Nicole M. Valenzuela

HLA Class I Antibodies Trigger Increased Adherence of Monocytes to Endothelial Cells by Eliciting an Increase in Endothelial P-Selectin and, Depending on Subclass, by Engaging FcγRs

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.

Antibody-mediated graft injury: complement-dependent and complement-independent mechanisms.

Purpose of reviewAntibody-mediated rejection (AMR) is emerging as the leading cause of chronic rejection and allograft failure. Traditionally, the mechanisms of graft injury mediated by donor-specific antibodies beyond complement activation were not well appreciated. However, an evolving paradigm of Fc-independent antibody functions, along with clinical recognition of C4d-negative AMR, has increased awareness of the action of antibodies leading to endothelial activation and dysfunction. Recent findingsHerein, we address current clinical trends, including the signature of microvascular inflammation in biopsies of grafts undergoing AMR, the prevalence of antibodies to human leukocyte antigen class II DQ locus (HLA-DQ) and non-HLA targets, and the functional characterization of HLA immunoglobulin G (IgG) subclasses and complement-fixing capacity. We also discuss recent experimental evidence revealing new mechanisms of endothelial and smooth muscle cell activation by HLA antibodies, which may contribute to vascular inflammation and chronic rejection. Finally, we touch upon novel discoveries of the interplay between antibodies, the complement system, and CD4 T-cell-mediated alloimmunity. SummaryThe current literature suggests that, although complement-fixing antibodies may have some prognostic value for graft outcome, complement-independent mechanisms of graft injury are increasingly relevant. Therapeutic strategies, which target endothelial activation induced by antibodies may ameliorate vascular inflammation and mononuclear cell infiltration characteristic of AMR.

Blockade of P-Selectin Is Sufficient to Reduce MHC I Antibody-Elicited Monocyte Recruitment In Vitro and In Vivo

Donor‐specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody‐mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody‐mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc‐independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody‐mediated rejection, in which B6.RAG1−/− recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P‐selectin, which supports monocyte binding. In the experimental model, donor‐specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL‐1‐Ig abolished antibody‐induced monocyte infiltration in the allograft, but had little effect on antibody‐induced endothelial injury. Our data suggest that antagonism of P‐selectin may ameliorate accumulation of macrophages in the allograft during antibody‐mediated rejection.

Antibodies in Transplantation: The Effects of HLA and Non-HLA Antibody Binding and Mechanisms of Injury

Until recently, allograft rejection was thought to be mediated primarily by alloreactive T cells. Consequently, immunosuppressive approaches focused on inhibition of T cell activation. While short-term graft survival has significantly improved and rejection rates have dropped, acute rejection has not been eliminated and chronic rejection remains the major threat to long-term graft survival. Increased attention to humoral immunity in experimental systems and in the clinic has revealed that donor specific antibodies (DSA) can mediate and promote acute and chronic rejection. Herein, we detail the effects of alloantibody, particularly HLA antibody, binding to graft vascular and other cells, and briefly summarize the experimental methods used to assess such outcomes.

Everolimus Inhibits Anti‐HLA I Antibody‐Mediated Endothelial Cell Signaling, Migration and Proliferation More Potently Than Sirolimus

Antibody (Ab) crosslinking of HLA I molecules on the surface of endothelial cells triggers proliferative and pro‐survival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy (TV). The purpose of this study was to investigate the role of mammalian target of rapamycin (mTOR) in HLA I Ab‐induced signaling cascades. Everolimus provides a tool to establish how the mTOR signal network regulates HLA I–mediated migration, proliferation and survival. We found that everolimus inhibits mTOR complex 1 (mTORC1) by disassociating Raptor from mTOR, thereby preventing class I–induced phosphorylation of mTOR, p70S6K, S6RP and 4E‐BP1, and resultant class I–stimulated cell migration and proliferation. Furthermore, we found that everolimus inhibits class I–mediated mTORC2 activation (1) by disassociating Rictor and Sin1 from mTOR; (2) by preventing class I–stimulated Akt phosphorylation and (3) by preventing class I–mediated ERK phosphorylation. These results suggest that everolimus is more effective than sirolimus at antagonizing both mTORC1 and mTORC2, the latter of which is critical in endothelial cell functional changes leading to TV in solid organ transplantation after HLA I crosslinking. Our findings point to a potential therapeutic effect of everolimus in prevention of chronic Ab‐mediated rejection.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Antibody‐mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor‐specific antibodies (DSA) binding donor Class I and II HLA (HLA‐I and HLA‐II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti‐C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA‐Ab)‐induced complement activation. Complement deposition induced by HLA‐Ab was evaluated using novel cell‐ and bead‐based assays. Human aortic endothelial cells (HAEC) were cultured with HLA‐Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA‐Ab and human complement. TNT003 inhibited HLA‐Ab mediated complement deposition on HAEC in a concentration‐dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI‐Ab)‐ and Class II (HLAII‐Ab)‐specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA‐I/II‐Ab in vitro.

