PROFESSIONAL ANTIGEN PRESENTING CELLS Express MHC class I and class II molecules Express co-stimulatory molecules (B7, CD40) Take up extracellular antigens B cells – soluble proteins, toxins (ADAPTIVE) Macrophages – extracellular pathogens (bacteria, yeast) INNATE – particles Dendritic cells – viruses, apoptotic cells

PROFESSIONAL ANTIGEN PRESENTING CELLS Express MHC class I and class II molecules Express co-stimulatory molecules (CD40, B7) Take up extracellular antigens B cells – soluble proteins, toxins ADAPTIVE – Ag specific Macrophages – extracellular pathogens (bacteria, yeast) Dendritic cells – viruses, apoptotic cells INNATE 3 – 6% ~1% ~25%

CHARACTERISTICS OF PROFESSIONAL ANTIGEN PRESENTING CELLS Macrophage Dendritic cellB - lymphocyte Ag uptake phagocytosis +++ phagocytosis +++ Ag-specific mIg virus infection MHC expression induced +/+++ constitutive ++++ constitutive +++ bacteria, cytokine immature/mature +++/++++ activation ++++ Pesented Ag particulate Ag protein soluble protein intra/extracellular virus protein, allergen toxin pathogens apoptotic cell Co-stimulation induced +/++ constitutive ++++ induced +/+++ éretlen/érett +++/++++ Localization lymphoid tissue lymphoid tissue lymphoid tissue connective tissue connective tissue peripheral blood body cavities epithelium Lymph node evenly immature – tissue follicles mature – T cell area

Morphology of plasmacytoid dendritic cells IPC/DC2 monocytepDC Scanning EMTransmission EM

Plasmacytoid DCs control the function of many immunocytes Role in immune response and in the pathogenesis of autoimmune diseases and cancer HIV infects PDC IFNα is impotant in SLE pathology

Migration Pathways of PDC/IPC versus mDC into a lymph node IPC: HEV mDC: afferent lymphatics Both migrate into the T-cell rich areas

Tissue DC Activated DC DC AND T CELLS ENCOUNTER T CELL ACTIVATION CIRCULATION Naive T cells Effector and memory T cells TISSUE LYMPH NODE TISSUE Lymphatics Inflammation Pathogen ACTIVATION AND MIGRATION OF DENDRITIC CELLS ANTIGEN

Huang et al Immunity 2004 Bone-marrow derived DCs (yellow) were pulsed with 1 µM Ova 4 peptide and 10 µM Ova for 1 hour at 37 o C, then injected into the footpad of a C57BL/6 recipient. This was followed 6 hours later by i.v. co-injection of OT-I CD8+ T cells (5 µM CFSE, green) and OT- II CD4+ T cells (5 µM SNARF, red). Rapid DC Migration in the Subcapsular Space Capture of an Ag-Specific T Cell by an Ag-Bearing DC Bone-marrow derived DCs (either 5 µM CFSE, green) or (50 µM Cell Tracker Blue, blue) were injected into the footpad of a C57BL/6 mouse, followed 18 hours later by intravenous injection of freshly isolated polyclonal CD4+ T cells (5 µM SNARF, red) and CD8+ T cells (5 µM CFSE and 5 µM SNARF, yellow). The draining LN was removed 6 hours after injection

ONE, TWO and DANGER signal hypothesis Matzinger, P. Science, (2002)

The danger hypothesis & co-stimulation Fuchs & Matzinger 1995 Full expression of T cell function and self tolerance depends upon when and where co-stimulatory molecules are expressed. Apoptotic cell death. A natural, often useful cell death. APC No danger Cell containing only self antigens Innocuous challenge to the immune system fails to activate APC and fails to activate the immune system

The danger hypothesis APC Necrotic cell death e.g. tissue damage, virus infection etc Pathogens recognised by microbial patterns DANGER APC that detect ‘danger’ signals express costimulatory molecules, activate T cells and the immune response

DIFFERENTIATION AND MATURATION OF T CELLS IN THE THYMUS

REGULATED T-CELL DIFFERENTIATION a a pre T cell pro T cell immature T cell NO ANTIGEN RECOGNIZING RECEPTOR SIGNALING RECEPTOR ANTIGEN RECOGNIZING RECEPTOR preT-  CD4+CD8+ TCR Epithelial cell APC

T- CELL DEVELOPMENT NK cell Pro-T  -rearrangement T Pre-T  -rearrangement Pre-T  Selection clonal deletion T T T Mature-T Lymphoid precursor Mature-B c-kit/CD44 H rearrangementSurrogate LL rearrangement Selection clonal deletion B B B B  Pro-B Pre-B RAG-1/RAG-2

1.Generation of NK cells – no TCR 2. Differentiation of γδ and αβ TCR carrying T cells 3. Selection of αβ TCR – positive selection – negative selection 4. Differentiation of CD4+ and CD8+ T cell lineages EVENTS OF T CELL DIFFERENTIATION IN THE THYMUS Early pre-T Pre-Tα- chain Lck signal β rearrangement γδ T-cell No selection αβ NKT-cell αβCD4+ αβCD8+ CD4+CD8+ IL-7-dependent proliferation Pro-T unsuccesful β- chain unsuccesful α-chain no positive selection negative selection α rearrangement Late pre-T CD4+CD8+

1.The primary T cell pool is biased to MHC-specificity (V genes) 1-2% for one allotype 2.Focusing the T cell pool to self MHC recognition (+) 3.Elimination of useless clones 4.Elimination of self agressive clones (-) 5.CENTRAL TOLERANCE 6.Focusing The T cell repertoire for recognition of non self 7.Individualized T cell repertoire is available in the periphery 8.CD4 and CD8 co-stimulatory molecules are involved in positive selection αβTCR CD4+ CD8+ SELECTION OF T LYMPHOCYTES IN THE THYMUS UNDER THE CAPSULE CORTEX CORTEX/ MEDULLA IL-7-dependent proliferation β+preTα CD4-CD8- DN CD4+CD8+ DP MEDULLA TCRαβ TCR(-) sMHC+sP sMHC+fP fMHC+fP  selection – selection  – AICD NO  PERIPHERAL TOLERANCE AICD – Activation Induced Apoptosis

CD4+CD8+ POSITIVE SELECTION OF DOUBLE POSITIVE (DP) T CELLS ALSO DIRECTS CD4 AND CD8 SINGLE POSITIVE (SP) T CELL COMMITMENT MHC-II + peptide complexes recruit CD4 Thymic epithelial cell MHC-I + peptide complexes recruit CD8 BARE LYMPHOCYTE SYNDROME (BLS) Lack of MHC class I – no CD8+ cellsLack of MHC class II – no CD4+ cells POSITIVE SELECTION FOR 3 – 4 DAYS, SUCCESSIVE α-GENE REARRANGEMENTS

a a Activated T-cell Mature naive T-cell Memory T-cell T-CELL DIFFERENTIATION IN THE PERIPHERY Ag CD4 TCR APC CD8 TCR APC CD4 TCR APC CD8 TCR APC CD4 TCR APC CD8 TCR APC Ag

POSITIVE SELECTION – Thymic education (no instruction for specificity) Low avidity interaction of MHC - self peptide - TCR Thymic epithelial cells Self peptide composition and concentration (foreign peptides are not present) Low peptide dose induces positive selection – special ligands 80-90% of DN (CD4-CD8-) T cells is NOT positively selected PASSIVE CELL DEATH BY NEGLECTION NEGATIVE SELECTION – Central self tolerance High avidity of MHC - self peptide - TCR interaction Ubiquitous and abundant self antigens are present in the thymus High peptide dose induces negative selection Any thymic antigen presenting cell: epithelial cells, bone marrow-derived macrophages, dendritic cells THE GENERATION OF SELF MHC + FOREIGN PEPTIDE SPECIFIC T CELLS REQUIRES WEAK INTERACTION WITH SELF MHC + SELF PEPTIDE SELF RESTRICTED AND TOLERANT PERIPHERAL T CELL REPERTOIRE PHYSIOLOGICAL TRESHOLD SELECTION OF THE T CELL REPERTOIRE – CENTRAL TOLERANCE

Normal tissue cells do not express MHC class II NO SIGNAL 1. for CD4+ Th activation Normal tissue cells do not express co-stimulatory molecules and do not produce T cell differentiating cytokines NO SIGNAL 2. for CD4+ Th activation Migration of naive T lymphocytes to normal tissues is limited Antigen presenting cells are not activated in normal tissues NO SIGNAL 3. for CD4+ Th activation PERIPHERAL TISSUES TOLERIZE THEMSELVES PERIPHERAL TOLERANCE IMMUNE RESPONSES ARE NOT INITIATED IN THE PERIPHERY

ANERGY – Functional unresponsiveness, no IL-2 secretion SIGNAL 1 Recognition of auto-antigen on tissue cell SIGNAL 2 No B7 and CD40 expression, no co-stimulation Tissue resident professional APC are not activated SIGNAL 3 Innate immunity is not activated No inflammation CLONAL DELETION – Activation induced cell death Requires persistant high antigen dose Fas – FasL interaction SUPPRESSION – Activity of other cells Cytokine-mediated balance Effector functions are inhibited by regulatory T cells CLONAL IGNORANCE No contact with the immune system Immunologically privileged sites Central nervous system, eye No recognition in the periphery MECHANISMS OF PERIPHERAL TOLERANCE

Homozygote Heterozygote HOMEOSTASIS OF POSITIVE AND NEGATIVE SELECTION IN THE DEVELOPMENT OF THE AVAILABLE T LYMPHOCYTE REPERTOIRE Number of MHC molecules Ratio of positive selection Ratio of negative selection increases with the number of MHC genes