1. Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

2. T CELL RECEPTOR SIGNALING Tímea Berki and Ferenc Boldizsár Signal transduction Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

3. TÁMOP-4.1.2-08/1/A-2009-0011 DN1 CD44 + CD25 - DN2 CD44 + CD25 + DN3 CD44 - CD25 + DN4 CD44 - CD25 - DP CD4 + CD8 + HSC CD4 - CD8 + SP CD4 + CD8 - SP Surface receptors Transcription factors HES-1, GATA-3 Sox4, HEB, NFATc Ikaros E2A, STAT5 TCF-1-Lef-1, NF-  B, p53 Frizzled receptor, Death receptor, pre-TCR Frizzled receptor, Death receptor, pre-TCR Notch- 1 c-Kit, IL-7R  -  c TCR  β CommitmentCommitment checkpoint checkpointPre-TCRcheckpointPre-TCRcheckpoint β -selection Role of transcription factors in thymocyte development

4. TÁMOP-4.1.2-08/1/A-2009-0011 CD4 + CD8 - CD4 + CD8 - CD8 + CD4 - Positive selection Lineage commitment Post-commitment TOX RUNX No TOX Apoptosis No GATA3 or MYB GATA3 MYB ThPOK GATA3 CD4 hi CD8 hi CD4 mid CD8 mid CD4 hi CD8 mid No ThPOK No RUNX Th - Tc cell decision

5. TÁMOP-4.1.2-08/1/A-2009-0011 CD43 CD44CD45 TCR-CD3-peptide-MHC CD28-CD80/CD86 CTLA4-CD80/CD86 Agrin PKC  CD4 LCK LFA1-ICAM1 Talin CD2-CD48/CD59 LCK CD44 CD45 CD4/CD8 CD43 CD28 CD80/CD86 CD2 CD48/CD59 LFA1 ICAM1 CTLA4 CD80/CD86 TCR Peptide-MHC CD3 APC T cell PI3K SHIP 2 ZAP70 Molecules of the „immunological synapse”

6. TÁMOP-4.1.2-08/1/A-2009-0011 Co-stimulatory pathways regulate the TcR signal TCR    CD28 Plasma membrane Calcium flux, NFAT activation Calcium flux, NFAT activation MAPK activation, AP1 formation MAPK activation, AP1 formation GRB2 SOS RAS GRP1 RAS GRP1 DAG GTPGTP NFAT IL-12 Nucleus ZAP70 AP1 CD3

7. TÁMOP-4.1.2-08/1/A-2009-0011 Binding of ZAP-70 kinase (Zeta-chain Associated Protein kinase 70 kDa) APC T cell TCR CD3  YY YY YY YY YY YY YYYYYYYY P P P P Peptide-MHCII CD4 ZAP-70 Lck TCR CD3   Y Y YY YY YY YY YY YYYYYYYY P P P P P Peptide-MHCII CD4 ZAP-70 Lck P P P P P P Downstream signals Gene transcription T cell activation Downstream signals Gene transcription T cell activation Activation through the T cell receptor (TcR) initiates various signaling pathways. The T and NK cell specific protein tyrosine- kinase ZAP-70 plays a crucial role in early TcR signalling. Phosphorylation on ZAP-70 leads to downstream signaling events, including phosphorylaton of the MAPK cascade and the increase of intracellular calcium level.

8. TÁMOP-4.1.2-08/1/A-2009-0011 Phosphorylation sites of the ZAP-70 kinase 0100200300400500600 NC Y69Y126Y178Y238 Y292 Y315/319Y474Y493 Y492 Y597/598 Function unclear Activating phosphorylation sites Inhibitory phosphorylation sites Y69Y315/319Y292 Y126Y474Y492 Y178Y493Y597/598 Y238

9. TÁMOP-4.1.2-08/1/A-2009-0011 The role of ZAP-70 ZAP-70 consists of two tandem SH2 domains, and a C-terminal kinase domain separated by interdomains A and B. It contains a total of 30 tyrosine (Tyr) residues, 11 of them have been identified as phosphorylation sites, some of them still with unknown function (Tyr069, Tyr126, Tyr178 and Tyr238). Interdomain B plays an important role in regulating ZAP-70 conformation. ZAP-70 is phosphorylated by p56Lck but autophosphorylation haa also been described on some of the Tyr residues. ZAP-70 is not only indispensible in T cell activation, but also an important prognostic marker in B-CLL. Moreover, in mice, spontaneous or induced ZAP-70 mutations lead to the development of autoimmunity.

10. TÁMOP-4.1.2-08/1/A-2009-0011 T cell activation pathwaysL R T E N/RE Apoptosis FADD Mort1 Fas TNF  R FasL TNF  Caspases 1-14 Activation Cytokine production CD28 CD80 CD86 PIP2Ras IP3 DAG Raf-1 MEK MAPK CD3  TCR  β CD4 Allopeptide Ras Raf-1 MEK MAPK LckFynZAP70 PIP2 PLC  1 CaM PKC  PKC β PKC  CaN IP3 DAG Ca 2+ NFATc, NF-  B, Jun/Fos, NFATn, c-Rel, I  B, Oct-1 Cellular proliferation IL-2R  IL-2R β IL-2R  IL-2 LykSyk FynSHC Raf-1 MEK MAPK STAT3 STAT5a STAT5b IP3 DAG Ras JAK1 JAK3 PIP2 Bcl-2, Bcl-x, Kip1, cyclinE/Cdk2

