Figure 1
Figure 1. miR-146a is regulated in the T-cell compartment after HCT and its deficiency in T cells enhances GVHD. (A) T cells were isolated from the spleens and inguinal LNs of untreated mice (day 0) or on days 2, 6, and 12 after (left) major MHC mismatch allo-HCT (C57BL/6→BALB/c), (center) minor histocompatibility antigen mismatch allo-HCT (BALB/B→C57BL/6), or (right) syngeneic HCT (BALB/c→BALB/c) by magnetic-activated cell sorting enrichment for CD4 and CD8. miR-146a expression was analyzed by qRT-PCR. Each data point represents an individual animal. (B-C) Allo-HCT was performed as described for the C57BL/6 into BALB/c combination. (B) Survival of BALB/c recipient mice transplanted with WT BM cells plus T cells from miR-146a−/− or miR-146a+/+ mice as indicated is shown. (C) The small intestine, colon, and liver were isolated from mice receiving T cells from miR-146a−/− or miR-146a+/+ mice on day 7 after allo-HCT and scored for GVHD severity. (D-E) Syngeneic HCT was performed as described for the C57BL/6 into C57BL/6 model. (D) Survival of C57BL/6 recipient mice transplanted with WT BM cells plus T cells from syngeneic miR-146a−/− or miR-146a+/+ mice as indicated is shown. (E) Small intestines, colons, and livers were isolated on day 50 after syngeneic HCT and scored for GVHD severity. (F-H) Allo-HCT was performed as described for the C57BL/6 into BALB/c combination. (F) T cells were isolated from the spleens of allo-HCT hosts receiving T cells from miR-146a−/− or miR-146a+/+ mice on day 7 after allo-HCT and analyzed for apoptosis/necrosis via Annexin-V/7-AAD staining. (G) Recipient mice receiving 2 × 105 miR-146a+/+ T cells or 25% less miR-146a−/− T cells as indicated were monitored for survival. (H) The indicated cytokines were measured in the serum of recipient mice receiving T cells from miR-146a−/− or miR-146a+/+ mice on day 7 after allo-HCT by cytometric bead array. (I) Proliferation of carboxyfluorescein diacetate succinimidyl ester-labeled T cells (C57BL/6) in response to coculture with LPS-stimulated BMDCs (BALB/c) for 96 hours at a 1:1 ratio is shown for T cells derived from miR-146a−/− compared with miR-146a+/+ mice. Data were normalized to the miR-146a+/+ control group. All data were pooled from 2 to 3 independent experiments.

miR-146a is regulated in the T-cell compartment after HCT and its deficiency in T cells enhances GVHD. (A) T cells were isolated from the spleens and inguinal LNs of untreated mice (day 0) or on days 2, 6, and 12 after (left) major MHC mismatch allo-HCT (C57BL/6→BALB/c), (center) minor histocompatibility antigen mismatch allo-HCT (BALB/B→C57BL/6), or (right) syngeneic HCT (BALB/c→BALB/c) by magnetic-activated cell sorting enrichment for CD4 and CD8. miR-146a expression was analyzed by qRT-PCR. Each data point represents an individual animal. (B-C) Allo-HCT was performed as described for the C57BL/6 into BALB/c combination. (B) Survival of BALB/c recipient mice transplanted with WT BM cells plus T cells from miR-146a−/− or miR-146a+/+ mice as indicated is shown. (C) The small intestine, colon, and liver were isolated from mice receiving T cells from miR-146a−/− or miR-146a+/+ mice on day 7 after allo-HCT and scored for GVHD severity. (D-E) Syngeneic HCT was performed as described for the C57BL/6 into C57BL/6 model. (D) Survival of C57BL/6 recipient mice transplanted with WT BM cells plus T cells from syngeneic miR-146a−/− or miR-146a+/+ mice as indicated is shown. (E) Small intestines, colons, and livers were isolated on day 50 after syngeneic HCT and scored for GVHD severity. (F-H) Allo-HCT was performed as described for the C57BL/6 into BALB/c combination. (F) T cells were isolated from the spleens of allo-HCT hosts receiving T cells from miR-146a−/− or miR-146a+/+ mice on day 7 after allo-HCT and analyzed for apoptosis/necrosis via Annexin-V/7-AAD staining. (G) Recipient mice receiving 2 × 105miR-146a+/+ T cells or 25% less miR-146a−/− T cells as indicated were monitored for survival. (H) The indicated cytokines were measured in the serum of recipient mice receiving T cells from miR-146a−/− or miR-146a+/+ mice on day 7 after allo-HCT by cytometric bead array. (I) Proliferation of carboxyfluorescein diacetate succinimidyl ester-labeled T cells (C57BL/6) in response to coculture with LPS-stimulated BMDCs (BALB/c) for 96 hours at a 1:1 ratio is shown for T cells derived from miR-146a−/− compared with miR-146a+/+ mice. Data were normalized to the miR-146a+/+ control group. All data were pooled from 2 to 3 independent experiments.

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