Inhibition of CD28/CD3-mediated costimulation of naive and memory human T lymphocytes by intracellular incorporation of polyclonal antibodies specific for the activator protein-1 transcriptional complex.
A number of indirect methods have been utilized in demonstrating activator protein-1 transcription factor function in IL-2 promoter activity. However, there has been no direct demonstration that activator protein-1 is involved in CD28-dependent costimulation of IL-2 gene transcription in freshly isolated naive and memory human T lymphocytes. To address this issue, the method of scrape loading was applied to purified peripheral blood T lymphocytes. Since scrape loading relies on adherent cells, peripheral blood human T (PB-T) cells were immobilized on the nonspecific cell attachment factor poly-L-lysine. Cells scraped off poly-L-lysine in the presence of Ig FITC efficiently incorporated Ig, with relatively uniform fluorescence. T cells retained their physical parameters as measured by forward and side light scatter, and functional activity as measured by costimulation of proliferation and IL-2 production after being scraped off this substrate. CD28/CD3-costimulated T cells produced intracellular IL-2 from all subsets measured (CD4+, CD4-, CD45RO+, and CD45RO-). IL-2 production and intracellular accumulation in nonscraped PB-T cells activated with CD28/CD3 coligation were skewed favoring CD45RO+ and CD4+ subsets, as was IL-2 production in scraped PB-T cells. The intracellular incorporation of Abs specific for c-Fos and c-Jun family members by scrape loading inhibited the production and intracellular accumulation of IL-2 within 6 h of costimulation with PMA/ionomycin, or costimulation by CD28 and CD3 ligation. Scrape loading thus provides an efficient mechanism for intracellular incorporation of macromolecules, and the first direct evidence that c-Fos and c-Jun are involved in transcription of the IL-2 gene within its correct chromosomal context, in resting human T lymphocyte subpopulations.
Woodside
D G
DG
Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
McIntyre
B W
BW
D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D018106:CD28 Antigens / Q000187:drug effects / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D002448:Cell Adhesion / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D016572:Cyclosporine / Q000494:pharmacology; D006801:Humans; D007156:Immunologic Memory; D007166:Immunosuppressive Agents / Q000378:metabolism*; D007376:Interleukin-2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007399:Interphase / Q000276:immunology; D007424:Intracellular Fluid / Q000276:immunology* / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D016760:Proto-Oncogene Proteins c-fos / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000276:immunology* / Q000502:physiology; D014158:Transcription, Genetic / Q000276:immunology
D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D015759:Ionomycin; D016572:Cyclosporine; D013755:Tetradecanoylphorbol Acetate