Abstract
Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. Here we found that NFAT5, an NFAT family transcription factor that lacks an AP-1 docking site, was highly expressed in exhausted CD8+ T cells in the context of chronic infections and tumors but was selectively required in tumor-induced CD8+ T cell exhaustion. Overexpression of NFAT5 in CD8+ T cells reduced tumor control, while deletion of NFAT5 improved tumor control by promoting the accumulation of tumor-specific CD8+ T cells that had reduced expression of the exhaustion-associated proteins TOX and PD-1 and produced more cytokines, such as IFNɣ and TNF, than cells with wild-type levels of NFAT5, specifically in the precursor exhausted PD–1+TCF1+TIM–3–CD8+ T cell population. NFAT5 did not promote T cell exhaustion during chronic infection with clone 13 of lymphocytic choriomeningitis virus. Expression of NFAT5 was induced by TCR triggering, but its transcriptional activity was specific to the tumor microenvironment and required hyperosmolarity. Thus, NFAT5 promoted the exhaustion of CD8+ T cells in a tumor-selective fashion.
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Data availability
Omics data are deposited and accessible under accession numbers GSE224913, GSE237313 and GSE209683. Source data are available for each figure or upon request to the corresponding author. Source data are provided with this paper.
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Acknowledgements
We thank H. Zdimerova for editing the paper, J. Guillaume for contribution to the plasmid production, and P.-C.Ho and T. Walzer for suggestions about the project. This work was funded by the University of Lausanne, and grants from the Max Cloëtta Foundation (G.V.), the Swiss National Science Foundation (G.V.: 310030_182680, 310030_215153, W.H.: 310030_200898), ISREC Institute (D.C.) and the Swiss Cancer Research foundation (G.V.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.
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L.T, D.C, W.H, M.I, G.C, and G.V conceived and designed the experiments. L.T., D.C., P.R., M.C., G.B., M.M.L., and G.V. performed the experiments. T.W. and J.L. analyzed the scRNA-seq data and M.A., S.C., S.N and I.C. performed bioinformatic analysis. C.L.R. provided Cd4-Cre; Nfat5fl/fl mice. L.T., D.C. and G.V. prepared the figures and wrote the paper with input from all authors. D.E.S., W.H., C.L.R. and S.C. reviewed the paper.
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Extended data
Extended Data Fig. 1 Characterization of NFAT5mCherry reporter mice.
a) Fold change of Nfat5 expression level in Tpex or Tex over indicated populations (left). Mean Nfat5 expression level of various CD8+ T cell populations in indicated studies assessed by TILatlas (right). b) NFAT5 reporter mouse strain: The TAG stop codon in exon 14 of the mouse Nfat5 gene was replaced by CRISPR/Cas-mediated genome engineering with the P2A-mCherry cassette to create a knock-in NFAT5-P2A-mCherry reporter model in C57BL/6 mice. c) Example of a gating strategy used throughout the study. d–f) Spleen (d), lymph node (e), and thymus (f) from WT, NFAT5m/wt and NFAT5m/m mice were collected and analyzed for their immune compartment, and thymic development. (e) by flow cytometry. Each dot represents one mouse. g, Relative expression (2-deltaCq) of mCherry and Nfat5 assessed by RT-PCR and normalized to β-2-microglobulin of NFAT5m/wt or NFAT5m/m CD8+ T cells cultured for 72 h under different hyperosmolar conditions ranging from 300 mOsm/kg to 500 mOsm/kg. Each dot represents the mean from a technical replicate from cells from one mouse. h) Histogram showing mCherry expression of NFAT5m/m CD8+ T cells from the lymph node in comparison to a littermate control mouse (WT). i) Western blot showing the total protein level of NFAT5flox/flox CD4-Cre−/− (WT), CD4-Cre+/− NFAT5flox/flox (NFAT5KO), NFAT5m/wt and NFAT5m/m P14 CD8+ T cells cultured in complete RPMI with 1 uM GP33 peptide and 20 U/ml rhIL-2 at 420 mOsm/kg- for 72 hours. β-actin serves as a housekeeping gene. Ratio of NFAT5 level on β-actin has been calculated after quantification of the bands with ImageJ. Biological duplicates shown for each condition. This experieent is representative of 3 independent experiments.
Extended Data Fig. 2 Overexpression of NFAT5 in tumor specific P1A cell led to reduced tumor control.
a) The four murine isoforms of NFAT5 were aligned on the software Geneious using the entries from GenBank and Ensemble. Domains described by Cheung et al. were aligned against mouse isoforms 203, whose length corresponds to human isoform C. NES: Nuclear export signal, AD1: Activation domain 1, AES: Auxiliary export signal, DRL: DNA recognition loop, DD: Dimerization domain, RHD: Rel homology region, AD2: Activation domain 2, AD3: Activation domain 3. b) Retroviral vector where the various isoforms were cloned. c) Nfat5 mRNA levels in CD8+ T cells transduced with a vector encoding for NFAT5 isoform A (NFAT5 A) (left) or control eGFP (right) and sorted by flow cytometry according to GFP expression. d) Timeline of the experiment. Activated TCRP1A CD8+ T cells were transduced, sorted on the base of GFP expression and transferred into Rag1−/−B10D2 mice seven days post P511 tumor engraftment. CD8+ T cells were sorted for RNA-sequencing on day 14. e) Tumor growth in mice transferred with TCRP1A CD8+ T cells transduced and sorted according to GFP expression with the indicated constructs (control eGFP, NFAT5 A, NFAT5 A lacking the DNA binding domain (DBD), NFAT5 D or NFAT1 CA-RIT). n = 5 mice per group (left) n = 5 mice eGFP, n = 6 mice NFAT5 IsoA, n = 7 mice NFAT5 noDBD, and n = 5 NFAT5 IsoD (middle, right). Two-ways ANOVA was performed. Error bars represent SEM. f) Bioluminescence of the luciferase-expressing TCRP1A CD8+ T cells after injection of the mice with luciferin. Each dot is representing one mouse. g) PC analysis of sorted GFP+ CD8+ TILs transduced with control eGFP, NFAT5 A, or NFAT1 CA-RIT. Each dot is representing one mouse. h, Venn diagram showing the number of genes upregulated in NFAT5 A transduced CD8+ TILs, NFAT1 CA-RIT transduced TILs, or both.
