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Functions of Cd45 in Tcr Signaling in Cd4+Cds+ Double-Positive Thymocytes

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Functions of Cd45 in Tcr Signaling in Cd4+Cds+ Double-Positive Thymocytes FUNCTIONS OF CD45 IN TCR SIGNALING IN CD4+CDS+ DOUBLE-POSITIVE THYMOCYTES Gordon W. Cheng A thesis submitted in conformity with the requirements for the degree of Master's of Science Graduate Department of Immunology University of Toronto O Copyright by Gordon W. Cheng (1997) Acquisitions and Acquisitions et Bibliographie Services semices bibliographiques 395 Wellington Street 395, rue Wellington Onawa ON KIA ON4 Onawa ON K1A ON4 Canada Canada Your fib Volro re:erence Our Ida Norre iefeisnce The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de rnicrofiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. - a Thymocytes Master's of Science (1997) Gordon W. Cheng Graduate Department of Immunology University of Toronto Abstract T ceIl receptor (TCR) signals are essential for normal T cell developrnent, and CD45 is thought 10 be essential for coupling TCR to the intracellular signaling rnachinery. However, T cell development is only partially compromised in CD45-deficient mice. This thesis describes a CD45-deficient CD4+CD8+double-posi tive (DP) thymoma called 3T7. 1 show that in 3T7 ce1 ls and DP thymocytes CD45 is necessary for the TCR-induced protein tyrosine phosphorylation, as well as changes in CD5, MG-1 and CD41CD8 expression levels. However, CO-aggregation of TCR and either CD4 or CD8 induced signaling events in a CD45-independent rnanner, providing a ralionale for the developmental phenotype observed in CD45-dericient mice. Surprisingly, 1 found no disferences in the overall phosphorylation slatc of Lck or Fyn in CD45 deficient cells versus CD45-positive cells. However, 1 show that loss of CD45 is accornpaiiied by a hyperphosphorylation of TCRS in 3T7 cells and converscly a hypophosphorylation of TCRC in CD45-deficient thyrnocytes, providing onc possible biochemical mcchanism for ~hc TCR signaling dcficits dcscribed. Collectively, these observations providc a conccplual and esperimental framcwork for understanding the role of CD45 in TCR signaling at the DP slagc of' T ce11 dcvelopment. Acknowledgments 1 would likc to thank al1 those without whom this work would not have been possible. First and foremost, 1 thank my supervisor Cindy, for her guidance and support. For sharing thc reagents rvhich allowed this work to be done: Drs. Pauline Johnson for the CD45 construct; Michael Julius for the a-Lck antisera; Phi1 Branton for the a-CD45 antisera; Andrc Veillcttc for the a-Fyn antisera; and Josef Penninger for the CD45-/-mice. To my cornmiitcc, Drs. Michacl Julius and Rob Rottapel for their scientific input. Jayne Danska, for her big piclure and conceptual input. The Danskonians (Danny, Case, Priscilla, Chns ti ne, Ildico) for their group supporUtherapy. To my sister, Serena, for always listening & understanding - 1 thank you. To my parents as always, your patience, lovc and confidence in me have allowcd mc to complctc this, and mainlain somc semblance of sanity. And last and most importantly, 1 mus1 lhank my Iab mütcs pas1 and prcscnl (a spial thanks to Tim, you wcrc thcre from thc bcginning to thc end and whüt a joumcy its becn!!; to thc rest, Dianne, Patti, Trang, BJ and thc oihers who movcd on) for making thc daily grind bearable, and dare 1 say, enjoyabie. Paqe Abstract II . Acknowledgements III Table of Contents iv-v List of Abbreviations List of Figures and Tables Chagter 1) INTRODUCTION A) T Cell Development i) Overview ii) Positive Selection and Negative Seiection iii) Markers of Positive Selection B) T Cell Receptor (TCR) Signal Transduction Mechanisms i) TCR Stucture ii) Proximal/Distal Evcnts in Mature T ce1 1s iii) TCR Signal Transduction in DP Thymocytes C) Regulation and Function of Tyrosine Phosphatase, CD45 i) Background ii) CD45-Deficient Cell Lines iii) CD45 Substrates iv) CD45 in T Cell Dcvelopment D) Thcsis Objective Chapter 2) MATERIALS AND METHODS A) Ce11 lines and Cell Cullurc B) Retroviral Gene Transfer C) Antibodies and Second Stage Reagents D) Flow cytomelry E) Stimulation of cells for assessment of tyrosine phosphorylation F) Stimulation of cells for assessment of surface phenotypic changcs G) Immunoprccipitations H) Cc11 Surfacc Biotinylation 1) SDS-PAGE and Immunoblotting J) RNA preparation and Northern analysis K) cDNA synthesis and RT-PCR analysis Chaptes 3) RESULTS 3 1-59 A) Charictcnzation of a CD45-dcficienl thymoma ccll linc, 