The therapeutic efficacy of adoptive T cell therapy is largely restricted by reduced viability and dysfunction of CD8⁺ T cells. Continuous antigen stimulation disrupts the expansion, effector function, and metabolic fitness of CD8⁺ T cells, leading to their differentiation into an exhausted state within the tumor microenvironment (TME). While the function of the cell cycle negative regulator p16 in senescent cells is well understood, its role in T cell exhaustion remains unclear. In this study, we demonstrated that TCR stimulation of CD8⁺ T cells rapidly upregulates p16 expression, with its levels positively correlating with TCR affinity. Chronic TCR stimulation further increased p16 expression, leading to CD8⁺ T cell apoptosis and exhaustion differentiation, without inducing DNA damage or cell senescence. Mechanistic investigations revealed that p16 downregulates mTOR, glycolysis, and oxidative phosphorylation (OXPHOS) associated gene expression, resulting in impaired mitochondrial fitness, reduced T cell viability, and diminished effector function. Furthermore, the deletion of p16 significantly enhances the persistence of CD8⁺ T cells within tumors and suppresses the terminal exhaustion of tumor-infiltrating T cells. Overall, our findings elucidate how increased p16 expression reshapes T cell intracellular metabolism, drives T cell apoptosis and exhaustion differentiation, and ultimately impairs T cell anti-tumor function.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs). They play pivotal roles in orchestrating innate and adaptive immune responses, particularly in cancer. In tumor-draining lymph nodes (tdLNs), de novo priming occurs, where DCs present antigens to naive T cells, activating them and initiating their clonal expansion. In the tumor microenvironment (TME), intratumoral DCs provide survival or co-stimulatory signals to shape T cell differentiation. However, the scarcity and dysfunctional states of DCs can greatly limit anti-tumor responses, and DCs can even be hijacked by tumor-related factors to promote tumor progression. Therefore, comprehensively understanding the anti- or pro-tumor activities of DCs is crucial. In this review, we discuss the ontogeny of DC lineages and the emerging complexity of intratumoral DCs states. Importantly, we emphasize the significant roles of DCs in priming and sustaining productive T cell anti-tumor immunity. In light of these findings, we also explore promising approaches for targeting DCs to boost anti-tumor immunity and overcome resistance to cancer immunotherapies. We propose that insights into the rational design of DC-based immunotherapeutic strategies against cancer hold immense, underexploited potential.

Exhausted CD8⁺ T cells with limited effector functions and high expression of multiple co-inhibitory receptors are one of the main barriers hindering antitumor immunity. The NADase CD38 has received considerable attention as a biomarker of CD8⁺ T cell exhaustion, but it remains unclear whether the increased CD38 directly promotes T cell dysfunctionality. Here, we surprisingly found that although Cd38 deficiency partially reverses NAD⁺ degradation and T cell dysfunction in vitro, the terminal exhausted differentiation of adoptively transferred CD8⁺ T cells in tumor is not impacted by either deficiency or overexpression of CD38. Monitoring the dynamic NAD⁺ levels shows that NAD⁺ levels are comparable between tumor infiltrated WT and Cd38^−/− OT-1 cells. Therefore, our results suggest that decreased NAD⁺ are correlated with T cell dysfunction, but deficiency of CD38 is not enough for rescuing NAD⁺ in tumor infiltrated CD8⁺ T cells and fails to increase the efficacy of antitumor T cell therapy.

Background: Compared to other subtypes, the CMS4 subtype is associated with lacking of effective treatments and poorer survival rates.Methods: A total of 24 patients with CRC were included in this study. DNA and RNA sequencing were performed to acquire somatic mutations and gene expression, respectively. MATH was used to quantify intratumoral heterogeneity. PPI and survival analyses were performed to identify hub DEGs. Reactome and KEGG analyses were performed to analyze the pathways of mutated or DEGs. Single-sample gene set enrichment analysis and Xcell were used to categorize the infiltration of immune cells.Results: The CMS4 patients had a poorer PFS than CMS2/3. CTNNB1 and CCNE1 were common mutated genes in the CMS4 subtype, which were enriched in Wnt and cell cycle signaling pathways, respectively. The MATH score of CMS4 subtype was lower. SLC17A6 was a hub DEG. M2 macrophages were more infiltrated in the tumor microenvironment of CMS4 subtype. The CMS4 subtype tended to have an immunosuppressive microenvironment.Conclusion: This study suggested new perspectives for exploring therapeutic strategies for the CMS4 subtype CRC.

Background: BRAF non-V600 mutation occupies a relatively small but critical subset in colorectal cancer (CRC). However, little is known about the biological functions and impacts of BRAF class III mutation in CRC. Here, we aim to explore how D594A mutation impacts on biological behaviors and immune related signatures in murine CRC cells. Methods: BRAF V600E (class I), G469V (class II) and D594A (class III) mutant cell lines were established based on MC38 cells. The biological behaviors of cells were evaluated in respect of cell growth, cell proliferation, cell apoptosis, cell migration and invasion by the methods of colony-forming assay, CCK-8 assay, Annexin V/PI staining and transwell assay. The concentrations of soluble cytokines were detected by ELISA. The membrane expression of immuno-modulatory molecules and the pattern of tumor infiltrating lymphocyte were evaluated by flow cytometry. The molecular mechanism was explored by RNA sequencing. Immunohistochemistry (IHC) staining was used for the detection of CD8α in tumor tissues. qRT-PCR and western blot were performed to assess the mRNA and protein expression. Anti-PD-L1 treatment and cytokines neutralization experiments were conducted in in vivo models. Results: D594A mutant cells displayed lower grade malignancy characteristics than V600E (class I) and G469V (class II) mutant cells. Meanwhile, D594A mutation led to evident immuno-modulatory features including upregulation of MHC Class I and PD-L1. In vivo experiments displayed that the frequency of infiltrated CD8⁺ T cells was significantly high within D594A mutant tumors, which may provide potential response to anti-PD-L1 therapy. RNA sequencing analysis showed that D594A mutation led to enhanced expression of ATF3 and THBS1, which thus facilitated CXCL9 and CXCL10 production upon IFN-γ treatment. In addition, CXCL9 or CXCL10 neutralization reduced the infiltration of CD8⁺ T cells into THBS1-overexpressing tumors. Conclusions: D594A mutant CRC exhibited lower aggressiveness and immune-activated phenotype. ATF3-THBS1-CXCL9/CXCL10 axis mediated functional CD8⁺ T cells infiltration into the microenvironment of D594A mutant CRC. Our present study is helpful to define this mutation in CRC and provide important insights in designing effective immunotherapeutic strategies in clinic.