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Importance of the CD3 Ectodomain Terminal -Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly¹

Maki Touma^*,, Zhen-Yu J. Sun, Linda K. Clayton^*,, Wilfred E. Marissen^2,, Ada M. Kruisbeek^3,, Gerhard Wagner and Ellis L. Reinherz^4,*,

^* Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute and Departments of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

CD3 and CD3 are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal -strands that are linked to individual subunit membrane proximal stalk segments. CD3, CD3, and CD3 stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal -strand, we created a CD3 double mutant CD3_C82S/C85S and a CD3 -strand triple mutant CD3_{Q76S/Y78A/Y79A} for use in retroviral transduction of lymphoid progenitors for comparison with CD3wt. Although both mutant CD3 molecules reduced association with CD3 in CD3 heterodimers, CD3_{Q76S/Y78A/Y79A} abrogated surface TCR expression whereas CD3_C82S/C85S did not. Furthermore, CD3_C82S/C85S rescued thymic development in CD3^–/– fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3_C82S/C85S transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3^–/– thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3wt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3, as evidenced by the loss of reactivity with CD3 N terminus-specific antisera. Single histidine substitution of either CD3 stalk cysteine failed to restore CD3 association and conformation in transient COS-7 cell transfection studies. Thus, CD3_C82 and CD3_C85 residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3_C80 and CD3_C83. The implications of these results for CD3 and TCR structure and signaling function are discussed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI19807 and AI37581.

² Current address: Crucell Holland BV, Archimedesweg 4, Leiden, The Netherlands.

³ Current address: VUmc-Amsterdam, Technology Transfer Office, De Boelelaan 1085, Kamer, F546, 1081 HV Amsterdam, The Netherlands.

⁴ Address correspondence and reprint requests to Dr. Ellis L. Reinherz, Dana-Farber Cancer Institute, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: ellis_reinherz{at}dfci.harvard.edu

⁵ Abbreviations used in this paper: DP, double positive; SP, single positive; DN, double negative; FTOC, fetal thymic organ culture; BM, blocking media; HA, hemagglutinin; TM, transmembrane; MFI, mean fluorescence intensity.