T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression.
CD28 is a homodimeric glycoprotein expressed on the surface of a major subset of human T cells that has recently been identified as a member of the immunoglobulin supergene family. The binding of monoclonal antibodies to the CD28 antigen on purified T cells does not result in proliferation; however, previous studies have shown that the combination of CD28 stimulation and protein kinase C activation by phorbol myristate acetate (PMA) results in T-cell proliferation that is independent of both accessory cells and activation of the T-cell receptor-CD3 complex. In the present study, effects of stimulation by anti-CD28 on cell cycle progression and on the interleukin 2 (IL-2) and IL-2 receptor system have been investigated on primary cultures of purified peripheral-blood CD28+ T cells. There was no measurable effect on cell size or on DNA synthesis after stimulation of resting (G0) cells by CD28 alone. After 3 h of activation of T cells by PMA alone, a slight (8%) increase in cell volume occurred that did not progress to DNA synthesis. In contrast, T-cell stimulation by CD28 in combination with PMA resulted in a progressive increase in cell volume in approximately 100% of cells at 12 to 14 h after stimulation. Northern blot (RNA blot) analysis revealed that CD28 stimulation alone failed to cause expression of the alpha chain of the IL-2 receptor or of IL-2 mRNA, and in accord with previous studies, stimulation by PMA alone resulted in the accumulation of IL-2 receptor transcripts but no detectable IL-2 mRNA. In contrast, T-cell stimulation by the combination of CD28 and PMA resulted in the appearance of IL-2 transcripts and enhanced expression of IL-2 receptor mRNA. Functional studies revealed that the proliferation induced by CD28 and PMA stimulation was entirely resistant to cyclosporine, in contrast to T-cell activation induced by the CD3-T-cell receptor complex. Cyclosporine was found not to affect the accumulation of IL-2 mRNA after CD28 plus PMA stimulation, although there was no detectable IL-2 mRNA after stimulation by CD3 in the presence of the drug. Furthermore, stimulation by CD28 in combination with immobilized CD3 antibodies caused a striking enhancement of IL-2 mRNA expression that was, in part, resistant to the effects of cyclosporine. These studies indicate that the CD28 molecule synergizes with protein kinase C activation to induce IL-2 gene expression and demonstrate that stimulation by the CD28 pathway can cause vigorous T-cell proliferation even in the presence of cyclosporine and that cyclosporine does not prevent transcription of 16-2 mRNA, as has been suggested previously. Moreover, these findings suggest that a potential role for the CD28 molecule in vivo may be to augment IL-2 production after stimulation of the CD3-T-cell receptor molecular complex and thereby to amplify an antigen-specific immune response. Finally, these results provide further evidence that the CD28 molecule triggers T-cell proliferation in a manner that differs biochemically from CD3-T-cell receptor-induced proliferation.
June
C H
CH
Naval Medical Research Institute, Bethesda, Maryland 20814.
Ledbetter
J A
JA
Gillespie
M M
MM
Lindsten
T
T
Thompson
C B
CB
D000328:Adult; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003524:Cyclosporins / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D008213:Lymphocyte Activation / Q000187:drug effects; D009693:Nucleic Acid Hybridization; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface; D003524:Cyclosporins; D007376:Interleukin-2; D012333:RNA, Messenger; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; D004247:DNA; D003513:Cycloheximide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate