Publications by authors named "Arend Mulder"

88 Publications

Tissue-specific endothelial cell heterogeneity contributes to unequal inflammatory responses.

Sci Rep 2021 Jan 21;11(1):1949. Epub 2021 Jan 21.

Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, 1000 Veteran Avenue, Room 1-520, Los Angeles, CA, 90095, USA.

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.
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http://dx.doi.org/10.1038/s41598-020-80102-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820348PMC
January 2021

PAKC: A novel panel of HLA class I antigen presentation machinery knockout cells from the same genetic origin.

Eur J Immunol 2021 Mar 1;51(3):734-737. Epub 2021 Feb 1.

Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands.

A single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I antigen presentation pathway. PAKC will accelerate HLA-I research in the fields of oncology, infectiology, and autoimmunity.
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http://dx.doi.org/10.1002/eji.202048599DOI Listing
March 2021

The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses.

Immunity 2021 Jan 2;54(1):132-150.e9. Epub 2020 Dec 2.

Department of Immunopathology, Sanquin Research, Amsterdam, the Netherlands; Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; Cancer Center Amsterdam, Amsterdam, the Netherlands. Electronic address:

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8 T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8 T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.
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http://dx.doi.org/10.1016/j.immuni.2020.11.003DOI Listing
January 2021

Antibody-induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection.

Am J Transplant 2020 10 22;20(10):2686-2702. Epub 2020 May 22.

Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California.

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') stimulated endothelium solely induced higher CD206 (M(HLA I F(ab') )). 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.
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http://dx.doi.org/10.1111/ajt.15934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529968PMC
October 2020

Recombinant human monoclonal HLA antibodies of different IgG subclasses recognising the same epitope: Excellent tools to study differential effects of donor-specific antibodies.

HLA 2019 11 25;94(5):415-424. Epub 2019 Aug 25.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.

In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.
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http://dx.doi.org/10.1111/tan.13664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851673PMC
November 2019

HLA Expression in Uveal Melanoma: An Indicator of Malignancy and a Modifiable Immunological Target.

Cancers (Basel) 2019 Aug 7;11(8). Epub 2019 Aug 7.

Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and gives rise to metastases in 50% of cases. The presence of an inflammatory phenotype is a well-known risk factor for the development of metastases. This inflammatory phenotype is characterized by the presence of high numbers of lymphocytes and macrophages, and a high expression of the HLA Class I and II antigens. An abnormal expression of HLA Class I may influence cytotoxic T lymphocyte (CTL) as well as Natural Killer (NK) cell responses. We provide a comprehensive review regarding the inflammatory phenotype in UM and the expression of locus- and allele-specific HLA Class I and of Class II antigens in primary UM and its metastases. Furthermore, we describe the known regulators and the role of genetics (especially chromosome 3 and BRCA-Associated Protein 1 (BAP1 status)), and, last but not least, the effect of putative therapeutic treatments on HLA expression.
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http://dx.doi.org/10.3390/cancers11081132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721545PMC
August 2019

Characterization of donor and recipient CD8+ tissue-resident memory T cells in transplant nephrectomies.

Sci Rep 2019 04 12;9(1):5984. Epub 2019 Apr 12.

Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Tissue-resident memory T (T) cells are characterized by their surface expression of CD69 and can be subdivided in CD103+ and CD103- T cells. The origin and functional characteristics of T cells in the renal allograft are largely unknown. To determine these features we studied T cells in transplant nephrectomies. T cells with a CD103+ and CD103- phenotype were present in all samples (n = 13) and were mainly CD8+ T cells. Of note, donor-derived T cells were only detectable in renal allografts that failed in the first month after transplantation. Grafts, which failed later, mainly contained recipient derived T cells. The gene expression profiles of the recipient derived CD8+ T cells were studied in more detail and showed a previously described signature of tissue residence within both CD103+ and CD103- T cells. All CD8+ T cells had strong effector abilities through the production of IFNγ and TNFα, and harboured high levels of intracellular granzyme B and low levels of perforin. In conclusion, our results demonstrate that donor and recipient T cells reside in the rejected renal allograft. Over time, the donor-derived T cells are replaced by recipient T cells which have features that enables these cells to aggressively respond to the allograft.
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http://dx.doi.org/10.1038/s41598-019-42401-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461670PMC
April 2019

Determining the extent of maternal-foetal chimerism in cord blood.

