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NaCl enhances CD8+ T cell effector functions in cancer immunotherapy

Nature Immunology volume 25, pages 1845–1857 (2024)Cite this article

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Abstract

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.

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Fig. 1: High salt boosts human CD8+ T cell effector differentiation.
Fig. 2: High-salt diet promotes CD8+ T cell-dependent tumor control.
Fig. 3: HSD-induced CD8+ T cells resemble those induced by anti-PD-1.
Fig. 4: CD8+ T cell reprogramming by NaCl is dependent on glutamine.
Fig. 5: NaCl enhances glutamine-dependent epigenetic remodeling.
Fig. 6: NaCl boosts CD8+ T cell function upon adoptive transfer.

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Data availability

Bulk RNA–seq, scRNA–seq, ATAC–seq and metabolomics data generated in this paper have been deposited in Zenodo at https://zenodo.org/doi/10.5281/zenodo.10012831 (ref. 80) and https://zenodo.org/doi/10.5281/zenodo.11207788 (ref. 81). Publicly available scRNA–seq data were also accessed from the Gene Expression Omnibus under accession code GSE200996. Gene sets of interest were retrieved from collections C2 and C7 in the Molecular Signatures Database ‘v2023.1.Hs.symbols’ (http://www.broadinstitute.org/gsea/msigdb/index.jsp). Source data are provided with this paper.

Code availability

All codes related to the analyses reported in this paper can be found on GitHub at https://github.com/luglilab/NaCl-enhances-CD8-T-cell-effector-functions-in-cancer-immunotherapy.

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Acknowledgements

We thank G. Natoli and S. Polletti (IEO, Milan) for providing the Tn5, and A. Inforzato, S. Marchini, F. De Paoli, G. Alvisi (Humanitas), the members of the Lugli lab and the ‘T cell club’ for insightful comments. This work was supported by the CRI Lloyd J. Old STAR (CRI award 3914) and the Associazione Italiana per la Ricerca sul Cancro (AIRC IG 2017–ID 20676, AIRC IG 2022–ID 27391 and AIRC 5×1000 program UniCanVax 22757) to E.L. E.L. is also supported by EU funding within the MUR PNRR Italian network of excellence for advanced diagnosis (INNOVA, project no. PNC-E3-2022-23683266 PNC-HLS-DA). E.M.C.M. is supported by the Associazione Italiana per la Ricerca sul Cancro (MFAG 26471). C.S. and S.P. were supported by Fellowships from the Fondazione Italiana per la Ricerca sul Cancro-Associazione Italiana per la Ricerca sul Cancro (FIRC-AIRC). The purchase of a FACSSymphony A5 was defrayed in part by a grant from the Italian Ministry of Health (Agreement 82/2015).

Author information

Author notes

  1. These authors contributed equally: Caterina Scirgolea, Rosa Sottile.

Authors and Affiliations

  1. IRCCS Humanitas Research Hospital, Milan, Italy

    Caterina Scirgolea, Rosa Sottile, Marco De Luca, Alberto Susana, Silvia Carnevale, Simone Puccio, Valentina Ferrari, Veronica Lise, Giorgia Contarini, Alice Scarpa, Eloise Scamardella, Simona Feno, Chiara Camisaschi, Gabriele De Simone, Gianluca Basso, Desiree Giuliano, Emilia Maria Cristina Mazza, Diletta Di Mitri, Matteo Simonelli, Agnese Losurdo, Davide Pozzi, Sara Timo, Giuseppe Martano, Maria Rescigno, Sebastien Jaillon & Enrico Lugli

