Hormonal Therapies Upregulate MANF and Overcome Female Susceptibility to Immune Checkpoint Inhibitor-Myocarditis

Abstract

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-PD-1 (programmed cell death 1) and anti-CTLA-4 (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 as well as 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 downregulation of MANF (Mesencephalic Astrocyte Derived Neurotrophic Factor) and HSPA5 (Heat Shock 70kDa Protein 5) in the heart; this downregulation was particularly striking 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-βbeta 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 β-specific agonist as well as androgen depletion therapy attenuated ICI-associated cardiac effects. Taken 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.

One Sentence Summary:

ICI inhibits estradiol-induced cardiac MANF/HSPA5 expression, leading to myocarditis that could be attenuated by recombinant MANF or hormone therapy.

INTRODUCTION

Immune checkpoint inhibitors (ICIs) have advanced options for nearly 50% of cancer patients (1–3). PD-1, PD-L1, and CTLA-4 are the most comprehensively characterized antibody-mediated targets of several successful drug therapies(4–7). ICIs are used in combination (for example, anti-PD-1 and anti-CTLA4) (8, 9), which results in enhanced anti-tumor efficacy, but a broad spectrum of immune-related adverse events (irAE) can occur(9, 10). Cardiovascular toxicities including myocarditis, are most problematic and result increased mortality(11–13). ICI-associated myocarditis is characterized by electrocardiographic abnormalities, particularly atrial fibrillation, and conduction disease, with systolic cardiac function occurring in about half of patients (14, 15). Endomyocardial biopsy, characterized by patchy inflammatory infiltrates and cardiomyocyte death, is the most definitive means of ICI-myocarditis diagnosis (16). Several pre-clinical models of ICI-myocarditis have been generated to enhance our understanding of underlying pathophysiology (17–21). These include pharmacological models as well as a genetic model whereby monoallelic loss of Ctla4 (encoding CTLA-4) in the context of complete genetic absence of Pdcd1 (encoding PD-1) leads to myocardial immune infiltration and severe electrocardiographic abnormalities, recapitulating the ICI-associated myocarditis observed in patients (18). In all these models, myocardial tissues show predominantly CD8⁺ T cells interspersed with CD4⁺ T cells and macrophages (22). However, the underlying mechanisms of ICI-associated myocarditis, in particular contributions from genetic and environmental factors, remain largely elusive. Clinically, females are more susceptible to ICI-associated myocarditis independent of other clinical parameters (23, 24). In the genetic mouse model of ICI-myocarditis, female Ctla4^+/− Pdcd1^−/− mice have greater mortality and a more fulminant course compared to male Ctla4^+/− Pdcd1^−/− mice (18). The emerging clinical and pre-clinical data with ICI-myocarditis are intriguing because in non-ICI-myocarditis (viral or autoimmune), male sex is considered an important risk factor and associates with a more fulminant course (25), which suggests a possible interaction of immune checkpoints, sex hormones and the heart, perhaps providing insights into the mechanisms of sex-based differences in heart disease.

While immune dysregulation plays a critical role in inflammatory cardiomyopathies such as ICI-myocarditis, it is less clear what roles myocardial compensatory and protective processes play in attenuating immune infiltration. Heart-specific proteins, especially secreted ones (“cardiokines”), have been identified in cardiac stress and injury models and include members of the transforming growth factor-β superfamily (e.g., Growth Differential Factors 15 and 8), Migration Inhibitory Factor (MIF), and endoplasmic reticulum (ER)-stress induced proteins (26–29). Cardiac intrinsic protective factors are important to explore in ICI-myocarditis, not just for scientific reasons but also for therapeutic purposes, since focusing on inhibiting the immune system may also dampen the anti-tumor effects of ICI and other immune-mediated therapies (13, 30).

Here, we developed a tumor-bearing, pharmacological model of ICI-associated myocarditis, replicating the clinical correlates in patients with ICI-myocarditis. In this model we concomitantly assessed tumor response following ICI treatment (anti-CTLA4 and anti-PD1 treatment). Using this pharmacological model, the genetic Ctla4^+/− Pdcd1^−/− mouse model and myocardial samples from patients with ICI-myocarditis, we studied underlying sex differences, specifically female predilection to ICI-myocarditis. Further, we explored cardiac-intrinsic responses that may play a role in regulating cardiac-immune interactions and tested whether targeting these factors would attenuate cardiac inflammation without disturbing anti-cancer effects of the ICI.

RESULTS

Immune checkpoint blockers induce cardiac complications

To investigate the mechanisms of ICI-associated myocarditis and other cardiovascular sequelae in cancer patients and to assess the interactions of tumors on these side-effects, we generated three mouse tumor models representing colorectal cancer (MC38) (31), melanoma (B16F10) (32), and breast cancer (EO771) (33) using both male and female C57BL/6J mice with tumor volumes reaching 200–250 mm³ (Fig. 1A). Age- and gender-matched wild type C57BL/6J mice (WT) without tumor inoculation were included as controls (Fig. 1A). Both tumor-bearing and non-tumor-bearing mice were subjected to combination anti-PD-1 and anti-CTLA-4 antibody treatment, using doses that have been previously used to assess tumor efficacy. Combined anti-PD-1 and anti-CTLA-4 antibody treatment inhibited tumor growth and increased cleavage of caspase 3 and immune cell infiltration in the tumor upon ICI treatment (fig. S1). In addition, multiple tissues showed evidence of T cell infiltration (particularly CD8⁺ T cells) including liver, kidneys, heart, skeletal muscle, and lung (fig. S1). ICI-associated irAEs were most physiologically problematic in the cardiovascular system. Affected mice exhibited myocarditis, electrocardiographic (ECG) disturbances, and mild cardiac systolic dysfunction. Histologically, mononuclear immune infiltration consisting of macrophages and CD8⁺ T cells was observed in ICI-treated hearts but not in IgG-treated hearts, using CD8 and F4/80 as markers respectively (Fig. 1, B and C, and fig. S2, A to D). These cardiac findings were observed in both tumor-bearing and non-tumor-bearing mice post ICI treatment, suggesting minimal effects of tumor on the ICI-associated myocarditis (Fig. 1D and fig. S2, A and B). In addition, focal areas with myocardial dropout and areas with TUNEL-positive staining and cleaved-caspase 3 were observed (fig. S2, E to H). These results are consistent with several other pre-clinical models of ICI-myocarditis and clinical forms of ICI-myocarditis observed in patients.

