Human intestinal and circulating invariant natural killer T cells are cytotoxic against colorectal cancer cells via the perforin–granzyme pathway

Abstract

Invariant natural killer T (iNKT) cells are lipid-specific T lymphocytes endowed with cytotoxic activities and are thus considered important in antitumor immunity. While several studies have demonstrated iNKT cell cytotoxicity against different tumors, very little is known about their cellkilling activities in human colorectal cancer (CRC). Our aim was to assess whether human iNKT cells are cytotoxic against colon cancer cells and the mechanisms underlying this activity. For this purpose, we generated stable iNKT cell lines from peripheral blood and colon specimens and used NK-92 and peripheral blood natural killer cells as cell-mediated cytotoxicity controls. In vitro cytotoxicity was assessed using a panel of well-characterized human CRC cell lines, and the cellular requirements for iNKT cell cytotoxic functions were evaluated. We demonstrated that both intestinal and circulating iNKT cells were cytotoxic against the entire panel of CRC lines, as well as against freshly isolated patient-derived colonic epithelial cancer cells. Perforin and/or granzyme inhibition impaired iNKT cell cytotoxicity, whereas T-cell receptor (TCR) signaling was a less stringent requirement for efficient killing. This study is the first evidence of tissue-derived iNKT cell cytotoxic activity in humans, as it shows that iNKT cells depend on the perforin-granzyme pathway and both adaptive and innate signal recognition for proper elimination of colon cancer cells.

Adoptive Immunotherapy With Engineered iNKT Cells to Target Cancer Cells and the Suppressive Microenvironment

“…Also in human cancers CD1d is mainly expressed by the hematopoietic ones ( 45 ), whereas very few solid tumors are CD1d-positive ( 53 – 55 ). Furthermore, cancer cells can downregulate CD1d surface expression, thereby limiting direct recognition by iNKT cells ( 55 – 57 ), even though they have shown to kill by perforin and/or granzymes at least colon cancer cells also in a CD1d-independent manner ( 58 ). The mechanisms underlying CD1d downregulation or loss by cancer cells are largely unknown.…”

Tumor infiltrating iNKT cells sustain neutrophil pro-tumorigenic functions influencing disease progression in human colorectal cancer

“…iNKT cell cytotoxicity assay iNKT cell cytotoxicity toward the human CRC cell lines Colo205 and RKO (American Type Culture Collection, ATCC) was performed as previously described 17 . Cytotoxicity was assessed using the Cytotoxicity Lactate Dehydrogenase (LDH) Assay Kit-WST (nonhomogeneous assay, Dojindo, EU) following the manufacturer's instructions.…”

“…iNKT cells are considered important in antitumor immunity 10 and their infiltration in tumoral lesions is a positive prognostic factor in different cancer types 14,15 . Moreover, iNKT cells possess cytotoxic properties 16 and are endowed with cell-killing activities towards different human CRC cell lines and primary patient’s derived cancer epithelial cells through the perforin– granzyme pathway 17 . However, their role in CRC progression has never been fully elucidated and it is still controversial 14,18 .…”

Overcoming the challenges in translational development of natural killer cell therapeutics: An opinion paper

“…Hence, several research efforts are geared towards unravelling possible NK mechanism of cytolysis like mitochondrial apoptosis (64) and release of perforin and granzymes (69); factors that may increase their cytotoxicity such as E26 transformation-specific transcription factor ELK3 expression by cancer cells (70); factors that attenuate cytolytic function like increased transforming growth factor-beta 1 (TGF-b1) (32), inhibition of O-GlcNAcylation (71), low surface expression and impaired function of transient receptor potential melastatin 3 (TRPM3) (32, 71-75); and addressing the complications accompanying rejection-prone cellular products (67,76,77).…”