Figure 4.
ICA treatment of allo-BMT recipients leads to sustained survival after removal of ICA and induces tolerance in allogeneic T cells. (A) Lethally irradiated B10.BR recipients were transplanted with TCD-BM in combination with purified T cells (TCD-BM + T) from C57Bl/6 (B6) mice to induce GVHD. Mice received daily oral gavage of 150 mg/kg ICA or vehicle through day 45 posttransplant, at which point ICA delivery was terminated. Weight loss and survival were tracked through day 60. (A) Treatment schema (top). Weight loss (middle). Survival curves representing a subset of vehicle-treated recipients (n = 9) and ICA-treated recipients (N = 10) from Figure 2, followed for an additional 15 days after ICA treatment was terminated (bottom). (B) Lethally irradiated B10.BR recipients were transplanted with TCD-BM in combination with purified T cells from C57Bl/6 mice to induce GVHD. Top panel: Treatment schema. Early ICA mice received daily oral gavage of ICA from day −2 to day 12 and vehicle day 13 to day 53. Late ICA mice received daily oral gavage of vehicle from day −2 to day 12 and ICA day 13 to day 53. Control mice received ICA or vehicle throughout. Middle panel: Weight loss. Lower panel: Kaplan-Meier survival curve. Early ICA and late ICA, n = 15 per group. Controls, n = 4 per group. (C-D) T cells were harvested from spleens of ICA-treated survivors (n = 8, from panel A) at 60 days posttransplant (15 days after termination of ICA delivery), labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and used for secondary transfer. Splenocytes were harvested from secondary recipients 3 days after transfer, and proliferation profiles assessed by Flow Cytometry. (C) CFSE proliferation profiles of CD8 T cells in control B6 → B6 syngeneic transfer (upper left), N = 3; control B6 → B10.BR allogeneic transfer (lower left), N = 4; marrow-derived T cells from ICA-treated survivors → B10.BR recipients (upper right) and donor spleen-derived T cells from ICA-treated survivors → B10.BR recipients (lower right), N = 4. Flow cytometry gating was used to quantify T cells which were marrow hematopoietic stem cell (HSC) derived vs donor splenic T-cell derived. (D) Replication indices for the CD8 CFSE profiles depicted in panel C as well as CD4 CFSE profiles. (E) Splenocytes from Early ICA and late ICA survivors at day 53 posttransplant (from panel B) were tested, along with splenocytes from B6 control and B6 → B10.BR control with chronic GVHD, in MLR. Stimulators included irradiated splenocytes from B6, B10.BR, and FVB. At 48 hours, culture wells were harvested and analyzed for donor-derived CD4 (left) and CD8 T-cells (right) and Ki67 expression. Error bars represent standard deviations. Statistics: Mantel Cox log-rank (survival curve), ANOVA, Student t test. **P = .001 to .01; *P = .01 to .05.

ICA treatment of allo-BMT recipients leads to sustained survival after removal of ICA and induces tolerance in allogeneic T cells. (A) Lethally irradiated B10.BR recipients were transplanted with TCD-BM in combination with purified T cells (TCD-BM + T) from C57Bl/6 (B6) mice to induce GVHD. Mice received daily oral gavage of 150 mg/kg ICA or vehicle through day 45 posttransplant, at which point ICA delivery was terminated. Weight loss and survival were tracked through day 60. (A) Treatment schema (top). Weight loss (middle). Survival curves representing a subset of vehicle-treated recipients (n = 9) and ICA-treated recipients (N = 10) from Figure 2, followed for an additional 15 days after ICA treatment was terminated (bottom). (B) Lethally irradiated B10.BR recipients were transplanted with TCD-BM in combination with purified T cells from C57Bl/6 mice to induce GVHD. Top panel: Treatment schema. Early ICA mice received daily oral gavage of ICA from day −2 to day 12 and vehicle day 13 to day 53. Late ICA mice received daily oral gavage of vehicle from day −2 to day 12 and ICA day 13 to day 53. Control mice received ICA or vehicle throughout. Middle panel: Weight loss. Lower panel: Kaplan-Meier survival curve. Early ICA and late ICA, n = 15 per group. Controls, n = 4 per group. (C-D) T cells were harvested from spleens of ICA-treated survivors (n = 8, from panel A) at 60 days posttransplant (15 days after termination of ICA delivery), labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and used for secondary transfer. Splenocytes were harvested from secondary recipients 3 days after transfer, and proliferation profiles assessed by Flow Cytometry. (C) CFSE proliferation profiles of CD8 T cells in control B6 → B6 syngeneic transfer (upper left), N = 3; control B6 → B10.BR allogeneic transfer (lower left), N = 4; marrow-derived T cells from ICA-treated survivors → B10.BR recipients (upper right) and donor spleen-derived T cells from ICA-treated survivors → B10.BR recipients (lower right), N = 4. Flow cytometry gating was used to quantify T cells which were marrow hematopoietic stem cell (HSC) derived vs donor splenic T-cell derived. (D) Replication indices for the CD8 CFSE profiles depicted in panel C as well as CD4 CFSE profiles. (E) Splenocytes from Early ICA and late ICA survivors at day 53 posttransplant (from panel B) were tested, along with splenocytes from B6 control and B6 → B10.BR control with chronic GVHD, in MLR. Stimulators included irradiated splenocytes from B6, B10.BR, and FVB. At 48 hours, culture wells were harvested and analyzed for donor-derived CD4 (left) and CD8 T-cells (right) and Ki67 expression. Error bars represent standard deviations. Statistics: Mantel Cox log-rank (survival curve), ANOVA, Student t test. **P = .001 to .01; *P = .01 to .05.

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