Figure 2.
Recipient TNFRSF25 and CD25 receptor stimulation before HSCT induces expansion of TR Tregs and a functionally suppressive environment in GVHD target tissues. (A-F) Assessment of GVHD target tissues of recipient BALB/c mice either untreated or treated with protocol C (TL1A-Ig+IL-2) and analyzed 8 days after initiation of treatment (day 0 of BMT). (A) Treg frequencies in nonhematopoietic tissue compartments, that is, the colon, liver, skin, lung, and ocular adnexa (conjunctiva and lacrimal gland), are shown. All GVHD target tissues show significant Treg expansion. (B-E) Measurement of suppressive capabilities of Treg cells isolated from GVHD target tissues on day 0, assessing in vitro proliferation using anti-CD3 mAb. (B) Cell number of colon LP cultures were counted at hour 72 from protocol C–treated and untreated mice. Untreated mice showed higher cell counts than protocol C–treated mice after in vitro anti-CD3 stimulation. (C-E) In vitro studies assessing the liver and the ocular adnexa. Each point represents 1 experiment, and each experiment had a minimum of 3 replicate wells for each group. On day 0, liver CD4+ T cells were enriched via EasySep beads and then plated with anti-CD3 mAb to induce proliferation. (C) Percent increase of enriched liver mononuclear cells at hour 120 shows no net proliferation in cell counts when mice were treated with protocol C. (D) Percent increase of total conjunctiva mononuclear cells at hour 120 if no treatment was given. (E) Percent increase of total lacrimal gland cells at hour 120 if no TL1A-Ig+IL-2LD treatment was given. (F) Representative flow plots depicting the initial frequency of FoxP3+ Tregs at the start of the cultures, and below representative images of well health at hour 120 between in vivo treated or untreated, respectively. (G-I) The S1PR modulator, FTY720, was used to delineate whether the TL1A-Ig+IL-2LD–expanded Tregs traffic from hematopoietic sites or proliferate in nonhematopoietic GVHD target tissues at day 8 after starting treatment (see “Methods”). (G) Gating strategy for resident FoxP3+ Tregs (CD69+CD103+) from the liver (upper) and cervical LNs (lower panel). (H) Absolute numbers of liver-resident (CD69+CD103+) Tregs are significantly increased after TL1A-Ig+IL-2LD treatment. (I) Most Tregs highly express the proliferation marker, Ki-67, indicating robust expansion of TR Tregs. Data represent the mean ± SEM, with ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001 defining significance levels.

Recipient TNFRSF25 and CD25 receptor stimulation before HSCT induces expansion of TR Tregs and a functionally suppressive environment in GVHD target tissues. (A-F) Assessment of GVHD target tissues of recipient BALB/c mice either untreated or treated with protocol C (TL1A-Ig+IL-2) and analyzed 8 days after initiation of treatment (day 0 of BMT). (A) Treg frequencies in nonhematopoietic tissue compartments, that is, the colon, liver, skin, lung, and ocular adnexa (conjunctiva and lacrimal gland), are shown. All GVHD target tissues show significant Treg expansion. (B-E) Measurement of suppressive capabilities of Treg cells isolated from GVHD target tissues on day 0, assessing in vitro proliferation using anti-CD3 mAb. (B) Cell number of colon LP cultures were counted at hour 72 from protocol C–treated and untreated mice. Untreated mice showed higher cell counts than protocol C–treated mice after in vitro anti-CD3 stimulation. (C-E) In vitro studies assessing the liver and the ocular adnexa. Each point represents 1 experiment, and each experiment had a minimum of 3 replicate wells for each group. On day 0, liver CD4+ T cells were enriched via EasySep beads and then plated with anti-CD3 mAb to induce proliferation. (C) Percent increase of enriched liver mononuclear cells at hour 120 shows no net proliferation in cell counts when mice were treated with protocol C. (D) Percent increase of total conjunctiva mononuclear cells at hour 120 if no treatment was given. (E) Percent increase of total lacrimal gland cells at hour 120 if no TL1A-Ig+IL-2LD treatment was given. (F) Representative flow plots depicting the initial frequency of FoxP3+ Tregs at the start of the cultures, and below representative images of well health at hour 120 between in vivo treated or untreated, respectively. (G-I) The S1PR modulator, FTY720, was used to delineate whether the TL1A-Ig+IL-2LD–expanded Tregs traffic from hematopoietic sites or proliferate in nonhematopoietic GVHD target tissues at day 8 after starting treatment (see “Methods”). (G) Gating strategy for resident FoxP3+ Tregs (CD69+CD103+) from the liver (upper) and cervical LNs (lower panel). (H) Absolute numbers of liver-resident (CD69+CD103+) Tregs are significantly increased after TL1A-Ig+IL-2LD treatment. (I) Most Tregs highly express the proliferation marker, Ki-67, indicating robust expansion of TR Tregs. Data represent the mean ± SEM, with ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001 defining significance levels.

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