Figure 7
Figure 7. Transduction of CTP-c-Cbl in EM cells results in lower functional response. (A) Limited amounts of CTP-GFP were transduced in memory cells and the transduction efficiency in CM and EM cells assessed by FACS. Thin and bold lines depict non-transduced and transduced cells, respectively. (B) Detection of c-Cbl following SDS-PAGE and immunoblotting of lysates of memory CD4 T cells previously transduced with wt or C381A CTP-c-Cbls and activated overnight with anti-CD3 and -CD28 antibodies. Vertical lines have been inserted to indicate repositioned gel lanes. (C) Proliferative capacities of CM and EM cells as measured by H3 thymidine incorporation (left). Memory cells were transduced with increasing amount of wt and C381A CTP-c-Cbls fusion proteins (AU: arbitrary units), stimulated for 16 hours with anti-CD3 (5 μg/mL) and -CD28 (1 μg/mL) antibodies and their proliferation measured after 3 days. CTP-mock transduction was used as negative control (). Shown are data from 4 cumulated subjects (right). (D) Effect of CTP-c-Cbls transduction on memory T-cell cytokine production in response to the stimulation described for panel C. Cytokine production was normalized to non-transduced cells and CTP-mock transduction used as negative control. Significant changes (P < .05) in the functions of transduced cells relative to control and mock-transduced cells are indicated (★) as well as P values for significant differences between wt and C381A mutant c-Cbls. (E) Effect of CTP-c-Cbl on the proliferation threshold of purified EM cells stimulated as described for panel C. One representative example out of 3 is shown. (F) Impact of CTP-c-Cbl transduction on the proliferation of EM cells in response to IL-2, anti-CD3 (3 μg/mL) and -CD28 (1 μg/mL) antibodies. Each set of linked dots correspond to a distinct subject and significant changes (P < .05) between IL-2–treated and untreated cells are shown. (G) Quantification of the amounts of CTP-c-Cbls transduced in memory T cells relative to endogenous expression of c-Cbl following 16 hours of activation.

Transduction of CTP-c-Cbl in EM cells results in lower functional response. (A) Limited amounts of CTP-GFP were transduced in memory cells and the transduction efficiency in CM and EM cells assessed by FACS. Thin and bold lines depict non-transduced and transduced cells, respectively. (B) Detection of c-Cbl following SDS-PAGE and immunoblotting of lysates of memory CD4 T cells previously transduced with wt or C381A CTP-c-Cbls and activated overnight with anti-CD3 and -CD28 antibodies. Vertical lines have been inserted to indicate repositioned gel lanes. (C) Proliferative capacities of CM and EM cells as measured by H3 thymidine incorporation (left). Memory cells were transduced with increasing amount of wt and C381A CTP-c-Cbls fusion proteins (AU: arbitrary units), stimulated for 16 hours with anti-CD3 (5 μg/mL) and -CD28 (1 μg/mL) antibodies and their proliferation measured after 3 days. CTP-mock transduction was used as negative control (). Shown are data from 4 cumulated subjects (right). (D) Effect of CTP-c-Cbls transduction on memory T-cell cytokine production in response to the stimulation described for panel C. Cytokine production was normalized to non-transduced cells and CTP-mock transduction used as negative control. Significant changes (P < .05) in the functions of transduced cells relative to control and mock-transduced cells are indicated (★) as well as P values for significant differences between wt and C381A mutant c-Cbls. (E) Effect of CTP-c-Cbl on the proliferation threshold of purified EM cells stimulated as described for panel C. One representative example out of 3 is shown. (F) Impact of CTP-c-Cbl transduction on the proliferation of EM cells in response to IL-2, anti-CD3 (3 μg/mL) and -CD28 (1 μg/mL) antibodies. Each set of linked dots correspond to a distinct subject and significant changes (P < .05) between IL-2–treated and untreated cells are shown. (G) Quantification of the amounts of CTP-c-Cbls transduced in memory T cells relative to endogenous expression of c-Cbl following 16 hours of activation.

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