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.

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.

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.

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.

'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.

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.

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.

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. Methods and results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells. Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

Cardiovascular oncology: exploring the effects of targeted cancer therapies on atherosclerosis.

PURPOSE OF REVIEW: Targeted cancer therapies have revolutionized the treatment of cancer in the past decade, but cardiovascular toxicity is a rising problem in cancer patients. Here we discuss the effects of targeted cancer therapies on atherosclerosis. Increasing the awareness of these adverse effects will promote the development of evidence-based preventive strategies in the emerging field of cardiovascular oncology. RECENT FINDINGS: Vascular endothelial growth factor inhibitors, immunomodulatory imide drugs, tyrosine kinase inhibitors and immune checkpoint inhibitors are successfully used as treatment for many types of solid and hematologic malignancies. However, clinical and experimental studies have demonstrated that these drugs can drive atherosclerosis, thereby causing adverse cardiovascular events such as myocardial infarction, stroke and peripheral arterial occlusive diseases. SUMMARY: In this review, we discuss how on-target and off-target effects of novel cancer drugs may affect atherosclerosis and we postulate how these cardiovascular adverse events can be prevented in the future.

CD40L Deficiency Protects Against Aneurysm Formation.

OBJECTIVE: The mechanisms underlying formation of arterial aneurysms remain incompletely understood. Because inflammation is a common feature during the progressive degeneration of the aortic wall, we studied the role of the costimulatory molecule CD40L, a major driver of inflammation, in aneurysm formation. APPROACH AND RESULTS: Transcriptomics data obtained from human abdominal aortic aneurysms and normal aortas revealed increased abundance of both CD40L and CD40 in media of thrombus-free and thrombus-covered human abdominal aortic aneurysms samples. To further unravel the role of CD40L in aneurysm formation, apolipoprotein E-deficient (Apoe-/-) and Cd40l-/-Apoe-/- mice were infused with angiotensin II for 7 and 28 days. Only a minority of Cd40l-/-Apoe-/- mice (33% and 17%) developed (dissecting) aneurysms compared with 75% and 67% of Apoe-/- littermates after 7 and 28 days of infusion, respectively. Total vessel area of the aorta at the suprarenal level was 52% smaller in angiotensin II-infused Cd40l-/-Apoe-/- mice compared with that in angiotensin II-infused Apoe-/- mice. Chimeric Apoe-/- mice repopulated with Cd40l-/-Apoe-/- bone marrow afforded a similar protection against dissecting aneurysm formation. Moreover, lack of CD40L protected mice from fatal aneurysm rupture. T helper cell and macrophage accumulation in aneurysmal tissue was reduced in Cd40l-/-Apoe-/- mice with a concomitant decrease in expression of proinflammatory chemo- and cytokines. In addition, aneurysms of Cd40l-/-Apoe-/- mice displayed reduced abundance of matrix metalloproteinase-13 and an increase in tissue inhibitor of metalloproteinase-3 while activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 was diminished. CONCLUSIONS: Deficiency of (hematopoietic) CD40L protects against dissecting aneurysm formation and reduces the incidence of fatal rupture. This is associated with a decreased accumulation and activation of inflammatory cells and a dampened protease activity in the arterial wall.

Targeting CD40-Induced TRAF6 Signaling in Macrophages Reduces Atherosclerosis.

BACKGROUND: Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. OBJECTIVES: This study evaluates the potential of TRAF-STOP treatment in atherosclerosis. METHODS: The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe-/-) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages. RESULTS: TRAF-STOP treatment of young Apoe-/- mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe-/- mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair "classical" immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and ß2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe-/- mice. CONCLUSIONS: TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

Exploring immune checkpoints as potential therapeutic targets in atherosclerosis.

In the past decades, the inflammatory nature of atherosclerosis has been well-recognized and despite the development of therapeutic strategies targeted at its classical risk factors such as dyslipidemia and hypertension, atherosclerosis remains a major cause of morbidity and mortality. Additional strategies targeting the chronic inflammatory pathways underlying the development of atherosclerosis are therefore required. Interactions between different immune cells result in the secretion of inflammatory mediators, such as cytokines and chemokines, and fuel atherogenesis. Immune checkpoint proteins have a critical role in facilitating immune cell interactions and play an essential role in the development of atherosclerosis. Although the therapeutic potential of these molecules is well-recognized in clinical oncology, the use of immune checkpoint modulators in atherosclerosis is still limited to experimental models. Here, we review recent insights on the role of immune checkpoint proteins in atherosclerosis. Additionally, we explore the therapeutic potential and challenges of immune checkpoint modulating strategies in cardiovascular medicine and we discuss novel therapeutic approaches to target these proteins in atherosclerosis.

Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates.

Macrophage accumulation in atherosclerosis is directly linked to the destabilization and rupture of plaque, causing acute atherothrombotic events. Circulating monocytes enter the plaque and differentiate into macrophages, where they are activated by CD4+ T lymphocytes through CD40-CD40 ligand signalling. Here, we report the development and multiparametric evaluation of a nanoimmunotherapy that moderates CD40-CD40 ligand signalling in monocytes and macrophages by blocking the interaction between CD40 and tumour necrosis factor receptor-associated factor 6 (TRAF6). We evaluated the biodistribution characteristics of the nanoimmunotherapy in apolipoprotein E-deficient (Apoe-/-) mice and in non-human primates by in vivo positron-emission tomography imaging. In Apoe-/- mice, a 1-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, which was due to the impaired migration capacity of monocytes, as established by a transcriptome analysis. The rapid reduction of plaque inflammation by the TRAF6-targeted nanoimmunotherapy and its favourable toxicity profiles in both mice and non-human primates highlights the translational potential of this strategy for the treatment of atherosclerosis.

Inhibition of CD40-TRAF6 interactions by the small molecule inhibitor 6877002 reduces neuroinflammation.

BACKGROUND: The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation. METHODS: Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice). RESULTS: We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models. CONCLUSIONS: Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.