T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells

Karen Hathcock1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.3
DOI:  10.1002/0471142735.im0303s28
Online Posting Date:  May, 2001
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T cells from mouse spleen and lymph node do not express the cell‐surface glycoproteins encoded for by MHC class II genes, whereas most non‐T cells in these organs do (i.e., B cells and accessory cells). This unique feature of T cells makes it possible to enrich for them using cytotoxic anti‐class II monoclonal antibodies and activating complement. Because there are two isotypes of MHC class II genes (A and E genes, located adjacent to each other in the MHC region originally called the I region), it is necessary to identify which of these two genes are expressed in the mouse strain used. This unit lists suitable antibody‐producing hybridomas available from ATCC, with antigen specificities (I‐A vs. I‐E) and corresponding mouse strains (H‐2 strains a, b, k, etc., indicated as superscripts). Other antibodies can be tested in pilot experiments using the procedure outlined. This unit describes T cell enrichment using cytotoxic antibodies, and UNIT describes the depletion of T cells and their subpopulations using the same approach. In the latter unit, T cell surface markers (Thy‐1, CD4, and CD8) are targeted by the cytotoxic antibodies.

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Table of Contents

  • Basic Protocol 1: Elimination of Non–T Cells Using Anti–MHC Class II Monoclonal Antibodies
  • Reagents and Solutions
  • Commentary
  • Tables
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Basic Protocol 1: Elimination of Non–T Cells Using Anti–MHC Class II Monoclonal Antibodies

  • Mouse spleen and lymph node
  • Complement‐fixing, anti‐I‐A/E MAb (or antibody cocktail; units 2.5 & 2.6) of known specificity (e.g., see Table 3.3.1) and cytotoxic titer
  • Complete RPMI‐5 medium ( appendix 2A)
  • recipeRabbit serum complement (stored in aliquots at −70°C and diluted just before use)
  • 15‐ml polypropylene tubes (e.g., Falcon #2059)
  • Sorvall H‐1000B rotor (or equivalent)
  • Additional reagents and equipment for preparing single‐cell suspensions and removing red blood cells (unit 3.1), counting viable cells ( appendix 3A), and determining cell composition by flow cytometry (unit 5.4)
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Literature Cited

Literature Cited
   Janeway, C.A., Conrad, P.J., Lerner, J., Babick, J., Wettstein, P., and Murphy, D.B. 1984. Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells. Possible role of T cell–bound Ia antigens as target of immunoregulatory cells. J. Immunol. 132:662‐669.
   Kappler, J.W., Skidmore, B., White, J., and Marrack, P. 1981. Antigen‐inducible, H‐2‐restricted, interleukin‐2‐producing T cell hybridomas. J. Exp. Med. 153:1198‐1214.
   Oi, V.T., Jones, P.P., Goding, J.W., and Herzenberg, L.A. 1978. Properties of monoclonal to mouse Ig allotypes, H‐2, and Ia antigens. Curr. Topics Microbiol. Immunol. 81:115‐129.
   Ozato, K. and Sachs, D.H. 1981. Monoclonal antibodies to mouse MHC antigens. III. Hybridoma antibodies reacting to antigens of the H‐2 haplotype reveal genetic control of isotype expression. J. Immunol. 124:533‐540.
   Ozato, K., Hansen, T.H., and Sachs, D.H. 1980a. Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H‐2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex. J. Immunol. 125:2473‐2477.
   Ozato, K., Mayer, N., and Sachs, D.H. 1980b. Hybridoma cell lines secreting monoclonal antibodies to mouse H‐2 and Ia antigens. J. Immunol. 124:533‐540.
   Sharrow, S.O., Mathieson, B.J., and Singer, A. 1981. Cell surface appearance of unexpected host MHC determinants on thymocytes from radiation bone marrow chimeras. J. Immunol. 126:1327‐1333.
Key References
   Hansen, T.H. and Sachs, D.H. 1989. The major histocompatibility complex.. In Fundamental Immunology, 2nd ed. (W.E. Paul, ed.) pp. 445‐487. Raven Press, New York.
  Excellent coverage of basic immunogenetic principles required for understanding the MHC complex, with emphasis on the mouse.
   Klein, J. 1986. Natural History of the Major Histocompatibility Complex.. John Wiley & Sons, New York.
  A broad overview of the structure and function of antigens encoded for by the MHC complex.
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