Invariant NKT Cells Recognize Leukemia Cells with T-Cell and NK Receptors in a CD1d-Independent Manner

Background: Invariant natural killer T (iNKT) cells are known as CD1d-restricted T cells that express the invariant T-cell receptors (TCR) Vα24 and Vβ11 in humans and specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells presenting glycolipid antigens and indirect cytotoxicity by activating other cytotoxic immune cells or regulating CD1d-positive immunosuppressive cells in the tumor microenvironment. Although we previously reported that αGalCer-activated NKT cells exert a potent perforin-dependent cytotoxic activity against a wide variety of human tumor cell lines, the direct recognition of CD1d-negative tumors is controversial and the mechanism is unknown. Here we clarify whether iNKT cells recognize and exhibit cytotoxicity toward leukemia cells in a CD1d-independent manner and identify the molecule that recognizes CD1d-negative leukemia cells.

Methods: Purified iNKT cells were generated from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteer donors. PBMCs were cultured in complete RPMI 1640 medium for 9-14 days in the presence of 100 U/mL of recombinant human IL-2 and 200 ng/mL of αGalCer. The iNKT cells were then isolated with an autoMACS Pro separator using FITC-labeled anti-Vα24 antibody (clone, C15) and anti-FITC microbeads. We evaluated the cytotoxic activity of iNKT cells toward CD1d-negative leukemia cells within four days after isolation using a CD107a assay for degranulation, cytometric bead array for cytokine production, and cytotoxicity assay in vitro and in vivo. For in vivo cytotoxicity assays, NOG mice were inoculated with 1 × 10⁶ K562-luc cells on day 0 and with 4 × 10⁶ human iNKT cells on day 1. Gene knock-out (KO) was performed using a CRISPR/Cas9 system. T-cell or NK receptor-KO iNKT cells were used for experiments three or four days after electroporation of the Cas9 protein and guide RNA CRISPR ribonucleoprotein complex. Patient-derived leukemia cells were obtained from PBMCs or bone marrow mononuclear cells of pre-treatment pediatric patients. All studies were approved by the institutional review board and the Animal Care and Use Committee of Chiba University.

Results: We observed that iNKT cells degranulated and released Th1 cytokines when co-cultured with CD1d-negative leukemia cells (K562, HL-60, REH, and CD1d-KO U937) as well as αGalCer-loaded CD1d-positive leukemia cells (Jurkat), and showed in vitro cytotoxicity toward these CD1d-negative leukemia cells. This CD1d-independent degranulation decreased over time after isolation and was not restored with re-stimulation by αGalCer. The cytotoxicity of iNKT cells toward K562 cells was confirmed in vivo by comparsion with survival curves of K562-inoculated NOG mice given iNKT cells or PBS alone (log-rank, p= 0.016). To identify the receptors contributing to the CD1d-independent recognition and cytotoxicity against CD1d-negative leukemia cells, we first focused on costimulatory receptors, which are also known as activating NK receptors and are expressed on iNKT cells such as NKG2D, DNAM-1, 2B4, LFA-1, and CD2, and analyzed cytotoxicity after blocking these receptors with antibodies. We found that all costimulatory receptors that we assessed contributed to cytotoxicity toward CD1d-negative leukemia cells. Next, we analyzed cytotoxicity of TCR-KO iNKT cells toward CD1d-negative leukemia cells to confirm the contribution of TCR to CD1d-independent recognition. Notably, TCR-KO iNKT cells showed decreased degranulation, Th1 cytokine release, and cytotoxicity toward K562 cells more so than iNKT cells with KO of NK receptors such as LFA-1(CD11a) or CD2. To assess the clinical application potential of adoptive iNKT cell immunotherapy for leukemia treatment, we analyzed degranulation of iNKT cells using patient-derived leukemia cells. We found iNKT cells degranulation using cells from four out of five myeloid leukemia cases, but only one out of eight BCP-ALL cases (p = 0.032).

Conclusion: Primary iNKT cells activated by αGalCer can recognize and show anti-tumor effects toward leukemia cells in an unrestricted manner via CD1d. The TCR also has an important role in recognizing CD1d-negative leukemia cells and multiple NK receptors assist in cytotoxicity. Adoptive iNKT cell immunotherapy may be effective in treating myeloid leukemia.