Fig. 1. Characterization of ICI-induced cardiotoxicities in mice.

(A) Graphic illustration of experimental setting. (B) Representative hematoxylin and eosin (H&E) staining of heart tissues from the female mice inoculated with B16F10 cells, followed by the IgG or ICI treatment. Scale bars, 100 μm. (C and D) Representative multiplex immunohistochemistry (mIHC) staining using the indicated antibodies (C) and statistical analysis of CD8 staining intensities (D) of heart tissues of the female and male wild type (WT), MC38-, B16F10- or EO771-tumor bearing mice respectively, treated with IgG or ICI (25mg/kg each) (D). Scale bars, 1 mm or 100 μm as indicated. Error bars, SD, n = 10 animals per experimental group, one-way ANOVA. (E and F) Representative monitoring lead II ECG traces (E) and statistical analysis of QRS duration (F) from the female mice inoculated with MC38, B16F10 or EO771 cells and treated with IgG or ICI. The implanted monitoring ECG shows sinus arrest in the ICI condition. Error bars, SD, n = 5 animals per experimental group, one-way ANOVA. (G) Kaplan-Meier survival curve of Ctla4^+/−/Pdcd1^−/− and littermate Ctla4^+/+/Pdcd1^−/− mice (male and female separately, n = 100, 100, 100, 100 animals). P value represents the result of the Mantel-Cox log-rank test. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

ICI-associated myocardial effects were preferentially observed in female mice compared to male mice. ICI-treated female hearts exhibited increased CD8⁺ T cell infiltration (Fig. 1D), electrocardiographic abnormalities (Fig. 1, E and F, and fig. S3, A to C), as well as depressed cardiac systolic dysfunction on echocardiography compared to male mice (fig. S3, D to J). These effects were not observed in either female or male isotype controls (Fig. 1F and fig. S3, D to J). Furthermore, mice treated with anti-PD-1, anti-CTLA-4 antibodies alone or in combination exhibited reduced ejection fraction and fractional shortening (fig. S3, K and L). To further ensure that the sex differences observed were not limited to our model of myocarditis, we assessed the recently described genetic model whereby Ctla4^+/− Pdcd1^−/− mice exhibited immune infiltration limited to the heart and led to a more fulminant form of myocarditis, characterized by premature mortality in affected mice (Fig. 1G). In this model, Ctla4^+/− Pdcd1^−/− female mice had a more fulminant course compared to Ctla4^+/− Pdcd1^−/− male mice (Fig. 1G). Both Ctla4^+/− Pdcd1^−/− mice and ICI-treated mice exhibited elevated serum Troponin I concentrations (fig. S3, M and N). Finally, recent data suggest that the serum levels of mmu-miR-721 are elevated in acute myocarditis, making this a more specific marker of myocarditis (vs. other cardiac injury such as ischemia) (34). Ctla4^+/− Pdcd1^−/− mice have elevated serum miR-721 levels in comparison to Ctla4^+/+ Pdcd1^+/+ female littermates (fig. S3O). The sex differences in ICI-myocarditis among patients have been harder to analyze given the increased proportion of male patients treated with ICI in clinical trials. However, the sex imbalance in our mice is consistent with emerging clinical data from patients where irAEs and myocarditis are more pronounced in females (23, 24). Taken together, our findings suggest that ICI-myocarditis preferentially manifests in female sex in multiple pre-clinical models, suggesting a possible connection between immune checkpoints, downstream immune or myocardial targets and sex hormones.

ICI modulates cardiac expression of MANF and HSPA5

To examine downstream affected pathways that are preferentially biased toward manifestation of myocarditis in female mice, we profiled the transcriptomes of the heart, kidneys, lungs, liver, and skeletal muscles following isotype IgG or ICI treatment (Fig. 2A, table S1, and GSE145573), focusing on differentially expressed genes (DEGs) in the heart (compared to other organs). Multiple genes and gene subsets were differentially expressed in the ICI-treated hearts compared to IgG-treated hearts including upregulation of gene set involved in reactive oxygen species (fig. S4A), suggesting that ICI treatment may impair the stress-response of cardiac muscles. Among heart-specific genes, Mesencephalic Astrocyte Derived Neurotrophic Factor (Manf) and Heat Shock 70kDa Protein 5 (Hspa5) transcription was decreased in expression upon ICI treatment in both the non-tumor-bearing and tumor-bearing hearts (Fig. 2B and fig. S4, B to E). Specifically, Manf downregulation was more pronounced in female hearts compared to male hearts post-ICI treatment (Fig. 2, C and D). Immunohistochemistry staining validated the downregulation of MANF and HSPA5 at the protein expression in the heart following ICI treatment (fig. S5, A to D), with confirmation by immunoblotting of a greater downregulation of MANF protein in female hearts compared to male hearts (fig. S5, E to G). To further expand these observations, we assessed Manf and Hspa5 expression in the genetic ICI-myocarditis model. The cardiac expression of Manf was downregulated in Ctla4^+/−/Pdcd1^−/− mice compared to Ctla4^+/+/Pdcd1^−/− and Ctla4^+/+/Pdcd1^+/+ mice (fig. S6A). Hspa5 expression was downregulated in both Ctla4^+/−/Pdcd1^−/− and Ctla4^+/+/Pdcd1^−/− compared to Ctla4^+/+/Pdcd1^+/+ mice (fig. S6B). Reduced MANF and HSPA5 protein expression negatively correlated with CD8⁺ staining intensity (Fig. 2E and fig. S6, C to H). Finally, MANF and HSPA5 protein expression were decreased in human heart tissues from patients with ICI-myocarditis compared to hearts of healthy donors (Fig. 2, F and G, and table S2). This downregulation was particularly striking in areas of more severe inflammation, correlating with elevated CD8⁺ T cell infiltration (fig. S6, I to L).

Fig. 2. ICI modulates cardiac expression of MANF and HSPA5 in human and mouse hearts.

