The clinical potential of current chimeric antigen receptor-engineered T (CAR-T) cell therapy is hampered by its autologous nature that poses considerable challenges in manufacturing, costs and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture method to differentiate gene-engineered hematopoietic stem and progenitor (HSP) cells into allogeneic invariant natural killer T (AlloNKT) cells and their CAR-armed derivatives (AlloCAR-NKT cells). We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate AlloCAR-NKT cells, including HSP cell engineering, HSP cell expansion, NKT cell differentiation, NKT cell deep differentiation and NKT cell expansion. In addition, we describe procedures for evaluating the pharmacology, antitumor efficacy and mechanism of action of AlloCAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature AlloCAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.
© 2025. Springer Nature Limited.

Given the time and resources invested in clinical trials, innovative prediction methods are needed to decrease late-stage failure in vaccine development. We identify combinations of early innate responses that predict neutralizing antibody (nAb) responses induced in HIV-Env SOSIP immunized cynomolgus macaques using various routes of vaccine injection and adjuvants. We analyze blood myeloid cells before and 24 h after each immunization by mass cytometry using a three-step clustering, and we discriminate unique vaccine signatures based on HLA-DR, CD39, CD86, CD11b, CD45, CD64, CD14, CD32, CD11c, CD123, CD4, CD16, and CADM1 surface expression. Various combinations of these markers characterize cell families positively associated with nAb production, whereas CADM1-expressing cells are negatively associated (p < 0.05). Our results demonstrate that monitoring immune signatures during early vaccine development could assist in identifying biomarkers that predict vaccine immunogenicity.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.
Copyright © 2019 Elsevier Inc. All rights reserved.

Allergic rhinitis (AR) is characterized by mucosal inflammation, driven by activated immune cells. Mast cells and TH2 cells might decrease epithelial barrier integrity in AR, maintaining a leaky epithelial barrier.
We sought to investigate the role of histamine and TH2 cells in driving epithelial barrier dysfunction in AR.
Air-liquid interface cultures of primary nasal epithelial cells were used to measure transepithelial electrical resistance, paracellular flux of fluorescein isothiocyanate-dextran 4 kDa, and mRNA expression of tight junctions. Nasal secretions were collected from healthy control subjects, AR patients, and idiopathic rhinitis patients and were tested in vitro. In addition, the effect of activated TH1 and TH2 cells, mast cells, and neurons was tested in vitro. The effect of IL-4, IL-13, IFN-γ, and TNF-α on mucosal permeability was tested in vivo.
Histamine as well as nasal secretions of AR but not idiopathic rhinitis patients rapidly decreased epithelial barrier integrity in vitro. Pretreatment with histamine receptor-1 antagonist, azelastine prevented the early effect of nasal secretions of AR patients on epithelial integrity. Supernatant of activated TH1 and TH2 cells impaired epithelial integrity, while treatment with anti-TNF-α or anti-IL-4Rα monoclonal antibodies restored the TH1- and TH2-induced epithelial barrier dysfunction, respectively. IL-4, IFN-γ, and TNF-α enhanced mucosal permeability in mice. Antagonizing IL-4 prevented mucosal barrier disruption and tight junction downregulation in a mouse model of house dust mite allergic airway inflammation.
Our data indicate a key role for allergic inflammatory mediators in modulating nasal epithelial barrier integrity in the pathophysiology in AR.
Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.