Sci Rep 2019 03 27;9(1):5247. Epub 2019 Mar 27.

Sanquin Research, Dept of Hematopoiesis, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

During pregnancy, maternal T cells can enter the foetus, leading to maternal-foetal chimerism. This phenomenon may affect how leukaemia patients respond to transplantation therapy using stem cells from cord blood (CB). It has been proposed that maternal T cells, primed to inherited paternal HLAs, are present in CB transplants and help to suppress leukaemic relapse. Several studies have reported evidence for the presence of maternal T cells in most CBs at sufficiently high numbers to lend credence to this idea. We here aimed to functionally characterise maternal T cells from CB. To our surprise, we could not isolate viable maternal cells from CB even after using state-of-the-art enrichment techniques that allow detection of viable cells in heterologous populations at frequencies that were several orders of magnitude lower than reported frequencies of maternal T cells in CB. In support of these results, we could only detect maternal DNA in a minority of samples and at insufficient amounts for reliable quantification through a sensitive PCR-based assay to measure In/Del polymorphisms. We conclude that maternal microchimerism is far less prominent than reported, at least in our cohort of CBs, and discuss possible explanations and implications.
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http://dx.doi.org/10.1038/s41598-019-41733-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437214PMC
March 2019

Measuring anti-HLA antibody active concentration and affinity by surface plasmon resonance: Comparison with the luminex single antigen flow beads and T-cell flow cytometry crossmatch results.

Mol Immunol 2019 04 16;108:34-44. Epub 2019 Feb 16.

CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Place Amélie Raba Léon, Bordeaux, France; Immuno ConcEpT, UMR CNRS 5164, 146 rue Léo Saignat, Bordeaux, France; Université de Bordeaux, 146 rue Léo Saignat, Bordeaux, France.

Background: Although the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant recipients, their semi-quantitative fluorescence read-out is not closely linked to graft outcome.

Methods: Surface plasmon resonance (SPR) was implemented to determine truly quantitative parameters of five human monoclonal anti-class I HLA antibodies (mAbs): first the active concentration and then the binding constants. The results were compared to those obtained with SAFB and T-cell FCXM (T-FCXM).

Results: The five mAbs displayed different rate and equilibrium constants for their cognate antigens. No correlation was evidenced between SAFB MFI or T-FCXM ratio and the binding parameters measured by SPR. Some mAbs amino acid substitutions within the epitope that influenced SAFB MFI resulted in affinity variations evidenced by SPR.

Conclusion: The SAFB MFI and T-FCXM ratio, both semi-quantitative parameters, only partially reflected the subtlety of the anti-HLA antibody/antigen interaction as it can be analyzed by SPR. Future clinical studies using SPR for anti-HLA antibodies characterization could bring novel insights into the understanding of HLA/anti-HLA interaction and therefore anti-HLA antibodies pathogenicity.
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http://dx.doi.org/10.1016/j.molimm.2019.02.006DOI Listing
April 2019

The Vacuolar Pathway of Long Peptide Cross-Presentation Can Be TAP Dependent.

J Immunol 2019 01 17;202(2):451-459. Epub 2018 Dec 17.

Ludwig Institute for Cancer Research, Brussels B-1200, Belgium;

The intracellular pathway of cross-presentation, which allows MHC class I-restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2-restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1-restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2-restricted peptide, cross-presentation of the HLA-A1-restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.
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http://dx.doi.org/10.4049/jimmunol.1800353DOI Listing
January 2019

Prolongation of allograft survival by passenger donor regulatory T cells.

Am J Transplant 2019 05 5;19(5):1371-1379. Epub 2019 Feb 5.

Department of Surgery, School of Clinical Medicine, University of Cambridge, Cambridge, UK.

Tissue resident lymphocytes are present within many organs, and are presumably transferred at transplantation, but their impact on host immunity is unclear. Here, we examine whether transferred donor natural regulatory CD4 T cells (nT-regs) inhibit host alloimmunity and prolong allograft survival. Transfer of donor-strain lymphocytes was first assessed by identifying circulating donor-derived CD4 T cells in 21 consecutive human lung transplant recipients, with 3 patterns of chimerism apparent: transient, intermediate, and persistent (detectable for up to 6 weeks, 6 months, and beyond 1 year, respectively). The potential for transfer of donor nT-regs was then confirmed by analysis of leukocyte filters recovered from ex vivo normothermic perfusion circuits of human kidneys retrieved for transplantation. Finally, in a murine model of cardiac allograft vasculopathy, depletion of donor CD4 nT-regs before organ recovery resulted in markedly accelerated heart allograft rejection and augmented host effector antibody responses. Conversely, adoptive transfer or purified donor-strain nT-regs inhibited host humoral immunity and prolonged allograft survival, and more effectively so than following administration of recipient nT-regs. In summary, following transplantation, passenger donor-strain nT-regs can inhibit host adaptive immune responses and prolong allograft survival. Isolated donor-derived nT-regs may hold potential as a cellular therapy to improve transplant outcomes.
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http://dx.doi.org/10.1111/ajt.15212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519070PMC
May 2019

No Evidence for Cross-reactivity of Virus-specific Antibodies With HLA Alloantigens.

Transplantation 2018 Nov;102(11):1844-1849

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.

Background: Antibodies directed against HLA can develop through pregnancy, blood transfusions, or organ transplants. Anecdotal evidence suggests that virus-specific antibodies may have the capacity to cross-react with HLA, a phenomenon called heterologous immunity, which is well described for T-cell alloreactivity.

Methods: To determine whether antibody cross-reactivity between viral antigens and HLA is common, we tested 51 virus-specific human monoclonal antibodies (mAbs) specific for human immunodeficiency virus, varicella zoster virus, cytomegalovirus, and parvovirus, for reactivity against HLA class I and class II in single-antigen bead assays. In addition, we tested the reactivity of 41 HLA-specific human mAbs against common viral antigens of cytomegalovirus, varicella zoster virus, human immunodeficiency virus, Epstein-Barr virus, and BK polyomavirus.

Results: No cross-reactivity of any of the virus-specific mAbs with either HLA class I or class II molecules, as well as no cross-reactivity of any of the HLA-specific mAbs with any of the viral antigens was observed.

Conclusions: These findings indicate that the frequency of cross-reactivity on the antibody level between viral antigens and HLA, if present at all, is low. The emergence of HLA antibodies upon viral infection or vaccination is therefore probably due to bystander activation of dormant HLA-specific memory B cells.
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http://dx.doi.org/10.1097/TP.0000000000002369DOI Listing
November 2018

The long and winding road towards epitope matching in clinical transplantation.

Transpl Int 2019 01 26;32(1):16-24. Epub 2018 Nov 26.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands.

Recent data suggest that HLA epitope matching is beneficial for the prevention of de novo donor specific antibody (DSA) formation after transplantation. In this review, different approaches to predict the immunogenicity of an HLA mismatch will be discussed. The parameters used in these models are often called epitopes but the actual antibody epitope is far more complex. Exact knowledge of the antibody epitope is crucial if epitope matching is also used as a tool to select compatible donors for (highly) sensitized patients. Evidence is provided that it is not always possible to give an exact definition of an antibody epitope. We conclude that HLA "epitope" matching is superior over HLA antigen matching with respect to the prevention of de novo DSA formation and will enhance the prediction of acceptable HLA mismatches for sensitized patients. However, epitope matching at our current level of knowledge will not solve all histocompatibility problems as unexpected antibody reactivity still may occur.
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http://dx.doi.org/10.1111/tri.13362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379527PMC
January 2019

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets.

Haematologica 2019 02 27;104(2):403-416. Epub 2018 Sep 27.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.
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http://dx.doi.org/10.3324/haematol.2018.201665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355480PMC
February 2019

A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation.