Extended Data Fig. 3 Tumor infiltrated NFAT5KO P14 cells had superior anti-tumor potential compared to TI NFAT5WT P14 cells in a co-transfer setting.
a) Timeline of the experiment: activated NFAT5WT (CD45.1) and NFAT5KO (CD45.2) P14 CD8+ T cells were co-transferred into B16-GP33−bearing CD45.1.2 mice. CD8+ TILs were analyzed 7 days post-ACT. b) Overlay of the GMFI of indicated proteins of NFAT5WT and NFAT5KO on the day of 1:1 transfer. c,d) Representative overlaid histograms of NFAT5WT, NFAT5KO P14, and endogenous CD8+ TILs of indicated proteins (up) with two-sided paired t-test comparison (below). Each pair of dots representing TILs analyzed from one mouse.
Extended Data Fig. 4 NFAT5 controled Tox expression in hyperosmolar conditions.
a) Analysis of scRNA-seq data of human melanoma tumors: Pearson correlation coefficients were calculated between NFAT5 mRNA levels and mRNA levels of indicated genes. b) Schematic representation of the protocol used to generate in vitro ‘exhausted’ CD8+ T cells (up). Comparison of NFAT5WT (black) and NFAT5KO (red) level of the indicated proteins below. n = 2 mice NFAT5WT and n = 3 mice NFAT5KO. Mann-Whitney test, 2 sided. c), ATACseq and CUT&RUN-seq analysis of the Tox locus. Enhancers 1 and 2 are indicated. d) Frequency of mCherry+ CD8+ cells among GFP+ transduced with reporter constructs for enhancer 1 and enhancer 2 (left). Representative contour plots for the TCR + NaCl conditions is shown (right). n = 2 culture wells (enhancer 1) and n = 3 culture wells (enhancer 2). Data from 2 pooled experiments (2 biological replicates per enhancer). Mann-Whitney test, 2 sided. e) Vectors used for the reporter constructs using enhancers 1 or 2. USCS’s representations of the 2 regions cloned into the promoter region with potential binding sites for NFAT1−5 (using JASPAR prediction tool).
Extended Data Fig. 5 scRNAseq analysis of NFAT5KO/WT P14 cells during LCMV cl13 infection showed no significant difference.
a) UMAP where cells are clustered using functions of the Seurat package. b) Cells are colored according to cluster ID, to see if one or the other cluster is enriched in cells from different origins. c) Marker genes used to annotate each cluster at resolution 0.1. d,e) We calculated a module score per cell for the genes that were found to be differentially expressed in the bulk RNA sequencing experiment of NFAT5KO vs NFAT5WT in tumors. The DEG genes with adj. p-value < 0.05 and FC > 1.5 were selected to calculate a score for each single cell. We calculated a signature enrichment score of the up-regulated (d) or down-regulated (e) genes obtained from the bulk RNA sequencing data using the AddModuleScore_UCell function of the UCell package. The score varies from 0 to 1, with 1 as the maximum enrichment of the signature.
Extended Data Fig. 6 TI NFAT5KO P14 Tpex cells showed a reduced exhaustion phenotype when co-transferred with NFAT5WT P14 cells.
a) Representative histograms of NFAT5-mCherry expression in P14 CD8+ T cells from acute (Armstrong) or chronic (clone 13) LCMV-infected mice at day seven or day 28 post-infection. b) Frequency of NFAT5WT and NFAT5KO P14 TI CD8+ among co-transferred cells from mice bearing tumors 7 days post-ACT. c) Frequency of Tpex and Tex among NFAT5WT and NFAT5KO P14 from the tumor 7 days post-ACT. d) GMFI of indicated protein expression in Tpex and Tex among NFAT5WT and NFAT5KO P14 from the tumor 7 days post-ACT. b-d) Two independent experiments were pooled. Paired students t-test, two sided. Each pair of dots is representing Tils analyzed from one mouse.
Extended Data Fig. 7 Constructs used to report NFAT5 transcriptional activity in vitro and in vivo.
a) Fold change of mCherry for FK506 treated over DMSO treated animals during indicated periods in the tdLN and ndLN with n = 10 mice per timepoint. Mann-Whitney test, two sided. b) Schematic representation of the luciferase NFAT5 activity reporter plasmid. The blue sequence represents the NFAT5 binding site (TonE). c) Schematic representation of the mCherry NFAT5 activity reporter plasmid. The blue sequence represents the NFAT5 binding site (TonE). d) Frequencies of GFP+ mCherry+ NFAT5WT and NFAT5KO P14 CD8+ T cells from the tdLN or the tumor from B16-GP33 bearing mice. Each pair of dots is representing cells analyzed from one mouse. Paired student t-test, two sided.
Supplementary information
Supplementary Table 1
Gene list of differentially expressed genes between TI P14 NFAT5KO and TI P14 NFAT5WT cells.
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Tillé, L., Cropp, D., Charmoy, M. et al. Activation of the transcription factor NFAT5 in the tumor microenvironment enforces CD8+ T cell exhaustion. Nat Immunol 24, 1645–1653 (2023). https://doi.org/10.1038/s41590-023-01614-x
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DOI: https://doi.org/10.1038/s41590-023-01614-x
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