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Ab-mediated rejection of organ transplants remains a stubborn, frequent problem affecting patient quality of life, graft function, and grant survival, and for which few efficacious therapies currently exist. Although the field has gained considerable knowledge over the last two decades on how anti-HLA Abs cause acute tissue injury and promote inflammation, there has been a gap in linking these effects with the chronic inflammation, vascular remodeling, and persistent alloimmunity that leads to deterioration of graft function over the long term. This review will discuss new data emerging over the last 5 y that provide clues into how ongoing Ab-endothelial cell interactions may shape vascular fate and propagate alloimmunity in organ transplants., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The adhesion and subsequent activation of T cells is a critical step in local inflammatory responses, particularly of alloreactive leukocytes in rejection of transplanted donor tissue. Interferon (IFN)γ is an adaptive cytokine that promotes endothelial cell (EC) expression of pro-adhesive factors and costimulatory molecules. We recently reported that IFNγ-induced endothelial cell antigen-presenting capacity was protracted after cytokine withdrawal. This study sought to determine what intracellular signaling mediates this chronic endothelial activation by IFNγ. The durability of interferon signaling in human aortic endothelial activation was tested. Pro-adhesive and costimulatory gene expression, phenotype, secretome, and Janus kinase (JAK)/STAT phosphorylation in human primary endothelial cells were measured under chronic and transient IFNγ stimulation, with various JAK inhibitors. IFNγ reporter cells were tested for STAT1 transcriptional activity with JAK inhibition and suppressors of cytokine signaling (SOCS) overexpression, under continuous and priming conditions. The consequences of even short exposure to IFNγ were long-lasting and broad, with sustained elevation of adhesion molecules and chemokines up to 48 h later. JAK/STAT and interferon response factor expression were likewise durable, dependent on new transcription but autonomous of continuous IFNγ. Persistent STAT new transcription and JAK signaling in the endothelium was required to maintain a pro-adhesive and proimmunogenic phenotype after IFNγ withdrawal since both could be prevented by cycloheximide but only by JAKinibs with potency against JAK2. Finally, the suppressor of cytokine signaling SOCS1 failed to emerge in primed endothelial cells, which likely accounted for prolonged inflammatory gene expression. The results reveal a sustained JAK-dependent perturbation of endothelial function and suggest that JAKinibs may have therapeutic benefits in dampening vascular inflammation and allogeneic leukocyte activation. NEW & NOTEWORTHY The central question investigated in this study is why vascular endothelium remains inflamed and what underlying signaling is responsible. The new results show that the resolution of endothelial-controlled inflammation may be impaired or delayed because Janus kinase (JAK)/STAT activation is maintained autonomous of interferon (IFN)γ presence, and the late phase negative regulator suppressors of cytokine signaling (SOCS)1 fails to be induced.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin β 4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Background: Vascular endothelial cells (EC) are critical for regulation of local immune responses, through coordination of leukocyte recruitment from the blood and egress into the tissue. Growing evidence supports an additional role for endothelium in activation and costimulation of adaptive immune cells. However, this function remains somewhat controversial, and the full repertoire and durability of an enhanced endothelial costimulatory phenotype has not been wholly defined., Methods: Human endothelium was stimulated with continuous TNFα or IFNγ for 1-48hr; or primed with TNFα or IFNγ for only 3hr, before withdrawal of stimulus for up to 45hr. Gene expression of cytokines, costimulatory molecules and antigen presentation molecules was measured by Nanostring, and publicly available datasets of EC stimulation with TNFα or IFNγ were leveraged to further corroborate the results. Cell surface protein expression was detected by flow cytometry, and secretion of cytokines was assessed by Luminex and ELISA. Key findings were confirmed in primary human endothelial cells from 4-6 different vascular beds., Results: TNFα triggered mostly positive immune checkpoint molecule expression on endothelium, including CD40, 4-1BB, and ICOSLG but in the context of only HLA class I and immunoproteasome subunits. IFNγ promoted a more tolerogenic phenotype of high PD-L1 and PD-L2 expression with both HLA class I and class II molecules and antigen processing genes. Both cytokines elicited secretion of IL-15 and BAFF/BLyS, with TNFα stimulated EC additionally producing IL-6, TL1A and IL-1β. Moreover, endothelium primed for a short period (3hr) with TNFα mostly failed to alter the costimulatory phenotype 24-48hr later, with only somewhat augmented expression of HLA class I. In contrast, brief exposure to IFNγ was sufficient to cause late expression of antigen presentation, cytokines and costimulatory molecules. In particular HLA class I, PD-1 ligand and cytokine expression was markedly high on endothelium two days after IFNγ was last present., Conclusions: Endothelia from multiple vascular beds possess a wide range of other immune checkpoint molecules and cytokines that can shape the adaptive immune response. Our results further demonstrate that IFNγ elicits prolonged signaling that persists days after initiation and is sufficient to trigger substantial gene expression changes and immune phenotype in vascular endothelium., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valenzuela.)

Endothelial cells (EC) coordinate vascular homeostasis and inflammation. In organ transplantation, EC are a direct alloimmune target. We posited that tissue specific heterogeneity of vascular EC may partly underlie the disparate organ-specific alloimmune risk. We examined the vascular endothelial response to inflammation across six primary endothelial beds from four major transplanted organs: the heart, lung, kidney and liver. First, we reanalyzed a public dataset of cardiac allograft rejection and found that endothelial inflammatory response genes were elevated in human cardiac allograft biopsies undergoing rejection compared with stable grafts. Next, the inducible inflammatory phenotypes of EC from heart, lung, kidney, and liver were characterized in vitro, focused on expression of adhesion molecules and chemokines, and recruitment of allogeneic peripheral blood mononuclear immune cells. Large vessel cardiac EC most highly upregulated VCAM-1, particularly compared with hepatic EC, supported greater leukocyte adhesion and had distinct chemokine profiles after stimulation with cytokines and complement. Differentially expressed gene candidates that are known regulators of cytokine signaling and inflammatory programming were verified in publicly available datasets of organ-specific endothelial transcriptomes. In summary, differential baseline expression of immune regulating genes may contribute to differential vascular inflammatory responses depending on organ.

HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68 + CD206 + CD163 + macrophages (M(HLA I IgG)), whereas HLA I F(ab') 2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab') 2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Transplant recipients developing donor-specific HLA class II (HLA-II) Abs are at higher risk for Ab-mediated rejection (AMR) and transplant vasculopathy. To understand how HLA-II Abs cause AMR and transplant vasculopathy, we determined the signaling events triggered in vascular endothelial cells (EC) following Ab ligation of HLA-II molecules. HLA-II expression in EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNF-α/IFN-γ. Ab ligation of class II stimulated EC proliferation and migration. Class II Ab also induced activation of key signaling nodes Src, focal adhesion kinase, PI3K, and ERK that regulated downstream targets of the mammalian target of rapamycin (mTOR) pathway Akt, p70 ribosomal S6 kinase, and S6 ribosomal protein. Pharmacological inhibitors and small interfering RNA showed the protein kinases Src, focal adhesion kinase, PI3K/Akt, and MEK/ERK regulate class II Ab-stimulated cell proliferation and migration. Treatment with rapalogs for 2 h did not affect HLA-II Ab-induced phosphorylation of ERK; instead, mTOR complex (mTORC)1 targets were dependent on activation of ERK. Importantly, suppression of mTORC2 for 24 h with rapamycin or everolimus or treatment with mTOR active-site inhibitors enhanced HLA-II Ab-stimulated phosphorylation of ERK. Furthermore, knockdown of Rictor with small interfering RNA caused overactivation of ERK while abolishing phosphorylation of Akt Ser 473 induced by class II Ab. These data are different from HLA class I Ab-induced activation of ERK, which is mTORC2-dependent. Our results identify a complex signaling network triggered by HLA-II Ab in EC and indicate that combined ERK and mTORC2 inhibitors may be required to achieve optimal efficacy in controlling HLA-II Ab-mediated AMR., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The signaling capacity of HLA molecules in vascular cells has been well established. Intracellular signaling and association with the coreceptor integrin β4 has been well-studied for HLA class I. However, little is known regarding HLA class II intracellular signaling in human endothelial cells. Investigation of HLA class II has been challenging due to the loss of HLA class II expression in cultured primary cells. Herein, we describe methods for inducing expression of endogenous alleles and loci of HLA class II molecules, as well as for studying intracellular signaling. This includes siRNA knockdown of proteins and coimmunoprecipitation of putative coreceptors for HLA in primary human aortic endothelial cells.

Background: Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling., Methods: Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003)., Results: Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement., Conclusions: Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.

Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions.

Purpose of Review: Advances in immunosuppression and patient management have successfully improved 1-year transplant outcome. Unfortunately, antibody-mediated rejection is a major barrier to long-term graft survival. This study summarizes the effects of antibodies on endothelial cell and smooth muscle cell (SMC) migration, proliferation and leukocyte recruitment, emphasizing the intracellular signaling pathways that orchestrate these distinct functional outcomes., Recent Findings: Several studies have provided further insight into the effects of human leukocyte antigen (HLA) class I antibodies on vascular cells. We found that HLA I molecules partner with integrin β4 to transduce proliferative signaling, and identified proteins that associate with the cytoskeleton after HLA class I crosslinking. Natural killer cells have been strongly implicated in a murine model of donor-specific major histocompatibility complex I antibody-triggered neointimal thickening. A recently developed human arterial graft model revealed the role of matrix metalloproteinases in SMC mitogenesis by HLA class I antibodies. Using a donor transgenic for HLA-A2, Fukami et al. investigated the mechanisms of accommodation induced by low titers of HLA class I antibodies., Summary: Ligation of HLA class I molecules with antibodies leads to the activation of intracellular signals in endothelial cells and SMCs, which in turn promote actin cytoskeletal remodeling, survival, proliferation, and recruitment of leukocytes.

BackgroundVascular endothelial cells (EC) are critical for regulation of local immune responses, through coordination of leukocyte recruitment from the blood and egress into the tissue. Growing evidence supports an additional role for endothelium in activation and costimulation of adaptive immune cells. However, this function remains somewhat controversial, and the full repertoire and durability of an enhanced endothelial costimulatory phenotype has not been wholly defined.MethodsHuman endothelium was stimulated with continuous TNFα or IFNγ for 1-48hr; or primed with TNFα or IFNγ for only 3hr, before withdrawal of stimulus for up to 45hr. Gene expression of cytokines, costimulatory molecules and antigen presentation molecules was measured by Nanostring, and publicly available datasets of EC stimulation with TNFα or IFNγ were leveraged to further corroborate the results. Cell surface protein expression was detected by flow cytometry, and secretion of cytokines was assessed by Luminex and ELISA. Key findings were confirmed in primary human endothelial cells from 4-6 different vascular beds.ResultsTNFα triggered mostly positive immune checkpoint molecule expression on endothelium, including CD40, 4-1BB, and ICOSLG but in the context of only HLA class I and immunoproteasome subunits. IFNγ promoted a more tolerogenic phenotype of high PD-L1 and PD-L2 expression with both HLA class I and class II molecules and antigen processing genes. Both cytokines elicited secretion of IL-15 and BAFF/BLyS, with TNFα stimulated EC additionally producing IL-6, TL1A and IL-1β. Moreover, endothelium primed for a short period (3hr) with TNFα mostly failed to alter the costimulatory phenotype 24-48hr later, with only somewhat augmented expression of HLA class I. In contrast, brief exposure to IFNγ was sufficient to cause late expression of antigen presentation, cytokines and costimulatory molecules. In particular HLA class I, PD-1 ligand and cytokine expression was markedly high on endothelium two days after IFNγ was last present.ConclusionsEndothelia from multiple vascular beds possess a wide range of other immune checkpoint molecules and cytokines that can shape the adaptive immune response. Our results further demonstrate that IFNγ elicits prolonged signaling that persists days after initiation and is sufficient to trigger substantial gene expression changes and immune phenotype in vascular endothelium.