  2. Institute of Genetic and Biomedical Research, UoS Milan, National Research Council, Milan, Italy

    Simone Puccio

  3. Division of Functional Immune Cell Modulation, Leibniz Institute for Immunotherapy (LIT), Regensburg, Germany

    Luca Gattinoni

  4. University of Regensburg, Regensburg, Germany

    Luca Gattinoni

  5. Center for Immunomedicine in Transplantation and Oncology (CITO), University Hospital Regensburg, Regensburg, Germany

    Luca Gattinoni

  6. Department of Pathology, University of Cambridge, Cambridge, UK

    Rahul Roychoudhuri

  7. Immunology Programme, Babraham Institute, Babraham Research Campus, Cambridge, UK

    Rahul Roychoudhuri

  8. Department of Biomedical Sciences, Humanitas University, Milan, Italy

    Emanuele Voulaz, Diletta Di Mitri, Matteo Simonelli, Davide Pozzi, Maria Rescigno & Sebastien Jaillon

  9. Division of Thoracic, IRCCS Humanitas Research Hospital, Milan, Italy

    Emanuele Voulaz

  10. The Peter Doherty Institute for Infection and Immunity and Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia

    Carlson Tsui & Axel Kallies

  11. Institute of Neuroscience, National Research Council of Italy (CNR) c/o Humanitas Mirasole S.p.A, Milan, Italy

    Giuseppe Martano

  12. Department of Sciences and Technological Innovation, University of Piemonte Orientale, Alessandria, Italy

    Elettra Barberis

  13. Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy

    Marcello Manfredi

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  1. Caterina Scirgolea
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  2. Rosa Sottile
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  33. Enrico Lugli
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Contributions

C.S., R.S. and E.L. conceived the study. C.S., R.S., M.D.L., A. Susana, S.C., S.P., V.F., V.L., G.C., A. Scarpa, E.S., S.F., E.M.C.M., C.T., A.K., S.T., G.M., E.B. and M.M. performed experiments. C.C. and G.D.S. performed cell sorting experiments. G.B. and D.G. performed genomic experiments. L.G., R.R., D.P. and D.D.M. provided critical assistance in data interpretation. E.V., M.S. and A.L. collected human samples and/or data. C.S., R.S., M.D.L., S.P., E.M.C.M., G.M., M.M. and E.L. analyzed the data. M.R., S.J. and E.L. supervised the study. C.S. and E.L. wrote the manuscript; all authors edited and approved the paper.

Corresponding author

Correspondence to Enrico Lugli.

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Competing interests

C.S and E.L. submitted a patent on the use of NaCl to improve adoptive T cell therapy (PCT/EP2023/063475). E.L. received research grants from Bristol Myers Squibb on a topic unrelated to this paper, royalties from the National Institutes of Health for a patent on methods to develop T memory stem cells and consulting fees from BD Biosciences, Biolegend and Swarm Therapeutics. The other authors declare no competing interests.

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Nature Immunology thanks Ping-Chih Ho and Tuoqi Wu for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Nick Bernard, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Identification of the NaCl effective dose on CD8+ T cells and evaluation of osmotic pressure.

a, Representative flow cytometry gating strategy to identify human CD8+ T cells. b, Mean ± s.e.m. % viable CD8+ T cells recovered at day 5 after activation of cells as in Fig. 1a and after treatment with increasing concentrations of NaCl (0 mM, n = 10; 20 mM, n = 3; 40 mM, n = 9; 60 mM, n = 3; 80 mM, n = 14; 100 mM, n = 2; 150 mM, n = 4). c, Mean ± s.e.m. summary of the levels (MFI) of CD27, GZMB, PD-1, TIM3 and T-bet on CD8+ T cells after activation with anti-CD3/28 + IL-2 in control medium (Ctrl) and with increasing concentrations of NaCl (n = 4 donors except 20 mM NaCl n = 2; two independent experiments; Kruskal-Wallis test). d, Representative flow cytometry analysis of % GZMB and T-bet expression in CD8+ T cells activated as in b and treated with excess NaCl (80 mM) or equiosmolar urea (176 mM final concentration) or mannitol (142 mM final concentration). e, Mean ± s.e.m. summary of data shown in d (n = 2, mannitol; n = 4, all other conditions; two independent experiments; Kruskal-Wallis test). f, As in e, but related to PD-1, TIM3 and CD27 expression (two independent experiments; Kruskal-Wallis test).

