TNFRSF25 and CD25 Stimulation Expands Tregs and ILC2s in the GI Tract: Recipient Modulation Pre-HSCT

Abstract

Our lab has shown that using a 2-pathway strategy of stimulating TNFRSF25 and CD25 with a TL1A-Ig fusion protein (FP) and low dose IL-2, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased. Tregs and innate lymphoid cells (ILCs) are critical to maintaining immune homeostasis and generating tolerance in the GI tract. In addition to a highly diverse population of Tregs, the GI also contains various populations of ILCs, recently shown to modulate GVHD (Bruce, 2017). ILCs express CD25 as well as TNFRSF25 and respond individually to IL-2 and TL1A administration respectively. Here we asked how our 2-pathway (FP+IL-2) approach affected Tregs and ILCs in the GI of healthy mice. First, we examined Tregs from B6-Nur77^GFP or B6-Nur77^GFPFoxP3^RFP mice to assess the Treg TcR signaling status in different tissues. Mice were administered FP, IL-2, or FP+IL-2 over 1 wk. The frequency of Treg/CD4 in the lamina propria (LP) of the large intestine (LI) reached levels >60% (1A). This elevation included FoxP3⁺RORyt⁻ and FoxP3⁺RORyt⁺ Tregs. In contrast to our previous findings in the LHC, treatment with FP alone elevated levels to the same extent as the FP+IL-2 in the LI/LP, whereas IL-2 treatment alone had only a modest effect on elevating the frequency of Tregs (1A), suggesting that the activation status of Tregs differs based on compartmental location. The frequency of GFP⁺ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LHC (<35%) (1C). An independent experiment verified these findings and found that only conjunctival Tregs had a similar activation (GFP) phenotype (1D). Interestingly, without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon (1B). Next, we evaluated FP+IL-2 treated B6-Nur77^GFP mice. Tregs - <i>but not CD8 T-cells</i> in the LHC had elevated GFP by frequency and MFI vs untreated mice (1E). Notably, we also found that FP+IL-2 treatment <i>in vivo</i> increases the relative frequency of both ILC2s and ILC3s (1F). Regarding ILC2s, FP+IL-2 increased their frequency greater than either TNFRSF25 or CD25 stimulation alone in the GI tract (1G). We then asked if our FP+IL-2 approach could be used to augment these populations as a potential strategy to protect HSCT recipients. FP+IL-2 stimulation prior to TBI in BALB/c mice significantly augmented Tregs and ILC2s in the GI tract for at least 1 wk post-irradiation (2A,2B). In total, these findings demonstrate 1) basal Treg activation status differs depending on the compartment and mucosal Tregs express highest TcR activation, 2) FP alone vs. FP+IL-2 stimulation results in equivalent expansion of GI Tregs, 3) only FP+IL-2 effectively expand ILC2s and 4) pre-TBI FP+IL-2 treatment results in elevated Treg and ILC2 levels post-irradiation. Ongoing experiments are examining GI GVHD and the role of Tregs/ILCs in TNFRSF25 +/- CD25 stimulated aHSCT recipients.