High-frequency activation of single CD4+ and CD8+ T cells to proliferate and secrete cytokines using anti-receptor antibodies and IL-2(1).
A high cloning efficiency single-cell culture system was developed to define the activation requirements of isolated CD4+ and CD8+ T cells to proliferate and secrete cytokines. T cells were triggered using solid-phase anti-CD3 and anti-CD4 or anti-CD8 antibodies plus rIL-2. Activation was measured by microscopic scoring of proliferation and by measurement of cytokine production using the cytokine-responsive cell lines FDC-P1, which responds to GM-CSF, IL-3, IFN-gamma and IL-4, and 32D clone 3 which responds to IL-3 only. Whilst anti-CD3 plus rIL-2 triggered only 4% of peripheral T cells to proliferate, anti-CD3 plus anti-CD8 mAb triggered about 40% of CD8+ T cells; 80% of the resultant clones secreted cytokine and 90% of these were IL-3+. Anti-CD3 plus anti-CD4 mAb triggered proliferation in about 20% of CD4+ T cells, of which 34% formed cytokine-producing clones with 47% of these secreting IL-3. In addition to responding at higher frequency, CD8+ T cells formed larger clones which produced higher levels of cytokines than CD4+ cells. Cell separation on the basis of Pgp-1 expression suggested that this culture system did not select for previously activated cells. Whereas Pgp-1+ T cells from keyhole limpet haemocyanin (KLH)-primed mice were enriched in KLH-specific cells, no significant differences were observed in the clonogenicity or cytokine-secreting capacity of Pgp-1+ and Pgp-1- T cells from normal mice.
Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria, Australia.
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Maraskovsky
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Pech
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Kelso
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology / Q000502:physiology; D017252:CD3 Complex; D015704:CD4 Antigens / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D016827:CD8 Antigens; D016207:Cytokines / Q000378:metabolism*; D007376:Interleukin-2 / Q000494:pharmacology*; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000502:physiology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D016207:Cytokines; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell