Separating the Wheat from the Chaff among HLA-DQ Eplets.

In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQß-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQß eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.

Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of HLA molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells produce IL-6 in the steady-state and this is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in endothelial cell immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered endothelial cell interactions with allogeneic PBMC and after anti-HLA or DSA binding to endothelial cells in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation, and C4d deposition were examined. Blockade of IL-6 reduced endothelial cell secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of endothelial cells by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Th17, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

Tumor Lysis Syndrome and AKI: Beyond Crystal Mechanisms.

BACKGROUND: The pathophysiology of AKI during tumor lysis syndrome (TLS) is not well understood due to the paucity of data. We aimed to decipher crystal-dependent and crystal-independent mechanisms of TLS-induced AKI. METHODS: Crystalluria, plasma cytokine levels, and extracellular histones levels were measured in two cohorts of patients with TLS. We developed a model of TLS in syngeneic mice with acute myeloid leukemia, and analyzed ultrastructural changes in kidneys and endothelial permeability using intravital confocal microscopy. In parallel, we studied the endothelial toxicity of extracellular histones in vitro. RESULTS: The study provides the first evidence that previously described crystal-dependent mechanisms are insufficient to explain TLS-induced AKI. Extracellular histones that are released in huge amounts during TLS caused profound endothelial alterations in the mouse model. The mechanisms of histone-mediated damage implicates endothelial cell activation mediated by Toll-like receptor 4. Heparin inhibits extracellular histones and mitigates endothelial dysfunction during TLS. CONCLUSION: This study sheds new light on the pathophysiology of TLS-induced AKI and suggests that extracellular histones may constitute a novel target for therapeutic intervention in TLS when endothelial dysfunction occurs.

Endothelial cell, myeloid, and adaptive immune responses in SARS-CoV-2 infection.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is an emerging respiratory pathogen that has rapidly spread in human populations. Severe forms of infection associate cytokine release syndrome and acute lung injury due to hyperinflammatory responses even though virus clearance is achieved. Key components of inflammation include immune cell recruitment in infected tissues, a step which is under the control of endothelial cells. Here, we review endothelial cell responses in inflammation and infection due to SARS-CoV-2 together with phenotypic and functional alterations of monocytes, T and B lymphocytes with which they interact. We surmise that endothelial cells function as an integrative and active platform for the various cells recruited, where fine tuning of immune responses takes place and which provides opportunities for therapeutic intervention.

Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of human leukocyte antigen (HLA) molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells (ECs) produce IL-6 in the steady-state that is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in EC immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered EC interactions with allogeneic PBMC and after anti-HLA or DSA binding to ECs in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation and C4d deposition were examined. Blockade of IL-6 reduced EC secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of ECs by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

Endothelial Cells Activated by Extracellular Histones Promote Foxp3+ Suppressive Treg Cells In Vitro.

Histones are widely recognized as pro-inflammatory mediators upon their release from the nucleus into the extracellular space. However, their impact on endothelial cell immunogenicity is unknown. Endothelial cells, Human Microvascular Endothelial cells 1 (HMEC1), have been exposed to recombinant histones in order to study their effect on the endothelial phenotype. We then studied the differentiation of CD4+-T lymphocytes subpopulations after three days of interaction with endothelial cells in vitro and observed that histone-treated endothelial cells differentiate a suppressive FoxP3+ T regulator subpopulation that expressed Human Leucocyte Antigen DR (HLA-DR) and Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA4). Toll-Like Receptor 4 (TLR4) inhibition significantly decreased the expansion of these Treg cells. Moreover, blockade of Interleukin (IL)-6 and Intercellular Adhesion Molecule (ICAM)-1 in cocultures significantly decreased the expansion of Tregs, suggesting an IL-6 and ICAM-1 dependent pathway. Thus, beyond their inflammatory effects, extracellular histones may induce an increase of immunosuppressive Treg population via their action on endothelial cells. Further studies are needed to evaluate the impact on immunosuppression of an increase of peripheral suppressive Treg via endothelial cell activation by histones in vivo.

T cell antigenicity and immunogenicity of allogeneic exosomes.

