Rapid differentiation of regulatory CD4⁺ T cells in the infarcted myocardium blunts in situ inflammation

Abstract

Background: Myocardial infarction (MI) is a sterile inflammatory condition associated with tissue injury that results in the activation of T helper cell targeting cardiac antigens. However, the differentiation trajectories and in situ activity of heart-specific CD4+T cells activated in the MI context remain poorly understood. Methods: Herein, we combined T-cell receptor transgenic models targeting myocardial protein, single-cell transcriptomics, and functional phenotyping to elucidate how the myosin-specific CD4+ T cells (TCR-M) differentiate in the murine infarcted myocardium and ultimately influence tissue repair. Furthermore, we adoptively transferred heart-specific T-cells that were pre-differentiated in vitro towards pro-inflammatory versus regulatory phenotypic states to dissect how they differentially regulate post-myocardial infarction (MI) inflammation. Results: Flow cytometry and single-cell transcriptomics findings reveled that transferred TCR-M cells rapidly acquired an induced regulatory phenotype (iTreg) in the infarcted myocardium and blunt local inflammation. Myocardial TCR-M cells differentiated into two main lineages enriched with cell activation and pro-fibrotic transcripts (e.g. Tgfb1) or with suppressor immune checkpoints (e.g. Pdcd1), which we also found in human myocardial tissue. These cells produced high levels of latency-associated peptide (LAP) and inhibited interleukine-17 (IL-17) responses. Notably, TCR-M cells that were pre-differentiated in vitro towards a regulatory phenotype maintained a stable in vivo FOXP3 expression and anti-inflammatory activity when adoptively transferred prior to MI induction. In contrast, TCR-M cells that were pre-differentiated in vitro towards a pro-inflammatory TH17 phenotype were partially converted towards a regulatory phenotype in the injured myocardium and blunted myocardial inflammation. Conclusions: These findings reveal that the myocardial milieu provides a suitable environment for iTreg differentiation and reveals novels mechanisms by which the healing myocardium shapes local immunological processes.

Myocardial-Treg Crosstalk: How to Tame a Wolf

“…In addition, heart recipients’ IL-17 production by CD4 + T-cells was abrogated in TCR-M-transferred mice, while recipients’ heart Treg numbers rose. Moreover, transferring in vitro -Treg-expanded TCR-M cells inhibited cardiac inflammatory responses ( 40 ). Remarkably, thymic epithelial cells do not express the MYHCA protein in either mice or humans; thus, central tolerance mechanisms are not functional for this antigen and peripheral presentation plays a fundamental role ( 41 – 43 ).…”

“…For instance, in vitro pre-differentiated T H 17 but not T H 1 TCR-M cells still acquired FOXP3 expression in the heart, albeit to a lower extent than naïve TCR-M cells. Conversely, Treg-expanded TCR-M cells kept FOXP3 expression in the heart, suggesting they do not become ex-Tregs ( 40 ). More importantly, increased TCR-M Treg conversion correlated with lower inflammatory responses in the heart, regardless of infarct size, illustrating that cardiac Treg tonus directly affects local inflammatory responses ( 40 ).…”

“…Conversely, Treg-expanded TCR-M cells kept FOXP3 expression in the heart, suggesting they do not become ex-Tregs ( 40 ). More importantly, increased TCR-M Treg conversion correlated with lower inflammatory responses in the heart, regardless of infarct size, illustrating that cardiac Treg tonus directly affects local inflammatory responses ( 40 ). Taken together, these findings suggest a strong regulatory tonus imposed in the myocardium during MI’s acute healing phase affects T-helper cells that in turn module tissue inflammation.…”

“…In a mouse model of MI, our team has characterized both the endogenous (polyclonal) and myosin-specific (TCR-M cells) T-cell responses at the single-cell level in the heart and MedLNs. Intriguingly, TCR-Ms clustered separately from bona fide Tregs and showed a transcriptome suggesting an induced Treg signature (enriched for Cd200, Pou2f2, Sox4 and Izumo1r ) ( 40 ). More detailed single cell transcriptomic analyses suggested that the TCR-M cells activated after MI differentiate into two main Treg transcriptional states: a subset enriched for transcripts associated to TCR activation, cell growth/cycling and pro-fibrotic responses ( Myc , Tnfrsf9 , Mif and Tgfb1 ) and another subset expressing high levels of immune checkpoint inhibitor transcripts ( Pdcd1, Lag3, Tigit) .…”

“…More detailed single cell transcriptomic analyses suggested that the TCR-M cells activated after MI differentiate into two main Treg transcriptional states: a subset enriched for transcripts associated to TCR activation, cell growth/cycling and pro-fibrotic responses ( Myc , Tnfrsf9 , Mif and Tgfb1 ) and another subset expressing high levels of immune checkpoint inhibitor transcripts ( Pdcd1, Lag3, Tigit) . However, further investigation is needed to validate these phenotypic states and eventually resolve the mechanisms underlying their differentiation and function ( 40 ) ( Figure 3 ). Overall, cardiac Treg responses stem from naturally occurring and locally induced regulatory T-cells that have features linked to tissue repair, extracellular matrix organization and potent immune suppression.…”