Figure 1.
Depletion of T cells and increase in MDSCs in BALB/c recipients after transplantation of C57BL/6 heart and bone marrow using TLI + ATG conditioning to establish mixed chimerism. (A) Experimental scheme used for murine and human transplantation. Recipients were given an organ graft on day 0, followed by 5 doses of TLI and 5 doses of rabbit anti-thymocyte antibodies (ATG for humans and ATS for mice) in the first week after transplantation and 5 doses of TLI during the second week. The donor cell infusion was given immediately after the completion of TLI (day 15 in mice and day 11 in humans). (B) Representative 2-color flow cytometric analysis of CD11b vs TCRβ staining of spleen cells and PBMCs from wild-type BALB/c mice that were left untreated (WT UNT) or from wild-type BALB/c mice that were recipients of combined transplants (WT TX). Gated CD11b+ cells were analyzed further for expression of Gr-1 vs CD11b. Recipient cells were obtained 5 days after the infusion of the donor cells. The percentage of cells in each box is shown. (C) Mean absolute number of TCRβ+ T cells and CD11b+Gr-1hi myeloid cells in the spleen from untreated BALB/c mice vs the number in the spleen obtained from transplant recipients 5 days after the donor cell infusion. (D) Mean percentage suppression of responder TCRβ+ T-cell proliferation in the BALB/c vs C57BL/6 MLR was calculated by comparing the percentage of dull CFSE staining, with or without the addition of sorted CD11b+Gr-1hi spleen cells. The latter cells were obtained from untreated BABL/c mice or from BALB/c recipient mice 5 days after the donor cell infusion. (E) Mean percentage of donor cell chimerism among gated TCRβ+ T cells in PBMCs from BALB/c recipients without administration of anti–Gr-1 mAb or with administration of anti–Gr-1 mAb to deplete MDSCs. PBMCs were obtained from recipients 28 days after the donor cell infusion. (F) Mean duration of graft survival after allogeneic heart and bone marrow cell transplant in nondepleted or MDSC-depleted or in MDSC-depleted with add back. Cessation of heartbeats in grafts was used as an indicator of heart graft rejection. (G) Mean absolute number of CD11b+Gr-1hi myeloid cells in the spleen from untreated BALB/c mice vs the number in the spleen obtained from transplant recipients on days 28 and 85 after the donor cell infusion. (H) Mean median fluorescence intensity (MFI) and percentages of LOX-1+ cells among gated CD11b+Gr-1hi spleen cells from wild-type BALB/c mice that were left untreated (UNT) or from wild-type BALB/c mice conditioned with 10 doses of TLI (TLI), 5 days after the end of conditioning. Recipient data are a representation of up to 8 independent experiments with ≥6 to 10 mice per group. *P < .05, **P < .01, ***P < .001, Mann-Whitney U test (2-group comparisons) and 1-way ANOVA (for 3-group comparisons). HTx, heart transplant; NS, not significant.

Depletion of T cells and increase in MDSCs in BALB/c recipients after transplantation of C57BL/6 heart and bone marrow using TLI + ATG conditioning to establish mixed chimerism. (A) Experimental scheme used for murine and human transplantation. Recipients were given an organ graft on day 0, followed by 5 doses of TLI and 5 doses of rabbit anti-thymocyte antibodies (ATG for humans and ATS for mice) in the first week after transplantation and 5 doses of TLI during the second week. The donor cell infusion was given immediately after the completion of TLI (day 15 in mice and day 11 in humans). (B) Representative 2-color flow cytometric analysis of CD11b vs TCRβ staining of spleen cells and PBMCs from wild-type BALB/c mice that were left untreated (WT UNT) or from wild-type BALB/c mice that were recipients of combined transplants (WT TX). Gated CD11b+ cells were analyzed further for expression of Gr-1 vs CD11b. Recipient cells were obtained 5 days after the infusion of the donor cells. The percentage of cells in each box is shown. (C) Mean absolute number of TCRβ+ T cells and CD11b+Gr-1hi myeloid cells in the spleen from untreated BALB/c mice vs the number in the spleen obtained from transplant recipients 5 days after the donor cell infusion. (D) Mean percentage suppression of responder TCRβ+ T-cell proliferation in the BALB/c vs C57BL/6 MLR was calculated by comparing the percentage of dull CFSE staining, with or without the addition of sorted CD11b+Gr-1hi spleen cells. The latter cells were obtained from untreated BABL/c mice or from BALB/c recipient mice 5 days after the donor cell infusion. (E) Mean percentage of donor cell chimerism among gated TCRβ+ T cells in PBMCs from BALB/c recipients without administration of anti–Gr-1 mAb or with administration of anti–Gr-1 mAb to deplete MDSCs. PBMCs were obtained from recipients 28 days after the donor cell infusion. (F) Mean duration of graft survival after allogeneic heart and bone marrow cell transplant in nondepleted or MDSC-depleted or in MDSC-depleted with add back. Cessation of heartbeats in grafts was used as an indicator of heart graft rejection. (G) Mean absolute number of CD11b+Gr-1hi myeloid cells in the spleen from untreated BALB/c mice vs the number in the spleen obtained from transplant recipients on days 28 and 85 after the donor cell infusion. (H) Mean median fluorescence intensity (MFI) and percentages of LOX-1+ cells among gated CD11b+Gr-1hi spleen cells from wild-type BALB/c mice that were left untreated (UNT) or from wild-type BALB/c mice conditioned with 10 doses of TLI (TLI), 5 days after the end of conditioning. Recipient data are a representation of up to 8 independent experiments with ≥6 to 10 mice per group. *P < .05, **P < .01, ***P < .001, Mann-Whitney U test (2-group comparisons) and 1-way ANOVA (for 3-group comparisons). HTx, heart transplant; NS, not significant.

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