Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea and pseudomembranous colitis, and there remains a significant unmet need for therapies specifically targeting C. difficile. The Pyrin inflammasome, activated by bacterial toxins, plays a critical role in driving macrophage-mediated intestinal inflammation during CDI. In this study, we report that myeloid-specific deficiency of Zbtb16 protects mice from C. difficile-induced colitis by attenuating IL-1β-dependent inflammatory signaling. Mechanistic studies revealed that Zbtb16 deletion disrupts ASC oligomerization and speck formation, thereby selectively inhibiting inflammasome assembly and reducing mature IL-1β production in macrophages stimulated with C. difficile culture supernatant or purified TcdB toxin. Importantly, pharmacological degradation of ZBTB16 using the cereblon E3 ligase modulating drug CC-3060 significantly ameliorated colitis severity in a murine model of CDI. Our findings establish ZBTB16 as a key regulator of Pyrin inflammasome activation in macrophages, highlighting the therapeutic promise of ZBTB16 degradation as a novel strategy for treating CDI.

Background/Objectives: Cholangiocarcinoma (CCA) is characterized by a heterogeneous immune microenvironment, where the prognostic significance of CD8⁺ tissue-resident memory T (T_RM) cell activation and spatial positioning remains to be fully elucidated. This study investigated how the activation phenotypes and their spatial distribution relative to tumor cells influence anti-tumor immunosurveillance and predict clinical outcomes in CCA. Methods: Multiplex immunohistochemistry (mIHC) and single-cell RNA sequencing (scRNA-seq) were employed to characterize naïve (PD-1⁻CD103⁺CD8⁺) and exhausted (PD-1⁺CD103⁺CD8⁺) subsets. G-cross function analysis was utilized to quantify the spatial proximity between these specific T_RM subsets and tumor cells, correlating the spatial interaction with patient overall survival. Results: scRNA-seq profiling revealed that PD-1⁻CD103⁺CD8⁺ T_RM cells were enriched in genes associated with lymphocyte activation and cytotoxicity, while PD-1⁺CD103⁺CD8⁺ T_RM cells exhibited an exhaustion signature. Spatially, PD-1⁻CD103⁺CD8⁺ T_RM cells exhibited increased interactions with tumor cells, whereas PD-1⁺CD103⁺CD8⁺ T_RM cells showed reduced engagement. Therefore, the close proximity of PD-1⁻CD103⁺CD8⁺ T_RM cells to tumor cells was identified as a significant predictor of favorable clinical outcomes. Conclusions: The activation state of CD8⁺ T_RM cells combined with their spatial localization constitutes a critical prognostic factor in CCA. Effective anti-tumor immunosurveillance relies on the direct engagement of naïve T_RM cells with tumor cells. These findings highlight the potential of PD-1-targeted immunotherapies to remodel the spatial proximity of the tumor microenvironment, potentially promoting the redistribution of effector cells into tumor-proximal regions.