(A) Venn diagrams present the number of significantly DEGs, which is unique to the specific tissues or shared between tissues (the ICI- versus IgG-treated mouse, see also GSE145573). (B) RT-qPCR analysis of the indicated genes in heart tissues from female WT, MC38-, B16F10- or EO771 tumor-bearing mice treated with the indicated treatment. Error bars, SD, n = 9 animals per experimental group, Student’s t-test. (C and D) Statistical analysis of Manf (C) and Hspa5 (D) gene expression in the hearts of male and female WT, MC38-, B16F10- or EO771 tumor-bearing mice treated with IgG or ICI. Error bars, SD, n = 5 animals per experimental group, Student’s t-test. (E) Representative mIHC staining of heart tissues of female Ctla4^+/−/Pdcd1^−/− or littermate Ctla4^+/+/Pdcd1^−/− mice using the indicated antibodies. Scale bars, 100 μm as indicated. (F and G) Representative mIHC staining images using the indicated antibodies (F) and statistical analysis of MANF and HSPA5 staining intensities (G) of normal human heart tissues or ICI induced myocarditis human heart tissues. Scale bars, 100 μm. Error bars, SD, n = 7, 5 independent visual fields in each experimental group, Student’s t-test. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

MANF is an evolutionary conserved protein related to unfolded protein response (UPR) with emerging data suggesting a protective role of MANF in ER stress-related diseases (35). While the role that MANF plays in the heart is only beginning to be explored, early data suggest that MANF is activated in cardiac ischemia and protects from cardiac injury via the UPR sensor and effector (35). HSPA5 (also known as ER chaperone BiP) has also been shown to be involved in protecting cardiomyocytes from injury (36). MANF directly interacts with HSPA5 in the ischemic heart (37). To assess whether MANF or HSPA5 plays regulatory roles in other forms of myocardial stress, we assessed the expression of Manf and Hspa5 in an experimental myocardial ischemia model using permanent coronary artery ligation. Manf was upregulated 3 days following coronary artery ligation in wild-type mice, although the expression subsided 28 days following ligation (fig. S7). On the other hand, Hspa5 was downregulated in the experimental myocardial ischemia model, both immediately after coronary ligation and later during cardiac remodeling (fig. S7). These findings contrast with our data in ICI-myocarditis where MANF, and possibly HSPA5, are both downregulated in mouse and human models. Collectively, our data suggest that MANF and HSPA5 may regulate the interactions between the cardiovascular and immune systems during myocardial stress.

Depletion of Manf sensitizes hearts to ICI-associated myocarditis

To explore the functional role that Manf downregulation may play in myocardial homeostasis, we deleted Manf in adult mouse hearts by taking advantage of adeno-associated virus (AAV9)-delivered, saCas9-mediated gene editing, whereby cardiac troponin (cTnT)-driven expression of saCas9 leads to cardiac-specific expression post-AAV delivery (38) (Fig. 3A). 3 weeks following AAV delivery, we confirmed reduced MANF protein expression in mice administered with AAV9-Manf-sagRNAs compared to mice administered with AAV9-Scr-sagRNAs or saline (Fig. 3, B and C, and fig. S8A). We also confirmed that saCas9 was expressed in the heart but marginally in other organs following administration (Fig. 3C and D, and fig. S8B). Manf-proficient and -deficient animals were further subjected to tumor inoculation and ICI treatment. Heart-specific depletion of Manf resulted in minimal effects on the tumor growth of B16F10 xenografts following IgG or ICI treatment (fig. S8C). While ICI-treatment led to increased CD8 staining in both Manf-proficient and deficient mice, this increase was pronounced in Manf-deficient mice (Fig. 3, E and F). Furthermore, Manf-deficient hearts exhibited increased electrocardiographic disturbances as well as decreased systolic cardiac functions following ICI treatment. (Fig. 3, G to J). Hence, our findings suggested the functional importance of MANF in ICI-associated myocarditis.

Fig. 3. Deficiency of Manf aggravated ICI treatment-induced cardiotoxicities in mice.

(A) Graphic illustration of experimental setting. (B and C) Representative mIHC images using the indicated antibodies (C) and statistical analysis of MANF staining intensities (D) of the hearts administrated with AAV-cTnT-saCas9 and indicated sagRNAs. Scale bars, 100 μm. Error bars, SD, n = 5 animals per experimental group, One-way ANOVA. (D) Representative IHC staining of the indicated mouse tissues upon cTnT-blank or -saCas9 expression using anti-Cas9 antibody. Scale bars, 100 μm. (E and F) Representative mIHC images using the indicated antibodies (E) and statistical analysis of CD8 staining intensities (F) of WT or B16F10 tumor-bearing female hearts administrated with AAV-cTnT-saCas9 and indicated sagRNAs, followed by the IgG or ICI treatment. Scale bars, 100 μm. Error bars, SD, n = 8 animals per experimental group, one-way ANOVA. WT mice delivered with AAV-cTnT-saCas9 were included as control. (G to J) Statistical analysis of QRS duration (G), QT interval (H), ejection fraction (%) (I) or fractional shortening (FS%) (J) of WT or tumor-bearing female mice administrated with AAV-cTnT-saCas9 and indicated sagRNAs, followed by the IgG or ICI treatment. Error bars, SD, n = 5 animals per experimental group, one-way ANOVA. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

MANF administration improves heart function following ICI treatment

Conversely, we asked whether MANF might protect against ICI-associated myocarditis. MANF has been suggested to associate with HSPA5 to form a macromolecular complex (37). Considering the potential fast degradation of recombinant proteins in human serum, we attempted to deliver recombinant MANF and HSPA5 proteins in a 1:1 ratio to achieve stabilized rMANF tissue pharmacokinetics (PK) in vivo. We first determined MANF serum concentrations, finding that wild type non-tumor-bearing mice harbor about 4 ng MANF per ml of serum (fig. S9A). Hence, we injected ~40 ng (2 μg/kg) rMANF and ~156 μg rHSPA5 (7.8 μg/kg) per animal to achieve a 1:1 molar ratio. To determine the PK of rMANF and rHSPA5, the heart tissues of non-tumor-bearing mice administered recombinant MANF and HSPA5 (2 μg/kg and 7.8 μg/kg respectively, i.p., daily) were harvested for immunohistochemistry staining to determine the presence of His-tagged-rMANF or c-Myc-tagged-HSPA5 (Fig. 4, A and B, and fig. S9, B and C). Immunofluorescence staining of the anti-His tag exhibited overlap with the anti-MANF antibody, suggesting the presence of His-tagged rMANF in heart (Fig. 4A and fig. S9A). Similarly, immunofluorescence staining of the c-Myc tag antibody exhibited overlap with the anti-HSPA5 antibody, suggesting the presence of c-Myc-tagged rHSPA5 in heart (Fig. 4B and fig. S9C). Animals treated with rMANF or rHSPA5 exhibited no obvious pathology. Specifically, rMANF or rHSPA5 treatment did not affect serum markers of liver or kidney function (fig. S10, A to D).