Haematologica 2018 10 1;103(10):1741-1752. Epub 2018 Jun 1.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, the Netherlands.

HLA antibodies are associated with refractoriness to platelet transfusion, leading to rapid platelet clearance, sometimes coinciding with clinical side effects such as fever and chills. The presence of HLA antibodies is not always manifested by clinical symptoms. It is currently unclear why refractoriness to platelet transfusion is only observed in a subset of patients. Here, we utilized the availability of a unique panel of human monoclonal antibodies to study whether these were capable of activating platelets. Three out of eight human HLA-specific monoclonal antibodies induced activation of HLA-matched platelets from healthy donors as evidenced by enhanced α-granule release, aggregation, and αb activation. The propensity of HLA monoclonal antibodies to activate platelets was independent of the HLA subtype to which they were directed, but was dependent on the recognized epitope. Activation was fully inhibited either by blocking FcγRIIa, or by blocking FcγRIIa-dependent signaling with Syk inhibitor IV. Furthermore, activation required the presence of the IgG-Fc part, as F(ab') fragments of HLA monoclonal antibodies were unable to induce platelet activation. Mixing experiments revealed that activation of platelets occurred in an intra-platelet dependent manner. Accordingly, a proportion of sera from refractory patients with HLA antibodies induced FcγRIIa-dependent platelet activation. Our data show that a subset of HLA antibodies is capable of crosslinking HLA and FcγRIIa thereby promoting platelet activation and enhancing these cells' phagocytosis by macrophages. Based on these findings we suggest that FcγRIIa-dependent platelet activation may contribute to the decreased platelet survival in platelet-transfusion-dependent patients with HLA antibodies.
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http://dx.doi.org/10.3324/haematol.2018.189365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165798PMC
October 2018

HLA Class I Antigen Expression in Conjunctival Melanoma Is Not Associated With PD-L1/PD-1 Status.

Invest Ophthalmol Vis Sci 2018 02;59(2):1005-1015

Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.

Purpose: Antitumor T cells need expression of HLA class I molecules but can be inhibited by ligands such as programmed death ligand 1 (PD-L1). We determined expression and regulation of these molecules in human conjunctival melanoma (CM) samples, cell lines, and murine xenografts.

Methods: Immunofluorescence staining was performed to examine the expression of HLA-A, HLA-B/C, and β-2-microglobulin (B2M) in 23 primary CM samples. HLA class I expression was compared with clinicopathologic characteristics, the presence of tumor-infiltrating leukocytes, and PD-L1/PD-1 status. The effect of interferon γ (IFN-γ) on HLA class I expression was tested on three CM cell lines using quantitative PCR and flow cytometry. Furthermore, HLA class I expression was determined in CM cell line-derived murine xenografts.

Results: One third of tumors had positive HLA-A, HLA-B/C, and B2M expression. A positive expression was especially seen in thin and epibulbar tumors but was not associated with recurrences. HLA class I expression was correlated with M2 macrophage density and tended to associate with CD8+ T-cell density but was independent of PD-L1 or PD-1 expression. IFN-γ upregulated HLA class I expression and genes involved in HLA transcription and transportation on CM cell lines. Murine xenografts showed a comparable HLA class I expression as their respective cell lines.

Conclusions: Our data indicate that subsets of CM have positive HLA class I expression, and HLA class I and PD-L1/PD-1 are expressed independently. When one considers immunotherapy, one should also analyze HLA class I expression, whose downregulation can limit the efficacy of T cell-mediated therapies.
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http://dx.doi.org/10.1167/iovs.17-23209DOI Listing
February 2018

Response to the comments on "Direct quantitative measurement of the kinetics of HLA-specific antibody interactions with isolated HLA proteins".

Hum Immunol 2018 02 5;79(2):130-131. Epub 2017 Dec 5.

Renal Department, UHCW NHS Trust, Coventry, UK; Warwick Medical School, University of Warwick, Coventry, UK.

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http://dx.doi.org/10.1016/j.humimm.2017.12.001DOI Listing
February 2018

Direct quantitative measurement of the kinetics of HLA-specific antibody interactions with isolated HLA proteins.

