Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis

Abstract

Background: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages.

Methods: We used an established murine ICI myocarditis model (Ctla4^+/-Pdcd1^-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies.

Results: We observed marked increases in CCR2 (C-C chemokine receptor type 2)⁺ monocyte-derived macrophages and CD8⁺ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2⁺ subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2⁺ monocytes. It is important that a similar macrophage population expressing CXCL9, CXCL10, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9⁺Cxcl10⁺ macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8⁺ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9⁺Cxcl10⁺ macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis.

Conclusions: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ-induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

Keywords: CXCL9 chemokine; IFN-gamma; T-cells; cytotoxic T lymphocyte-associated antigen 4-immunoglobulin; macrophages; myocarditis; programmed cell death protein 1.

Figure 1.. Accumulation of CCR2⁺ monocytes and macrophages in Ctla4^+/−Pdcd1^−/− mouse hearts.

A, Representative images of H&E and CD68 immunofluorescent staining (red) in wild-type (Ctla4^+/+Pdcd1^+/+), Ctla4^+/+Pdcd1^−/−, and Ctla4^+/−Pdcd1^−/− hearts. Quantification of CD68⁺ cells. Data collected from 2 independent experiments. Ctla4^+/+Pdcd1^+/+ (n=5), Ctla4^+/+Pdcd1^−/− (n=4), Ctla4^+/−Pdcd1^−/− (n=10), Welch's t-test, 2-tailed. Scale bar for H&E staining images, 50 μm. Scale bar for CD68 staining images, 100 μm. B, Quantification of CD45⁺, CD64⁺, CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ cells in the heart by flow cytometry. Data collected from 4 independent experiments. Ctla4^+/+Pdcd1^−/− (n=22), Ctla4^+/−Pdcd1^−/− (n=14), Mann-Whitney test, 2-tailed. C, Quantification of CCR2⁺ macrophages and LY-6C^high monocytes by flow cytometry. Data collected from 4 independent experiments. Ctla4^+/+Pdcd1^−/− (n=22), Ctla4^+/−Pdcd1^−/− (n=14), Mann-Whitney test, 2-tailed. D, Ccr2 (green) and Cd68 (red) expression detected in Ctla4^+/+Pdcd1^−/− and Ctla4^+/−Pdcd1^−/− mouse hearts by RNA in situ hybridization. Left, Representative images, scale bar, 50 μm. Right, Quantification of the percentage of Cd68⁺Ccr2⁺cells in Cd68⁺cells and Ccr2⁺cells, respectively, as well as the cell numbers per 10× field. Ctla4^+/+Pdcd1^−/− (n=5), Ctla4^+/−Pdcd1^−/− (n=7), Mann-Whitney test, 2-tailed. E, In vivo cardiac CCR2 signal was detected with a CCR2 specific radiotracer, ⁶⁴Cu-DOTA-ECL1i, using positron emission tomography. Representative CCR2 positron emission tomography–computed tomography (PET/CT) images (left) and quantification of CCR2 tracer uptake (right). Data collected from 2 independent experiments, Ctla4^+/+Pdcd1^+/+ (n=4), Ctla4^+/+Pdcd1^−/− (n=12), Ctla4^+/−Pdcd1^−/− (n=17), Mann-Whitney test, 2-tailed. CCR2 indicates C-C chemokine receptor type 2; H&E, Hematoxylin and Eosin; and LY-6C, lymphocyte antigen 6C.

Figure 2.. Expansion of Cxcl9⁺Cxcl10⁺ macrophages in Ctla4^+/−Pdcd1^−/− mouse hearts.