Fig. 4. Recombinant MANF/HSPA5 attenuated ICI-induced cardiotoxicities.

(A and B) Statistical analysis of staining intensities of His-tag or anti-MANF antibodies (A), anti-c-Myc-tag or anti-HSPA5 antibodies (B) in the heart treated with the vehicle or recombinant MANF+HSPA5 (2 μg/kg and 7.8 μg/kg respectively). Error bars, SD, n = 5 animals per group, Student’s t-test. (C) Graphic illustration of experimental setting. (D) Representative monitoring lead II ECG traces from the female B16F10 tumor-bearing mice with the indicated treatment. (E and F) Statistical analysis of QRS duration (E) or QT interval (F) of monitoring lead II ECG traces from the female B16F10-bearing mice with the indicated treatment. Error bars, SD, n = 5 animals per experimental group, one-way ANOVA. (G and H) Statistical analysis of EF% (G) or FS% (H) of the female B16F10 tumor-bearing mice with the indicated treatment. Error bars, SD, n = 5 animals per experimental group, one-way ANOVA. (I and J) Statistical analysis of CD8 staining intensities (I) and representative mIHC staining (J) of the hearts from the female B16F10 tumor-bearing mice with the indicated treatment. Scale bars, 1mm or 100 μm as indicated. Error bars, SD, n = 5 animals per experimental group, one-way ANOVA. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

We then determined whether rMANF co-administered with rHSPA5 can attenuate ICI-associated myocarditis. Female mice were pre-treated with rMANF+rHSPA5, followed by inoculation with B16F10 cells (Fig. 4C). When tumors reached 200–250 mm³, the animals were treated with IgG or ICI as described above (Fig. 4C). ICI treatment inhibited the growth of B16F10 tumors; rMANF+rHSPA5 treatment had no effects on tumor growth nor did it affect the anti-tumor effects of ICI treatment (fig. S10E). Histologically, treatment with rMANF+rHSPA5 had no effects on tumor infiltrating CD8⁺ T cells (fig. S10, F and G). However, treatment with rMANF+rHSPA5 attenuated macrophage and CD8⁺ T cell infiltration in the heart, decreased arrhythmogenicity and conduction disease, and improved cardiac function as assessed through echocardiography (Fig. 4, D to J, and fig. S10, H and I). rMANF+rHSPA5-treated mice also exhibited reduced cleaved caspase 3 foci (as a marker of cardiac injury) following ICI treatment (fig. S11). Hence, our findings suggested that administration of recombinant MANF attenuated myocyte death and immune infiltration in ICI-associated myocarditis, signifying the functional importance of MANF in this disease.

To explore the underlying mechanisms of MANF in attenuating myocarditis, we examined the transcriptional profiles of vehicle or rMANF/rHSPA5-treated hearts based on the ICB treatment (fig. S12A and GSE213468). Compared to the vehicle-treated group, rMANF/rHSPA5-treated hearts increased genes responsible for junction complex organization, Wnt signaling, cell adhesion, and inhibition of epithelial mesenchymal transition (EMT), cellular processes that promote tissue protection against injury (fig. S12, B and C; GSE213468). In addition, enhanced cell adhesion/junction complex organization and reduced EMT are known to improved cell-cell adhesion, inhibiting leukocyte migration (39). Indeed, previous studies indicated that Manf-deficiency leads to downregulated genes mostly pertaining to cell adhesion and differentiation (40). Wnt and downstream β-catenin play essential roles in the proliferation and anti-apoptosis of cardiomyocytes (41). Furthermore, data from other models of myocardial injury suggest that diminished Wnt signaling leads to arrhythmogenic cardiomyopathy (42, 43). We next determined the phosphorylation of LRP6 (Low-density lipoprotein receptor-related protein 6) and β-catenin at S33/S37/T41 upon Wnt3α stimulation in the presence of rMANF or rHSPA5, finding that rMANF, but not rHSPA5 facilitated the Wnt-induced phosphorylation of LRP6 at S1490 and β-catenin at S33/S37/T41 (fig. S12, D to F). The presence of rMANF enhanced the phosphorylation status of these sites (fig. S12, D to F).

ICI effects on sex hormones and transcriptional regulation of MANF and HSPA5

Previous literature has shown a critical role for female hormones, specifically 17 β-estradiol, and male hormones (testosterones) in immune regulation and cardio protection in other settings (44, 45). Since we did not observe any pathology at baseline in our mouse models, we wondered whether ICI treatment might affect the serum concentrations of 17 β-estradiol and testosterone in both male and female animals. Surprisingly, we found that ICI treatment significantly reduced serum 17 β-estradiol concentrations in both males and females 2 weeks after treatment. This effect was observed in both tumor-bearing (female) and non-tumor-bearing (male and female) animals (Fig. 5, A and B). ICI administration showed no effects on the serum concentrations of testosterone progesterone in either male or female animals (Fig. 5, C and D, and fig. S13, A and B). Ovaries and mammary gland tissues of female mice following vehicle or ICI treatment showed no obvious damage (fig. S13C).

Fig. 5. ICI treatment decreased 17 β-estradiol in mouse serum.

(A and B) Serum 17 β-estradiol concentration measurement of the female (A) and male (B) WT and tumor-bearing mice that received IgG or ICI treatment. Error bars, SD, n = 8 animals per experimental group, two-way ANOVA. (C and D) Serum testosterone concentration measurement of the female (C) or male (D) WT or tumor-bearing mice that received IgG or ICI treatment. Error bars, SD, n = 8 animals per experimental group, two-way ANOVA. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

