Soluble HLA Research Articles

HLA-A23/HLA-A24 serotypes and dementia interaction in the elderly: Association with increased soluble HLA class I molecules in plasma.

MHC class I molecules regulate brain development and plasticity in mice and HLA class I molecules are associated with brain disorders in humans. We investigated the relationship between plasma-derived soluble human HLA class I molecules (sHLA class I), HLA class I serotypes and dementia. A cohort of HLA class I serotyped elderly subjects with no dementia/pre-dementia (NpD, n = 28), or with dementia (D, n = 28) was studied. Multivariate analysis was used to examine the influence of dementia and HLA class I serotype on sHLA class I levels, and to compare sHLA class I within four groups according to the presence or absence of HLA-A23/A24 and dementia. HLA-A23/A24 and dementia, but not age, significantly influenced the level of sHLA class I. Importantly, the concurrent presence of HLA-A23/A24 and dementia was associated with higher levels of sHLA class I (p < 0.001). This study has shown that the simultaneous presence of HLA-A23/HLA-A24 and dementia is associated with high levels of serum sHLA class I molecules. Thus, sHLA class I could be considered a biomarker of neurodegeneration in certain HLA class I carriers.

HLA antibody affinity determination: From HLA-specific monoclonal antibodies to donor HLA specific antibodies (DSA) in patient serum.

Organs transplanted across donor-specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA-affinity as well as DSA-concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA-specific monoclonal antibodies and assessed the technology-specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio-layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow-induced dispersion analysis (FIDA). While the first three (solid-phase) technologies revealed comparable high binding-strengths, suggesting measurement of avidity, the latter (in-solution) approach revealed slightly lower binding-strengths, presumably indicating measurement of affinity. We believe that our newly developed in-solution FIDA-assay is particularly suitable to provide useful clinical information by not just measuring DSA-affinities in patient serum samples but simultaneously delivering a particular DSA-concentration. Here, we investigated DSA from 20 pre-transplant patients, all of whom showed negative CDC-crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA-concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449-fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre-transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA-concentration and DSA-affinity.

Abstract 4376: A correlation of soluble HLA serum levels with clinical data and survival in patients with invasive breast cancer after neoadjuvant treatment

Abstract Invasive breast cancer (IBC) is the most common malignant disease in women worldwide. In recent years, the landscape of treatment options has rapidly evolved. With the success of checkpoint inhibitors in triple negative IBC, immunotherapy has become standard of care highlighting the strength of T cell-based strategies for anti-tumor therapy. Naturally, the targets of T cell-mediated immune responses are represented by short peptides presented on human leukocyte antigen (HLA) molecules. For several years, huge effort has been made to analyze and characterize HLA-presented tumor antigens to further understand anti-tumor immunity and to identify suitable targets for novel immunotherapy approaches. Aside from membrane-bound HLA molecules, soluble HLA-peptide complexes (sHLA) can be found in a variety of body fluids. These molecules are suggested to impact cellular immune responses, but it is still a matter of debate, whether shifts in sHLA levels contribute to disease outcome or are observed as a consequence of disease. Whereas the role of the non-classical sHLA-G in IBC has been studied showing elevated levels in patients compared to a healthy cohort, data on classical sHLA class I molecules is not yet available. Here, we report on sHLA class I serum levels in patients with IBC after neoadjuvant chemotherapy or combined chemotherapy with antibody treatment (n = 52). We observed higher serum levels of sHLA in neoadjuvant-treated IBC patients without histopathological complete response after neoadjuvant therapy (n = 36) compared to healthy volunteers (n = 84). Patients with locally advanced tumors, according to TNM classification, had a tendency towards lower sHLA levels than patients with small size tumors. Furthermore, sHLA levels were decreased in patients with sentinel node or disseminated axillary lymph node metastases compared to non-metastatic patients. Of note, sHLA levels tended to be elevated in patients with Her2-overexpression (based on immunohistochemistry staining and fluorescence in situ hybridization), whereas for histological hormone receptor expression a tendency towards lower sHLA levels in patients with high expression was observed. Furthermore, sHLA levels were reduced in patients with disseminated tumor cells in bone marrow aspiration. No significant difference in sHLA levels were seen in terms of grading of tumor, prevalence of distant metastasis, menopausal status and elevated tumor markers. Interestingly, we observed a tendency to prolonged progression-free survival in patients with high serum concentrations of sHLA, which will be updated with data from additional patients at the AACR meeting. Taken together, these data provides first insights about sHLA class I in patients with IBC after neoadjuvant treatment suggesting an impact of sHLA and T cell-mediated immune surveillance on survival in patients with IBC. Citation Format: Christian M. Tegeler, Jonas Rieth, André Koch, Dominik Dannehl, Léa L. Volmer, Sabine Matovina, Markus Hahn, Tobias Engler, Andreas D. Hartkopf, Sara Y. Brucker, Juliane S. Walz, Jonas S. Heitmann, Annika Nelde. A correlation of soluble HLA serum levels with clinical data and survival in patients with invasive breast cancer after neoadjuvant treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4376.