Extracellular vesicles, including exosomes, are regularly released by allogeneic cells after transplantation. Recipient antigen-presenting cells (APCs) capture these vesicles and subsequently display donor MHC molecules on their surface. Recent evidence suggests that activation of alloreactive T cells by the so-called cross-dressed APCs plays an important role in initiating the alloresponse associated with allograft rejection. On the other hand, whether allogeneic exosomes can bind to T cells on their own and activate them remains unclear. In this study, we showed that allogeneic exosomes can bind to T cells but do not stimulate them in vitro unless they are cultured with APCs. On the other hand, allogeneic exosomes activate T cells in vivo and sensitize mice to alloantigens but only when delivered in an inflammatory environment.

Cytokines in Liver Transplantation.

Cytokines, soluble mediators of the immune system, play a critical role in the pathogenesis of autoimmune, allergic and infectious diseases. They are also implicated in the initiation and development of allograft rejection. During recent years, there have been considerable advances in generating novel anti-cytokine agents with promoted efficacy and safety, which could be administrated for managing dysregulated cytokine secretion; besides, gene therapy for overexpression of immunomodulatory cytokines has shown substantial improvements. Liver transplantation has been established as a life-saving treatment for end-stage hepatic diseases but the growing number of recipients urge for improved post-transplant care including tolerance induction, infection control and resolving immunosuppressant drugs adverse effects. Cytokines with a wide range of proinflammatory and regulatory properties might be considered as potential therapeutic targets for selective suppression or enhancement of the immune responses in recipients. In the present review, we aimed to summarize the positive and negative effects of cytokines on liver allograft in addition to their prognostic and therapeutic values.

HLA-DQ alloantibodies directly activate the endothelium and compromise differentiation of FoxP3high regulatory T lymphocytes.

Development of donor-specific antibodies is associated with reduced allograft survival in renal transplantation. Recent clinical studies highlight the prevalence of human leukocyte antigen (HLA)-DQ antibodies amongst de novo donor-specific antibodies (DSAs), yet the specific contribution of these DSAs to rejection has not been examined. Antibody-mediated rejection primarily targets the microvasculature, so this study explored how patient HLA-DQ alloantibodies can modulate endothelial activation and so immunoregulation. HLA-DQ antibodies phosphorylated Akt and S6 kinase in microvascular endothelial cells. This activation prior to culture with alloreactive lymphocytes increased IL-6 and RANTES secretion. The antibody-mediated upregulation of IL-6 was indeed Akt-dependent. The binding of HLA-DQ antibodies to endothelial cells selectively reduced T cell alloproliferation and FoxP3high Treg differentiation. In clinical studies, detection of HLA-DQ DSAs with other DSAs is associated with worse graft survival than either alone. Endothelial cells stimulated with HLA-DR and HLA-DQ antibodies showed a synergistic increase in pro-inflammatory cytokine secretion and a decrease in Treg expansion. HLA-DQ antibodies strongly promote pro-inflammatory responses in isolation and in combination with other HLA antibodies. Thus, our data give new insights into the pathogenicity of HLA-DQ DSAs.

Markers of Endothelial-to-Mesenchymal Transition: Evidence for Antibody-Endothelium Interaction during Antibody-Mediated Rejection in Kidney Recipients.

Antibody-mediated rejection (ABMR) is a leading cause of allograft loss. Treatment efficacy depends on accurate diagnosis at an early stage. However, sensitive and reliable markers of antibody-endothelium interaction during ABMR are not available for routine use. Using immunohistochemistry, we retrospectively studied the diagnostic value of three markers of endothelial-to-mesenchymal transition (EndMT), fascin1, vimentin, and heat shock protein 47, for ABMR in 53 renal transplant biopsy specimens, including 20 ABMR specimens, 24 cell-mediated rejection specimens, and nine normal grafts. We validated our results in an independent set of 74 unselected biopsy specimens. Endothelial cells of the peritubular capillaries in grafts with ABMR expressed fascin1, vimentin, and heat shock protein 47 strongly, whereas those from normal renal grafts did not. The level of EndMT marker expression was significantly associated with current ABMR criteria, including capillaritis, glomerulitis, peritubular capillary C4d deposition, and donor-specific antibodies. These markers allowed us to identify C4d-negative ABMR and to predict late occurrence of disease. EndMT markers were more specific than capillaritis for the diagnosis and prognosis of ABMR and predicted late (up to 4 years after biopsy) renal graft dysfunction and proteinuria. In the independent set of 74 renal graft biopsy specimens, the EndMT markers for the diagnosis of ABMR had a sensitivity of 100% and a specificity of 85%. Fascin1 expression in peritubular capillaries was also induced in a rat model of ABMR. In conclusion, EndMT markers are a sensitive and reliable diagnostic tool for detecting endothelial activation during ABMR and predicting late loss of allograft function.