Hum Immunol 2018 Feb 31;79(2):122-128. Epub 2017 Oct 31.

Renal Department, UHCW NHS Trust, Coventry, UK; Warwick Medical School, University of Warwick, Coventry, UK.

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (k 3.63-24.25 × 10 per mol per second) and dissociation rates (k, 0.99-10.93 × 10 per second). Dissociation constants (K) ranged from 5.9 × 10 M to 3.0 × 10 M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA.
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http://dx.doi.org/10.1016/j.humimm.2017.10.012DOI Listing
February 2018

Platelets from donors with consistently low HLA-B8, -B12, or -B35 expression do not undergo antibody-mediated internalization.

Blood 2018 01 1;131(1):144-152. Epub 2017 Nov 1.

Department of Product and Process Development, Sanquin Blood Bank, Amsterdam, The Netherlands.

Patients refractory to platelet transfusions because of alloimmunization require HLA-matched platelets, which is only possible if a large HLA-typed donor pool is available. However, even then, patients with broad immunization or rare haplotypes may not have suitable donors. In these patients, transfusions with platelets showing low HLA class I expression may be an alternative to fully HLA-matched transfusions. In this study, we quantified the proportion of donors with consistently low HLA-B8, -B12, and -B35 expression on platelets using human monoclonal antibodies specific for these antigens. Furthermore, as model for in vivo clearance, antibody-mediated internalization of these platelets by macrophages was investigated. The expression of HLA-B8, -B12, or -B35 on platelets was extremely variable between individuals (coefficients of variation, 41.4% to 73.6%). For HLA-B8, but not for HLA-B12 or -B35, this variation was in part explained by zygosity. The variation was most pronounced in, but not exclusive to, platelets. Expression within one donor was consistent over time. Remarkably, 32% of 113 HLA-B8, 34% of 98 HLA-B12, and 9% of 66 HLA-B35 donors showed platelet antigen expression that was not or only minimally above background. Antibody-mediated internalization of platelets by macrophages correlated with antibody opsonization and antigen expression and was absent in platelets with low or minimal HLA expression. In conclusion, our findings indicate that a substantial proportion of donors have platelets with consistently low expression of specific HLA class I antigens. These platelets may be used to treat refractory patients with antibodies directed against these particular antigens, despite HLA mismatches.
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http://dx.doi.org/10.1182/blood-2017-07-799270DOI Listing
January 2018

KIR2DS2 recognizes conserved peptides derived from viral helicases in the context of HLA-C.

Sci Immunol 2017 09;2(15)

Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, Tremona Road, Southampton S016 6YD, UK.

Killer cell immunoglobulin-like receptors (KIRs) are rapidly evolving species-specific natural killer (NK) cell receptors associated with protection against multiple different human viral infections. We report that the activating receptor KIR2DS2 directly recognizes viral peptides derived from conserved regions of flaviviral superfamily 2 RNA helicases in the context of major histocompatibility complex class I. We started by documenting that peptide LNPSVAATL from the hepatitis C virus (HCV) helicase binds HLA-C*0102, leading to NK cell activation through engagement of KIR2DS2. Although this region is highly conserved across HCV isolates, the sequence is not present in other flaviviral helicases. Embarking on a search for a conserved target of KIR2DS2, we show that HLA-C*0102 presents a different highly conserved peptide from the helicase motif 1b region of related flaviviruses, including dengue, Zika, yellow fever, and Japanese encephalitis viruses, to KIR2DS2. In contrast to LNPSVAATL from HCV, these flaviviral peptides all contain an "MCHAT" motif, which is present in 61 of 63 flaviviruses. Despite the difference in the peptide sequences, we show that KIR2DS2 recognizes endogenously presented helicase peptides and that KIR2DS2 is sufficient to inhibit HCV and dengue virus replication in the context of HLA-C*0102. Targeting short, but highly conserved, viral peptides provide nonrearranging innate immune receptors with an efficient mechanism to recognize multiple, highly variable, pathogenic RNA viruses.
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http://dx.doi.org/10.1126/sciimmunol.aal5296DOI Listing
September 2017

Multiple E2 ubiquitin-conjugating enzymes regulate human cytomegalovirus US2-mediated immunoreceptor downregulation.

