Influenza infection following allogeneic hematopoietic cell transplantation (allo-HCT) can result in severe complications. The effectiveness of the annual vaccine depends on age, immune competence and the antigenic potential of the 3 strains included (1). . We hypothesized that a second vaccine dose, the standard of care for vaccine-naïve children, might improve post-HCT immune responses. Patients >60 days post-HCT were randomized to receive either 1 (n=33) or 2 (n=32) influenza vaccine doses separated by one month. The primary endpoint was whether two vaccinations induced superior immunity, however, we found no difference. Secondary endpoints were to identify variables associated with responses. Both hemagglutination inhibition (HAI) (p<0.005) and ELISpot responses (p=0.03) were greater for patients vaccinated ≥1 year post transplant. UCB recipients showed less IFN-γ responses (p=<0.001). Interestingly, there was a positive correlation between the total number of CD19+ cells prior to vaccination and seroconversion (p=0.01) and an inverse correlation for IFN-γ responses (p=0.05). Variables not associated with vaccine responses included: pre-vaccine CD4+ cell counts (total, naïve or memory), steroid usage at vaccination, age, or conditioning intensity. Time from transplantation to vaccination and absolute CD19+ cell counts were the strongest predictors of vaccine responses. Methods to improve influenza vaccine responses after allo-HCT are needed.

1,25-dihydroxy1,25(OH)2D3 [1,25(OH)2D3] is the biologically active form of Vitamin D and is immunoregulatory. 1,25(OH)2D3 binds the Vitamin D Receptor (VDR) complex present in many immune populations and can illicit transcriptional responses that vary amongst different immune subsets. The effects of 1,25(OH)2D3 on mature and developing human natural killer (NK) cells are not well characterized. Here we studied the influence of 1,25(OH)2D3 using an established NK cell differentiation system. Briefly, UCB CD34+ cells were isolated and cultured in conditions optimal for natural killer (NK) cell differentiation and varying concentrations of 1,25(OH)2D3 were administered. At physiological concentrations (10 nM),1,25(OH)2D3 impaired NK cell development. Moreover, the NK cells that did develop under the influence of 1,25(OH)2D3 showed a significant reduction in function (cytotoxicity and cytokine production). Conversely,1,25(OH)2D3 strongly induced hematopoietic stem cells to differentiate along a myeloid pathway, giving rise to CD14+ cells. Mechanistically, 1,25(OH)2D3 drives hematopoietic progenitor cells to rapidly upregulate monocyte genes (i.e. C/EBPα and CD14). There were no effects of 1,25(OH)2D3 on mature NK cytotoxicity or cytokine production. Collectively, these studies provide novel data showing the negative regulatory effect of 1,25(OH)2D3 on NK cell development.

Natural killer (NK) cells differentiated from hematopietic stem cells (HSCs)may have significant clinical benefits over those from adult donors, including the ability to choose allo-reactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study NK differentiation from HSCs. Stroma and cytokines support NK differentiation, but may have considerable regulatory hurdles. Recently, a clinical grade heparin-based method has been reported and could serve as an alternative. How these two compare in NK generating efficiency or functionis unknown. We show that compared to heparin-based cultures, stromasignificantly increases the yield of HSC-derived NK cells by differentiating less committed progenitors into the NK lineage. NK cells generated by both approaches were similar for most NK activating or inhibitor receptors. While both approaches resulted in a phenotype consistent with CD56brightstage IV NK cells, heparin-based cultures favored the development of CD56+CD16+ cells, while stromaproducedmore KIR-expressing NK cells, both markers of terminal maturation. At day 21, stromal-based cultures showed significantly more IL-22 production and both methods yielded similar amounts of IFN-γ production and cytotoxicity (day 35). Thus, heparin-based cultures are an effective replacement for stroma and may facilitate clinical trials testing HSC-derived NK cells.

Death receptor 3 (DR3, TNFRSF25) is expressed by activated lymphocytes and signaling by its ligand, TL1A, enhances cytokine expression and proliferation. Recent studies show that DR3 is also present on murine type 2 innate lymphoid cells (ILC2s). Here, we show that DR3 is expressed by IL-22 producing human group 3 innate lymphoid cells (ILC3s). Stimulation of ILC3 cells with exogenous TL1A alone had no impact on cytokine production or proliferation. Addition of TL1A to IL-1β + IL-23 significantly enhanced the amount IL-22 produced by ILC3 cells as well as the percentage IL-22 and IL-8 producing cells. Addition of TL1A to IL-1β + IL-23 also augmented ILC3 proliferation in short term (5 day assays). Mechanistically, this occurred through the up-regulation of CD25 and responsiveness to IL-2 stimulation. The combination of TL1A, IL-1β+ IL-23 and IL-2 expanded ILC3 cells (39.3 fold) while IL-1β+ IL-23 did not increase proliferation above controls. After two weeks of expansion, ILC3 cells maintained their phenotype, transcription factor expression and function (IL-22 production). These findings identify DR3 as a costimulatory molecule on ILC3 cells that can be exploited for ex vivo expansion and clinical use.

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. Disclosures: Miller: Coronado Biosciences: Scientific Advisory Board Other.