Ovarian cancer (OC) is highly lethal due to late detection and frequent recurrence. Initial treatments, comprising surgery and chemotherapy, lead to disease remission but are invariably associated with subsequent relapse. The identification of novel therapies and an improved understanding of the molecular and cellular characteristics of OC are urgently needed. Here, we conducted a comprehensive analysis of primary tumor cells and their microenvironment from 16 chemonaive and 10 recurrent OC patient samples. Profiling OC tumor biomarkers allowed for the identification of potential molecular targets for developing immunotherapies, while profiling the microenvironment yielded insights into its cellular composition and property changes between chemonaive and recurrent samples. Notably, we identified CD1d as a biomarker of the OC microenvironment and demonstrated its targeting by invariant natural killer T (iNKT) cells. Overall, our study presents a comprehensive immuno-profiling of OC tumor and microenvironment during disease progression, guiding the development of immunotherapies for OC treatment, especially for recurrent disease.
© 2023 The Author(s).

Cytomegalovirus (CMV) reactivation remains a common complication and leads to high mortality in patients who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Early natural killer (NK) cell reconstitution may protect against the development of human CMV (HCMV) infection post-HSCT. Our previous data showed that ex vivo mbIL21/4-1BBL-expanded NK cells exhibited high cytotoxicity against leukemia cells. Nevertheless, whether expanded NK cells have stronger anti-HCMV function is unknown. Herein, we compared the anti-HCMV functions of ex vivo expanded NK cells and primary NK cells. Expanded NK cells showed higher expression of activating receptors, chemokine receptors and adhesion molecules; stronger cytotoxicity against HCMV-infected fibroblasts; and better inhibition of HCMV propagation in vitro than primary NK cells. In HCMV-infected humanized mice, expanded NK cell infusion resulted in higher NK cell persistence and more effective tissue HCMV elimination than primary NK cell infusion. A clinical cohort of 20 post-HSCT patients who underwent adoptive NK cell infusion had a significantly lower cumulative incidence of HCMV infection (HR = 0.54, 95% CI = 0.32-0.93, p = 0.042) and refractory HCMV infection (HR = 0.34, 95% CI = 0.18-0.65, p = 0.009) than controls and better NK cell reconstitution on day 30 post NK cell infusion. In conclusion, expanded NK cells exhibit stronger effects than primary NK cells against HCMV infection both in vivo and in vitro.
© 2023. The Author(s), under exclusive licence to CSI and USTC.

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.
© 2023 The Author(s).

Allo-HSCT is a curative therapy for hematologic malignancies owing to GvL effect mediated by alloreactive T cells; however, the same T cells also mediate GvHD, a severe side effect limiting the widespread application of allo-HSCT in clinics. Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity. Here, we report the successful generation of third-party HSC-engineered human iNKT (3rdHSC-iNKT) cells using a method combining HSC gene engineering and in vitro HSC differentiation. The 3rdHSC-iNKT cells closely resembled the CD4-CD8-/+ subsets of endogenous human iNKT cells in phenotype and functionality. These cells displayed potent anti-GvHD functions by eliminating antigen-presenting myeloid cells in vitro and in xenograft models without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia, supporting 3rdHSC-iNKT cells as a promising off-the-shelf cell therapy candidate for GvHD prophylaxis.
© 2022 The Authors.

Cell-based immunotherapy has become the new-generation cancer medicine, and "off-the-shelf" cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.© 2021 The Authors.