Henrique Borges da Silva

Cholera toxin (CTx) is a potent enterotoxin and widely used mucosal adjuvant, yet the acute host response to CTx itself, in the absence of a co-administered antigen, remains vague. In this study, we integrated physiological and metabolic phenotyping with intestinal transcriptomics, epithelial imaging, immune profiling, hepatic gene-expression analysis, and paired microbiome profiling to characterize the response to a single oral CTx exposure in mice. Within 24 h, CTx remodeled immune-cell composition in both epithelial and lamina propria compartments, with responses shaped by sex and genetic background. Recurrent changes included increased epithelial group 3 innate lymphoid cells, reduced lamina propria RORγt+ regulatory T cells, and decreased epithelial ILC1/NK cells in males of both strains. CTx also increased villus DCLK1+ tuft cells and reduced MUC2 immunoreactivity. Jejunal transcriptomics revealed induction of inflammatory defense, glycolytic, autophagy, and lipid-utilization programs together with suppression of proliferative and mitochondrial programs, consistent with coordinated stress adaptation. These local changes were accompanied by rapid restructuring of the fecal microbial community and by systemic hypometabolism, despite markedly different hypothermic responses across strains. CTx also elicited a selective hepatic stress and metabolic program, including conserved suppression of Cyp7a1 across sexes and strains. Together, these findings show that CTx alone rapidly reorganizes the intestinal immune, epithelial, metabolic, and microbial environment while suppressing whole-body metabolism. This work provides an integrated framework for understanding the acute host response to CTx and for interpreting its widespread use as a mucosal adjuvant.

Durable T cell immunity against cancer depends on the continual replenishment of effector CD8⁺ T cells. Thymic output has been associated with favorable prognosis in cancer patients across a range of ages, suggesting that the thymus is an important source for replenishing T cells capable of controlling cancer progression. However, whether CD8⁺ T cells acquire effector potential within the thymus, and how thymic output of effector CD8⁺ T cells contribute to peripheral tumor immunity, remain unclear. In this study, we discover that thymic single-positive (SP) CD8⁺ T cells undergo latent effector differentiation following thymic selection, but this process is subject to PD-1 regulation. We further demonstrate that PD-1 limits the contribution of thymic output of CD8⁺ T cells in shaping the TCR repertoire within the tumor tissues for tumor immunosurveillance. Although PD-1 inhibition facilitates the expansion of effector CD8⁺ T cells in the periphery, these cells gradually lose antitumor activity within tumors due to accelerated exhaustion in the absence of PD-1. Thus, while latent effector differentiation of thymic CD8⁺ T cells enables a rapid response to malignant cells in the periphery, PD-1 restrains this process to prevent overt or terminal effector differentiation, which may compromise balanced and durable peripheral immunity.

Fig. S4. Effects of CD4+ T-cell intrinsic P2RX7 on tumor growth and Treg accumulation in the lungs.

Fig. S3. Effects of T-cell intrinsic P2RX7 on the growth of tumors in the lung.

Fig. S5. Effects of P2RX7 expression on Tregs in lung cancer experimental model.

Lung cancer is the leading cause of cancer-related deaths worldwide, and despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4+ T cells (CD4+ Teff) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Treg), which accumulate in lung tumors and mediate suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Herein, we identify the extracellular adenosine 5-triphosphate receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell–specific P2RX7-knockout (P2RX7-KO) mice, reduced Treg infiltration was accompanied by increased CD4+ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared with P2RX7-KO Tregs, which failed to inhibit type 1 and follicular helper T–like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared with P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

Fig. S6. Effects of Treg-specific P2RX7 ablation on Teffs in experimental lung cancer model.