NK cells promote cardiac cell death and regulate myelopoiesis in myocardial infarction

Domain: biology
Published: 2026-04-01
Editorial outcome: minor_revision

Abstract
Ischemic heart failure remains a major clinical challenge, underscoring the need to better understand post-infarction immune mechanisms and identify new therapeutic targets. Both innate and adaptive immunity contribute to adverse cardiac remodeling following myocardial infarction (MI), yet the role of cytotoxic cells such as natural killer (NK) cells remains poorly defined. Here, we show that after acute MI in mice, NK cells are recruited to the ischemic myocardium in a CCR2-dependent manner and become activated. Activated NK cells locally release granzyme B, promoting cardiomyocyte apoptosis, adverse ventricular remodeling, and impaired cardiac function. Genetic deletion or pharmacological depletion of NK cells reduces cardiomyocyte death, attenuates inflammation, limits myocardial injury, and improves cardiac function. In contrast, NK cell activation using an anti-NKG2A monoclonal antibody exacerbates ischemic heart failure. We further demonstrate that NK cells regulate bone marrow myelopoiesis through local GM-CSF production. Finally, we identify a distinct NK cellular and transcriptomic signature in human ischemic heart tissue at early stages. Together, these findings reveal a detrimental role for NK cells following acute MI and highlight NK cells as potential therapeutic targets to limit adverse cardiac remodeling.

Introduction
Despite significant advances in the early management of acute myocardial infarction (MI), ischemia-related heart failure remains a persistent clinical and social burden, including a heightened risks of arrhythmias, recurrent hospitalizations, disabilities and mortality1. Recent epidemiological evidence, including a study using nationwide medico-administrative data from the French National Hospitalization Database (2013-2018), underscores the rising incidence of ischemic heart failure, especially among young adults 2. This trend underscores the need for intensified efforts to deepen our understanding of post-ischemic pathophysiology and develop treatments targeting specific pathways involved in cardiac tissue remodeling.

Several human and experimental studies have highlighted both beneficial and harmful effects of inflammation following acute MI and reperfusion injury 1. Large numbers of immune cells are rapidly recruited to the heart to facilitate the clearance of dead cardiac myocytes and debris, and to orchestrate tissue repair 3. However, blood-derived leukocytes also contribute to long-term cardiac damage. Recruited immune cells, activated by Danger/Damage Associated Molecular Patterns (DAMPS), release signaling molecules, including chemokines, cytokines, and reactive oxygen species, which contribute to detrimental remodeling of the heart by modulating the production and degradation of extracellular matrix proteins, and impairing the functions of local cardiac and non-cardiac cells 4. Notably, recent research from our group has identified CD8+ T cells, a subset of T lymphocytes with cytotoxic activity, as key players in post-ischemic cardiac remodeling, facilitating cardiomyocyte death through the local release of Granzyme B 5,6.

Natural Killer (NK) cells represent another powerful cytotoxic population. NK cells represent the largest subset of the innate lymphoid cell (ILC) family and share many common features with ILC1 cells 7,8. Despite lacking somatic rearrangements of antigen receptors, NK cells are responsive to multiple immune signals necessary for pathogen defense and tissue homeostasis 9. However, the role of NK cells in post-ischemic cardiac remodeling remains poorly understood.

Here, utilizing a combination of gain-of-function and loss-of-function approaches in a murine model of MI, we demonstrated that NK cells are activated and are recruited to the ischemic heart in a CCR2-dependent manner. Locally, NK cells induce cardiomyocyte death through the release of Granzyme B, which exacerbates myocardial inflammation, tissue injury, and reduces myocardial function. Additionally, we revealed that NK cells modulate myelopoiesis in the bone marrow (BM) through the release of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, immunotherapy based on the administration of neutralizing antibodies targeting NK cells promoted cardiac repair in a murine model of MI.

Results
NK cells are recruited and activated in the ischemic heart after acute MI
First, we investigated the trafficking of NK cells following acute MI. To achieve this, we employed an experimental model of MI in C57BL/6J mice, induced by permanent coronary artery ligation. Subsequently, we analyzed cell suspensions obtained from digested hearts at various time points following the onset of ischemia using flow cytometry. Our observations revealed accumulation of CD3-NK1.1+NKp46+ NK cells accumulate in the injured myocardium, starting as early as day 3 and peaking at day 7 after MI (Figure 1.a-b & Supplementary Fig.1). Notably, the infiltration of NK cells was around twice as high as that of CD8+ T cells (Supplementary Fig.2a). Further immunofluorescent staining and transcriptomic studies (Figure 1c-d & Supplementary Fig.3) confirmed the heightened presence of NK cells in both peri-infarct and infarcted areas of the heart following MI, as compared to sham-operated animals. To further characterize NK cell populations in the context of MI, we reanalyzed previously published data on cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) 10 of total CD45+ cells from control and ischemic hearts at day 5 post-MI 11. Cells corresponding to T and NK cells were extracted and separately reclustered. The infiltrating NK cells predominantly exhibited a mature (Itgam+) CD11b+ phenotype (Figure 1e-f), along with a cytotoxic transcriptomic signature, as evidenced by enrichment for Gzma, Gzmb, and Perforin mRNA within this population (Figure 1g-h). Mature NK cells also express high Itga4 mRNA (CD49d), an integrin involved in adhesion (Supplementary Fig.4). NK cells within ischemic hearts were found to express Ncr1 (encoding NKp46), Klrc1 (encoding NKG2A), and Klrk1 (encoding NKG2D) receptors, which are crucial for regulating their activation (Figure 1i-l). We then investigated the underlying mechanisms governing NK cell trafficking, focusing on CCR2 and CCR5, as both chemokine receptors are expressed by infiltrating NK cells (Figure 1m-n & Supplementary Fig.5ab). Deletion of Ccr2, while not impacting CCR5 expression, resulted in impaired NK cell recruitment to the heart in the context of MI (Supplementary Fig.5c-e). Conversely, deletion of Ccr5, which is associated with an up-regulation of CCR2, led to an increase in NK cell infiltration within the ischemic heart tissue (Supplementary Fig.5f-i). Given that global Ccr2 deletion affects multiple immune cell populations, we performed additional experiments to specifically validate the critical role of CCR2 for NK cell recruitment to ischemic heart tissue. To this end, Nkp46iCre+/-R26RDTA (referred to as NKKO) mice were repopulated with purified Wild-type or Ccr2-/- NK cells 3 days before MI (Figure 1o). The purity of NK cells and the NK cell deficiency in NKKO mice were confirmed by flow cytometry (Supplementary Fig.5j-6). As depicted in figure 1p, at day 3 post-MI, NK cell frequencies were comparable in the blood across groups but were significant lower in the hearts of MI-operated mice repopulated with Ccr2-/- NK cells (Figure 1qr). Collectively, these findings suggest that mature cytotoxic NK cells are recruited into the myocardium following MI partly via CCR2, thus implicating a potential role for NK cell-mediated immune responses in this setting.

NK depletion or deficiency prevents deleterious adverse ventricular remodeling after acute MI in mice.
To directly investigate the impact of NK cells on cardiac remodeling following MI, we administered an NK1.1 monoclonal antibody 12 to deplete NK cells one hour after the onset of MI. This treatment resulted in a rapid and substantial depletion of NK cells (>95%) within 6 hours post-injection in the peripheral blood, bone marrow (BM), spleen and cardiac tissue (Supplementary Fig.7a-c & Figure 2a). NK1.1 mAb had no impact on CD8+ T cells (Supplementary Fig.7d). It is noteworthy that NK1.1 mAb treatment also effectively depleted NKT cells, despite their being a small population in the ischemic heart (Supplementary Fig.2b). Complete NK cell depletion was confirmed by immunofluorescence in the ischemic heart (Figure 2b). Echocardiography performed 21 days post-MI revealed a significant improvement in left ventricular (LV) ejection fraction (Figure 2c) and cardiac output (Figure 2d) in mice subjected to NK1.1 mAb-induced NK cell depletion, compared to isotype-treated control mice. The improvement in cardiac function following NK1.1 mAb treatment was accompanied by a reduction in adverse LV remodeling. Both infarct size (Figure 2e) and interstitial fibrosis (Figure 2f), as assessed by collagen content, were significantly diminished in NK1.1 mAb-treated mice (p=0.004 and p=0.036, respectively) compared to isotype-treated control animals at day 21 post-MI. Importantly, the protective effect of NK cell depletion was evident in female as well as male C57BL/6J mice (Supplementary Fig.8). NKI cell depletion correlated with decreased collagen synthesis, as evidenced by reductions in Col1a1 and Col3a1 mRNA levels (Figure 2g).
To further corroborate the pathogenic role of NK cells in post-ischemic cardiac remodeling, we employed Nkp46iCre+/-R26RDTA (referred to as NKKO) mice, littermate Nkp46iCre- R26RDTA mice served as controls (NKWT). At day 21 post-MI, NKKO mice had improved LV systolic function (Figure 2i) and higher cardiac output (Figure 2j), compared to control NKWT mice. Additionally, both infarct size (Figure 2k) and interstitial fibrosis (Figure 2l) were significantly decreased in NK-deficient mice, further emphasizing the pathogenic role of NK cells in adverse post-ischemic cardiac remodeling.

NK activation aggravates post-ischemic cardiac damage in experimental MI.
To further evaluate the role of NK cells in post-ischemic cardiac remodeling, we utilized a complementary in vivo gain-of-function approach involving a neutralizing anti-NKG2A monoclonal antibody (or IgG isotype control) in C57BL/6J mice (Figure 3a) 12. NKG2A is an inhibitory receptor, and its blockade enhances NK cell activity. Increased activation of NK cells following NKG2A mAb treatment was confirmed by flow cytometry in the spleen and BM at day 3 after acute MI (Supplementary Fig. 9). NKG2A mAb treatment increased total CD45+ leucocytes, but had no impact on NK cell numbers in the heart at day 5 after MI (Figure 3b-d). However, NKG2A mAb treatment significantly aggravated cardiac damage induced by acute ischemia, with increased heart weight (Figure 3e), greater LV dilation (Figure 3f) and reduced LV ejection fraction (Figure 3g) at day 21 compared to isotype-treated control mice. The worsening of cardiac dysfunction following NKG2AR mAb treatment was associated with exacerbated adverse LV remodeling. Both infarct size (Figure 3h) and interstitial fibrosis (Figure 3i & Supplementary Fig.10), as assessed by collagen content, were significantly increased in NKG2A mAb-treated mice compared to isotype-treated control animals at day 21 post-MI. Consistent with these findings, cardiac Col1a1 mRNA levels in heart tissue were significantly increased in the NKG2A group (Figure 3j).
Given the NKG2A receptor is also expressed by CD8+ T cells, the pathogenic effects of the NKG2A mAb may not be solely due to NK cell activation. To specifically evaluate the impact of NK cell activation in the context of MI, we repeated anti-NKG2A monoclonal antibody or IgG isotype treatments in CD8-depleted C57BL/6J mice (Figure 3k). CD8 depletion was confirmed by flow cytometry (Figure 3l). As shown in Figure 3m-o, in the absence of CD8+ T cells, NKG2A mAb still aggravated post-ischemic cardiac damage, resulting in reduced LV systolic function, larger infarct size and increased fibrosis, compared to isotype treatment.

Pathogenic activity of NK cells after MI is not due to NKp46 receptor
Our findings indicated that NK cells play a role in harmful post-ischemic cardiac remodeling. We then examined which receptors govern NK cell pathogenic activity, initially focusing on NKp46, as its role in cardiac remodeling is not well understood. Previous studies have shown that NKp46 regulates the growth of tumors such as melanoma 13, lymphoma 14 or carcinoma. To study NKp46 in vivo, we utilized Ncr1 knockout mice, in which the Ncr1 gene (coding for NKp46) was replaced by a green fluorescent protein (Gfp) reporter (Ncr1gfp/gfp) 15 (Supplementary Fig.11a). NK cell counts in the circulation were similar between control Ncr1+/+ and Ncr1gfp/gfp mice (Supplementary Fig.11b). Notably, Ncr1 deficiency did not affect NK cell infiltration into the ischemic heart at day 5 post-MI (Supplementary Fig. 11c) and had no significant impact on cardiac function (Supplementary Fig. 11d), infarct size (Supplementary Fig. 11e) or interstitial fibrosis (Supplementary Fig. 11f) at day 21. Taken together, these results suggest that the pathogenic activity of NK cells in the context of MI do not require the engagement of the NKp46 receptor.