A, UMAP clustering of 23 606 cells from 14 mouse hearts (Ctla4^+/+Pdcd1^−/−, n=4; Ctla4^+/−Pdcd1^−/−, n=10), showing 8 major cell types. B, UMAP clustering of 3209 the myeloid cells spilt by experimental group highlighting 5 transcriptionally distinct subclusters. C, The proportion of each myeloid subcluster in Ctla4^+/+Pdcd1^−/− and Ctla4^+/−Pdcd1^−/− mice. D, Dot plots of differentially expressed genes in each myeloid subcluster. E, Z score feature plot of enriched genes in each myeloid subcluster and density plot of Ccr2 expression. Cell state marker genes (black) were selected based on robust enrichment in their respective subclusters. Bhlhe40 indicates basic helix-loop-helix family member e40; Ccl24, C-C motif chemokine ligand 24; Ccr7, C-C motif chemokine receptor 7; Chil3, Chitinase-like 3; Clec9a, C-type lectin domain containing 9A; DC, dendritic cell; Flt3, fms related receptor tyrosine kinase 3; Mono, monocyte; Plac8, Placenta specific 8; Serpinb6b, serine (or cysteine) peptidase inhibitor, clade B, member 6b; and UMAP, Uniform Manifold Approximation and Projection.

Figure 3.. Cxcl9⁺Cxcl10⁺ macrophages exhibit an activated phenotype in immune checkpoint inhibitor myocarditis.

A, Volcano plot of differentially expressed genes amongst myeloid cells from Ctla4^+/+ Pdcd1^−/− and Ctla4^+/−Pdcd1^−/− hearts obtained by Wilcoxon rank-sum test using R package Seurat (v4). B, Zscore feature plot of the top 10 upregulated genes in Ctla4^+/+ Pdcd1^−/− myeloid cells compared with Ctla4^+/−Pdcd1^−/− myeloid cells split by experimental group. Differentially expressed genes are selectively expressed in Cxcl9⁺Cxcl10⁺ macrophages. C, Increased Cxcl9, Cxcl10, Gbp2b, Ccl8, and Fcgr4 mRNA expression in Ctla4^+/+ Pdcd1^−/− compared with Ctla4^+/−Pdcd1^−/− heart tissue measured by RT-PCR. Data collected from 2 independent experiments, Ctla4^+/+Pdcd1^−/− (n=8), Ctla4^+/−Pdcd1^−/− (n=6), Mann-Whitney test, 2-tailed. D, Coexpression of Cxcl9 and Cxcl10 with Ccr2 in mouse hearts visualized by RNA in situ hybridization. Left, Representative images in each condition; scale bar, 50 μm. Right, Quantification of the number of Cxcl9⁺Ccr2⁺ cells or Cxcl10⁺Ccr2⁺ cells per 10× field in each condition as well as the percentage of Cxcl9⁺Ccr2⁺ or Cxcl10⁺Ccr2⁺ cells in Cxcl9⁺ or Cxcl10⁺ cells. Ctla4^+/+Pdcd1^+/+ (n=4), Ctla4^+/+Pdcd1^−/− (n=4), Ctla4^+/−Pdcd1^−/− (n=9), Mann-Whitney test, 2-tailed. E, Quantification of FCGR4 protein expression on CD64⁺ macrophages by flow cytometry. Data collected from 4 independent experiments, Ctla4^+/+Pdcd1^−/− (n=20), Ctla4^+/−Pdcd1^−/− (n=14), Mann-Whitney test, 2-tailed. F, Gene ontology pathway enrichment analysis of upregulated genes in Ctla4^+/−Pdcd1^−/− myeloid cells. The top 5 enriched pathways in Ctla4^+/−Pdcd1^−/− myeloid cells are displayed. Genes used in the analysis were selected from Seurat differential expression with P < 0.05 and log₂FC > 0.5. P value calculated using hypergeometric distribution and corrected for multiple comparisons. G, Z score feature plots of enriched genes involved in response to IFN-γ (interferon gamma), cytokine-mediated signaling pathway, myeloid leukocyte migration, antigen processing, and presentation pathways in myeloid cells. Arf3 indicates ADP ribosylation factor 3; AW112010, expressed sequence AW112010; Ly6a, lymphocyte antigen 6 family member A; Marcksl1, MARCKS like 1; Nfkbia, NFKB inhibitor alpha; and Snx3, sorting nexin 3.

Figure 4.. Cxcl9⁺Cxcl10⁺ macrophages originate from monocytes.

