Graft endothelial cell (EC) injury is central to the pathogenesis of antibody-mediated rejection (AMR). The ability of donor-specific antibodies (DSA) to bind C1q and activate the classical ...complement pathway is an efficient predictor of graft rejection highlighting complement-dependent cytotoxicity as a key process operating during AMR. In the past 5 y, clinical studies further established the cellular and molecular signatures of AMR revealing the key contribution of other, IgG-dependent and -independent, effector mechanisms mediated by infiltrating NK cells and macrophages. Beyond binding to alloantigens, DSA IgG can activate NK cells and mediate antibody-dependent cell cytotoxicity through interacting with Fcγ receptors (FcγRs) such as FcγRIIIa (CD16a). FcRn, a nonconventional FcγR that allows IgG recycling, is highly expressed on ECs and may contribute to the long-term persistence of DSA in blood. Activation of NK cells and macrophages results in the production of proinflammatory cytokines such as TNF and IFNγ that induce transient and reversible changes in the EC phenotype and functions promoting coagulation, inflammation, vascular permeability, leukocyte trafficking. MHC class I mismatch between transplant donor and recipient can create a situation of "missing self" allowing NK cells to kill graft ECs. Depending on the microenvironment, cellular proximity with ECs may participate in macrophage polarization toward an M1 proinflammatory or an M2 phenotype favoring inflammation or vascular repair. Monocytes/macrophages participate in the loss of endothelial specificity in the process of endothelial-to-mesenchymal transition involved in renal and cardiac fibrosis and AMR and may differentiate into ECs enabling vessel and graft (re)-endothelialization.
As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte ...trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.
Notch signaling is a major pathway in cell fate decisions. Since the first reports showing the major role of Notch in embryonic development, a considerable and still growing literature further ...highlights its key contributions in various pathological processes during adult life. In particular, Notch is now considered as a major player in vascular homeostasis through the control of key cellular functions. In parallel, confounding evidence emerged that inflammatory responses regulate Notch signaling in vitro in endothelial cells, smooth muscle cells or vascular infiltrating cells and in vivo in vascular and inflammatory disorders and in cardiovascular diseases. This review presents how inflammation influences Notch in vascular cells and, reciprocally, emphasizes the functional role of Notch on inflammatory processes, notably by regulating key cell functions (differentiation, proliferation, apoptosis/survival, activation). Understanding how the disparity of Notch receptors and ligands impacts on vasculature biology remains critical for the design of relevant and adequate therapeutic strategies targeting Notch in this major pathological context.
Notch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis. In mammals, four Notch receptors (Notch 1-4) can interact with ...five distinct ligands Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4. We previously reported that Notch activation is modulated in endothelial cells and monocytes during inflammation and showed that inflammation upregulates DLL4 on endothelial cells. DLL4 promotes differentiation of blood monocytes into proinflammatory M1 macrophages. Here, we further investigated the ability of DLL4 to interfere with the polarization of blood monocytes into immunosuppressive M2 macrophages.
Human blood monocytes were differentiated in vitro into M0 macrophages and then polarized into M1 or M2 macrophages with LPS/IFNγ and IL-4, respectively. Polarization steps were performed in the presence of immobilized recombinant DLL4. Immune phenotype and apoptosis of macrophage subsets were analyzed and quantified by flow cytometry. Regulatory effects of DLL4 on gene expression, cell signaling and apoptotic pathways were investigated by QPCR and western blots.
The phenotype of M2 macrophages was subject to specific alterations in the presence of recombinant DLL4. DLL4 inhibits the upregulation of IL-4 induced M2 markers such as CD11b, CD206, and CD200R. Survival of macrophages upon M2 polarization was also strongly reduced in the presence of DLL4. DLL4 induces a caspase3/7-dependent apoptosis during M2 but not M1 macrophage polarization. The Notch ligand DLL1 has no apoptotic effect. Both DLL4 signaling via Notch1 as well as DLL4-mediated apoptosis are Notch-dependent. Fully differentiated M2 macrophages became resistant to DLL4 action. Mechanistically, DLL4 selectively upregulates gene expression in macrophages upon M2 polarization, thereby affecting the Notch pattern (Notch1, 3, Jag1), activity (HES1), and transcription (IRF5, STAT1). The pro-apoptotic effectors Bax and Bak and the BH3-only proteins Bid and Bim seem to convey DLL4 apoptotic signal.