NK cells aggravate cardiac cell death through Granzyme B
We next explored the mechanisms through which NK cells influence cardiac remodeling and function, focusing on their well-known cytotoxic activity, which has been documented in cancers and viral infections 16. ScRNA-seq analysis showed that infiltrating NK cells express Granzyme A, Granzyme B, and Perforin (Figure 4a). Following MI, Gzma mRNA levels in the heart remained relatively constant, but Gzmb levels significantly increased between days 3 and 7, coinciding with the infiltration of CD11b+ NK cells (Figure 4b-c). Depletion of NK cells with the NK1.1 mAb treatment resulted in reduced Gzmb mRNA levels in ischemic heart tissue (Figure 4d) and a complete absence of Gzmb+ NKp46+ NK cells (Figure 4e). Of note, a population of GzmB+ NKp46- cells, corresponding to CD8+ T cells, remained detectable in the ischemic heart (Figure 4e). The decrease in cardiac Granzyme B content was associated with a significant reduction in both TUNEL+ cells and infarct size 3 days post-MI (Figure 4f & Supplementary Fig. 12). In contrast, NK cell activation using anti-NKG2A treatment increased Gzmb mRNA levels in ischemic heart tissue (Figure 4g), elevated GzmB+ NKp46+ NK cell number (Figure 4h), and led to a substantial increase in both TUNEL+ cells and infarct size 3 days post-MI (Figure 4i & Supplementary Fig. 12). The cytotoxic role of NK cells was confirmed in NK-deficient mice, where tissue necrosis was significantly reduced (Figure 4j).
To further investigate the role of NK cell-derived Granzyme B, we reconstituted NK-deficient mice with purified NK cells from wild-type or Gzmb-/- mice (Figure 4k) 48 hours before MI. Immunofluorescent staining confirmed the successful transfer of both wild-type or Gzmb-/- NK cells (Supplementary Fig.13). Transferring wild-type NK cells into NK-deficient mice resulted in increased heart tissue necrosis 3 days post-MI, whereas the transfer of Gzmb-/- NK cells did not result in increased necrosis (Figure 4l). These findings suggest that Granzyme B has direct cytotoxic effects on cardiomyocytes. To test this hypothesis, we co-cultured mouse cardiomyocytes with purified wild-type or Gzmb-/- NK cells (Figure 4m-o & Supplementary Fig.14). After 24 hours, NK cells were removed, and cardiomyocyte apoptosis was monitored for an additional 24 hours using a caspase-3 fluorescent dye. Incubation with non-activated NK cells only modestly affected cardiomyocyte survival, while incubation with activated NK cells caused cardiomyocyte apoptosis, with the effect being more pronounced at higher NK/cardiomyocyte ratios (Figure 4o). The cytotoxicity of NK cells was significantly reduced when Granzyme B was deficient, as shown by decreased apoptosis rate in cardiomyocytes (Figure 4p). Finally, to assess the human in vitro relevance of our findings, we co-cultured human 3D cardiac rings with purified human NK cells, and measured contractility parameters before and 12 hours after co-culture (Figure 4q & Supplementary Fig.15a). Non-activated NK cells did not affect 3D cardiac ring function. However, activated NK cells which aggregated on rings, impaired both organoid contractility and relaxation in a dose-dependent manner (Figure 4r & Supplementary Fig.15b). Overall, these results strongly suggest that infiltrating NK cells impair cardiomyocyte function and survival in the context of MI through the release of Granzyme B.

NK cells orchestrate myeloid cell trafficking and inflammatory responses
Modulating NK cells had a significant impact on the inflammatory profile of the heart at day 7 post-MI. In NK-depleted mice, the mRNA levels of Il-1b and Tnf were significantly lower in infarct hearts compared to controls (Figure 5a & Supplementary Fig.16a), along with a marked reduction in metalloproteinase activity (Figure 5b-c). NK cell depletion also limited monocyte recruitment in the heart at days 5 and 7 post-MI (Figure 5d and Supplementary Fig.17-18) and decreased the content of CCR2+ macrophages at day 7 (Supplementary Fig.18). To further evaluate the impact of NK depletion on the transcriptomic phenotype of myeloid cells, scRNA-seq was performed on heart CD45+ cells at day 5 post-MI in animals treated or not with anti-NK1.1 depleting antibody (Supplementary Fig.19a). Following clustering and extraction of myeloid cells populations, we identified monocytes, 5 macrophages subsets, resident macrophages, and 3 neutrophil subsets (Supplementary Fig.19b-c). Effective NK depletion in anti NK1.1-treated mice was confirmed (Supplementary Fig.19d). Using pseudobulk analysis, we identified differentially expressed genes in monocyte and macrophage populations after NK cell depletion and assessed their expression across total cells per group. Of note, Cd36 and Hdac9 were upregulated, whereas P2rx7, a gene involved in inflammasome activation, was downregulated in the NK depleted group (Supplementary Fig.19e). Moreover, Tnf expression was reduced in the main populations of monocytes, macrophages and neutrophils in the depleted group (Supplementary Fig.19f). Altogether, these results indicate that NK cell depletion promotes a shift in macrophages toward a less inflammatory phenotype. In contrast, anti-NKG2A treatment, which activates NK cells, drives a shift toward a more inflammatory cardiac phenotype, characterized by higher Il-1b, Il-6 mRNA levels, lower Il-10 mRNA levels (Figure 5e), and greater local metalloproteinase activity (Figure 5f-g). Consistently, Anti-NKG2A-mediated NK activation increased the recruitment of neutrophils and monocytes (Figure 5h & Supplementary Fig.18f-g) in the heart at day 5 post-MI and increased the number of CCR2+ macrophages at day 7 (Supplementary Fig.18h).
We next examined how NK cell modulation affected monocyte content in the heart. We found that NK cell depletion significantly reduced monocyte counts in the spleen and BM within the first days post-MI (Supplementary Fig.18d-e). In addition, granulocyte/macrophage progenitor cell (GMP) populations in the BM were reduced in NK-depleted mice (Figure 5i & Supplementary Fig.17). NK modulation did not affect Ccl2, Ccl3, Cxcl1 and CxcL2 gene expression in the heart (Supplementary Fig.16). Based on these findings and previous studies showing that NK cells produce GM-CSF 17, we hypothesized that NK cells control BM myelopoiesis through GM-CSF production during MI. We confirmed that NK cells are an important source of GM-CSF in the BM of MI-operated mice (Figure 5j-k) and observed decreased plasma levels of GM-CSF at day 3 post-MI in NK-depleted mice compared to isotype-treated controls (Figure 5l).
To specifically investigate the role of GM-CSF produced by NK cells in myelopoiesis during cardiac ischemia, we crossbred mice carrying an Nkp46 Cre allele with mice carrying a floxed Csf2 allele. GM-CSF deficiency was confirmed by ELISA in the supernatant of stimulated purified NK cells from Nkp46Cre+/-Csf2lox/lox mice (Figure 5m). Monocytes and neutrophils populations were similar between Nkp46Cre+/-Csf2lox/lox and littermate Nkp46Cre-Csf2lox/lox control mice at baseline as well as cardiac function (Supplementary Fig.20). MI was then induced in both groups of mice, and myeloid populations were analyzed at day 5 (Figure 5n). NK cell counts in the BM were similar between Nkp46Cre+/-Csf2lox/lox and Nkp46Cre-Csf2lox/lox MI-operated mice (Supplementary Fig.21). Notably, we observed a significant reduction in GMP count and monocytes in the BM of GM-CSF-deficient mice (Figure 5o), associated with a decrease in neutrophils (Supplementary Fig.22), monocytes, and CCR2+ macrophages in ischemic heart tissue (Figure 5p). Finally, we observed higher left ventricle ejection fraction in Nkp46Cre+/-Csf2lox/lox mice when compared to littermate Nkp46 Cre-Csf2lox/lox controls (Figure 5q).

NK cells in human MI
To evaluate the relevance of our findings to human disease, we performed transcriptomic analysis of human heart biopsies using Nanostring Technology targeting a panel of 758 genes related to inflammatory pathways. We compared gene expression in healthy heart tissue with ischemic cardiac tissue at early stages (median time 7 (2-13) days) and late stages (median time 39 (3-108) months) after MI (Supplementary Fig.23). The transcriptomic profile of the heart tissue was profoundly altered by ischemia, with clear distinctions in the principal component analysis between the 3 groups (Figure 6a). We analyzed 3 NK cell subsets in heart tissue, previously characterized at the transcriptomic level 18: active NK cells, characterized by up-regulation of inflammatory genes (NR4A2, DUSP1, FOSB, FOS, JUN, and JUNB); mature NK cells, expressing genes associated with cytotoxic activity (PRF1, GZMA, GZMB, GZMH); and terminal NK cells, which display a transcriptional profile similar to the mature NK subset but express the transcription factor ZEB2, indicative of terminal maturation. As depicted in figure 6b, Mature NK cell transcriptomic signature was significantly upregulated at early stages post-MI, whereas active NK cell transcriptomic signature was significantly upregulated at late stages post-MI. Immunostaining further confirmed the infiltration of NK cells in the heart tissues collected from patients with a recent coronary occlusion (Figure 6c-d). In addition, we re-analyzed single-nucleus RNA sequencing data from human hearts (Kuppe et al. 2022). Samples were obtained from the necrotic area or the unaffected LV myocardium regions (remote zone) of patients with acute myocardial infarction, as well as from fibrotic zones of post-MI hearts at a later stage. By calculating module scores for cytotoxic and inflammatory pathways, we quantified the transcriptional activity of these pathways in individual NK cells (see methods). This approach allowed us to identify subtle but significant shifts in pathway activity. As shown in Figure 6e, we identified both pro-inflammatory and cytotoxic transcriptional signatures in NK cells infiltrating both ischemic and remote areas of the heart at day 10. By day 30, cytotoxic activity and inflammation were more pronounced in remote areas. Finally, by day 100, the fibrotic zone exhibited heightened cytotoxic activity.
Finally, we investigated the chemokines/chemokines receptors in the human data from Kuppe’s work 19 . Cells were categorized into cardiomyocytes, non-cardiomyocytes (non-CM), NK cells and other immune cells. As shown in Supplementary Fig.24, all major cell types exhibited high expression of chemokines CCL2 and CCL5. In contrast, CCR2 and CCR5 were predominantly expressed in immune cells and NK cells, respectively. Cardiomyocytes and non-CM cells showed minimal or no expression of these receptors, suggesting that immune and NK cells are the primary targets.

Methods
Mice models. All experiments were conducted according to the French veterinary guidelines and those formulated by the European Community for experimental animal use and were approved by the Institut National de la Santé et de la Recherche Médicale. Permission was granted to perform animal experiments by the appropriate committee (the ethical committee CEEA34 Université de Paris, APAFIS #29531- 2021020416255418v2). All the animals were maintained under identical standard conditions (housing, regular care and normal chow). Mice were maintained in isolated ventilated cages under specific pathogen-free conditions. Experimental/control animals were bred separately.

Myocardial infarction. All mice used in this study were of the C57Bl/6J strain. Specifically, C57BL/6J mice (sourced from Janvier, France, Stock designation: C57BL/6JRj), Gzmb-/- mice (from Jackson Laboratory, USA, Stock No. 002248), Nkp46iCre+/- R26RDTA mice (provided by E. Vivier’s lab in Marseille, France 38), Ncr1gfp/gfp mice (from O. Mandelboim’s lab in Jerusalem, Israel 15), and Csf2Lox/lox mice (from I.P. Wicks at WEHI in Melbourne, Australia 17) were used. Ccr2-/- mice were obtained from Jackson Laboratory (USA) and Apoe-/-Ccr5-/- (named Ccr5-/- in the manuscript) came from Y. Döring’s Lab 39 (Switzerland). All mice were aged 9-10 weeks at the time of myocardial infarction (MI) induction. All the experiments were performed on 8 to 10-week old male mice. The protective effect of NK depletion using anti-NK1.1 mAb was also validated on female mice. No sample size calculation were performed.
The MI was induced by ligating the left anterior descending coronary artery, as described by Santos-Zas et al. 6. Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), intubated, and ventilated using a small animal respirator (SAR830A/P, CWE). After shaving the chest wall, a thoracotomy was performed in the fourth left intercostal space to expose the left ventricle. The pericardial sac was removed, and the left anterior descending artery was permanently ligated with a 7/0 non-absorbable monofilament suture (Peters Surgical, France) at the point where it emerges under the left atrium. A successful coronary occlusion was confirmed by significant color changes in the ischemic area. The thoracotomy was closed with 6/0 non-absorbable monofilament sutures (Peters Surgical, France). Sham-operated controls underwent the same procedure without tying the ligature. After the surgery, the endotracheal tube was removed when spontaneous breathing resumed, and the mice were placed on a warming pad at 37°C until fully awake. To ensure compliance with ARRIVE guidelines and our institutional ethical board, animal welfare was monitored via a clinical scoring system (0–3) evaluating body weight, physical appearance, behavior, and wound healing. Mandatory euthanasia was performed via cervical dislocation under isoflurane sedation for any single category score of 3 or a cumulative score of 6, while intermediate scores (3–5) were managed with buprenorphine analgesia and systematic re-evaluation within 24 to 72 hours.
To study NK cell recruitment and activation in the ischemic heart post-MI, C57BL/6J wild type mice underwent permanent coronary ligation. Sham-operated C57BL/6J wild type mice served as controls, undergoing the same procedure without the coronary ligation. Mice were euthanized by cervical dislocation by trained and experienced staff, at various time points for flow cytometry and histological analysis. To evaluate the role of NK cells in post-ischemic cardiac remodeling, C57BL/6J Wild type mice were injected i.p. with a depleting mouse anti-mouse NK1.1 monoclonal antibody (200g/mouse, BioxCell BE0036 clone PK136) or with a mouse IgG2a isotype control (200g/mouse BioxCell BE0085 clone 2A3) 1 hour after coronary artery ligation. In order to maintain cell depletion for an extended period, mice were re-injected at day 7 and day 14 after MI. Animals were euthanized 24 hours, 3, 5, 7, 14 and 21 days after surgery for flow cytometry and histological analysis. To activate in vivo NK cells, C57BL/6J wild type mice were injected i.p. with a neutralizing rat anti-mouse NKG2A/C/E monoclonal antibody (200g/mouse, BioXCell BE0321, clone 20D5) or with a rat IgG2a isotype control (200g/mouse BioxCell BE0085 clone 2A3) 1 hour after coronary artery ligation. In order to maintain cell activation for an extended period, mice were re-injected once a day every day until day 7 after MI. To explore the role of NKG2D in the context of MI, isotype- or anti-NK1.1-treated C57BL/6J mice were injected i.p. with a neutralizing Armenian hamster anti-mouse anti-NKG2D monoclonal antibody (200g/mouse, BioxCell BE0111, Clone HMG2D).
Next, 8 week-old NK deficient Nkp46iCre+/- R26R DTA mice were used as recipients for NK cell repopulation studies 3 days prior MI. Purified NK cells, obtained from C57BL/6J WT mice or Gzmb-/- mice, were resuspended in phosphate-buffered saline (2x106 cell/ 100 μl/ mouse) and transferred to NK deficient mice by i.v injection 3 days before MI. Repopulation was checked at day 3 following MI in the heart.

Echocardiographic measurements. Transthoracic echocardiography was performed at 21 days after surgery using an echocardiograph (VEVO2100, Visualsonics) and a 18 to 38-MHz transducer (MS400). The investigator was blinded to group assignment. Animals were anesthetized by isoflurane inhalation. Two-dimensional parasternal long-axis views of the left ventricle were obtained for guided M-mode measurements of the LV internal diameter at end diastole (LVDD) and end systole (LVDS), as well as the interventricular septal wall thickness, and posterior wall thickness at the same points. Ejection fractional percentage (% EF) was calculated using Simpson method.

NK cell purification and transplantation. NK cells were isolated from C57BL/6J, Gzmb-/- spleens and purified using a mouse NK cell isolation kit (130-115-818 Miltenyi Biotec; Paris, France) according to the manufacturer’s protocol. In brief, NK cells were negatively selected using a cocktail of antibody-coated magnetic beads followed by cell separation using LS magnetic columns (Miltenyi Biotec; Paris, France), yielding NK cells with >90% purity. Then cells were: (a) intravenously injected 3 days prior MI in Nkp46iCre+/- R26RDTA mice (b) cultured for in vitro experiments.

NK cell culture in vitro. NK cells were isolated as described above and cultured in activation medium containing RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin (Gibco, Thermofisher) 5 ng/ml IL15 (R&D Systems; Abingdon, United Kingdom). To confirm GM-CSF deficiency in Nkp46Cre+/-Csf2lox/lox mice, purified NK cells were cultured at 37°C with 5% CO2 in activation medium containing RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin (Gibco, Thermofisher), rIL-15 (10 ng/ml), rIL-18 (50 ng/ml), and rIL-12 (50 pg/ml, R&D Sytems). After 48 hours, the supernatant was recovered and GM-CSF levels were measured by ELISA (MGM00, R&D Systems), following manufacturers protocol.

HL1 cardiomyocytes culture. Cells were seeded into 24-well plates (coated with gelatin-fibronectin (Sigma-Aldrich) overnight, 37ºC) at a density of 20 x 103 cells/ml/well and maintained during 2 days in Growth Medium containing Claycomb Medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin (Gibco, Thermofisher), 1% norepinephrine 10mM (Sigma-Aldrich) and 1% L-Glutamine 100X (Sigma-Aldrich).

Co-culture and Apoptosis experiment. Activated (rIL-15, 10 ng/ml) and non-activated NK cells were co-cultured directly with HL1 cardiomyocytes in different ratios (1:1, 1:5 and 1:10) with respect to cardiomyocyte count. Following co-culture for 24h, NK cells were removed and HL1 cardiomyocytes were labeled with the addition of 5 M Caspase-3/7 Green Apoptosis Reagent (Incucyte, Welwyn Garden, United Kingdom) to the growth medium. After 30 min at 37°C in dark, apoptosis in real time was evaluated with a microscope Nikon Eclipse, Ti. Time-lapse images were captured at 10 min intervals. To evaluate GM-CSF production by NK cells ex vivo, cells were stimulated with rIL-12 (50 microg/ml), IrIL-15 (10 ng/ml) and rIL-18 (50ng/ml) 17.

3D cardiac rings generation and recording
3D cardiac rings were generated as previously described 40. The tissues were generated on a commercial SmartHeart® 96-well plate (4DCell, Montreuil, France), which contains a molded hydrogel in each well that induces the seeded cells to self-organize into ring-shaped tissues. On day 22 of differentiation, human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CMs) were dissociated and mixed with human dermal fibroblasts at a ratio of 1:3 (hiPSC-CM:fibroblast). Nine days after their generation, the tissues were imaged at baseline using brightfield microscopy with a high-speed CCD camera (PLD672MU, Pixelink) mounted on Primovert microscope (Zeiss) at 10X magnification. The tissues were then incubated for 12 hours before recording with either non-activated or IL-2-activated human Natural Killer cells, at hiPSC-CMs:NK ratios of 1:1 and 1:5. Automated video analysis was performed using a Matlab script to recover contractility parameters such as contraction frequency, contraction stress and the maximum contraction and relaxation speeds. The protocol for human iPS was approved by the local institutional ethics committee (Comité de Protection des Personnes Ile de France XI IRB number 11-015). Human NK cells were isolated from blood donors (Collaboration Etablissement Français du sang) using kits (Miltenyi biotech, Paris, France, (catalog #130-092-657) and next were activated or not in vitro overnight with IL-2r (100 IU/mL) (IRB 2022-2026-046 Comité de Coordination de la Protection des Sujets et des Locaux Imagine). Next, cells were washed and co-cultured with 3D cardiac rings at different ration during 12 hours. All the volunteers and blood donors gave informed consents.

Histopathological and immunofluorescence analyses. Cardiac healing following myocardial infarction was assessed at different time points. Hearts were excised, rinsed in PBS and frozen in liquid nitrogen. For tissue morphology analysis after MI, hearts were cut along their length into 7 μm thick cardiac muscle cryosections (CM 3050S, Leica). Serial sections were mounted on microscope slides (n=10), each section being spaced from the next one by 400 μm. In this way, 10 sections / hearts allowed entire heart tissue analysis. Masson’s trichrome and Sirius Red stainings were performed for infarct size and interstitial fibrosis evaluation, respectively. Infarct size was calculated as a percentage of infarct area to total LV surface. The collagen volume fraction was calculated as the ratio of the total area of interstitial fibrosis to the myocyte area in the entire visual field of the section.
Heart sections for immunofluorescence analysis were fixed with paraformaldehyde 4%, permeabilized using 0.2% Triton X100 in Phosphate Buffer Solution (PBS) 30 minutes at room temperature, blocked with PBS-T (0.2% Triton X100, 10% goat serum, 0,2% BSA in PBS) for 1 hour, and incubated with primary antibodies diluted in PBS-T overnight at 4ºC: NK cell infiltration was detected using an anti-NKp46 antibody (Abcam ab233558, 1:200); Granzyme B detection in ischemic heart tissue was performed using an anti-Granzyme B antibody (R&D Systems AF1865, 1:100); Sections were washed with PBS and incubated with a mixture of appropriate secondary antibodies for 1 hour at room temperature: Cyanine 3 Goat anti-rat and Cyanine 5 Donkey anti-goat, respectively (Jackson Immunoresearch, 1:250). 4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain cell nuclei.
To evaluate cell apoptosis (day 3 post-MI), immunofluorescence analyses were performed using a TUNEL assay kit (Roche Diagnostis, Meylan, France) according to manufacturer’s instructions. The digital images of immunofluorescence were acquired with a Zeiss Axioimager Z2 Apotome. Six microscopic fields in both sides of peri-infarct area from each heart were examined using ImageJ64.

TTC staining. 2,3,5-Triphenyltetrazolium chloride was used to evaluate infarct size at early time points (3 days after MI). TTC staining identifies metabolically active tissue and is helpful to measure tissue viability. Hearts were removed and sectioned into 2 mm thick slices in a semi frozen state. The slices were then immersed 40 min into 1% TTC solution at 37 ºC. Once the color had been established, slices were fixed in 4% PAF overnight at room temperature. Slices were photographed and infarcted heart tissue areas were calculated using ImageJ64 blindly.

Ex vivo reflectance epifluorescence imaging. Mice were anaesthetized with isoflurane and received intravenously 150 µL of a fluorescent imaging probe MMPsense 680 (NEV 10126, PerkinElmer) 24 h before euthanasia. This agent is optically silent in its un-activated state and becomes highly fluorescent following activation by MMPs including MMP-2, -3, -9, and -13. Images of ischemic hearts were acquired using a fluorescence molecular imaging system (FMT 2500TM, VisEn Medical) 41.

Cell suspension preparation for Flow Cytometry. Mice were euthanized on days 1, 3, 5, 7, 14 and 21 post myocardial infarction. Peripheral blood was drawn via inferior vena cava puncture using a syringe primed with heparin solution. For blood staining, erythrocytes were lysed using BD FACS lysis solution (BD Biosciences). Spleens were surgically removed and dissociated obtaining a single cell suspension that was filtered through 40-m nylon mesh (BD Biosciences). Cell suspensions were centrifuged at 400g for 15 min at 4°C. Red blood cells were removed using a red blood lysis buffer (Sigma-Aldrich) and splenocytes were washed with PBS supplemented with 3% FBS. Left ventricle of infarcted and control mice were harvested, minced with fine scissors, digested using a solution of collagenase II and IV (both at 10mg/mL) for 30 min at 37°C. Upon digestion, tissue is gently passed through a 100-μm nylon mesh (BD Biosciences) followed by a 40-μm nylon mesh (BD Biosciences) and centrifuged at 400g for 15 min. Red blood cells were removed by incubation in red blood cell lysis buffer (Sigma-Aldrich) and single cell suspension were washed with PBS supplemented with 3% FBS.

Flow Cytometry. General characteristics of antibodies are summarized in the supplementary Table 1. Surface stainings were performed before permeabilization when intracellular labeling was necessary. Cells were incubated in Fixation/Permeabilization buffer (eBiosciences) 45 min, washed with permeabilization buffer (eBiosciences) and stained with the appropriate marker for intracellular staining. Forward scatter (FSC) and side scatter (SSC) parameters were used to gate live cells excluding red blood cells, debris, and cell aggregates in total splenocytes. Cells were analyzed BD LSR FORTESSA flow cytometer (BD Biosciences).

Quantitative real-time PCR. Quantitative real-time PCR was performed on a Step-one Plus (Applied Biosystems) qPCR machine. Gapdh was used to normalize gene expression. The primer sequences are described below :
Gapdh Forward 5ʹ-CGTCCCGTAGACAAAATGGTGAA-3ʹ, Reverse 5ʹ-GCCGTGAGTGGAGTCATACTGGAACA-3ʹ; Gzmb Forward 5ʹ-GTGCGGGGGACCCAAAGACCAAAC-3ʹ, Reverse: 5ʹ-GCACGTGGAGGTGAACCATCCTTATAT-3ʹ; Il1 Forward 5’-GAAGAGCCCATCCTCTGTGA-3’, Reverse 5’-GGGTRGTGCCGTCTTTCATTA-3’; Il6 Forward 5ʹ-TGACAACCACGGCCTTC CCTA-3ʹ, Reverse: 5ʹ-TCAGAATTGCCATTGCACAACTCTT-3’; Il10, Forward 5ʹ-ACTTCCCAGTCGGCCAGAGCCACAT-3ʹ, Reverse 5ʹ-GATGACAGCGCCTCAGCCGCATCCT-3ʹ; Tnf Forward 5’-GATGGGGGGCTTCCAGAACT-3’, Reverse 5’-GATGGGGGGCTTCCAGAACT-3’ ; Mmp9 Forward 5’-GCGTCATTCGCGTGGATAAGGAGT-3’, Reverse 5’-GTAGCCCACGTCGTCCACCTGGTT-3’; Gmcsf Forward 5’-TACGGGGCAATTTCACCAAACTC-3’, Reverse 5’-ATGAAATCCGCATAGGTGGTAACTTGT-3’; Ccl2 Forward 5’-ATGCTTCTGGGCCTGCTGTTCA-3’, Reverse 5’-GAGTGGGGCGTTAACTGCATCTG-3’; Mmp2 Forward 5’-CCGAGACCGCTATGTCCACTGT-3’, Reverse 5’-CCGGTCATCATCGTAGTTGGTTGT-3’; Gzma, Forward 5’-GTGCAGGGGACCTCCGTGGTGGAAAG-3’, Reverse 5’-GCCATCGGCGATCTCCACACTTCTCT-3’; Ncr1 Forward 5’-CAGCCTTGCACCTACCGACCCTAC-3’, Reverse 5’-GATCCCAGAAGGCGGAGTCCTTTT-3’; Csf2 Forward : 5' AACCTCCTGGATGACATGCCTG 3', Reverse : 5' AAATTGCCCCGTAGACCCTGCT 3'; Col1a1 Forward 5’-TGCCCTCCTGACGCATGGCCAAGA-3’, Reverse 5’-CCTCGGGTTTCCACGTCTCACCATT-3’; Col3a1 Forward 5’-CACCCCGAACTCAAGAGTGGAGAA-3’, Reverse 5’-ATGGGGCTGGCATTTATGCATGTT-3’