J Cell Sci 2017 Sep 25;130(17):2883-2892. Epub 2017 Jul 25.

Dept. Medical Microbiology, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands

Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2-induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system.
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http://dx.doi.org/10.1242/jcs.206839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612228PMC
September 2017

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003.

Transplantation 2017 07;101(7):1559-1572

1 UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA.2 Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.3 True North Therapeutics Inc., South San Francisco, CA.

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.
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http://dx.doi.org/10.1097/TP.0000000000001486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482566PMC
July 2017

NLRP2 is a suppressor of NF-ƙB signaling and HLA-C expression in human trophoblasts†,‡.

Biol Reprod 2017 Apr;96(4):831-842

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.
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http://dx.doi.org/10.1093/biolre/iox009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803763PMC
April 2017

Selective graft-versus-leukemia depends on magnitude and diversity of the alloreactive T cell response.

J Clin Invest 2017 Feb 9;127(2):517-529. Epub 2017 Jan 9.

Patients with leukemia who receive a T cell-depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.
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http://dx.doi.org/10.1172/JCI86175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5272193PMC
February 2017

Identification of Donor Origin and Condition of Transplanted Islets In Situ in the Liver of a Type 1 Diabetic Recipient.

Cell Transplant 2017 01 10;26(1):1-9. Epub 2016 Oct 10.

Transplantation of islet allografts into type 1 diabetic recipients usually requires multiple pancreas donors to achieve insulin independence. This adds to the challenges of immunological monitoring of islet transplantation currently relying on surrogate immune markers in peripheral blood. We investigated donor origin and infiltration of islets transplanted in the liver of a T1D patient who died of hemorrhagic stroke 4 months after successful transplantation with two intraportal islet grafts combining six donors. Immunohistological staining for donor HLA using a unique panel of human monoclonal HLA-specific alloantibodies was performed on liver cryosections after validation on cryopreserved kidney, liver, and pancreas and compared with auto- and alloreactive T-cell immunity in peripheral blood. HLA-specific staining intensity and signal-to-noise ratio varied between tissues from very strong on kidney glomeruli, less in liver, kidney tubuli, and endocrine pancreas to least in exocrine pancreas, complicating the staining of inflamed islets in an HLA-disparate liver. Nonetheless, five islets from different liver lobes could be attributed to donors 1, 2, and 5 by staining patterns with multiple HLA types. All islets showed infiltration with CD8+ cytotoxic T cells that was mirrored by progressive alloreactive responses in peripheral blood mononuclear cells (PBMCs) to donors 1, 2, and 5 after transplantation. Stably low rates of peripheral islet autoreactive T-cell responses after islet infusion fit with a complete HLA mismatch between grafts and recipient and exclude the possibility that the islet-infiltrating CD8 T cells were autoreactive. HLA-specific immunohistochemistry can identify donor origin in situ and differentiate graft dysfunction and immunological destruction.
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http://dx.doi.org/10.3727/096368916X693437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657695PMC
January 2017

Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia.

Sci Transl Med 2016 09;8(357):357ra123

Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.

Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell antileukemia potential is unclear. Cytokine-induced memory-like NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for weeks to months after preactivation. We hypothesized that memory-like NK cells exhibit enhanced antileukemia functionality. We demonstrated that human memory-like NK cells have enhanced interferon-γ production and cytotoxicity against leukemia cell lines or primary human AML blasts in vitro. Using mass cytometry, we found that memory-like NK cell functional responses were triggered against primary AML blasts, regardless of killer cell immunoglobulin-like receptor (KIR) to KIR-ligand interactions. In addition, multidimensional analyses identified distinct phenotypes of control and memory-like NK cells from the same individuals. Human memory-like NK cells xenografted into mice substantially reduced AML burden in vivo and improved overall survival. In the context of a first-in-human phase 1 clinical trial, adoptively transferred memory-like NK cells proliferated and expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.
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http://dx.doi.org/10.1126/scitranslmed.aaf2341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436500PMC
September 2016

Complex MHC Class I Gene Transcription Profiles and Their Functional Impact in Orangutans.

