Death Receptor 3 (DR3) Is Expressed By Innate Lymphoid Cells (ILC) and Ligation By Tumor Like Antigen-1 (TL1A) Leads To Costimulation and Significant ILC Expansion

ROR-gt expressing innate lymphoid cells (ILCs) are unique lymphocytes that play important roles in the immune system and have applications to hematopoietic cell transplantation. In fetal life, ILCs orchestrate lymph node organogenesis, while in adults they facilitate the repair of damaged lymphoid structures and mediate mucosal immunity. ILCs function through the production of IL-22 and the expression of TNF-superfamily molecules (lymphotoxin, BAFF, and OX40L) that act on stroma or other lymphocytes, respectively. ILCs are almost exclusively found in the secondary lymphoid tissues (i.e., lymph nodes) and are essentially absent from the peripheral blood, making the study of these cells difficult and clinical application nearly impossible. To overcome these limitations, we devised a method to generate ILCs from hematopoietic stem cells (HSCs) cultured on irradiated stroma in the presence of cytokines (IL-7, IL-15, SCF and FLT3L) [Blood 11:4052-5, 2011]. After ∼21 days of culture, functional ILCs and conventional NK cells differentiate. While this system robustly leads to the ILCs differentiation, GMP-compatible methods of expansion will be required for clinical translation. We sought to identify factors involved in ILC growth and proliferation. After screening gene expression arrays on purified ILCs, the TNF superfamily receptor, known as Death Receptor 3 (DR3, TNFRSF25) was selected for further study. DR3 expression was confirmed using quantitative PCR. Compared to cNK cells, ILCs expressed significant quantities of mRNA for DR3 (p<0.001). While DR3 contains death receptor signaling domains, under some conditions it can also mediate T cell growth. We assessed DR3 functionality in purified ILCs by stimulating them with recombinant TL1A (TNSF15), the only reported ligand for DR3. Consistent with known effect of TL1A on NF-kB activation in T cells, NK-kB phosphorylation was also observed in ILCs. To determine the impact of TL1A on ILC function, IL-22 production was tested. When TL1A was added to ILCs, there was surprisingly no IL-22 production by intracellular cytokine staining or ELISA. We reasoned that TL1a may costimulate ILC activation and combined TL1A with IL-1b and IL-23, known to activate IL-22 production in ILCs. Compared to IL-1b and IL-23, the addition of TL1A led to a significantly higher percentage of IL-22 producing ILCs (9.3% vs. 23.3%, n=8, p<0.001) and more IL-22 production as determined by ELISA (3399 pg/ml vs. 8757 pg/ml, n=3, p<0.001). Nearly identical results were obtained for IL-8 production (p<0.001). Prior studies in T cells show that DR3 signaling increases the expression of the high affinity IL-2 receptor (CD25). While TL1A alone did not increase CD25 on resting ILCs and IL1b + IL-23 only marginally increased CD25 expression (∼1.7x increase from baseline), the combination of TL1A and IL1b + IL-23 led to significantly higher amounts of CD25 on the surface of ILCs (∼3.8x induction). We then tested whether IL-2 could be used to expand ILCs in vitro. Purified ILCs were treated in the following conditions: media (control), TL1A alone, IL1b + IL-23, or the combination of TL1A + IL-1b + IL-23 for 16 hours. Cells were then washed and cultured in media containing IL-2 (1,000U/ml). In short term (5 day) cultures, there was significantly more proliferation with TL1A + IL-1b + IL-23 (14.4% vs. 21.2% vs. 17.4% vs. 42%, n=7, p<0.001) as measured by CSFE dilution. When ILCs were cultured for 14 days, TL1A + IL-1b + IL-23 resulted in significantly greater expansion than IL1-b + IL23 cultured cells (39.3x vs. 14x, n=7, p=0.007). Since TL1A has been associated with skewing of T cells to IL-17 production and the onset of inflammatory conditions (Crohn’s disease), we assessed the expanded ILCs for the loss of IL-22 and/or the acquisition of IL-17. ILCs expanded in the presence of TLA1 + IL-1b + IL-23 had no change in their surface phenotype or capacity to produce IL-22. Importantly, neither IL-17 nor changes in RORgt or AHR mRNA expression were detected after expansion. Collectively, these studies identify a novel axis where DR3/TL1A signaling costimulates IL1b and IL-23 induced production of IL-22 and results in the expression of IL-2R (CD25) along with the associated proliferative response to IL-2. These studies significantly advance our ability to devise GMP-compliant methods to generate ILCs and pave the way for adoptive transfer experiments using ILCs in humans.