3T7 B) TCR signaling defcct in 3T7 celis II l2illy CVG111,4 - IIIUULLIUII U1 IYlUblilC: ~IIUh~IlUI~liLLII~Il ii) Latc cvcnts - phcnotypic maturation, RAG- 1 downmodulation Differential ability of CO-rcccptorsto ovcrcome TCR signaling defect Presence of other phosphatases in 3T7 cells Defect in CD45 gene espression in 3T7 cclls Re-espression of exogenous CD45 in 3T7 cells Rescue of TCR signaling defects by CD45 i) Early events - induction of tyrosine phosphorylation ii) iate events - phenotypic maturation, RAG- 1 downmodulation Biochemical basis for rescue of TCR signaling by CD45 Analysis of thymocytes from ~~45-I-mice Chapter 4) DISCUSSION A) CD45-Dependent versus CD45-Independent TCR Signaling Pathways B) Molecular Targets of CD45 in TCR Signal Transduction C) Role of CD45 in Positive Selection of DP Thymocytes D) Future Studies Chapter 5) REFERENCES BSA: Bovine >ci-iiriialhiin~iri cDN A: Coinplcniciirary cicox yrihoiliickic acid CTL: Cytotoxic T lymphocyic DAG: Diacylglyccnd DEPC: Dictiiyl pyiocaihoilatc DMF: Dinlcthyl Iormmidc DN: Doublc ncptivc dNTP: Dcoxyribonuclcosidc 5'-triphosphate DP: Doublc posirivc DR: Dilhiothrcilol ECL: Enhanccci chcmilumincscci~sc EDTA: Ethylcncdiaminc tctrüacictiç ad FACS: Fluorcsccncc aciivatcd cc11 sostcr FCS: Fctial cdl' scrum FITC: Fluorescein isothiocyanak GM: Growth nicdium HBS: HEPES bufkrcd saline MEPES: N-2-hydroxycthylpipcr~~inc-NI-2-clhancsulli~nicacid HRP: Horsc radish peroxidasc IP: immunoprccipilii~ion IPj: Inositol tri-phosphate ITAM: Iinm~inorcccptortyrosine-hascd activation motif LB: Lysis buflCr mAb: Monoclunal aiilibudy MAE: MOPS, sodiiim iicciatc, EDTA 2-ME: 7-1ncrcaptoctl1~111ol MFI: nican 17~ioscsccnccinlcnsily MI IC 1: Ma,ior Iiistocc,nipa~ibililyccii~~plcx class 1 MHC II: Maior histoconipatibilily complcx class II mRNA: Mcsscngcr RNA MOPS: 3-(N-niorpho1ino)-propanc sulfonic acid PBS: Phosphate bull'ci'cd salitic PCR: Polyincrasc chain iciictiori PE: Phycocrytliiin PMA: Phorbol 12-niyi-istritc 13-acclatc L '0. ' ",LU" 'J"'.""" """"7" PTf'iisc: Pi'0t~iiliyili~ili~ ~iho~~)hiilii~~ p-tyr: Phospho~yrosiiic K AG: Rcuvnhin:isc ircriv;itirig pc RNA: Kibonuclcic acid RT: Rcvcrsc lranscripli~w SDS-PAGE: Sodium dodccyl sullà~cpolyacrylamidc gcl clcctrophorcsis SHI : Src-homology 1 domain SH3: Src-honmlogy 7 domain SH3: Src-homology 3 domain SP: Singlc positive TCR: T-cc11 antigcn rcccptor Tyr: Tyrosinc V(D)J: Variahic, divcrsily, joining List of Figures Fur1 : Surlàcc phcnotype of 317 cclls. Figurc 2: ElTcct of CD45 dcficicncy on TCR-rilcdiatcd sisna1 ~ransduciionin 3T7 cclls. (A) Induclion of tyrosine phosphorylation in 377 cclls aller TCRP or TCRP + co- rcccpior crosslinking. (B) CD5 induction in 3T7 cclls alter TCRp or TCRP + co-rcccpior crosslinking. (C) CDS induclion in 3T7 cclls ~rcatcdwith thc PTK inliibilor. Hcrhimycin A. Figure 3: Molccular basis OC difl'crcntial signaling hctwccn CD4 and CD8 co-rcccptrirs in 3T7 cclls. (A) Difkrcntial association of Lck wi~tiCD4 and CD8 co-rcccp~orsin 3T7 cclls. (B) Ev;ilu;ilion of CD8 corcccpor isoforms cxpi-csscd in Yi7 cclls. Figure 4: El'lcci oc thc tyrosine phosphatase inhibitor, pcrvanadatc on 3T7 cclls. (A) Induction of lyrosinc phosphoryla~ionin 3l7 cclls aficr pcrvanadaic &calmeni. (B) Induction of CD5 in 3T7 cclls akcr pcrvanadalc trcatrncnt. Figurc 5: Analysis or CD45 gctic cxprcssion in 3T7 cclls.. (A) Northcrn analysis of CD45 mRNA expression in 3T7 cclls. (B) Surlàcc CD45 inducibly cxprcsscd in 3T7 cclls. (C) RT-PCR anülysis of CD45 isoforms inducibly cxprcsscd in 3T7 cclls. Figurc 6: Re-expression of cndogcnous CD45 coirclatcs with rcstorütion of TCR rcsponsivcncss. Figure 7: Expression of cxogcniius CD45 in 317 cclls by rctrovirnl-nicdialcd gcnc 1ransCcr. (A) Sclicrnatic rcprcscnlation of CD45 rclroviral construcl. (B) Wcstci-n blot anülysis of CD45 prorcin lcvcls in 3T7 inl'cçtan~s. (C) Surface CD45 expression in (3418-rcsis~ani3T7 infcctants. Figure 8: El'l'cct of exogciious CD45 cxprcssion on 'TCR-mcdiatcd signal trarisduc~ionin 3T7 cclls. (A) Exogcnously cxprcsscd CD45 rcstorcs TCR-mcdiatcd protcin Lyrosinc phosphorylaiion in 317 cdls. (B) Exogci~uuslycxprcsscd CD45 rcstorcs TCR-induced changes in cc11 surfacc phcnolypc. (C)Extigcnously cxprcsscd CD45 rcstores TCR-induccd RAG- 1 downmodulalion. Figurc 9: Tyrosinc phosphorylalion indcx of potcntial CD45 suhstraics in 3T7 celIs. (A) Aniilysis of TCR phosphorylation status. (B) AnaIysis oSLck phosphosphorylntion status. (C) Andysis of Fyn phosphorylation status. Figure 10: Anülysis of'TCR< tyrosinc phosphorylation
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