BRD4 sustains immunosuppressive erythroid precursors to compromise CAR-T cell function

Abstract Introduction: Emerging evidence suggests that the immunosuppressive tumor microenvironment is a major limiting factor impairing CAR-T cell function. Recent studies have identified immunosuppressive erythroid precursor cells (EPCs) – a novel cell population that accumulates in pathological contexts (including neonates, cancer-bearing hosts, and infected individuals) – which potently suppress immune responses and diminish upon maturation. In this study, we aim to investigate the impact of these immunomodulatory EPCs on CAR-T cell function and explore potential strategies to overcome their suppressive effects, thereby enhancing CAR-T cell efficacy. Methods: We established KA539 lymphoma models in irradiated C57BL/6 mice and collected clinical peripheral blood samples from hematologic malignancy patients to quantify EPCs accumulation in extramedullary tissues. Functional profiling was performed via RNA-seq on FACS-sorted CD45⁺ versus CD45⁻ EPCs, validated by scRNA-seq. To assess EPCs-mediated CAR-T cell suppression, CD19 CAR-T cells were co-cultured with EPCs followed by multiparameter analysis including proliferation, cytokine secretion, exhaustion, and tumor-killing capacity of CAR-T cells. Mechanistically, we performed RNA-seq and Cut&Tag with erythroid precursors upon BRD4 inhibition, and genetic validation in erythroid-specific BRD4 cKO mice to demonstrate that BRD4 regulates the immunity of CD45+ EPCs. Results: We confirmed that both tumor-bearing mice and patients with hematologic malignancies showed significantly increased EPCs proportions in extramedullary tissues, including peripheral blood, spleen, and liver. RNA-seq analysis revealed that CD45+ EPCs exhibited elevated expression of immunosuppressive genes, including VISTA, PD-L1, LGALS3. Notably, tumor-bearing mice exhibited increased EPCs levels following CAR-T treatment, particularly in the poor-response group, suggesting a potential link between EPCs expansion and therapeutic resistance. Coculture with CD45+ EPCs significantly impaired CAR-T cell function, reducing both TNF-α secretion and proliferative capacity. Consistent with our previous findings on BRD4's role in blocking erythroid maturation, we observed that BRD4 similarly impeded EPCs' maturation in tumor-bearing mice and patient-derived samples. Furthermore, BRD4 inhibition downregulated immune-related genes and pathways in EPCs. Integrated analysis of Cut&Tag and RNA-seq data revealed that BRD4 directly transcriptionally activated immune regulatory genes in EPCs, including PD-L1 and VISTA. Besides, experiments with BRD4conditional knockout mice further confirmed that BRD4 inhibition promoted maturation of CD45+ EPCs and repressed expression of novel immune checkpoint VISTA. Importantly, flow cytometry analysis demonstrated that BRD4 inhibition significantly alleviated EPCs-induced CAR-T cell exhaustion, as evidenced by reduced expression of the exhaustion markers PD-1 and LAG-3, supporting BRD4 inhibition as a viable strategy to potentiate CAR-T cell function by modulating EPCs' immunosuppressive activity. Conclusion: Our study establishes that immunosuppressive EPCs directly impair CAR-T cell function. We demonstrate that BRD4 transcriptionally activates VISTA expression in EPCs, and targeting BRD4 reverses EPC-mediated CAR-T suppression. These findings unveil a novel mechanism of the immunosuppressive microenvironment in CAR-T therapy and provide a potential approach to enhance clinical efficacy against hematologic malignancies.