Since adequate expression of costimulatory adhesion molecules is essential for proper T cell responses, be it cell survival, activation or proliferation, we decided to analyze the expression levels of various cytokine receptors, cell adhesion molecules, and cell surface receptors in a cell population treated with fairly TSA. Splenocytes from C57BL 6 mice were stimulated with sol uble anti CD3 antibody or IL 2 and exposed to 100 nM TSA. Samples were taken after 4 and 20 hours, and expression of cell surface markers examined by flow cytometry. CD4 T cells stimulated with IL 2 and exposed to TSA for 4 hours, exhibited lower levels of expression of CD11c, CD69, CD28, CD40, and CD40L, and higher levels of expression of IL2R with all others remaining constant.

After 20 hours of exposure IL2R, ICAM 1, CD8a, CD11c, CD45RB, CD69, CD28, CD40L, CD80, CD86 and CD95 were downregulated to various extents, but none of the examined molecules was upregulated. In non CD4 cells stimulated with IL 2 and exposed to TSA for 4 hours, the pattern of effects was very similar. Thus, expression of CD11c, CD69, CD28, CD40, and CD40L was downregu lated, whereas expression of IL2R was increased. After 20 hours of exposure, IL2R, IL2R, LFA 1, ICAM 1, CD8a, CD11b, CD11c, CD45RB, CD69, CD28, CD40, CD40L, CD48, CD86, and CD95 were downregulated to various extents, but of the examined molecules only CD80 was upregulated. Stimulation of PBMCs with anti CD3 led to profound alterations in the profile of TSA mediated cell surface molecule expression.

CD4 T cells exposed to TSA for 4 hours, exhibited higher levels of expression of CD11c and ICAM 1 with all others remaining constant. After 20 hours of exposure, IL2R, IL2R, LFA 1, CD45RB, CD28, CD40, CD40L, and CD48 were down regulated, whereas ICAM 1 and CD69 were upregulated. In non CD4 cells treated with TSA for 4 hours levels of expression of CD11c were increased. After 20 hours of exposure, IL2R, IL2R, LFA 1, ICAM 1, CD8a, CD11c, CD28, CD40L, and CD95 were downregulated, and CD11b, CD40, and CD86 were upregulated. These results demonstrate that HDAC inhibitors may modulate T cell function in part through changes in the expression of cell surface proteins. The differences in expression of cell surface molecules, such as CD95 Fas, in PBMCs in the two cell subpopula tions we examined suggested to us that the various cell types might be affected to different extents by TSA.

To clarify this point, we cultured PBMCs in the presence of either soluble anti CD3 antibody or IL 2 and treated with TSA or DMSO for 20 hours. Subsequently, cells were analyzed by flow cytometry to determine cell viabil ity and the ratio of CD4 T cells versus non CD4 cells. Plotting of the percentage of total viable cells in the pop ulation shows that TSA induces apoptosis in PBMCs Brefeldin_A to a similar extent in anti CD3 or IL 2 stimulated cells.