T Cell Receptors

1. T Cell Receptors By: Alexander Ko, David Lam, Caddie Sze and Vicky Yee Date: November 4th, 2013 PHM142 Fall 2013 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson

2. What is T Cell Receptor • T Cell Receptor are receptors found on T Cells (also called T Lymphocytes) • They bind to and recognize antigens that are bound to a MHC molecules (major histocompatibility complex molecules) • Source: • RusselP, Wolfe S, Hertz P, et al. Biology: Exploring the Diversity of Life1st Edition. Toronto: Nelson Education Ltd, 2010. Print.

3. Generation of T-Cell Receptors (TCRs)

4. Generation of T-Cell Receptors • Occurs in the thymus • By mechanism of somatic recombination • Most TCRs contain an α-chain and a β-chain • Variability arises due to the unique gene sequences in the α-chain and β-chain and the diversity of the junctions between gene sequences

5. Somatic Recombination • The mechanism of genetic recombination in the early stages of TCR generation • The same mechanism for immunoglobulin (Ig) generation • Combines 3 types of gene segments: • V – Variable • D – Diverse • J – Joining

6. Major players in Somatic Recombination • Recombinant Signal Sequences (RSSs)– Nucleotide sequence which attaches to the ends of the gene sequences used for DNA cleavage • VDJ Recombinase – Group of enzymes that cleave DNA at the RSSs • Terminal deoxynucleotidylTransferase(TdT) – An enzyme that randomly joins gene sequences

7. Mechanism of Recombination • The V, D, & J gene sequences are flanked by Recombinant Signal Sequences (RSSs) • Then they are recognized by a group of enzymes known as VDJ recombinase which cleaves the DNA at the RSSs • Finally terminal deoxynucleotidyltransferase (TdT) randomly adds nucleotides to the ends

8. Structure of T Cell Receptors

9. Structure of the T Cell Receptor • An α and β protein chains • carbohydrate side chains • A constant region and variable region • Disulfides bonds that connect the α and β • Trans membrane domain on both the α and β chain • A small cytoplasmic tail • Figure 3.12 – Structure of the T Cell Receptor • Source: • http://www.ncbi.nlm.nih.gov/books/NBK27145/

10. Antigen Binding Site • Only 1 antigen binding site • T Cell Receptor combines with a CD3 complex • complex acts as a signal transduction mechanism Figure 3.13The crystal structure of an α:β T-cell receptor resolved at 2.5 Å Source: . http://www.ncbi.nlm.nih.gov/books/NBK27145/

11. T-Cell Receptor Signaling

12. Recall from undergrad: • Kinase (i.e. Lck , Fyn, and Zap 70)– molecules that adds a phosphate • Phophatase(i.e. CD45) – molecules that remove phosphates • MHC 2 is an antigen presenting cell

13. T-Cell Receptor Signaling • The process is low affinity binding and degenerate • Not very selective, but at the same time, signal does not persist for a prolonged period of time What would happen if pathogens bound at relatively high affinities?

14. Therefore, • Many T-Cell Receptors will recognize the same antigen complex • Different types of antigens can bind to the same T-Cell Receptor • Produce a large immune response when needed

15. General Signaling Pathway

16. Step 1 • MHC II recognizes that a pathogen is foreign to the body • Generates a complex with the foreign material (think of this as now substrate) Source: Morrison, L.A., Lukacher, A.E., Braciale, V.L., Fan, D.P. and Braciale, T.J. (1986) Differences in antigen presentation to MHC class I- and MHC Class II-restricted influenza virus-specific cytolytic T-lymphocyte clones. Journal of Experimental Medicine. 163: 903

17. Step 2 • T-Cell Receptor will recognize MHC II molecule with its bound antigen Source: "Major Histocompatibility Complex." Protein Data Bank. N.p., Feb. 2005. Web. 15 Oct. 2013.

18. Step 3Activation of Kinases • Intracellular Src family tyrosine kinases (Fyn) will be activated by CD45 • CD45 has a cytosolic tyrosine phosphatase to remove phosphates

19. Step 4Phosphorylation of ITAMS • Activated Fyn kinase phosphorylates ITAMS on the cytosolic side of the T-Cell Receptor • Phosphorylated ITAMS are a binding site for a secondary tyrosine kinase, Zap 70

20. Step 5Zap 70 Binding • Zap 70 binds to the phosphorylated ITAMs • Further amplifies the signal • Requires phosphorylation to be activated

21. Step 6T-Cell Activation • Leads to activation of multiple pathways • Induces effector functions • Ultimately leads to an immune response in the body against the foreign substance present

22. T Cell Receptor: Deficiency and Dysfunction

23. T-Cell Receptor (TCR)Deficiency & Dysfunction • Defects that occur during various stages of T-cell maturation can result in deficiencies and dysfunctions of TCRs and its associated molecules • Consequences of TCR deficiency and dysfunction varies from increased susceptibility of infections to autoimmunity and severe combined immunodeficiency (SCID) Source 1: http://harunyahya.com/en/Books/989/the-miracle-of-the-immune/chapter/3768 Source 2: http://arthritis-research.com/content/11/1/202/figure/f1?highres=y

