T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

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.

Differences in Cardiometabolic Proteins in Pregnancy Prioritize Relevant Targets of Preeclampsia.

BACKGROUND: Preeclampsia is a hypertensive disorder of pregnancy characterized by widespread vascular inflammation. It occurs frequently in pregnancy, often without known risk factors, and has high rates of maternal and fetal morbidity and mortality. Identification of biomarkers that predict preeclampsia and its cardiovascular sequelae before clinical onset, or even before pregnancy, is a critical unmet need for the prevention of adverse pregnancy outcomes. METHODS: We explored differences in cardiovascular proteomics (Olink Explore 384) in 256 diverse pregnant persons across 2 centers (26% Hispanic, 21% Black). RESULTS: We identified significant differences in plasma abundance of markers associated with angiogenesis, blood pressure, cell adhesion, inflammation, and metabolism between individuals delivering with preeclampsia and controls, some of which have not been widely described previously and are not represented in the preeclampsia placental transcriptome. While we observed a broadly similar pattern in early (<34 weeks) versus late (≥34 weeks) preeclampsia, several proteins related to hemodynamic stress, hemostasis, and immune response appeared to be more highly dysregulated in early preeclampsia relative to late preeclampsia. CONCLUSIONS: These results demonstrate the value of performing targeted proteomics using a panel of cardiovascular biomarkers to identify biomarkers relevant to preeclampsia pathophysiology and highlight the need for larger multiomic studies to define modifiable pathways of surveillance and intervention upstream to preeclampsia diagnosis.

Proteomic Biomarkers of Quantitative Interstitial Abnormalities in COPDGene and CARDIA Lung Study.

Rationale: Quantitative interstitial abnormalities (QIAs) are early measures of lung injury automatically detected on chest computed tomography scans. QIAs are associated with impaired respiratory health and share features with advanced lung diseases, but their biological underpinnings are not well understood. Objectives: To identify novel protein biomarkers of QIAs using high-throughput plasma proteomic panels within two multicenter cohorts. Methods: We measured the plasma proteomics of 4,383 participants in an older, ever-smoker cohort (COPDGene [Genetic Epidemiology of Chronic Obstructive Pulmonary Disease]) and 2,925 participants in a younger population cohort (CARDIA [Coronary Artery Disease Risk in Young Adults]) using the SomaLogic SomaScan assays. We measured QIAs using a local density histogram method. We assessed the associations between proteomic biomarker concentrations and QIAs using multivariable linear regression models adjusted for age, sex, body mass index, smoking status, and study center (Benjamini-Hochberg false discovery rate-corrected P ⩽ 0.05). Measurements and Main Results: In total, 852 proteins were significantly associated with QIAs in COPDGene and 185 in CARDIA. Of the 144 proteins that overlapped between COPDGene and CARDIA, all but one shared directionalities and magnitudes. These proteins were enriched for 49 Gene Ontology pathways, including biological processes in inflammatory response, cell adhesion, immune response, ERK1/2 regulation, and signaling; cellular components in extracellular regions; and molecular functions including calcium ion and heparin binding. Conclusions: We identified the proteomic biomarkers of QIAs in an older, smoking population with a higher prevalence of pulmonary disease and in a younger, healthier community cohort. These proteomics features may be markers of early precursors of advanced lung diseases.

Capillary blood self-collection for high-throughput proteomics.

In this study, we sought to compare protein concentrations obtained from a high-throughput proteomics platform (Olink) on samples collected using capillary blood self-collection (with the Tasso+ device) versus standard venipuncture (control). Blood collection was performed on 20 volunteers, including one sample obtained via venipuncture and two via capillary blood using the Tasso+ device. Tasso+ samples were stored at 2°C-8°C for 24-hs (Tasso-24) or 48-h (Tasso-48) prior to processing to simulate shipping times from a study participant's home. Proteomics were analyzed using Olink (384 Inflammatory Panel). Tasso+ blood collection was successful in 37/40 attempts. Of 230 proteins included in our analysis, Pearson correlations (r) and mean coefficient of variation (CV) between Tasso-24 or Tasso-48 versus venipuncture were variable. In the Tasso-24 analysis, 34 proteins (14.8%) had both a correlation r > 0.5 and CV < 0.20. In the Tasso-48 analysis, 68 proteins (29.6%) had a correlation r > 0.5 and CV < 0.20. Combining the Tasso-24 and Tasso-48 analyses, 26 (11.3%) proteins met these thresholds. We concluded that protein concentrations from Tasso+ samples processed 24-48 h after collection demonstrated wide technical variability and variable correlation with a venipuncture gold-standard. Use of home capillary blood self-collection for large-scale proteomics should be limited to select proteins with good agreement with venipuncture.

