Antibody-Mediated Inhibition of CTLA4 Aggravates Atherosclerotic Plaque Inflammation and Progression in Hyperlipidemic Mice

Abstract

T cell-driven inflammation plays a critical role in the initiation and progression of atherosclerosis. The co-inhibitory protein Cytotoxic T-Lymphocyte Associated protein (CTLA) 4 is an important negative regulator of T cell activation. Here, we studied the effects of the antibody-mediated inhibition of CTLA4 on experimental atherosclerosis by treating 6–8-week-old Ldlr−/− mice, fed a 0.15% cholesterol diet for six weeks, biweekly with 200 μg of CTLA4 antibodies or isotype control for six weeks. 18F-fluorodeoxyglucose Positron Emission Tomography—Computed Tomography showed no effect of the CTLA4 inhibition of activity in the aorta, spleen, and bone marrow, indicating that monocyte/macrophage-driven inflammation was unaffected. Correspondingly, flow cytometry demonstrated that the antibody-mediated inhibition of CTLA4 did not affect the monocyte populations in the spleen. αCTLA4 treatment induced an activated T cell profile, characterized by a decrease in naïve CD44−CD62L+CD4+ T cells and an increase in CD44+CD62L− CD4+ and CD8+ T cells in the blood and lymphoid organs. Furthermore, αCTLA4 treatment induced endothelial activation, characterized by increased ICAM1 expression in the aortic endothelium. In the aortic arch, which mainly contained early atherosclerotic lesions at this time point, αCTLA4 treatment induced a 2.0-fold increase in the plaque area. These plaques had a more advanced morphological phenotype and an increased T cell/macrophage ratio, whereas the smooth muscle cell and collagen content decreased. In the aortic root, a site that contained more advanced plaques, αCTLA4 treatment increased the plaque T cell content. The short-term antibody-mediated inhibition of CTLA4 thus accelerated the progression of atherosclerosis by inducing a predominantly T cell-driven inflammation, and resulted in the formation of plaques with larger necrotic cores and less collagen. This indicates that existing therapies that are based on αCTLA4 antibodies may promote CVD development in patients.

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability

“…Immunohistochemistry was performed for MAC3 (BD Pharmingen), alpha smooth muscle actin (Sigma-Aldrich), CD3 (AbD Serotec), CD8 (eBioscience), Ki67 (Abcam) and TUNEL ( in situ apoptosis detection kit, Roche). The stability index was defined as (% α smooth muscle actin/% necrotic core; Poels et al, 2020 ). Double immunohistochemical stainings were performed with rabbit anti-mouse PFKFB3 antibody (Abcam, AB2617178) in combination with CD31 (Dianova, Hamburg, Germany), and the previously mentioned MAC3, CD3 and alpha smooth muscle actin.…”

Immune checkpoint inhibitor treatment and atherosclerotic cardiovascular disease: an emerging clinical problem

“…Both T cell-specific overexpression of CTLA4 or treatment with the CTLA4-Ig fusion protein Open access abatacept reduced atherosclerosis in hyperlipidemic mice, whereas antibody-mediated inhibition of CTLA4 increased atherosclerotic burden in Ldlr −/− mice. [50][51][52][53] Blockage of CTLA4 induced an activated CD4 + and CD8 + T-cell profile in the circulation and lymphoid organs and promoted activation of the aortic endothelium. 53 Atherosclerotic lesion size increased on CTLA4 inhibition and plaques contained more T cells, which promoted necrotic core formation and subsequent plaque progression towards an advanced, clinically unfavorable phenotype.…”

“…[50][51][52][53] Blockage of CTLA4 induced an activated CD4 + and CD8 + T-cell profile in the circulation and lymphoid organs and promoted activation of the aortic endothelium. 53 Atherosclerotic lesion size increased on CTLA4 inhibition and plaques contained more T cells, which promoted necrotic core formation and subsequent plaque progression towards an advanced, clinically unfavorable phenotype. 53 As combined inhibition of CTLA4 and PD1 is a therapeutic strategy for several malignancies, we recently investigated the effects of dual antibody-mediated inhibition of CTLA4 and PD1 on atherosclerosis in Ldlr −/− mice.…”

CD8+ T Cells in Atherosclerosis

“…Several human and murine studies already demonstrated accelerated immune processes after blocking or depleting PD-1 or its ligand PD-L1, with parallel increased lesional CD4 + and CD8 + T cell levels in atherosclerosis [ 68 , 91 , 92 ]. The combined treatment of mice with immune checkpoint inhibitors targeting both PD-1 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), or CTLA inhibition alone, increased both CD4 + effector T cell and CD8 + cytotoxic T cell numbers in the arterial wall of hyperlipidemic mice [ 93 ]. A recent clinical study furthermore demonstrated that cardiovascular events were higher after initiation of immune checkpoint inhibitor treatment, potentially mediated by accelerated progression of atherosclerosis [ 94 ].…”