Human leukocyte antigen (HLA)-induced signaling in endothelial and smooth muscle cells causes dramatic cytoskeletal rearrangement, increased survival, motility, proliferation, adhesion molecule and chemokine expression, and adhesion of leukocytes. These mechanisms are directly related to endothelial activation, neointimal proliferation, and intragraft accumulation of leukocytes during antibody-mediated rejection (AMR) and chronic rejection. Clustering of HLA by ligands in trans, such as in antigen-presenting cells at the immune synapse, triggers physiological functions analogous to HLA antibody-induced signaling in vascular cells. Emerging evidence has revealed previously unknown functions for HLA beyond antigen presentation, including association with coreceptors in cis to permit signal transduction, and modulation of intracellular signaling downstream of other receptors that may be relevant to HLA signaling in the graft vasculature. We discuss the literature regarding HLA-induced signaling in vascular endothelial and smooth muscle cells, as well as under endogenous biological conditions, and how such signaling relates to functional changes and pathological mechanisms during graft injury.

Background: Correlation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved. Methods: We retrospectively assessed patients with transplant biopsies scored following Banff'15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA. Results: One-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent predictors of ABMRhDSApos. Conclusions: In conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage. [ABSTRACT FROM AUTHOR]

Transplant recipients developing donor-specific HLA class II (HLA-II) Abs are at higher risk for Ab-mediated rejection (AMR) and transplant vasculopathy. To understand how HLA-II Abs cause AMR and transplant vasculopathy, we determined the signaling events triggered in vascular endothelial cells (EC) following Ab ligation of HLA-II molecules. HLA-II expression in EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNF-α/IFN-γ. Ab ligation of class II stimulated EC proliferation and migration. Class II Ab also induced activation of key signaling nodes Src, focal adhesion kinase, PI3K, and ERK that regulated downstream targets of the mammalian target of rapamycin (mTOR) pathway Akt, p70 ribosomal S6 kinase, and S6 ribosomal protein. Pharmacological inhibitors and small interfering RNA showed the protein kinases Src, focal adhesion kinase, PI3K/Akt, and MEK/ERK regulate class II Ab-stimulated cell proliferation and migration. Treatment with rapalogs for 2 h did not affect HLA-II Ab-induced phosphorylation of ERK; instead, mTOR complex (mTORC)1 targets were dependent on activation of ERK. Importantly, suppression of mTORC2 for 24 h with rapamycin or everolimus or treatment with mTOR active-site inhibitors enhanced HLA-II Ab-stimulated phosphorylation of ERK. Furthermore, knockdown of Rictor with small interfering RNA caused overactivation of ERK while abolishing phosphorylation of Akt Ser473 induced by class II Ab. These data are different from HLA class I Ab-induced activation of ERK, which is mTORC2-dependent. Our results identify a complex signaling network triggered by HLA-II Ab in EC and indicate that combined ERK and mTORC2 inhibitors may be required to achieve optimal efficacy in controlling HLA-II Ab-mediated AMR. [ABSTRACT FROM AUTHOR]

Chronic vascular inflammation underlies many diseases, including atherosclerosis, autoimmune vasculitides and transplant rejection. The resolution of inflammation is critical for proper healing and restoration of homeostasis, but the timing and signaling mechanisms involved in the return to a non-inflamed state are not well understood. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells was measured in primary human aortic endothelium under chronic TNFα stimulation, and after short-term TNFα priming followed by withdrawal. The effects of NFκB, MAPK and JAK1/2 inhibitors on TNFα-induced gene expression were tested. The majority of inducible TNFα effectors, such as E -selectin, VCAM-1 and most chemokines, required continuous exposure for reinforcement of the altered phenotype, and were NFκB dependent. However, 3 h priming with TNFα induced late phase STAT activation and interferon response genes after 18 h, as well as enhanced ICAM-1, BST2 and CXCR3 ligand expression. Chronic activation was autonomous of continuous TNFα, and could be blocked by the JAK1/2 inhibitor ruxolitinib. The results demonstrate that NFκB is not a significant driver of the later phase of endothelial cell activation by TNFα, but that sustained inflammation is JAK1/2-dependent and characterized by adaptive chemokines. [Display omitted] [ABSTRACT FROM AUTHOR]

