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Abstract: The ability of tetrameric major histocompatibility complex (MHC) class I-peptide complexes (tetramers) to detect antigen-specific T lymphocyte responses has yielded significant information about the generation of in vivo immunity in numerous antigenic systems. Here we present a novel method for rapid validation of tetrameric HLA molecules based on the presence of allodeterminants. Human monoclonal antibodies (mAbs) recognizing polymorphic determinants on HLA class I were immobilized on polystyrene microparticles and used to probe the structural integrity of tetrameric HLA class I molecules by flow cytometry. A total of 22 tetramers, based on HLA-A1, A2, A3, A24, B7 and B8 were reactive with their counterpart mAbs, thus confirming their antigenic integrity. A positive outcome of this mAb test ensures that tetrameric HLA class I can be used with greater confidence in subsequent functional assays. [Copyright &y& Elsevier]

Objective: Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack human leukocyte antigen (HLA)-matched donors. However, the low cell dose adversely affects hematopoietic recovery and therefore limits application of UCB transplantation in adults. Transplantation of multiple UCB units could be a strategy to overcome cell dose limitations. Materials and Methods: To investigate the effect of double cord transplantation, nonobese diabetic/severe combined immunodeficient mice were transplanted with human hematopoietic progenitor cells (CD34+) derived from two UCB units with HLA disparity. Human cell engraftment and donor origin was determined by flow cytometry. Results: Double CB transplantation resulted in increased engraftment levels in the bone marrow and peripheral blood in comparison with recipients of a single unit. Because this effect could be due to the higher cell dose (2.105 vs 1.105 cells), double CB transplantation was compared with single units containing equal cell numbers (2.105). In some cases, engraftment levels in recipients of single units containing 2.105 cells were significantly higher than after transplantation of 1.105 cells. These engraftment levels were similar to those observed after double CB transplantation. Chimerism analysis indicated that increased engraftment in recipients of two units was predominantly derived from one unit, whereas in other cases the contribution of the two units was similar. Conclusion: These results indicate that engraftment may be enhanced by addition of a second unrelated CB that might be attributed to a cell dose effect or due to a graft-facilitating effect. [Copyright &y& Elsevier]

Abstract: A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient’s cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface. [Copyright &y& Elsevier]

Although HLA matching is beneficial in clinical transplantation, it is not feasible to select a completely HLA matched donor for every potential recipient because of the enormous polymorphism of the HLA system. As a consequence, the majority of the recipients will be transplanted with a mismatched donor organ or hematopoietic stem cell transplant. For this large group of patients it is important to take advantage of the differential immunogenicity of HLA mismatches and to select for them a donor with HLA mismatches of low immunogenicity, the so-called acceptable mismatches. The differential immunogenicity of HLA mismatches can be determined by either retrospective analysis of graft survival data or by in vitro assays measuring T-cell and B-cell alloreactivity. A recently developed computer algorithm (HLAMatchmaker) can be instrumental in selecting donors with HLA mismatches, which do not lead to alloantibody formation. The theoretical background and practical implications of this acceptable mismatch approach are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Definition of the antibody specificity in the serum of patients waiting for a renal transplant or in need for platelet transfusion is a crucial step for finding adequate donors. Confounding factors are the complexity of the serum antibodies and the expression of several, up to six, different human leukocyte antigens (HLA) on peripheral blood lymphocytes used as target cells in the antibody screening. Single antigen-expressing (SAL) cell lines were generated by transfecting human major histocompatibility complex (MHC) class I sequences into K562, an erythroleukemia-derived cell line lacking MHC class I and II expression. Thirty-seven different SALs have been generated so far. In this study, we present the validation of 16 of those SALs by flow cytometry against a panel of 84 human HLA-specific monoclonal antibodies (30 HLA-A [8 IgG/22 IgM], 45 HLA-B [18 IgG/27 IgM], 6 HLA-A, B [3 IgG/3 IgM], and 3 HLA-C [all IgM]) developed in our laboratory. The SALs proved to be suitable tools to determine acceptable mismatches for highly sensitized patients. This concept of transfecting target sequences in immortalized cell lines opens up new avenues in the definition of serum and cellular reactivity for sensitized patients awaiting a suitable organ or blood component. [Copyright &y& Elsevier]