In parallel, we established an in vitro model using human induced pluripotent stem (iPS) cells-derived cardiomyocytes (iPS-CMs) to further explore the myocyte-specific regulation of MANF/HSPA5 transcription and the potential role of sex hormones in this process. We initially hypothesized that estrogens may suppress MANF/HSPA5, thus predisposing female mice to ICI-myocarditis. Surprisingly, in iPS-CMs, estrogen (E₂) robustly induced mRNA and protein expression of MANF and HSPA5. Conversely, dihydrotestosterone (DHT) inhibited the expression of MANF and HSPA5 (Fig. 6, A to C). In comparison, treatment of iPS-CMs with pro-inflammatory cytokines, such as IL-6, IFN-γ, or TNF-α, which have been shown to recapitulate inflammatory-mediated cardiac damage, minimally affected MANF/HSPA5 at the mRNA and protein expression (Fig. 6, A to C). The promoters of MANF and HSPA5 contain consensus sequences for androgen receptor (AR) and estrogen receptor (ER) binding elements (Fig. 6, D and E, and fig. S14, A and B, top panels). Chromatin immunoprecipitation (ChIP) assay indicated the recruitment of AR to the promoters of MANF and HSPA5 in iPS-CMs following DHT treatment (Fig. 6D and fig. S14A). The recruitment of AR to the transcription units following DHT treatment might trigger activation or repression of transcription (46). We confirmed the recruitment of transcription co-repressors NcoR and REST (47) to the promoters of MANF/HSPA5 upon ligand stimulation (Fig. 6D and fig. S14A). Furthermore, ERβ, but not ERα, was recruited to the promoter regions of MANF and HSPA5 respectively upon E₂ stimulation (Fig. 6E and fig. S14B). The E₂-induced expression of mouse Manf/Hspa5 occurred in an Esr2-dependent manner (Fig. 6, F to H). Similarly, knockout of Ar abolished the DHT-triggered repression of Manf/Hspa5 in neonatal mouse cardiomyocytes (Fig. 6, I to K). Taken together, our findings suggest that ICI treatment results in reduced female hormone concentrations in blood, serving as a fundamental mechanism underlying the sex disparities of ICI-associated myocarditis. On other hand, estrogens induce MANF/HSPA5 expression in cardiomyocytes. Therefore, one may hypothesize that hormone-directed therapies following ICI treatment may serve as a therapeutic strategy in attenuating ICI-myocarditis.

Fig. 6. ICI treatment inhibits estrogen-dependent transcription of Manf and Hspa5.

(A) Immunoblotting (IB) of MANF and HSPA5 in hiPS-CMs with indicated stimulation. ACTC1 was used as a loading control to normalize the relative intensities of MANF and HSPA5 (shown as fold change). (B and C) RT-qPCR analysis of MANF (B) or HSPA5 (C) mRNA expression in hiPS-CMs following the indicated stimulation. Error bars, SD, n = 3 independent experiments, Student’s t-test. (D and E) ChIP-qPCR analysis of the occupancy of indicated proteins on MANF promoter in hiPS-CMs after the indicated stimulation. Error bars, SD, n = 3 independent experiments, one-way ANOVA. (F) IB of ERβ and ERα in Esr2^+/+ or Esr2^−/− mouse fetal cardiomyocytes. GAPDH was used as a loading control. (G and H) RT-qPCR analysis of Manf (G) or Hspa5 (H) mRNA expression in Esr2^+/+ or Esr2^−/− mouse fetal cardiomyocytes with indicated stimulation. Error bars, SD, n = 7 animals per experimental group, Student’s t-test. (I) IB of AR in Ar-proficient or Myh6-Ar knockout mouse fetal cardiomyocytes with indicated stimulation. (J and K) RT-qPCR analysis of Manf (J) or Hspa5 (K) mRNA expression in Ar-proficient or Myh6-Ar knockout mouse fetal cardiomyocytes with indicated stimulation. Error bars, SD, n = 6 animals per experimental group, Student’s t-test. (L) IB of the indicated proteins in hiPS-CMs harboring indicated shRNAs. (M to N) RT-qPCR analysis of HSPA5 (M) or MANF (N) mRNA expression in hiPS-CMs harboring indicated shRNAs, that received indicated treatments. Error bars, SD, n = 5 independent experiments, one-way ANOVA. (O to P) RT-qPCR detection of HSPA5 (O) or MANF (P) in hiPS-CMs treated with indicated stimuli. Error bars, SD, n = 3 independent experiments, one-way ANOVA. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

We next explored whether hormone-directed therapies may affect the expression of MANF and HSPA5 using our in vitro system. We specifically focused on ERβ, since perturbation of ERα may affect anti-tumor effects of cancer therapies, especially since ERα promotes the growth of multiple types of tumors including breast cancer and melanoma (48). On the other hand, it has been suggested that ERβ may act as a tumor suppressor (49). Indeed, LY500307, an ERβ-specific agonist, has been shown to have anti-tumor effects in pre-clinical models of triple negative breast cancer (TNBC) (49). Treatment with LY500307 promoted MANF and HSPA5 expression, which was diminished upon ERβ depletion (Fig. 6, L to N). On the other hand, apalutamide (ARN-509), which associates with AR’s ligand binding domain and inhibits AR-dependent transcriptional activity, has recently been approved by the FDA for treating metastatic castration-sensitive prostate cancer (50). ARN-509 antagonizes the DHT-dependent repression of MANF and HSPA5 (Fig. 6, O and P). Hence, the results of our study indicated that the expression of MANF and HSPA5 was activated by E₂ and repressed by DHT, whereby the expression of MANF and HSPA5 were restored upon ERβ agonist or AR deprivation therapy.

Hormone therapies alleviate immunotherapy-associated cardiotoxicity

Our previous experiments showed that transcriptional activity of MANF is activated by ERβ and repressed by AR. To test whether hormone therapy can attenuate myocarditis following ICI treatment, tumor-bearing mice were treated with LY500307 or ARN-509 (apalutamide) to stimulate the activation of ERβ signaling or inhibit AR signaling, respectively (Fig. 7A). LY500307 has been suggested to up-regulate the expression of ERβ and diminishes tumor resistance to ICI therapy (51). Female mice harboring EO771 tumors, which represent human triple negative breast cancer, were administered with LY500307 at the time of ICI treatment (Fig. 7A and fig. S15A). LY500307-treated hearts exhibited increased MANF/HSPA5 protein expression, reduced cleaved caspase 3 staining, reduced CD8⁺ T cell infiltration, and enhanced heart function (Fig. 7, B to F, and fig. S15, B to E). Conversely, RM1 murine prostate cancer-bearing mice were treated with ARN-509 (apalutamide) following ICI treatment. ARN-509 treatment did not affect tumor inhibition by ICI in the RM1-bearing male mice (fig. S15F). ARN-509-treated hearts showed enhanced MANF/HSPA5 expression, decreased cleaved caspase 3 staining intensity, diminished CD8⁺ T cell infiltration, and improved cardiac contraction (Fig. 7, G to K, and fig. S15, G to J). Taken together, our data suggested that hormone therapy improves the expression of MANF while inhibiting ICI-associated cardiac irAEs.