Aim We sought to understand the incidence of antibodies (Ab) to HLA, MICA, AT1R or endothelial cells (EC) in pre- and post-transplant patients, and whether such Ab present concurrently in patients and in single serum samples. Methods We retrospectively analyzed clinical results from pre- and post-transplant patients tested for AT1R, MICA, HLA, and EC Ab, and evaluated single serum samples tested for multiple types of Ab to determine whether these Ab occur together. AT1R Ab were measured by quantitative ELISA; HLA and MICA Ab were detected by Luminex single antigen; Ab to EC were detected in a flow cytometric crossmatch against mature primary aortic EC (ECXM). Results In transplant patients, the overall incidence of patients with Ab to EC was 43.2%, MICA Ab were seen in 9.9%, and 57.1% had AT1R Ab in at least one test. Regraft patients were more likely to exhibit EC or MICA Ab, particularly those with a deceased donor transplant compared with a living donor. HLA Ab were also correlated with anti-EC and MICA Ab. 78 serum samples were tested for all 3 non-HLA Ab. 26.9% of sera were negative for all three tests, while 1.3% were positive for all. There was no correlation between Ab to MICA and anti-EC Ab tested in the same serum. However, a majority of samples positive for ECXM also had AT1R Ab. AT1R Ab were significantly higher in sera that were positive for ECXM (28.8U/mL in ECXM+ vs. 19.4U/mL in ECXM-); however, 21.9% of samples without AT1R Ab were positive in ECXM, while 19% of samples negative for ECXM had very strong (>40 U/mL) AT1R Ab. Finally, in patients who had both ECXM testing and XM-ONE against potential donors, there was no apparent correlation between Ab to mature EC and donor specific endothelial precursors. Conclusion HLA sensitized patients and regraft patients were more likely to have either EC or MICA Ab. The incidence of AT1R Ab was high; however, testing tended focus on high risk patients and may not reflect true incidence in the general transplant population. Serum with high AT1R Ab also tended to be positive for ECXM, but there were cases with strong AT1R Ab but negative ECXM. These results suggest that Ab to MICA, AT1R and EC are manifestations of different immune responses and a panel of non-HLA Ab testing is informative to identify different types of non-HLA Ab that may be detrimental graft survival. [ABSTRACT FROM AUTHOR]

Aim A hallmark of chronic antibody-mediated rejection is the presence of leukocytes infiltrates within the allograft. We tested the hypothesis that ligation of HLA class II on EC mediates myeloid cell recruitment by inducing p-selectin presentation through Weibel Palade body exocytosis. Methods Class II expression was induced by adenoviral transfection of EC with CIITA. Western blots were used to quantitate protein phosphorylation. Monocyte adherence was measured by counting CFSE-labeled monocytes (MonoMac 6) recruited to HLA-class II Ab stimulated EC in the presence/absence of Fc receptor blockade with human IgG. Transendothelial cell migration was determined using a transwell assay. P-selectin expression was detected by Cell ElISA. Results Class II ligation by antibody (Ab) against a monomorphic epitope determinant on class II antigens significantly increased monocyte transendothelial migration in a Fc-independent manner. Class II Ab-mediated myeloid cell transendothelial migration was attenuated by blockading with recombinant PSGL-1 (rPSGL-1). Class II Ab caused a 1.54 ± 0.11 fold increases in monocyte adherence that was inhibited by pretreatment with rPSGL-1. In addition, pretreatment of EC with Src inhibitor PP2 and ERK inhibitor U0126 resulted in decreased myeloid cell recruitment (Fig. 1). Furthermore, PP2 and U0126 diminished class II induced p-selectin cell surface expression (Fig. 2). Conclusions We provide evidence that class II DSA promotes a MAPK-mediated monocyte adherence by upregulating EC p-selectin expression, stimulating monocyte adherence and infiltration into allografts, leading to transplant vasculopathy. [ABSTRACT FROM AUTHOR]

