the rates of histone removal and assembly are altered in the

ChA6 mAb induces apoptosis in A6 bright CD4 T cells To determine whether the inhibition of proliferation was caused 3-Deazaneplanocin A by depletion of responder T cells, the ability of chA6 mAb to induce T cell apoptosis was investigated. Overnight,incubation of CD4 T cells with chA6 mAb in the presence or absence of anti CD3 and anti CD28 mAb resulted in in creased percentages of early apoptotic cells. The percentages of annexin VPI cells were signifi cantly higher in cultures incubated with 10 gml or 1 g ml of chA6 mAb than in cultures incubated with an iso type control mAb, The mean value of ED50 for the induction of apoptosis was 20. 6 8. 7 gml, Apoptosis induced by chA6 mAb was not signifi cantly enhanced when CD4 T cells were activated with anti CD3 and anti CD28 mAbs, Double staining of CD4 T cells with annexin V Organism FITC and A6 PE mAb re vealed that apoptosis was induced mainly in CD4 A6bright cells, which represent the CD45 RORB bright cells, These results indicate that chA6 mAb induces apoptosis in CD4 T cells in a dose dependent manner, which does not require T cell activation, and selec tively depletes CD4 CD45 RORB bright T cells, which rep resent the CD4 effector memory T cell population. Cross linking of CD45RO or CD45RA isoforms by specific mAb did not induce apoptosis on human CD4 T cells, indicating the specific effect of the cross link of CD45 RORB isoform by chimeric A6 mAb. ChA6 mAb failed to induce apoptosis of CD8 T cells and of non T cells at concentrations up to 10 gml, indicating a specific effect on CD4 T cells, To verify whether the apoptosis mediated by chA6 mAb was targeting preexisting CD4 A6bright responding T cells, we examined the effect of chA6 mAb on cells preincubated with chA6 mAb and depleted of annexin V cells. As ex pected, depletion of annexin V cells resulted in a reduced percentage of CD4 A6bright T cells, whereas the proportion of CD4 A6low T GSK923295 cells increased, Annexin V depleted CD4 T cells reexpressed the A6 epitope on the cell surface and subsequently became suscepti ble to apoptosis induced by chA6 mAb, Together, these data show that ligation of CD45 RBRO isoforms by chA6 mAb leads to the death of preexisting and de novo induced CD4 A6bright memory T cells. The obser vation that chA6 mAb inhibited primary allogeneic prolifer ative responses of freshly isolated CD4 T cells and annexin V,depleted CD4 T cells in a comparable fashion indicates that the immunosuppressive effect of chA6 mAb is caused by the induction of apoptosis of preexisting CD4 A6 bright T cells and of newly activated effector cells, which expressed the A6 epitope at high levels. ChA6 mAb induces apoptosis through the intrinsic pathway We investigated the mechanism involved in the apoptosis induced by chA6 mAb by analyzing the expression and acti vation of several caspases, including caspase 3, one of the key molecules involved in apoptosis.