Abstract
Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.
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Data availability
Single-cell sequence data have been deposited in ENA under accession code PRJEB52332. All other data are present in the article and Supplementary Information or from the corresponding authors upon reasonable request. Source data are provided with this paper.
Code availability
Methods for the bioinformatic analyses of scRNA and TCR-seq data can be found on GitLab (https://gitlab.univ-nantes.fr/gourain-v-1/cxcr6_lungsepsis) and Zenodo (https://doi.org/10.5281/zenodo.10715057)73.
Change history
07 October 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41590-024-02000-x
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Acknowledgements
We thank L. Brusselle for helping with the preparation of single-cell libraries, M. Papot for preparing the L–R analyses and E. Sechet for administrative support (all from Nantes Université, INSERM, Center for Research in Transplantation and Translational Immunology). We thank L. Legentil and V. Ferrières (Univ. Rennes, Ecole Nationale Supérieure de Chimie de Rennes) for providing the laminarin and C. Louvet (Nantes Université, INSERM, Center for Research in Transplantation and Translational Immunology) for providing B16-OVA and LLC cell lines. We thank staff at the Cytometry Facility ‘Cytocell’, University of Nantes and the Genomics and Bioinformatics Core Facility of Nantes (GenoBiRD, Biogenouest) for their technical support. We acknowledge the IBISA MicroPICell facility (Biogenouest), a member of the national infrastructure France-Bioimaging supported by the French National Research Agency (ANR-10-INBS-04). We thank the biological resource center for biobanking (CHU Nantes). A.B. was supported by La Ligue contre le Cancer (Comites 22, 44 85) and INCA (grant 16690). S.C. and F.M.C. were supported by the Association pour la Recherche sur le Cancer (grant SIGN’IT20181007792). A.R. was supported by Agence National de la Recherche (grant ANR JCJC Progr-AM). We gratefully acknowledge the financial support of LabEx IGO.
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Contributions
A.B. performed experiments, contributed to the study design, data analyses, interpretation of results and writing and revising the manuscript. V.G. generated scRNA-seq data, performed bioinformatics analysis, contributed to the study design, data analyses, interpretation of results and writing and revising the manuscript. T.G. performed the epidemiological study and contributed to interpreting results, writing, and revising the manuscript. V.L.M., D.S., M.A., C.J., F.P.M., C.P., P.M., M.D., L.B., M.P., C.F., M.C., T.L., M.G., J.F.M. and T.C. performed experiments and contributed to the interpretation of results and revision of the manuscript. L.L. and V.F. provided laminarin and contributed to interpreting results, writing and revising the manuscript. H.E.G.M., T.L., C.H., E.S., S.C., F.M.C., N.M., J.A.V., P.A.G. and K.A. contributed to the interpretation of results and revision of the manuscript. J.P. and A.R. contributed to the study design, data analyses, result interpretations and manuscript writing and revision.
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Extended data
Extended Data Fig. 1 Raw data of risk of cancer up to ten years after cure from sepsis and SIRS in humans.
A-C. Cumulative incidences of cancers in sepsis survivors and infection survivors after ten years of follow-up in the (A) crude sample of cohort 1, (B) narrow matching criteria samples, and (C) in patients with low (IGS-II < 25) vs. high severity score (IGS-II > 25). Curves represented as calculated incidence +/- SD. D. Relative risk (RR) of cancer in sepsis vs. infection survivors (green line) and nonseptic SIRS vs. no SIRS (orange line) at the indicated time after the index medical condition. RR > 1 means increased risk in the severe condition, and a RR < 1 means a reduced risk of cancer. Results were obtained in the matched samples of cohorts 1 and 2. E-G. Cumulative incidence of cancers in nonseptic SIRS and no-SIRS survivors after ten years of follow-up in the (E) crude, (F) matched and (G) narrow matching criteria samples. Curves represented as calculated incidence +/- SD, and comparisons are obtained using ANCOVA (A-C, E-G) or RR with a 95% confidence interval (D).
Extended Data Fig. 2 Sepsis-cured mice model and scRNA-seq validations.
A-B. Bacterial load in digested lung tissue and weight loss at the indicated time after E. coli intratracheal instillation (n = 2 for uninfected, n = 16 for day 1, n = 14 for day 2, n = 7 for day 3, n = 8 for day 7, n = 7 for day 10). Graph represents median ± IQR and is pooled data from 2 independent experiments. C. Lung section of uninfected mice, and at 3 and 7 days after E. coli pneumonia. Representative of 3 independent experiments. D. Respiratory sepsis was induced by intratracheal instillation of E. coli. LLC was injected iv. in uninfected and sepsis-cured wild-type or Cxcr6-deficient mice (infected 28 days prior), and the surfaces of lung tissue and metastasis were measured 4 weeks after LLC injection. Wild-type mice: n = 6 for uninfected, n = 7 for sepsis-cured. Cxcr6−/− mice: n = 6 for uninfected, n = 9 for sepsis-cured. Graphs represent median ± IQR pooled from 3 independent experiments. E. Rationale of single-cell RNA sequencing assay. F. Quality controls of the single-cell RNA sequencing data representing the distribution of detected genes (left panel), mapped reads (middle panel), and reads mapped against mitochondrial genes (right panel) per cell. G. Heatmap of top 10 markers per cluster. Cxcr6 and Cd3g are characteristic of cluster 5. Statistical significance was assessed by One-way analysis of variance with Sidak correction for multiple comparisons (D).
Extended Data Fig. 3 Lung CXCR6 T cells characteristics.
A. CXCR6 staining on γδ tissue-resident T cells in sepsis-cured wild-type or Cxcr6-deficient mice expressing GFP as gene reported. Representative of 10 independent experiments. B. Clustering (numbers) and annotation (colors) of immune cells from the scRNA-seq in mouse lung. C. Expression of Ccr7 (top) and S1pr1 (bottom) in immune cells of mice lung. D-E. PE-conjugated anti-CD45 antibody was injected intravenously 3 and 5 days after E. coli sepsis. Brilliant Violet (bv)480 conjugated anti-CD45 antibody was injected immediately before sampling to remove immune cells adherent to the endothelium. (D) The percentages of blood-recruited (bv480- PE + ) CXCR6− and CXCR6 + T cells were measured on day 7 (n = 3 uninfected and 5 septic mice). Graph represents median ± IQR and are pooled data from 2 independent experiments. (E) Percentages of blood-recruited (bv480- PE + ) myeloid F4/80+ cells were measured on day 3 after pneumonia (n = 3 mice/group). (F) Percentage of blood PE + T cells 60 minutes after anti-CD45-PE iv. injection. Representative of three independent experiments. G. Expression of CXCR6 on CD8, CD4, NK1.1+ and γδ T cells (left panel) and linear regression of T cell subsets increase and their CXCR6 expression in 7 days infection-cured mice (right panel). H. Number of CXCR6− and CXCR6+ tissue-resident T cells in uninfected and sepsis-cured mice. N= for uninfected and n = 7 for sepsis-cured mice. Graph represents median ± IQR, pooled data from 2 independent experiments. Statistical significance was assessed by Mann–Whitney test (D-E) and one-way analysis of variance with Bonferroni correction was used for multiple comparisons (H). Correlation was tested by the Pearson test (G).
Extended Data Fig. 4 Activated pathways in lung T cell clusters.
A-B. Expression of genes associated with the Wikipathway «Chemokine signaling» (WP2292) during (A) and after (B) sepsis. C-D. Expression of genes of TGFb pathway (KEGG mmu:04350) at (C) day 3 and (D) day 7 post-sepsis in T cell compartment. (E) Expression of the interferon type II pathway effector genes in the T cell compartment. The colors represent the cell–cell communication probability for each test R-L and the circle radius the significance. (F) Number of resident CXCR6+γδT cells in uninfected and sepsis-cured wild-type and IfnγR deficient mice. (n = 7 for wild-type and n = 11 for IfnγR deficient mice per group). Graphs represent median ± IQR and are representative of 2 independent experiments. The color gradient represents the level of expression, and the circle radius the percentage of expressing cells. Statistical significance was assessed by one-way analysis of variance with Bonferroni correction for multiple comparisons (F).
Extended Data Fig. 5 scRNA-sequencing of CXCR6pos and CXCR6neg T cells.
A. Expression of cytokine and chemokine signaling genes in the T cell compartment after sepsis. The color gradient represents the level of expression, and the circle radius the percentage of expressing cells. B. Cell-sorting strategy for scRNA sequencing of tissue- resident T cells in septic-cured wild-type and Cxcr6−deficient mice. C. Expression of the genes associated with tissue residency (list from literature review). The colors represent average expression for each and the percentage of expressing cells. Graph represents median ± IQR and is pooled data from 2 independent experiments. D. Heatmap of differentially expressed genes in CD69high or CD69low resident γδ, CD4 + , CD8 + T cells from wild-type vs. Cxcr6−deficient sepsis-cured mice (from scRNA-seq). E. Differential pathway expression levels between CD69high or CD69low resident γδ T cells from wild-type vs. Cxcr6−deficient sepsis-cured mice. F. CXCR6 expression after 18 hours in vitro stimulation of T cells sorted from uninfected T cells with the stated CCR2 agonists. (n = 4 for untreated and n = 6 for treated conditions). Graph represents median ± IQR and is pooled data from 2 independent experiments. Statistics were calculated with the two-sided Wilcoxon test (D). P values were corrected with the Bonferroni test with the confident interval of 95%. Gene set enrichments were calculated with the permutation test of ranked values and corrected with the Benjamini-Hochberg test (E).
Extended Data Fig. 6 CXCL16 blocking and resident AM depletion.
A. Number of CXCR6+ and CXCR6− resident γδ, CD4 and CD8 T cells in sepsis-cured mice treated with isotype and anti-CXCL16 blocking antibody (n = 5 mice/group). Graphs represent median ± IQR and are pooled from two experiments. B. Ki67 expression by CXCR6− and CXCR6 + T cells in septic-cured mice treated with anti-CXCL16 blocking antibody or isotype control. (n = 3 mice/group). Graph represents median ± IQR and is pooled data from 2 independent experiments. C. PE staining of resident AM from sepsis-cured mice by anti-CXCL16 antibody or isotype control (Representative of 3 independent experiments). D. Percentages of Cxcl16 expressing cells (pi-charts, n = 2 mice per time point) and Cxcl16 RNA expression levels (box-plots) in the clusters at the indicated time point after E. coli pneumonia. The ratio of positive (red) and negative (blue) cells is represented in the upper panel. (n = 2 mice per time point). E. Expression of cytokine receptors on CXCR6 T cells and cognate cytokines in the different cell-type compartments during (left panel) and after (right panel) sepsis. Pairs of cytokine and cytokine receptors were initially extracted from the KEGG database. The colors represent the average expression level, and the circle radius represents the percentage of expressing cells. Significance of R-L interactions was evaluated with the one-sided permutation test. F. Gating strategy to identify resident alveolar macrophages and monocyte-derived macrophages (mo-AM). Representative of 10 experiments. G. Number of resident AM and mo-AM in uninfected and sepsis-cured wild-type and Ccr2-deficient mice. For resident AM (top panel): n = 6 mice per group. For mo-AM (bottom panel): n = 13 mice per group for all groups except wild-type day 7: n = 9 mice. Graphs represent median ± IQR and are pooled from two experiments. H. Numbers of resident AM, and mo-AM, in uninfected and sepsis- cured mice treated on day 3 and day 5 blocking anti-CXCL16 antibody or isotype control. N = 2 mice per group for uninfected. n = 3 mice per group in sepsis-cured. Graphs represent median ± IQR and are one experiment. I. Numbers of resident AM in sepsis-cured mice injected with LLC and treated with blocking anti-CXCL16 antibody or isotype control. N = 6 mice per group for all groups except isotype Ab group: n = 5 mice. Graph represents median ± IQR and is pooled data from 2 independent experiments. J. Number and percentage of resident AM depletion in untreated mice, and 2, 7 and 14 days post clodronate injection (n = 6 / group). Graphs represent median ± interquartile range and are pooled data from 2 independent experiments. Statistical significance was assessed by Mann–Whitney test (A) and One-way analysis of variance with Sidak’s correction for multiple comparisons (B, G, H, I, J).
Extended Data Fig. 7 CXCR3 and CCR2 expressions by lung tissue resident T cells, and epigenic regulation of resident AM.
A. Percentage of CXCR3+ on antigen-experienced resident memory T cells (CD44 + CD69 + T cells) and antigen-experienced non-resident memory T cells (CD44 + CD69- T cells) in the lungs of uninfected, primary infection-cured and secondary infection-cured mice. n = 2 mice per group in uninfected and n = 4 mice per group in sepsis-cured group. Graphs represent median ± interquartile range and are pooled data from 2 independent experiments. B. Mice were infected with E. coli; 14 d later, the mice were treated with anti-CXCR3 antibody (200 μg twice) or isotype control and were finally assessed 7 days after the induction of secondary lung sepsis. Enumeration of antigen-experienced non-resident memory T cells (CD44 + CD69- T cells) in the lungs of uninfected mice or of mice cured from a secondary sepsis (n = 2 mice per group in uninfected, n = 5 in isotype group and n = 4 in anti-CXCR3 group). Graphs represent median ± interquartile range and are pooled data from 2 independent experiments. C. Pie-charts represent percentages of Ccr2 expressing cells and Ccr2 expression levels in the T cell compartment at the indicated time point during primary and secondary sepsis. The ratio of positive (red) and negative (blue) cells is represented in the upper panel. (n = 2 mice per time point). D. Promoter and enhancer activity at Il7, Ccl3, and Ccl9 loci in resident alveolar macrophages before (black) and after (red) sepsis. Statistical significance was assessed by one-way analysis of variance with Bonferroni correction for multiple comparisons (A-B), two-sided Wilcoxon test (C).
