T cell specific deletion of Casitas B lineage lymphoma-b reduces atherosclerosis, but increases plaque T cell infiltration and systemic T cell activation.

Introduction: Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. Methods: To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Results: Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. Conclusion: In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.

Tumour-infiltrating lymphocyte therapy for patients with advanced-stage melanoma.

Immunotherapy with immune-checkpoint inhibitors (ICIs) and targeted therapy with BRAF and MEK inhibitors have revolutionized the treatment of melanoma over the past decade. Despite these breakthroughs, the 5-year survival rate of patients with advanced-stage melanoma is at most 50%, emphasizing the need for additional therapeutic strategies. Adoptive cell therapy with tumour-infiltrating lymphocytes (TILs) is a therapeutic modality that has, in the past few years, demonstrated long-term clinical benefit in phase II/III trials involving patients with advanced-stage melanoma, including those with disease progression on ICIs and/or BRAF/MEK inhibitors. In this Review, we summarize the current status of TIL therapies for patients with advanced-stage melanoma, including potential upcoming marketing authorization, the characteristics of TIL therapy products, as well as future strategies that are expected to increase the efficacy of this promising cellular immunotherapy.

The emerging role for CAR T cells in solid tumor oncology.

In recent years, treatment with chimeric antigen receptor (CAR) T-cells has revolutionized the outcomes of patients with relapsed or refractory hematological malignancies with long-term remissions in >30% of patients. Similarly, the introduction of immune checkpoint inhibitor therapy changed the therapeutic landscape for several solid malignancies also leading to impressive long-term remission in patients. However, so far CAR T-cell therapy in solid tumors has shown low response rates and especially a lack of long-term remissions. This review focuses on the latest clinical advances and discusses promising results seen with CAR T-cells exploring new target antigens. We then review relevant challenges limiting long-term responses with CAR T-cell therapy in solid tumors like CAR T-cell persistence and target antigen expression. In addition, there is an increasing understanding on T-cell function and dysfunction within the immunosuppressive tumor microenvironment. This comprises of inhibitory cytokines and checkpoint molecules limiting the killing capacity of CAR T-cells. Finally, we will discuss how this deeper knowledge can be used to develop CAR T-cell therapies overcoming these inhibitory factors and results in CAR T-cell products with higher efficacy and safety. These technological developments will hopefully lead to enhanced clinical activity and improved solid tumor patient outcomes in the near future.

Peritoneal Metastases From Prostate Carcinoma Treated With 177 Lu-PSMA-I&T.

ABSTRACT: A 71-year-old man was referred for 177 Lu-PSMA therapy. He had metastatic castration-resistant prostate cancer, progressive on several treatment lines, with current PSA 260 µg/L and deteriorating condition. CT showed ascites with omental and peritoneal metastases, all positive on PSMA PET/CT. He was treated with 4 cycles of 7.4 GBq (0.2 Ci) 177 Lu-PSMA-I&T. Posttherapy scans showed good targeting of all metastases. After 4 cycles, PSA had dropped to 44 µ/L. Four months after the fourth cycle, the patients' general condition had significantly improved, and PSA had decreased to 7.0 µg/L.

Immuno-PET Imaging of Atherosclerotic Plaques with [89Zr]Zr-Anti-CD40 mAb-Proof of Concept.

Non-invasive imaging of atherosclerosis can help in the identification of vulnerable plaque lesions. CD40 is a co-stimulatory molecule present on various immune and non-immune cells in the plaques and is linked to inflammation and plaque instability. We hypothesize that a 89Zr-labeled anti-CD40 monoclonal antibody (mAb) tracer has the potential to bind to cells present in atherosclerotic lesions and that CD40 Positron Emission Tomography (PET) can contribute to the detection of vulnerable atherosclerotic plaque lesions. To study this, wild-type (WT) and ApoE-/- mice were fed a high cholesterol diet for 14 weeks to develop atherosclerosis. Mice were injected with [89Zr]Zr-anti-CD40 mAb and the aortic uptake was evaluated and quantified using PET/Computed Tomography (CT) imaging. Ex vivo biodistribution was performed post-PET imaging and the uptake in the aorta was assessed with autoradiography and compared with Oil red O staining to determine the tracer potential to detect atherosclerotic plaques. On day 3 and 7 post injection, analysis of [89Zr]Zr-anti-CD40 mAb PET/CT scans showed a more pronounced aortic signal in ApoE-/- compared to WT mice with an increased aorta-to-blood uptake ratio. Autoradiography revealed [89Zr]Zr-anti-CD40 mAb uptake in atherosclerotic plaque areas in ApoE-/- mice, while no signal was found in WT mice. Clear overlap was observed between plaque areas as identified by Oil red O staining and autoradiography signal of [89Zr]Zr-anti-CD40 mAb in ApoE-/- mice. In this proof of concept study, we showed that PET/CT with [89Zr]Zr-anti-CD40 mAb can detect atherosclerotic plaques. As CD40 is associated with plaque vulnerability, [89Zr]Zr-anti-CD40 mAb has the potential to become a tracer to detect vulnerable atherosclerotic plaques.