Complement-binding anti-HLA antibodies and kidney-allograft survival.

BACKGROUND: Anti-HLA antibodies hamper successful transplantation, and activation of the complement cascade is involved in antibody-mediated rejection. We investigated whether the complement-binding capacity of anti-HLA antibodies plays a role in kidney-allograft failure. METHODS: We enrolled patients who received kidney allografts at two transplantation centers in Paris between January 1, 2005, and January 1, 2011, in a population-based study. Patients were screened for the presence of circulating donor-specific anti-HLA antibodies and their complement-binding capacity. Graft injury phenotype and the time to kidney-allograft loss were assessed. RESULTS: The primary analysis included 1016 patients. Patients with complement-binding donor-specific anti-HLA antibodies after transplantation had the lowest 5-year rate of graft survival (54%), as compared with patients with non-complement-binding donor-specific anti-HLA antibodies (93%) and patients without donor-specific anti-HLA antibodies (94%) (P<0.001 for both comparisons). The presence of complement-binding donor-specific anti-HLA antibodies after transplantation was associated with a risk of graft loss that was more than quadrupled (hazard ratio, 4.78; 95% confidence interval [CI], 2.69 to 8.49) when adjusted for clinical, functional, histologic, and immunologic factors. These antibodies were also associated with an increased rate of antibody-mediated rejection, a more severe graft injury phenotype with more extensive microvascular inflammation, and increased deposition of complement fraction C4d within graft capillaries. Adding complement-binding donor-specific anti-HLA antibodies to a traditional risk model improved the stratification of patients at risk for graft failure (continuous net reclassification improvement, 0.75; 95% CI, 0.54 to 0.97). CONCLUSIONS: Assessment of the complement-binding capacity of donor-specific anti-HLA antibodies appears to be useful in identifying patients at high risk for kidney-allograft loss.

Human endothelial cells generate Th17 and regulatory T cells under inflammatory conditions.

Organ transplantation represents a unique therapeutic option for irreparable organ dysfunction and rejection of transplants results from a breakdown in operational tolerance. Although endothelial cells (ECs) are the first target in graft rejection following kidney transplantation, their capacity to alloactivate and generate particular T lymphocyte subsets that could intervene in this process remains unknown. By using an experimental model of microvascular endothelium, we demonstrate that, under inflammatory conditions, human ECs induced proliferation of memory CD4(+)CD45RA(-) T cells and selectively amplified proinflammatory Th17 and suppressive CD45RA(-)HLA-DR(+)FoxP3(bright) regulatory CD4(+) T lymphocytes (Tregs). Although HLA-DR expression on resting microvascular ECs was sufficient to induce proliferation of memory CD4(+) T cells, Treg amplification was dependent on the interaction with CD54, highly expressed only under inflammatory conditions. Moreover, expansion of Th17 cells was dependent on IL-6 and STAT-3, and inhibition of either specifically impaired Th17, without altering Treg expansion. Collectively these data reveal that the HLA-DR(+) ECs regulate the local inflammatory allogeneic response, promoting either an IL-6/STAT-3-dependent Th17 response or a contact-CD54-dependent regulatory response according to the cytokine environment. Finally, these data open therapeutic perspectives in human organ transplantation based on targeting the IL-6/STAT-3 pathway and/or promoting CD54 dependent Treg proliferation.

HLA-G inhibition of NK-cell cytolytic function is uncoupled from tumor cell lipid raft reorganization.

HLA-G is a non-classical HLA class I molecule with tolerogenic properties and restricted tissue distribution. The expression of HLA-G can be induced by tumors thus providing an efficient way to escape the anti-tumoral immune response. Although lipid rafts regulate diverse immunological mechanisms their relationship with HLA-G remains controversial. Our results show that HLA-G-mediated inhibition of both the interaction between NK and tumor cells, and of intracellular calcium flux in NK cells conjugated to their target cells were independent of lipid raft integrity. In addition, cytotoxicity assays indicated that HLA-G continued to efficiently inhibit NK-cell cytolytic function in several different tumor cells independently of lipid raft integrity. Confocal microscopy with 3D reconstruction combined with biochemical analysis showed that HLA-G was mainly localized outside the lipid rafts of tumor cells after cross-linking with specific antibody and remained excluded from lipid rafts during interaction with the ILT2 inhibitory receptor of NK cells. This study indicates that the inhibitory function of HLA-G is uncoupled from lipid raft organization, further distinguishing HLA-G from classical HLA molecules and providing novel information in the understanding of tumor immune escape mechanism mediated through HLA-G.

