CD7 activation regulates cytotoxicity-driven pathology in systemic sclerosis, yielding a target for selective cell depletion.

OBJECTIVES: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

Differences in local immune cell landscape between Q fever and atherosclerotic abdominal aortic aneurysms identified by multiplex immunohistochemistry.

Background: Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue. Methods: Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune systems. Results: Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs, the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+CD8-FoxP3+ regulatory T cells. Finally, Q fever AAAs did not contain any well-defined granulomas. Conclusions: These findings demonstrate that despite the presence of pro-inflammatory effector cells, persistent local infection with C. burnetii is associated with an immune-suppressed microenvironment. Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716].

Circulating Apoptotic Microparticles in Systemic Lupus Erythematosus Patients Drive the Activation of Dendritic Cell Subsets and Prime Neutrophils for NETosis.

OBJECTIVE: Circulating chromatin-containing apoptotic material and/or neutrophil extracellular traps (NETs) have been proposed to be an important driving force for the antichromatin autoimmune response in patients with systemic lupus erythematosus (SLE). The aim of this study was to determine the exact nature of microparticles in the circulation of SLE patients and to assess the effects of the microparticles on the immune system. METHODS: We analyzed microparticles isolated from the plasma of patients with SLE, rheumatoid arthritis (RA), and systemic sclerosis (SSc), as well as from healthy subjects. The effects of the microparticles on blood-derived dendritic cells (DCs) and neutrophils were assessed by flow cytometry, enzyme-linked immunosorbent assay, and immunofluorescence microscopy. RESULTS: In SLE patients, we identified microparticles that were highly positive for annexin V and apoptosis-modified chromatin that were not present in healthy subjects or in RA or SSc patients. These microparticles were mostly CD31+/CD45- (endothelial), partly CD45+/CD66b+ (granulocyte), and negative for B and T cell markers. Microparticles isolated from the plasma of SLE patients increased the expression of the costimulatory surface molecules CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines interleukin-6, tumor necrosis factor, and interferon-α by blood-derived plasmacytoid DCs (PDCs) and myeloid DCs (MDCs). SLE microparticles also primed blood-derived neutrophils for NETosis. Microparticles from healthy subjects and from RA or SSc patients exhibited no significant effects on MDCs, PDCs, and NETosis. CONCLUSION: Circulating microparticles in SLE patients include a population of apoptotic cell-derived microparticles that has proinflammatory effects on PDCs and MDCs and enhances NETosis. These results underline the important role of apoptotic microparticles in driving the autoimmune response in SLE patients.

Clinical Implications of Co-Inhibitory Molecule Expression in the Tumor Microenvironment for DC Vaccination: A Game of Stop and Go.

The aim of therapeutic dendritic cell (DC) vaccines in cancer immunotherapy is to activate cytotoxic T cells to recognize and attack the tumor. T cell activation requires the interaction of the T cell receptor with a cognate major-histocompatibility complex-peptide complex. Although initiated by antigen engagement, it is the complex balance between co-stimulatory and co-inhibitory signals on DCs that results in T cell activation or tolerance. Even when already activated, tumor-specific T cells can be neutralized by the expression of co-inhibitory molecules on tumor cells. These and other immunosuppressive cues in the tumor microenvironment are major factors currently hampering the application of DC vaccination. In this review, we discuss recent data regarding the essential and complex role of co-inhibitory molecules in regulating the immune response within the tumor microenvironment. In particular, possible therapeutic intervention strategies aimed at reversing or neutralizing suppressive networks within the tumor microenvironment will be emphasized. Importantly, blocking co-inhibitory molecule signaling, often referred to as immune checkpoint blockade, does not necessarily lead to an effective activation of tumor-specific T cells. Therefore, combination of checkpoint blockade with other immune potentiating therapeutic strategies, such as DC vaccination, might serve as a synergistic combination, capable of reversing effector T cells immunosuppression while at the same time increasing the efficacy of T cell-mediated immunotherapies. This will ultimately result in long-term anti-tumor immunity.

Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial.

BACKGROUND AIMS: RNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients. METHODS: CD14(+) cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilm's tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients. RESULTS: In a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14(+) cells (94.5+/-3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25+/-10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients. CONCLUSIONS: Our method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe.