1. Background on Zap70 SCID Slides from Arthur Weiss GEMS Journal Club August 2012

2. Case Presentation A 1 year old Hispanic female was referred to UCSF for the possibility of a bone marrow transplantation. She had a history of: recurrent pneumonias chronic gastroenteritis failure to thrive oral/cutaneous candidiasis history of disseminated varicella Her parents were first cousins. One of her three sibs (brother) died at age 9 months. Physical Exam was unremarkable

3. Laboratory Evaluation B cell number: Normal Immunoglobulin levels: Decreased IgM, IgG and IgA Abnormal lymphocyte phenotype: CD3 76%; CD4 74%; CD8 0-2% T cells are polyclonal In vitro proliferation studies: B cells proliferate normally to mitogens CD4 T cells: Fail to respond to PHA or anti-CD3 mAb Positive proliferative response to IL-2 Positive proliferative response to PMA plus ionomycin

4. Severe Combined Immunodeficiency Syndrome SCID Primary Defect - Absent T cell function - Variable T cell counts (absent to elevated) Associated Defects - Decreased B cell responses (intrinsic or extrinsic) - Variable B cell counts (often normal)

5. Multiple Molecular Causes of SCID X-linked Common  chain (IL-2, IL-4, IL-7, IL-9, and IL-15 receptors) - 50% Jak 3 tyrosine kinase IL-7 receptor alpha chain Adenosine Deaminase (ADA) Purine Nucleoside Phosphorylase (PNP) Recombinase Activating Genes (Rag 1 and Rag 2) CD3  and  CD45 tyrosine phosphatase Bare lymphocyte syndrome (absent MHC molecules) ZAP-70 tyrosine kinase

6. Causes of severe T cell deficiency Liston, Enders, Siggs NRI, 2008

7. Unusual Phenotype of ZAP-70 Deficient SCID: Absence of Peripheral CD8 T Cells Chan, et al., Science, 1994

8. TCR Stimulation Patient Normal Western Blot anti- phosphotyrosine ZAP-70 Deficient CD4 T Cells Fail to Signal in Response to TCR Stimulation Chan, et al., Science, 1994

9. ZAP-70 Mutations Accounting for SCID

10. What is the Function of ZAP-70?

11. TCR Signaling is Initiated by Coordinated Actions of Two Cytoplasmic Tyrosine Kinases: Lck and ZAP-70 Wang, et al, CSHL, 2009

12. How Does the Absence of ZAP-70 Affect T cell Development ?

13. CD4 SP DP CD8 SP DN CD8 CD4 A Brief Introduction to T Cell Development  Pre-TCR Positive & selection Negative Selection T Cell Developmental Checkpoints

14. CD4 CD8 Thymocytes Lymph node cells Complete T cell Developmental Arrest in ZAP-70 Deficient Mice Normal ZAP-70 KO

15. Decreased Function of ZAP-70 Can Lead to Autoimmunity

16. Spontaneous Development of Erosive Arthritis in SKG mice Incidental finding in Balb/c mouse colony 100% penetrant, males and females affected Onset at 2 months (joint swelling and hyperemia) or following activation of the innate immune response Interphalangeal joints -> symmetrical involvement including wrist and ankle joints. Spares large joints and spine (except base of tail) Destruction of joint space. RF+, mediated by CD4 T cells Sakaguchi, et al, Nature, 2003

17. Hypomorphic Mutant Allele of ZAP-70 with Decreased Catalytic Activity Associated with ANA Positive Autoimmunity in Mice Siggs, et al., Immunity, 2007

18. Thymocyte selection is dependent on signaling through the TCR Signal strength = Affinity X TCR signaling intensity Signal strength

19. Thymocyte selection is dependent on signaling through the TCR Signal strength = Affinity X TCR Signaling Intensity Signal strength Decreasing TCR Signaling by ZAP-70 mutation alters the TCR repertoire of thymocytes surviving both positive and negative selection

20. Asymmetric effects on immunogenic and tolerogenic TCR signaling may account for autoimmunity with partial immunodeficiency Liston, Enders, Siggs NRI, 2008