Clonal hematopoiesis of indeterminate potential and outcomes after heart transplantation: A multicenter study.

Cardiac allograft vasculopathy (CAV) is a leading cause of late graft failure and mortality after heart transplantation (HT). Sharing some features with atherosclerosis, CAV results in diffuse narrowing of the epicardial coronaries and microvasculature, with consequent graft ischemia. Recently, clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a risk factor for cardiovascular disease and mortality. We aimed to investigate the relationship between CHIP and posttransplant outcomes, including CAV. We analyzed 479 HT recipients with stored DNA samples at 2 high-volume transplant centers, Vanderbilt University Medical Center and Columbia University Irving Medical Center. We explored the association between the presence of CHIP mutations with CAV and mortality after HT. In this case-control analysis, carriers of CHIP mutations were not at increased risk of CAV or mortality after HT. In a large multicenter genomics study of the heart transplant population, the presence of CHIP mutations was not associated with an increased risk of CAV or posttransplant mortality.

Early Assessment of Cardiac Allograft Vasculopathy Risk Among Recipients of Hepatitis C Virus-infected Donors in the Current Era.

BACKGROUND: Transplantation of hearts from hepatitis C virus (HCV)-positive donors has increased substantially in recent years following development of highly effective direct-acting antiviral therapies for treatment and cure of HCV. Although historical data from the pre-direct-acting antiviral era demonstrated an association between HCV-positive donors and accelerated cardiac allograft vasculopathy (CAV) in recipients, the relationship between the use of HCV nucleic acid test-positive (NAT+) donors and the development of CAV in the direct-acting antiviral era remains unclear. METHODS AND RESULTS: We performed a retrospective, single-center observational study comparing coronary angiographic CAV outcomes during the first year after transplant in 84 heart transplant recipients of HCV NAT+ donors and 231 recipients of HCV NAT- donors. Additionally, in a subsample of 149 patients (including 55 in the NAT+ cohort and 94 in the NAT- cohort) who had serial adjunctive intravascular ultrasound examination performed, we compared development of rapidly progressive CAV, defined as an increase in maximal intimal thickening of ≥0.5 mm in matched vessel segments during the first year post-transplant. In an unadjusted analysis, recipients of HCV NAT+ hearts had reduced survival free of CAV ≥1 over the first year after heart transplant compared with recipients of HCV NAT- hearts. After adjustment for known CAV risk factors, however, there was no significant difference between cohorts in the likelihood of the primary outcome, nor was there a difference in development of rapidly progressive CAV. CONCLUSIONS: These findings support larger, longer-term follow-up studies to better elucidate CAV outcomes in recipients of HCV NAT+ hearts and to inform post-transplant management strategies.

Limited Balloon Atrial Septostomy for Left Ventricular Unloading in Peripheral Extracorporeal Membrane Oxygenation.

BACKGROUND: This study describes the authors' experience with a limited balloon atrial septostomy technique, using a median balloon size of 15 mm, as a left ventricular (LV) unloading strategy in venoarterial extracorporeal membrane oxygenation (VA-ECMO). There has been increasing use of VA-ECMO in cardiogenic shock. Although LV unloading strategies have been suggested to improve outcomes, it is unclear which strategy is optimal. METHODS AND RESULTS: We performed a retrospective study of patients who underwent a limited balloon atrial septostomy for LV unloading in peripheral VA-ECMO at a single center. The goal of this study was to define the procedural outcomes and clinical characteristics of these patients. Of the 12 patients identified, none had complications related to the procedure. There was a significant decrease in the mean left atrial pressure and the majority of patients had radiologic improvement in pulmonary vascular congestion. Of the 12 patients, 58.3% survived to discharge. CONCLUSIONS: Limited BAS is an elegant and safe method for unloading the LV in peripheral VA-ECMO.