Maternally induced allotolerance both in clinical and experimental organ transplantation appears to require both in utero and oral exposure to noninherited maternal antigens (NIMA). Soluble major histocompatibility complex (MHC) class I antigens were studied in 18 mother-baby pairs in order to determine the extent of neonatal exposure to NIMA. Enzyme-linked immunoabsorbent assay (ELISA) analysis of cord blood from three genetically HLA-A2 negative babies born to HLA-A2+ mothers and from two HLA-A3 negative babies born to HLA-A3+ mothers revealed significant NIMA HLA-A levels in cord plasma. The level of NIMA-A2 or -A3 in cord blood were approximately 10% of the predicted value for a baby genetically positive for that allele. HLA-A2 or –A3 was undetectable (<1.0 ng/ml) in cord blood from HLA-A2 or -A3 negative babies whose mothers were also HLA-A2 or -A3 negative. Breast milk from HLA-A2+ mothers contained soluble HLA-A2 (sHLA-A2) at levels averaging 36.2 ng/ml, resulting in milligram quantities of ingested antigen over 3 months of nursing. Western blot analysis of cord plasma confirmed that bands corresponding to NIMA HLA-A protein were present. This study demonstrates that oral and intravenous exposure to NIMA sHLA in the fetus and newborn is much higher than previously thought, and emphasizes the importance of nursing in the overall antigen dose achieved. [Copyright &y& Elsevier]

In our initial study of liver transplant recipients using the trans vivo delayed-type hypersensitivity (DTH) assay, we found that in donor derived B-LCL or sonicates of donor leukocytes triggered linked suppression of the response to recall antigens tetanus toxoid (TT) or Epstein-Barr virus (EBV). Since both donor antigen sources contain HLA class I and class II proteins, we wished to determine which donor HLA proteins were responsible for the linked suppression effect. PBMC from four liver transplant recipients with donor-specific DTH regulation were studied. Surprisingly, we found that single donor HLA-A or B antigens (4/4 patients) but not single HLA-DR (0/4) donor antigens triggered linked suppression of DTH. A dose response study of two patients revealed that donor-type HLA-DR antigens (0.5–500ng) were not capable of triggering any linked suppression; however, as little as 500pg of donor-type HLA-class I protein triggered linked suppression of DTH response to a recall antigen. These findings may have implications for the differential impacts of class I vs class II mismatching in organ transplantation. On a practical level, they indicate that soluble HLA-A and B antigens are the proper choice for detection of DTH regulation as part of a “tolerance assay” in human liver transplant recipients. [Copyright &y& Elsevier]

Human leukocyte antigen (HLA) class I expression at the allelic level was analyzed in 397 acute myeloid leukemia (AML) and 186 acute lymphoid leukemia (ALL) using a complement-dependent cytotoxicity assay. Impaired recognition possibly due to HLA downregulation was observed in 2% of the patients with AML and ALL in complete remission, and in 8%–15% in the groups with blasts. In 15 instances of diminished cytotoxicity, leukemic cells and control PHA blasts from the same patients were further analyzed using flow cytometry. In 4/6 ALL and 4/9 AML patients HLA downregulation or complete loss (2 patients) of cell surface expression could be confirmed. No genomic abnormalities were observed. In addition, 12 AML and 13 ALL patients were tested during relapse using flow cytometry. In 1/12 AML patients and 1/13 ALL patients allelic downregulation of cell surface expression was found. In two patients tested, downregulation or loss of cell surface expression of HLA class I antigens corresponded with impaired T cell mediated lysis by HLA restricted cytotoxic T lymphocyte.Treatment of the cells with α- or γ-interferon could restore HLA class I expression and T-cell recognition. In conclusion, downregulation of cell surface expression of HLA class I expression at the allelic level in AML and ALL is infrequent but functionally relevant. HLA downregulation was reversible and T-cell recognition could be restored by α- or γ-interferon. [Copyright &y& Elsevier]

