Periodic Reporting for period 4 - ANIMATE (Adaptive Immunity in Human Atherosclerosis: Understanding its Cellular Basis to Define Novel Immunomodulatory Therapies)
Berichtszeitraum: 2024-07-01 bis 2024-12-31
Cardiovascular disease (CVD) and its complications, myocardial infarction (MI) and stroke, represent the leading cause of mortality worldwide and cause an estimated 17.9 million deaths annually (~31% of all worldwide death cases). The most common underlying pathology for CVD is atherosclerosis, a chronic disease of the wall of medium-sized and large arteries that leads to the build-up of lipid- and leukocyte-rich atherosclerotic plaques. The continuous growth of atherosclerotic plaques eventually restricts the blood flow, which may cause a total occlusion of the vessel. Atherosclerosis is accompanied by an inflammatory and autoimmune response with immune cells (CD4+ T-helper cells) that falsely respond to endogenous proteins in the body (self-antigens), including ApoB-100 (ApoB), the main protein in low-density lipoprotein (LDL) cholesterol particles. The existence and function of such self-reactive CD4+ T cells remains elusive. For instance, it is not clear whether these are anti-inflammatory and protect from atherosclerosis or pro-inflammatory and pathogenic. In this project, the autoimmune response carried out by T-helper cells will be comprehensively studied by state-of-the-art cellular and molecular tools in mice and humans. Insights of this research will be used to identify novel immunomodulatory strategies to therapeutically stabilize the population of protective ApoB-specific T-helper cells, or to prevent their transformation into pathogenic T cell phenotypes by vaccination. In clinical association studies, a direct correlation of autoimmunity and clinical atherosclerosis will be tested. This project was designed to decipher traits of protective immunity in atherosclerosis and help to build the conceptual framework to define novel therapeutic strategies for patients in the future.
In this action, we found that ApoB-reactive T cells protect against early atherosclerosis, initially exhibiting a regulatory T cell (Treg) phenotype. Under hypercholesterolemia, they shift to pathogenic Th1/Th17 cells, driving inflammation. Using an MHC II tetramer, we identified these cells as an oligoclonal population with mixed Th1/Th17/Treg phenotypes in healthy mice. Single-cell RNA sequencing revealed overlapping pro- and anti-inflammatory transcripts. In atherosclerosis, ApoB-reactive T cells increased, shifting toward pathogenic Th1/Th17-like cells. Adoptive transfer showed converted ApoB+ T cells failed to protect, and circulating ApoB-reactive CD4+ T cells were more frequent in humans with atherosclerosis. This suggests an initially protective autoimmune response that becomes pathogenic, identifying ApoB-reactive T cells as a therapeutic target. Loss of FoxP3 drives the switch from protective to pathogenic phenotypes. Vaccination with ApoB-peptides boosts atheroprotection. Single-cell RNA sequencing revealed human plaques are T cell-dominated (over 60% of leukocytes), unlike myeloid-driven mouse plaques, highlighting a limited translatability of mouse models. Antibodies against ApoB-peptides correlated with clinical disease, implicating adaptive immunity in human atherosclerosis. B cells are essential for atheroprotective vaccination. Immunization with ApoB-peptides induced IL-10 in CD4+ T cells, but B cells were required for protection. HELMET Apoe-/- mice, lacking polyclonal B cells, failed to generate anti-ApoB antibodies or protection. Adding the cognate antigen HEL restored atheroprotection without anti-ApoB antibodies, showing T cell activation and BCR engagement are both necessary. IL-10 production, linked to BCR signaling, was critical, underscoring the importance of T and B cell collaboration. Immune cell transcriptomes predict cardiovascular outcomes. Single-cell and bulk RNA sequencing identified activation, memory formation, and proliferation pathways in CAD patients. Proliferating T and NK cells were associated with an increased risk, demonstrating immune cell transcriptomics' potential to refine risk prediction. Immune checkpoint inhibitors (ICIs) accelerated atherosclerosis, as shown by increased arterial [18F]FDG uptake in patients. This highlights ICIs' role in low-grade arterial inflammation but also [18F]FDG's variability in capturing vascular inflammation. ApoB-specific CD4+ T cells, enriched in circulating memory T cells (TCM), displayed a transitional immunophenotype, expressing both anti-inflammatory (IL-10) and pro-inflammatory (IFN-γ, TNF-α) cytokines. Their abundance correlated with cardiovascular risk, suggesting a role in atherogenesis. Plaque-resident T cells resembled ApoB+ T cells and plaque Treg cells with a Th17 phenotype, linking Treg destabilization to ApoB+ T cell function. Endothelial CD40 and CD40L promote plaque instability by recruiting inflammatory cells. Mice with endothelial-specific CD40 or CD40L deficiency developed smaller, more stable plaques with reduced macrophage content and increased collagen. Targeting these molecules may offer therapeutic strategies for atherosclerosis.