Single-cell RNA-sequencing identifies unique cell-specific gene expression profiles in high-grade cardiac allograft vasculopathy.

Background: Cardiac allograft vasculopathy (CAV), a diffuse thickening of the intima of the coronary arteries and microvasculature, is the leading cause of late graft failure and mortality after heart transplantation (HT). Diagnosis involves invasive coronary angiography, which carries substantial risk, and minimally-invasive approaches to CAV diagnosis are urgently needed. Using single-cell RNA-sequencing in peripheral blood mononuclear cells (PBMCs), we sought to identify cell-specific gene expression profiles in CAV. Methods: Whole blood was collected from 22 HT recipients with angiographically-confirmed CAV and 18 HT recipients without CAV. PBMCs were isolated and subjected to single-cell RNA-sequencing using a 10X Genomics microfluidic platform. Downstream analyses focused on differential expression of genes, cell compositional changes, and T cell receptor repertoire analyses. Results: Across 40 PBMC samples, we isolated 134,984 cells spanning 8 major clusters and 31 subclusters of cell types. Compositional analyses showed subtle, but significant increases in CD4+ T central memory cells, and CD14+ and CD16+ monocytes in high-grade CAV (CAV-2 and CAV-3) as compared to low-grade or absent CAV. After adjusting for age, gender, and prednisone use, 745 genes were differentially expressed in a cell-specific manner in high-grade CAV. Weighted gene co-expression network analyses showed enrichment for putative pathways involved in inflammation and angiogenesis. There were no significant differences in T cell clonality or diversity with increasing CAV severity. Conclusions: Unbiased whole transcriptomic analyses at single-cell resolution identify unique, cell-specific gene expression patterns in CAV, suggesting the potential utility of peripheral gene expression biomarkers in diagnosing CAV.

A prognostic molecular signature of hepatic steatosis is spatially heterogeneous and dynamic in human liver.

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing in parallel with an obesity pandemic, calling for novel strategies for prevention and treatment. We defined a circulating proteome of human MASLD across ≈7000 proteins in ≈5000 individuals from diverse, at-risk populations across the metabolic health spectrum, demonstrating reproducible diagnostic performance and specifying both known and novel metabolic pathways relevant to MASLD (central carbon and amino acid metabolism, hepatocyte regeneration, inflammation, fibrosis, insulin sensitivity). A parsimonious proteomic signature of MASLD was associated with a protection from MASLD and its related multi-system metabolic consequences in >26000 free-living individuals, with an additive effect to polygenic risk. The MASLD proteome was encoded by genes that demonstrated transcriptional enrichment in liver, with spatial transcriptional activity in areas of steatosis in human liver biopsy and dynamicity for select targets in human liver across stages of steatosis. We replicated several top relations from proteomics and spatial tissue transcriptomics in a humanized "liver-on-a-chip" model of MASLD, highlighting the power of a full translational approach to discovery in MASLD. Collectively, these results underscore utility of blood-based proteomics as a dynamic "liquid biopsy" of human liver relevant to clinical biomarker and mechanistic applications.

Cardiomyocyte-derived circulating extracellular vesicles allow a non-invasive liquid biopsy of myocardium in health and disease.

The ability to track disease without tissue biopsy in patients is a major goal in biology and medicine. Here, we identify and characterize cardiomyocyte-derived extracellular vesicles in circulation (EVs; "cardiovesicles") through comprehensive studies of induced pluripotent stem cell-derived cardiomyocytes, genetic mouse models, and state-of-the-art mass spectrometry and low-input transcriptomics. These studies identified two markers ( POPDC2 , CHRNE ) enriched on cardiovesicles for biotinylated antibody-based immunocapture. Captured cardiovesicles were enriched in canonical cardiomyocyte transcripts/pathways with distinct profiles based on human disease type (heart failure, myocardial infarction). In paired myocardial tissue-plasma from patients, highly expressed genes in cardiovesicles were largely cardiac-enriched (vs. "bulk" EVs, which were more organ non-specific) with high expression in myocardial tissue by single nuclear RNA-seq, largely in cardiomyocytes. These results demonstrate the first "liquid" biopsy discovery platform to interrogate cardiomyocyte states non-invasively in model systems and in human disease, allowing non-invasive characterization of cardiomyocyte biology for discovery and therapeutic applications.