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. • Iterative KO screens reveal a new pathway controlling HLA-I antigen presentation • SPPL3 suppresses B3GNT5 activity affecting the cell surface GSL repertoire • B3GNT5-generated GSLs limit the capacity of HLA-I to interact with natural ligands • Inhibition of GSL synthesis in glioma enhances anti-tumor immune activation Numerous glycosphingolipids (GSLs) are expressed at the cell surface; however, their functions remain mostly unknown. Jongsma et al. identify that the protease SPPL3 destroys the GSL synthesis enzyme B3GNT5, whose GSL products negatively affect HLA-I-mediated immune responses. This pathway represents a potential therapeutic target in cancer, infection, and autoimmunity. [ABSTRACT FROM AUTHOR]

Aim The primary focus of HLA antibody testing for transplant patients is to assess a given patient’s potential risk for graft loss by determining the immunological status before and after transplantation. Since the physiological responses of the adaptive immune system against an HLA target is highly variable and antibody composition is distinct to each individual, accurate determination of specificities, titer and strengths of antibodies is of uttermost importance. Although MFI values provide a pragmatic approach in ranking responses, the method makes no attempt to explain titer and affinity influences in evaluating a response. To overcome such limitations, our goal was to demonstrate that titration analysis will allow better interpretation of individual antibody-HLA target interactions which will result in a better indicative risk assessment than MFI values. Methods By introduction of recombinant technology, soluble HLA ABC locus alleles were produced by a mammalian expression system and utilized to create a unique 120 single antigen assay. Utilizing this novel assay, a dose-response model was evaluated by performing multiple serological titration experiments probing the interaction of single HLA antigens with HLA antibodies. Results Results showed that the assay follows the principles of the dose-response concept not only demonstrating the binding of HLA antibodies as effectors to specific HLA targets with sigmoidal functions but also delivering potency values of their interaction. Potency analysis is a clinically relevant evaluation approach to determine titer in combination with strength of HLA antibodies independent of antibody efficacy levels. Reducing dose-response data to relative half maximum effect values showed the advantages of a fine-tuned data output by decreasing titration complexity, allowing semi-quantitative analysis and becoming independent of MFI values. Conclusions We believe that dose-response information will greatly support the identification of factors that can contribute to the individual characterization of sera antibodies and offer the potential of direct physical meaning that will resonate with clinical outcomes. This technique will likely lead to a better assessment of HLA titers and generate new insights for pre- and post-transplant monitoring. [ABSTRACT FROM AUTHOR]

Approximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against human leukocyte antigens (HLA) and, to a lesser extent, against human platelet antigens (HPA). These antibodies can lead to the rapid clearance of donor platelets, presumably through IgG-Fc receptor (FcγR)-mediated phagocytosis or via complement activation, resulting in platelet refractoriness. Strikingly, not all patients with anti-HLA or -HPA antibodies develop platelet refractoriness upon unmatched platelet transfusions. Previously, we found that IgG Fc glycosylation of anti-HLA antibodies was highly variable between patients with platelet refractoriness, especially with respect to galactosylation and sialylation of the Fc-bound sugar moiety. Here, we produced recombinant glycoengineered anti-HLA and anti- HPA-1a monoclonal antibodies with varying Fc galactosylation and sialylation levels and studied their ability to activate the classical complement pathway. We observed that anti-HLA monoclonal antibodies with different specificities, binding simultaneously to the same HLA-molecules, or anti-HLA in combination with anti-HPA-1a monoclonal antibodies interacted synergistically with C1q, the first component of the classical pathway. Elevated Fc galactosylation and, to a lesser extent, sialylation significantly increased the complement-activating properties of anti-HLA and anti-HPA-1a monoclonal antibodies. We propose that both the breadth of the polyclonal immune response, with recognition of different HLA epitopes and in some cases HPA antigens, and the type of Fc glycosylation can provide an optimal stoichiometry for C1q binding and subsequent complement activation. These factors can shift the effect of a platelet alloimmune response to a clinically relevant response, leading to complement-mediated clearance of donor platelets, as observed in platelet refractoriness.