Interplay between the DLL4/Notch and IL-4/IL-4R signaling pathways impairs M2 differentiation. Thus, DLL4 may drive a Notch-dependent selection process not only by promoting M1 macrophage differentiation but also by preventing M2 macrophage differentiation through inhibition of M2-specific gene expression and apoptotic cell death.
Lnk (SH2B3) emerges as a key regulator in hematopoeitic and vascular cells moderating growth factor, integrin and cytokine receptor-mediated signaling.
A better knowledge of the process by which ...inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.
Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. ...Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of β2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.
HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for ...various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA
tetramer complexes (HLA-E
, HLA-E
, and HLA-A2
) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV
and HCMV
individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E
and HLA-A2
CD8 T cells, and expansion of γδT and NK subsets including δ2
γT and memory-like NKG2C
CD57
NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.
Human leukocyte antigen (HLA)–E belongs, with HLA-G and HLA-F, to the nonclassic major histocompatibility complex (MHC) class I (Ib) molecules, broadly defined by a limited polymorphism and a ...restricted pattern of cellular expression. In contrast to HLA-G, the expression and function of HLA-E and HLA-F in physiologic and pathologic processes remain poorly established. In the present study, we show that HLA-E protein expression in normal human nonlymphoid organs is mainly restricted to endothelial cells (ECs). HLA-E is also basally expressed by B and T lymphocytes, natural killer (NK) cells and by macrophages. We demonstrate that tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interferon γ (IFNγ) up-regulate the cell-surface expression of HLA-E on ECs in vitro and induce the release of soluble HLA-E (sHLA-E). HLA-E up-regulation protects IFNγ-activated ECs from NK-mediated cell lysis, while sHLA-E protects bystander cells. Finally, sHLA-E is not detected in normal sera, and increased serum levels correlate with disease activity in patients with antineutrophil cytoplasmic antibody–associated systemic vasculitis. Thus, HLA-E expression and release of sHLA-E are features of EC activation and emphasize immunoregulatory functions of the endothelium. The present identification of soluble HLA-E molecules may have important implications in understanding the pathogenesis of immune-mediated vascular diseases and for the diagnosis and monitoring of patients.
This study investigated the frequency and peptide specificity of long-lasting HCMV-specific CD8 T cells in a cohort of 120 cytomegalovirus seropositive (HCMV+) healthy carriers with the aim of ...deciphering the relative contribution of unconventional HLA-E- versus conventional HLA-A2-specific CD8 T cells to long-term T cell memory expansion in HCMV immunity. The presence of HCMV-specific CD8 T cells was investigated by flow cytometry using five MHC/peptide tetramer complexes (HLA-A2/pp65, HLA-A2/IE1 and three different HLA-E/UL40). Here, we report that 50% of HCMV+ healthy individuals possess HCMV-specific CD8 T cells, representing ≥0.1% of total blood CD8 T cells years post-infection. Around a third (30.8%) of individuals possess HLA-A2-restricted (A2pp65 or A2IE1) and an equal proportion (27.5%) possess an HLA-E/UL40 CD8 T response. Concomitant HLA-E- and HLA-A2-reactive CD8 T cells were frequently found, and VMAPRTLIL peptide was the major target. The frequency of HLA-E/VMAPRTLIL among total blood CD8 T cells was significantly higher than the frequency of HLA-A2pp65 T cells (mean values: 5.9% versus 2.3%,
= 0.0354). HLA-EUL40 CD8 T cells display lower TCR avidity but similar levels of CD3 and CD8 coreceptors. In conclusion, HLA-E-restricted CD8 T cells against the VMAPRTLIL UL40 peptide constitute a predominant subset among long-lasting anti-HCMV CD8 T cells.
Binding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, ...including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules ("missing self") on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR.
Among 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay (
=73, 54%) had a higher risk of transplant failure (
=0.002). Among the remaining patients with complement-independent chronic AMR (
=62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival (
=0.02). In multivariable analysis, only proteinuria (HR: 7.24;
=0.01) and the presence of missing self (HR: 3.57;
=0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage.
The assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure.
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