Clinical-transcriptional prioritization of the circulating proteome in human heart failure.

Given expanding studies in epidemiology and disease-oriented human studies offering hundreds of associations between the human "ome" and disease, prioritizing molecules relevant to disease mechanisms among this growing breadth is important. Here, we link the circulating proteome to human heart failure (HF) propensity (via echocardiographic phenotyping and clinical outcomes) across the lifespan, demonstrating key pathways of fibrosis, inflammation, metabolism, and hypertrophy. We observe a broad array of genes encoding proteins linked to HF phenotypes and outcomes in clinical populations dynamically expressed at a transcriptional level in human myocardium during HF and cardiac recovery (several in a cell-specific fashion). Many identified targets do not have wide precedent in large-scale genomic discovery or human studies, highlighting the complementary roles for proteomic and tissue transcriptomic discovery to focus epidemiological targets to those relevant in human myocardium for further interrogation.

Influenza infection induces neuroinflammation, alters hippocampal neuron morphology, and impairs cognition in adult mice.

Influenza is a common and highly contagious viral pathogen, yet its effects on the structure and function of the CNS remain largely unknown. Although there is evidence that influenza strains that infect the brain can lead to altered cognitive and emotional behaviors, it is unknown whether a viral strain that is not neurotropic (A/PR/8/34) can result in a central inflammatory response, neuronal damage, and neurobehavioral effects. We hypothesized that neuroinflammation and alterations in hippocampal neuron morphology may parallel cognitive dysfunction following peripheral infection with live influenza virus. Here, we show that influenza-infected mice exhibited cognitive deficits in a reversal learning version of the Morris water maze. At the same time point in which cognitive impairment was evident, proinflammatory cytokines (IL-1ß, IL-6, TNF-α, IFN-α) and microglial reactivity were increased, while neurotrophic (BDNF, NGF) and immunomodulatory (CD200, CX3CL1) factors were decreased in the hippocampus of infected mice. In addition, influenza induced architectural changes to hippocampal neurons in the CA1 and dentate gyrus, with the most profound effects on dentate granule cells in the innermost portion of the granule cell layer. Overall, these data provide the first evidence that neuroinflammation and changes in hippocampal structural plasticity may underlie cognitive dysfunction associated with influenza infection. In addition, the heightened inflammatory state concurrent with reduced neurotrophic support could leave the brain vulnerable to subsequent insult following influenza infection. A better understanding of how influenza impacts the brain and behavior may provide insight for preventing inflammation and neuronal damage during peripheral viral infection.

Expansion of Disease Specific Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

Background: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1/PD-L1 or CTLA4 have revolutionized cancer management but are associated with devastating immune-related adverse events (irAEs) 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. While much has been learned regarding the role of T-cells in ICI-myocarditis, little is understood regarding the identity, transcriptional diversity, and functions of infiltrating macrophages. Methods: We employed 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 and molecular imaging and antibody neutralization studies. Results: We observed marked increases in CCR2 + 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 , Cxcl10 , Gbp2b , and Fcgr4 that originated from CCR2 + monocytes. Importantly, a similar macrophage population expressing CXCL9 , CXCL10 , and CD16α (human homologue of mouse FcgR4) was found selectively expanded in patients with ICI myocarditis compared to other forms of heart failure and myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9 + Cxcl10 + macrophages via IFN-γ and CXCR3 signaling pathways. Depleting CD8 + T-cells, 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. Conclusion: 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.

Outcomes of Heart Transplant Donation After Circulatory Death.

