American Society of Hematology

Figure 5.

$Figure 5. Functional activity of dividing and nondividing hT cells after 4 days of culture. CD3-purified hT cells were initially stained with CFSE and then cultured in the presence of CD3 + IL-2 (▪); CD3/CD28 + IL-2 (▦); and CD3/CD28 (□). At day 4 of culture, dividing and nondividing cells were sorted in order to assay their functionality. (A) A typical CFSE analysis of day 4 cultured T cells with CD3 + IL-2, indicating how dividing (CFSElow) and nondividing (CFSEhigh) cells were sorted. (B) Proliferation of nondividing and dividing T-cell fractions in response to allogeneic EBV B-cell stimulation. Noncultured T cells (▨) are also shown. The cell proliferation was measured by 3H-Tdr incorporation at day 4 of culture. Results are presented as mean cpm ± SEM of triplicate wells. *Significant values as compared to those of nondividing cells (P < .01, unpaired Student t test). (C) Suppressive activity of CFSElow sorted T cells on the proliferation of autologous CFSEhigh T cells in response to allogeneic EBV B-cell stimulation. The cell proliferation was measured by 3H-Tdr incorporation at day 4. Results from triplicates were normalized for each culture condition to the positive control (ie, proliferation of CFSEhigh cells alone in the presence of allogeneic EBV B-cell stimulation is considered as 0% of the suppression activity). One representative of 2 independent experiments performed with cells from 2 different donors is shown. (D) Dose-dependent suppressive activity of CFSElow cells sorted from T cells cultured in the presence of CD3 + IL2 for 4 days. CFSElow sorted cells were mixed at various cell ratios with autologous nondividing (CFSEhigh) cells stimulated by 50 Gy-irradiated allogeneic EBV B cells. Cell proliferation was measured by 3H-Tdr incorporation 4 days later. Results are presented as mean cpm ± SEM of triplicate wells. (E) Role of the cell-cell contact on the suppressive activity of CFSElow sorted T cells on the proliferation of autologous CFSEhigh T cells in response to allogeneic EBV B-cell stimulation. MLR suppressive T-cell assays were performed as in panel C, cells being separated (+) or not separated (-) using transwell plates. One representative of 2 independent experiments performed in triplicate is shown.$

Functional activity of dividing and nondividing hT cells after 4 days of culture. CD3-purified hT cells were initially stained with CFSE and then cultured in the presence of CD3 + IL-2 (▪); CD3/CD28 + IL-2 (▦); and CD3/CD28 (□). At day 4 of culture, dividing and nondividing cells were sorted in order to assay their functionality. (A) A typical CFSE analysis of day 4 cultured T cells with CD3 + IL-2, indicating how dividing (CFSE^low) and nondividing (CFSE^high) cells were sorted. (B) Proliferation of nondividing and dividing T-cell fractions in response to allogeneic EBV B-cell stimulation. Noncultured T cells (▨) are also shown. The cell proliferation was measured by ³H-Tdr incorporation at day 4 of culture. Results are presented as mean cpm ± SEM of triplicate wells. ^*Significant values as compared to those of nondividing cells (P < .01, unpaired Student t test). (C) Suppressive activity of CFSE^low sorted T cells on the proliferation of autologous CFSE^high T cells in response to allogeneic EBV B-cell stimulation. The cell proliferation was measured by ³H-Tdr incorporation at day 4. Results from triplicates were normalized for each culture condition to the positive control (ie, proliferation of CFSE^high cells alone in the presence of allogeneic EBV B-cell stimulation is considered as 0% of the suppression activity). One representative of 2 independent experiments performed with cells from 2 different donors is shown. (D) Dose-dependent suppressive activity of CFSE^low cells sorted from T cells cultured in the presence of CD3 + IL2 for 4 days. CFSE^low sorted cells were mixed at various cell ratios with autologous nondividing (CFSE^high) cells stimulated by 50 Gy-irradiated allogeneic EBV B cells. Cell proliferation was measured by ³H-Tdr incorporation 4 days later. Results are presented as mean cpm ± SEM of triplicate wells. (E) Role of the cell-cell contact on the suppressive activity of CFSE^low sorted T cells on the proliferation of autologous CFSE^high T cells in response to allogeneic EBV B-cell stimulation. MLR suppressive T-cell assays were performed as in panel C, cells being separated (+) or not separated (-) using transwell plates. One representative of 2 independent experiments performed in triplicate is shown.

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