![Figure 5. DR3 activation after alloantigen exposure promotes donor T-cell activation. (A, i) Representative results of DR3 expression in immune cells by viSNE analysis are shown. Whole splenocytes were isolated and stained with various antibodies. CD4+ T cells (CD4+TCRβ+Foxp3−), CD8+ T cells (CD8+TCRβ+), B cells (TCRβ-B220+), NK cells (NK1.1+TCRβ−B220−), NKT cells (NK1.1+TCRβ+), and Treg (CD4+TCRβ+Foxp3+) were categorized as shown in the panel (supplemental Figure 4). The colors were scaled to the expression level of DR3. (ii) The expression level of DR3 in CD4+Foxp3− T cells, CD8+T cells, Treg after CD3/CD28 beads activation at various ratios (T cells:beads 1:80-1:10) are shown (n = 6). (B) T cells were cultured with CD3/CD28 beads together with αDR3 or isotype control Abs. After 3 days in culture, Foxp3 (i) and T-bet (ii-iii) expression were analyzed (n = 6; ns = not significant; *P < .05; **P < .01). (C) MLR using freshly isolated T cells from nontreated WT B6 mice and γ-irradiated splenocytes from WT Balb/c mice with or without αDR3 (1-10 μg/mL) was performed. The ratio of responder (T cell):stimulator (irradiated splenocytes) was from 1:0 to 1:2, as shown in the graph. [3H] thymidine incorporation was measured and analyzed (*P < .05; **P < 0.01; ***P < .001). Representative data from 2 independent experiments (each experiment was performed in triplicate) are shown. (D) T cells were isolated from the GVHD mice on day 7. DR3 expression on donor T cells was analyzed (n = 5; **P < .01; ****P < .0001). (E) TCD-BM from WT B6 mice and T cells from B6-luc mice were transplanted into lethally irradiated Balb/c mice on day 0. αDR3 or isotype control Ab was injected on day 3. (F) T cells from the spleen were isolated on day 7 after transplant, and the frequencies of Foxp3+ cells from donor- or recipient-derived CD4+ T cells were analyzed (n = 4; ns = not significant; ***P < .001). (G) Donor T-cell proliferation in mice treated on day 3 with αDR3 was assessed by BLI. Representative images on day 7 are shown in (i). The summary of BLI was shown in (ii) (n = 10; *P < .05). (H) Survival curve is assessed (*P < .05; ***P < .001).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/128/24/10.1182_blood-2016-06-723783/4/blood723783f5.jpeg?Expires=1735516146&Signature=obOrWSoCrOrn07qJCFHKDzJrwQn0YZ~-YLsIXfXBEXj16ygKCbyFfGWJ4MHjT58YyQY-w9hRuhc44WsH-qvqsLnWUGApB1xe4XxpXhbjisgpOh5sJ~6P~p09eawC1OpJn5-bhpcQ38zvcVqehEXtkEZRjzdhfCgcGlHGB2ntAMjTKd--BMmgHqjKGqqhuwAao1R6yEvW1RknNvaHzpqujvO75YKxG6T6xS8c7ei~6Cr~xtzDD6zMLylvaUXa~78BsYqL-qJA2hBm4DSZgzZrRdJYeRd7cRLUj0a2QFn8eRVx1l3lkpI3F2Dy9eYWPUDlzkAKrEyzR-OWMpuOPBAeZQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
DR3 activation after alloantigen exposure promotes donor T-cell activation. (A, i) Representative results of DR3 expression in immune cells by viSNE analysis are shown. Whole splenocytes were isolated and stained with various antibodies. CD4+ T cells (CD4+TCRβ+Foxp3−), CD8+ T cells (CD8+TCRβ+), B cells (TCRβ-B220+), NK cells (NK1.1+TCRβ−B220−), NKT cells (NK1.1+TCRβ+), and Treg (CD4+TCRβ+Foxp3+) were categorized as shown in the panel (supplemental Figure 4). The colors were scaled to the expression level of DR3. (ii) The expression level of DR3 in CD4+Foxp3− T cells, CD8+T cells, Treg after CD3/CD28 beads activation at various ratios (T cells:beads 1:80-1:10) are shown (n = 6). (B) T cells were cultured with CD3/CD28 beads together with αDR3 or isotype control Abs. After 3 days in culture, Foxp3 (i) and T-bet (ii-iii) expression were analyzed (n = 6; ns = not significant; *P < .05; **P < .01). (C) MLR using freshly isolated T cells from nontreated WT B6 mice and γ-irradiated splenocytes from WT Balb/c mice with or without αDR3 (1-10 μg/mL) was performed. The ratio of responder (T cell):stimulator (irradiated splenocytes) was from 1:0 to 1:2, as shown in the graph. [3H] thymidine incorporation was measured and analyzed (*P < .05; **P < 0.01; ***P < .001). Representative data from 2 independent experiments (each experiment was performed in triplicate) are shown. (D) T cells were isolated from the GVHD mice on day 7. DR3 expression on donor T cells was analyzed (n = 5; **P < .01; ****P < .0001). (E) TCD-BM from WT B6 mice and T cells from B6-luc mice were transplanted into lethally irradiated Balb/c mice on day 0. αDR3 or isotype control Ab was injected on day 3. (F) T cells from the spleen were isolated on day 7 after transplant, and the frequencies of Foxp3+ cells from donor- or recipient-derived CD4+ T cells were analyzed (n = 4; ns = not significant; ***P < .001). (G) Donor T-cell proliferation in mice treated on day 3 with αDR3 was assessed by BLI. Representative images on day 7 are shown in (i). The summary of BLI was shown in (ii) (n = 10; *P < .05). (H) Survival curve is assessed (*P < .05; ***P < .001).
