IL-2/IL-2R, TL1A/TNFRSF25 or Their Combined Stimulation Results in Distinct CD4+FoxP3+ Regulatory T Cell Phenotype and Suppressive Function

Abstract

CD4+Foxp3+ regulatory T cells (Tregs) are non-redundant mediators of immunity and tolerance. Transfer of Tregs has emerged as a promising therapy for graft-versus-host disease (GVHD) following allogeneic HSCT (aHSCT). Our prior work demonstrated that a 2-pathway in vivo strategy targeting TNFRSF25 (with TL1A-Ig) and IL-2 receptor (with low-dose IL-2, IL-2LD) can elicit a strong increase in Treg numbers and function. In fact, very low numbers of these in vivo expanded donor Tregs suppressed GVHD post-aHSCT. Based on these findings and the success of IL-2 in pre-clinical and clinical studies, we asked: are there phenotypic/functional differences between Tregs expanded via IL-2 LD, TL1A-Ig or their combined application? Mice were administered IL-2LD, TL1A-Ig or TL1A-Ig+IL-2LD over 6 days. Splenic and lymph node (LN) Treg phenotype was determined by flow cytometry. Treg functionality was assessed with sorted populations using an in vivo MHC-mismatched aHSCT. Treatment of B6-FoxP3RFP mice with TL1A-Ig+IL-2LD vs. IL-2LD only resulted in significantly higher levels of Treg activation/differentiation and functional markers, i.e. KLRG1, CD103, Nrp1, ICOS (Fig. 1A). Ki67 expression was higher in 2-pathway vs. IL-2LD stimulation alone (Fig.1B). Notably, TL1A-Ig treatment alone induced the above phenotype. CD25 (IL-2Rα) expression was reduced after TL1A-Ig vs. IL-2-mediated expansion further emphasizing the difference between the TNFRSF25 and IL-2 receptor stimulation (Fig. 1A). Results were corroborated using a second independent mouse strain, BALB/c, following use of these protocols. To test if the observed phenotypic differences could be related to more potent Treg function in vivo, an MHC-mismatched aHSCT (B6→BALB/c) was performed using 200,000 sorted Tregs (>98% purity by CD4+FoxP3+ selection from C57BL/6-FoxP3RFP reporter mice) from donor unexpanded, IL-2LD, or TL1A-Ig+IL-2LD treated mice + T cells. As anticipated, transfer 200,000 TL1A-Ig+IL-2LD stimulated Tregs ameliorated acute GVHD (Fig. 1C). Interestingly, lower GVHD clinical scores were obtained using the same number of IL-2LD only vs.TL1A-Ig+IL-2LD stimulated Tregs (Fig. 1C). Moreover, early post-transplant, higher LN and splenic CD4/CD8 ratios were detected in aHSCT recipients treated with IL-2LD expanded Tregs vs. TL1A-Ig+IL-2LD (Fig. 1D). Overall, TL1A-Ig+IL-2LD promotes a more activated/differentiated and proliferative Treg phenotype accompanied by less effective long-term GVHD inhibition vs. IL-2LD only stimulated Tregs. We posit that these different 1- and/or 2-pathway driven Tregs may promote development of new strategies utilizing one or both populations dependent on the desire for GVHD prophylaxis or therapeutic treatment following aHSCT.