Figure 1.
Optimizing in vivo Treg expansion protocol with TL1A-Ig+IL2LD for Treg survival after radiation. (A) qpTIFF CODEX images illustrating a subset of cell populations (7 selected markers) highlighting diminished Treg numbers in colonic tissue from BALB/c mice transplanted with BM + T cells experiencing GVHD vs BM transplanted alone; white arrows show Treg locations in the images. (B) Select immune cell frequencies separated by non-GVHD (BM only, black bars) and GVHD (BM + T cells, gray bars) from the whole colonic tissue, n = 2 per group. (C) Mean nuclear Ki-67 expression of Tregs separated by groups. Mice without GVHD (BM alone) contained a greater number of Tregs than animals with GVHD (BM+T) with ∼45% (compared with 0% in mice without GVHD) having moderate to high Ki-67 expression indicating proliferation. (D) BALB/c mice were treated with TL1A-Ig+IL-2LD. Treg expansion in colonic LP at day 7 without detectable increase in colonic TH17 CD4+ T cells is shown. (E-F) BALB/c mice were assessed on day 12, 6 days after completing TL1A-Ig+IL-2LD treatment. (E) The colon LP shows a persistence in elevated total Tregs as well as Treg subsets (SP: CD4+FoxP3+RORγt-; DP: CD4+FoxP3+RORγt+) at day 12, and (F) this persistence is greatest in the colon vs other tissues. (G-M) BALB/c mice were treated with TL1A-Ig+IL-2LD using different protocols (supplemental Figure 2C) and received TBI (8.5 Gy) on day −1. On day 4, immune cells were assessed for Treg frequencies in the spleen (G) and colonic LP (H). Protocol C demonstrated significantly higher Treg frequencies and numbers than protocols in panels A-B, and no treatment (panels G-I). Protocol C also shows no difference in TH17 (J) and CD4+ Tconv (K) numbers of the colon LP. Panels E-K: data points represent individual animals. (L-M) The liver exhibited a greater Treg frequency (L) when using protocol C. (M) Representative flow cytometry plots gating of CD4+FoxP3+Treg cells using cell isolates from the liver. Data represent the mean ± standard error of the mean (SEM) with ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001 defining significance levels. DAPI, 4′,6-diamidino-2-phenylindole; FSC-A, forward scatter-area; mLN, mesenteric lymph nodes; ns, not significant; pLN, peripheral lymph nodes; T, T cells.

Optimizing in vivo Treg expansion protocol with TL1A-Ig+IL2LD for Treg survival after radiation. (A) qpTIFF CODEX images illustrating a subset of cell populations (7 selected markers) highlighting diminished Treg numbers in colonic tissue from BALB/c mice transplanted with BM + T cells experiencing GVHD vs BM transplanted alone; white arrows show Treg locations in the images. (B) Select immune cell frequencies separated by non-GVHD (BM only, black bars) and GVHD (BM + T cells, gray bars) from the whole colonic tissue, n = 2 per group. (C) Mean nuclear Ki-67 expression of Tregs separated by groups. Mice without GVHD (BM alone) contained a greater number of Tregs than animals with GVHD (BM+T) with ∼45% (compared with 0% in mice without GVHD) having moderate to high Ki-67 expression indicating proliferation. (D) BALB/c mice were treated with TL1A-Ig+IL-2LD. Treg expansion in colonic LP at day 7 without detectable increase in colonic TH17 CD4+ T cells is shown. (E-F) BALB/c mice were assessed on day 12, 6 days after completing TL1A-Ig+IL-2LD treatment. (E) The colon LP shows a persistence in elevated total Tregs as well as Treg subsets (SP: CD4+FoxP3+RORγt-; DP: CD4+FoxP3+RORγt+) at day 12, and (F) this persistence is greatest in the colon vs other tissues. (G-M) BALB/c mice were treated with TL1A-Ig+IL-2LD using different protocols (supplemental Figure 2C) and received TBI (8.5 Gy) on day −1. On day 4, immune cells were assessed for Treg frequencies in the spleen (G) and colonic LP (H). Protocol C demonstrated significantly higher Treg frequencies and numbers than protocols in panels A-B, and no treatment (panels G-I). Protocol C also shows no difference in TH17 (J) and CD4+ Tconv (K) numbers of the colon LP. Panels E-K: data points represent individual animals. (L-M) The liver exhibited a greater Treg frequency (L) when using protocol C. (M) Representative flow cytometry plots gating of CD4+FoxP3+Treg cells using cell isolates from the liver. Data represent the mean ± standard error of the mean (SEM) with ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001 defining significance levels. DAPI, 4′,6-diamidino-2-phenylindole; FSC-A, forward scatter-area; mLN, mesenteric lymph nodes; ns, not significant; pLN, peripheral lymph nodes; T, T cells.

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