Figure 3.
CD8+ T cells initially increase efflux capacity in antigen-stimulated conditions or when cultured alone in vitro and then lose efflux capacity as they proliferate. (A-F) Human T cells were stimulated in MLC as in Figure 1, stimulated with anti-CD3/CD28 beads, or cultured without allogeneic stimulators or beads. At 3 or 7 days of in vitro culture, Rh-123 effluxing was assessed as per Figure 2. The “+CsA” designation refers to culturing with CsA only during the effluxing to serve as a noneffluxing control, but these cells were not cultured with CsA during the 3 or 7 days of in vitro culture. In parts (A-E), T cells were labeled with CellTrace Violet before in vitro culture. Shown are combined results from 3 independent experiments for parts (A-B,D) and 2 independent experiments for parts (C,E). (A) The efflux capacity of CD8+ T cells in MLC was higher than with anti-CD3/CD28 bead stimulation. **P = .0075 on paired t test. (B) However, proliferation on day 3 was much less extensive after MLC, with negligible numbers of CD8+ T cells in MLC proliferating >1 generation. Example plots of proliferation as measured by CellTrace dilution are shown for T cells from a healthy donor treated either in MLC or with anti-CD3/CD28 beads. (C-E) On both day 3 (D) and day 7 (C,E) of in vitro culture, efflux capacity was increased in nonproliferating cells or those that had only divided a single generation regardless of MLC or anti-CD3/CD28 stimulation but decreased in CD8+ T cells that had proliferated more extensively. This divergence may explain the overall decreased effluxing seen in (A) after bead stimulation because of much more extensive proliferation with that treatment. On day 3 of MLC, effluxing was considered nonevaluable (NE) in the cells proliferating more than a single generation because of extremely low numbers of cells in that group. Representative flow cytometric plots are shown in (C). *P < .05, **P < .01, and ****P < .0001 on nonrepeated-measure one-way ANOVA followed by the Holm-Sidak post hoc test. (F) CD8+ T cells cultured with media alone increased effluxing capacity similarly compared with CD8+ T cells stimulated in MLC, and this was not augmented with treatment with common γ-chain cytokines. The combined results of 2 independent experiments are shown. *P < .05, **P < .01, and ns, not significantly different on repeated-measure one-way ANOVA followed by the Holm-Sidak post hoc test. (G) Human CD8+ T cells retrieved from the blood of patients (n = 5) on day 3 after allogeneic hematopoietic cell transplantation also had increased efflux capacity (P = .035 on paired t test) compared with effluxing seen in recipient or donor cells on day 0. These patients were not treated with any immunosuppression after transplant (PTCy or other) before sample collection on day 3.

CD8+ T cells initially increase efflux capacity in antigen-stimulated conditions or when cultured alone in vitro and then lose efflux capacity as they proliferate. (A-F) Human T cells were stimulated in MLC as in Figure 1, stimulated with anti-CD3/CD28 beads, or cultured without allogeneic stimulators or beads. At 3 or 7 days of in vitro culture, Rh-123 effluxing was assessed as per Figure 2. The “+CsA” designation refers to culturing with CsA only during the effluxing to serve as a noneffluxing control, but these cells were not cultured with CsA during the 3 or 7 days of in vitro culture. In parts (A-E), T cells were labeled with CellTrace Violet before in vitro culture. Shown are combined results from 3 independent experiments for parts (A-B,D) and 2 independent experiments for parts (C,E). (A) The efflux capacity of CD8+ T cells in MLC was higher than with anti-CD3/CD28 bead stimulation. **P = .0075 on paired t test. (B) However, proliferation on day 3 was much less extensive after MLC, with negligible numbers of CD8+ T cells in MLC proliferating >1 generation. Example plots of proliferation as measured by CellTrace dilution are shown for T cells from a healthy donor treated either in MLC or with anti-CD3/CD28 beads. (C-E) On both day 3 (D) and day 7 (C,E) of in vitro culture, efflux capacity was increased in nonproliferating cells or those that had only divided a single generation regardless of MLC or anti-CD3/CD28 stimulation but decreased in CD8+ T cells that had proliferated more extensively. This divergence may explain the overall decreased effluxing seen in (A) after bead stimulation because of much more extensive proliferation with that treatment. On day 3 of MLC, effluxing was considered nonevaluable (NE) in the cells proliferating more than a single generation because of extremely low numbers of cells in that group. Representative flow cytometric plots are shown in (C). *P < .05, **P < .01, and ****P < .0001 on nonrepeated-measure one-way ANOVA followed by the Holm-Sidak post hoc test. (F) CD8+ T cells cultured with media alone increased effluxing capacity similarly compared with CD8+ T cells stimulated in MLC, and this was not augmented with treatment with common γ-chain cytokines. The combined results of 2 independent experiments are shown. *P < .05, **P < .01, and ns, not significantly different on repeated-measure one-way ANOVA followed by the Holm-Sidak post hoc test. (G) Human CD8+ T cells retrieved from the blood of patients (n = 5) on day 3 after allogeneic hematopoietic cell transplantation also had increased efflux capacity (P = .035 on paired t test) compared with effluxing seen in recipient or donor cells on day 0. These patients were not treated with any immunosuppression after transplant (PTCy or other) before sample collection on day 3.

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