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Extended Data Fig. 2 NaCl potentiates TCR signaling and acts in part via SGK1.

a, Representative flow cytometry of % pERK, pS6, and GZMB expression by CD8+ T cells activated as in Fig. 1a and restimulated at day 5 with plate-boundanti-CD3 and soluble anti-CD28 for 30 min. b, Mean ± s.e.m summary of the data as in a (indicated as % expression of the marker among total CD8+; n = 6, two independent experiments; two-sided paired t-test). c, Schematic representation of the experiment in d. d, Mean ± s.e.m summary of flow cytometric analysis of specific (combinations of) markers by Ctrl or NaCl-treated CD8+ T cells (n = 3, one experiment; two-sided paired t-test). Data are expressed as % expression or median fluorescence intensity (MFI) of the marker among total CD8+ T cells. e, Summary of flow cytometric analysis of specific markers by CD8+ T cells activated as in Fig. 1a but in the presence or 2μM SGK1 inhibitor GSK 650394 (n = 6, two independent experiments; paired one-way ANOVA with Dunn’s post test). Data are expressed as MFI.

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Extended Data Fig. 3 High-salt diet increases the infiltration of CD8+, CD4+ and NK cells in tumors without altering the frequency of CD4+ Treg cells.

a, Mean ± s.e.m. CD4+ and CD8+ T cell count/gram of MC38 tumor and b, absolute count of CD4+ and CD8+ T cells in the spleen of tumor-bearing B6 male mice fed with normal salt diet (NSD, n = 16) or high-salt diet (a: HSD, n = 15; b, HSD, n = 14; in b, one spleen sample was removed because of faulty antibody staining). Data are from three independent experiments, two-sided unpaired t-test. c, Frequency of TCM (central memory, CD44hiCD62Lhi), TEM (effector memory, CD44hiCD62Llo) and TTE (terminal effectors, CD44loCD62Llo) among total CD8+ T cells in the tumor (NSD, n = 11; HSD, n = 12) and d, of FOXP3+ cells among CD4+ T cells in the spleen and in the tumor of B6 male mice fed as in a (NSD, n = 10; HSD, n = 6). Data are from two independent experiments; two-sided unpaired t-test. Data from one experiment was excluded due to faulty FOXP3 antibody staining. e. NK cell count/gram of MC38 tumor from B6 male mice fed as in a (NSD, n = 16; HSD, n = 15; three independent experiments; two-sided unpaired t-test).

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Extended Data Fig. 4 Decreasing glutamine concentration in cell culture affects CD8+ T cell activation but not viability.

a, Mean ± s.e.m. % live CD8+ T cells (determined as Zombie– by flow cytometry), and b,c, GZMB and TIM3 MFI by flow cytometry in CD8+ T cells activated as in Fig. 1a but with progressively decreasing concentrations of glutamine. Statistically significant differences (p values) are shown only for the NaCl condition for simplicity (n = 4; two-way ANOVA with Geisser-Greenhouse correction; two independent experiments).

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Extended Data Fig. 5 Identification of NaCl and glutamine-specific transcriptional programs in CD8+ T cells.

a, Numbers of differentially expressed genes (DEGs) following pairwise comparisons of mRNA-seq data in the indicated experimental conditions. Data refer to CD8+ T cells from Fig. 4d. b, Schematic representation of the comparisons to identify transcripts specifically upregulated by 80 mM NaCl or glutamine (Gln) from mRNA-seq experiment in Fig. 4d. Ctrl: anti-CD3/28 + IL-2. FC: fold change; FDR: false discovery rate (quasi-likelihood f-test, Benjamini–Hochberg correction). c, Number of genes obtained by overlapping gene lists from b. Genes of interest depicted in the figure were manually selected from Supplementary Table 10. d, Gene signature overrepresentation analysis (Gprofiler) of gene lists identified in c. Gene signatures of interest were manually selected from Supplementary Table 11.

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Scirgolea, C., Sottile, R., De Luca, M. et al. NaCl enhances CD8+ T cell effector functions in cancer immunotherapy. Nat Immunol 25, 1845–1857 (2024). https://doi.org/10.1038/s41590-024-01923-9

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