Alan D. Friedman

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.

Epidermal Growth Factor Receptor (EGFR) is often present on the cell surface of a wide variety human malignancies, including non-small-cell lung cancer (NSCLC), glioblastoma (GBM), pancreatic ductal carcinoma (PDC), and castration-resistant prostate cancer (CRPC). Efforts to optimize immunotherapies targeting EGFR are limited by murine intolerance of human EGFR. To overcome this obstacle, we developed C57BL/6 mice in which a truncated variant of human EGFR (hEGFRt), lacking the ligand binding domain and cytoplasmic domain, is expressed from the CAG regulatory elements comprised of the CMV enhancer, β-actin promoter, and β-globin poly-adenylation signals. Cetuximab, a high-affinity, clinically available anti-human EGFR antibody, retains affinity for hEGFRt. The hEGFRt cDNA in the CAG-hEGFRt transgene is flanked by lox P sites to enable its excision thereby reducing interaction of hEGFRt-directed immunotherapies with normal tissues. The CAG-hEGFRt(f/f) transgene is abundantly expressed in hematopoietic lymphoid and myeloid cells, with low-level expression evident also in non-hematopoietic liver, lung, kidney, and brain. Mx1-Cre-mediated transgene excision in adult mice reduces hEGFRt ~ 5-fold in blood mononuclear cells. CAG-hEGFRt(f/f) adult mice are tolerant of syngeneic NSCLC, GBM, prostate, and PDC lines expressing hEGFRt. These mice retain hEGFRt tolerance after transgene deletion. CAG-hEGFRt(f/f) mice provide a new and important tool for the development of immunotherapies targeting hEGFR.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by epidermal barrier dysfunction and dysregulated immune responses, particularly an imbalance between T helper type 1 (Th1) and type 2 (Th2) cytokines. Natural products with immunomodulatory activity have attracted increasing attention as potential strategies for regulating allergic inflammation. In this study, we investigated the immunomodulatory effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F). In vitro assays using human B cells, mast cells, and keratinocytes were conducted to evaluate IgE production, mast cell degranulation, and epithelial inflammatory mediator release. The efficacy of the BRB-F:BFR-F mixture was further evaluated in BALB/c mice with 2,4-dinitrochlorobenzene (DNCB)/Dermatophagoides farinae extract (DFE)-induced AD-like dermatitis. BRB-F and BFR-F suppressed IgE production, attenuated mast cell degranulation and thymic stromal lymphopoietin (TSLP) release, and reduced keratinocyte-derived inflammatory mediators (thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and IL-6). In mice, dietary supplementation with the BRB-F:BFR-F mixture (10–80 mg/kg/day) dose-dependently improved clinical skin lesions and histopathological changes, with serum IgE reduced by up to 87.1% at the highest dose. The treatment significantly suppressed Th2 cytokine mRNA expression in ear tissue (IL-4, IL-5, and IL-13) by 37.2%, 32.7%, and 34.0%, respectively, compared with the positive control. In contrast, splenic Th1 cytokine mRNA expression (IL-2, IL-12, and IFN-γ) was partially restored by 37.1%, 22.5%, and 18.7%, respectively. These findings indicate that BRB-F and BFR-F modulate multiple immune pathways and help restore Th1/Th2 immune balance, suggesting their potential as functional materials for regulating immune dysregulation associated with AD.

Background Atopic dermatitis (AD) is a chronic inflammatory skin disorder frequently associated with dysregulated Th1/Th2 immune balance and impaired regulatory T (Treg) activity. Although conventional treatments can reduce clinical symptoms, concerns regarding long-term adverse effects have led to increased interest in complementary and functional dietary materials with immunomodulatory potential. Bioprocessed black rice bran (BRB-F) and balloon flower root fractions (BFR-F) have individually demonstrated anti-inflammatory activities, but their combined oral efficacy and mechanisms in AD remain insufficiently characterized. Methods Mechanistic in vitro assays were first performed using a human B-cell line to measure IgE secretion, mouse and/or human mast cells to evaluate degranulation and TSLP release, and keratinocyte cultures to quantify TARC, MDC, and IL-6 production. These assays were used to determine whether BRB-F and BFR-F individually or cooperatively modulate immune and epithelial cell activation relevant to AD pathology. Subsequently, an AD-like phenotype was induced in BALB/c mice by topical application of 1-chloro-2,4-dinitrobenzene followed by mite extract. Animals received dietary supplementation with a 3:1 BRB-F:BFR-F mixture (10–80 mg/kg/day), and clinical skin inflammation, ear histopathology, and cytokine profiles were analyzed to assess Th1/Th2 balance and Treg-associated markers. Results Dietary supplementation with the BRB-F:BFR-F binary combination resulted in dose-dependent improvement in AD-like skin lesions and reduced histopathological severity. The mixture suppressed Th2 cytokines (IL-4, IL-5, IL-13) in ear tissue and increased splenic Th1 cytokines (IL-2, IL-12, IFN-γ), accompanied by dose-dependent elevation of galectin-9, suggesting activation of Treg-associated pathways. IL-10 expression was also reduced in vivo. In vitro, BRB-F and BFR-F cooperatively inhibited IgE production by B cells, attenuated mast cell activation and TSLP release, and lowered keratinocyte secretion of TARC, MDC, and IL-6. Conclusions The combined oral administration of bioprocessed BRB-F and BFR-F exerts multi-targeted anti-atopic activity that includes modulation of Th1/Th2 immune balance, activation of Treg-associated signaling, and suppression of B-cell, mast cell, and keratinocyte responses. These findings, confirmed by histopathology of skin tissues before and after treatment, provide preclinical support for the development of BRB-F:BFR-F as a complementary functional material for managing immune dysregulation associated with AD, although validation in human patients will be required.

Adoptive transfer of immature myeloid cells lacking the repressive NF-κB p50 subunit (p50-IMC) slows the growth of syngeneic murine prostate cancer and other tumors. Directing p50-IMC to tumors using Fc receptor-bound antibodies (Abs) or surface chimeric antigen receptors (CARs) may increase tumor localization and subsequent phagocytosis of cancer cells by their mature myeloid progeny, potentiating anti-tumor T cell activation. PSMA and EGFR are found on aggressive human prostate cancers, and p50-IMC express receptors that bind the antibody Fc domain. p50-IMC combined with PSMA Ab, EGFR Ab (Cetuximab), or fully humanized PSMA.CAR10.3 manifest increased localization to Myc-CaP murine prostate cancer tumors expressing PSMA or EGFR. Tumor localization is further increased when myelo-depleting 5-fluorouracil precedes p50-IMC administration. Additionally, we find that PSMA Ab, EGFR Ab, or PSMA.CAR10.3 increase in vitro phagocytosis of Myc-CaP cells expressing PSMA or EGFR by p50-IMC-derived macrophages, including in M2-promoting IL-4, which is a component of the immune-suppressive tumor microenvironment. Lack of tolerance of human PSMA or EGFR by immune-competent mice and lack of expression of human PSMA protein in the prostate of AR₂-Probasin-hPSMA transgenic mice precluded our ability to determine whether human-specific PSMA or EGFR antibody or PSMA.CAR10.3 increases anti-tumor efficacy of murine p50-IMC. Nevertheless, this study indicates the potential clinical utility of adding a tumor-directing antibody or CAR, including the novel, fully humanized PSMA.CAR10.3, to proinflammatory p50-IMC to optimize the activation of anti-tumor immunity in prostate cancer and other malignancies, and understanding PSMA toxicity in normal but not malignant prostate epithelium may reveal a novel therapeutic opportunity.