Fig. 7. Hormone therapy minimized ICI-induced cardiotoxicities in mice.

(A) Graphic illustration of experimental setting. (B to F) Representative mIHC and IHC staining images using the indicated antibodies (B), and staining intensity statistical analysis of MANF (C), HSPA5 (D), CD8 (E) or cleaved caspase 3 (F) in the hearts of EO771 tumor-bearing female mice that received indicated treatments. Scale bars (B), 100 μm. Error bars (C-F), SD, n = 5 animals per experimental group, one-way ANOVA. (G to K) Representative mIHC and IHC staining using the indicated antibodies (G), and staining intensity statistical analysis of MANF (H), HSPA5 (I), CD8 (J) or cleaved caspase 3 (K) in the hearts of RM1 tumor-bearing male mice that received the indicted treatments. For C to F and H to K, blue dot and red square represents IgG and ICI treatment respectively in both vehicle and LY500307 or ARN-509 groups. Scale bars (G), 100 μm. Error bars (H-K), SD, n = 5 animals per experimental group, one-way ANOVA. n.s.: P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

DISCUSSION

Although immune checkpoint blockade has shown remarkable efficacy in treating multiple cancer types, cardiac irAEs remain a serious concern (52). Alleviating the adverse side effects of ICI treatment without affecting anti-tumor immunity is essential. In this study, we developed tumor-bearing mouse models of ICI-myocarditis and noted a female predilection to myocarditis, as noted previously in a genetic model of ICI-myocarditis and from preliminary data in the human population. We investigated the underlying cardiac compensatory mechanisms that modulate the cardiac-immune interaction. We identified two factors, MANF and HSPA5, related to unfolded protein response (UPR) which were significantly downregulated in ICI-myocarditis, especially in females. We provided data whereby MANF plays a causal role in modulating myocarditis in these models. We showed that ICI treatment significantly reduced serum concentrations of 17 β-estradiol, especially in female mice, resulting in downregulation of Manf and Hspa5 in the heart. In addition, hormonal therapy using ERβ agonist or androgen deprivation therapy (ADT) attenuated myocarditis via upregulating Manf and Hspa5 expression (fig. S15K). Our findings provide clinical considerations that may broaden the utility and viability of ICI therapies, which could allow this treatment strategy to benefit a wider range of cancer patients.

The mechanism of ICI-associated myocarditis remains elusive. Multiple possible immune-mediated mechanisms have been proposed including shared antigens between the tumor and cardiomyocytes (53). In addition, immune checkpoints including PD-1/PD-L1 (and possibly CTLA-4) pathways regulate T cell interactions with the heart under physiological conditions. However, the mechanisms underlying the gender disparities of myocarditis are currently unclear. Here we identify MANF and HSPA5 are critical in attenuating myocardial inflammation in ICI-myocarditis. We show that MANF and HSPA5 were transcriptionally enhanced by female hormones. Conversely, male hormones inhibited the expression of these genes. Administration of recombinant MANF restored the heart function of ICI-treated mice. Though HSPA5 expression was also decreased, our findings suggested that HSPA5 were downregulated similarly in both male and female ICI-treated hearts. Hence, our findings suggested that MANF might play a critical role in the gender disparities of ICI-associated myocarditis in our mouse model. The presence of rMANF and rHSPA5 exhibited minimal effects on tumor infiltrating CD8⁺ T cells and macrophages, leading to unaffected anti-tumor immunity. However, a role for HSPA5 in ICI-associated cardiotoxicity cannot be ruled out.

Our findings support that hormone therapies, including ERβ agonists or androgen depletion, effectively restored the cardiac expression of MANF/HSPA5, reduced the infiltration of CD8⁺ T cells and macrophages in the heart, and prevented the apoptosis of the myocardium. Genetic evidence indicated that mice with ERβ knockout, but not ERα knockout, are susceptible to ischemia/reperfusion injury (54). Erβ-specific agonists have been shown to be cardioprotective against ischemia-reperfusion injury (55). Hence, combining an ERβ-specific agonist, or androgen deprivation therapies with immunotherapy might improve anti-tumor effects of ICIs and reduce the incidence of ICI-associated cardiotoxicity (56, 57).

The irAEs of immunotherapy involve endocrinopathies, which include primary adrenal insufficiency, hypophysitis, thyroid dysfunction, diabetes, and others (58). Chemotherapies also reduce 17 β-estradiol concentration (59). Although testosterone and E₂ concentrations in cancer patients’ serum are not typically evaluated in clinical settings, we demonstrated the tumor burden-independent downregulation of mouse serum E₂ concentration in female mice following ICI treatment. The potential mechanisms of ICI-induced reduction of estradiol are unknown. One possibility is that ICI could result in hormone suppression via the ovaries or pituitaries, but this would mean a more general change in hormone concentration and clearly requires further investigation. Monitoring the hormone concentration of cancer patients undergoing ICI treatments could therefore be a crucial clinical consideration. The potential difference of rodent and human sex hormone metabolism (60) shall be considered.

Several lines of evidence suggest that MANF has tissue-protective properties. MANF has been shown to negatively correlate with atrial apoptosis in atrial fibrillation (61). The expression of MANF is induced by ischemic conditions and protects against ischemic damage (35, 62). Expression of exogenous MANF via an AAV-mediated method has been suggested to improve neuronal functional recovery post-stroke (63). Recombinant MANF alleviates simulated ischemia reperfusion-associated injury (62) and promotes neuronal differentiation post-stroke (64). Cardiokine therapies could serve as potential strategies for treating cardiac irAE. Three strategies could be considered for restoring the expression of Manf/Hspa5 in the heart: AAV-mediated gene therapy, administration of recombinant protein, and transcriptional upregulation of these genes with hormone therapies. Given potential concerns about unspecific incorporation of therapeutic genes into the genome (65) and the successful application of recombinant MANF against neurodegenerative disease (64), we considered that in vivo delivery of recombinant MANF might be suitable for cancer patients who have a high risk of heart failure and are undergoing ICI treatment. Preclinical PK and toxicology studies suggested that injection of MANF protein results in robust infusion in vivo (64). The tissue half-life of MANF could be 5.5h in rats (66). Repeated infusion of MANF showed well-tolerated clinical signs, undetectable pathology parameters, and normal neurological evaluations with minimal carcinogenicity in male and female rhesus macaques (66). Hence, the administration of recombinant MANF might provide a convenient, safe, and effective strategy for combatting ICI-induced heart defects. Building upon this, future work will explore the mechanisms by which not only ICI treatment decreases estrogen signaling / 17β-estradiol but also MANF/HSPA5 decreases apoptosis and lymphocyte infiltration in hear tissue.

