Invariant Natural Killer T Cells from Donor Lymphocyte Infusions (DLI-iNKTs) Contribute to Anti-Tumor Immunity after Allogeneic Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting potent graft-versus-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients. We showed previously that invariant natural killer T (iNKT) cells promote anti-tumor immunity in murine models of allogeneic HCT without exacerbating graft-versus-host disease (GVHD).

We therefore studied iNKT cells in DLIs and investigated how culture-expanded iNKT cells from such DLIs (DLI-iNKTs) could lyse leukemia cells. We analyzed 63 cryopreserved DLI samples by flow cytometry. iNKT cells were identified using the PBS57-loaded CD1d tetramer. Under steady state conditions, iNKT cells represent 0.04% (range, 0.01-0.6) of live donor lymphocytes and need to undergo ex vivo expansion for further experiments and clinical application. We established a two-week protocol resulting in a 300-fold expansion of functional DLI-iNKTs with a purity of 94%. Interestingly, we observed a preferential expansion of CD4+ DLI-iNKTs. This subset turned out to be critical for tolerance induction and prevention of GVHD after allogeneic HCT. For degranulation and leukemia cell lysis assays, culture-expanded DLI-iNKTs were co-cultured with leukemia cells. CD107a as a marker of degranulation and iNKT-cell activity was upregulated on DLI-iNKTs upon engagement with leukemia cells that were subsequently lysed in a dose-dependent manner. We also observed an increased release of cytokines like IFN-γ (85 vs. 7 pg/ml, p=0.04). Moreover, the cytotoxic effects of culture-expanded DLI-iNKTs were CD1d-dependent: blocking the interaction between the MHC-I-like molecule CD1d and the T-cell receptor of DLI-iNKTs abrogated iNKT-cell degranulation and efficient leukemia cell lysis.

Our results suggest that iNKT cells from DLIs are involved in anti-tumor immunity after allogeneic HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival. Prior expansion of iNKT cells could promote beneficial effects on tumor control, immune tolerance and overall survival.