11. TÁMOP-4.1.2-08/1/A-2009-0011 2 signals are required for T-cell activation Signal 1: Signal 1: TCR-CD3 complex Antigen specific Signal 2: co-stimulation signal CD28 - B7 interaction NOT antigen specific Signal 1: Signal 1: TCR-CD3 complex Antigen specific Signal 2: co-stimulation signal CD28 - B7 interaction NOT antigen specific T-cell differentiation and proliferation T-cell differentiation and proliferation Effector and memory T-cells Effector and memory T-cells

12. TÁMOP-4.1.2-08/1/A-2009-0011 Initial steps of T cell activation 1 1ITAM: Immunoreceptor Tyrosine-based Activation Motif 2 2TCR crosslinking results inthe association of co-receptors (CD4, CD3, CD45) closer to TcR 3 3Protein tyrosine kinase (PTK) Lck and Fyn activation: CD45 phosphatase removes an inhibitory phosphate (Pi) + addition of a phosphate (Pa) 4 4Fyn and Lck phosphorylate ITAMs of CD3 complex 5 5Docking of ZAP-70 PTK to  -chain ITAMs and its phosphorylation

13. TÁMOP-4.1.2-08/1/A-2009-0011 Th differentiationNaive CD4 + T cell Treg IL-2 FOXP3 RUNX1 Th2 Th2 cytokine locus Th2 cytokine locus IL-12R β 2 GATA3 Th17 IL-17 ROR  t RUNX1 Th1 IFN  T-bet RUNX3 Th2 cytokine locus Th2 cytokine locus

14. TÁMOP-4.1.2-08/1/A-2009-0011 Anergy TCR    CD28 Calcium flux, NFAT activation Calcium flux, NFAT activation GRB2 SOS RAS GRP1 RAS GRP1 DAG GDP NFAT IL-12 Nucleus ZAP70 CD3 P DGKa Phosphatidic acid Phosphatidic acid

15. TÁMOP-4.1.2-08/1/A-2009-0011 Similarities in TcR and FceR signaling Lyn Lck   IgEITAM  Fc  RI Antigen-specific receptor Src-family kinase Syk-family kinase ZAP-70 expression is restricted to T cells, NK cells and a subpopulation of CLL Syk is present in most hematopoetic cell types TCR CD3   ITAM Syk

16. TÁMOP-4.1.2-08/1/A-2009-0011 Characteristics of Src and Syk family kinases Src kinases consist of eight non-receptor tyrosine kinases (Src, Fyn, Yes, Lck, Lyn, Hck, Fgr and Blk) that interact with the intracellular domains of growth factor/cytokine receptors, GPCRs and integrins. Members of the Src kinase family have a very similar domain structure. ZAP-70 is an abbreviation for Zeta-chain-associated protein kinase 70 (70 is the molecular weight in kDa). The protein is a member in the Syk protein- tyrosine kinase family. ZAP-70 is normally expressed in T cells and natural killer cells and has a critical role in the initiation of T-cell signaling. ZAP-70 in B cells is used as a prognostic marker in identifying different forms of chronic lymphocytic leukemia (CLL). ZAP70 deficiency results in a form of immune deficiency.

17. TÁMOP-4.1.2-08/1/A-2009-0011 Non-receptor tyrosin kinases Myr Fes/Fer Syk/Zap70 Jak Tec Fak Ack Src Csk Srm Rak/Frk Brk/Sik Abl SH3 binding region DNAActin Cdc-42 Y Myr PH Y Y Y Y Y Y Y Y Y Y Y Y Y Y SH2 PTK catalytic SH3 Integrin-binding/JEF domain Kinase-like domain

18. TÁMOP-4.1.2-08/1/A-2009-0011 Characteristics of Src family kinases Src family memberPattern of expressionIsoformsOncogenic forms BlkB cells FgrMyeloid cells, B cells Oncogenic fusion with gag sequences in feline sarcoma virus; overexpressed in some leukemias and lymphomas FynUbiquitousT-cell-specific isoform (Fyn T) HckMyeloid cellsTwo different translational starts LckT cells, NK cells, brain Overexpressed in T-cell acute lymphocytic leukemias Lyn Brain, B cells, myeloid cells Two alternatively spliced forms SrcUbiquitousNeuron-specific isoforms Mutated and truncated in retroviruses; truncated in colon cancer; overexpressed in mammary, pancreatic and other cancers YesUbiquitous Oncogenic fusion with gag sequences in avian sarcoma viruses; highly expressed in colon, malignant melanoma and other cancers YrkUbiquitous

19. TÁMOP-4.1.2-08/1/A-2009-0011 Overview of TcR/CD3 signaling pathway Erk PKC  Ras GRP Ras GRP PIP 3 lBlB lBlB NF-  B Calcineurin MKK3/6 p38 MKK4/7 JNK Akt NFAT Raf MEK1/2 CaM Ras lBβlBβ Fos Jun Rel NIK MEKKK Wasp Calpain Rac/ cdc42 Rac/ cdc42 MEKK Ca 2+ Proteasomal degradation Proteasomal degradation Proteasomal degradation Proteasomal degradation  β  Ca 2+ Carma1 Malt1 Bcl 10 TCRCD4CD45 PI3K CD28 Cytoplasm Nucleus Nuclear membrane IL-2 Gene CRAC Channel F-Actin Clustering Tali n LFA1 Actininin ER Intracellular Ca 2+ store Intracellular Ca 2+ store Zap70 Lck DAG PIP 2 IP 3