Single-cell RNA-sequencing (Mouse)
Gene expression patterns in NK cells were analyzed in previously published single-cell RNA-seq data of total CD45+ cells from control and infarcted C57BL6/J mouse hearts (day 5 after MI) from reference 11. Briefly, cells corresponding to T and NK cells as identified in 11 were extracted and reclustered in Seurat v4 42, with batch correction of separate scRNA-seq libraries performed using harmony 43. Clustering was performed using 30 principal components and a 0.6 resolution. NK cells were identified based on the expression of surface NK1.1 as measured by cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) 10.
C57BL/6J wild type mice underwent permanent coronary ligation and were injected i.p. with a depleting mouse anti-mouse NK1.1 monoclonal antibody (200microg/mouse, BioxCell BE0036 clone PK136) or with isotype 1 hour after coronary artery ligation. Five days after MI, the mice were injected i.v. with 2.5µg anti-CD45.2 APC (clone 104, Biolegend, cat. #109814) under isoflurane anesthesia, 5 minutes before being killed. The heart was perfused with PBS, excised, and the infarct, peri-infarct area, and adjacent viable myocardium collected, weighed and digested for 1 hour at 37°C under agitation in RPMI containing 450U/ml collagenase I (Sigma-Aldrich, #C0130), 125U/ml Collagenase XI (Sigma-Aldrich, C7657), 60U/ml Hyaluronidase (Sigma-Aldrich, H3506), 60U/ml DNAse (Roche #11284932001). After digestion, the cell suspensions were passed through a 70µm cell strainer and washed twice in MACS buffer (PBS+0.5% BSA+2mM EDTA) and incubated for 5 minutes on ice with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101320, 10µg/ml). Afterwards, cells were incubated with mouse CD45 microbeads (Miltenyi, 130-052-301, 1:10 final concentration), and anti-mouse CD45.2-AlexaFluor488. After 10 minutes incubation at 4°C, TotalSeq-A hashtag antibodies (Biolegend, see table for CITE-seq panels) were added as follows: hashtag 1 to 5 controls; hashtags 6 to 10: NK-depleted. Cells were incubated for an additional 15 minutes at 4°C, washed twice in MACS buffer, pooled, and magnetic selection was performed using LS Columns (Miltenyi (# 130-042-401) and MidiMACS separators (Miltenyi # 130-042-302). Sorted CD45+ cells were washed twice with PBS containing 0.04% BSA (Sigma-Aldrich A1595) and incubated for 25 minutes at 4°C in PBS/0.04% FCS containing Fixable Viability Dye eFluor™ 780 (ThermoFisher #65-0865-14, 1:1000) and anti-mouse TotalSeqA-Antibodies (see CITE-seq panels summary below). Cells were washed with PBS/0.04% FCS, resuspended and sorted using a BD FACS Aria III with a 100µm nozzle. Cells were resuspended, counted (final concentration 1,650 cells/µl) and loaded in the 10x Genomics Chromium using Chromium Next GEM Single Cell 3' High Throughput (HT) Reagent Kits v3.1.
Samples were prepared for CITE-Seq/Hashing according to the manual until the cDNA amplification step in which 1 μl (0.2 μM) ADT PCR additive primer to capture Antibody-derived tags (ADTs) and 1 μl (0.1 μM) HTO PCR additive primer to capture Hashtag oligos (HTOs) were added. After cDNA amplification 60 μl (0.6x) SPRI beads (Beckman Coulter) were added to separate the supernatant fraction that contains the ADT/HTO-derived cDNAs (<180bp) and the bead fraction that contains the mRNA-derived cDNAs (>300bp). The mRNA-derived cDNAs (bead fraction) were processed following the standard 10x Genomics protocol. The ADT/HTO-derived cDNAs (supernatant fraction) were purified twice with 2x SPRI. After the two purifications, half the amount of the eluted cDNAs were amplified for ADTs and half for hashtags. In the same reactions, the ADTs were indexed using TruSeq Small RNA primers and the hashtags using modified TruSeq DNA primers. The libraries were purified once more with 1.6x SPRI (160 μl). All libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNSA kit (Agilent). Sequencing was performed with S1 or S2 100bp flowcell with Novaseq 6000 platform (Illumina) and the reads for CITE-Seq/Hashing sample were allocated as follow: 5% for the hashtags, 10% for the ADTs and 85% for the mRNAs. 10x Genomics data, including HTO and ADT libraries, was demultiplexed using Cell Ranger software (version 7.0.1). Mouse GRCm38 (mm10) reference genome was used for the alignment and counting steps. To evaluate the expression of the cell surface proteins alongside the transcriptome level in our main data set, --feature-ref flag of the Cell Ranger software was used which creates a matrix that contains gene expression counts alongside the expression of cell surface proteins. The gene-barcode matrix obtained from Cell Ranger was further analyzed using R 4.5.1 and Seurat 5.3.0. 44. Clustering was performed using 20 principal components and a 0.4 resolution. Cells were identified based on their top10 DEG and classical markers. Using hashing identification, samples containing less than 2000 cells were excluded, further analysis were conducted with 6 samples in total (3 control and 3 depleted). Myeloid cells (Monocytes, macrophages and neutrophils) were extracted for further analysis. Pseudo bulk analysis were run for each cell population to identified DEG across control and depleted group with DESeq2. Among them, Cd36, Hdac9 and P2rx7 were identified, and their global expressions were assessed in Monocytes, Mac Selenop, Mac Spp1 and Mac resident, and presented with violin plots, where statistics (t-test) were calculated for each cell type between control and depleted group with SeuratExtend. Tnf expression was assessed in Monocytes, Mac Selenop, Mac Spp1 and Mac resident, Neutro SiglecF and Neutro Lcn2, and presented with violin plots, where statistics (t-test) were calculated for each cell type between control and depleted group with SeuratExtend. 45.

Transcriptomic analysis (Nanostring)
RNA extraction. Up to three 20-μm thick sections were collected from FFPE tissue blocks to obtain 100 ng of RNA required for analyses. RNA was isolate and extracted using the tissue RNEasy FFPE kit #73504 (Qiagen, Hilden, Germany). The concentration and quality of isolated RNA were assessed using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
RNA sequencing. Total RNA from each sample was hybridized with the nCounter® Human Banff Organ Transplant gene panel (B-HOT, NanoString Technologies, Seattle, WA, USA). This panel evaluates mRNA expression of 758 target genes and 12 selected housekeeping genes for data normalization. Internal quality reference for each assay was ensured by including a Panel Standard in each run: a pool of synthetic DNA oligonucleotides corresponding to the endogenous probes, allowing normalization of user, instrument, and lot-to-lot variations.
Quality control and data normalization. Raw counts of gene expression data were analyzed with the nSolver Analysis Software (version 4.0.70) and subjected to normalization using housekeeping genes. Background correction was applied, and the means of the supplied controls and housekeeping genes were used to normalize the measured expression values.
To evaluate the accuracy and quality of the gene expression input data, we applied quality control (QC) metrics (nSolver documentation) in all the samples included in the study. The QC assays refer to imaging, binding density, linearity of the positive controls, and limit of detection (LOD). If one or more of the four QC evaluations flagged an issue, the sample was removed from downstream analysis. Quantity and quality of isolated RNA were adequate in all cases and all the samples met the QC metrics.
All the samples passed the QC metrics which were defined as binding density > 0.05 and < 2.25 probes/µm2, % fields of view > 0.75, positive control linearity > 0.95, and sufficient assay efficiency. Per-sample LOD was defined as the mean of the counts of negative control probes for each sample and was ensured by using internal quality control reference probes. Raw gene expression data were normalized using the Remove Unwanted Variation (RUV) approach following quality control and housekeeping assessment.

Single-cell RNA-sequencing (Human)
As described in the original work 19, using single-nucleus RNA sequencing, we analyzed human heart data including thirty-one samples from twenty-three different individual hearts (Kuppe et al. 2022). The samples were taken from the necrotic area (ischaemic zone from 12 samples), the unaffected LV myocardium regions (remote zone from 6 samples), patients with acute myocardial infarction, and the human heart specimen from the late stage of myocardial infarction (fibrotic zone from 6 samples). These areas were identified based on discrete cardiac myocardial time points from the patients. There were 191 795 cells in all over 29 126 features in the data. We subtracted the data on natural killer cells because we were primarily interested in immune cells, and the result was 696 cells. Based on area, the data was further separated into 10, 30, and 100 days. We divided the patients into several time periods based on distinct zones of myocardial infarction in order to ascertain the function of NK cells in inflammation and cytotoxicity. 10, 30, and 100 days were the three distinct time periods into which the data was split comprising of 8, 1, and 2 patient samples, respectively. Next, we gathered the pathways linked to cytotoxicity or inflammation and used the AddModuleScore tool to get the scores for each cell. The scores for each pathway that displayed the gene expression trend linked with them were then obtained.

Immunostaining of human ischemic heart tissue. Normal heart tissue was obtained from Creative Bioarray company (United States). The pathological human cardiac tissue samples were collected in the IRB-approved CVMR biobank in our institution (IRB-approval CVMR-PRB HEGP authorization Comité de Protection des Personnes Ile de France 2 2016-13-09 MS2). All the patients gave informed consents for the surgical procedure. All the patients had left ventricle assist device (HeartMate II or HeartWare) implantation for severe cardiac failure. For implantation of the device the apex of the left ventricle was open and tissue samples were excised. Tissues were fixed in formalin and paraffin embedded. The tissue sections were stained with H&E for pathological diagnostic procedure allowing dating of myocardial infarction in addition to clinical dating. On remnant sections immunohistochemistry was performed with anti-NKp46 (clone 8E5B, Innate Pharma).

