Figure 6
Figure 6. Differential impact of AT on RAS, RAP-1, and Rho-B prenylation, T-bet, GATA3, and c-MAF expression. (A) CD4+ T cells (H-2Kq) were exposed to irradiated allogeneic APCs (H-2Kd) of each cell type, 2 × 106 T cells/flat-bottomed, 6-well plate for 48 hours in the presence or absence of AT (10 μM), harvested and analyzed by intracellular cytokine staining with anti–IL-10, –IL-4, –TNF-α, or –IFN-γ. Mean fluorescence for the representative cytokines of CD4+H-2Kq+ gated cells is presented. (B) Cells cultured under the conditions described in panel A were then washed and responder T cells (H-2Kq) were re-isolated by MACS with anti–H-2Kq-FITC and anti-FITC beads. Protein samples from in vitro–treated T cells were subject to Western blot analysis. AT is effective at inhibiting the prenylation of Ras, Rap-1, and Rho-B in the allogeneic activation culture. Increased amounts of nonprenylated proteins are found when AT is present (solid arrow). (C) T cells were harvested from AT-treated donors on day 0 (left panel) or on day 3 (right panel) after BMT from the recipients' secondary lymphoid organs and were analyzed by Western blot for the indicated total or phosphorylated proteins. (D) Mean fluorescence for the representative pSTATs is analyzed by phospho-flow analysis of CD4+H-2Kq+ gated cells, presented for the respective day prior to or after BMT. MFI for pSTAT4 was 11.4 (± 0.7) versus 47.4 (± 3.4) for the AT versus the PBS group, respectively, prior to transfer into the irradiated host (P < .05). MFI for pSTAT4 was 97.2 (± 2.1) versus 541.4 (± 11.3) for the AT versus the PBS group, respectively, on day 3 after BMT (P < .01). (E) Donor type T cells derived from AT-treated donors are presented for the respective day prior to or after BMT. Mean fluorescence for c-MAF, T-bet, and GATA-3 within CD4+H-2Kq+ gated cells is presented. MFI for T-bet was 12.4 (± 0.8) versus 98.4 (± 6.3) for the AT versus the PBS group, respectively, prior to transfer into the irradiated host (P < .01). MFI for T-bet was 137.2 (± 4.1) versus 21.4 (± 5.2) for the PBS versus the AT group, respectively, on day 3 after BMT (P < .01).

Differential impact of AT on RAS, RAP-1, and Rho-B prenylation, T-bet, GATA3, and c-MAF expression. (A) CD4+ T cells (H-2Kq) were exposed to irradiated allogeneic APCs (H-2Kd) of each cell type, 2 × 106 T cells/flat-bottomed, 6-well plate for 48 hours in the presence or absence of AT (10 μM), harvested and analyzed by intracellular cytokine staining with anti–IL-10, –IL-4, –TNF-α, or –IFN-γ. Mean fluorescence for the representative cytokines of CD4+H-2Kq+ gated cells is presented. (B) Cells cultured under the conditions described in panel A were then washed and responder T cells (H-2Kq) were re-isolated by MACS with anti–H-2Kq-FITC and anti-FITC beads. Protein samples from in vitro–treated T cells were subject to Western blot analysis. AT is effective at inhibiting the prenylation of Ras, Rap-1, and Rho-B in the allogeneic activation culture. Increased amounts of nonprenylated proteins are found when AT is present (solid arrow). (C) T cells were harvested from AT-treated donors on day 0 (left panel) or on day 3 (right panel) after BMT from the recipients' secondary lymphoid organs and were analyzed by Western blot for the indicated total or phosphorylated proteins. (D) Mean fluorescence for the representative pSTATs is analyzed by phospho-flow analysis of CD4+H-2Kq+ gated cells, presented for the respective day prior to or after BMT. MFI for pSTAT4 was 11.4 (± 0.7) versus 47.4 (± 3.4) for the AT versus the PBS group, respectively, prior to transfer into the irradiated host (P < .05). MFI for pSTAT4 was 97.2 (± 2.1) versus 541.4 (± 11.3) for the AT versus the PBS group, respectively, on day 3 after BMT (P < .01). (E) Donor type T cells derived from AT-treated donors are presented for the respective day prior to or after BMT. Mean fluorescence for c-MAF, T-bet, and GATA-3 within CD4+H-2Kq+ gated cells is presented. MFI for T-bet was 12.4 (± 0.8) versus 98.4 (± 6.3) for the AT versus the PBS group, respectively, prior to transfer into the irradiated host (P < .01). MFI for T-bet was 137.2 (± 4.1) versus 21.4 (± 5.2) for the PBS versus the AT group, respectively, on day 3 after BMT (P < .01).

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