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

Antibody-mediated blockade of co-inhibitory molecules such as cytotoxic T lymphocyte-associated protein 4, PD1 and PDL1 elicits potent antitumor responses and improves the prognosis of many patients with cancer. As these immune checkpoint inhibitors (ICIs) are increasingly prescribed to a diverse patient population, a broad range of adverse effects is emerging. Atherosclerosis, a lipid-driven chronic inflammatory disease of the large arteries, may be aggravated by ICI treatment. In this review, we discuss recent clinical studies that analyze the correlation between ICI use and atherosclerotic cardiovascular disease (CVD). Indeed, several studies report an increased incidence of atherosclerotic CVD after ICI administration, with the occurrence of pathologies such as myocardial infarction, ischemic stroke and coronary artery disease significantly higher after ICI use. Increased awareness and better monitoring of ICI-treated patients can elucidate risk factors that contribute to ICI-induced aggravation of atherosclerosis and identify promising treatment strategies. For now, optimal cardiovascular risk assessment is required to protect ICI-receiving patients and long-term survivors of cancer from the detrimental effects of ICI therapy on atherosclerotic CVD.

Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

The CD40-CD40L Dyad as Immunotherapeutic Target in Cardiovascular Disease.

Chronic inflammation drives the development of atherosclerosis. Despite optimal treatment of classical cardiovascular risk factors, a substantial portion of the population has elevated inflammatory biomarkers and develops atherosclerosis-related complications, indicating that a residual inflammatory risk drives atherosclerotic cardiovascular disease in these patients. Additional anti-inflammatory therapeutic strategies are therefore required. The co-stimulatory molecule CD40 and its ligand CD40L (CD154) have a central role in the regulation of the inflammatory response during the development of atherosclerosis by modulating the interaction between immune cells and between immune cells and non-immune cells. In this review, we discuss the role of the CD40-CD40L dyad in atherosclerosis, and we discuss recent studies on the therapeutic potential of novel CD40-CD40L targeting strategies in cardiovascular medicine.

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

AIMS: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. METHODS AND RESULTS: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6-8 weeks old male Ldlr -/- mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. CONCLUSION: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis.

'Nur'turing tumor T cell tolerance and exhaustion: novel function for Nuclear Receptor Nur77 in immunity.

The nuclear receptor Nur77 is expressed in a multitude of tissues, regulating cell differentiation and homeostasis. Dysregulation of Nur77 signaling is associated with cancer, cardiovascular disease, and disorders of the CNS. The role of Nur77 in T cells has been studied for almost 30 years now. There is a clear appreciation that Nur77 is crucial for apoptosis of self-reactive T cells. However, the regulation and function of Nur77 in mature T cells remains largely unclear. In an exciting development, Nur77 has been recently demonstrated to impinge on cancer immunotherapy involving chimeric antigen receptor (CAR) T cells and tumor infiltrating lymphocytes (TILs). These studies indicated that Nur77 deficiency reduced T cell tolerance and exhaustion, thus raising the effectiveness of immune therapy in mice. Based on these novel insights, it may be proposed that regulation of Nur77 activity holds promise for innovative drug development in the field of cellular immunotherapy in cancer. In this review, we therefore summarize the role of Nur77 in T cell selection and maturation; and further develop the idea of targeting its activity in these cells as a potential strategy to augment current cancer immunotherapy treatments.

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

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.

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) drives atherosclerosis in mice and is associated with an unstable plaque phenotype and cerebrovascular events in humans.

AIMS: GITR-a co-stimulatory immune checkpoint protein-is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). METHODS AND RESULTS: GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr-/-Apoe-/- mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr-/-Apoe-/- and Apoe-/- monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr-/-Apoe-/- monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity. CONCLUSION: Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.

E3 Ubiquitin Ligases as Immunotherapeutic Target in Atherosclerotic Cardiovascular Disease.

Chronic low-grade inflammation drives atherosclerosis and despite optimal pharmacological treatment of classical cardiovascular risk factors, one third of the patients with atherosclerotic cardiovascular disease has elevated inflammatory biomarkers. Additional anti-inflammatory strategies to target this residual inflammatory cardiovascular risk are therefore required. T-cells are a dominant cell type in human atherosclerotic lesions. Modulation of T-cell activation is therefore a potential strategy to target inflammation in atherosclerosis. Ubiquitination is an important regulatory mechanism of T-cell activation and several E3 ubiquitin ligases, including casitas B-lineage lymphoma proto-oncogene B (Cbl-B), itchy homolog (Itch), and gene related to anergy in lymphocytes (GRAIL), function as a natural brake on T-cell activation. In this review we discuss recent insights on the role of Cbl-B, Itch, and GRAIL in atherosclerosis and explore the therapeutic potential of these E3 ubiquitin ligases in cardiovascular medicine.

Short-term regulation of hematopoiesis by lipoprotein(a) results in the production of pro-inflammatory monocytes.

