Off the Shelf Stem Cell Derived Innate Lymphoid Cells Ameliorate Gvhd through IL-9 Induced-T Cell Senescence

Innate lymphoid cells (ILCs) are tissue resident lymphocytes that play a key role in tissue homeostasis and the regulation of gastrointestinal tract inflammation. Prior studies have linked ILCs to allogeneic hematopoietic cell transplantation (allo-HCT) outcomes. For instance, ILC3-derived IL-22 drives intestinal stem cell regeneration and thymic epithelial cell protection from irradiation. Adoptive transfer of IL-25 and IL-33 mobilized ILC2s can protect some mice from lethal GVHD, and in human studies, high quantities of activated (CD69+) peripheral blood ILC2s and ILC3s have been correlated with less acute GVHD. Despite these studies, the precise mechanisms underlying ILC modulation of T cell inflammation are incompletely understood. Leveraging our established method for in vitro ILC differentiation from hematopoietic stem cells (HSCs), we investigated the interplay between HSC-derived ILCs and allogeneic T cells. Using xenogeneic GVHD models, 3^rd party human ILC2 (p=0.0007) or ILC3 (p=0.002) cells could significantly prolong survival. Likewise, in murine GVHD models (B6"Balb/c) adoptive transfer of either donor (p=0.04) or recipient (p=0.01) ILCs could also prolong survival. In 7 day co-culture experiments the addition of allogeneic ILC2 and ILC3 cells to CD3xCD28 activated T cells showed significant attenuation in T cell proliferation. Furthermore, comparing T cell cultures to co-culture with ILCs, there was a significant reduction in naïve T cells and an increase in the Tcm compartment. To further characterize the impact of ILCs on T cells, we performed transcriptomic analysis (RNAseq) and used gene set enrichment analysis (GSEA) to identify T cell pathways that were activated or inhibited with ILC co-culture. Of a total of 15 inhibited pathways with ILC2 coculture and 16 inhibited pathways with ILC3 coculture, 13 were shared. Among these were reductions in the proliferation-related gene sets (E2F Targets, MYC Targets V1, MYC Targets V2, G2M checkpoint and DNA repair hallmarks). Interestingly, the oxidative phosphorylation pathway was also inhibited in T cells following co-culture with ILC2s or ILC3s and seahorse assays confirmed these findings in the cocultured T cells. Our findings also revealed that ILC2s and ILC3s induced senescence in activated T cells as evidenced by reduced T cell proliferation, heightened cytokine production (Th1, Th2 and Th17), and upregulation of senescence-associated surface proteins (CD57, KLRG1, TIGIT, and TIM3). Moreover, these ILC-exposed T cells exhibited increased expression of senescence-related proteins (p16, p21, p53, GATA4, and NF-kB). Additional ILC and T cell coculture experiments showed similar findings even when contact was inhibited by a transwell membrane, suggesting that soluble molecules mediate these observations. Interrogating ILC2 and ILC3 for cytokine production demonstrated that IL-9 was produced by both populations and ILC-derived IL9 emerged as key regulator of T cell proliferation and senescence induction. More specifically, the inhibition of proliferation seen with ILCs was ameliorated with IL9 blocking antibodies (p<0.001) and replicated when T cells were cultured (in the absence of ILCs) with exogenous rhIL9 (p<0.01). Likewise, rhIL-9 upregulated the expression of senescence associated proteins in T cells (p21, p53, GATA4 and NFKb) and in short-term cultures rhIL-9 reduced the naïve T cell fraction while increasing the proportion of Tcm cells. Collectively, we show that human and mouse HSC-derived ILC2 and ILC3 cells mitigate GVHD through the induction of IL-9 mediated T cell senescence, thus offering promising insights into the development of off the shelf cellular therapies for GVHD.