Unveiling the impact of tertiary lymphoid structures on immunotherapeutic responses of clear cell renal cell carcinoma.

Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that form under pathological conditions. However, the predictive value of TLS in clear cell renal cell carcinoma (ccRCC) for immunotherapies remains unclear. We comprehensively assessed the implications for prognosis and immunological responses of the TLS spatial and maturation heterogeneity in 655 ccRCC patients. A higher proportion of early-TLS was found in peritumoral TLS, while intratumoral TLS mainly comprised secondary follicle-like TLS (SFL-TLS), indicating markedly better survival. Notably, presence of TLS, especially intratumoral TLS and SFL-TLS, significantly correlated with better survival and objective reflection rate for ccRCC patients receiving anti-Programmed Cell Death Protein-1 (PD-1)/Programmed Cell Death-Ligand-1 (PD-L1) immunotherapies. In peritumoral TLS cluster, primary follicle-like TLS, the proportion of tumor-associated macrophages, and Treg infiltration in the peritumoral regions increased prominently, suggesting an immunosuppressive tumor microenvironment. Interestingly, spatial transcriptome annotation and multispectral fluorescence showed that an abundance of mature plasma cells within mature TLS has the capacity to produce IgA and IgG, which demonstrate significantly higher objective response rates and a superior prognosis for ccRCC patients subjected to immunotherapy. In conclusion, this study revealed the implications of TLS spatial and maturation heterogeneity on the immunological status and clinical responses, allowing the improvement of precise immunotherapies of ccRCC.

Stimuli-responsive nanodelivery systems for amplifying immunogenic cell death in cancer immunotherapy.

Immunogenic cell death (ICD) is a special pattern of tumor cell death, enabling to elicit tumor-specific immune response via the release of damage-associated molecular patterns and tumor-associated antigens in the tumor microenvironment. ICD-induced immunotherapy holds the promise for completely eliminating tumors and long-term protective antitumor immune response. Increasing ICD inducers have been discovered for boosting antitumor immunity via evoking ICD. Nonetheless, the utilization of ICD inducers remains insufficient owing to serious toxic reactions, low localization efficiency within the tumor microenvironmental niche, etc. For overcoming such limitations, stimuli-responsive multifunctional nanoparticles or nanocomposites with ICD inducers have been developed for improving immunotherapeutic efficiency via lowering toxicity, which represent a prospective scheme for fostering the utilization of ICD inducers in immunotherapy. This review outlines the advances in near-infrared (NIR)-, pH-, redox-, pH- and redox-, or NIR- and tumor microenvironment-responsive nanodelivery systems for ICD induction. Furthermore, we discuss their clinical translational potential. The progress of stimuli-responsive nanoparticles in clinical settings depends upon the development of biologically safer drugs tailored to patient needs. Moreover, an in-depth comprehending of ICD biomarkers, immunosuppressive microenvironment, and ICD inducers may accelerate the advance in smarter multifunctional nanodelivery systems to further amplify ICD.

Heterogeneity in tertiary lymphoid structures predicts distinct prognosis and immune microenvironment characterizations of clear cell renal cell carcinoma.

