Wei Hu

Regulatory T (T _reg ) cells, expressing the transcription factor Foxp3, are obligatory gatekeepers of immune responsiveness, yet the mechanisms by which Foxp3 governs the T _reg transcriptional network remain incompletely understood. Using a novel chemogenetic system of inducible Foxp3 protein degradation in vivo, we found that while Foxp3 was indispensable for the establishment of transcriptional and functional programs of newly generated T _reg cells, Foxp3 loss in mature T _reg cells resulted in minimal functional and transcriptional changes under steady state. This resilience of the Foxp3-dependent program in mature T _reg cells was acquired over an unexpectedly long timescale; however, in settings of severe inflammation, Foxp3 loss led to a pronounced perturbation of T _reg cell transcriptome and fitness. Furthermore, tumoral T _reg cells were uniquely sensitive to Foxp3 degradation, which led to impairment in their suppressive function and tumor shrinkage in the absence of pronounced adverse effects. These studies demonstrate a context-dependent differential requirement for Foxp3 for T _reg transcriptional and functional programs.

Regulatory T (Treg) cells, expressing the transcription factor Foxp3, are obligatory gatekeepers of the immune responsiveness. While Foxp3 essential role in Treg l differentiation is well established, the mechanisms by which Foxp3 governs the Treg-specific transcriptional network remain incompletely understood. Here, we employed a novel chemogenetic system of inducible, time-controlled degradation of Foxp3 protein in vivo to dissect its Treg stage stage-specific functions. While Foxp3 was indispensable for the establishment of the Treg transcriptional program and suppressive function during thymic Treg progenitors and newly generated peripheral Treg cells, degradation of Foxp3 in mature Treg cells resulted in unexpectedly minimal transcriptional changes largely limited to direct Foxp3 targets and largely preserved suppressive capacity. However, tumoral Treg cells were uniquely sensitive to Foxp3 degradation, which led to impaired suppressive function and tumor growth restraint absent pronounced adverse effects. These studies demonstrate context-dependent differential requirement for Foxp3 for Treg transcriptional and functional programs.

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Following infection or vaccination, activated B cells at extrafollicular sites or within germinal centers (GCs) undergo vigorous clonal proliferation. Proliferating lymphocytes have been shown to undertake lactate dehydrogenase A (LDHA)-dependent aerobic glycolysis; however, the specific role of this metabolic pathway in a B cell transitioning from a naïve to a highly proliferative, activated state remains poorly defined. Here, we deleted LDHA in a stage-specific and cell-specific manner. We find that ablation of LDHA in a naïve B cell did not profoundly affect its ability to undergo a bacterial lipopolysaccharide-induced extrafollicular B cell response. On the other hand, LDHA-deleted naïve B cells had a severe defect in their capacities to form GCs and mount GC-dependent antibody responses. In addition, loss of LDHA in T cells severely compromised B cell-dependent immune responses. Strikingly, when LDHA was deleted in activated, as opposed to naïve, B cells, there were only minimal effects on the GC reaction and in the generation of high-affinity antibodies. These findings strongly suggest that naïve and activated B cells have distinct metabolic requirements that are further regulated by niche and cellular interactions.

While regulatory T (T_reg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct T_reg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral T_reg cell ‘connectivity’ to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual T_reg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared T_reg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon T_reg cell deprivation in either setting, as well as in T_reg cell-poor versus T_reg cell-rich human lung adenocarcinomas. Accordingly, punctual T_reg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.

Purpose: Tumor-associated macrophages (TAMs) are known to contribute to adaptive resistance to anti-vascular endothelial growth factor (VEGF) antibody (AVA) therapy in ovarian cancer. BET (bromodomain and extra-terminal domain) inhibitors (BETi) may have unique roles in targeting TAMs. Our objective was to examine the effects of BETi on TAMs, especially in the context of enhancing the efficacy of AVA therapy. Methods: We conducted a series of in vitro (MTT assay, apoptosis, flow cytometry, and RNA sequencing) and in vivo (xenograft ovarian cancer model) experiments to determine the biological effects of BETi combined with AVA in ovarian cancer. For statistical analysis, a two-tailed Student’s t test (equal variance) or ANOVA was used for multiple groups’ comparison, and p < 0.05 was considered significant. Results: BETi resulted in a dose-dependent decrease in cell viability and induced apoptosis (p < 0.01) in ovarian cancer cells (SKOV3ip1, OVCAR5, and OVCAR8). Treatment with BETi significantly increased apoptosis in THP-1 monocytes and macrophages (PMA-differentiated THP-1; p < 0.01). Furthermore, BETi selectively induced greater apoptosis in M2-like macrophages (PMA and IL-4, IL-13-differentiated THP-1) (31.3%-36.1%) than in M1-like macrophages (PMA and LPS-differentiated THP-1) (12.4%-18.5%) (p < 0.01). Flow cytometry revealed that the percentage of M1-like macrophages (CD68⁺/CD80⁺) was significantly increased after treatment with low-dose BETi (ABBV-075 0.1 µM; p < 0.05), whereas the percentage of CD68⁺/CCR2⁺ macrophages was significantly decreased (p < 0.001); these findings suggest that BETi may selectively inhibit the survival of CCR2⁺ macrophages and re-polarize the macrophages into an M1-like phenotype. RNA-seq analysis revealed that BETi selectively targeted macrophage infiltration-related cytokines/chemokines in ovarian cancer (adjusted p < 0.05 and Log2 fold change ≥ 1.5). Finally, using in vivo ovarian cancer models, compared with control or monotherapy, the combination of BETi (ABBV-075) and bevacizumab resulted in greater inhibition of tumor growth and macrophage infiltration (p < 0.05) and longer survival of tumor-bearing mice (p < 0.001). Conclusions: Our findings indicate a previously unrecognized role for BETi in selectively targeting CCR2⁺ TAMs and enhancing the efficacy of AVA therapy in ovarian cancer.