Invariant natural killer T cells in hematopoietic stem cell transplantation: killer choice for natural suppression

Abstract

Invariant natural killer T cells (iNKTs) are innate-like lipid-reactive T lymphocytes that express an invariant T-cell receptor (TCR). Following engagement of the iTCR, iNKTs rapidly secrete copious amounts of Th1 and Th2 cytokines and promote the functions of several immune cells including NK, T, B and dendritic cells. Accordingly, iNKTs bridge the innate and adaptive immune responses and modulate susceptibility to autoimmunity, infection, allergy and cancer. Allogeneic hematopoietic stem cell transplantation (HSCT) is one of the most effective treatments for patients with hematologic malignancies. However, the beneficial graft versus leukemia (GvL) effect mediated by the conventional T cells contained within the allograft is often hampered by the concurrent occurrence of graft versus host disease (GvHD). Thus, developing strategies that can dissociate GvHD from GvL remain clinically challenging. Several preclinical and clinical studies demonstrate that iNKTs significantly attenuate GvHD without abrogating the GvL effect. Besides preserving the GvL activity of the donor graft, iNKTs themselves exert antitumor immune responses via direct and indirect mechanisms. Herein, we review the various mechanisms by which iNKTs provide antitumor immunity and discuss their roles in GvHD suppression. We also highlight the opportunities and obstacles in manipulating iNKTs for use in the cellular therapy of hematologic malignancies.

Figure 1.

Antitumor mechanisms of iNKTs. (a) Indirect mechanism of iNKT cytotoxicity. The cross-talk between iNKTs and Ag-presenting cells (such as DCs), presenting a tumor-derived glycolipid, leads to the activation of iNKTs, IFN-γ and CD40 stimulation. This iNKT-derived IFN-γ induces DC production of IL-12, which further augments IFN-γ production by iNKT and NK cells, and serves to stimulate CD8⁺ T cell- and NK cell-dependent killing of tumor cells. (b) iNKTs recognize glycolipid Ags presented by CD1d on tumor cells and mount direct cytotoxicity via perforin/granzyme exocytosis or Fas–Fas ligand (Fas L) interactions. (c) iNKTs can also limit tumor growth via their interactions with immunosuppressive cells that promote tumor growth such as TAMs and IL-10-producing neutrophils. Although iNKTs can directly kill TAMs, they alleviate the suppressive effect of the neutrophils via CD40–CD40L interactions.

Figure 2.

iNKTs protect from GvHD but retain the GvL activity. Reduced intensity host-conditioning with TLI and ATG allows host iNKT expansion. Both host and donor iNKTs interact with APCs via CD1d to produce IL-4 that in turn skews donor T cells toward a Th2 cytokine bias. Both host and donor iNKTs also promote nTreg expansion in an IL-4-dependent manner that can inhibit donor T-cell proliferation and migration to GvHD target organs. Recently described, third-party CD4⁺ iNKTs also inhibit T-cell proliferation, promote Th2-biased cytokine response as well as expansion of donor MDSCs. These donor MDSCs are crucial for nTreg expansion and protection from GvHD lethality. Importantly, iNKTs (host, donor or third party) do not abrogate donor T-cell anti-leukemia activity. Furthermore, iNKTs can also directly kill the leukemia cells and contribute to the GvL effect. Taken together, attenuation of GvHD without loss of the GvL activity decreases tumor burden and improves overall survival of the host.

Figure 3.

In vitro expansion and isolation of human iNKTs. (a) Human PBMC are cultured in complete medium (Aim-V, 10% fetal calf serum; recombinant human (rh) IL-2 (50 U/mL)) and αGC (500 ng/mL)). After 4 days, cultures are supplemented with rhIL-15 (10 ng/mL) and rh IL-2 (10 U/mL) and 4–5 days later, iNKTs are purified by MACS sorting, based on expression of Vα24, the α-chain of the iNKT TCR. Using this approach, we observe that iNKTs can be expanded 500–1000-fold and it is therefore very feasible to obtain the large number of cells within a week. (b) Representative FACS plots demonstrate how iNKTs can be successfully expanded from the blood and isolated to >99% purity. Alternatively, iNKTs can be first isolated from PBMCs by MACS or high-speed cell sorting and then expanded in vitro in the presence of αGC and cytokines (IL-2, IL-7 and IL-15). For long-term cultures, iNKTs can be restimulated every 8–12 days with αGC-pulsed, irradiated autologous PBMC in the presence of cytokines.