The following list gives an overview of the most important key findings:
1. Phenotype and function of ApoB-reactive T Cells in Atherosclerosis: One paper published, 4 more in preparation (ApoB-specific T cells in humans and correlation with clinical data / Systematic characterization of T cell phenotypes in mouse atherosclerosis / Inhibition of aged Tregs in mice / transcriptomes of aged Tregs in humans)
2. Immune Cell Transcriptomics and Cardiovascular Risk: 1 paper submitted, 1 more in preparation.
3. Impact of B Cells on Atheroprotective Vaccination: 1 paper published, 1 paper in revision, 2 more papers expected.
4. Differences in Human and Mouse Atherosclerosis: 1 paper published. 1 paper in submission.
5. Endothelial CD40 and CD40L in Atherosclerosis: 1 paper published, 1 paper in submission.
The following list gives an overview of the most important key findings:
1. Phenotype and function of ApoB-reactive T Cells in Atherosclerosis: One paper published, 4 more in preparation (ApoB-specific T cells in humans and correlation with clinical data / Systematic characterization of T cell phenotypes in mouse atherosclerosis / Inhibition of aged Tregs in mice / transcriptomes of aged Tregs in humans)
2. Immune Cell Transcriptomics and Cardiovascular Risk: 1 paper submitted, 1 more in preparation.
3. Impact of B Cells on Atheroprotective Vaccination: 1 paper published, 1 paper in revision, 2 more papers expected.
4. Differences in Human and Mouse Atherosclerosis: 1 paper published. 1 paper in submission.
5. Endothelial CD40 and CD40L in Atherosclerosis: 1 paper published, 1 paper in submission.
Our observation that peptide vaccination can boost protective parts of the ApoB-peptide response, open a new field for novel therapies in the future. We will optimize therapeutic strategies that may be tested in first clinical studies in humans after the completion of projects and papers. In particular, this project has opened a path for employing novel vaccination strategies beyond peptide vaccination, potentially including mRNA-based vaccination.
It was unexpected to see a broad applicability of circulating ApoB-specific T cells and transcriptomics as future biomarkers in cardiovascular disease. Circulating ApoB-T cells are directly related to Apolipoprotein B (ApoB), the major component of low-density lipoprotein (LDL) cholesterol, a key driver of atherosclerosis. Their presence and activity (e.g. cytokine production) could reflect the extent and activity of the immune response against atherosclerotic plaques. High levels or specific activation profiles may indicate ongoing inflammation and plaque instability, thus acting as a predictive marker for cardiovascular events.
It was unexpected to see a broad applicability of circulating ApoB-specific T cells and transcriptomics as future biomarkers in cardiovascular disease. Circulating ApoB-T cells are directly related to Apolipoprotein B (ApoB), the major component of low-density lipoprotein (LDL) cholesterol, a key driver of atherosclerosis. Their presence and activity (e.g. cytokine production) could reflect the extent and activity of the immune response against atherosclerotic plaques. High levels or specific activation profiles may indicate ongoing inflammation and plaque instability, thus acting as a predictive marker for cardiovascular events.