BACKGROUND: Heart transplantation using donation after circulatory death (DCD) allografts is increasingly common, expanding the donor pool and reducing transplant wait times. However, data remain limited on clinical outcomes. OBJECTIVES: We sought to compare 6-month and 1-year clinical outcomes between recipients of DCD hearts, most of them recovered with the use of normothermic regional perfusion (NRP), and recipients of donation after brain death (DBD) hearts. METHODS: We conducted a single-center retrospective observational study of all adult heart-only transplants from January 2020 to January 2023. Recipient and donor data were abstracted from medical records and the United Network for Organ Sharing registry, respectively. Survival analysis and Cox regression were used to compare the groups. RESULTS: During the study period, 385 adults (median age 57.4 years [IQR: 48.0-63.7 years]) underwent heart-only transplantation, including 122 (32%) from DCD donors, 83% of which were recovered with the use of NRP. DCD donors were younger and had fewer comorbidities than DBD donors. DCD recipients were less often hospitalized before transplantation and less likely to require pretransplantation temporary mechanical circulatory support compared with DBD recipients. There were no significant differences between groups in 1-year survival, incidence of severe primary graft dysfunction, treated rejection during the first year, or likelihood of cardiac allograft vasculopathy at 1 year after transplantation. CONCLUSIONS: In the largest single-center comparison of DCD and DBD heart transplantations to date, outcomes among DCD recipients are noninferior to those of DBD recipients. This study adds to the published data supporting DCD donors as a safe means to expand the heart donor pool.

Clonal hematopoiesis and vascular disease.

Somatic mutations in hematopoietic stem cells are common with aging and can result in expansion of clones harboring mutations, termed clonal hematopoiesis. This results in an increased risk of blood cancers but has also been linked with chronic inflammatory disease states. In recent years, clonal hematopoiesis has been established to have a causative role in atherogenesis and cardiovascular disease. Additionally, as the effector cells have been identified to be immune cells, there is ongoing interest in assessing whether dysregulated immune function plays a role in other chronic inflammatory conditions such as rheumatologic disease. Here, we summarize current understanding of clonal hematopoiesis with a focus on cardiovascular disease and inflammation while outlining the potential, yet unexplored, relationship between clonal hematopoiesis and autoimmune disease. Hematopoietic stem cells (HSCs) continually regenerate blood cells. Acquisition of a somatic mutation that provides a selective advantage, a driver mutation, can result in clonal expansion. Clonal hematopoiesis of indeterminate potential, where somatic mutations in certain cancer-associated genes result in clonal expansion in the absence of overt malignancy, can result in atherosclerotic cardiovascular disease in multiple vascular beds, inflammation, and may also contribute to the pathogenesis of autoimmune disease. Many questions remain unanswered regarding the relationship between clonal hematopoiesis and inflammatory disorders.

Hormonal therapies up-regulate MANF and overcome female susceptibility to immune checkpoint inhibitor myocarditis.

Immune checkpoint inhibitors (ICIs) have been increasingly used in combination for cancer treatment but are associated with myocarditis. Here, we report that tumor-bearing mice exhibited response to treatment with combinatorial anti-programmed cell death 1 and anti-cytotoxic T lymphocyte antigen-4 antibodies but also presented with cardiovascular toxicities observed clinically with ICI therapy, including myocarditis and arrhythmia. Female mice were preferentially affected with myocarditis compared to male mice, consistent with a previously described genetic model of ICI myocarditis and emerging clinical data. Mechanistically, myocardial tissue from ICI-treated mice, the genetic mouse model, and human heart tissue from affected patients with ICI myocarditis all exhibited down-regulation of MANF (mesencephalic astrocyte-derived neurotrophic factor) and HSPA5 (heat shock 70-kDa protein 5) in the heart; this down-regulation was particularly notable in female mice. ICI myocarditis was amplified by heart-specific genetic deletion of mouse Manf and was attenuated by administration of recombinant MANF protein, suggesting a causal role. Ironically, both MANF and HSPA5 were transcriptionally induced by liganded estrogen receptor ß and inhibited by androgen receptor. However, ICI treatment reduced serum estradiol concentration to a greater extent in female compared to male mice. Treatment with an estrogen receptor ß-specific agonist and androgen depletion therapy attenuated ICI-associated cardiac effects. Together, our data suggest that ICI treatment inhibits estradiol-dependent expression of MANF/HSPA5 in the heart, curtailing the cardiomyocyte response to immune injury. This endocrine-cardiac-immune pathway offers new insights into the mechanisms of sex differences in cardiac disease and may offer treatment strategies for ICI myocarditis.