Scalable expansion of human iNKT cells: single-cell profiling and in vivo control of GvHD with preserved GvL activity
- 1IMoPA Universite de Lorraine CNRS
Abstract
Invariant natural killer T (iNKT) cells can limit graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), but their scarcity in peripheral blood limitstherapeutic development. Current clinical-grade human iNKT expansion protocols mainly rely on IL-2, require prior iNKT-cell sorting, last 6-8 weeks, and predominantly expand CD4+ iNKT cells, whereas human CD4- iNKT cells are more strongly associated with GVHD control in patients and uniquely regulate antigen-presenting cells and T-cell activation. We developed a scalable culture system to preferentially expand human CD4- iNKT cells directly from total peripheral blood mononuclear cells (PBMCs) using alpha galactosylceramide (-GalCer) and optimized cytokine conditions. IL-15 was the most effective cytokine. The optimized 14-day protocol generated a mean of 3.8x107 iNKT cells from 2x107 PBMCs, including 74% CD4- iNKT cells. Single-cell transcriptomic profiling identified eight major iNKT subsets, differentiation trajectories during expansion, and distinct IL-2- versus IL-15-associated transcriptional programs. IL-15-expanded iNKT cells induced apoptosis of monocyte-derived dendritic and leukemic cells in vitro, controlled xeno-GVHD, and preserved graft-versus-leukemia (GVL) activity in preclinical mouse models. This platform enables reproducible production of human CD4- iNKT cells at clinically relevant scale and position IL-15-expanded iNKT cells as a compelling immunotherapy candidate for allo-HSCT.
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BibTeX
@misc{brouard2026scalable,
title = {{Scalable expansion of human iNKT cells: single-cell profiling and in vivo control of GvHD with preserved GvL activity}},
author = {J. Brouard and C. Caraiman and G. FIEVET and L. Monchablon and T. Coman and S. Hergalant and S. Pagliuca and D. MOULIN and M. T. Rubio},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.11.750594},
url = {https://www.biorxiv.org/content/10.64898/2026.09.11.750594v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Scalable expansion of human iNKT cells: single-cell profiling and in vivo control of GvHD with preserved GvL activity
AU - J. Brouard
AU - C. Caraiman
AU - G. FIEVET
AU - L. Monchablon
AU - T. Coman
AU - S. Hergalant
AU - S. Pagliuca
AU - D. MOULIN
AU - M. T. Rubio
PY - 2026
DA - 2026/09/11
PB - bioRxiv
DO - 10.64898/2026.09.11.750594
UR - https://www.biorxiv.org/content/10.64898/2026.09.11.750594v1
AB - Invariant natural killer T (iNKT) cells can limit graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), but their scarcity in peripheral blood limitstherapeutic development. Current clinical-grade human iNKT expansion protocols mainly rely on IL-2, require prior iNKT-cell sorting, last 6-8 weeks, and predominantly expand CD4+ iNKT cells, whereas human CD4- iNKT cells are more strongly associated with GVHD control in patients and uniquely regulate antigen-presenting cells and T-cell activation. We developed a scalable culture system to preferentially expand human CD4- iNKT cells directly from total peripheral blood mononuclear cells (PBMCs) using alpha galactosylceramide (-GalCer) and optimized cytokine conditions. IL-15 was the most effective cytokine. The optimized 14-day protocol generated a mean of 3.8x107 iNKT cells from 2x107 PBMCs, including 74% CD4- iNKT cells. Single-cell transcriptomic profiling identified eight major iNKT subsets, differentiation trajectories during expansion, and distinct IL-2- versus IL-15-associated transcriptional programs. IL-15-expanded iNKT cells induced apoptosis of monocyte-derived dendritic and leukemic cells in vitro, controlled xeno-GVHD, and preserved graft-versus-leukemia (GVL) activity in preclinical mouse models. This platform enables reproducible production of human CD4- iNKT cells at clinically relevant scale and position IL-15-expanded iNKT cells as a compelling immunotherapy candidate for allo-HSCT.
ER -