2. Cellular and Molecular Immunology: Peripheral B and T cell differentiation Christoph Mueller; Institute of Pathology christoph.mueller@pathology.unibe.ch General principles: - functional subsets - plasticity vs. stability of phenotype Molecular basis of lymphocyte differentiation: transcription factors Soluble factors and cognate interactions involved in the differentiation of lymphoid cells Experimental approaches to study B/T cell differentiation Consequences of impaired T and B cell differentiation

4. RAG-1, 2 RAG1 and RAG2 (“Recombination Activation Genes”) are essential for the rearrangement of the Ig and TCR genes Mice deficient for either RAG1 and/or RAG2 are deficient for both T and B cells (but may still have some NK cells) Mice deficient for either RAG1 and/or RAG2 are deficient for both T and B cells (but may still have some NK cells) to prevent the later generation of autoreactive T and B cells, the expression of these two genes needs to be tightly regulated to prevent the later generation of autoreactive T and B cells, the expression of these two genes needs to be tightly regulated

5. Regulation of T cell receptor gene rearrangement Experimental approach for determining the regulation of RAG gene expression

6. Experimental set-up Mouse, transgenic for TCR , recognizing the LCMV peptide gp33 in the context of H-2 D b -In a C57BL/6 (H-2 b ) genetic background: positive selection of TCR  tg T cells, tg TCR  expressed on thymocytes) - In a BALB/c (H-2 d ) background (no positive selection of TCR  tg T cells; no tg TCR  expressed on thymocytes) Working hypothesis: recognition of a MHC/Ag complex via a positively selecting TCR  down-regulates RAG expression in the differentiating T cells, and thus, terminates TCR  rearrangements

7. In situ hybridisation for the detection of RAG-1 mRNA Thymus, wild type mouse (C57Bl/6 mouse) Cortex Medulla

8. Murine Thymus, TCR  tg mouse with a positively selecting MHC haplotype: transcription of RAG1 gene is suppressed Medulla Cortex

9. Medulla Cortex Murine Thymus, TCR  tg mouse with a non - selecting MHC haplotype: transcription of RAG1 gene is still active in the cortex

11. CD8 T cell differentiation

12. Functional Heterogeneity of CD4 T Lymphocytes naive CD4 T Cell Th1 Th0Th2 ThO: IL2, IL3, IL4, IL5, IL6, IL9, IL10, IFN  Th1:IL2, IFN , TNF  lymphotoxin Th2:IL4, IL5, IL6, IL9, IL10

13. Grogan & Locksley Curr Opinion Immunol 14: 366-72; 2002 Th 2 Th 1 Naïve CD4 IL 12 IL 4 IFN 

14. Leprosy Chronic - progressive infectious disease, affecting the skin, peripheral nerves and occasionally the respiratory tractChronic - progressive infectious disease, affecting the skin, peripheral nerves and occasionally the respiratory tract Infectious agent: Mycobacterium lepraeInfectious agent: Mycobacterium leprae Globally, approx. 10-20 million patients infected, endemic in tropical areas (e.g. Southeast Asia; India, South America, Subsaharan Africa)Globally, approx. 10-20 million patients infected, endemic in tropical areas (e.g. Southeast Asia; India, South America, Subsaharan Africa)

15. Leprosy: Prevalence

16. Leprosy: different clinical forms of the disease Lepromatous Leprosy: Multiple, nodular lesions of the skin, in particular, of the face (”lion face"). Persistent bacteriemia, foamy cell-like lesions with numerous M. leprae present Tuberculoid Leprosy: Singular, small macular lesions of the skin. Peripheralnerves (e.g. N. ulnaris, peronealis, N. auricularis) are often affected sensory neuropathy. Granuloma are frequent (with only low numbers of M. leprae present)

17. naïve CD4 T cellsTh1 Th0 Th0Th2 cellularimmunity humoralimmunity Immunological Spectrum of Leprosy Tuberculoid leprosyLepromatous leprosy Granuloma formation Persistence of M. leprae Tissue damage may ensueDisfiguring disorder

