Figure 6.
Experimental targeting of the GPR39 receptor improves thymic repair and T-cell reconstitution after allo-HCT. (A) Six- to 8-week-old male C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO4) for 21 days, at which point mice were given a lethal dose of TBI (2 × 550 cGy) along with T-cell depleted BM from female C57BL/6 mice. Mice were maintained on ZnSO4 in drinking water for the duration of the study. Total thymic cellularity at days 7, 21, 28, and 42 after HCT (n = 5-10/group/timepoint combined from 2 independent experiments). (B) Six- to 8-week-old male C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO4) for 21 days, at which point mice were given a lethal dose of TBI (2 × 550 cGy) along with T-cell depleted BM from female RAG2-GFP mice. Splenic T cells were analyzed for GFP expression on day 53 (n = 4-5/group). (C) Six- to 8-week-old female C57BL/6 mice were given 550 cGy of TBI and either vehicle or TC-G 1008 (20 mg/kg per mouse per day) by guided feeding daily from day 0 until day 10, when thymus cellularity was assessed (n = 8-9 combined from 2 independent experiments). (D-H) Six- to 8-week-old female BALB.B mice were given a lethal dose of TBI (900 cGy) along with 10 × 106 T-cell depleted BM from female C57BL/6 mice and either vehicle or TC-G 1008 (20 mg/kg per mouse per day) by guided feeding daily from day 0 until day 8 and then biweekly. Thymuses were harvested and analyzed at day 14 (n = 10/group from 2 separate experiments) and at day 40 (n = 6/group from 2 separate experiments)(D) Total thymic cellularity at day 14 and 40 after HCT. (E) Spleen harvested at day 40 after HCT, and numbers of GFP+ T cells were calculated (n = 6/group, from 2 separate experiments). (F) Number of T-cell subsets. (G-H) CD3+ T cells were isolated from spleen at day 40 after HCT and stimulated for 72 hours with anti-CD3 and anti-CD28. (G) Proliferation of all T cells (n = 6/group from 2 separate experiments). (H) Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α on CD4+ and CD8+ (n = 6/group from 2 separate experiments). Graphs represent mean ± SEM; each dot represents a biologically independent observation. *P < .05; **P < .01; ***P < .001.

Experimental targeting of the GPR39 receptor improves thymic repair and T-cell reconstitution after allo-HCT. (A) Six- to 8-week-old male C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO4) for 21 days, at which point mice were given a lethal dose of TBI (2 × 550 cGy) along with T-cell depleted BM from female C57BL/6 mice. Mice were maintained on ZnSO4 in drinking water for the duration of the study. Total thymic cellularity at days 7, 21, 28, and 42 after HCT (n = 5-10/group/timepoint combined from 2 independent experiments). (B) Six- to 8-week-old male C57BL/6 mice were given supplemental Zn in drinking water (300 mg/kg per day of ZnSO4) for 21 days, at which point mice were given a lethal dose of TBI (2 × 550 cGy) along with T-cell depleted BM from female RAG2-GFP mice. Splenic T cells were analyzed for GFP expression on day 53 (n = 4-5/group). (C) Six- to 8-week-old female C57BL/6 mice were given 550 cGy of TBI and either vehicle or TC-G 1008 (20 mg/kg per mouse per day) by guided feeding daily from day 0 until day 10, when thymus cellularity was assessed (n = 8-9 combined from 2 independent experiments). (D-H) Six- to 8-week-old female BALB.B mice were given a lethal dose of TBI (900 cGy) along with 10 × 106 T-cell depleted BM from female C57BL/6 mice and either vehicle or TC-G 1008 (20 mg/kg per mouse per day) by guided feeding daily from day 0 until day 8 and then biweekly. Thymuses were harvested and analyzed at day 14 (n = 10/group from 2 separate experiments) and at day 40 (n = 6/group from 2 separate experiments)(D) Total thymic cellularity at day 14 and 40 after HCT. (E) Spleen harvested at day 40 after HCT, and numbers of GFP+ T cells were calculated (n = 6/group, from 2 separate experiments). (F) Number of T-cell subsets. (G-H) CD3+ T cells were isolated from spleen at day 40 after HCT and stimulated for 72 hours with anti-CD3 and anti-CD28. (G) Proliferation of all T cells (n = 6/group from 2 separate experiments). (H) Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α on CD4+ and CD8+ (n = 6/group from 2 separate experiments). Graphs represent mean ± SEM; each dot represents a biologically independent observation. *P < .05; **P < .01; ***P < .001.

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