Abstract Background. Graft versus host disease (GVHD) remains a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplant (HSCT) and improved GVHD prevention is critical. Invariant natural killer T (iNKT) cells are rare lymphocytes with roles in anti-microbial defense, cancer immunity, and immune regulation, namely, GVHD suppression. The invariant T-cell receptor (iTCR) recognizes glycolipids presented by CD1d, a monomorphic MHC class I-like molecule, thus, iNKT cells are incapable of allorecognition and causing GVHD (O'Neal,2024), a critical distinction from non-iNKT αβ T cells. Human correlative (Chaidos,2012) and murine studies show IL-4 dependent GVHD reduction with increased iNKT cells (Schneidawind,2014). Importantly, allogeneic iNKT cell transfer was shown to be safe in clinical trials to treat malignancies (Ramos,2021). These studies strongly support the novel clinical use of iNKT cell adoptive transfer for GVHD prevention. Notably, frozen regulatory T cell products lose CD62L expression causing a detrimental functional loss in preventing GVHD (Florek,2015); this may also apply to iNKT cells which have improved in vivo survival when CD62L+ (O'Neal,2024). We hypothesize it is feasible to produce an iNKT cell product with desired purity (90%) and cell counts (2e8) with retained cytokine production for use in a phase I study of ex vivo expanded iNKT cell-mediated GVHD prevention following allo-HSCT. Methods. Peripheral blood mononuclear cells (PBMCs) isolated from leukoreduction system (LRS) chambers were enriched for iNKT cells using iTCR antibody-conjugated magnetic beads (Miltenyi Biotech) and manual columns. Enriched iNKT cells and irradiated PBMCs (40Gy) were co-cultured with α-GalCer (100ng/mL) and IL-2 (200IU/mL). After 14 days of expansion, fresh cells were analyzed or cryopreserved. IL-4 secretion was measured after PMA/I stimulation with an IL-4 secretion assay PE detection kit (Miltenyi Biotech). Starting iNKT cell purity was defined as pre-enrichment %iNKT of T cells. Post-expansion total cell counts were extrapolated for 1e9 starting cells. The goal of 5e7 cells/LRS chamber equates to ≥2e8 cells/apheresis product, our highest planned cell dose. Results. We found 11 of 15 donors reached 90% post-expansion purity. Starting and post-expansion iNKT purity did not correlate (r=0.3, p=0.27), but all donors with 90% post-expansion purity started with 0.07% iNKT cell purity. Starting iNKT cell purity ≥0.07% positively correlated with ≥5e7 cells post-expansion (6 of 7 donors, r=0.75, p=0.0498); the outlier was expanded from frozen. With double column iNKT enrichment, we found 8 of 12 donors expanded to 90% purity with starting iNKT purities ≥0.031% (r=0.22, p=0.48) but only 4 of these 8 donors had 5e7 cells post-expansion. We found a mean of 5.3e6 αβ (non-iNKT) T cells (n=7, range 1.4-11.6e6) in our expanded products (extrapolated to a cell dose of 2e8), well below our estimated maximum of 8e6 αβ T cells (≤1e5 αβ T cells/kg for an average 80kg adult). Only 1 of our 7 donors would require product reduction based on αβ T cell content. Secreted IL-4 was detected (n=4) in a mean of 50.4% iNKT cells (range 33.4-73.3%), 0.4% in unstimulated. Upon cryopreservation and thawing, Cryostor had reduced cell loss, mean 29.3%, compared to Synth-A-Freeze, mean 35.2% (p=0.05, SEM 2.6, n=10). CD62L expression peaked at day 14, mean 62.2%, decreased immediately post-thaw, mean 40%, and returned to a mean 55.4% after 24hrs rest. Conclusions. Our preclinical feasibility analysis of expanding iNKT cells for a clinical trial found that donor starting iNKT cell purity was a critical factor to predicting expansion success and double column iNKT cell enrichment was inconsistently successful. Thus, we concluded a donor cut-off of ≥0.07% iNKT of T cells with single column enrichment will consistently obtain the desired post-expansion purity and cell counts, with few products requiring dose reduction due to αβ T cell content at our highest dose level. We showed retained IL-4 secretion capacity post-expansion and similar CD62L expression post-freeze/thaw in rested iNKT cells, suggesting these cells maintain viability and immuno-suppressive functions. Future work will assess the feasibility of expanding iNKT cells from apheresis products with current Good Manufacturing Practice (cGMP) reagents and automated iNKT enrichment. The resultant iNKT cell expansion protocol will be used in our phase I clinical trial.
Busken et al. (Mon,) studied this question.