T cell signatures and modulation by regulatory T cells

CD4 T cells are crucial coordinators of protective immune responses against microbes and tumors. However, immune responses misdirected towards self-antigens are the hallmarks of autoimmune diseases. Regulatory T cells (Tregs) maintain peripheral immune tolerance, mainly by suppressing effector T cells. Unfortunately, immunosuppression by Tregs can also favor cancer. Regulating the susceptibility of T cells towards Tregmeditated immunosuppression presents an exciting approach to reconcile between immune activation and tolerance in a disease-specific way. However, less is known about the regulation and mechanism of Treg-mediated suppression in target T cells. The work presented in this thesis is dedicated to unraveling the modulation of signaling cascades in T cells upon T cell receptor (TCR) stimulation and suppression by Tregs. By using unbiased phosphoproteomics, we have mapped the global phosphoproteome of T cells upon TCR stimulation and suppression by Tregs. Our data indicate that Tregs suppress T cells mainly by opposing activation-induced phosphorylation. We discovered that Tregs revert activation-induced phosphorylation of DEF6 at specific sites (T 595 / S 597). Utilizing phospho-mutants, we discovered novel functions of these phosphorylations in disrupting the interaction of DEF6 with the IP3R and regulating T cell signaling via modulation of NFAT activation and transcriptional regulation of T cell cytokines. Upon further exploration of the phosphoproteomic candidate list, we observed similarly altered phospho-regulation of protein phosphatase 1, regulatory subunit 11 (PPP1R11) by Tregs. We revealed that silencing of PPP1R11 renders T cells resistant to Treg-mediation suppression. Our data indicates PPP1R11 to be a novel regulator of T cell activation and Treg-mediated suppression. In light of several reports on resistant T cells in multiple diseases, the phosphoproteomic mapping of suppressed T cells and the insights into novel roles of DEF6 and PPP1R11 from our study may aid in regulating the sensitivity of T cells towards immunosuppression by Tregs in cancer and autoimmune diseases. Besides phosphorylation of proteins, the subcellular localization of proteins is also an important regulatory mechanism of protein activity. Protein localization and activity is modulated by TCR stimulation and was shown for well-known TCR signaling proteins such as NFAT to be affected by Tregs as well. However, these mechanisms are not understood on a global level. Since there is no global data set available on subcellular protein localization and TCR stimulation-induced translocation in primary human T cells, as the first milestone in this field, we have performed a comprehensive mapping of the spatial proteome of T cells and TCR-induced subcellular protein translocation. Besides exploring the target T cell side, in this work we also studied novel methods to induce Tregs, which represents the “other side” of Treg-based immune therapies. We present a novel method of inducing iTregs by using supernatants from M2 macrophages. Our data demonstrate that M2 macrophages induce iTregs by binding and re-releasing TGF-β, which may be explored for Treg induction in situ in the future. Together, by presenting a global picture of T cell protein signaling yet with fine resolution, our work provides new mechanisms and data sets to revisit the role of T cells in therapy, especially in the context of T cell suppression by Tregs. LIST OF SCIENTIFIC PAPERS I. Joshi, R.N. , Binai, N.A. , Marabita, F. , Sui, Z. , Altman, A. , Heck, A.J.R. , Tegnér, J., and Schmidt, A. (2017). Phosphoproteomics reveals regulatory T cell-mediated DEF6 dephosphorylation that affects cytokine expression in human conventional T cells. Front. Immunol. 8. II. Joshi R.N. , Fernandes S.J., Shang M.M. , Kiani N.A. , Gomez-Cabrero D. , Tegnér J., Schmidt A. Phosphatase inhibitor PPP1R11 modulates resistance of human T cells towards Treg-mediated suppression of cytokine expression. (Accepted for publication in Journal of Leukocyte Biology) III. Schmidt, A. , Zhang, X. -M. , Joshi, R.N. , Iqbal, S. , Wahlund, C. , Gabrielsson, S. , Harris, R.A. , and Tegnér, J. (2016). Human macrophages induce CD4 + Foxp3 + regulatory T cells via binding and re-release of TGF-β. Immunol. Cell Biol. 94. IV. Joshi R.N. , Vesterlund M. , Stadler C. , Tegnér J., Schmidt A. , Lehtio J. TcellSubC: an atlas of the subcellular proteome of human T cells. (Manuscript) ADDITIONAL PUBLICATION Publications during doctoral studies that are not included in this thesis 1. Schmidt, A. , Éliás, S*. , Joshi, R.N. *, and Tegnér, J. (2016a). In vitro differentiation of human CD4+FOXP3+induced regulatory T cells (iTregs) from naïve CD4+T cells using a TGF-β-containing protocol. J. Vis. Exp. 2016. *shared second authors CONTENTS