Monocyte recruitment by HLA IgG-activated endothelium: the relationship between IgG subclass and FcγRIIa polymorphisms.

It is currently unclear which donor specific HLA antibodies confer the highest risk of antibody‐mediated rejection (AMR) and allograft loss. In this study, we hypothesized that two distinct features (HLA IgG subclass and Fcγ receptor [FcγR] polymorphisms) which vary from patient to patient, influence the process of monocyte trafficking to and macrophage accumulation in the allograft during AMR in an interrelated fashion. Here, we investigated the contribution of human IgG subclass and FcγR polymorphisms in monocyte recruitment in vitro by primary human aortic endothelium activated with chimeric anti‐HLA I human IgG1 and IgG2. Both subclasses triggered monocyte adhesion to endothelial cells, via a two‐step process. First, HLA I crosslinking by antibodies stimulated upregulation of P‐selectin on endothelium irrespective of IgG subclass. P‐selectin‐induced monocyte adhesion was enhanced by secondary interactions of IgG with FcγRs, which was highly dependent upon subclass. IgG1 was more potent than IgG2 through differential engagement of FcγRs. Monocytes homozygous for FcγRIIa‐H131 adhered more readily to HLA antibody‐activated endothelium compared with FcγRIIa‐R131 homozygous. Finally, direct modification of HLA I antibodies with immunomodulatory enzymes EndoS and IdeS dampened recruitment by eliminating antibody‐FcγR binding, an approach that may have clinical utility in reducing AMR and other forms of antibody‐induced inflammation.

The link between major histocompatibility complex antibodies and cell proliferation.

Experimental evidence indicates that donor-specific antibodies targeting major histocompatibility complex classes I and II molecules can elicit the key features of transplant vasculopathy by acting on the graft vasculature in 3 ways: directly activating proliferative, prosurvival, and migratory signaling in the target endothelial and smooth muscle cells; increasing expression of mitogenic factors in vascular endothelial cells, creating a potential proliferative autocrine loop; and promoting recruitment of inflammatory cells that produce mitogenic factors and elicit chronic inflammation, proliferation, and fibrosis. Here, we review the experimental literature showing the complement and Fc-independent effects of major histocompatibility complex classes I and II antibodies on graft vascular cells that may directly contribute to the proliferative aspect of transplant vasculopathy.

Phosphorylated S6 kinase and S6 ribosomal protein are diagnostic markers of antibody-mediated rejection in heart allografts.

BACKGROUND Anti-MHC Class I alloantibodies have been implicated in the processes of acute and chronic rejection. These antibodies (Ab) bind to endothelial cells (EC) and transduce signals leading to the activation of cell survival and proliferation pathways, including Src, FAK and mTOR, as well as downstream targets ERK, S6 kinase (S6K) and S6 ribosomal protein (S6RP). We tested the hypothesis that phosphorylation of S6K, S6RP and ERK in capillary endothelium may serve as an adjunct diagnostic tool for antibody-mediated rejection (AMR) in heart allografts. METHODS Diagnosis of AMR was based on histology or immunoperoxidase staining of paraffin-embedded tissue, consistent with 2013 ISHLT criteria. Diagnosis of acute cellular rejection (ACR) was based on ISHLT criteria. Endomyocardial biopsies from 67 heart transplant recipients diagnosed with acute rejection [33 with pAMR, 18 with ACR (15 with Grade 1R, 3 with Grade ≥2R), 16 with pAMR and ACR (13 with 1R and 3 with ≥2R)] and 40 age- and gender-matched recipients without rejection were tested for the presence of phosphorylated forms of ERK, S6RP and S6K by immunohistochemistry. RESULTS Immunostaining of endomyocardial biopsies with evidence of pAMR showed a significant increase in expression of p-S6K and p-S6RP in capillary EC compared with controls. A weaker association was observed between pAMR and p-ERK. CONCLUSIONS Biopsies diagnosed with pAMR often showed phosphorylation of S6K and S6RP, indicating that staining for p-S6K and p-S6RP is useful for the diagnosis of AMR. Our findings support a role for antibody-mediated HLA signaling in the process of graft injury.

HLA class I antibody-mediated endothelial and smooth muscle cell activation.

Purpose of reviewAdvances 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 findingsSeveral 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 &bgr;4 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. SummaryLigation 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.