BACKGROUND: Lipoproteins are important regulators of hematopoietic stem and progenitor cell (HSPC) biology, predominantly affecting myelopoiesis. Since myeloid cells, including monocytes and macrophages, promote the inflammatory response that propagates atherosclerosis, it is of interest whether the atherogenic low-density lipoprotein (LDL)-like particle lipoprotein(a) [Lp(a)] contributes to atherogenesis via stimulating myelopoiesis. METHODS & RESULTS: To assess the effects of Lp(a)-priming on long-term HSPC behavior we transplanted BM of Lp(a) transgenic mice, that had been exposed to elevated levels of Lp(a), into lethally-irradiated C57Bl6 mice and hematopoietic reconstitution was analyzed. No differences in HSPC populations or circulating myeloid cells were detected ten weeks after transplantation. Likewise, in vitro stimulation of C57Bl6 BM cells for 24 h with Lp(a) did not affect colony formation, total cell numbers or myeloid populations 7 days later. To assess the effects of elevated levels of Lp(a) on myelopoiesis, C57Bl6 bone marrow (BM) cells were stimulated with lp(a) for 24 h, and a marked increase in granulocyte-monocyte progenitors, pro-inflammatory Ly6high monocytes and macrophages was observed. Seven days of continuous exposure to Lp(a) increased colony formation and enhanced the formation of pro-inflammatory monocytes and macrophages. Antibody-mediated neutralization of oxidized phospholipids abolished the Lp(a)-induced effects on myelopoiesis. CONCLUSION: Lp(a) enhances the production of inflammatory monocytes at the bone marrow level but does not induce cell-intrinsic long-term priming of HSPCs. Given the short-term and direct nature of this effect, we postulate that Lp(a)-lowering treatment has the capacity to rapidly revert this multi-level inflammatory response.

Author Correction: Ablation of CD8α+ dendritic cell mediated cross-presentation does not impact atherosclerosis in hyperlipidemic mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cancer patients receiving immune checkpoint inhibitor therapy are at an increased risk for atherosclerotic cardiovascular disease.

The widespread clinical use of immune checkpoint inhibitors (ICI) has increased our knowledge on their adverse effects on chronic inflammatory diseases. Atherosclerosis, a low-grade lipid-driven inflammatory disease of the larger arteries, is commonly present in cancer patients. A major concern is the adverse effect of ICI on atherosclerosis-related cardiovascular disease, resulting in cardiovascular events, such as myocardial infarction or ischaemic stroke. The effects of ICI on atherosclerosis in cancer patients are incompletely understood, but it is well known that immune checkpoint proteins orchestrate the inflammatory response underlying atherogenesis. This paper addresses the hypothesis that ICI therapy puts cancer patients at an increased risk for atherosclerosis-related cardiovascular disease, that might only become apparent years after ICI therapy. Until clinical and experimental studies have addressed this hypothesis, optimal cardiovascular risk management in ICI-treated patients is opportune to reduce the occurrence of cardiovascular disease in cancer patients and long-term cancer survivors.

Immune Checkpoint Inhibitor Therapy Aggravates T Cell-Driven Plaque Inflammation in Atherosclerosis.

BACKGROUND: Immunotherapy has revolutionized cancer treatment. However, immune checkpoint inhibitors (ICIs) that target PD-1 (programmed cell death protein-1) and/or CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) are commonly associated with acute immune-related adverse events. Accumulating evidence also suggests that ICIs aggravate existing inflammatory diseases. OBJECTIVES: As inflammation drives atherosclerotic cardiovascular disease, we studied the propensity of short-term ICI therapy to aggravate atherosclerosis. METHODS: We used 18F-FDG (2-deoxy-2-[fluorine-18]fluoro-D-glucose) positron emission tomography-computed tomography to detect macrophage-driven vascular and systemic inflammation in pembrolizumab and nivolumab/ipilimumab-treated melanoma patients. In parallel, atherosclerotic Ldlr -/- mice were treated with CTLA-4 and PD-1 inhibition to study the proinflammatory consequences of immune checkpoint inhibition. RESULTS: ICI treatment did not affect 18F-FDG uptake in the large arteries, spleen, and bone marrow of melanoma patients, nor myeloid cell activation in blood and lymphoid organs in hyperlipidemic mice. In contrast, we found marked changes in the adaptive immune response (i.e., increased CD4+ effector T cell and CD8+ cytotoxic T cell numbers in lymphoid organs and the arterial wall of our hyperlipidemic mice). Although plaque size was unaffected, plaques had progressed toward a lymphoid-based inflammatory phenotype, characterized by a 2.7-fold increase of CD8+ T cells and a 3.9-fold increase in necrotic core size. Increased endothelial activation was observed with a 2.2-fold and 1.6-fold increase in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, respectively. CONCLUSIONS: This study demonstrates that combination therapy with anti-CTLA-4 and anti-PD-1 antibodies does not affect myeloid-driven vascular and systemic inflammation in melanoma patients and hyperlipidemic mice. However, short-term ICI therapy in mice induces T cell-mediated plaque inflammation and drives plaque progression.