Background: Cross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown., Methods: In this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function., Results: Here, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression., Conclusion: These data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of de novo donor-specific antibody formation. However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data., Competing Interests: The authors declare 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 © 2022 Bezstarosti, Bakker, Kramer, de Fijter, Reinders, Mulder, Claas and Heidt.)

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR + TCR deficient human Tregs maintained both Treg phenotype and function in vitro . Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR + TCR deficient Tregs did not impair the function of these HLA-A2 + islets, whereas similarly engineered A2-CAR + TCR deficient CD4 + conventional T cells rejected the islets in less than 2 weeks. A2-CAR + TCR deficient Tregs delayed graft- versus -host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance., Competing Interests: A provisional patent on A2-CAR Tregs has been submitted. QT is a co-founder and scientific advisor of Sonoma Biotherapeutics. JB is a co-founder and the Chief Executive Officer and President of Sonoma Biotherapeutics. JW is co-Founder of Soteria Biotherapeutics developing small molecule switchable biologics, on the SAB of Spotlight, and recipient of sponsored research from Bristol Myers Squibb. The remaining authors declare 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 Muller, Ferreira, Ronin, Ho, Nguyen, Faleo, Zhou, Lee, Leung, Skartsis, Kaul, Mulder, Claas, Wells, Bluestone and Tang.)

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., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU., Competing Interests: The authors declare 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 Venema, Hiddingh, de Boer, Claas, Mulder, den Hollander, Stratikos, Sarkizova, van der Veken, Janssen, van Veelen and Kuiper.)

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.)

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., (© 2018 The Authors American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

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 vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors., (Copyright © 2019 Ferrata Storti Foundation.)

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.

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 α IIb b 3 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') 2 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., (Copyright © 2018 Ferrata Storti Foundation.)

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.

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., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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.

Background: Double-cord-blood transplantation (DCBT) in patients is typically accompanied by predominance of a single unit. The causative mechanism, however, is unknown. Identifying the dynamics of mixed donor chimerism in general and in specific subpopulations may help to resolve this question. We conducted studies in a mouse model to develop a new analytic method using anti-human HLA Class I allele-specific monoclonal antibodies (HLA-MoAbs) in flow cytometry., Study Design and Methods: Single-cord-blood transplantation or DCBT from HLA-mismatched donors was performed in NOD/SCID mice. Bone marrow (BM) and peripheral blood were collected from 3 to 20 weeks after transplantation. Donor chimerism was determined quantitatively within human platelets (hPLTs), human CD45+ (hCD45+) cells, and human myeloid and lymphocyte subsets by flow cytometry., Results: Both cord donors stably engrafted in NOD/SCID. The sensitivity to detect chimerism measured with all HLA-MoAbs was 1% (>10 cells/µL). In mouse BM, the percentage of human cells measured with hCD45+ versus HLA-MoAbs correlated excellently (r = 0.999). Donor origin could be defined with HLA-MoAbs for nearly all (>93.6%) human cells in mouse peripheral blood and BM in all lineages. Chimerism of hPLTs in peripheral blood correlated well with hCD45+ cells in BM enabling frequent measurement of chimerism from early after transplantation onward., Conclusion: This approach using HLA-MoAbs enables longitudinal analysis of double-mixed human chimeric populations despite low absolute concentrations of human hematopoietic cell subsets in peripheral blood and BM in mice. Lacking reactivity with mouse cells, the HLA-MoAbs are suitable for use in other mouse models and in humans to identify the mechanisms involved in DCBT., (© 2012 American Association of Blood Banks.)

