Mtb HLA-E-tetramer-sorted CD8+ T cells have a diverse TCR repertoire.

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFß/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206-) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFß-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice.

Cardiac Progenitor Cell-Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation.

Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI.

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction.

AIMS: Hereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFß co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery. METHODS AND RESULTS: After inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (Eng+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4+ MNCs residing in the infarcted Eng+/- hearts (Eng+/- 73.17±12.67 vs. Eng+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (Eng+/- 46.60±9.33% vs. Eng+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in Eng+/- mice. An increased number of capillaries (Eng+/- 61.63±1.43 vs. Eng+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (Eng+/- 11.88±0.63 vs. Eng+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in Eng+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. Eng+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels. CONCLUSIONS: In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in Eng+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.

Glycosylated Cell Surface Markers for the Isolation of Human Cardiac Progenitors.

The aim of stem cell therapy after cardiac injury is to replace damaged cardiac tissue. Human cardiac progenitor cells (CPCs) represent an interesting cell population for clinical strategies to treat cardiac disease and human CPC-specific antibodies would aid in the clinical implementation of cardiac progenitor-based cell therapy. However, the field of CPC biology suffers from the lack of human CPC-specific markers. Therefore, we raised a panel of monoclonal antibodies (mAb) against CPCs. Of this panel of antibodies, we show that mAb C1096 recognizes a progenitor-like population in the fetal and adult human heart and partially colocalize with reported CPC populations in vitro. Furthermore, mAb C1096 can be used to isolate a multipotent progenitor population from human heart tissue. Interestingly, the two lead candidates, mAb C1096 and mAb C19, recognize glycosylated residues on PECAM1 (platelet and endothelial cell adhesion molecule 1) and GRP78, respectively, and de-N-glycosylation significantly abolishes their binding. Thereby, this report describes new clinically applicable antibodies against human CPCs, and for the first time demonstrates the importance of glycosylated residues as CPCs specific markers.

Human fetal and adult epicardial-derived cells: a novel model to study their activation.

BACKGROUND: The epicardium, a cell layer covering the heart, plays an important role during cardiogenesis providing cardiovascular cell types and instructive signals, but becomes quiescent during adulthood. Upon cardiac injury the epicardium is activated, which includes induction of a developmental gene program, epithelial-to-mesenchymal transition (EMT) and migration. However, the response of the adult epicardium is suboptimal compared to the active contribution of the fetal epicardium to heart development. To understand the therapeutic value of epicardial-derived cells (EPDCs), a direct comparison of fetal and adult sources is paramount. Such analysis has been hampered by the lack of appropriate culture systems. METHODS: Human fetal and adult EPDCs were isolated from cardiac specimens obtained after informed consent. EPDCs were cultured in the presence of an inhibitor of the TGFß receptor ALK5. EMT was induced by stimulation with 1 ng/ml TGFß. PCR, immunofluorescent staining, scratch assay, tube formation assay and RT2-PCR for human EMT genes were performed to functionally characterize and compare fetal and adult EPDCs. RESULTS: In this study, a novel protocol is presented that allows efficient isolation of human EPDCs from fetal and adult heart tissue. In vitro, EPDCs maintain epithelial characteristics and undergo EMT upon TGFß stimulation. Although similar in several aspects, we observed important differences between fetal and adult EPDCs. Fetal and adult cells display equal migration abilities in their epithelial state. However, while TGFß stimulation enhanced adult EPDC migration, it resulted in a reduced migration in fetal EPDCs. Matrigel assays revealed the ability of adult EPDCs to form tube-like structures, which was absent in fetal cells. Furthermore, we observed that fetal cells progress through EMT faster and undergo spontaneous EMT when TGFß signaling is not suppressed, indicating that fetal EPDCs more rapidly respond to environmental changes. CONCLUSIONS: Our data suggest that fetal and adult EPDCs are in a different state of activation and that their phenotypic plasticity is determined by this activation state. This culture system allows us to establish the cues that determine epicardial activation, behavior, and plasticity and thereby optimize the adult response post-injury.

Mononuclear cells and vascular repair in HHT.

Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and activin receptor-like kinase 1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs) fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing) of MNCs following tissue damage is regulated by the stromal cell derived factor 1 (SDF1). MNCs that express the C-X-C chemokine receptor 4 (CXCR4) migrate toward the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4). Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing toward damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair.