Targeting the IL-2/CD25 and TL1A/TNFRSF25 Pathways: A New Approach to Markedly Expand Donor Tregs in Multiple Compartments Leads to in Situ Immune Regulation

Regulatory T cells (Treg) are critical for the maintenance of self-tolerance and Treg expansion is being investigated as a promising therapy for graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. Because the numbers of donor graft Tregs necessary for maximal efficacy are significant, ex vivo expansion presents both practical and scientific challenges. We and others have demonstrated that in situ expansion of CD4⁺ FoxP3⁺ Tregs using IL-2 may be used to inhibit alloreactive T cells that mediate GVHD. Recently, we developed a novel combination approach to expand donor Tregs, wherein we administered a fusion protein (TL1A-Ig) and IL-2, which target, respectively, TNF receptor super family 25 (TNFRSF25) and CD25, the high affinity IL-2 receptor. Following a 6-day sequence of Tl1A-Ig and IL-2, we observed markedly greater peripheral Treg expansion (5-8 fold) vs. either reagent alone (2-3 fold). The resulting Treg expansion in healthy animals is transient, peaking by 7-8 days, and results in no long-term complications, as assessed by changes in blood counts, immune phenotype / function and tissue pathology 6 months following cessation of treatment. Analyses of animals 1 week following sequential therapy revealed Treg expansion in blood, spleen, lymph nodes, and the colon but not bone marrow. Expanded Tregs were highly activated (Ki67⁺) with an elevated KLRG1⁺ fraction. High functional activity was also reflected in their hypomethylated FoxP3 promoter region, elevated pSTAT5 expression and the ability of sorted Tregs to mediate functional suppression of lymphocyte proliferation. Notably, assessment of the splenic vs. LN Treg compartment indicated that there were consistently higher Treg frequencies an d greater levels of Treg effector proteins (Gzmb/TNFa/IFNg/IL-10) in the splenic Treg compartment. To assess the functional significance of this difference, we stimulated splenocytes and lymph node cells from TL1A-Ig/IL-2 treated animals with anti-CD3 mAb and LPS, and observed significantly greater suppression in the spleen vs LN cultures. Importantly, even when cultures of LN cells contained the same or greater numbers of Tregs compared with the spleen, greater suppression was detected in the splenic cultures. Based on the heightened suppressive environment in spleens post-Treg expansion, we performed HSCT across a complete MHC mismatch using the B6àBALB/c donor/recipient combination. Groups of 8.5 Gy TBI-conditioned mice received T cell depleted B6 bone marrow and either spleen or LN cells from TL1A-Ig/IL-2 treated or untreated B6 mice. While GVHD was reduced in recipients of either spleen or lymph node T cells, relative to recipients of control (non-expanded) donors, suppression of GVHD was significantly greater in recipients of spleen cells (by weight loss, clinical score and survival). In summary, we have identified a novel strategy to rapidly and transiently expand donor Treg cell numbers in situ. The splenic compartment contained increased numbers and more functionally active Tregs compared to lymph nodes. Overall, TL1A-Ig/IL-2 expanded Tregs demonstrated excellent suppressive function both ex vivo and in vivo, as assessed by GVHD incidence and severity following allogeneic transplantation. This approach may be a promising strategy for GVHD prevention or therapy in the clinical setting.

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