PMC

Role of miR-17-92 on donor T-cell migration and expression of chemokine receptor and integrin. BMT was carried out as outlined in , in which 1 × 10⁶/mouse donor T cells were transplanted. Fourteen days post-BMT, recipient spleens and livers were collected and mononuclear cells were isolated and subjected to cell counting and FACS staining. Absolute numbers of donor H2K^b+Ly5.1^–CD4⁺ or CD8⁺ WT (gray symbols) and KO (black symbols) T cells were shown in spleen (A) or liver (B). % CXCR3⁺ cells were shown on gated H2K^b+Ly5.1^–CD4⁺ or CD8⁺ T cells in spleen (C-D). % α4β7⁺ cells were shown on gated H2K^b+Ly5.1^–CD4⁺ or CD8⁺ T cells in spleen (E-F). Data shown were pooled from 2 replicated experiments (N = 10/group). *P < .05, **P < .01, ***P < .001.

Effects of miR-17-92 on the differentiation of donor T cells after allo-BMT. BMT was carried out as outlined in . Two weeks post-BMT, recipient spleens were collected and subjected to cell counting and FACS staining. (A) The expression of IFNγ (Th1 cytokine), IL-4/5 (Th2 cytokine), and Foxp3 (Treg markers) were shown on gated donor H2K^b+Ly5.1^–CD4⁺ cells as shown in the gating strategy; (B) absolute numbers of IFNγ⁺, IL-4/5⁺, or Foxp3⁺ cells among donor H2K^b+Ly5.1^–CD4⁺ cells were shown. Few cells differentiated into IL-17–producing Th17 cells and there was no difference between WT and KO T cells (data not shown). (C) The levels of cytokines in recipient serum were measured using cytokine beads assay and displayed as mean ± SD. Data shown were pooled from 2 replicated experiments (N = 10/group). **P < .01, ***P < .001.

Effects of miR-17-92 on T-cell proliferation, activation, and survival. Purified T cells from WT or miR-17-92 KO mice (miR-17-92^fl/fl CD4-Cre⁺) on B6 background were labeled with CFSE and transferred into lethally irradiated BALB/c mice at 2 × 10⁶/mouse. Four days after cell transfer, recipient spleens were collected and subjected to cell counting and fluorescence-activated cell sorting (FACS) staining. (A-B) Percentage (as showed by representative flow figures) and absolute numbers of donor-derived (H2K^b+) CD4 and CD8 T cells on gated live cells; (C-D) representative flow figures of CFSE dilution and percentage of CFSE-diluted cells on gated donor cells; (E-F) percentage and absolute numbers of IFNγ⁺ cells on gated donor CD4 and CD8 T cells; (G) cell death along with cell division on gated donor T cells; and (H) percentage of dead cells among fast-dividing (CFSE^low) or slow-dividing (CFSE^high) on gated donor T cells. Data were shown of 1 representative mouse (A,C,E,G) or mean ± standard deviation (SD) of 4 mice per group (B,D,F,H). Two replicate experiments were performed with a total of 8 mice. *P < .05, **P < .01, ***P < .001.

Role of miR-17-92 on donor T cells in the induction of GVHD. BALB/c mice were lethally irradiated and transplanted with 5 × 10⁶/mouse T cell–depleted marrow cells (TCD-BM, Ly5.1⁺) or plus purified T cells (Ly5.2⁺) from WT or KO mice on B6 background at the doses indicated. Recipient mice were monitored for survival (A) and body weight change (B) until 80 days after transplant. Five to seven mice were included in each group at each cell dose. Statistical analysis was performed to compare the same dose of WT and KO T cells. In separate experiments, pathology injuries (C) and scores (D) were shown 14 days post-BMT; N = 14 per group. SI, small intestine; LI, large intestine. BALB/b mice were lethally irradiated and transplanted with TCD-BM (Ly5.1⁺) alone or plus purified T cells at 3 × 10⁶/mouse from WT or KO mice on B6 background. Recipient mice were monitored for survival (E) and body weight change (F) until 90 days after BMT. Data shown were pooled from 2 replicated experiments with 10 to 12 mice per group. *P < .05, **P < .01, ***P < .001.