Statistical analysis. Values are expressed as mean ± s.e.m. Differences between values were examined using the non-parametric Mann-Whitney test or Kruskal-Wallis test and were considered significant at P<0.05. Shapiro-Wilk test was used for assessment of normality. All statistical tests were two-sided and performed using SAS software version 9.4.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Current data on the role of NK cells in myocardial ischemia are generally insufficient, and the prevailing hypothesis is that\nNK cells, early after myocardial ischemia, are detrimental due to their promotion of excessive inflammation and direct\ncytotoxic effects. Nevertheless, at later stages, NK cell activity appears to exert beneficial effects by facilitating the clearance\nof dead cells and promoting repair.\nThe study by Raphael Cohen et al. sheds new light by further corroborating the detrimental role of NK cells in the setting of\nacute ischemia in a mouse model of myocardial infarction induced by permanent coronary artery ligation. The authors also\npresent an analysis of human myocardial tissue that correlates with their findings in mice.\nUnfortunately, the study remains largely descriptive, despite employing several elegant in vivo approaches. It lacks a clear\nelucidation of the cellular mechanisms underlying the detrimental effects of NK cells in the acute phase of myocardial\ninfarction. Most of the experiments rely on monoclonal antibodies and site-specific recombinases, which are often treated as\nfoolproof tools but in reality require rigorous controls to ensure true specificity for the targeted cells.\nThe main weakness of this manuscript lies in the insufficient exploration of cellular and molecular mechanisms. The\ndetrimental role of NK cells is correlated to Granzyme B activity; however, this merely replicates findings previously reported\nby the same group a few years ago. One would instead expect NK cells to modulate the broader immune response within\nthe ischemic environment, necessitating analysis of other immune cells such as macrophages and monocytes, as suggested\nby the CCR2 experiments. Cell-to-cell immune interactions should therefore be systematically assessed. Additionally, the\nmechanisms through which NK cells influence infarct size remain unexplored. If the effect is mediated by cardiomyocyte\napoptosis, this should be directly demonstrated. Similarly, the impact of NK modulation on interstitial fibrosis should be\nclarified—does it involve direct interactions between NK cells and fibroblasts?\nAnother important limitation concerns the specificity of the monoclonal antibodies used to deplete NK cells. Specifically,\nNK1.1 is not exclusively expressed on NK cells. Invariant NKT (iNKT) cells—a special subset of T cells expressing TCRs\nand recognizing lipid antigens—also express NK1.1. Moreover, under inflammatory conditions, some activated T cells\n(especially CD8+ T cells) can transiently upregulate NK1.1, and a subset of ILC1s (type 1 innate lymphoid cells) also\nexpresses NK1.1. The CD8 depletion experiments are confusing, as the CD8 monoclonal antibody removes all CD8+ cells\nwithout discriminating between specific subsets.\nA further important issue is that while it is plausible that removing a pro-inflammatory and pro-fibrotic cellular response\nimproves myocardial remodeling, it remains unclear whether this is merely a secondary effect or whether it is associated\nwith a true switch toward a pro-reparative, pro-regenerative response. Does NK cell depletion promote cardiomyocyte\nrenewal and myocardial regeneration overall?\nFinally, the human tissue analysis is purely descriptive. While it is understandable that human tissues have intrinsic\nlimitations that make mechanistic studies difficult, there are human-based models, such as organoids, that could offer more\nsuitable platforms to study the underlying molecular and cellular mechanisms in human myocardial tissues.", "reviewer_2": "Acute myocardial infarction (AMI) often leads to rapid patient mortality; however, our understanding of its\nimmunopathological mechanisms is not as developed as in the field of tumor immunology. The manuscript's primary\ncontribution lies in expanding the research scope of NK cells in pathological diseases. The authors aim to demonstrate that\nNK cells play a destructive role in myocardial infarction (MI) tissue, directly leading to myocardial cell apoptosis and the\ngeneration of inflammatory myeloid cells. Despite this, the manuscript lacks rigor, which significantly limits its quality.\nMajor Comments:\nMechanism of NK cells vs CD8+ T cells in MI tissue: What is the difference in the mechanisms of NK cells and CD8+ T cells\nin MI tissue? Both are suggested to utilize GZMB as their key effector molecule. How do the authors exclude the roles of\nIFN-γ and TNF-α, especially in the induction of fibrosis?\nNK cell migration to infarct tissue: How do NK cells in circulation sense the infarcted tissue? CCR2 appears to be a\nnecessary but insufficient condition. More adhesion molecules should be considered. Additionally, can NK cells leave the\ninfarct tissue and home back to the bone marrow to release GM-CSF? Based on the current results, GM-CSF levels in serum\nappear minimal.\nNeutrophils in MI tissue: 70% of circulating human leukocytes are neutrophils. Why are they not considered as directly\ninvolved in the response to MI tissue, instead of relying on NK cells to release GM-CSF?\nTargeting NK cells in MI therapy: The authors suggest targeting NK cells in MI treatment. According to results from the\nmouse MI model, NK cell counts peak only in the early stages (day 7, reaching ~75 cells/mg), and significantly decrease to\nbaseline levels by day 14. However, heart function assessments are performed at days 21-28. Early MI is often difficult to\ndetect.\nOverall manuscript quality: The manuscript appears to be pieced together, and its rigor needs significant improvement.\nSuggestions for Improving Rigor:\nFigure 1C: Is \"inf\" an abbreviation for infarct? How was the infarct area identified in the fluorescence immunohistochemistry\nslices?\nFigure 1D: The \"p<.001\" is not standardized.\nFigure 1G-H: Are the sequencing results from the subgroups in Figure 1I? If so, this introduces a logical order confusion.\nAdditionally, for the non-myeloid single-cell transcriptomics, what molecular markers were used for sampling?\nFigure 1P: There is no figure legend. The staggered offset could be misleading, and a histogram\nwould be more suitable. More chemokines and adhesion molecules should be considered.\nFigure 2B: Is the positioning of \"INF\" incorrect?\nFigure 3K: The IgG control group is missing in the experimental design.\nFigures 2G, 3J: No figure legends.\nFigure 4A: Is this image reused?\nFigures 4B-D: The authors perform transcript-level detection. How should we interpret the differences between sequencing\nand PCR sequencing? How should \"Relative to Gapdh\" be understood? Does it represent the percentage of GAPDH\nexpression?\nFigure 4GJK: What is the biological significance of the y-axis? How is myocardial damage assessed? Are the white areas\nnecrotic? Why are they mainly concentrated in the vessel walls rather than the infarct area?\nNK cell manipulation terminology: There is confusion in the terminology used for NK cell manipulation (e.g., NK cell\ndepletion, ANTI-NK1.1, NK-KO, No-NK). The authors need to standardize and consistently use the correct terms.\nFigure 4M: Data is missing.\nFigures 4N-R: HL1 cells cannot be considered primary cardiomyocytes. The labeling of \"Cardiomyocyte apoptosis\"\nseems to be an over-interpretation. Additionally, how were NK cells pre-activated? Do pre-activated NK cells express GZMA? How\nwas CASP3/7 activation detected? Is it phosphorylated CASP? Why are the time points so densely arranged?\nFigure 5C: Is the y-axis correct?\nFigure 5D: Was the cell loading amount consistent for flow cytometry? \"Cellss\" is a clear grammatical error.\nFigure 5I: Is the mouse model used for MI?\nFigure 5M: How were NK cells pre-stimulated?\nFigure 6A: Were human NK cells isolated from the infarct tissue or peripheral blood? How were control samples obtained?\nFigure 6D: What markers were used for NK cell staining?\nFigure 6E: What is the biological significance of the \"Mean expression\" in the signaling pathway? How is it calculated?", "reviewer_3": "Comments to Authors:\nThe manuscript by Cohen et al., entitled “NK cells promote cardiac cell death and regulate myelopoiesis in myocardial\ninfarction”, investigates the role of NK cells in promoting apoptosis and impaired myocardial function. The authors\ndemonstrate that treatment with an anti-NKG2A monoclonal antibody exacerbates ischemic heart failure. While the study\naddresses an important question, the manuscript can be strengthened by addressing the following concerns:\n1. Figure 1: The authors demonstrate that increased infiltration of NK cells, notably CCR2+ NK cells, in the infarcted\nmyocardium compared to sham. However, it remains unclear which specific chemokines mediate the recruitment of NK cells\nto the MI region, especially considering that monocyte-derived macrophages also express high levels of CCR2. Are the\nsame chemokines responsible for recruiting CCR2-positive monocyte-derived macrophages to the infarct zone? Clarification\nis needed.\n2. Figures 1/2: The specificity of NK cell depletion must be addressed. Since CCR2 and CCR5 are expressed by other\nimmune cells, including macrophages. The use of a more specific NK cell marker, such as NKp46, would strengthen the\nstudy. This issue is evident in Figures 2b and 3d.\n3. Figure 2: It would be informative to evaluate how NK cell depletion influences other immune cell populations and\nchemokine expression in MI.\n4. As the authors are claiming that worse cardiac function and fibrosis are due to NK cell-mediated cytotoxicity, it’d be useful\nto demonstrate immune cell infiltration in the myocardium as well as increased cell death by TUNEL staining around day 3-\n7d following LAD ligation when the NK cells/mg heart tissue are at peak.\n5. Can the authors discuss whether the interaction between NK cells and cardiomyocytes is direct or mediated via other cell\ntypes?\n6. Figure 4: the immunostaining for GZMB shows significant GZMB even in NK depleted tissues. An explanation would be\nrequired. Additionally, details regarding co-culture conditions would be needed. Were the cells separated by inset/outlet or\ndid they have any direct contacts? Since HL1 cells are proliferating\ncardiomyocytes, they may not be an ideal model for cardiomyocyte cell death, which should be at least acknowledged.\n7. Could the authors provide insight into whether human NK cells are likely to exhibit similar behavior in the context of MI?\nRegarding Figure 6, how did the authors differentiate active vs mature vs terminal NK? Also, I am having a very difficult time\nunderstanding what Figure 6e is trying to convey.\nMinor comments\n· Figure labeling: Ensure consistent formatting (e.g., Figure 1G – capital vs. lowercase letters).\n· Page 6, Line 128: Revise “We then we investigated” to “We then investigated.”", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to reviewers\nReviewer #1 (Remarks to the Author):\nCurrent data on the role of NK cells in myocardial ischemia are generally insufficient, and the\nprevailing hypothesis is that NK cells, early after myocardial ischemia, are detrimental due to\ntheir promotion of excessive inflammation and direct cytotoxic effects. Nevertheless, at later\nstages, NK cell activity appears to exert beneficial effects by facilitating the clearance of dead\ncells and promoting repair.\nResponse: We fully concur with the Reviewer that the role of NK cells has been\ninsufficiently investigasted. To the best of our knowledge, “the hypothesis that NK cells,\nearly after myocardial ischemia, are detrimental“ has only been mentioned sporadically\nin general reviews addressing immune responses in MI, and, so far, has not been directly\nsupported by experimental evidence, as demonstrated in our study.\nWe also appreciate the Reviewer’s reference to the potential beneficial effects of NK cells\nat later stages, which we presume relates to the study by Ayach et al. (Proc Natl Acad Sci\nU S A. 2006 Feb 14;103(7):2304-9). We would like to respectufully point out that this study\nused a rabbit anti-mouse polyclonal NK blocker from Cedarlane Laboratory, which is an\nanti-asialo GM1 antibody that is known to lack specificity for NK cells. Our laboratory\nhas previously shown that anti-asialo GM1 depletes not only NK cells but also NKT cells\nand CD8+ T cells (see Table 1 in Nour-Eldine et al. Circ Res. 2018 Jan 5;122(1):47-57).\nTherefore, the conclusions from that study should, in our view, be interpreted with\ncaution, given the non-specific nature of the depletion strategy employed.\nUnfortunately, the study remains largely descriptive, despite employing several elegant in vivo\napproaches. It lacks a clear elucidation of the cellular mechanisms underlying the detrimental\neffects of NK cells in the acute phase of myocardial infarction. Most of the experiments rely on\nmonoclonal antibodies and site-specific recombinases, which are often treated as\nfoolproof tools but in reality require rigorous controls to ensure true specificity for the targeted cells.\nResponse: We would like to respectfully disagree with the reviewer’s comment. In our\nstudy, we employed multiple, complementary loss- and gain-of-function approaches to\ninvestigate, in a mechanistic manner, the critical role of NK cells in the context of MI.\nSpecifically, we used genetically modified mouse models as well as pharmacological\nmodulators in immunocompetent animals. Acknowledging the inherent limitations\nassociated with each individual model, we intentionally combined several complementary\nexperimental strategies to enhance the robustness and reliability of our findings.\nThe main weakness of this manuscript lies in the insufficient exploration of cellular and\nmolecular mechanisms. The detrimental role of NK cells is correlated to Granzyme B activity;\nhowever, this merely replicates findings previously reported by the same group a few years\nago.\nResponse: We agree with the Reviewer that our group has previously demonstrated the\ndeleterious effect of CD8+-derived Granzyme B on post-MI cardiac remodeling. In the\npresent study, we further show that granzyme B derived from NK cells also plays a\nmajor role in this process. However, the Reviewer suggests that we have insufficiently\nexplored the cellular and molecular mechanisms involved. We respectfully disagree with\nthis assessment, as our findings provide substantial evidence that:\n1. NK cells contribute to deleterious post-ischemic cardiac remodeling independently of\nCD8+ T cells;\n2. NK cells promote cardiomyocyte cell death through the release of granzyme B;\n3. NK cells regulate myeloid cell trafficking by releasing GM-CSF within the bone\nmarrow;\n4. Mature cytotoxic NK cells are recruited into the myocardium post-MI, in part via\nCCR2; and\n5. NK cells promote a pro-inflammatory phenotype in macrophages.\nImportantly, Figures 3K-O specifically illustrate the pathogenic role of NK cells in the\nabsence of CD8+ T cell activity, thereby supporting the conclusion that NK cell function\nin this context is independent of CD8+ T cells. Taken together, we believe that our study\nprovides a novel mechanistic insight into how NK cells contribute to post-MI cardiac\nremodeling,a process that, to our knowledge, has not been previously characterized.\nOne would instead expect NK cells to modulate the broader immune response within the\nischemic environment, necessitating analysis of other immune cells such as macrophages and\nmonocytes, as suggested by the CCR2 experiments. Cell-to-cell immune interactions should\ntherefore be systematically assessed.