Could chimeric antigen receptor-T cell treatment of atherosclerosis in mice translate to a human therapy?

Cardiovascular disease (CVD) is caused by atherosclerosis, a complex, chronic, inflammatory condition initially characterised by the recruitment and growth of lipid-filled immune cells causing fatty streaks within the arterial intima. In time, the fatty streaks develop into complex atherosclerotic plaques composed of lipid-filled macrophages or foam cells, smooth muscle cells and fibroblasts, T-cells, neutrophils and other inflammatory cells. Necrosis of the foam cells causes the formation of an acellular necrotic core, which becomes mechanically unstable, resulting in plaque erosion and rupture into the artery lumen. Atherosclerosis is difficult to treat, especially as the patients first presentation is often a serious event (heart attack, stroke) late in the progression of the disease. The paper by Schwab et al. (1) presents a novel approach to reducing atherosclerosis by constructing a chimeric antigen receptor T regulatory (CAR-Treg) cell targeting a form of oxidised low-density lipoprotein (oxLDL) which is involved in foam cell formation.

The trapping of oxLDL within the subendothelial matrix of the artery wall appears to be a key process in plaque development (2,3). It is not agreed exactly how oxLDL forms, but its composition is complex, being made up of oxidised cholesterol and cholesterol esters, oxidised phospholipids, lipid aldehydes, and a large array of protein oxidation products formed from the major LDL protein, apolipoprotein B100 (apoB100) (4-6). Macrophages in the arterial intima endocytose the oxLDL via the scavenger receptors, predominately ScR-A1, which binds lysine-rich regions on apoB100 that have been derivatised by lipid aldehydes, and CD36, which binds oxidised phosphatidylserine on the oxLDL surface (7,8). As there is no down-regulation of the scavenger receptors, lipid material from the oxLDL accumulates, and lipid droplets within the cytoplasm accumulate, giving the cells a foamy appearance. The resulting combination of oxidised lipids, immune cells and necrotic cell debris appears to drive inflammation within the plaque.

The genetic engineering of chimeric antigen receptor T (CAR-T) cells provides a means of generating large numbers of T cells specific for a single antigen. Classical CAR-T therapies target cancer-associated surface antigens and trigger cytotoxicity (9). However, it is also possible to generate CAR-Treg cells. These cells have an immunosuppressive rather than effector function and have attracted increasing interest as a treatment in diseases associated with inflammation. In the context of atherosclerosis, Tregs are present at relatively low frequencies, suggesting that impaired Treg-mediated suppression may play a role in plaque development (10). In the current study, a CAR-Treg therapy was developed with the aim of delivering a localised anti-inflammatory response at the site of foam cell formation to reduce atherosclerotic plaque formation. The CAR-Treg was generated from CD4⁺ T cells by transduction with FOX-P3 in combination with a second-generation chimeric antigen receptor (CAR) that specifically recognises aldehyde-derivatised oxLDL.

The antigen specificity of this construct was provided by a sequence corresponding to the variable chains of the 2DO3 monoclonal antibody. The 2DO3 monoclonal antibody was raised against malondialdehyde-derivatised low-density lipoprotein (MDA-LDL) (11). Malondialdehyde-derivatised (MDA) is one of many products resulting from the oxidation of the polyunsaturated cholesterol esters and phospholipids that occurs within the oxLDL particle. The breakdown of lipid hydroperoxides by β-scission reactions releases the aldehyde, MDA, which rapidly reacts with lysine residues within the apoB100 protein moiety of the LDL particle (12). Aldehyde-modified LDL is rapidly taken up by macrophages via the ScR-A1/2 (13-15), resulting in the formation of lipid-loaded foam cells. Interestingly, the injection of the anti-MDA-LDL-2D03 antibodies had previously been shown to reduce atherosclerotic plaque growth in rLDL^−/−, Apobec^−/− mice (16).

Schwab et al. initially showed that when the 2D03-CAR-Treg cells were co-incubated with human and mouse primary macrophages, the uptake of MDA-LDL and lipid-loaded foam cell formation was greatly reduced without the loss of cell numbers, indicating a regulatory response. Indeed, the authors found that in the presence of MDA-LDL, the 2D03-CAR-Tregs expressed a number of anti-inflammatory cytokines, including transforming growth factor-β (TGF-β). Furthermore, they found that the addition of anti-TGF-β antibodies blocked the effect of the 2D03-CAR-Treg cells with the macrophages returning to accumulating MDA-LDL.

The infusion of the mouse 2D03-CAR-Treg cells into rLDL^−/−, Apobec^−/− mice and subsequent tracking of the T-cells up to 42 days established that the therapy was sustainable. The therapy significantly reduced plaque formation in the arteries of the treated mice as indicated by both en face lipid staining and histological assessment of the aortic arch, which also indicated an increased collagen content indicative of an enhancement in plaque stability. This was a highly significant demonstration that 2D03-CAR-Treg could slow and maybe inhibited fatty streak and possible plaque formation.

