Engineering IL-4 resistant proinflammatory human myeloid cells for cancer immunotherapy.

Adoptive transfer of proinflammatory myeloid cells enhances antitumor T-cell responses and limits tumor growth in murine models. Previous work identified transcription factors NF-κB p50 and STAT6 as key regulators whose inactivation enhances the inflammatory potential of murine myeloid cells. Herein, we translate murine findings to human cells and establish methods to generate proinflammatory myeloid cells from human marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPCs). Using CRISPR-Cas9, we disrupted NFKB1, STAT6, both NFKB1 and STAT6, or NFKB1 and NFKB2 (encoding NF-κB p52), achieving robust loss of target proteins. We determined conditions for expanding HSPCs and differentiating them into monocytes. To evaluate proinflammatory potential under immune-suppressive conditions, edited monocytes were differentiated into macrophages, exposed to IL-4, and analyzed for gene expression. RNA sequencing revealed enhanced proinflammatory pathway activation in STAT6- and p50/STAT6-deficient macrophages, but not in p50- or p50/p52-deficient macrophages. Transcriptomic analysis was validated by measuring protein secretion of a subset of inflammatory mediators. These findings establish a strategy to generate proinflammatory human myeloid cells with potential clinical application in cancer immunotherapy. Future clinical evaluation will be required to determine whether STAT6-IMC or p50/STAT6-IMC exhibit superior therapeutic efficacy.