\nResponse : In Figure 4, we examined the impact of NK cell depletion or activation on local\ninflammatory responses following MI. In NK cell-depleted mice, mRNA levels of Il-1b\nand Tnfa were significantly reduced in infarcted hearts compared to controls (Figure 5a),\nalong with a marked decrease in metalloproteinase activity (Figure 5b-c). NK cell\ndepletion also led to reduced monocyte recruitment in the heart at days 5 and 7 post-MI\n(Figure 5d; Extended Data Fig.17 and 18a-b) and a lower proportion of CCR2+\nmacrophages at day 7 (Extended Data Fig.18c).\nConversely, NK cell activation using an anti-NKG2A mAb promoted a shift towards a\nmore pro-inflammatory cardiac environment. This was characterized by elevated Il-1b\nand Il-6 mRNA levels, decreased Il-10 mRNA levels (Figure 5e), and increased local\nmetalloproteinase activity (Figure 5f-g). Anti-NKG2A-mediated NK activation also\nenhanced the recruitment of neutrophils and monocytes in the heart at day 5 post-MI\n(Figure 5h; Extended Data Fig.18f-g) and increased the number of CCR2+ macrophages\nat day 7 (Extended Data Fig.18h).\nTaken together, these data provide a comprehensive analysis of the local inflammatory\nresponse modulated by NK cell activity. In response to the reviewer’s concern regarding\ncell-cell interactions, we performed scRNA-seq analysis on CD45+ cells isolated from\nischemic heart tissue to further elucidate the cellular dynamics and interactions involved.\nAs shown in the new supplemental figure 19, pseudobulk analysis revealed differentially\nexpressed genes in monocyte and macrophage populations following NK cell depletion,\nand their expression was assessed across total cells per group. Notably, Cd36 and Hdac9\nwere upregulated, whereas P2rx7, which is involved in inflammasome activation, was\ndownregulated in the NK-depleted group (Extended Data Fig.19e). Moreover, Tnf\nexpression was reduced in the main populations of monocytes, macrophages and\nneutrophils in the depleted group (Extended Data Fig.19f). Altogether, these results\nindicate that NK cell depletion promotes a shift in macrophage profile toward a less\ninflammatory phenotype, highlighting the significant role of NK cells in modulating post-\nMI local immune responses.\nAdditionally, the mechanisms through which NK cells influence infarct size remain unexplored.\nIf the effect is mediated by cardiomyocyte apoptosis, this should be directly demonstrated.\nResponse: We have substantial experience in quantifying apoptosis in cardiac tissue in\nthe context of MI. In our view, TTC staining remains the most comprehensive and\nintegrative method for evaluating tissue necrosis in the heart. This approach is widely\naccepted and commonly used in the field to assess infarct size and myocardial injury (e.g.\nYang et al. Circulation 2006; Leuschner et al. Circ Res 2010; Santos-Zas et al. Nat Comm\n2021).\nTo directly address the reviewer’s concern, we conducting additional TUNEL staining\nexperiments in two relevant models: NK cell depletion (using NK1.1 mAb) and NK cell\nactivation (Anti-NKG2A). As shown in the new supplemental figure 12, we found that NK\ncell depletion reduced TUNEL+ cells in the ishemic heart when compared to isotype\ntreated mice, whereas anti-NKG2A mediated NK cell activation increased TUNEL + cells\nin the ischemic heart. These findings are fully consistent with the results from TTC\nstaining further supporting the role of NK cells in modulating cardiomyocyte death\nfollowing MI.\nSimilarly, the impact of NK modulation on interstitial fibrosis should be clarified—does it\ninvolve direct interactions between NK cells and fibroblasts ?\nResponse: In our models, interstitial fibrosis appears to follow the extent of infarct size\nand associated tissue necrosis. Specifically, we observed reduced fibrosis in conditions of\nNK cell depletion, where infarct size was smaller, and increased fibrosis following NK cell\nactivation, which led to larger infarcts. Moreover, we found that modulation of the infarct\nsize by NK cells also influenced the inflammatory signature in heart tissue, particularly\nthe expression of key cytokines such as IL-1 and TNF-, which are well-established\ndrivers of fibrotic remodeling. Notably, a recent study published in Nature (PMID:\n39443792) demonstrated that IL-1 plays a direct role in regulating fibroblast activity in\nthe heart.\nWe agree that potential direct interactions between NK cells and fibroblasts in the setting\nof MI are of interest are of great interest. While exploring this question is beyond the\nscope of the current study, we hope the Reviwer will appreciate our focus on other\nmechanistic aspects. Importantly, we have highlighted this point in the discussion section\nof the revised manuscript as a promising avenue for future research.\nAnother important limitation concerns the specificity of the monoclonal antibodies used to\ndeplete NK cells. Specifically, NK1.1 is not exclusively expressed on NK cells. Invariant NKT\n(iNKT) cells—a special subset of T cells expressing TCRs and recognizing lipid antigens—\nalso express NK1.1. Moreover, under inflammatory conditions, some activated T cells\n(especially CD8+ T cells) can transiently upregulate NK1.1, and a subset of ILC1s (type 1\ninnate lymphoid cells) also expresses NK1.1.\nResponse: We appreciate the Reviewer’s comment regarding NKT cells. While NKT cells\ndo express NK1.1, this population is minimal in the context of murine MI, approximately\n10-fold lower than NK cells, as shown in Extended Data Fig.2. Importantly, anti-NK1.1\nmAb treatment did not affect the CD8+ T population, and these data are now included as\na new Supplemental figure 7d in the revised manuscript.\nRegarding ILC1s, our scRNA-seq analysis revealed that this population is extremely rare\nand virtually undetectable in heart tissue under ischemic conditions, suggesting that they\nare unlikely to contribute significantly to the observed phenotype.\nAs noted in the manuscript, we are fully aware that anti-NK1.1 is not entirely specific for\nNK cells. To address this limitation and minimize potential confounding factors, we\nemployed multiple complementary approaches. Specifically, we used Nkp46iCre+/-\nR26RDTA mice, which allow for specific depletion of NK cells, and further confirmed NK\ncell-specific effects through repopulation experiments with purified NK cells. These\ncomplementary strategies reinforce the specificity and validity of our conclusions\nregarding NK cell function in post-ischemic cardiac remodeling.\nThe CD8 depletion experiments are confusing, as the CD8 monoclonal antibody removes all\nCD8+ cells without discriminating between specific subsets.\nResponse: In the context of MI, our scRNA-seq analysis at day 5 post-MI showed that\nCD8 expression was restricted to CD3+ CD8+ T cells and a very small subset of dendritic\ncells. Importantly, we did not detect significant CD8α expression in other cardiac immune\ncell populations at this stage. Based on these observations, we consider our anti-CD8\ndepleting strategy to be both valid and appropriately specific for targeting CD8+ T cells\nin this experimental context.\nA further important issue is that while it is plausible that removing a pro-inflammatory and\npro-fibrotic cellular response improves myocardial remodeling, it remains unclear whether\nthis is merely a secondary effect or whether it is associated with a true switch toward a pro-\nreparative, pro-regenerative response. Does NK cell depletion promote cardiomyocyte\nrenewal and myocardial regeneration overall ?\nResponse: We appreciate the Reviewer’s comment regarding the potential role of NK cells\nin cardiac regenaration. While this is indeed an intriguing area of research, it falls outside\nthe scope of the current study, which was focused specifically on the contribution of NK\ncells to acute injury and adverse post-schemic remodeling. Furthermore, the hypothesis\nthat NK cells may contribute to heart repair following MI is primarily based on the study\nby Ayach et al. (PNAS 2006), which should be interpreted with caution due to the non-\nspecific depletion strategy employed, as discussed above.\nFinally, the human tissue analysis is purely descriptive. While it is understandable that human\ntissues have intrinsic limitations that make mechanistic studies difficult, there are human-based\nmodels, such as organoids, that could offer more suitable platforms to study the underlying\nmolecular and cellular mechanisms in human myocardial tissues.\nResponse: We thank the Reviewer for raising the important point regarding the human\nrelevance of our findings. We were fortunate to access rare human heart tissue samples\nfrom MI patients, which allowed us to identify and characterize the local NK cell\npopulation. Using spatial transcriptomic analysis, we were able to describe both the\nphenotype and spatial distribution of NK cell subsets over time across distinct cardiac\nregions.\nWhile cardiac organoids may become a valuable tool in the future, they are not fully\nestablished for studying human cardiac pathophysiology, particularly with respect to\nimmune cell-tissue interactions. Current organoid systems do not recapitulate the\ncomplex in vivo interactions between hematopoietic cells and cardiac tissue, which are\nessential for investigating the role of NK cells during myocardial infarction.\nTo further address this point, we we co-cultured human 3D cardiac ring organoids with\npurified human NK cells and measured contractility parameters before and 12 hours after\nco-culture (new Figure 4r & Extended Data Fig.15a). Non-activated NK cells had no effect\non cardiac organoid function, whereas activated NK cells impaired both contractility and\nrelaxation in a dose-dependent manner (new Figure 4s & Extended Data Fig.15b),\nproviding experimental support for the relevance of our findings in the human setting.\nReviewer #2 (Remarks to the Author):\nAcute myocardial infarction (AMI) often leads to rapid patient mortality; however, our\nunderstanding of its immunopathological mechanisms is not as developed as in the field of\ntumor immunology. The manuscript's primary contribution lies in expanding the research scope\nof NK cells in pathological diseases. The authors aim to demonstrate that NK cells play a\ndestructive role in myocardial infarction (MI) tissue, directly leading to myocardial cell\napoptosis and the generation of inflammatory myeloid cells. Despite this, the manuscript lacks\nrigor, which significantly limits its quality.\nMajor Comments:\nMechanism of NK cells vs CD8+ T cells in MI tissue: What is the difference in the mechanisms\nof NK cells and CD8+ T cells in MI tissue? Both are suggested to utilize GZMB as their key\neffector molecule.\nResponse: As shown in Supplemental figure 2, the kinetics of CD8+T cell and NK cell\nrecruitment differ in the context of acute MI. CD8+ T/NK ratio was >1 at day 1 but\nbecame <1 at days 3-5-7. Both cell types exhibit cytotoxic activity via Granzyme B\nproduction. In addition, in this study we found that NK cells modulate myelopoiesis\nthrough the production of GM-CSF.\nHow do the authors exclude the roles of IFN-γ and TNF-α, especially in the induction of fibrosis\n?\nResponse: We thank the reviewer for his/her insightful comment regarding cytokines and\nfibrosis, which have been further elaborated in the Discussion section.\nIn our work, fibrosis closely followed infarct size and the extent of myocardial necrosis.\nIn conditions where infarct size was reduced (i.e. following NK depletion), fibrosis was\nalso decreased. Converseley, in settings where infarct size was exacerbated (i.e. after NK\nactivation), we observed an increase in fibrosis. In addition, we found that the modulation\nof infarct size correlated with the inflammatory profile in cardiac tissue, particularly IL-\n1 expression. Specifically, Il1b mRNA levels were decreased in the NK depletion model\nand increased in the NK activation model. These findings are in line with a recent study\npublished in Nature (PMID: 39443792), which demonstrated that IL-1 regulates\nfibroblast activity in the heart. Interestingly, although fibrosis was elevated in the NK cell\nactivation experiment, we did not observe any significant change in Tnfa mRNA\nexpression (figure 5e) suggesting that TNF- may not play a central role in fibrosis in our\nexperimental models.\nTo adress the reviewer’s comment regarding the role of IFN-γ, we have quantified T-bet\nmRNA levels in both the NK depletion and NK activation experiments, Tbet being a key\nregulator of IFN-γ production (Huang et al Front Immunol 2021). As shown in the new\nextended data figure 16, in experiments with NK cell activation (anti-NKG2A), we did not\nobserve any change in Tbet gene expression between isotype treated and anti-NKG2A-\ntreated mice. Given that fibrosis was more pronounced in the heart of anti-NKG2A-\ntreated mice, these results suggest that IFN-γ may not play a central role in fibrosis in our\nexperimental models. These additional results have been added in the discussion section\nof the revised manuscript.\nNK cell migration to infarct tissue: How do NK cells in circulation sense the infarcted tissue?\nCCR2 appears to be a necessary but insufficient condition. More adhesion molecules should\nbe considered.\nResponse: As shown in Supplemental fig.4, we used scRNA-seq to analyze the expression\nof 22 chemokine receptors in infiltrating leukocytes. We found that, in the context of MI,\ninfiltrating NK cells exclusively expressed CCR2, CCR5 and CXCR4. This observation\nled us to specifically investigate the roles of CCR2 and CCR5 in NK cell recruitment,\nusing KO mouse models. We found that Ccr2 global deletion led to reduced NK cell\ninfiltration in the ischemic heart at day 3. Given that global CCR2 deletion impacts on\nseveral immune population, we performed an additional series of experiments to support\nthe critical role of CCR2 for NK cell recruitment in the ischemic heart tissue. We\nrepopulated Nkp46iCre+/- R26RDTA (referred to as NKKO) mice with purified Wild-type\nor Ccr2-/- NK cells 3 days before MI (new Figure 1O). NK deficiency in NKKO mice was\nvalidated by flow cytometry (Extended Data Fig.5j). As shown in figure 1P, at day 3 post-\nMI, NK cell repopulation in the blood was similar between groups. However, NK cell\nnumbers were significant lower in the heart of MI-operated mice repopulated with Ccr2-\n/- NK cells compared to those repopulated with Ccr2+/+ NK cells(New Figure 1QR).\nFinally, additional transcriptomic analyses performed on human tissus from MI patients\nconfirmed that infiltrating NK cells express CCR2 (new Extended Data Fig.24)\nRegarding the reviewer’s concern about adhesion molecules, we described in the original\nversion of the manuscript that a subset of mature NK cells, named NK1, which are\nrecruited during the first days post-MI express high levels of CD11b (Itgam). CD11b an\nimportant member of the β2-integrin subunit, which affects leukocyte migration and\ntissue infiltration. Additional analyses revealed that this N1 subset also expressed higher\nlevels of CD49d (New Extended Data Fig 4).\nTo further address the reviewer’s concern, we quantified Ccl2, Ccl3, Cxcl1 and Cxcl2\nmRNA levels in the ischemic heart of controls NK-depleted (Anti-NK1.1) and NK-\nactivated (Anti-NKG2A) groups. As shown in the new supplemental figure 16, NK\ndepletion or NK activation had no effect on Ccl2, Ccl3, Cxcl1 and Cxcl2 mRNA levels.\nThese new results have been added in the revised version of the manuscript.\nAdditionally, can NK cells leave the infarct tissue and home back to the bone marrow to\nrelease GM-CSF? Based on the current results, GM-CSF levels in serum appear minimal.\nResponse: We would like to thank the reviewer for this insightful question. Several\nhypothesis could be proposed to explain how NK cells modulate myelopoiesis. One\npossibility is NK cell recirculation from the ischemic heart to the BM (as suggested by the\nreviewer). Alternatively, NK cells may be activated directly within the BM by circulating\nfactors. Parabiosis experiments have demonstrated that factors released during MI can\nmodulate hematopoiesis (Cremer et al. JACC 2020). Other studies have proposed that\nDAMPs and/or cardiomyocyte-derived vesicles may also be involved. While we\nacknowledge these potential mechanisms, we believe that a thorough investigation of this\nquestion would require a separate, dedicated study.