24. Deficiencies of TCR subunits • Mutation of the T-cell receptor alpha constant (TRAC) gene • Defective TCRα chain • ↓ expression of TCRαβ T-cells • ↑ expression of TCRγδ T-cells • Causes autoimmunity and ↑ susceptibility to infections Defective VDJ recombination of TCR • Occurs between the pro-T cell to pre-T cell stage • Mutations in the genes encoding for RAG1 and RAG2 enzymes • Absence of T cells or decreased TCR functionality • Can result in either SCID or Omenn Syndrome Source: http://www.jle.com/e-docs/00/04/2C/57/article.phtml?fichier=images.htm

25. Defective VDJ recombination of TCR • Defects in the genes encoding for Artemis, DNA ligase IV, and DNA-protein kinase catalytic subunit (DNA-PKC) involved in DNA repair • Causes radiation sensitive SCID (RS-SCID) • ↑risk of DNA damage and developing cancer Defective binding of TCR & MHC • MHC Class I deficiency • Mutation of TAP1 or TAP2 genes • Absence of surface MHC class I molecules • Lack of TCRαβ and ↑ susceptibility to infections • MHC Class II deficiency • Mutation of CIITA, RFXANK, RFX5 or RFXAP genes • Lack of expression of MHC class II molecules • Severe immunodeficiency

26. Defective TCR Signalling • Mutations of genes encoding for CD3 subunits and CD45 • Defective CD3 complex/CD45 essential for TCR signalling • Causes SCID • Defective ZAP-70 protein kinase • Absence of CD8 and CD4 T-cells & dysfunctional TCR signalling cascade 1 Adapted from “Janeway’sImmunobiology 8th edition,” by K. Murphy, 2012, Garland Science p. 522

27. Severe Combined Immunodeficiency (SCID) • Absence or trivial numbers of functional B-Cells and T-cells • Infections are severe and sometimes life-threatening • Several forms of SCID • i.e. X-linked SCID, “Bubble boy disease” • Bone marrow transplant is the most common treatment Source: http://www.allmovie.com/movie/bubble-boy-v250493

28. T Cell Receptors Summary • T Cell Receptors bind to and recognize antigens bound to a MHC molecules • Generation of T Cell Receptors: • T-cell Receptors are generated in the thymus by method of somatic recombination • Due to the seemingly random recombination, there is lots of variability in cell surface receptors • T Cell Receptor Structure: • made out of an α and β chain (protein chains) and each chain has a constant and variable region • Receptor only has 1 antigen binding site • T Cell Receptor combines with a CD3 complex and the complex acts as a signal transduction mechanism • T Cell Receptor Signaling (General Pathway): • Step 1: Recognition - MHCII recognizes foreign entity in body and generates a complex. T-Cell Receptor will recognize MHCII-complex  • Step 2: Activation of Kinases - Src family tyrosine kinases (Fyn) will be activated by cd45, a phosphatase that removes inhibitory phosphates • Step 3: Phosphorylation of ITAMS - Activated Fyn kinase phosphorylates ITAMS on cytosolic side of TCR, creating binding sites for Zap70 a secondary tyrosine kinase • Step 4: Zap 70 Binding - Binds to phosphorylated ITAMS to further amplify signal (Zap 70 is activated by Lck kinase) to initiate a variety of intracellular responses.  • Step 5: Further signalling pathways in cell are then activated to induce appropriate immunological responses towards antigen. • T Cell Receptor – Deficiency and Dysfunction: • Defects of genes encoding for TCR subunits and its associated molecules can lead to deficient and dysfunctional t-cell receptors • Consequences of such defects range from increased risk of infection to autoimmunity and severe combined immunodeficiency

29. References • Ballas, Zuhair K. “CD3/T Cell Receptor Complex Disorders Causing Immunodeficiency.” UpToDate. 10 Dec. 2013. Web. 28 Oct. 2013. • Clark, Mike. "T-Cell Receptor (TCR)." University of Cambridge. Department of Immunology, n.d. Web. 20 Oct 2013. • Huse, Morgan. "The T-Cell Receptor Signalling Network." Cell Science at Glance(2009): n. pag. Web. • JanewayCA Jr, Travers P, Walport M, et al. Immunology: The Immune System in Health and Disease 5th Edition. New York: Gardland Science, 2001. NCBI. Web. 28 October 2013. <http://www.ncbi.nlm.nih.gov/books/NBK27145/>. • Murphy, Kenneth. Janeway’sImmunobiology 8th Edition. New York: Garland Science, 2012. Print. • RusselP, Wolfe S, Hertz P, et al. Biology: Exploring the Diversity of Life1st Edition. Toronto: Nelson Education Ltd, 2010. Print. • Severe Combined Immunodeficiency. The SCID Homepage.Calao Designs, 2013. Web. 28 Oct. 2013. <http://www.scid.net/>. • T-Cell Receptor (TCR) Overview." T Cell Receptor (TCR) Overview. N.p., n.d. Web. 20 Oct 2013.