Expanded Human NKT Type II Cells: Effective Against Colon Cancer Cells, Safe across Major MHC Barriers and Successfully Expandable after Non-Myeloablative HCT for Solid Tumors.
Authors: Sangiolo Dario , Martinuzzi Emanuela , Carnevale-Schianca Fabrizio , Capaldi Antonio , Geuna Massimo , Vitaggio Katiuscia , Aglietta Massimo , Cignetti Alessandro 
Published: April 2020

Abstract
Cytokine activated cells (CIK) are ex-vivo expanded and activated cells expressing both T and NK surface markers (NKT type II, NKT-II). These cells have a broad non-MHC restricted antitumor cytotoxicity against autologous and allogeneic hematopoietic malignancies, while few data are available about their effectiveness against solid tumors. Murine studies demonstrated a reduced GVHD capacity of NKT-II cells when transplanted across major MHC barriers, making them a potential tool for adoptive therapy strategies after allogeneic hematopoietic cell transplant (HCT). Alloreactive potential of human NKT-II has been less investigated as well as their expansion from patients receiving an immunosuppressive treatment after HCT. We explored the alloreactive potential of human NKT- II and investigated NKT-II cytotoxicity against colon cancer cells. Additionally we looked at the possibility of expanding NKT-II from patients with solid tumors during immunosuppressive treatment after nonmyeloablative HCT (NM-HCT). NKT-II were obtained from PBMC, cultured for 3–4 weeks with timed addition of IL2, IFN-γ and anti-CD3. NKT-II with a CD3+/CD56+ phenotype were successfully expanded (median 140 folds, range 26–500) from both healthy donors (n=6) and patients (n=5); CD56+/CD3+ cells were either CD8+ (median 60%, range 18–86), CD4+ (7%, 1–27) or CD4/CD8 double negative (14%, 2–73). Expanded NKT-II cells efficiently proliferated in response to allogeneic irradiated PBMC as measured by 3H thymidine incorporation assay (n=2); this alloreactivity was comparable to a classic allogeneic mixed lymphocyte reaction used as control. Expanded NKT-II efficiently lysed 3 different colon cancer cell lines in standard chromium release assay (n=9). Interestingly, when NKT-II cultures were purified by positive selection of CD56+ cells, the CD56+ fraction did not either proliferate or have cytolitic activity against allogeneic PBMC, but fully retained the tumor killing capacity. Conversely, the CD56- fraction maintained the alloreactive potential but did not show any antitumor activity. These data suggest that the observed alloreactivity in the bulk NKT-II population is driven by CD56-/CD3+ cells, while the antitumor activity is due to CD56+/CD3+ T cells. The presence of pure NK cells (CD56+/CD3−) was negligible. When NKT-II cells were expanded after NM-HCT from patients affected by solid cancers (4 colorectal carcinoma, 1 osteosarcoma), they did not show significant differences as compared to NKT-II cells from healthy donors in terms of expansion and tumor killing. NKT-II were expanded from patients’ PBMC collected at a median of 42 days after a NM-HCT, when all patients were receiving cyclosporine as immunosuppression. In 2 cases analyzed, the degree of donor chimerism on expanded cells was comparable to the pre-expansion levels. In conclusion our data suggest that human NKT-II are able to kill colon cancer cells, even when expanded from chimeric patients treated with cyclosporine after allogeneic NM-HCT. The CD56+/CD3+ fraction is responsible for the antitumor activity and does not have a significant alloreactive potential across major MHC barriers. The presence of alloreactive CD56-/CD3+ cells in the bulk NKT-II population after the expansion should be taken into consideration for future application in the allogeneic setting, where a preventive CD56+ cell purification is advisable.