MATERIALS AND METHODS

Study design

The objective of this study was to develop a preclinical strategy to overcome female susceptibility to ICI-associated myocarditis using both pharmacological and genetic mouse models that recapitulated this complication. We defined the molecular mechanism by which ICI induces the heart-specific downregulation of MANF and HSPA5. Preclinical studies were further performed to evaluate the therapeutic effects of hormonal therapies, which restore the MANF and HSPA5 levels upon alleviation of ICI-associated myocarditis. A comprehensive approach that included RNA-seq, genetic knockout mouse model, iPSC-differentiated cardiomyocytes, mouse ECG assays, and so forth was applied to increase the rigor of this study. We obtained primary human ICI-Myocarditis heart tissues, approved by the Institutional Review Board of Vanderbilt University and UCSF. All in vivo experiments were performed following the guidance of the Institutional Animal Care and Use Committee. Mice were randomized before each experiment, but investigators were not blinded to group allocation. For other assays, at least two independent experiments were performed with biological replicates or triplicates. Sample size in experiments was specified in each figure legend. We did not exclude samples or animals. For every figure, statistical tests are justified as appropriate.

In vivo xenograft murine models and treatment

All animal-based research was performed in accordance with the guidelines and requirements set forth by the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals, the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act of 1966 as amended by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas M.D. Anderson Cancer Center (MDACC). Male and female 6–8 weeks old wild type C57/BL6J mice, Esr2 knockout mice (B6.129P2-Esr2tm1Unc/J) and conditional Ar knockout mice (B6.129S1-Artm2.1Reb/J) were obtained from the Jackson Laboratory. The animals were grouped randomly, and 3–5 mice were put into each cage. The animals were implanted with respective tumor cells in the unit of cages, which were randomly selected. Tumor size was measured every 3 days using a caliper, and tumor volume was calculated using the standard formula: 0.54×L×W², where L is the longest diameter and W is the shortest diameter. Immune checkpoint blockade treatment was started when tumor volume reached 200–250 mm³ using anti-mouse PD-1 [25 mg/kg, intraperitoneal (i.p.), every 3 days (q3)] combined with anti-mouse CTLA-4 antibody (25 mg/kg, i.p., q3). The control group was injected with an equal dosage of rat IgG2a isotype (50 mg/kg, i.p., q3).

HEK293T cells-purified recombinant c-Myc-tagged mouse HSPA5 (aa. 1–655) was obtained from Origene Technologies inc (Cat#: TP509794) and was purified with immobilized metal affinity chromatography (IMAC), with over 90% purity. HEK293-purified recombinant His-tagged mouse MANF (aa. 1–179) was obtained from Sino Biological (Cat#: 50954-M08H), with over 90% purity. C-Myc-tagged HSPA5 [7.8 μg/kg, i.p., everyday (qd)] and His-tagged MANF protein (2 μg/kg, i.p., qd) were resuspended in PBS pH 7.4 and were used to treat the mice from 7 days before tumor cell inoculation till the end of the experiment.

The female mice were treated with PBS or LY500307 (ip injection, 5 mg/kg) from the same day of tumor inoculation until the end of the experiment. The male mice were treated with the vehicle or ARN-509 (10 mg/kg, oral gavage, daily) from the same day of tumor cell inoculation until the end of the experiment. All animals were housed with a 12 h light/ 12 h dark cycle in the animal facility with free access to water and food. To obtain unbiased and reliable results, at least five mice were used in each group. Sample size was indicated in corresponding figure legend.

The genetic Ctla4 haploinsufficiency in the absence of Pdcd1 mouse model

Mice derived from a Ctla4^+/− Pdcd1^−/− by Ctla4^+/+ Pdcd1^−/− breeding performed at the Vanderbilt University Medical Center (VUMC) vivarium to generate progeny animals. All mice were genotyped at 3 weeks old, and their date of death were recorded as previously exhaustively described (18). For the generation of survival curve, events were defined as either death (i.e., mice found dead) or identification of mice by veterinary staff as requiring euthanasia (e.g., due to lethargy, moribund, dyspnea). All experiments were performed in accordance with the Vanderbilt University Medical Center IACUC guidelines.

Statistical analysis

The experiment was set up to use 3–12 samples/repeats per experiment/group/condition. Each of these experiments was independently repeated 3–8 times. Results are reported as mean ± standard error of the mean (S.E.M.) or standard deviation (SD). Each exact n value is indicated in the corresponding figure legend. Pearson’s correlation analysis was implemented for statistical analysis of the correlation between the intensity of cells marker and individual protein concentration. Kaplan-Meier survival curves were compared using the log-rank test. Statistical analysis was performed using GraphPad Prism version 9.0 (GraphPad software). The difference in multiple groups was estimated by one-way or two-way ANOVA analysis, and Student’s t-test was used to estimate of the difference in only two groups. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001; ns is not significant, as indicated in individual figures. Individual data value is reported in data file S1.

Murine model of acute ischemic cardiac injury

Myocardial infarction was performed by ligating the left anterior descending (LAD) coronary artery following open thoracotomy as described (67).

Samples of human ICI-myocarditis

Our group has established an international registry of patients with ICI-associated myocarditis using a Research Electronic Data Capture web-based platform with Institutional Review Board approval (Institutional Review Board No. 181337; URL: http://www.clinicaltrials.gov; Unique identifier: NCT04294771) (15, 68). At Vanderbilt University Medical Center and at University of California, San Francisco (UCSF), we have obtained formalin-fixed paraffin embedded tissue from patients with various forms of myocarditis including ICI-associated myocarditis as well as unused healthy donor heart samples (Institutional Review Board no. 201926), as previous described (69). The clinical information of human tissue samples was included in the table S2. Antibodies are listed in table S3.