A, tSNE force-directed layout plot of myeloid cells. Cells are colored by cell cluster annotations. B, Pseudotime and entropy values of myeloid cells. Cxcl9⁺Cxcl10⁺ macrophages (Cxcl9 Cxcl10 Mac) have high pseudotime and low entropy values, suggesting that they represent a differentiated cell state. C, Terminal state probability of cell states predicted as differentiated populations: Cxcl9 Cxcl10 Mac; Cd163 resident Mac; and DCs. D, Box plots of entropy (upper) and Cxcl9 Cxcl10 Mac terminal state probability (lower) of myeloid subclusters split by experimental group. E, Percentage of LY-6C^high monocytes and CCR2⁺ macrophages of cardiac CD64⁺ cells from vehicle or MC-21 antibody-treated mice quantified by flow cytometry. Displayed cells are CD45⁺LY-6G⁻CD64⁺. Data collected from 4 independent experiments. Vehicle group (n=12), MC-21–treated group (n=8), Mann-Whitney test, 2-tailed. F, Representative images (upper) and quantification (lower) of Cxcl9 and Cxcl10 positive cells in the heart 6 days after MC-21 antibody treatment. Data collected from 4 independent experiments. Vehicle group (n=12), MC-21–treated group (n=8), Mann-Whitney test, 2-tailed. DC indicates dendritic cell; and Mono, monocyte.

Figure 5.. CXCL9⁺CXCL10⁺ macrophages in human ICI associated myocarditis.

A, Expression of CXCL9and CXCL10 by RNA in situ hybridization in human heart tissue from patients with immune checkpoint inhibitor myocarditis (ICI, n=7), lymphocytic myocarditis (LM, n=5), ischemic cardiomyopathy (ICM, n=5), or dilated cardiomyopathy (DCM, n=6), and donor control subjects (n=6). Quantification of the number of CXCL9⁺ and CXCL10⁺ cells, Mann-Whitney test, 2-tailed. Scale bar, 50 μm. B, Immunofluorescent staining of CD16α (green), CCR2 (red), CD68 (white), and DAPI (blue) in human heart tissue from patients with ICI (n=8), LM (n=5), ICM (n=5), or DCM (n=6), and donor control subjects (n=6). Quantification of cell number and the percentage of CD68⁺CD16a⁺ cells in all CD68⁺ cells, Mann-Whitney test, 2-tailed. Scale bar, 50 μm.

Figure 6.. T-cells are the primary source of IFN-γ in immune checkpoint inhibitor myocarditis mouse hearts.

A, Increased Ifng mRNA expression in Ctla-4^+/−Pdcd1^−/− mouse hearts measured by RT-PCR. Data collected from 2 independent experiments, Ctla4^+/+Pdcd1^−/− (n=8), Ctla4^+/−Pdcd1^−/− (n=6), Mann-Whitney test, 2-tailed. B, Feature plot of Ifng expression in all cell types recovered from the heart showing specific expression in the NK/T-cell cluster. C, Feature plots of Ifng, Cd8a, and Cd4 expression in NK&T-cells showing CD8 T-cell expansion and enriched Ifng expression in CD8 T-cells from Ctla4^+/−Pdcd1^−/− hearts. D, Percentages of IFN-γ⁺CD4⁺, IFN-γ⁺CD8⁺ T-cells, IFN-γ⁺ NK-cells, and IFN-γ⁺CD64⁺ macrophages analyzed by flow cytometry. Data collected from 2 independent experiments, Ctla4^+/+Pdcd1^−/− (n=8), Ctla4^+/−Pdcd1^−/− (n=5), Mann-Whitney test, 2-tailed. E, The proportion of each NK&T subcluster per experimental group. F, Z score feature plots of top 10 upregulated genes in Ctla4^+/−Pdcd1^−/− NK&T-cells compared with Ctla4^+/+Pdcd1^−/− NK&T cells split by group. G, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched pathways using genes upregulated in Ctla4^+/−Pdcd1^−/− NK&T cells compared with Ctla4^+/+Pdcd1^−/− NK&T cells. Genes used in the analysis were selected from Seurat differential expression with P < 0.05 and log₂FC > 1. P values calculated by hypergeometric distribution using R package ClusterProfiler. Btg1 indicates B-cell translocation gene 1 protein; IFN-γ, interferon gamma; Klrd1, killer cell lectin like receptor D1; Lag3, lymphocyte activating 3; Lars2, probable leucyl-tRNA synthetase, mitochondrial; and Nrn1, neuritin 1.

