Cellular and Molecular Immunology:Peripheral B and T cell differentiation

Christoph Mueller; Institute of Pathologychristoph.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

RAG-1, 2RAG-1, 2

• RAG1 and RAG2 (“Recombination Activation Genes”) RAG1 and RAG2 (“Recombination Activation Genes”) are essential for the rearrangement of the Ig and TCR are essential for the rearrangement of the Ig and TCR genesgenes

• Mice deficient for either RAG1 and/or RAG2 are Mice deficient for either RAG1 and/or RAG2 are deficient for both T and B cells (but may still have some deficient for both T and B cells (but may still have some NK cells) NK cells)

• to prevent the later generation of autoreactive T and B to prevent the later generation of autoreactive T and B cells, the expression of these two genes needs to be cells, the expression of these two genes needs to be tightly regulatedtightly regulated

Regulation of T cell receptor gene rearrangement

Experimental approach for determining the regulation of RAG gene expression

Experimental set-up

Mouse, transgenic for TCR, recognizing the LCMV peptide gp33 in the context of H-2 Db

-In a C57BL/6 (H-2b) genetic background: positive selection of TCR tg T cells, tg TCRexpressed on thymocytes)

- In a BALB/c (H-2d) 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

In situ hybridisation for the detection of RAG-1 mRNAThymus, wild type mouse (C57Bl/6 mouse)

Cortex

Medulla

Murine Thymus, TCR tg mouse with a positively selecting MHC haplotype: transcription of RAG1 gene is suppressed

MedullaMedulla

CortexCortex

MedullaMedulla

CortexCortex

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

CD8 T cell differentiation

Functional Heterogeneity of CD4 T Lymphocytes

naive CD4 T Cell

Th1 Th1 Th0Th0 Th2Th2

ThO: IL2, IL3, IL4, IL5, IL6, IL9, IL10, IFN

Th1:IL2, IFN , TNFlymphotoxin

Th2:IL4, IL5, IL6, IL9, IL10

Grogan & Locksley Curr Opinion Immunol 14: 366-72; 2002

Th 2Th 2 Th 1Th 1

Naïve CD4

IL 12IL 4IFN

Leprosy

• Chronic - progressive infectious disease, affecting Chronic - progressive infectious disease, affecting the skin, peripheral nerves and occasionally the the skin, peripheral nerves and occasionally the respiratory tractrespiratory tract

• Infectious agent: Infectious agent: Mycobacterium lepraeMycobacterium leprae

• Globally, approx. 10-20 million patients infected, Globally, approx. 10-20 million patients infected, endemic in tropical areas (e.g. Southeast Asia; endemic in tropical areas (e.g. Southeast Asia; India, South America, Subsaharan Africa)India, South America, Subsaharan Africa)

Leprosy: Prevalence

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 presentTuberculoid Leprosy: • Singular, small macular lesions of the skin. • Peripheral nerves (e.g. N. ulnaris, peronealis, N.

auricularis) are often affected sensory neuropathy.

• Granuloma are frequent (with only low numbers of M. leprae present)

naïve CD4 T cells

Th1Th1 Th0Th0 Th2Th2cellularcellularimmunityimmunity

humoral humoral immunityimmunity

Immunological Spectrum of Leprosy

Tuberculoid leprosy Lepromatous leprosy

Granuloma formation Persistence of M. leprae

Tissue damage may ensue Disfiguring disorder

Lepromatous leprosy

Tuberkuloid leprosy

Type IV Hypersensitivity reactions

Fig. 5-11Kumar 6th edition

Pathogens may influence the resulting adaptive immune response

Science 302: 993-4; 2003

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). Information1, 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 promoter1Nature 430, 150 - 151 (08 July 2004)

Publications on Suppressor T cells and Regulatory T cells

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Suppressor T cells

Regulatory T cells

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 (TR1) cells, which secrete high levels of IL-10, no IL-4 and no or low levels of IFN-; and T helper 3 (TH3) 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

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.

Peripheral naive CD4+ T cell precursor cells (THp) can differentiate into three subsets of effector T cells (TH1, TH2 and TH-17) and several subsets of Treg cells, including induced Treg cells (iTreg), Tr1 cells and TH3 cells. Naturally occurring Treg cells (nTreg) 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.

CD4 T cell differentiation (for beginners)

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

B cells ….

B-Gedächtniszelle

CD40LCD40L

CD40CD40

A

A

naive B - Zelle

2.Signal:Quervernetzung derIg durch Antigen oderAktivierung durch CD40L

T-Zell-Hilfe durch Zytokine

CD4 T-Zelle

A1. Signal:Bindung des Antigen an Ig

kein 2. Signal

2. Signal

MantelzoneMantelzone

Keim-zentrum

B-Zellen (CD19)B-Zellen (CD19)

T-Zellen (CD3)T-Zellen (CD3)

FDZ FDZ (CD21)(CD21)

proliferierende Zellenproliferierende Zellen (Ki-67)(Ki-67)

MakrophagenMakrophagen (CD68)(CD68)

PlasmazellenPlasmazellen

Mantel-zone Keimzentrum

MantelzoneMantelzoneKeimzentrumKeimzentrum

aa bb

cc dd

ee ff

„„dark zone“dark zone“„„light zone“light zone“

• 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: