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The immune checkpoint regulator CD40 potentiates myocardial inflammation

Nature Cardiovascular Research volume 4, pages 458–472 (2025)Cite this article

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Abstract

Immune checkpoint therapeutics including CD40 agonists have tremendous promise to elicit antitumor responses in patients resistant to current therapies. Conventional immune checkpoint inhibitors (PD-1, PD-L1 and CTLA-4 antagonists) are associated with serious adverse cardiac events including life-threatening myocarditis. However, little is known regarding the potential for CD40 agonists to trigger myocardial inflammation or myocarditis. Here we leverage genetic mouse models, single-cell sequencing and cell depletion studies to show that an anti-CD40 agonist antibody reshapes the cardiac immune landscape through activation of CCR2+ macrophages and subsequent recruitment of effector memory CD8+ T cells. We identify a positive feedback loop between CCR2+ macrophages (positive for the chemokine receptor CCR2) and CD8+ T cells driven by IL-12b, TNF and IFNγ signaling that promotes myocardial inflammation and show that previous exposure to CD40 agonists sensitizes the heart to secondary insults and accelerates left ventricular remodeling. Collectively, these findings highlight the potential for CD40 agonists to promote myocardial inflammation and potentiate heart failure pathogenesis.

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Fig. 1: Expansion of CCR2+ macrophages and effector memory T cells in anti-CD40 agonist antibody-treated hearts.
Fig. 2: Dynamics of CCR2+ macrophage and T cell expansion and dependence on monocyte recruitment.
Fig. 3: CD40 activation promotes myocardial inflammatory cytokine and chemokine RNA production.
Fig. 4: CD40 signaling drives transcriptional shifts in mononuclear phagocytes, T cells and NK cells.
Fig. 5: CD40 signaling in CCR2+ macrophages drives expansion and activation of CCR2+ macrophages and CD8+ T cells.
Fig. 6: Cardiac immune cell expansion occurs through an IFNγ-dependent feed-forward loop.
Fig. 7: IL-12b and TNF regulate CD8+ T cell IFNγ production.
Fig. 8: CD40 agonist treatment accentuates cardiac injury and remodeling.

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Data availability

Raw sequencing files and processed normalized data can be found on the Gene Expression Omnibus (GSE290479). All other data supporting the findings in this study are included in the article and associated files. Source data are provided with this paper.

Code availability

All scripts used for analysis in this paper can be found on GitHub (https://github.com/jamrute/2025_NCVR_Jimenez_CD40).

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Acknowledgements

J.J. is supported by funding provided by the National Institutes of Health (1K08HL163518-01A1, R25 HL105400), the Paul and Patti Eisenberg Scholar Award of Washington University in the Division of Cardiology, Institutional Research Grant IRG-21-133-64 from the American Cancer Society and R25HL105446 Small Research Project. K.J.L. is supported by the Washington University in St. Louis Rheumatic Diseases Research Resource-Based Center grant (NIH P30AR073752), the National Institutes of Health (R01 HL138466, R01 HL139714, R01 HL151078, R01 HL161185, R35 HL161185), Leducq Foundation Network (number 20CVD02), Burroughs Wellcome Fund (1014782), Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CH-II-2015-462, CH-II-2017-628, PM-LI-2019-829), Foundation of Barnes-Jewish Hospital (8038-88) and gifts from Washington University School of Medicine. A.H. was supported by National Institute of Diabetes and Digestive and Kidney diseases (R01DK121200) and the VA merit award (I01BX005322). Y.K. was supported by the Japan Society for the Promotion of Science (JSPS) Overseas Research Fellowship (number 202360162) and The Uehara Memorial Foundation Overseas Research Fellowship (number 202140023). We thank E. Lutgens (Amsterdam University Medical Centre) for providing us with Cd40flox/flox mice. We are thankful to the Mouse Cardiovascular Phenotyping Core facility at Washington University for performing mouse echocardiography and the reperfused myocardial infarction surgeries. We thank the Genome Technology Access Center at the McDonnell Genome Institute for help with genomic analysis, the Digestive Diseases Research Core Center (DDRCC) for histology services and the Washington University Center for Cellular Imaging (WUCCI) at Washington University School of Medicine.

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Authors and Affiliations

  1. Center for Cardiovascular Research, Department of Medicine, Cardiovascular Division, Washington University School of Medicine, St. Louis, MO, USA

    Jesus Jimenez, Junedh Amrute, Pan Ma, Xiaoran Wang, Shibali Das & Kory J. Lavine

  2. Cardio-Oncology Center of Excellence, Department of Medicine, Cardiovascular Division, Washington University School of Medicine, St. Louis, MO, USA

    Jesus Jimenez

  3. Washington University, St. Louis, MO, USA

    Raymond Dai

  4. Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

    Yohei Komaru & Andreas Herrlich

  5. John Cochran Division, VA Saint Louis Health Care System, St. Louis, MO, USA

    Yohei Komaru & Andreas Herrlich

  6. Division of Nephrology, Department of Medicine, University Hospital Regensburg, Regensburg, Germany

    Matthias Mack

  7. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

    Kory J. Lavine

  8. Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA

    Kory J. Lavine

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Contributions

J.J., J.A. and K.J.L. were responsible for the conceptualization of the study. J.J., J.A., P.M., X.W., R.D., S.D. and K.J.L. contributed to the experimental design, acquired data and performed the data analysis. Y.K. and A.H. provided serum from anti-CCR2 antibody-treated mice. M.M. provided the anti-CCR2 antibodies. J.J. and J.A. performed the statistical analysis. J.J. and K.J.L. wrote the original draft of the paper, and all authors approved the final version.

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Correspondence to Kory J. Lavine.

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Extended data

Extended Data Fig. 1 No myocardial injury or change in baseline heart chamber size or function in CD40 agonist treated mice.

a, High sensitivity (hs) troponin T levels were not detectable (n.d.) in mice treated with Rat IgG (n = 4) or α-CD40 (n = 4) for 7 days. b, Quantification of left ventricular internal diameter during diastole (LVID,d), c, left ventricular internal diameter during systole (LVID,s), and d, % ejection fraction using 2-dimensional echocardiography in mice treated with Rat IgG (n = 6) or α-CD40 (n = 6) for 28 days. Two-tailed unpaired Student’s t-test performed for all statistical analysis. Error bars indicate means ± s.e.m.

Source data

Extended Data Fig. 2 Representative cardiac immune cell flow analysis gating strategies.

a, Gating strategy for macrophages starting with gating for immune cells (CD45+), followed by doublet exclusion, then live/dead exclusion, then macrophages (CD11b+, CD64+), then CCR2 macrophages (MHChiCCR2-GFP+). b, Gating strategy for T-cells starting with gating for immune cells (CD45+), followed by doublet exclusion, then T-cells (CD4+ or CD8+), then effector memory phenotype (CD4+CD44+ or CD8+CD44+). Gating strategies for c, dendritic cells (Zbtb46-GFP+), d, neutrophils (Ly6G+), and e, B cells (CD19+) are also shown.

Source data

Extended Data Fig. 3 Flow analysis in dendritic cells, neutrophils, and B cells.

a, Quantification of Zbtb46-GFP+ dendritic cells in the heart by flow cytometry comparing Rat IgG (n = 5) and α-CD40 (n = 5). b, Quantification of neutrophils (Ly6G+) in the heart by flow cytometry comparing Rat IgG (n = 4) and α-CD40 (n = 7). c, Quantification of B cells (CD19+) in the heart by flow cytometry comparing Rat IgG (n = 3) and α-CD40 (n = 6). Two-tailed unpaired Student’s t-test performed for all statistical analysis. Error bars indicate means ± s.e.m.

Source data

Extended Data Fig. 4 Histopathological changes following anti-CD40 agonist antibody treatment in various tissues.

Representative hematoxylin and eosin (H&E) staining in wildtype mice comparing Rat IgG isotype control to α-CD40 agonist. a, Heart shows increased immune cells. b, Liver shows increased immune cells. c, Kidney shows increased immune cells and renal intratubular casts (asterisk). d, Brain cortex appears unchanged. e, Skeletal muscle shows increased immune cells. f, Lung shows increased immune cells. g, Ileum shows a reduction of goblet cells (arrowhead). h, Spleen shows white pulp nodule expansion (asterisk) into the red pulp space (arrowhead). i, Ifng, and j, Cxcl9 mRNA expression measured by RT-qPCR from wildtype bulk heart, liver, kidney, brain, skeletal muscle, lung, ileum, or spleen tissue comparing Rat IgG (n = 4) and α-CD40 (n = 4). Representative CD45 cell immunohistochemical staining (brown) in k, heart (Rat IgG n = 4, α-CD40 n = 3), l, liver (Rat IgG n = 4, α-CD40 n = 4), m, kidney (Rat IgG n = 4, α-CD40 n = 3), n, brain (Rat IgG n = 4, α-CD40 n = 4), o, skeletal muscle (Rat IgG n = 4, α-CD40 n = 4), p, lung (Rat IgG n = 3, α-CD40 n = 4), q, ileum (Rat IgG n = 4, α-CD40 n = 4), and r, spleen tissue (Rat IgG n = 4, α-CD40 n = 4) with quantification of CD45+ cells per 20x field. Unable to quantify spleen CD45 staining. Scale bars 20 or 50 µm. Two-tailed unpaired Student’s t-test performed for all statistical analysis. Error bars indicate means ± s.e.m.

Source data

Extended Data Fig. 5 Low levels of anti-drug antibodies with minimal impact on the inflammatory response from serial injections.

a, Quantification of mouse anti-rat IgG levels from serum in naïve mice (n = 3), mice treated with 100 μg Rat IgG (n = 4) or 100 μg α-CD40 (n = 4) for 7 days, or mice treated with 20 μg α-CCR2 (n = 3) for 4 days. b, Experimental model of mice receiving 1 single or 3 serial anti-CD40 agonist antibody injections (Created in BioRender. Lavine, K. (2025) https://BioRender.com/e68h639). c, Ifng, and d, Cxcl9 mRNA expression measured by RT-qPCR from cardiac tissue in Rat IgG (n = 3), 1 injection of α-CD40 (n = 5), and 3 injections of α-CD40 (n = 4). One-way ANOVA with Sidak correction. Error bars indicate means ± s.e.m.

