Agonistic Anti-DR3 Antibody Expands Treg and Reduces Experimental Acute Graft-Versus-Host Disease

Regulatory T cells with the phenotype of CD4+CD25+FoxP3+ (Treg) decrease acute graft-versus-host disease (GVHD) while preserving graft-versus-leukemia effects. However, their low frequency limits clinical translation. One approach has been to expand Treg ex vivo using cytokines (IL-2/TGFb) and antibodies (anti-CD3/anti-CD28). Another approach may be to develop strategies to expand Treg in vivo. Death Receptor 3 (DR3) is a member of the TNF superfamily and its agonistic antibody (4C12) was reported to preferentially activate Treg. We investigated the role of DR3 stimulation in expanding Treg and decreasing acute GVHD in a murine model. Two approaches were investigated. The first approach was to utilize 4C12 to treat B6 donor mice (CD45.1/luc+) and isolate conventional T cells (Tcon) four days later from lymph nodes and spleen using anti-CD4 and anti-CD8 beads. Tcon from 4C12-treated donors were consistently enriched with an increased percentage and absolute numbers of Foxp3+CD4+ cells compared to Tcon from isotype-treated donors (25.6% vs. 12.6%, p<0.01). In the mixed lymphocyte reaction Tcon from 4C12-treated donors were less proliferative compared to Tcon from isotype-treated donors. Moreover, CD4+CD25+ cells from 4C12-treated donors showed stronger suppressive effects suggesting that not only were Treg expanded but that they were also activated and more functional. Tcon (1x10^6) from 4C12 vs isotype treated luc+ C57Bl/6 donors were injected into lethally irradiated Balb/c recipients along with 5x10^6 T cell-depleted BM cells from wild-type B6 mice. Animals that received 4C12 treated Tcon had significantly lower GVHD-related score and better survival than Balb/c mice that received Tcon from isotype-treated donors (n=10 for each group, p<0.01). Bioluminescence imaging (BLI) showed recipient mice that received Tcon from 4C12-treated donors had decreased Tcon expansion early after transplantation compared to recipients of Tcon from isotype-treated donors (Day 3, p=0.04; Day 6, p=0.04). Flow cytometric analysis of lymph nodes and spleens harvested from recipients of Tcon from 4C12-treated donors confirmed that significantly higher Treg levels were maintained in donor Tcon on Day 4 and Day 7 after transplantation. In other experiments, recipients of Tcon from 4C12-treated donors still had significantly higher Treg levels in donor Tcon even 20 days after infusion.

The second approach was to treat lethally irradiated recipient Balb/c mice with 4C12 or isotype antibody intraperitoneally one hour after the injection of 5x10^6 T cell-depleted BM cells from wild-type B6 mice. Tcon (1x10^6) from CD45.1 luc+ B6 mice were injected on Day 2. Recipient mice treated with 4C12 had significantly lower GVHD-related score and better survival than isotype-treated recipients (n=5 for each group, p<0.01). BLI also showed 4C12-treated mice had decreased T cell expansion (Day 8, p=0.03; Day 20, p<0.01). Lymph nodes and spleens were harvested on Day 5 after in vivo BrdU labeling for flow cytometric analysis. The host CD4 T cell population from 4C12-treated recipients had more Foxp3+ cells that also incorporated significantly more BrdU.

In summary, stimulation of Treg through DR3 resulted in enhanced number and function of Treg which was associated with less GVHD by either treating the donor mice or recipients at the time of transplant. This approach of Treg stimulation could serve as an alternative approach to ex vivo Treg expansion to enhance Treg function resulting in less morbidity and better survival with reduced GVHD risk. Together our data suggest that agonistic anti-DR3 antibody stimulation can effectively activate and expand Treg resulting in decreased acute GVHD in a murine GVHD model.