Figure 7.. T-cells are predicted to orchestrate the expansion and activation of Cxcl9⁺Cxcl10⁺ macrophages.

A, Cell to cell communication analysis using CellChat predicted that T-cells signal to macrophages through IFN-γ. Violin plot showing the expression distribution of IFN-γ pathway ligand and receptors in T-cells and macrophages. B, Heatmap showing the relative importance of each cell state based on the computed network of IFN-γ signaling. C, Circle plot summarizing the inferred intercellular communication network between T-cells and macrophages for IFN-γ signaling. D, Dot plot showing the strength of interaction between T-cell and macrophage cell states for IFN-γ signaling. P values were calculated using R package CellChat. E, Violin plot showing the expression distribution of signaling genes involved in the inferred reciprocal CXCL signaling network (Cxcl9/Cxcl10-Cxcr3) between macrophages and T-cells. F, Heatmap displaying the relative importance of each cell state based on the computed network of CXCL signaling. G, Circle plot depicting the inferred intercellular communication network between macrophage and T-cell states for CXCL signaling (Cxcl9/Cxcl10-Cxcr3). H, Quantification of cardiac Cxcl9⁺ and Cxcl10⁺ cells 6 days after anti-CD8 antibody treatment. Data collected from 3 independent experiments. Vehicle (n=15), anti-CD8 (n=7), Mann-Whitney test, 2-tailed. Cxcr3 indicates C-X-C motif chemokine receptor 3; Ifng, interferon gamma; and Ifngr, interferon gamma receptor.

Figure 8.. IFN-γ blockade and macrophage depletion reduce cardiac Cxcl9⁺Cxcl10⁺ macrophages and prolong the survival of Ctla4^+/−Pdcd1^−/− mice.

A, Survival of Ctla4^+/− Pdcd1^−/− mice treated with vehicle (isotype control) or anti–IFN-γ antibody (R46A2). Data collected from 4 independent experiments vehicle (n=25); anti–IFN-γ (n=26), log-rank test. B, Representative images (left) and quantification (right) of cardiac Cxcl9⁺ and Cxcl10⁺ cells as determined by RNA in situ hybridization 23 days after vehicle or anti–IFN-γ antibody treatment. Data collected from 5 independent experiments. Vehicle (n=15), anti–IFN-γ (n=21), Mann-Whitney test, 2-tailed. C, Survival of Ctla4^+/−Pdcd1^−/− mice treated with vehicle (isotype control) or anti-CSF1R antibody (AFS98). Vehicle (n=39), anti-CSF1R (n=32), log-rank test. D, Representative images (left) and quantification (right) of cardiac Cxcl9⁺ and Cxcl10⁺ cells as determined by RNA in situ hybridization 23 days after vehicle or anti-CSF1R antibody treatment. Data collected from 4 independent experiments. Vehicle (n=14), anti-CSF1R (n=10), Mann-Whitney test, 2-tailed. E, Cardiac CD64⁺ macrophages depletion was verified by flow cytometry. Representative images (left) and quantification (right) of CD64⁺ cells as determined by flow cytometry 60 days after first vehicle or anti-CSF1R antibody treatment. Displayed cells (left) are gated CD45⁺ cells. Data collected from 3 independent experiments. Vehicle (n=7), anti-CSF1R (n=7), Mann-Whitney test, 2-tailed. CSR1R indicates colony stimulating factor 1 receptor; IFN-γ, interferon gamma; and LY-6G, lymphocyte antigen 6 family member G.