Evgeniya Solodova 01.12.08

1. Evgeniya Solodova 01.12.08

2. Introduction: • Regulatory cytokine transforming growth factor-β (TGF-β): • is a secreted protein that exists in three isoforms called TGF-β1, TGF-β2 and TGF-β3 • TGF-β1 is the founding and predominant member of this family • controls proliferation, differentiation, and other functions in many cell types • acts as a negative autocrine growth factor • specific receptors for TGF-β activation trigger apoptosis when activated • many cells synthesize TGF-β and almost all of them have specific receptors for this peptide

3. Introduction: • T lymphocytes are the key components of the adaptive immune system • express T cell receptors (TCR) which recognize antigens in association with molecules of MHC • selected in the thymus according to the affinity to self-antigens to prevent auto immune response • differentiate upon infection into effector T cells – CD4+ helper T cells or CD8+ cytotoxic T cells – to combat the invading pathogen All these crucial processes of T cell development, homeostasis, tolerance to self antigens and differentiation are highly dependent on TGF-β regulation

4. Understand the regulation of T cells by TGF-β

5. Synthesized in inactive form in association with latency-associated protein (LAP) Secreted as such Bind to latent-TGF-β-binding protein (LTBP) targeting TGF-β to ECM

6. cells that produce TGF-β activator can differ from those that secrete TGF-β integration of signals from multiple cell types to regulate cellular responses

7. Binding to complex of TGF-β type I • (TGF-βRI) and type II (TGF-βRII) receptors • active signalling pathways • Phosphrilation of transcriptional factors • Smad2 and 3 translocation to the • nucleus with Smad4 or TIF1γ • - Binding to the regulatory sequences in • target genes regulation of gene • expression • OR • - Activation of Smad-independent • signalling pathways

8. The plasticity of Smad proteins in transcriptional regulation and the diversity of Smad-independent pathways enable TGF-β to exert its pleiotropic actions

9. TGF-β Regulates T Cell Development • Using different mice models (TGF-βRII- and TGF-βRI-deficient mice, TGF-βRII-deficient H-Y TCR transgenic mice) it was shown that TGF-β signalling in T cells : • promotes CD8+ and CD1d-dependent natural killer (NKT) T cell differentiation • reveals opposing functions on the CD4+Foxp3+ regulatory T (nTreg) cell development depending on the mice age

10. TGF-β regulates naive T cell homeostasis TGF-β signalling in T cells is essential for maintaince of peripheral T cell tolerance - How TCR specificity modulates T cell responses in TGF-β-deficient mice or mice with T cell-specific inactivation of TGF-β receptors? - What is the reason for naive T cell loss when TGF-β signalling is disrupted? • TGF-βRII-deficient OT-II cells (T cell TCRs have high binding affinity only to non-self MHC II antigen) undergo a high cell death and are largerly depleted in peripheral lymphoid organs • Lack of TGF-β signalling in CD8+ cells in H-Y TCR-transgenic mice led to diminished mature T cell numbers in female mice

11. TGF-β regulates naive T cell homeostasis In OT-II mice, deficient for Tgfbr2 gene, T cells actively differentiate into T helper 1 (Th1) or Th2 effector T cells, in contrast to normal polyclonal T cell population Th1 cells secrete IFNγ and lymphotoxin to combat intracellular pathogens through activation of adaptive immune system Th2 cells produce IL-4, IL-5 and IL-13 which direct antibody production to control extracellular pathogens TGF-β-deficient mice with other single nonself TCR affinities (TEα transgenic T cells and DO11.10 T cells)

12. TGF-β may have an essential role in promoting the survival of both CD4+ and CD8+ naïve T cells that interact with low affinity to self-antigens • Modulates immune tolerance by inhibiting high-affinity CD4+ and CD8+ T cell proliferation and differentiation into Th1 and Th2 and cytotoxic T lymphocytes

13. Active immune suppression by cytokine TGF-β1 or CD4+Foxp3+ Treg cells is a pivotal mechanism of peripheral T cell tolerance • Mice lacking either TGF-β1 or Foxp3, the transcription factor required for Treg cell function, develop multifocal inflammatory disorders What is the mechanism underlying TGF-β regulation of T cell tolerance?

14. TGF-βRII-, TGF-β1-deficient mice, bone marrow chimera and T cell transfer experiments Lack of TGF-β signalling leads to: -Reduction of nTreg cell -More activated and differentiated phenotype of T cell populations

15. T cells Foxp3 expression induced Treg (iTreg) cells in periphery thymic nTreg cells TGF-β • recruiment of downstream transcriptional factor Smad3 to a • Foxp3 enhancer element • T cell produced IL-2 activates STAT5 for binding to Foxp3 • promoter Differentiation of iTreg cells

16. T cells RORγt expression Foxp3 expression Th17 cells induced Treg (iTreg) cells in periphery thymic nTreg cells TGF-β • recruitment of downstream transcriptional factor Smad3 to a • Foxp3 enhancer element • T cell produced IL-2 activates STAT5 for binding to Foxp3 • promoter Differentiation of iTreg cells • IL-6 activates STAT3 induction of ROPγt expression Differentiation of Th17 cells • Foxp3 interacts with ROPγt and suppresses its function • mechanism for reciprocal differentiation of iTreg and Th17 cells Th17 cells secrete IL-17A, IL-17F, IL-22, that act on a broad range of innate immune and nonhematopoietic cells to protect the host from extracellular pathogens RORγt - transcription factor that orchestrates the differentiation of Th17 lineage

17. TGF-β inhibits Th1, Th2 and CTL proliferation and differentiation TGF-β • Block of TCR-induced Tec kinase Itk activation and Ca2+ influx in T cells • Downregulation of NFAT, T-bet and GATA-3 expression • Inhibition of IL-2, IFNγ and granzyme B transcription via • association of TGF-β with Smad2/3 complexes Inhibition of Th1, Th2 and CTL cell proliferation and differentiation Itk kinase - Interleukin-2 inducible T-cell protein tyrosine kinase NFAT - Nuclear factor of activated T-cells Th1 - specific T box transcription factor granzyme B -granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1

18. TGF-β engages multiple signalling pathways to control T cell development

19. A Three-Cell Model for TGF-β1-Dependent Regulation of T cells • Antigen recognition and presentation by DCs • Activation of Treg cells • Production of latent form of TGF-β1 • Association with LAP (and LTBP) • Activation and release of TGF-β1 • Inhibition of CD4+ T cell differentiation into Th1 and Th2 cells • Promotion of CD4+ T cell differentiation into iTreg or Th17 cells • Low production of TGF-β1 by activated CD4+ T cells that potentially regulates T cell differentiation through an autocrine route

20. Summary and Future Perspectives TGF-β: • regulates thymic T cell development and peripheral T cell survival, proliferation and differentiation • ensures the maintenance of divers and self-tolerant T cell repertoire and the initiation of appropriate T cell responses essential for an effective adaptive immune system • may have an important role in more ancient biological processes such as embryonic development and carcinogenesis New insights into the control of T cell responses by TGF-β will help to illuminate the fundamental principles of T cell regulation and facilitate the employment of TGF-β to treat a variety of immune-related disorders

21. Thank you for your attention!