J Immunol 2016 Jan 18;196(2):750-8. Epub 2015 Dec 18.

Comparative Genetics and Refinement, Biomedical Primate Research Centre, 2288 GJ Rijswijk, the Netherlands; Theoretical Biology and Bioinformatics, Utrecht University, 3584 CH Utrecht, the Netherlands.

MHC haplotypes of humans and the African great ape species have one copy of the MHC-A, -B, and -C genes. In contrast, MHC haplotypes of orangutans, the Asian great ape species, exhibit variation in the number of gene copies. An in-depth analysis of the MHC class I gene repertoire in the two orangutan species, Pongo abelii and Pongo pygmaeus, is presented in this article. This analysis involved Sanger and next-generation sequencing methodologies, revealing diverse and complicated transcription profiles for orangutan MHC-A, -B, and -C. Thirty-five previously unreported MHC class I alleles are described. The data demonstrate that each orangutan MHC haplotype has one copy of the MHC-A gene, and that the MHC-B region has been subject to duplication, giving rise to at least three MHC-B genes. The MHC-B*03 and -B*08 lineages of alleles each account for a separate MHC-B gene. All MHC-B*08 allotypes have the C1-epitope motif recognized by killer cell Ig-like receptor. At least one other MHC-B gene is present, pointing to MHC-B alleles that are not B*03 or B*08. The MHC-C gene is present only on some haplotypes, and each MHC-C allotype has the C1-epitope. The transcription profiles demonstrate that MHC-A alleles are highly transcribed, whereas MHC-C alleles, when present, are transcribed at very low levels. The MHC-B alleles are transcribed to a variable extent and over a wide range. For those orangutan MHC class I allotypes that are detected by human monoclonal anti-HLA class I Abs, the level of cell-surface expression of proteins correlates with the level of transcription of the allele.
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http://dx.doi.org/10.4049/jimmunol.1500820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707110PMC
January 2016

Selective downregulation of HLA-C and HLA-E in childhood acute lymphoblastic leukaemia.

Br J Haematol 2016 08 3;174(3):477-80. Epub 2015 Nov 3.

Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University, Düsseldorf, Germany.

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http://dx.doi.org/10.1111/bjh.13777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854806PMC
August 2016

Usefulness of the Nonself-Self Algorithm of HLA Epitope Immunogenicity in the Specificity Analysis of Monospecific Antibodies Induced during Pregnancy.

Front Immunol 2015 26;6:180. Epub 2015 May 26.

Leiden University Medical Center , Leiden , Netherlands.

Background: HLAMatchmaker is a program to analyze the epitope specificities of HLA antibodies. It considers each HLA allele as a string of eplets. Intralocus and interlocus comparisons between donor and recipient alleles offer a structural assessment of compatibility and an analysis of allele panel reactivity patterns can generate information about epitope specificities of HLA antibodies. However, HLAMatchmaker cannot always generate conclusive interpretations of reactivity patterns of all monospecific antibodies, which by definition recognize single epitopes.

Hypothesis: We have therefore developed a new antibody analysis approach that utilizes the nonself-self algorithm of HLA epitope immunogenicity. It is based on the concept that HLA antibodies originate from B-cells with immunoglobulin receptors to self-HLA epitopes on one given allele and which can be activated by epitopes defined by a few nonself residue differences whereas the remainder of the structural epitope of the immunizing allele consists of self residues.

Methods: Three human monoclonal class I antibodies from HLA typed women sensitized during pregnancy were tested in Ig-binding assays with single alleles on a Luminex platform.

Findings: Three new HLA epitopes were identified; they are defined by combinations of nonself- and self-residues for one allele of the antibody producer.

Conclusion: The nonself-self paradigm of HLA epitope immunogenicity offers a second approach to analyze HLA antibody specificities.
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http://dx.doi.org/10.3389/fimmu.2015.00180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443772PMC
June 2015