Source data

Extended Data Fig. 6 Quality control for CITE-sequencing and gene expression signatures of major cell types and subclusters.

a, Dot plot of differentially expressed genes for the major cell types. b, Violin plot of percentage mitochondrial reads per cell. Cells were filtered to include those with 1,000 < RNA unique molecular identifier (UMI) count < 20,000, and mitochondrial reads less than 5%. c, Heatmaps showing relative expression profiles of major cell types, d, mononuclear phagocytes, and e, T-cells and NK-cells.

Source data

Extended Data Fig. 7 CITE-seq expression of Ccr2 and inflammatory cytokines and chemokines.

a, Density plot showing expression of Ccr2, b, Cxcl9, c, Il12b, and d, Tnf in UMAP embedding in mononuclear phagocytes from hearts treated with anti-CD40 agonist antibody. e, Dot plot of Cxcl9 in each major cell type.

Source data

Extended Data Fig. 8 No change in CCR2+ macrophage expansion with B cell depletion.

a, In mice receiving depleting α-CD19 (B cell) with concurrent α-CD40 agonist or Rat IgG isotype antibodies for 7 days, CCR2+ macrophages were quantified in the heart by flow cytometry with comparison between Rat IgG (n = 3), Rat IgG+α-CD19 (n = 4), α-CD40 (n = 5), and α-CD40+α-CD19 (n = 4). One-way ANOVA with Sidak correction. b, Quantification of B cells (CD19+) in the spleen by flow cytometry confirmed B cell depletion with comparison between α-CD40 (n = 5) and α-CD40+α-CD19 (n = 4). Unpaired Student’s t-test, two-tailed. Error bars indicate means ± s.e.m.

Source data

Extended Data Fig. 9 No change in intracellular IFN-γ following CD40 agonist treatment with concurrent single agent IL12b or TNF neutralizing antibodies, and no change in CCR2+ macrophage expansion with CD4 T-cell depletion.

a, In mice receiving neutralizing α-IL12b with concurrent α-CD40 agonist treatment antibodies for 7 days, IFN-γ+CD44+ CD8 T-cells were quantified in the heart by flow cytometry with comparison between α-CD40 (n = 5), and α-CD40+α-IL12b (n = 5). b, In mice receiving neutralizing α-TNF with concurrent α-CD40 agonist treatment antibodies for 7 days, IFN-γ+CD44+ CD8 T-cells were quantified in the heart by flow cytometry with comparison between α-CD40 (n = 4), and α-CD40+α-TNF (n = 4). c, In mice receiving depleting α-CD8 with concurrent α-CD40 agonist treatment antibodies for 7 days, CD8+ T-cells were quantified in the spleen by flow cytometry to confirm CD8 T-cell depletion when comparing α-CD40 (n = 6) and α-CD40+α-CD8 (n = 6). d, In mice receiving depleting single agent α-CD4 or combined α-CD4 with α-CD8 and concurrent α-CD40 agonist for 7 days, CCR2+ macrophages were quantified in the heart by flow cytometry with comparison between Rat IgG (n = 3), α-CD40 (n = 6), α-CD40+α-CD4 (n = 6), and α-CD40+α-CD4+α-CD8 (n = 6). One-way ANOVA with Sidak correction. Two-tailed unpaired Student’s t-test performed for all statistical analysis except as noted. Error bars indicate means ± s.e.m.

Source data

Extended Data Fig. 10 Representative images of negative controls for immunofluorescent staining and RNA in situ hybridization experiments.

a, Negative controls of CD64 immunostaining with Opal 570 pertaining to Fig. 1c. b, Negative controls of CD8 immunostaining with Opal 570 pertaining to Fig. 1d. c, Negative controls of Ki67 immunostaining with Alexa 647 and CCR2-GFP immunostaining with Alexa 555 pertaining to Fig. 2i. d, Negative controls of Cxcl9 with Opal 570 and Ccr2 with Opal 520 by RNA in situ hybridization pertaining to Fig. 3b. e, Negative controls of CD68 immunostaining with Alexa 647 pertaining to Fig. 7h. f, Negative controls of Cxcl9 with Opal 570 by RNA in situ hybridization pertaining to Fig. 7j. g, Negative controls of CD8 immunostaining with Alexa 555 pertaining to Fig. 8b.

Source data

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Source Data Extended Data Figs. 1–10 (download XLSX )

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Jimenez, J., Amrute, J., Ma, P. et al. The immune checkpoint regulator CD40 potentiates myocardial inflammation. Nat Cardiovasc Res 4, 458–472 (2025). https://doi.org/10.1038/s44161-025-00633-1

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