Extended Data Fig. 8 CXCR6 expression by T cells during S. aureus pneumonia, systemic sepsis and CpG.
A. Numbers of CXCR6− and CXCR6 + T cells in the lungs 7 days after S. aureus pneumonia (n = 3 uninfected, n = 2 sepsis-cured mice). Graphs represent median ± IQR and is issued from 1 experiment. B. Numbers of CXCR6− and CXCR6+ tissue-resident T cells in the lungs and the spleen 7 days after S. aureus septicemia (n = 7 mice/group). Graphs represent median ± IQR and are pooled data from 2 independent experiments. C. Numbers of CXCR6− CD3+ and CXCR6 + CD3+ cells in the bone marrow, the gut, and spleen 7 days after CpG iv administration (n = 6 mice per time point). Graph represents median ± IQR, pooled data from 2 independent experiments. One-way analysis of variance with Bonferroni correction was used for multiple comparisons (A-C).
Extended Data Fig. 9 scRNA-sequencing of lung immune cells to traumatic brain injury.
A. Heatmap of top 10 markers in monocyte-derived alveolar macrophages (mo-AM) at day 3 and day 7 after E. coli pneumonia, and interstitial Macrophages (IntMac), resident alveolar macrophages (Res-AM) and macrophages 1 day after traumatic brain injury (TBI) by single-cell RNA sequencing analyses of lung CD45+ cells. B. IL-1a and IL-1b expression levels in the monocytes/macrophages cluster at the indicated time point after E. coli sepsis or after TBI (n = 2 independent biological replicate per time point). C. CD45+ cells were sorted from the lungs of control mice, mice with acute traumatic brain injury and 3–7 days after E. coli pneumonia. UMAP representing the macrophage compartment in mouse lung CD45+ cells annotated from the scRNA-seq. 10,542 single cells from 2 mice from all time points were analyzed. D. Volcano plots of the DEG in the macrophage compartment after TBI vs. 3 days (left) or 7 days (right) after E. coli pneumonia. E. Pie-chart representing percentages of Cxcl16 expressing cells and Cxcl16 expression levels in the monocytes/macrophages cluster at the indicated time point after E. coli sepsis or after TBI. (n = 2 mice per time point). F-G. Numbers of CXCL16+ and CCL2+ res-AM and mo-AM in uninfected mice after traumatic brain injury, 7 days after E. coli pneumonia or 7 days after S. aureus pneumonia. (n = 3-5 mice/group). Graphs represent median ± IQR and pooled from 2 independent experiments. H. Numbers of CXCR6+ and CXCR6− T cells in the lungs 7 days after traumatic brain injury (n = 3 sham, =5 TBI mice). I. Mice were depleted or not in res-AM (clodronate i.t.), then treated with laminarin (i.p.) or vehicle, and injected with LLC 7 days later. Mice survival was monitored up to 14 days post LLC- inoculation. The lung tumor surface was measured 4 weeks later. (n = 10 mice for vehicle and 11 for laminarin, and 4 for clodronate with lamarin). Graphs represent median ± IQR and are pooled from 2 independent experiments. Statistics were calculated with the Kruskal-Wallis test (B), the two-sided Wilcoxon test P values were corrected with the Bonferroni test (D), one-way analysis of variance with Bonferroni correction for multiple comparisons (E-H), and log-rank test for survival (E).
Extended Data Fig. 10 CXCR6 expression by T cells in human cancer samples.
A. Expression levels of Cxcl16, Ccl7, and Ccl2 in the macrophages compartment from BAL of healthy humans and patients with moderate (infection) or severe (sepsis) COVID- 19 pneumonia from GSE145926. B-C. Immunostained sections. Data are representative of a single experiment. (B) four human lung cancer, and (C) five colon adenocarcinoma. Green: CD3, Red: CXCR6, Blue: DAPI. D. Correlation between Cxcr6 level and lung tumor TRM, lung TRM, and dermis TRM gene signatures from publicly available datasets. GSE111898 (33) for lungs and GSE83637 (34) for the dermis. E. Projection of the gene signature of human dermis tissue-resident T cells (from GSE83637) on the UMAP representation of scRNA-seq of lung tissue from septic-cured mice. F. Definition of a Cxcr6 tissue-resident T cells signature in sepsis-cured mice. Significance of difference was evaluated with the two-sided Wilcoxon test (F).
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Supplementary Tables 1–5, Online Methods and Clinical Study Protocol.
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Broquet, A., Gourain, V., Goronflot, T. et al. Sepsis-trained macrophages promote antitumoral tissue-resident T cells. Nat Immunol 25, 802–819 (2024). https://doi.org/10.1038/s41590-024-01819-8
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DOI: https://doi.org/10.1038/s41590-024-01819-8