Tissue collection and immunohistochemistry (IHC) staining

Unless otherwise indicated, the mice were fasted for 4–6 h, anesthetized with isoflurane, blood was collected by heart puncture, and then mice were euthanized. H&E staining was performed to characterize cardiac muscle pathology. Fluorescent multiplex immunohistochemistry (mIHC) and regular immunohistochemistry (IHC) staining were performed by using antibodies as indicated in table S3. All immune-stained slides were scanned with the Vectra Polaris Quantitative Pathology Imaging System (PerkinElmer) for quantification by a digital image analysis. The quantification of IHC staining density was performed by Image-pro plus 6.0 software (Media Cybernetics) and calculated based on the average staining intensity.

AAV-meditated Manf KO

To evaluate the cardiac protective function of MANF, we knocked out Manf in adult C57BL/6J mice using AAV as previously described with minor modification (70). saCas9 under the control of the cardiac muscle troponin T promoter (cTnT) (AAV-cTnT-saCas9), and the sagRNA with scramble sequence, or targeting Manf exon 2, under the control of U6 promoter (AAV-Scr-sagRNA, AAV-Manf-sagRNA) were generated (AAV vector construction services provided by VectorBuilder). Scramble and Manf sagRNA sequence were included in the table S4 These viral vectors were sent to the Institutional Vector Core at the MD Anderson Cancer Center for packaging (AAV9 serotype, 1.5 × 10¹³ vg ml⁻¹). For AAV intra-cardiac injection, 2 × 10¹¹ viral genomes of each AAV (AAV9 serotype, 1.5 × 10¹³ vg ml⁻¹) were diluted in 50 μl saline, and were injected directly into the left ventricle of each mice at age of 5-week-old. 3 weeks post the AAV administration, the animals were subcutaneously inoculated with B16F10 cells. ICI and the IgG isotope treatment was conducted when the tumor volume reach to 200–250 mm³.

Tumor cell line culture

The mouse breast adenocarcinoma cell line EO771, mouse colon adenocarcinoma cell line MC38, mouse melanoma cell line B16F10, and mouse prostate cancer RM1 cells were purchased from the American Type Culture Collection (ATCC), and the tumor cells were maintained in the respective optimal complete growth medium according to the instruction of ATCC. 1×10⁵ EO771 were injected into the mammary fat pad. 1×10⁵ MC38, 0.5×10⁵ B16F10 or 1×10⁵ RM1 cells were inoculated subcutaneously into the right flank of the mice.

Induced pluripotent stem cells (iPSCs) maintenance and differentiation

The human iPSC studies were approved by the HEIP Stem Cell committee of the University of Texas, MD Anderson Cancer Center. The HSCC-022iPS cell line from an 11-year-old healthy female donor was obtained from the Human Stem Cell Core (HSCC) (Baylor College of Medicine). It was cultured in hESC-Qualified Matrigel (Corning)-coated plates and maintained in mTeSR plus medium (Stemcell Technologies). The iPS cells were then differentiated to cardiomyocytes by using the STEMdiff Cardiomyocyte Differentiation Kit (Stemcell Technologies) according to manufacture instruction. HiPS-CMs were treated with β-Estradiol (10 nM, Sigma-Aldrich), 5α-Dihydrotestosterone (10 nM, Sigma-Aldrich), IL-6 (10 ng/ml PeproTech), TNF-α (10 ng/ml PeproTech), IFN-γ (10 ng/ml PeproTech), LY500307 (10 nM, APExBIO), or ARN-509 (10 nM, Cayman Chemical) for 1 hr or as indicated in the figure legends.

Bulk RNA-seq, data analysis and QPCR

RNA-seq was performed by Illumina HiSeq 2000 with a 150bp paired-end read. Reads were aligned to the mouse genome (Ensembl mm10 mouse genome) using HISAT2 (V 2.1.0) (71) with default parameters, and reads were assigned to genes using feature Counts (V 1.6.5) (71). Differential expression analysis and FPKM (fragments per kilo-base of exon per million fragments) calculation of genes in each sample were performed by edgeR (72). Genes with an FDR (false discovery rate) adjusted by the method of Benjamini and Hochberg < 0.1 and a fold change > 1.5 were assigned as significantly differentially expressed genes (DEGs) between the ICI and IgG samples. Significantly DEGs were subjected to pathway enrichment by R package “clusterProfiler” (73). Venn diagrams shown the overlap of significantly DEGs were completed by R package “venn”. Heatmaps of significantly DEGs were plotted by R package “heatmap”. The raw RNA-seq data for this manuscript is available at GEO under the accession number GSE145573 and GSE213468. For bulk RNA-seq of mice following myocardial infarct, the data GSE151834 in NCBI GEO dataset (74) were analyzed. Reads were aligned to the mouse reference genome (mm10) using STAR aligner and used to quantify normalized expression values (RPKM) for annotated genes (Ensembl v.86). Normalized expression levels (RPKM) were used for gene expression visualizations.

RNA isolation, qRT-PCR, cell lysis, ChIP and immunoblotting

Total RNA was isolated from cells using RNeasy Mini Kit (QIAGEN) following the manufacturer’s protocol. First-strand cDNA synthesis from total RNA was carried out using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad). Alternatively, cDNAs were generated using iScript cDNA Synthesis Kit (BioRad) and qPCR was performed. Primer sequences are described in table S4. ChIP assays were performed as previously described (75). Primers using for ChIP assays were included in table S4. Cells were homogenized in 1×RIPA buffer (EMD Millipore) supplemented with Protease/ Phosphatase Inhibitor Cocktail (Pierce, Thermo Scientific), Panobinostat (Selleck chemicals) and Methylstat (Sigma-Aldrich). Lysates were cleared by centrifugation at 13,000 rpm for 15 minutes at 4 °C. Supernatants were analyzed for immunoprecipitation with the indicated antibodies as listed in table S3 and the immunoprecipitated proteins were further subjected to immunoblotting detection.

Data and material availability:

All data associated with this study are in the paper or supplementary materials. The raw RNA-seq data for this manuscript is available at GEO under the accession number GSE145573 and GSE213468. The hiPSs were obtained from the Coriell Institute for Medical Research and are subject to restrictions on redistribution and sharing. Ctla4- and Pdcd1-knockout transgenic mice are available from J.J.M. under a material transfer agreement with the University of Texas MD Anderson Cancer Center.