18. Lepromatous leprosy

19. Tuberkuloid leprosy

20. Type IV Hypersensitivity reactions Fig. 5-11 Kumar 6th edition

21. Pathogens may influence the resulting adaptive immune response Science 302: 993-4; 2003

22. Figure 1 Stimulating the Th1 or Th2 response. In both pathways, dendritic cells internalize the pathogen. They present its antigens to T cells, which recognize antigens through their T-cell receptors (TCR). a, Organisms such as intracellular bacteria or viruses are recognized by the Toll-like receptors on dendritic cells; the resulting signals induce the secretion of interleukin-12 (IL-12) and differentiation of CD4 T cells into the Th1 lineage that produces gamma interferon (IFN-). b, How dendritic cells recognize larger pathogens, such as parasitic worms, is not known. But the end result is differentiation of Th2 effector cells regulated by T-cell-produced interleukin-4 (IL-4). Information 1, 2 on the link between dendritic cells and T cells suggests that the former express different Notch ligands — Delta or Jagged — under different conditions. Jagged is specifically induced by stimuli known to induce Th2 differentiation. Notch signals (Notch-IC) can induce transcription of IL-4 through direct binding of RBPJ to the IL-4 promoter 1 Nature 430, 150 - 151 (08 July 2004)

23. Publications on Suppressor T cells and Regulatory T cells 197419761978 198019821984198619881990199219941996199820002002 # Publications per Year (PubMed) 100 150 200 250 300 0 50 Suppressor T cells Regulatory T cells

24. Natural regulatory T cells express the cell-surface marker CD25 and the transcriptional repressor FOXP3 (forkhead box P3). These cells mature and migrate from the thymus and constitute 5–10% of peripheral T cells in normal mice. Other populations of antigen-specific regulatory T cells can be induced from naive CD4 + CD25 - or CD8 + CD25 - T cells in the periphery under the influence of semi- mature dendritic cells, interleukin-10 (IL-10), transforming growth factor- (TGF-) and possibly interferon- (IFN-). The inducible populations of regulatory T cells include distinct subtypes of CD4 + T cell: T regulatory 1 (T R 1) cells, which secrete high levels of IL-10, no IL-4 and no or low levels of IFN-; and T helper 3 (T H 3) cells, which secrete high levels of TGF-. Although CD8 + T cells are normally associated with cytotoxic T-lymphocyte function and IFN- production, these cells or a subtype of these cells can secrete IL-10 and have been called CD8 + regulatory T cells. Rregulatory T cell subsets

25. Mechanism(s) of suppression. Various molecular and cellular events have been described to explain how Treg can suppress immune responses. They include: IL-2 gene expression inhibition, modulation of costimulatory molecules on APCs and interaction of LAG3 with MHC class II molecules (a), immunosuppressive cytokine secretion (b), induction of tryptophan catabolism through CTLA-4 (c) and cytotoxicity (d). However, none of those mechanisms can explain all aspects of suppression. It is probable that various combinations of several mechanisms are operating, depending on the milieu and the type of immune responses. It is also possible that there might be a single key mechanism that has not been found yet (e). Abbreviations: APC, antigen presenting cell; TCR, T cell receptor.

26. Peripheral naive CD4 + T cell precursor cells (T H p) can differentiate into three subsets of effector T cells (T H 1, T H 2 and T H -17) and several subsets of T reg cells, including induced T reg cells (iT reg ), Tr1 cells and T H 3 cells. Naturally occurring T reg cells (nT reg ) are generated from CD4 + thymic T cell precursors. The differentiation of these subsets is governed by selective cytokines and transcription factors, and each subset accomplishes specialized functions.

27. CD4 T cell differentiation (for beginners)

28. CD4 T cell differentiation (for specialists) (Keiji Hirota, Bruno Martin and Marc Veldhoen, 2010):

31. B cells ….

32. B-Gedächtniszelle CD40L CD40 A A naive B - Zelle 2.Signal:Quervernetzung derIg durch Antigen oderAktivierung durch CD40L T-Zell-Hilfe durch Zytokine CD4 T-Zelle A 1. Signal: Bindung des Antigen an Ig kein 2. Signal

37. Mantelzone Keim- zentrum B-Zellen (CD19) T-Zellen (CD3) FDZ FDZ(CD21) proliferierende Zellen (Ki-67) (Ki-67) Makrophagen (CD68) (CD68) Plasmazellen  Mantel- zone Keimzentrum Mantelzone Keimzentrum ab cd ef „dark zone“ „light zone“

38. Molecular mechanisms of immune tolerance Central tolerance induction in the B cell and T cell compartment Immune tolerance in the periphery Immunopathology vs. Autoimmunity Immune tolerance vs. Immune privilege vs. Immune ignorance Program of my next lectures: