Adaptive Immunity (I) PDF

Adaptive immunity (I) T cells – development and functions Prof. David Voehringer, Department of Infection Biology, UKER T cells Development and subsets Antigen recognition Effector functions T cell - microscopic image...

Adaptive immunity (I) T cells – development and functions Prof. David Voehringer, Department of Infection Biology, UKER T cells Development and subsets Antigen recognition Effector functions T cell - microscopic image NIAID Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 2  Hematopoiesis and T cell development Origin of T cells 2 lineages - myeloid and lymphoid ILCs Dendritic cell Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 3 Evolution of the adaptive immune system Jawless vertebrates Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 4 Thymus organogensis and involution T cells develop and mature in the thymus, a primary lymphoid organ next to the heart. Their function was not known until the 1970s. Patients with T-cell deficiencies suffer from recurrent infections. in the emryonic stage Thymus develops from the third pharyngal pouch and is composed of ectodermal (=>Cortex) and endodermal (=>Medulla) tissue. The transcription factor Foxn1 is essential for thymic development. Foxn1-ko mice lack thymus and fur (nude mice). Nude mice are used fro xenograft transplantation experiemts more T cells are generated Thymic involution: The size of the thymus reaches its maximum (~35g) in puberty and then declines to ~5g at the age of 70 years. is replaced by fat tissue Univ Michigan www.jax.org Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 5  T cells develop in the thymus - Precursor cells (CLPs) are generated in the bone marrow because of the attractionof the CCR9 - Migration to the thymus (chemokine receptor CCR9) - T cells develop in the thymus in distinct stages quality control - Mature T cells leave the thymus - Migration to peripheral (secondary) lymphoid organs then migration to diff organs after infection Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 6  Maturation of thymocytes to T cells TCRβ-rearrangement T cell receptor (TCRβ+preTα = preTCR) quality control TCRα-rearrangement (αβTCR) Functional γδ RA prevents αβ RA ETP: early thymocyte precursor (c-Kit/CD117+, IL-7R+) DN: double negative (CD4-CD8-) DP: double positive (CD4+CD8+) SP: single positive (CD4+ or CD8+) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 7  Major thymocyte subsets CD4 single-positive each spot depicts a single cell/ event. mature Double-positive (CD4+CD8+) immature CD8 single-positive mature Flow cytometry plot Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 8 What is the function of the thymus? Generation of T cells that are tolerant against „Self“ and responsive to „Foreign“ antigens. This is achieved by: killing of autoreactive / non- functional cells Negative Selection of autoreactive or non-functional T cells („central tolerance“) T cells that can recognise antigens and show activity against them Positive Selection of T cells that recognized self-antigens with low avidity (weak activating signal) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 9 Clonal selection of lymphocytes (T and B cells) - negative selection Sir Frank MacFarlane Sir Peter Medawar Burnet Nobel prize 1960 1. Every T or B cell produces only one spec. receptor 2. Receptor binding is required for activation 3. Daughter cells maintain the receptor specificity - clonal expansion of T cells 4. Cells with autoreactive receptors die during development Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 10  Positive and negative selection Positive selection Treg cells (regulatory Cell death T cells can suppress (death by autoreactive T cells) Chance for neglect) the T cell to survive selection in Negative Selection the thymus TCR affinity for self-antigens Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 11  How many different TCRs are encoded in the genome? What is the molecular composition of the TCR? ??? 12 Generation of TCR diversity by recombination V (variable) D (diversity) J (joining) C (constant) α β Recombination of different segments generates a huge variety of different TCRs Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 13  Segments that can be used for recombination -Vδ-Dδ-Jδ-Cδ- around 80 diff chains for variable domain, 60 diff chains for joining segment and 1 constant 2 diff diversity segments and 2 diff constants Variable, Diversity and Joining segments get recombined by specific enzymes. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 14  Recombination of TCR alpha and beta chains Replacement of preTα by α at DP stage Expression of β with preTα stops rearr. and induces proliferation. DN=>DP Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 15  The 12-23 rule of VDJ recombination Recombination signal sequences (RSS) spacer -23 C. Hewitt Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 16  The 12-23 rule of VDJ recombination bending of the DNA due to 23 spacers C. Hewitt Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 17  Rag 1 and Rag 2 make DNA breaks at RSS The DNA recombinases Rag1 and Rag2 bind to specific recognition sequences (RSS), endonucleases bring together 23nt and 12nt sequences and cleave the DNA. has to be sealed again are released Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 18  Repair of coding joints The DNA double strand breaks get stabilized by Ku70 and Ku80. DNA-protein kinase and Artemis open the hairpin. TdT adds N-nucleotides to the open DNA ends. DNA ligase IV and XRCC4 ligate the DNA ends. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 19  P- and N-nucleotides modify the joint region Palindromes TCGA TATA AGCT ATAT Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 20  P- and N-nucleotides modify the joint region Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 21  Combinatorial diversity of αβTCR vs BCR BCR αβTCR Even though more combinations are possible 22 But we have only about 1010 lymphocytes Evolution of the adaptive immune system Jawless vertebrates Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 23 VLR receptors in Lamprey (agnatha) Variable lymphocyte receptors (VLRs) Brook lamprey BBC Lampreys feed on fish in the Great Lakes of Northern America Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 24 Antigen recognition by T cells What does the TCR recognize? Is antigen recognition the same for CD4 and CD8 T cells? Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 25  Antigen recognition by αβT cells 20% of T cells in liver Vα14Jα18/Vβ8.2 or 7 (mouse) Vα24JαQ /Vβ11 (human) they recognise CD4 CD8 NKT cell glycolipids and not peptides αβ αβ αβ Some human γδ CC CC T cells also see CC T cell CD1d V V receptor VV VV Glycolipid Peptide peptide recognition site CD1d side binding CD4 peptide recognition site CD8 MHC I (αGalactosyl- Coreceptor Ceramide, …) Coreceptor MHC II β2m β2m Antigen-presenting cell MHC = major histocompatibility complex Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 26  Antigen recognition by T cells T cell docks on the surface of the peptide APC Abbas et al. 2007 Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 27 Crystal structure of TCR-peptide-MHC complex TCR the highlighted segments are very imp for docking View from top MHC-I Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 28  TCR recognizes peptide+MHC (MHC restriction) TCR X Y X MHCa MHCa MHCb Recognition No No Recognition Recognition Nobel-prize 1996 for Zinkernagel and Doherty Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 29  MHC locus is subdivided in class I, II and III MHC class I encodes: -Peptide-presenting proteins and „non-classical“ MHCs MHC class II encodes: -Peptide-presenting proteins -Subunits of immunoproteasomes (LMP2, LMP7) -Peptid transport proteins (TAP) -Chaperones for peptide presentation in MHC class II MHC „class III“ encodes: -Proteins of the complement system (C2, C4, factor B) -Cytokines (TNFα, LT-α, LT-β) -heat shock proteins Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 30  Which cells express MHC molecules? expressed on MHC class I on all nucleated cells (not on erythrocytes) more selective and only expressed on MHC class II only on antigen- presenting cells (APCs) like dendritic cells, macrophages and B cells. But also on thymic epithelial cells Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 31  Peptide presentation by MHCs Diff in structures: imp both contain antiparallel alpha helix sheets MHC I this opening is more narrow than MHC II Not encoded in the MHC MHC II Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 32  Space-filling models Closed on both sides. Open on both sides. Binds only short peptides (8-10AS) Binds longer peptides (10-20AS) accomodation - Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 33 Nomenclature of MHCs in mouse and human MHC Class is called H2 in mice Mouse MHC class I: H2-K, -D, -L (L not always present) MHC class II: H2-A, -E (also named I-A, I-E) depends on the diff mice strain Allotypes of inbread mouse strains indicated by superscript letters (C57BL/6: H-2KbDb, BALB/c: H-2KdDdLd,…) Human MHC class I: HLA-A, -B, -C MHC class II: HLA-DP, -DQ, -DR HLA = Human Leukocyte Antigen Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 34  MHC immunogenetics A B C Maternal allele Paternal allele three genes that serve the function and can be inherited from both parents Polygeny: many genes (loci) with the same function (A, B, C) Polymorphism: many alleles for one gene in the whole population (e.g. yellow and red for A) no preference for expression Co-dominance: Both alleles (maternal and paternal) for each MHC locus are expressed. combination of diff NHC genes in diff alleles Haplotype: Set of MHC loci of a given individual Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 35  Working of MHC Endogenous pathway (MHC-I) Ubiquitination degraded Proteasom Antigen in cytoplasm MHC-I TAP transport into ER - Golgi ATP dependent Tapasin Peptid ERp57 Calreticulin „cross MHC-I Calnexin β2m presentation“ CLIP chaperon removes this Peptide Invariant HLA-DM chain MHC-II need to stabilise the immature Endoplasmatic MHC class II reticulum Proteases generate peptides MHC-II Degraded invariant chain Endosome Exogenous Exogenous pathway (MHC-II) Antigen Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 36  CD8 T cells (cytotoxic T cells) Activation Killing of virus-infected Proliferation cells and tumor cells Differentiation naive v activ aktiv TCR ated Perforin MHC-I Granzyme B IFN-γ TNF-α FasL Apoptosis LYMPH NODE TISSUE Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 37 Composition of the T cell receptor TCRαβ -α and β chains determine the antigen-specificity - KD ~2-10µM (quite low affinity) cytoplasmic chains too short -no cytoplasmic domains therefore : -signal transduction via CD3-ε, γ, δ and TCR-ζ chains contains tyrosine that can be phosphorylated by kinases -Immunotyrosine activation motifs (ITAMs) get phosphorylated by kinases (Lck, Fyn) serve as docking sites for SH2-domain- containing proteins (ZAP70 kinase, SLP-76 adaptor protein,…) ITAM: YxxL/Ix(6-8)YxxL/I Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 38  PIP2 LAT PLC-γ kinases SLP-76 cleaves phospholipids in membrane and generaten signals DAG IP3 Calci- PKC MAPK neurin NFkB AP-1 NFAT imp for T cell activation 39  APC (dendritic cell or macrophage) MHC-I B7 (CD80, CD86) CD8 TCR CD3 CD28 IL-2R phosphorylation of kinases is imp for the activation of ITAM Lck PI3K ITAM JAK1 Ras JAK3 Immunoreceptor Fyn Signal 2 STAT5 tyrosine-based activation domain Signal 1 (costim.Signal) Signal 3 NFκB, NFAT, AP-1 T cell is dependent on IL -2 for proliferation T cell Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 40  importance of three levels of activation: anergy What happens if T cells get only signal 1? 41  Lack of co-stimulation results in anergy MHC B7 TCR CD28 Activation kind of safety mechanism Anergy reversible stage of inactivation Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 42  The immunological synapse SMAC: supramolecular adhesion complex Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 43  Formation of the immunological synapse c-SMAC Grakoui et al. Science 1999 400-700 TCR molecules are in a c-SMAC dynamic process 44  CD8 T cells (cytotoxic T cells) Activation Killing of virus-infected Proliferation cells and tumor cells Differentiation naive v activ activ TCR ated ated Perforin MHC-I Granzyme B IFN-γ TNF-α FasL Apoptosis LYMPH NODE TISSUE Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 45  :on release can form pores on the Plasma mem FasL - Ligand on the surface of T cell FasD - Receptor on the surface of... 46  Kinetic of acute viral infection Virus-specific CD8 T cells log Viral titer Immunological memory takes a few days fro T cells to multiply 0 4 8 14 28 56 112 Days after infection Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 47  Differentiation to effector and memory cells naive effector memory Oxidative phosphorylation faster but less efficient way to generate energy Fatty acid oxidation glycolysis Oxidative phosphorylation Fatty acid oxidation T-bet Blimp-1 Eomes Bcl-6 IL-7Rα CCR7 CD62L :entering receptor needed for lymph nodes IL-15Rα CD44 Granzyme B IFN-γ IL-2Rα only detectable in highly activated T cells Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 48  Subsets of memory T cells CXCR3 CD103 Tissue-resident memory CD62L− CCR7− (TRM), CD8 in epidermis, CD4 in dermis CD62L− Effector memory CD62L+ (TEM) retain low expression of CCR7− CD62L and CCR7 Immediate effector function CCR7+ Limited proliferative capacity naive effector CD62L+ CCR7+ Better proliferative potential CD62L: L-Selectin Reside in lymph nodes CCR7: chemokine receptor Central memory (TCM) go back to lymph nodes and retain the memory Required for recirculation through lymph nodes from Blood via high endothelial venules (HEV) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 49  Detection of virus-specific CD8 T cells Tetramer-Färbung Intrazelluläre Zytokinfärbung CTL-assay APC Peptid-MHC Streptavidin Biotin + Fluoreszenz Stimulation Brefeldin A Fixierung Permeabilisierung Intrazelluläre Ak- 51Cr Freisetzung Färbung Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 50 The lymphocytic choriomeningitis virus (LCMV) Infection model in mice - 1933 in St. Louis isolated - Natural host: Rodents - Family: Arenaviruses - Serum prevalence humans: 5-10% - enveloped - Transmission: Organs, birth - segmented ssRNA Virus - Healthy adults: Flu-like symptoms - Non-cytopathic does not directly destroy cell but can multiply - Pregnant: Risk of misscarriage immunocompromised patients - also dangerous CD8 T cell epitopes in C57BL/6 mice (H-2b) GP33-41 (dominant epitope) GP276-286 (weak epitope) NP396-405 (dominant epitope) Lapova et al. (2013). Acta virologica Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 51 Kinetic of the CD8 T cell response against LCMV effector cytokine Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 52 Immunopathology caused by LCMV infection LCMV i.v. Newborn Tolerates infection tolerates infection and becomes and becomes carrier carrier LCMV i.v. Adult Eliminates virus eliminates virus withinwithin a few a few days days by CD8 by T cells CD8 T cells LCMV intracranial Adult Mouse dies mouse dies due due to CD8 to CD8-mediated - mediated immunopathology immunopathology Carrier Transfer of LCMV- specific CD8 T cells Mouse dies due to CD8-mediated Adult mouse dies due to CD8 - mediated immunopathology immunopathology Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 53  CD4 T cells (Helper T cells) Functions: CD40L CD40 Activation Proliferation Differentiation Cytokines Polarisation Plasma cells Ig-Class switch Rec. GERMINAL CENTER Somat. Hypermutation CD40L CD40 naive vactiv TCR ated MHC-II TNF ROI, RNI IFN-γ Antimicrob. Peptide TISSUE acidic pH phagolysosome CD40L CD40 Help for CD8 T cells activation 4-1BBL 4-1BB LYMPH NODE LN 54 Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU)  Th1/Th2 polarization the beginning of the diversification IFN-γ but there are many more: TNF (tumor necrosis factor) -Stimulates macrophages for elimination of intracellular stimulate naive cells in the presence Th1 pathogens. of IL-4/12 produces : IL-12 -Stimulates B cells for class switch recombination to IgG2a Naive IL-4 IFN-γ CD4 T cell IL-4 Th2 -Stimulates B cells for CSR to IgE and IgG1 -Increases the numbers of eosinophils, basophils and mast cells in tissues IL-4 IL-5 IL-13 IL-25 1980s Mosmann & Coffman Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 55  Germinal center Current diversification- not complete IL-21 Bcl6 Naive TFH TGF-β IL-12 IL-6 Differentiation IL-4 dampen/ suppress TGF-β other T cells - prevent Master Transcription overshooting factor T-bet Gata-3 RORγt Foxp3 Th1 Th2 Th17 iTreg Viruses Parasites Fungi Chronic infection Bacteria (Fungi) (Bacteria) Autoimmunity Fungi Allergies Autoimmunity Intracell. parasites Autoimmunity IFN-γ IL-4 IL-17A TGF-β Effector TNF IL-5 IL-17F IL-10 cytokines Lymphotoxin IL-13 IL-22 IL-35 IL-25 activate infected induce Function pro-inflamm. Suppressive macrophages eosinophilia function on other induce IgG2a in B induce IgE in B induce IgA in B T cells cells cells cells Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 56  Summary - T cells develop in the thymus by positive and negative selection - CD4 T cells see peptide Ags in MHC-II, CD8 T cells in MHC-I recombination - The TCR is generated by rearrangement of V-, D-, J- and C-segments minor population - The TCR consists of the alpha/beta chains (or gamma/delta) and the CD3 complex is required for signal transduction - Naive T cells need three signals to get activated otherwise - anergy - Signal 1: MHC-TCR - Signal 2: Co-stimulation by B7-CD28 - Signal 3: Cytokine receptors (mainly IL-2) - CD8 T cells are cytotoxic and kill cells with cytoplasmic pathogens or tumor cells. - CD4 T cells have helper functions for B cells, macrophages and CD8 T cells. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 57