BACKGROUND: Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop postnatally in non-lymphoid tissues and are associated with pathological conditions. TLS typically comprise B-cell follicles containing and are encompassed by T- cell zones and dendritic cells. The prognostic and predictive value of TLS in the tumor microenvironment (TME) as potential mediators of antitumor immunity have gained interest. However, the precise relationship between localization and maturation of TLS and the clinical outcome of their presence in clear cell renal cell carcinoma (ccRCC) is yet to be elucidated. METHODS: Immunohistochemistry and multispectral fluorescence were used to evaluate the TLS heterogeneity along with TME cell-infiltrating characterizations. A thorough investigation of the prognostic implications of the TLS heterogeneity in 395 patients with ccRCC from two independent cohorts was conducted. Associations between TLS heterogeneity and immunologic activity were assessed by quantifying the immune cell infiltration. RESULTS: Infiltrated TLS were identified in 34.2% of the ccRCC samples (N=395). These TLS were found to be tumor-proximal, tumor-distal, or both in 37.8%, 74.1%, and 11.9% of the TLS-positive cases, respectively. A higher proportion of early TLS was found in tumor-distal TLS (p=0.016), while tumor-proximal TLS primarily comprised secondary follicle-like structures (p=0.004). In the main study cohort (Fudan University Shanghai Cancer Center, N=290), Kaplan-Meier analyses revealed a significant correlation between the presence of tumor-proximal TLS and improved progression-free survival (PFS, p<0.001) and overall survival (OS, p=0.002). Conversely, the presence of tumor-distal TLS was associated with poor PFS (p=0.02) and OS (p=0.021). These findings were further validated in an external validation set of 105 patients with ccRCC. Notably, the presence of mature TLS (namely secondary follicle-like TLS, with CD23+ germinal center) was significantly associated with better clinical outcomes in patients with ccRCC. Furthermore, novel nomograms incorporating the presence of tumor-proximal TLS demonstrated remarkable predictability for the 8-year outcomes of resected ccRCC (area under the curve >0.80). Additionally, ccRCC samples with tumor-distal TLS enriched with primary follicle-like TLS exhibited higher programmed death-ligand 1 tumor-associated macrophages levels and regulatory T cells infiltration in the tumor-distal region, indicative of a suppressive TME. CONCLUSION: This study for the first time elucidates the impact of TLS localization and maturation heterogeneities on the divergent clinical outcomes of ccRCC. The findings reveal that most TLS in ccRCC are located in the tumor-distal area and are associated with immature, immunosuppressive characterizations. Furthermore, our findings corroborate previous research demonstrating that tumor-proximal TLS were associated with favorable clinical outcomes.

Five Inhibitory Receptors Display Distinct Vesicular Distributions in Murine T Cells.

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to a persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3, and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct, with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

Five inhibitory receptors display distinct vesicular distributions in T cells.

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

Development and Validation of Risk Scores for All-Cause Mortality for a Smartphone-Based "General Health Score" App: Prospective Cohort Study Using the UK Biobank.

BACKGROUND: Given the established links between an individual's behaviors and lifestyle factors and potentially adverse health outcomes, univariate or simple multivariate health metrics and scores have been developed to quantify general health at a given point in time and estimate risk of negative future outcomes. However, these health metrics may be challenging for widespread use and are unlikely to be successful at capturing the broader determinants of health in the general population. Hence, there is a need for a multidimensional yet widely employable and accessible way to obtain a comprehensive health metric. OBJECTIVE: The objective of the study was to develop and validate a novel, easily interpretable, points-based health score ("C-Score") derived from metrics measurable using smartphone components and iterations thereof that utilize statistical modeling and machine learning (ML) approaches. METHODS: A literature review was conducted to identify relevant predictor variables for inclusion in the first iteration of a points-based model. This was followed by a prospective cohort study in a UK Biobank population for the purposes of validating the C-Score and developing and comparatively validating variations of the score using statistical and ML models to assess the balance between expediency and ease of interpretability and model complexity. Primary and secondary outcome measures were discrimination of a points-based score for all-cause mortality within 10 years (Harrell c-statistic) and discrimination and calibration of Cox proportional hazards models and ML models that incorporate C-Score values (or raw data inputs) and other predictors to predict the risk of all-cause mortality within 10 years. RESULTS: The study cohort comprised 420,560 individuals. During a cohort follow-up of 4,526,452 person-years, there were 16,188 deaths from any cause (3.85%). The points-based model had good discrimination (c-statistic=0.66). There was a 31% relative reduction in risk of all-cause mortality per decile of increasing C-Score (hazard ratio of 0.69, 95% CI 0.663-0.675). A Cox model integrating age and C-Score had improved discrimination (8 percentage points; c-statistic=0.74) and good calibration. ML approaches did not offer improved discrimination over statistical modeling. CONCLUSIONS: The novel health metric ("C-Score") has good predictive capabilities for all-cause mortality within 10 years. Embedding the C-Score within a smartphone app may represent a useful tool for democratized, individualized health risk prediction. A simple Cox model using C-Score and age balances parsimony and accuracy of risk predictions and could be used to produce absolute risk estimations for app users.

PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and target tumor cells within the tumor.

The killing of tumor cells by CD8+ T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programmed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To find cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8+ tumor-infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL-tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca2+ signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8+ T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell-tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.