The peptide content of MHC class I molecules present at the cell surface is monitored by surveilling CD8(+) cytotoxic T cells. In case of a viral infection, a proportion of the MHC class I molecules will carry peptides derived from viral proteins. This allows the CD8(+) T cells to recognize and eliminate virus-infected cells. This highly sensitive detection system of the host is counteracted by viruses, which have acquired functions to downregulate cell surface expression of MHC class I molecules. In this chapter, we describe a flow cytometry-based method to identify viral gene product(s) responsible for evasion from MHC class I-restricted antigen presentation. To this end, cells are transiently transfected using polyethylenimine (PEI) as a transfection reagent, followed by cell surface staining with MHC class I-specific monoclonal antibodies. Once viral proteins responsible for MHC class I downregulation have been identified, their mechanism of action can be characterized. Identification and characterization of virus-encoded MHC class I inhibitors augments our understanding of virus-host interactions and often provides new insights into antigen processing and presentation pathways, including related cellular processes such as protein trafficking and degradation.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour-reactive T and/or natural killer cells may improve the treatment of advanced-stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNgamma-induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele-specific antigens. Expression of antigen processing pathway components and beta-2 microglobulin (beta2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT-PCR, transduction assays, and flow cytometry. beta2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNgamma in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP-1/-2, tapasin, LMP-2/-7, and the beta2m/HLA class I complex by IFNgamma suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNgamma-inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced-stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced-stage EWS.

To present virus and tumor Ags, HLA class I molecules undergo a complex multistep assembly involving discrete but transient folding intermediates. The most extensive folding abnormalities occur in cells lacking the class I L chain subunit, called beta(2)-microglobulin (beta(2)m). Herein, this issue was investigated taking advantage of eight conformational murine mAbs (including the prototypic W6/32 mAb) to mapped H chain epitopes of class I molecules, four human mAbs to class I alloantigens, as well as radioimmunoprecipitation, in vitro assembly, pulse-chase, flow cytometry, and peptide-pulse/ELISPOT experiments. We show that endogenous (HLA-A1, -A66, and -B58) as well as transfected (HLA-A2) heavy chains in beta(2)m-defective Burkitt lymphoma Daudi cells are capable of being expressed on the cell surface, although at low levels, and exclusively as immature glycoforms. In addition, HLA-A2 is: 1) partially folded at crucial interfaces with beta(2)m, peptide Ag, and CD8; 2) receptive to exogenous peptide; and 3) capable of presenting exogenous peptide epitopes (from virus and tumor Ags) to cytotoxic T lymphocytes (bulk populations as well as clones) educated in a beta(2)m-positive environment. These experiments demonstrate a precursor-product relationship between novel HLA class I folding intermediates, and define a stepwise mechanism whereby distinct interfaces of the class I H chain undergo successive, ligand-induced folding adjustments in vitro as well as in vivo. Due to this unprecedented class I plasticity, Daudi is the first human cell line in which folding and function of class I HLA molecules are observed in the absence of beta(2)m. These findings bear potential implications for tumor immunotherapy.

CTLs specific for hematopoietic system-restricted minor histocompatibility antigens (mHags) can serve as reagents for cellular adoptive immunotherapy after allogeneic stem cell transplantation (SCT). In the HLA-mismatched setting, CTLs specific for hematopoietic system-restricted mHags expressed solely by the non-self 'allo' HLA molecules could be used to treat relapse after HLA-mismatched SCT. The generation of mHag-specific allo-HLA-restricted CTLs requires antigen-presenting cells (APCs) expressing low numbers of endogenous peptides to avoid co-induction of undesired allo-HLA reactivities. In this study, we exploited viral evasion strategies to generate APCs expressing a controlled set of endogenous peptides. Herpesviruses persist lifelong following primary infection due to expression of viral gene products that hamper T-cell recognition of infected cells. The herpesvirus-derived proteins US6, ICP47 and UL49.5 down-regulate endogenous antigen presentation in human APCs via inhibition of the transporter associated with antigen processing. EBV-transformed B cell lines transduced with retroviral vectors encoding US6, ICP47 or UL49.5 exhibited a stable decrease in cell-surface HLA class I expression and were protected from lysis by mHag-specific CTLs. Exogenous addition of mHag peptide fully restored target cell recognition. UL49.5 showed the most pronounced inhibitory effect, reducing HLA class I expression and mHag-specific lysis up to 99%. UL49.5 also significantly diminished allo-HLA reactivities mediated by allo-HLA-specific CTLs. In conclusion, UL49.5 could be a powerful new tool to study and modulate endogenous antigen presentation.

