Soluble factors in iNKT cell cultures mediate degranulation of iNKT cells toward K562 cells. A, Jurkat and K562 cells were stained with an anti-CD1d Ab and analyzed by flow cytometry. B–E, PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. iNKT cells were then treated with IL2 until analysis. B, iNKT cells maintained for 3 days after sorting were cocultured with K562, Jurkat, or α-GalCer–pulsed Jurkat cells at an E/T ratio of 2:1 and then a CD107a assay was performed. C and D, iNKT cells maintained overnight after sorting were washed and cultured in fresh medium or maintained in the same medium (control) for 2 days. C, iNKT cells were cocultured with K562 cells at an E/T ratio of 2:1 and then a CD107a assay was performed. D, iNKT cells were cocultured with K562 cells at 10:1, 5:1, and 2.5:1 E/T ratios. An LDH release assay was performed and the percentage cytotoxicity was calculated. E, Sorted iNKT cells were maintained overnight and then the culture supernatant was collected. iNKT cells maintained overnight after sorting were washed and then maintained for 4 days. The culture supernatant was added to an iNKT cell culture overnight prior to coculture with K562 cells for the CD107a assay. Data are representative of two independent experiments. Data represent the mean ± SEM. ***, P < 0.001; ****, P < 0.0001. HD, healthy donor.
Funding
MEXT | Japan Society for the Promotion of Science (JSPS)
Takeda Science Foundation (TSF)
Chiba University Futuristic Medical Fund
ARTICLE ABSTRACT
Invariant natural killer T (iNKT) cells play an essential role in antitumor immunity by exerting cytotoxicity and producing massive amounts of cytokines. iNKT cells express invariant T-cell receptors (TCR) to recognize their cognate glycolipid antigens such as α-galactosylceramide (α-GalCer) presented on CD1d. We recently reported that iNKT cells recognize CD1d-negative leukemia cell line K562 in a TCR-dependent manner. However, it remains controversial how iNKT cells use TCRs to recognize and exhibit cytotoxic activity toward CD1d-negative tumors cells without CD1d restriction. Here, we report that iNKT cells exerted cytotoxicity toward K562 cells via a carried over anti-Vα24 TCR mAb from positive selection by magnetic bead sorting. We found that addition of the anti-Vα24Jα18 TCR mAb (6B11 mAb) rendered iNKT cells cytotoxic to K562 cells in an FcγRII (CD32)-dependent manner. Moreover, iNKT cells treated with 6B11 mAb became cytotoxic to other CD32+ cell lines (U937 and Daudi). In addition, iNKT cells treated with 6B11 mAb suppressed K562 cell growth in a murine xenograft model in vivo. These data suggest that anti-iNKT TCR mAb treatment of iNKT cells can be applied as a therapeutic strategy to treat CD32+ cancers such as leukemia, lymphoma, and lung cancer.
Our findings unveiled that iNKT cells recognize and kill CD1d-negative target tumors via the anti-iNKT TCR mAb bound to CD32 at the tumor site, thereby bridging iNKT cells and CD1d-negative tumors. These findings shed light on the therapeutic potential of anti-iNKT TCR mAbs in NKT cell–based immunotherapy to treat CD1d-negative CD32+ cancers.
