CD8⁺ T Cells in Doxorubicin Cardiotoxicity

Corresponding Author

Anthracycline class drugs such as doxorubicin are widely used chemotherapeutic agents in treating multiple types of cancer. Although anthracyclines are largely effective for treating cancer, they exert significant cardiotoxicity in a subset of patients.^1,2 This cardiotoxicity often results in chronic heart failure characterized by reduced cardiac function, increased fibrosis, and heightened inflammation.^1,3 Current heart failure therapies frequently fail to address anthracycline-induced cardiotoxicity, highlighting the need to better define how anthracyclines disrupt distinct cardiac cell populations.

Anthracycline-induced cardiotoxicity is widely attributed to effects on cardiomyocytes, as anthracyclines are known to cause double-stranded breaks, disrupt mitochondrial function, and increase reactive oxygen species in cardiomyocytes.⁴ However, emerging evidence in the field indicates that the effects of anthracycline-induced cardiotoxicity are mediated through an interplay of cells including endothelial cells, macrophages, neutrophils, T cells, and fibroblasts, among others.^5,6 Prior work has demonstrated that doxorubicin treatment stimulates cardiac fibroblasts to increase intercellular adhesion molecule 1 expression resulting in increased interferon gamma expression and cytotoxic granzyme degranulation in CD8⁺ T cells through fibroblast–T-cell direct interaction, suggesting a central and emerging role for T-cell activation in doxorubicin-induced cardiac fibrosis.⁷ In this issue of JACC: CardioOncology, Bayer et al⁸ further investigate the phenotypic shift occurring in T cells upon doxorubicin treatment in humans and mice to identify a novel matrix remodeling T-cell subset and elucidate the molecular mechanism driving this T-cell phenotype.

Bayer et al⁸ began the study by investigating transcriptomic data of CD8⁺ T cells from anthracycline-treated patients, along with single nucleus RNA sequencing from hearts of patients treated with doxorubicin. From these analyses, they identified a T-cell transcriptional signature driven by doxorubicin treatment that was characterized by collagen synthesis and extracellular matrix (ECM)–remodeling pathways including elevated upregulation of lysyl oxidases (LOX), matrix metalloproteinases (MMP), and collagen isoforms. This observation suggested that doxorubicin induces a profibrotic T-cell phenotype termed “matrix remodeling T cell” (mxT cell). Compared with other CD8⁺ T cells, mxT cells showed reduced classical activation but increased ECM-remodeling gene expression. With the discovery of this novel ECM remodeling T-cell population, Bayer et al⁸ sought to characterize the functional impact of these cells on the doxorubicin-treated heart. Mechanical testing revealed that hearts from doxorubicin-treated mice were stiffer and more fibrotic compared to hearts from control treated mice which correlated with lower ejection fraction. In accordance, CD8⁺ T cells from the circulation and spleen of doxorubicin-treated mice showed increased expression of matrix remodeling genes including Lox, Loxl1, and Mmp9. CD8⁺ T cell expression of LOX significantly correlated with ECM stiffness, suggesting a functional impact of these cells in the context of doxorubicin-induced adverse matrix remodeling.

To test whether doxorubicin was signaling directly to CD8⁺ T cells to increase matrix remodeling profiles, primary murine splenic CD8⁺ T cells were treated with doxorubicin. This resulted in increased levels of Lox, Loxl1, Mmp9, Mmp13, and Col1a1. In agreement with these data, LOX, a secreted enzyme that can stiffen ECM through crosslinking collagen fibrils,⁹ was also elevated at the protein level following doxorubicin treatment. Bayer et al⁸ also tested whether primary cardiac fibroblasts responded to direct doxorubicin treatment by activating LOX expression and found that doxorubicin did not influence Lox or Lox1 expression, suggesting that T cells selectively upregulate LOX in response to doxorubicin.