miR-17-92 affects T-cell response intrinsically. Lethally irradiated BALB/c mice were transplanted with 5 × 10⁶/mouse TCD-BM (Thy1.1⁺) together with 1 × 10⁶ /mouse WT (Ly5.1⁺ Thy1.2⁺) and 1 × 10⁶ /mouse miR-17-92 KO T cells (Ly5.2⁺ Thy1.2⁺) from B6 mice. Fourteen days after BMT, recipient mice were killed and spleen, liver, lung, and intestine were harvested. Mononuclear cells were isolated from these organs and subjected for flow staining. (A) The percentage of KO (Ly5.2⁺) and WT (Ly5.1⁺) CD4 or CD8 T cells on gated on H2K^b+Thy1.2⁺ donor cells were shown in the representative flow figures. (B) BrdU was injected into recipients (2 mg/mouse) 24 hours before euthanasia of the recipients. Percentage of BrdU⁺ cells in WT and KO cells T cells were displayed on gated Ly5.1⁺ or Ly5.2⁺ cells as shown in (A). (C) Based on the percentage of WT and KO T cells in (A), we calculated the ratio of KO to WT T cells in spleen as well as in GVHD target organs. The first bar indicates 1:1 mixture of WT and KO T cells before BMT. (D) Percentages of IFNγ⁺, IL-4/5⁺, and Foxp3⁺ cells among WT or KO CD4⁺ T cells are shown. Data shown are from 1 of 2 replicate experiments (N = 10/group). *P < .05, **P < .01, ***P < .001.

Effects of anti-miR-17 or anti-miR-19 LNA antagomirs on donor T-cell alloresponse and GVHD. BALB/c mice were lethally irradiated and transplanted with 2 × 10⁶ purified and CFSE-labeled T cells from WT B6 mice. Recipient mice were injected IV with scrambled, anti-miR-17, or anti-miR-19 at 25 mg/kg on day 0 and 5 mg/kg on day 2. Recipients were killed on day 4 after cell transfer, and splenocytes were counted and stained for surface expression of H2K^b (donor marker), CD4, CD8 on gated live cells (A), and intracellular IFNγ along with CFSE profile gated on H2K^b+ CD4⁺ or CD8⁺ cells (C). Absolute numbers of CD4⁺ or CD8⁺ donor T cells (B), and CFSE-diluted IFN-γ⁺ cells (D) were shown. Four mice were used in each group. In a separate experiment, BALB/c mice were lethally irradiated and transplanted with 5 × 10⁶ TCD-BM alone or with T cells from B6 mice at 1 × 10⁶/mouse. Recipient mice were treated with individual antagomirs as indicated twice per week from 25 mg/kg on day 0 and then 5 mg/kg until day 21 after BMT. Recipient survival (E) was shown on 6 mice per group. Using the same BMT model, recipient mice were also infused with 5000 luciferase-transduced A20 cells at the day of BMT, and treated with individual antagomirs as indicated. Recipient survival (F) and mortality caused by tumor relapse (G) were shown on 4 to 7 mice per group. *P < .05, **P < .01, ***P < .001.

Role of miR-17-92 on donor T cell–mediated GVL and cytolytic activities. BDF1 mice were lethally irradiated and transplanted with 5 × 10⁶/mouse TCD-BM alone or plus purified T cells from WT or KO B6 mice at 4 × 10⁶/mouse. Recipient mice were also infused with 5 × 10³ luciferase-transduced p815 cells at the day of BMT. Recipients were monitored for survival (A) and body weight change (B) until 80 days after transplant. (C) Tumor growth was monitored using bioluminescence imaging on the dates indicated. Data were pooled from 2 replicate experiments (N = 5-10/group). Using the same BMT model, we increase the dose of p815 cells to 1 or 2.5×10⁴/mouse, respectively. Recipients were monitored for survival (D) and mortality caused by tumor relapse (E) until 80 days after transplant (N = 6/group). In a separate experiment, recipient spleens were collected 14 days post-BMT and stained for the expression of granzyme B (GrZB), CD107a, IFNγ, and TNFα on gated CD8⁺ donor T cells. Representative flow figures were shown on gated donor CD8 T cells (F), and the bar graph indicates absolute numbers of total CD8 T cells or CD8 T cells that express each of these molecules (G). ***P < .001.