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In Vivo Depletion of CD4‐ and CD8‐Specific T Cells

Ada M. Kruisbeek1

1Netherlands Cancer Institute, Amsterdam, The Netherlands, Netherlands

Publication Name: 
Current Protocols in Immunology
Unit Number: 
UNIT 4.1
DOI: 
10.1002/0471142735.im0401s01
Online Posting Date: 
May, 2001
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Abstract

In vivo depletion of CD4- and CD8-specific T cells is a means of studying the role of these subpopulations in the initiation and effector phases of particular in vivo immune responses. In this unit, a protocol is provided for harvesting anti-CD4 or anti-CD8 monoclonal antibody- producing ascites fluid or tissue culture supernatant from rat or mouse T cell hybridomas. The antibody (preferably IgG) is then purified and injected intraperitoneally into adult mice. Depletion of the appropriate subset of T cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on in vivo immune responses. The depleted condition is maintained by repeated injections of the monoclonal antibody.

     
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Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
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Materials

 Basic Protocol
 Materials
  • Ascites fluid or tissue culture supernatant containing anti-CD4 MAb from rat
  • GK1.5 hybridoma or anti-CD8 from 2.43 or 53-6.72 rat hybridomas (or tested alternative; see reagents and solutions)
  • Adult mice (>6 weeks old)
  • HBSS or PBS (appendix 2A)
  • Spectrophotometer
  • 4-ml round-bottom polystyrene tubes (Falcon #2052)
  • Additional reagents and equipment for purifying IgG MAb from ascites or supernatant (unit 2.7), intraperitoneal injection and euthanasia of mice (units 1.6 & 1.7), removal of organs (unit 1.9), preparing single-cell suspensions from spleen and lymph node (unit 3.1), enrichment of T cells (units 3.2 & 3.3), staining cells (unit 5.3), and flow cytometry analysis (unit 5.4)
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Literature Cited

Literature Cited
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    Bluestone, J.A., Cron, R.Q., Cotterman, M., Houlden, B.A., and Matis, L.A. 1988. Structure and specificity of T cell receptor on major histocompatibility complex antigen-specific CD3+CD4–CD8– T lymphocytes. J. Exp. Med. 168:1899-1916.
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