Summary Donor lymphocyte infusion (DLI) is an effective method to establish full donor chimerism or to prevent and treat relapse after allogeneic haematopoietic cell transplantation (allo‐HCT). Usually, DLIs are collected from naïve donors as steady‐state lymphocytes. When donor lymphocytes are collected during stem cell apheresis, donors are pre‐treated with granulocyte colony‐stimulating factor (G‐CSF). However, the impact of G‐CSF stimulation and the resulting composition of DLIs on beneficial anti‐leukaemic responses and survival remain elusive. Therefore, we performed a retrospective analysis to evaluate the role of G‐CSF‐DLIs: 44 patients received either steady‐state DLIs or G‐CSF‐DLIs to prevent and treat relapse or establish full donor chimerism after allo‐HCT. The G‐CSF‐DLI patient cohort showed an improved conversion to full donor chimerism and a lower cumulative incidence of relapse or disease progression without a significantly increased cumulative incidence of graft‐versus‐host disease (GVHD). CD34+ cells, monocytic myeloid‐derived suppressor cells and monocytes as well as donor age and the subsequent occurrence of chronic GVHD were identified as risk factors that significantly improve overall survival after DLI administration. In conclusion, our data suggest that administration of G‐CSF‐DLIs results in graft‐versus‐leukaemia effects without exacerbating GVHD, therefore, improving survival after DLIs.

Graft-versus-host disease (GVHD) is a major cause of significant morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses, protect from lethal GVHD, and promote graft-versus-leukemia effects in murine studies. Since iNKT cells constitute less than 0.5% of human peripheral blood mononuclear cells (PBMCs), in vitro expansion with their glycolipid ligands is required before they can be used for cytotherapy and experimental purposes. Three weeks of cell culture and autologous restimulation with either KRN7000, PBS44, or PBS57 resulted in a robust proliferation of iNKT cells from human PBMCs. Next, iNKT cells were sorted to a purity higher than 90% being crucial for further experimental and clinical applications. These iNKT cells significantly decreased activation and proliferation of allogeneic CD3+ T lymphocytes. In addition, leukemia cell lines and primary leukemia cells were efficiently lysed by culture-expanded iNKT cells. Importantly, culture-expanded donor iNKT cells promoted robust antileukemia activity against HLA-matched allogeneic patient leukemia cells. Our data indicate that the adoptive transfer of culture-expanded iNKT cells could be a powerful cytotherapeutic approach to induce immune tolerance and prevent leukemia relapse after allogeneic HCT in humans.

Allogeneic hematopoietic cell transplantation (allo-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 graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.

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. Disclosures Handgretinger: Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Bethge:Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding; Neovii GmbH: Honoraria, Research Funding.