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CD8 + T cells control tumors but inevitably become dysfunctional in the tumor microenvironment. Here, we show that sodium chloride (NaCl) counteracts T cell dysfunction to promote cancer regression. NaCl supplementation during CD8 + T cell culture induced effector differentiation, IFN-γ production and cytotoxicity while maintaining the gene networks responsible for stem-like plasticity. Accordingly, adoptive transfer of tumor-specific T cells resulted in superior anti-tumor immunity in a humanized mouse model. In mice, a high-salt diet reduced the growth of experimental tumors in a CD8 + T cell-dependent manner by inhibiting terminal differentiation and enhancing the effector potency of CD8 + T cells. Mechanistically, NaCl enhanced glutamine consumption, which was critical for transcriptional, epigenetic and functional reprogramming. In humans, CD8 + T cells undergoing antigen recognition in tumors and predicting favorable responses to checkpoint blockade immunotherapy resembled those induced by NaCl. Thus, NaCl metabolism is a regulator of CD8 + T cell effector function, with potential implications for cancer immunotherapy., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Flow cytometry (FCM) can investigate dozens of parameters from millions of cells and hundreds of specimens in a short time and at a reasonable cost, but the amount of data that is generated is considerable. Computational approaches are useful to identify novel subpopulations and molecular biomarkers, but generally require deep expertize in bioinformatics and the use of different platforms. To overcome these limitations, we introduce CRUSTY, an interactive, user-friendly webtool incorporating the most popular algorithms for FCM data analysis, and capable of visualizing graphical and tabular results and automatically generating publication-quality figures within minutes. CRUSTY also hosts an interactive interface for the exploration of results in real time. Thus, CRUSTY enables a large number of users to mine complex datasets and reduce the time required for data exploration and interpretation. CRUSTY is accessible at https://crusty.humanitas.it/ ., (© 2023. Springer Nature Limited.)

CD4 + T regulatory cells (Tregs) are a specialized subset of T lymphocytes, which promote immune homeostasis and tumor immunosuppression by restricting effector T cell immune responses. The characterization of context-specific Treg phenotypic heterogeneity is pivotal to determine their potential contributions to diseases. In the recent years, high-dimensional single-cell technologies, such as single-cell RNA sequencing, mass cytometry, or polychromatic flow cytometry, have played a central role in elucidating the heterogeneity of the Treg compartment at the cellular and molecular levels. Here we describe an example of high-dimensional flow cytometry analysis capable of defining an effector Treg subpopulation that positively correlates with cancer progression. Moreover, we provide a workflow template of high-dimensional single-cell analysis that is readily applicable to any leukocyte subpopulation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CD8 + T cells are a major prognostic determinant in solid tumors, including colorectal cancer (CRC). However, understanding how the interplay between different immune cells impacts on clinical outcome is still in its infancy. Here, we describe that the interaction of tumor infiltrating neutrophils expressing high levels of CD15 with CD8 + T effector memory cells (T EM ) correlates with tumor progression. Mechanistically, stromal cell-derived factor-1 (CXCL12/SDF-1) promotes the retention of neutrophils within tumors, increasing the crosstalk with CD8 + T cells. As a consequence of the contact-mediated interaction with neutrophils, CD8 + T cells are skewed to produce high levels of GZMK, which in turn decreases E-cadherin on the intestinal epithelium and favors tumor progression. Overall, our results highlight the emergence of GZMK high CD8 + T EM in non-metastatic CRC tumors as a hallmark driven by the interaction with neutrophils, which could implement current patient stratification and be targeted by novel therapeutics., (© 2022. The Author(s).)

Luminal-like breast cancer (BC) constitutes the majority of BC subtypes, but, differently from highly aggressive triple negative BC, is poorly infiltrated by the immune system. The quality of the immune infiltrate in luminal-like BCs has been poorly studied, thereby limiting further investigation of immunotherapeutic strategies. By using high-dimensional single-cell technologies, we identify heterogeneous behavior within the tissue-resident memory CD8+ T (Trm) cells infiltrating luminal-like tumors. A subset of CD127- CD39 hi Trm cells, preferentially present in the tumor compared to the adjacent normal breast tissue or peripheral blood, retains enhanced degranulation capacity compared to the CD127+ CD39 lo Trm counterpart ex vivo, and is specifically associated with positive prognosis. Nevertheless, such prognostic benefit is lost in the presence of highly-suppressive CCR8 hi ICOS hi IRF4+ effector Tregs. Thus, combinatorial strategies aiming at boosting Trm function and infiltration while relieving from Treg-mediated immunosuppression should be investigated to achieve proper tumor control in luminal-like BCs., (© 2021. The Author(s).)