\nDetecting and quantifying GM-CSF protein remains technically challenging. In our study,\nwe observed that NK cells produce GM-CSF in the context of MI, based on protein\ndetection in the blood and mRNA expression in the BM. More importantly, we\ninvestigated the functional relevance of NK cell-derived GM-CSF using Nkp46Cre+/-\nCsf2lox/lox mice. Our results indicate that the selective deletion of GM-CSF in NK cells\nalters myelopoiesis and affects post-ischemic cardiac remodeling (figure 5). This in vivo\nexperiment provides direct causal evidence supporting the role of NK cell-derived GM-\nCSF in the regulation of myelopoiesis.\nNeutrophils in MI tissue: 70% of circulating human leukocytes are neutrophils. Why are they\nnot considered as directly involved in the response to MI tissue, instead of relying on NK\ncells to release GM-CSF?\nResponse: In our study, we did not rule out a potential role for neutrophils in post-MI\nremodeling. Rather, we identified that NK cells contribute, at least in part, to the\nmodulation of myelopoiesis. Specifically, NK cell depletion led to a reduction in monocyte\ncontent within the ischemic heart, whereas NK cell activation increased both monocyte\nand neutrophil infiltration into cardiac tissue. Further analysis revealed that NK cells in\nthe bone marrow produce GM-CSF, a key driver of myelopoiesis in the context of acute\nMI, but not in sham-operated mice (a new Supplemental figure 20 has been added to\nillustrate this point). Importantly, specific deletion of GM-CSF in NK cells led to reduced\nmyelopoiesis and attenuated adverse post-ischemic cardiac remodeling.\nTaken together, our data demonstrate for the first time that NK cells promopte adverse\ncardiac remodeling through two complementary mechanisms:\n1. Directly, by modulating cardiomyocyte apoptosis; and\n2. Indirectly, by influencing myelopoiesis.\nTargeting NK cells in MI therapy: The authors suggest targeting NK cells in MI treatment.\nAccording to results from the mouse MI model, NK cell counts peak only in the early stages\n(day 7, reaching ~75 cells/mg), and significantly decrease to baseline levels by day 14.\nHowever, heart function assessments are performed at days 21-28. Early MI is often difficult to\ndetect.\nResponse: We found the reviewer’s comment somewhat difficult to interpret. In our\nstudy, we demonstrated that NK cells are actively recruited into cardiac tissue following\nMI, with a peak infiltration occurring between days 5 and 7. Once in the heart, NK cells\nexacerbate local tissue damage. This early increase in cardiomyocyte death contributes to\nmore severe cardiac dysfunction at later stages of post-MI remodeling.\nFrom a therapeutic perspective, these findings suggest that targeting NK cells during the\nacute phase of MI could be beneficial. Specifically, mAb-mediated NK cell depletion\ninitiated around the time of PCI and maintained during the first 10 days post-MI might\noffer a clinically relevant window. In humans, the onset of MI is typically easy to identify\ndue to the presence of chest pain, and recent studies report that the average time between\nsymptom onset and PCI is approximately 2 hours (ex :PMID: 39211964). We have\nclarified this point further in the revised Discussion section.\nSuggestions for Improving Rigor:\nFigure 1C: Is \"inf\" an abbreviation for infarct? How was the infarct area identified in the\nfluorescence immunohistochemistry slices?\nResponse: Immunofluorescent staining was performed to identify NKp46+ NK cells. The\ninfarct zone was delineated based on tissue architecture, including alterations in nucleus\nmorphology and the presence of immune cell infiltration.\nFigure 1D: The \"p<.001\" is not standardized.\nResponse : The manuscript has been be revised accordingly to incorporate this\nmodification.\nFigure 1G-H: Are the sequencing results from the subgroups in Figure 1I? If so, this introduces\na logical order confusion. Additionally, for the non-myeloid single-cell transcriptomics, what\nmolecular markers were used for sampling?\nResponse : The figure has been adjusted accordingly. The scRNA-seq data originates\nfrom a previously published paper, where the identification of immune cell lineages in the\ncardiac immune infiltrate is described in detail [PMID: 35950218]. Briefly, immune cell\nlineages were defined based on the expression of canonical marker genes and surface\nmarkers (measured by CITE-seq) of mouse immune cells. In these data, a population of\ncells corresponding to T cells, NK cells and also comprising other lymphoid subsets (e.g.\nILC2) formed a distinct cluster expressing markers of T and NK cells at the protein (e.g.\nCD3, NK1.1) and transcriptomic (e.g. Tbrc1, Nkg7) level, and devoid of myeloid cell (e.g.\nLy6G, F4/80) or B cell (CD19) markers. This subset of cells was here extracted and\nreanalyzed with higher granularity to reveal subsets of T and NK cells.\nTo address the review ‘s concern, the reference has been added in the manuscript.\nFigure 1P: There is no figure legend. The staggered offset could be misleading, and a histogram\nwould be more suitable. More chemokines and adhesion molecules should be considered.\nResponse: The figure has been be replaced by a histogram for greater clarity.\nAs shown in Supplemental figure 5, we analyzed the expression of 22 chemokine receptors\nin heart tissue at day 5 post-MI using scRNA-seq. We found that infiltrating NK cells\nexpressed only CCR2, CCR5 and CXCR4 mRNA. To assess the functional relevance of\nthese receptors, we performed experiments using CCR2 and CCR5 KO mice. Our data\nindicate that CCR2 contributes, at least in part, to NK cell recruitment into the heart.\nUnfortunately, CXCR4 KO mice cannot be used in this context, as CXCR4 is a critical\nregulator of global hematopoiesis and its global deletion is embryonically lethal.\nGiven that global CCR2 deletion impacts on several immune population and to address\nthe reviewer’s concern, we performed an additional series of experiments to support the\ncritical role of CCR2 for NK cell recruitment in the ischemic heart tissue. We\nrepopulated Nkp46iCre+/- R26RDTA (referred to as NKKO) mice with purified Wild-type\nor Ccr2-/- NK cells 3 days before MI (new Figure 1O). NK deficiency in NKKO mice was\nvalidated by flow cytometry (Extended Data Fig.5j). As shown in figure 1P, at day 3 after MI,\nNK cell repopulation in the blood was similar between groups. However, NK cell number was\nsignificant lower in the heart of MI-operated mice repopulated with Ccr2-/- NK cells\ncompared to animals repopulatd with Ccr2+/+ NK cells(New Figure 1QR).\nTo further address the reviewer’s concern, we quantified Ccl2, Ccl3, Cxcl1 and Cxcl2\nmRNA levels in the ischemic heart of controls NK depleted (Anti-NK1.1) and NK\nactivated (Anti-NKG2A) groups. As depicted in the new supplemental figure 16, NK\ndepletion or NK activation had no effect on Ccl2, Ccl3, Cxcl1 and Cxcl2 mRNA\nlevels.These new results have been added in the revised version of the manuscript.\nFigure 2B: Is the positioning of \"INF\" incorrect?\nResponse: Thank you for this comment. The position of “INF” has been modified\naccordingly.\nFigure 3K: The IgG control group is missing in the experimental design.\nResponse: With all due respect, we disagree with the reviewer’s concern. The objective of\nthis experiment was to specifically assess the impact of NK cell activation in the absence\nof CD8+ T cells. Both experimental groups treated with an anti-CD8 depleting antibody\nto ensure effective CD8+ T cell depletion, which was confirmed by flow cytometry. The\nNK cell activated group additionally received an anti-NKG2A antibody, while the control\ngroup received an isotype control. Our data show that NK cell activation exerts a\npathogenic effect in the absence of CD8+T cells.\nIncluding an IgG control group would not address the specific question under\ninvestigation and would therefore not provide additional relevant information in this\ncontext.\nFigures 2G, 3J: No figure legends.\nResponse: We apologize for the oversight. The figure legends have been updated\naccordingly.\nFigure 4A: Is this image reused ?\nResponse: Indeed, this is the same UMAP visualization of scRNA-seq profiles from non-\nmyeloid immune cells in figure 1i. This visualization is intended to assist readers in\nanalyzing the results of Figure 4B results, specifically regarding the gene expression of\nPerforin, Granzyme A and Granzyme B.\nFigures 4B-D: The authors perform transcript-level detection. How should we interpret the\ndifferences between sequencing and PCR sequencing? How should \"Relative to Gapdh\" be\nunderstood? Does it represent the percentage of GAPDH expression?\nResponse: ScRNA-seq (Fig 4B) enables the detection and quantification of transcripts in\ndifferent cell populations. In figure 4CD, the quantification of mRNA levels in ischemic\nheart tissue was performed using qPCR at various time points post-MI. mRNA levels were\nnormalized to Gapdh expression, with Gapdh serving as an internal control for gene\nexpression.\nFigure 4GJK What is the biological significance of the y-axis? How is myocardial damage\nassessed? Are the white areas necrotic? Why are they mainly concentrated in the vessel walls\nthan the infarct area?\nResponse: As detailed in the Methods section and figure legends, 2,3,5-\nTriphenyltetrazolium chloride (TTC) staining was used to evaluate the infarct size at\nearly time points (3 days after MI). TTC staining is a well-established method for\nidentifying metabolically active tissue and is widely used to assess tissue viability (see Yang\nCirculation 2006; Leuschner et al. Circ Res 2010; Santos-Zas et al Nat Comm 2021).\nNecrotic tissue appears white after staining.\nWe are uncertain about the reviewer’s comment regarding the vessel wall, as figures 4G,\nJ and K clearly show left ventricle sections taken 3 days after MI.\nTo further support the pro-apoptotic role of NK cells, we quantified TUNEL+ cells in the\nischemic heart at day 3 in CTR, NK depleted (anti-NK1.1) and NK activated (anti-\nNKG2A) treated mice. We found that NK depletion led to decreased number of TUNEL+\ncells in the heart, whereas NK activation increased TUNEL+ cell numbers (See new\nsupplemental fig12).\nNK cell manipulation terminology: There is confusion in the terminology used for NK cell\nmanipulation (e.g., NK cell depletion, ANTI-NK1.1, NK-KO, No-NK). The authors need to\nstandardize and consistently use the correct terms.\nResponse : We apologize for the oversight. The terminology has been standardized\naccordingly.\nFigure 4M: Data is missing.\nResponse: This mistake occurred during the PDF conversion of the PPT figure. It has\nbeen corrected accordingly.\nFigures 4N-R: HL1 cells cannot be considered primary cardiomyocytes. The labeling of\n\"Cardiomyocyte apoptosis\" seems to be an over-interpretation. Additionally, how were NK\ncells pre-activated? Do pre-activated NK cells express GZMA? How was CASP3/7 activation\ndetected? Is it phosphorylated CASP? Why are the time points so densely arranged?\nResponse: The figure legends have been modified accordingly: cardiomyocytes (HL1)\napoptosis.\nNK cells were pre-activated overnight with cytokines (rIL-15 at 5 ng/ml), then washed\nand co-cultured with HL1 cardiomyocytes. This procedure is described in the Methods\nsection.\nIn our study, we did not investigate the role of Granzyme A in NK cell cytotoxic activity\nin vitro, as we found that NK cell depletion did not affect Gzma mRNA levels in ischemic\nheart tissue (see figure 4c).\nActivated and non-activated NK cells were co-cultured with HL1 cardiomyocytes in\ndifferent ratios (1:1, 1:5 and 1:10, based on cardiomyocyte count). After 24h of co-culture,\nNK cells were removed and HL1 cardiomyocytes were labeled with 5 M Caspase-3/7\nGreen Apoptosis Reagent (Incucyte, Welwyn Garden, United Kingdom), which was\nadded to the growth medium. After a 30 min incubation at 37°C in the dark, apoptosis\nwas evaluated in real time, using a Nikon Eclipse Ti microscope. Time-lapse images were\ncaptured at 10-min intervals. The IncuCyte Caspase-3/7 Green Apoptosis Assay is a live-\ncell imaging system that enables real-time, quantitative assessment of apoptosis in cell\ncultures. This assay uses a fluorogenic substrate that becomes fluorescent upon cleavage\nby caspase-3/7, key executioners of apoptosis.\nFigure 5C: Is the y-axis correct?\nResponse: Sorry, this was an error, and the y-axis has been corrected to « fluorescent\nintensity » accordingly.\nFigure 5D: Was the cell loading amount consistent for flow cytometry? \"Cellss\" is a clear\ngrammatical error.\nResponse: The grammatical error has been corrected accordingly. Flow cytometry\nanalysis after heart tissue digestion is routinely performed in our laboratory and others.\nWe are able to detect 5.000 to 50.000 CD45+ cells per flow cytometry sample, depending\non the timepoint after MI induction. This yields cells roughly 500 to 10.000 CD45+ cells\nper mg of cardiac tissue. Those numbers are numbers consistent with the literature\n(PMID: 30586758, 32901075, 35950218)\nFigure 5I: Is the mouse model used for MI?\nResponse : Indeed, analysis has been done at day 3 post-MI. This point has been clarified\nin the legends\nFigure 5M: How were NK cells pre-stimulated?\nResponse: As previously described by Louis et al. (PMID 32097462) who investigated the\nrole of GM-CSF producing NK cells in experimental arthritis, NK cells were pre-\nstimulated with rIL-12 (50 microg/ml), rIL-15 (10 ng/ml) and rIL-18 (50ng/ml). This\nprocedure has been clarified in the Methods section.\nFigure 6A: Were human NK cells isolated from the infarct tissue or peripheral blood? How\nwere control samples obtained?\nResponse: As detailed in the Methods section, we measured the global transcriptomic\nsignature of heart tissue sections using Nanostring technology. Subsequently,\nbioinformatic analysis was performed to identify NK cell-related pathways. Normal heart\ntissue was obtained from Creative Bioarray (United States).\nFigure 6D: What markers were used for NK cell staining ?\nResponse: For immunostaining, we used an anti-NKp46 monoclonal mAb (clone 8E5B,\nInnate Pharma).\nFigure 6E: What is the biological significance of the \"Mean expression\" in the signaling\npathway ? How is it calculated ?\nResponse : The mean expression of pathway represents the “calculated module scores\nusing the AddModuleScore function in Seurat”. For each cell, the average expression of a\ngiven gene set (here, genes associated with inflammatory and cytotoxic pathways), is\ncomputed relative to control genes of similar expression. This yields a relative activity\nscore for the pathway in each cell. We used 50 randomly selected control genes to\nnormalise the score.\nBiological significance : The module score provides a measure of the transcriptional\nactivity of the cytotoxic and inflammatory pathways in each cell. This allows us to detect\nsubtle but significant shifts in pathway activity. Notably, the distribution of module scores\nacross different time points and treatments highlights cell populations undergoing\npotential state transitions, thereby, showcasing the differential regulation of these\npathways.\nThis explanation has been clarified in the revised manuscript.\nReviewer #3 (Remarks to the Author):\nComments to Authors:\nThe manuscript by Cohen et al., entitled “NK cells promote cardiac cell death and regulate\nmyelopoiesis in myocardial infarction”, investigates the role of NK cells in promoting apoptosis\nand impaired myocardial function. The authors demonstrate that treatment with an anti-NKG2A\nmonoclonal antibody exacerbates ischemic heart failure. While the study addresses an\nimportant question, the manuscript can be strengthened by addressing the following concerns:\n1. Figure 1: The authors demonstrate that increased infiltration of NK cells, notably CCR2+\nNK cells, in the infarcted myocardium compared to sham. However, it remains unclear which\nspecific chemokines mediate the recruitment of NK cells to the MI region, especially\nconsidering that monocyte-derived macrophages also express high levels of CCR2. Are the\nsame chemokines responsible for recruiting CCR2-positive monocyte-derived macrophages to\nthe infarct zone? Clarification is needed.\nResponse: As shown in Supplementary Figure 5A and well documented in the literature,\neach cell type expresses a distinct set of chemokine receptors, although several receptors\nare shared among different cell types. For example, CCR2 is expressed by both NK cells\nand classical monocytes.\nGiven that global CCR2 deletion impacts on several immune population and to address\nthe reviewer’s concern, we performed an additional series of experiments to support the\ncritical role of CCR2 for NK cell recruitment in the ischemic heart tissue. We\nrepopulated Nkp46iCre+/- R26RDTA (referred to as NKKO) mice with purified Wild-type\nor Ccr2-/- NK cells 3 days before MI (new Figure 1O). NK deficiency in NKKO mice was\nvalidated by flow cytometry (Extended Data Fig.5j). As shown in the new figure 1P, at day 3 post-\nMI, NK cell repopulation in the blood was similar between groups. However, NK cell\nnumber was significant lower in the heart of MI-operated mice repopulated with Ccr2-/-\nNK cells compared to animals repopulatd with Ccr2+/+ NK cells(New Figure 1QR). These\nresults confirmed the key role of CCR2 in the recruitment of NK cells in the ischemic\nheart.\n2. Figures 1/2: The specificity of NK cell depletion must be addressed. Since CCR2 and\nCCR5 are expressed by other immune cells, including macrophages. The use of a more\nspecific NK cell marker, such as NKp46, would strengthen the study. This issue is evident in\nFigures 2b and 3d.\nResponse: As shown in figure 1a, NK cells were defined for flow cytometry analysis as\nCD11b-CD3-NK1.1+ NKp46+. For immunostainings, NK cells were identified as NKp46+\ncells (yellow staining).\n3. Figure 2: It would be informative to evaluate how NK cell depletion influences other immune\ncell populations and chemokine expression in MI.\nResponse: To address the reviewer’s concern, we performed scRNA-seq analysis on\nCD45+ cells isolated from ischemic heart tissue to further elucidate the cellular dynamics\nand interactions involved. As shown in the new supplemental figure 19, pseudobulk\nanalysis revealed differentially expressed genes in monocyte and macrophage populations\nfollowing NK cell depletion, assessed across total cells per group. Of note, Cd36 and Hdac9\nwere upregulated, whereas P2rx7, involved in inflammasome activation, was\ndownregulated in the NK depleted group (Extended Data Fig.19e). Moreover, Tnf\nexpression in the main monocyte, macrophage and neutrophil populations was reduced\nupon NK cell depletion (Extended Data Fig.19f). Altogether, these results indicate that\nNK cell depletion promotes a shift in macrophage profiles toward a less inflammatory\nphenotype.\nTo further address the reviewer’s concern, we quantified Ccl2, Ccl3, Cxcl1 and Cxcl2\nmRNA levels in the ischemic heart of control, NK depleted (Anti-NK1.1) and NK activated\n(Anti-NKG2A) groups. As shown in the new supplemental figure 16, NK depletion or NK\nactivation had no impact on Ccl2, Ccl3, Cxcl1 and Cxcl2 mRNA levels.These new results\nhave been added in the new version of the manuscript.\n4. As the authors are claiming that worse cardiac function and fibrosis are due to NK cell-\nmediated cytotoxicity, it’d be useful to demonstrate immune cell infiltration in the myocardium\nas well as increased cell death by TUNEL staining around day 3-7d following LAD ligation\nwhen the NK cells/mg heart tissue are at peak.\nResponse: We have extensive experience in quantifying apoptosis in heart tissue in the\ncontext of MI, and consider that TTC staining the most comprehensive and integrative\nmethod for assessing cardiac tissue necrosis and viability (e.g. Yang Circulation 2006;\nLeuschner et al. Circ Res 2010; Santos-Zas et al Nat Comm 2021). In previous studies, we\nperformed TUNEL staining and observed very few TUNEL+ cells in the heart after MI,\nas the transition from apoptosis to necrosis occurs rapidly.\nHowever, to address the reviewer’s concern, we performed TUNEL staining at day 3 post-\nMI in both NK depletion (NK1.1) and NK activation (anti-NKG2A) groups. NK depletion\nwas associated with a significant reduction of TUNEL+ cells in the ischemic heart,\nwhereas NK activation was led to an increase, consistent with the TTC staining results\n(New supplemental figure 12).\n5. Can the authors discuss whether the interaction between NK cells and cardiomyocytes is\ndirect or mediated via other cell types ?\nResponse: As suggested by the reviewer, this question has been discussed in detail in the\nDiscussion section. We have gathered substantial evidence demonstrating that NK cells\ndirectly promote cardiomyocyte death through Granzyme B release in the context of MI,\nsupported by both in vitro and in vivo experiments. Moreover, since we have shown that\nNK cells also contribute to myelopoiesis during MI and that myeloid cells exhibit pro-\napoptotic activity, we propose that NK cells exert an indirect pro-apoptotic effect though\nthe regulation of myelopoiesis.\n6. Figure 4: the immunostaining for GZMB shows significant GZMB even in NK depleted\ntissues. An explanation would be required.\nResponse: As detailed in the manuscript, the detection of GZMB+ cells in the ischemic\nheart of NK-depleted mice is attributed to the presence of CD8+ T cells, which also\nproduce GZMB (see Santos-Zas et al. Nat Comm 2021).\nAdditionally, details regarding co-culture conditions would be needed. Were the cells separated\nby inset/outlet or did they have any direct contacts? Since HL1 cells are proliferating\ncardiomyocytes, they may not be an ideal model for cardiomyocyte cell death, which should be\nat least acknowledged.\nResponse: To adress the reviewer’s concern, the description of the co-culture method has\nbeen revised. The cells were not separated by a membrane, allowing for direct cell-cell\ncontact interactions. Futhermore, we demonstrated a direct pro-apoptotic role of\nGranzyme B, a soluble protein, as cardiomyocyte death was reduced in the co-cultures\nwith GzmB-/- NK cells.\n7. Could the authors provide insight into whether human NK cells are likely to exhibit similar\nbehavior in the context of MI? Regarding Figure 6, how did the authors differentiate active vs\nmature vs terminal NK? Also, I am having a very difficult time understanding what Figure 6e\nis trying to convey.\nResponse: In figure 6, NK cell subsets were classified based on previously published work\n(Yang et al Nat Com 2019 PMID: 31477722). Active NK cells were defined by a higher\nmodule score, characterized by up-regulated DEGs associated with “Inflamed NK” cells,\nincluding several immediate early genes (IEGs), such as NR4A2, DUSP1, FOSB, FOS,\nJUN, and JUNB. Mature NK cells were characterized by high expression of molecules\nimportant for cytotoxic function and cytolytic molecules, including perforin (PRF1) and\ngranzymes (GZMA, GZMB, GZMH). Terminal NK cells express functional molecules\nsimilar to the “Mature NK” subset but show high expression of the transcription factor\nZEB2, indicating terminal maturity. This clarification has been clarified in the revised\nmanuscript.\nFigure 6E is based on spatial transcriptomics of human heart tissue at different time\npoints after MI. Using this method and bioinformatic analysis, we were able to\ncharacterize NK subsets in different areas over time. We found that NK cells with an\ninflammatory and cytotoxic profile were detected at early stages in both the ischemic and\nremote zones. At later stages, NK cells with a cytotoxic profile were detcted in firotic areas.\nTo adress the reviewer’s concern, the results in figure 6 have been described in greater\ndetail in the revised manuscript.\nMinor comments\nFigure labeling: Ensure consistent formatting (e.g., Figure 1G – capital vs. lowercase letters).\nResponse: The modification has been made accordingly.\nPage 6, Line 128: Revise “We then we investigated” to “We then investigated.”\nResponse: The modification has been made accordingly."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have overall strengthened their mechanistic case that NK cells exacerbate acute post-MI remodeling via Granzyme B–dependent cardiomyocyte death and NK-derived GM-CSF–driven myelopoiesis. In the revised version of the manuscript, they provide additional controls addressing the specificity of NK depletion. They have also included new human data using 3D cardiac rings with human NK cells.\nHowever, the human data, while improved, remain limited and largely correlative. The readout of the 3D rings is acute contractility over 12 hours only and does not relate directly to cardiac remodeling or cardiomyocyte death. In addition, there is no demonstration of Granzyme B involvement or of the specific NK-dependent pathways implicated in the mouse model. NK cells are derived from healthy donors, are IL-2–activated in vitro, and are not obtained from MI patients. These limitations are substantial and should be experimentally addressed.\nFinally, while the authors argue that the role of NK cells in long-term cardiac remodelling remains to be fully elucidated and that addressing cardiac regeneration is outside the scope of this article, these points are not reflected in the revised manuscript. At a minimum, they should temper their “NK = bad” conclusion and explicitly acknowledge the possibility of later protective NK functions as a limitation and/or future direction. In parallel, they should discuss the possibility of cardiomyocyte renewal / cardiac regeneration as a potential contributing factor to the beneficial effects observed with acute NK depletion.", "reviewer_2": "The authors have made commendable improvements to the manuscript, and the overall quality has notably increased.\nHowever, we suggest the following revisions to further strengthen the work:\nThe discussion would benefit from a more detailed explanation of what initiates NK cell activation in the context of myocardial infarction. Please summarize available evidence indicating that NK cell activation occurs earlier than that of myeloid-derived pro-inflammatory cells.\nFigure 4a, as it stands, appears repetitive and does not sufficiently stand on its own. We recommend either removing it or merging it into Figure 4b, with a corresponding note in the figure legend.", "reviewer_3": "The revised manuscript by Cohen et al., entitled “NK cells promote cardiac cell death and regulate myelopoiesis in myocardial infarction”, investigates the role of NK cells in promoting apoptosis and impaired myocardial function. Authors have adequately addressed our concerns.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to reviewers\nReviewer #1 (Remarks to the Author):\nThe authors have overall strengthened their mechanistic case that NK cells exacerbate acute\npost-MI remodeling via Granzyme B–dependent cardiomyocyte death and NK-derived GM-\nCSF–driven myelopoiesis. In the revised version of the manuscript, they provide additional\ncontrols addressing the specificity of NK depletion. They have also included new human data\nusing 3D cardiac rings with human NK cells.\nResponse : We would like to thank the reviewer for his/her comments that helped to improve\nthe manuscript.\nHowever, the human data, while improved, remain limited and largely correlative. The readout\nof the 3D rings is acute contractility over 12 hours only and does not relate directly to cardiac\nremodeling or cardiomyocyte death. In addition, there is no demonstration of Granzyme B\ninvolvement or of the specific NK-dependent pathways implicated in the mouse model. NK\ncells are derived from healthy donors, are IL-2–activated in vitro, and are not obtained from MI\npatients. These limitations are substantial and should be experimentally addressed.\nResponse : Our in vitro experiments using 3D cardiac rings showed that activated NK rapidly\nimpaired human cardiomyocyte functions, consistent with all the results obtained from murine\nmodels and co-culture experiments. Regarding the underlying mechanisms, we acknowledge\nthe reviewer's concern about demonstrating granzyme B involvement in the human system. To\nthe best of our knowledge, functional blocking or neutralizing antibodies against human\ngranzyme B are not commercially available as research tools. While detection antibodies exist,\nvalidated functional inhibitors have not been developed commercially. Alternative approaches,\nsuch as small-molecule inhibitors would require substantial optimization and validation within\nour 3D tissue model. We also acknowledge that NK cells derived from healthy donors may not\nfully recapitulate the phenotype of NK cells from MI patients. However, obtaining sufficient\nnumbers of viable NK cells from MI patients for functional assays presents significant logistical\nand ethical challenges. In France, ethical approval for such experiments would require\napproximately 8-9 months.\nTo address the reviewer’s concern, an additional limitation has\nbeen added to the discussion section (Page 18).\nFinally, while the authors argue that the role of NK cells in long-term cardiac remodelling\nremains to be fully elucidated and that addressing cardiac regeneration is outside the scope of\nthis article, these points are not reflected in the revised manuscript. At a minimum, they should\ntemper their “NK = bad” conclusion and explicitly acknowledge the possibility of later\nprotective NK functions as a limitation and/or future direction. In parallel, they should discuss\nthe possibility of cardiomyocyte renewal / cardiac regeneration as a potential contributing factor\nto the beneficial effects observed with acute NK depletion.\nResponse : To address the reviewer’s concern, the discussion has been modified as follows:\n« Our study highlights a global pathogenic role for NK cells in the immune-inflammatory\nresponses contributing to tissue damage following acute MI. However, we cannot rule out a\nprotective role for specific NK cell subsets at later stages, as previous studies have reported\npro-angiogenic activities of NK cells {Bouchentouf, 2010 #38569} » (Page 21).\nReviewer #2 (Remarks to the Author):\nThe authors have made commendable improvements to the manuscript, and the overall\nquality has notably increased.\nResponse : We would like to thank the reviewer for his/her comments that helped to improve\nthe manuscript.\nHowever, we suggest the following revisions to further strengthen the work. The discussion\nwould benefit from a more detailed explanation of what initiates NK cell activation in the\ncontext of myocardial infarction. Please summarize available evidence indicating that NK cell\nactivation occurs earlier than that of myeloid-derived pro-inflammatory cells.\nResponse : The discussion regarding factors that may activate NK cells in the context of\nmyocardial infarction has been expanded in the Discussion section (Page 21). To our\nknowledge, no published data currently explore the very early timing of NK versus myeloid\ncell activation following cardiac tissue ischemia. Further studies will be required to specifically\naddress this point.\nFigure 4a, as it stands, appears repetitive and does not sufficiently stand on its own. We\nrecommend either removing it or merging it into Figure 4b, with a corresponding note in the\nfigure legend.\nResponse : Figure 4a has been modified accordingly\nReviewer #3 (Remarks to the Author):\nThe revised manuscript by Cohen et al., entitled “NK cells promote cardiac cell death and\nregulate myelopoiesis in myocardial infarction”, investigates the role of NK cells in promoting\napoptosis and impaired myocardial function. Authors have adequately addressed our concerns.\nResponse : We would like to thank the reviewer for his/her comments that helped to improve\nthe manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse : We would like to thank the reviewer for his/her comments that helped to improve\nthe manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse : We would like to thank the reviewer for his/her comments that helped to improve\nthe manuscript."}]

Decision letter