MDA-LDL is a reasonable model for oxLDL and antibodies against it have been found in the circulation of subjects with CVD (17). Furthermore, these antibodies have been shown to reduce disease progression in mouse models of atherosclerosis (16). The MDA-lysine derivatisation is only one of a large range of lipid and protein oxidation products within oxLDL formed in vivo. MDA-lysine-specific antibodies do recognise oxLDL formed by metal-catalysed oxidation (18) and species of oxLDL apparent in advanced lesions (19). MDA-LDL though lacks many of the atherogenic properties of metal-oxidised oxLDL, which is chemotactic and cytotoxic (5,20,21).

The mechanism(s) of exactly how the CAR-Treg cells reduce atherosclerosis is a major area of interest. The authors clearly establish that the presence of CAR-Treg is associated with changes in macrophage activity and this appears to be mediated by release of TGF-β. Changes identified in the study included the appearance of M2 macrophages and a reduction in macrophage-mediated oxLDL uptake. Although not investigated in this study, TGF-β can downregulate CD36 and ScR-A1, the two main scavenger receptors responsible for oxLDL uptake by macrophages (22-24). It is therefore possible that the observed CAR-Treg-induced reduction in macrophage oxLDL uptake reflects, at least in part, TGF-β-driven reduction of scavenger receptors. Whether the action of TGF-β was totally localised to developing plaques was not clear from the paper as circulating levels in plasma were not measured.

This raises the question of where exactly does the oxLDL CAR-Treg interaction take place? OxLDL taken up by the macrophages is no longer on the cell surface for the CAR-T cells to bind to. LDL does bind to the subendothelial collagen matric and glycosaminoglycans (GAG), the likely site of oxidation, before being taken up by macrophages (25,26). 2D03-CAR-Treg cells are likely to be binding to the GAG-bound oxLDL. It is also possible a ScR-oxLDL-D03-CAR-Treg structure forms between the Treg cells and the macrophages, though it is not clear how the macrophage would then respond.

The work raises the obvious question of whether the same CAR-Treg therapy could be used to treat patients? The work shows that the 2D03-CAR-Treg construct is stably maintained in transduced T-cells adopted into mice. The authors also show that the construct invokes an anti-inflammatory response in cultured human T-cells, therefore could potentially be successfully applied to humans. Whether this would result in a reduction in atherosclerosis in vivo could be dependent on the timing of treatment. The mouse model used here mimics fatty stretch formation, a very early stage in plaque progression, before significant cell necrosis from oxLDL ensues and plaques become unstable. As mentioned earlier, patients are often not detected until the later stages of atherosclerotic disease when plaques are becoming unstable. As indicated by the authors, it would therefore be pertinent to test the CAR-Treg therapy in older mice with more advanced lesions, and ideally in a more advanced animal model with plaque instability such as the cholesterol-fed rabbit (27), before taking into human trials (28).

An additional issue is, if the oxLDL is not being taken up within artery wall via the macrophage scavenger receptors, could it then cause other interactions? OxLDL is cytotoxic to macrophages through nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activation and the release of superoxide (20,29). Could TGF-β inhibition of oxLDL uptake result in oxLDL levels that were cytotoxic. The short time frame of the CAR-Treg experiments was not able to test this, though no cell death was apparent.

This study provides an important proof of principle with regards to the use of CAR-Treg in the treatment of atherosclerosis. CAR-Treg therapies are evolving rapidly, and further advances in therapy design, such as 4^th generation CAR incorporating expression of cytokines, have the potential to further increase efficacy (30). Recent advances in the area of in vivo CAR-T engineering raise the possibility that CAR-T and CAR-Treg therapies could be delivered to patients without ex vivo manipulation or patient pre-conditioning (31,32).

The therapy presented here may potentially have broader applications beyond CVD. MDA-lysine specific epitopes are also found in other oxidised proteins in several different pathologies (33). Patients with early rheumatoid arthritis exhibit elevated autoantibody titres against mildly oxidized low-density (34). Could the 2D03-CAR-Treg also release TGF-β within the joints of rheumatoid arthritis patients to elicit beneficial effects?

The use of a CAR-Treg construct could make it possible to deliver specific cytokines to defined area of chronic inflammation. This would offer a more targeted approach than the systemic blocking of cytokines by monoclonal antibodies, which has not always proven beneficial. For example, injections of anti-interleukin-1β (anti-IL-1β) antibodies to counteract the proinflammatory effect of IL-1β did reduce the incidence of serious CVD events but increased the rate of death and serious illness during infections, proving that the systemic block of inflammation is a double-edged sword (35). The alternative approach shown here, where anti-inflammatory cytokines could be delivered locally via CAR-Treg cells, may be a viable future treatment option for a range of inflammatory-based conditions.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article did not undergo external peer review.

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0132/coif). S.G. reports Heart Foundation of New Zealand (3-year project grant unrelated to this publication) and Consultant to MARS Bioimaging Ltd., NZ at 0.05EFT (imaging work unrelated to this manuscript). M.O. reports a PhD Scholarship from University of Canterbury, New Zealand. S.M. reports funding from University of Otago Research and Study Leave Funding and Grants from the Heart Foundation, Health Research Council and Royal Society of New Zealand (Marsden Fund). The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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