The authors had shown that doxorubicin could induce a unique expression profile in CD8⁺ T cells, characterized by upregulation of LOX. To determine whether T-cell–secreted LOX influenced cardiac ECM stiffness, decellularized cardiac ECM was treated with conditioned media from doxorubicin- or control-treated CD8⁺ T cells. ECM treated with conditioned media from control CD8⁺ T cells decreased stiffness, whereas conditioned media form the doxorubicin-treated CD8⁺ T cells resulted in more than a 2-fold increase in ECM stiffness. This increased stiffness was lost when a LOX inhibitor was added to the conditioned media, implicating LOX as a mechanistic mediator of cardiac ECM stiffness. Collectively, these data demonstrate that doxorubicin directly signals to CD8+ T cells to drive LOX secretion and subsequent ECM stiffening, highlighting the detrimental role of mxT cells in doxorubicin induced cardiotoxicity.

Mechanistic analysis focused on how anthracyclines drive CD8⁺ T cells toward a profibrotic state. Upstream regulator prediction across human anthracycline and murine doxorubicin datasets identified TP53 and SMAD3, implicating transforming growth factor β (TGFβ) signaling. TGFB1 transcripts were increased in CD8⁺ T cells 3 months after anthracycline treatment in humans and 8 weeks after doxorubicin in mice. Cardiac CD8⁺ T cells from doxorubicin-treated mice showed elevated phosphorylated SMAD family member 2 (p-SMAD2) by flow cytometry, and splenic CD8⁺ T cells demonstrated the same increase by western blot. Direct doxorubicin treatment of isolated CD8⁺ T cells in vitro induced Tgfb1 expression and p-SMAD2, indicating that doxorubicin triggers autocrine TGFβ signaling. Neutralizing TGFβ during doxorubicin exposure prevented LOX upregulation, and conditioned-media experiments showed that doxorubicin no longer increased ECM stiffness when TGFβ was blocked. Together, these findings show that autocrine TGFβ signaling is required for doxorubicin-induced matrix-remodeling programs in CD8⁺ T cells.

The subsequent pathway analysis in murine and human sequencing datasets determined how doxorubicin could increase TGFβ activity in CD8⁺ T cells. This analysis revealed hypoxia induced factor 1α (HIF1α) and P53 as strongly enriched pathways in both datasets. To assess whether these were activated downstream of doxorubicin, CD8⁺ T cells were plated in vitro and treated with doxorubicin over a time course. HIF1α levels were elevated from 1 to 4 hours then declined, and P53 phosphorylation increased from 4 to 8 hours followed by latent SMAD2 phosphorylation. This successive pattern of expression and phosphorylation suggests that HIF1α and P53 are activating TGFβ, which subsequently leads to phosphorylation of SMAD2. This relationship was also supported in vivo, as mouse CD8⁺ T cells had increased P53 and decreased HIF1α following doxorubicin treatment. When HIF1α was inhibited by echinomycin, doxorubicin treatment no longer increased LOX or Tgfb1 in vitro, and conditioned media did not increase ECM stiffness as strongly as with doxorubicin alone. This effect of echinomycin was reversed when a P53 activator was added, resulting in rescued ECM stiffness and LOX expression. This collective data suggest that P53 is indeed downstream of HIF1α in this pathway.

Overall, this study demonstrates that doxorubicin can directly polarize CD8⁺ T cells to a matrix remodeling state, where endogenous LOX expression leads to increased ECM stiffness and reduced cardiac function. Presented findings highlight the novel role of doxorubicin signaling through an axis of HIF1α-P53-TGFβ to directly stimulate LOX and other matrix remodeling genes specifically in CD8⁺ T cells. This study sheds insights into the complex and emerging role of immune cells in anthracycline cardiotoxicity and shows the importance of CD8⁺ T cells as key players in doxorubicin-mediated heart failure. This work sets the stage for future studies designed to further characterize mxT cells and to determine the comprehensive impact of T cells in cardiac ECM remodeling in response to doxorubicin treatment. Additional in vivo studies, assessing the impact of T-cell expressed LOX in the context of doxorubicin treatment would solidify its role in pathological cardiac remodeling implicated in the present study. Importantly, this study reveals a previously unrecognized role for T cells as direct regulators of ECM remodeling, overturning the prevailing view of how matrix stiffness is established and maintained in the heart following doxorubicin treatment.

Funding Support and Author Disclosures

The authors have reported that they have no relationships relevant to the contents of this paper to disclose.