Human leukocyte antigen (HLA) expression is important for the elimination of tumor cells by the immune system and immunotherapy. Activated T cells directed against tumor-associated antigens are fully capable of recognizing and eradicating neoplastic cells. Therefore, HLA expression loss is considered to be a main factor in tumor development. We report for the first time HLA-A and HLA-B allele-specific expression analysis by immunohistochemical staining of fresh tumor tissue and 9 lymph node metastases of 15 patients with head-and-neck squamous cell carcinoma. Heterogeneous HLA expression and HLA expression loss was detected in 13 tumor patients. Approximately 50% of the tumors had allele-specific expression loss, which would have remained undetected using HLA monomorphic and locus-specific antibodies. In the majority of the patients with head-and-neck squamous cell carcinoma, HLA allele-specific expression loss differed between primary lesions and metastases. This is important for the efficacy of immunotherapy in these patients. It can be concluded that it is crucial to study HLA expression at the allele-specific level of primary lesions and metastases. It increases and refines our knowledge of HLA expression loss in tumorgenesis, which will improve the development of specific immunotherapy.

Five HLA-A3 reactive human monoclonal antibodies (mAbs) originating from a parous woman were screened against HLA-typed panels by means of complement-dependent lymphocytotoxicity, high-definition ELISA, and flow cytometry with single antigen beads. Antibody reactivity profiles were compared with triplet amino acid sequence polymorphisms identified by HLAMatchmaker, and a three-dimensional structural modeling program (Cn3D of the National Center for Biotechnology Information) was used to determine the topography of epitopes recognized by each mAb. These mAbs originated from a woman who during pregnancy developed antibodies to the paternal HLA-A3 antigen of her child. Each mAb was specific for one mismatched triplet on HLA-A3, and the reactivity patterns of these IgM-type mAbs were practically the same in lymphocytotoxicity and antigen-binding assays. One mAb was specific for 163dT, a unique triplet present only on A3. The other mAbs reacted with 62Qe, 142mI, or 144tKr; these triplets are present on different groups of HLA-A alleles, some of which, however, did not react. Topographic modeling of triplet-defined epitopes identified clusters of polymorphic surface residues that were shared between reactive alleles. These clusters may serve as primary contact sites for the specificity-determining complementarity-determining region (CDR) loops of antibody. The reactivity with these mAbs required also the presence of self-sequence elsewhere on the HLA molecular surface as a critical secondary contact site for antibody, likely through another CDR loop. For instance, the reactivity of the 62Qe-specific mAb required the presence of a glycine residue in position 56 and the reactivity of the 142mI-specific mAb required the presence of the GTLRG sequence in positions 79-83. Conversely, there were many other amino acid differences between the mAb-reactive alleles and HLA-A3 that did not prevent antibody binding. For instance, the 62Qe-specific mAb-reactive alleles had 35 and the 142mI-reactive alleles had 50 of such "permissive" residue differences. An HLAMatchmaker-based analysis of the reactivity of human mAbs will increase our understanding of the structural definition of HLA epitopes and their reactivity with alloantibodies.

The Ig-genes of the heavy chains in anti-D-specific hybridomas and Fab/scFv-fragments selected from phage-display libraries are restricted to a group of closely related genes (IGHV3s genes). We analyzed the Ig-gene repertoire in anti-D-specific B cells of two hyperimmunized donors using a completely different method. Single B cells were cultured for 10 days in an EL4.B5 culture system. mRNA from anti-D-producing B cells was reverse transcribed into cDNA. Heavy- and light-chain gene rearrangements were amplified by PCR reactions, sequenced and cloned into a pNUT-vector system, thereby allowing the production of complete IgG and IgM. Eleven anti-D-specific B-cell clones were isolated and analyzed. Eight of these clones (including IgM-producing clones) had IGHV3s genes. We demonstrated that functional anti-D-specific IgM (4 clones) and IgG (2 clones) was produced. Using a new method, we analyzed the IGHV gene repertoire of anti-D-specific B cells of hyperimmunized donors and showed that it is indeed restricted. Moreover, we found a high frequency (1:100 and 1:500) of anti-D-specific B cells in the peripheral B cells of hyperimmunized donors. We suggest that this approach could be applied for the selection of human mAbs from immunized donors and for the analysis of Ig-gene repertoires at the single-B cell level.