dominant negative TGF^ receptor II in T cells (Gore-lik and Flavell 2000). In these mice, T cells were found to differentiate spontaneously into effector T cells, which led to the development of autoimmunity and enhanced tumor immunity and immune responses to pathogens. In addition, we took a gain-of-function approach to study TGFP regulation of autoimmune diabetes by overexpressing this cytokine in the islets of the pancreas (Peng et al. 2004). We demonstrated that a short pulse of TGF^ expression could expand the regulatory T cell population and inhibit diabetes in NOD mice. These studies have thus identified multiple mechanisms by which TGF^ regulates T cell tolerance.

9.2 Materials and Methods

9.2.1 Transgenic Mice

Mice expressing a dominant negative mutant of TGF^ receptor II under the control of CD4 promoter (DNR mice) were described previously (Gorelik and Flavell 2000). All mice were kept under SPF conditions at Yale animal facility according to the approved protocols.

9.2.2 Tumor Model

The B16-F10 melanoma tumor cell line syngeneic to the C57BL/6 background was provided by A. Garen. EL4 cells were obtained from the American Type Culture Collection (Manassas, VA). Wild-type andDNR mice on C57BL/6 background were challenged with either B16-F10 cells i.v. or EL4 cells i.p. and monitored for tumor growth.

9.2.3 Infection Model

DNR mice were backcrossed onto the BALB/c background as described (Gorlik et al. 2002). Wild-type and DNR mice were infected in the right hind foot with Leishmania major promastigotes of the WR309 substrain and monitored for disease progression.

9.2.4 Diabetes Model

Under the control of a rat insulin promoter (RIP), tetracycline-controlled transactivator (TTA) is expressed specifically in insulin-producing cells to ensure regulated TGF|3 expression in TTA/TGF|3 NOD mice, as previously described (Peng et al. 2004). NOD transgenic mice were fed with normal or doxycycline-supplemented food and monitored for disease development.

9.3 Results

9.3.1 TGFP and Effector T Cells

Mice with T cell-specific blockade of TGF^ signaling developed im-munopathology in multiple organs including lung, colon, and kidney. Both CD4+ and CD8+ T cells from DNR mice differentiated readily into effector/memory T cells and secreted cytokines upon in vitro stimulation. Levels of T-dependent classes of immunoglobulins in the sera were consistently found to be increased. These observations revealed an essential function for TGF^ in T cell tolerance (Gorelik and Flavell 2000). To investigate how TGF^ regulates tumor immunity, we challenged mice with syngeneic B16-F10 or EL4 tumor cells. In both cases, DNR mice were found to be resistant to the tumor challenge. Blockade of TGFP signaling in CD8+ T cells was essential for this protection, and was associated with the expansion of tumor-specific CD8+ T cells (Gorelik and Flavell 2001). CD4+ T cells play a critical role in orchestrating both the humoral and cellular arms of immune responses. CD4+ T cells from BALB/c mice selectively differentiate to a skewed Th2 phenotype, which renders these mice susceptible to infection by intracellular pathogens such as Leishmania. To study the function of TGF|3 signaling in pathogen infection, DNR mice were backcrossed to the

BALB/c background and investigated for their response to Leishmania major infection. Significantly, DNR mice were found to be resistant to the infection, because they developed an enhanced Thl-type cytokine response. Interestingly, Th2 cytokines were also found to be elevated in the DNR mice (Gorelik et al. 2000). These observations suggested that TGFP inhibits both arms of effector T cell differentiation. T helper cell differentiation is regulated by environmental cues and associated with epigenetic changes in the cytokine loci. Master regulators including GATA-3 for Th2 and T-bet for Thl differentiation have been identified by us (Zheng and Flavell 1997) and Szabo et al. (2000). Significantly, TGFP inhibited the expression of both factors which explains its regulation of help T cell differentiation (Gorelik et al. 2000, 2002).

9.3.2 TGFP and Regulatory T Cells

Several studies have demonstrated that CD4+CD25+ regulatory T cells produce elevated levels of TGF^ (Green et al. 2003). The fact that enhanced TGFP signaling receptors reside on the membrane of CD4+ CD25+ regulatory T cells underscores the potential for autocrine and/or paracrine receptor-ligand interactions in these cells. In this study, we provide direct evidence that TGF^ is a positive regulator of CD4+CD25+ regulatory T cell expansion in vivo. We generated mice in which TGF|3 expression can be induced temporally by the tetracycline regulatory system (Peng et al. 2004). Data from RT-PCR and histochemistry studies showed that TGF^ gene and protein expression can be efficiently turned on and off in the islets within l week after changing the diet to a doxycycline-containing food source. Using this system, we could control and target the expression of the transgene at specific stages of diabetes development. Transgenic mice from all groups were monitored for diabetes development in comparison to transgene-negative control littermates for 60 weeks. We found that TGF^ expression completely blocked the development of diabetes in NOD transgenic mice. Furthermore, a short pulse of TGF^ in the islets during the priming phase of the disease was sufficient to provide protection by promoting the expansion of the intra-islet CD4+CD25+ T cell pool. Approximately 40%-50% of intra-islet CD4+ T cells expressed the CD25 marker and exhibited characteristics of regulatory T cells, including small size, high level of intracellular CTLA-4, expression of Foxp3, and the ability to transfer protection against diabetes. Results from in vivo incorporation of BrdUrd revealed that the generation of a high frequency of regulatory T cells in the islets is due to in situ expansion as a result of TGF^ expression. These findings demonstrated that TGF^ inhibits autoimmune diseases via regulation of the size of the CD4+CD25+ regulatory T cell pool in vivo, a previously uncharacterized mechanism.

9.4 Conclusions

Understanding the role of TGF^ signaling in T-cell regulation has come a long way since the initial discovery of its ability to regulate T cell proliferation. Our recent work has demonstrated that TGF^ downregulates effector T cell differentiation by inhibiting the expression of transcription factors T-bet and GATA-3. The role of TGF^ in regulatory T cell biology warrants further investigation. Insights into the biology of regulatory T cells might provide important insights into how the different roles of TGFP in regulatory T-cell function relate to heterogeneity of the regulatory T cell population or another regulatory T cell property. The complex role of TGF^ in the regulation of immune homeostasis is likely to fascinate investigators as they decipher the many mysteries of this family of molecules for many years to come (Fig. 1).

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