CD3/CD28 Co-Stimulated Activation of PI3K-mTORC2-AKT Metabolic Programming Augments Granzyme B Expression and Direct Cytotoxicity in Expanded Human iNKT Cells

Invariant natural killer T (iNKT) lymphocytes are rare but potent effectors of immune regulation and tumor immunosurveillance. iNKT cells are MHC-non-restricted and thus ideal candidates for separation of GVHD and GVT after either MHC-matched or mismatched allogeneic hematopoietic cell transplantation (HCT). We have optimized a reproducible therapeutic expansion protocol for human iNKT cells from peripheral blood pheresis units and used this platform to delineate targetable pathways to augment cytokine secretion and direct cytotoxicity of expanded CD3⁺Vα24⁺Vβ11⁺ iNKT cells against lymphoid malignancies. Expanded, anti-CD2/CD3/CD28-stimulated iNKT cells expressed significantly higher (> 1500 pg/mL) levels of Th2 cytokines IL-4, IL-5, and IL-13, and GM-CSF, and substantially higher intracellular granzyme B (GrB) and PI3-kinase downstream phospho-AKT (p-AKT) at both the mRNA and protein levels as compared to iNKT cells treated with vehicle (P < 0.01), α-galactosylceramide (α-GalCer) (P < 0.01), or anti-Vα24 CDR3 loop stimulating antibody clone 6B11 (P < 0.01) (n = 4-6 serial expansions). In vitro iNKT cytotoxicity against NALM/6 Ph+ B-lineage acute lymphoblastic leukemia (B-ALL) was significantly augmented by CD2/CD3/CD28 activation (mean ± SEM % cytotoxicity in BADTA^® assay at 1:1 iNKT: target ratio 60.8 ± 2.8, P < 0.001, n = 12) but not α-GalCer stimulation (11.5 ± 5.0, P = 0.98 n = 12) or 6B11 treatment (11.0 ± 4.1, P = 0.99, n = 12) as compared to vehicle control. Potent augmentation of tumor clearance in vivo after CD2/CD3/CD28 but not α-GalCer or 6B11 stimulation was confirmed in luciferase+ NALM/6 xenograft-bearing C.B17 SCID mice (n = 25 mice/group; P < 0.001). Specific GrB inhibition by iNKT pre-treatment with the non-competitive inhibitor Z-AAD-CMK abrogated CD2/CD3/CD28 augmented iNKT cytotoxicity, both in vitro and in vivo. Using permutations of CD2, CD3, and CD28 stimulation combined with specific inhibitors of the PI3K-AKT-mTORC pathway, we defined that CD3/CD28-co-stimulated, PI3K-downstream, mTORC2-driven AKT activation directly up-regulates GrB protein expression and augments GrB-dependent cytotoxicity in expanded human iNKT cells. Notably, CD2/CD3/CD28-induced augmented iNKT killing was not inhibited by cyclosporin A, rapamycin, or tacrolimus. These data demonstrate the novel potential for augmentation of cytokine secretion and cytotoxicity in human iNKT cells through AKT signaling independent of mTORC1 or NF-AT, defining a key pathway for control of iNKT effector functions with broad implications for immunotherapy, vaccine augmentation, and anti-infective therapies. As our studies utilized random donor human iNKT cells expanded using a clinically applicable protocol, these data are directly relevant to therapeutic human iNKT cell manipulation to separate GVHD and GVT in the peri-transplant or non-transplant setting of human iNKT immunotherapy.