Jesús Jiménez

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte–associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model ( Ctla4 +/– Pdcd1 –/– mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2) + monocyte-derived macrophages and CD8 + T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2 + subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2 + monocytes. It is important that a similar macrophage population expressing CXCL9 , CXCL10 , and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9 + Cxcl10 + macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8 + T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9 + Cxcl10 + macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ–induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

Sex steroids control the proliferation of their target cells through two different pathways: 1) proliferative response (Step-1); and 2) inhibition of cell proliferation (Step-2). Mechanisms of cell proliferation regulation are incompletely understood; however, there is general agreement with the notion that sex steroid receptors play an important role in the control of the proliferation of sex steroid target cells. To test this hypothesis, a full human androgen receptor (AR) vector was transfected into human breast cancer MCF7 cells. The cloned cells that stably express the AR, called MCF7-AR1 cells, contained approximately five times more AR than the wild-type MCF7 cells from which they were derived. These AR-transfected cells retained their capacity to proliferate when estrogens were added to 10% charcoal-dextran stripped human serum but did not acquire the ability to proliferate when androgens were added to this medium. In serumless medium (ITDME), these cells proliferated maximally, as MCF7 cells did; however, natural and synthetic androgens prevented the AR-transfected cells from proliferating. Inhibition of cell proliferation occurred when physiological androgen concentrations (1 nM) were added to ITDME; this effect was almost completely reversed by Casodex, a synthetic androgen antagonist. Under the effect of androgens added to ITDME, MCF7-AR1 cells were arrested in the G0/G1 phase within 24 h. These data suggest that: 1) the androgen-induced inhibition of cell proliferation (Step-2) is AR-mediated; and 2) the AR may be necessary, but not sufficient, to mediate the androgen-induced proliferative response (Step-1).