Aim Leukocyte extravasation plays a major rolein the development of allograft rejection and transplant vasculopathy and consists of multiple processes, including leukocyte capture, firm adhesion, transmigration, and loss of endothelial cell integrity causing barrier dysfunction. Ligation of HLA class I molecules by antibodies (Ab) elicits signaling events in endothelial cells (EC) leading to EC cell migration, proliferation and leukocyte recruitment. We hypothesize that ligation of HLA class II molecules on EC by Ab contributes to chronic rejection by transducing intracellular signaling cascades leading to cytoskeletal remodeling, increase in EC permeability and monocyte transmigration. Methods Class II expression on primary EC was achieved by infection with adenoviral recombinant Class II Transactivator (CIITA) subcloned in pAd/PL-DEST. EC were treated with murine mAb against class II (F26C6G1). Stress fiber formation was assessed using Texas Red-phalloidin staining. Protein phosphorylation was assessed by Western blot. Changes in EC permeability were measured by VE-cadherin staining and FITC-dextran permeability. Monocyte MM6 recruitment to EC was measured by immunofluorescence microscopy and monocyte transendothelial migration was determined using a transwell assay. Results Anti-class II Ab significantly increased ERK phosphorylation, stimulated stress fiber formation, reduced VE-cadherin localization in EC junctions and enhanced FITC-dextran permeability. ERK inhibitor UO126 pretreatment abrogated class II-induced stress fiber formation, reversed VE-cadherin expression in EC junctions, and attenuated class II-stimulated FITC-dextran permeability. In addition, treatment of EC with anti-class II antibodies induced a 2-4-fold increase in the recruitment and transmigration of monocytes, which was attenuated by treatment with the ERK inhibitor U0126 or recombinant PSGL1 that blocks monocyte capture. Conclusions Ab ligation of HLA class II molecules on EC stimulates activation of the ERK signaling pathway and actin cytoskeleton remodeling leading to barrier dysfunction and monocyte transendothelial migration. Elucidation of the class II signaling pathways has the potential to identify novel therapeutic targets for treatment of chronic rejection. [ABSTRACT FROM AUTHOR]

Aim: HLA antibodies (Ab) activate endothelial cells (EC) and trigger monocyte (Mo) adhesion through P-selectin and FcγRs. FcγRIIa is dimorphic, at position H131R. We investigated whether FcγRIIa allotype or IgG subclass affect HLA I Ab-induced Mo adherence. Methods: Mo binding to immobilized or soluble IgG was measured by microscopy and flow cytometry, respectively. The variable regions of mouse anti-HLA I (W6/32) Ab were cloned onto human constant regions to generate chimerized HLA I hIgG1 and hIgG2. Human aortic EC were stimulated with murine or chimeric HLA I Ab and the number of adherent Mo was counted. Results: H131 homozygous Mono Mac 6 (MM6) 1) did not bind mIgG1, 2) had enhanced capacity to bind hIgG2, and 3) bound hIgG1 through FcγRI and FcγRIIa. FcγRIIa-R131 homozygous U937 1) had enhanced capacity to bind mIgG1, 2) had reduced capacity to bind hIgG1 and mIgG2a, 3) did not bind hIgG2, and 4) bound hIgG1 through only FcγRI [Table 1]. HLA I mIgG1 and mIgG2a-activated EC recruited a comparable number of U937. FcγR inhibition significantly reduced adhesion of U937 to HLA I mIgG1 and mIgG2a-activated EC. In contrast, dramatically more MM6 were recruited in response to mIgG2a compared with mIgG1. FcγR blockade effectively inhibited adhesion of MM6 to HLA I mIgG2a, but not mIgG1, treated EC. Heterozygous THP-1 Mo displayed an intermediate phenotype. Conclusions: FcγR polymorphisms in DSA+ transplant patients may influence outcome. Recipients with FcγRIIa-H131 allele may experience greater infiltration of FcγR-bearing myeloid cells during AMR. DSA of the IgG2 subclass, while not complement fixing, may be clinically significant in H131 homozygous patients. Thus, strategies such as IVIG which reduce interaction of HLA I IgG with FcγRs are likely to diminish macrophage, neutrophil and NK trafficking to the graft. [Copyright &y& Elsevier]