Assessing the allogenic realness of the Cw1/12/15 pattern occurring in the LABScreen single antigen assay.

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.

Endothelial CD34 expression and regulation of immune cell response in-vitro.

Endothelial cells cover the lining of different blood vessels and lymph nodes, and have major functions including the transport of blood, vessel homeostasis, inflammatory responses, control of transendothelial migration of circulating cells into the tissues, and formation of new blood vessels. Therefore, understanding these cells is of major interest. The morphological features, phenotype and function of endothelial cells varies according to the vascular bed examined. The sialomucin, CD34, is widely used as an endothelial marker. However, CD34 is differentially expressed on endothelial cells in different organs and in pathological conditions. Little is known about regulation of endothelial CD34 expression or function. Expression of CD34 is also strongly regulated in-vitro in endothelial cell models, including human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC). We have therefore analysed the expression and function of CD34 by comparing CD34high and CD34low endothelial cell subpopulations. Transcriptomic analysis showed that CD34 gene and protein expressions are highly correlated, that CD34high cells proliferate less but express higher levels of IL-33 and Angiopoietin 2, compared with CD34low cells. Higher secretion levels of IL-33 and Angiopoietin 2 by CD34high HUVECs was confirmed by ELISA. Finally, when endothelial cells were allowed to interact with peripheral blood mononuclear cells, CD34high endothelial cells activated stronger proliferation of regulatory T lymphocytes (Tregs) compared to CD34low cells whereas expansion of other CD4+-T cell subsets was equivalent. These results suggest that CD34 expression by endothelial cells in-vitro associates with their ability to proliferate and with an immunogenic ability that favours the tolerogenic response.

Endothelial cells: major players in acute myeloid leukaemia.

The role of the vascular microenvironment is increasingly studied in acute myeloid leukaemia (AML). Complex interactions between endothelial cells (ECs) and pre-leukaemic cells may contribute to the clonal evolution of pre-leukaemic stem cells in the bone marrow niche and to the proliferation, survival and chemoresistance of leukaemic cells. Through the expression of different adhesion molecules, ECs play a key role in the development of specific acute complications of AML, including leukostasis, acute respiratory failure, acute kidney injury or neurological complications. Moreover, in newly diagnosed patients, leukaemic cells promote endothelial activation and subsequent disseminated intravascular coagulation. Mechanisms of this bi-directional dialogue between leukaemic cells and ECs will reveal possible therapeutic targets to be explored to improve the survival of AML patients.

Relevance of Anti-HLA Antibody Strength Underestimation in Single Antigen Bead Assay for Shared Eplets.

BACKGROUND: HLAs contain combinations of multiple eplets, sometimes shared between numerous HLA alleles. Some authors suggested that single antigen bead (SAB) assays may underestimate the signal of anti-HLA antibodies (Ab) when several beads share the targeted eplet. However, this assumption has not yet been validated experimentally. METHODS: We selected 5 eplets shared by 1-24 beads of the routine SAB kits: the eplet 163LS/G; the 3 eplets 127K, 62GE, and 62GRN thereafter called cross-reactive group 2C; the 82LR eplet, well-known as Bw4; the locally called QB2A5 eplet associated with the DQA1*05:01/DQB1*02:01 combination; and the 40GR DQ eplet. We selected a dozen of sera for each eplet with Ab mean fluorescence intensity (MFI) between 1000 and 15 000 for the beads carrying the targeted eplet. We tested them with the classical SAB panel (SABp), with an isolated bead carrying the eplet (isolated SAB [SABi]) and with a mixture of both (SABp+i). RESULTS: No significant difference in MFI was detected among SABi, SABp, and SABp+i conditions for all the eplets. CONCLUSIONS: We noticed only a nonsignificant difference in the Ab MFI signal due to eplet sharing on the SAB assay. We, therefore, conclude that this phenomenon should no longer be considered as a significant risk factor during patient follow-up pre- or posttransplantation.