Increased soluble HLA in COVID-19 present a disease-related, diverse immunopeptidome associated with Tcell immunity.

HLA-presented antigenic peptides are central components of Tcell-based immunity in infectious disease. Beside HLA molecules on cell surfaces, soluble HLA molecules (sHLA) are released in the blood suggested to impact cellular immune responses. We demonstrated that sHLA levels were significantly increased in COVID-19 patients and convalescent individuals compared to a control cohort and positively correlated with SARS-CoV-2-directed cellular immunity. Of note, patients with severe courses of COVID-19 showed reduced sHLA levels. Mass spectrometry-based characterization of sHLA-bound antigenic peptides, the so-called soluble immunopeptidome, revealed a COVID-19-associated increased diversity of HLA-presented peptides and identified a naturally presented SARS-CoV-2-derived peptide from the viral nucleoprotein in the plasma of COVID-19 patients. Of interest, sHLA serum levels directly correlated with the diversity of the soluble immunopeptidome. Together, these findings suggest an inflammation-driven release of sHLA in COVID-19, directly influencing the diversity of the soluble immunopeptidome with implications for SARS-CoV-2-directed Tcell-based immunity and disease outcome.

Soluble HLA peptidome of pleural effusions is a valuable source for tumor antigens

BackgroundSoluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and...

Abstract 6625: Cancer neoepitope identification in soluble HLA from patient plasma

Abstract The roles of the Major Histocompatibility Complexes (MHC) in anti-cancer immunity are well established. Cytotoxic immune cells can recognize MHC presentation of unique peptides from altered proteins on the surface of tumor cells (tumor neoantigens) or the downregulation of MHC itself and kill the affected cells. One of the hallmarks of cancer progression is the evasion of anti-tumor immunity by various mechanisms, including the upregulation of checkpoint proteins. Checkpoint inhibitors, which have recently seen dramatic success in the clinic in some cancers, act by inducing potent immune responses to tumor neoantigens. In addition, the promise of personalized medicine lies in the theoretical ability to teach the immune system through vaccination to recognize and kill tumor cells bearing MHC-presented neoantigens. Aside from MHC on cell surfaces, the presence of soluble MHC (sMHC) has been shown in a variety of bodily fluids, including blood. Soluble MHC may be an important component of the tumor arsenal for inhibiting anti-tumor immune responses. The precise function of sMHC may be disease-dependent, but it has been postulated to play an inhibitory role, presumably through signaling cognate TCRs in the absence of a cellular signal (signal 2). Presence of sMHC carrying cancer-associated neoantigens could predict success of vaccination therapy or checkpoint therapy. Furthermore, sMHC-associated peptides could help to identify neoantigens that have already induced T cell anergy and would hence not be good targets. Here, data are presented describing the plasma immunopeptidome of advanced non small-cell lung cancer subjects under a variety of treatment paradigms. Citation Format: Julie M. Rumble, Michael Pisano, Richard Jones, Michael Ford. Cancer neoepitope identification in soluble HLA from patient plasma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6625.

Prognostic Value of Soluble HLA-G and CD38 Expression in Patients with Chronic Lymphocytic Leukemia in Egypt

Background: Chronic lymphatic leukemia (CLL) is the most common hematological malignancy in adults presenting with varied clinical courses. There is an increased demand for establishing known prognostic factors for stratifying CLL patients. Aim of the work: To investigate the expression of soluble form of human leucocyte antigen-G (HLA-G) & CD 38 in CLL and to correlate our findings with a variety of clinical and laboratory variables. Patients and methods: The study included thirty newly diagnosed CLL patients. They were 18 males and 12 females with age ranged from 49–65 (a mean of 57.5 years old). Diagnosis of CLL was confirmed by flow cytometric immunophenotyping using standard lymphoma panel. CD38 expression was determined by flow cytometry. S. HLAG was measured by ELISA method. Results: Positive CD38 expression was significantly associated with lower Hb and platelets (p = 0.008 and 0.025 respectively) also with higher Beta2 microglobulin (p = 0.012). Soluble HLA G was positively correlated with the platelets count(r = 0.400, p = 0.02). Higher level of soluble form HLA-G was significantly more frequent in patients of 60 years or older (p = 0.001). The cumulative overall survival was 83.3%. Positive CD 38 expression was associated with significantly worse survival (p < 0.005). HB count < 10 gm/dl was associated with significantly worse survival (p < 0.001). Conclusion: CD38 expressions are considered powerful prognostic markers in predicting overall survival for Egyptian CLL patients and they should be assessed to decide the patient's therapy and to determine disease prognosis. Expression of soluble form of HLA-G in CLL patients by ELISA get no extra prognostic importance.

Sex as a Determinant of Responses to a Coronary Artery Disease Self-Antigen Identified by Immune-Peptidomics.

A significant body of work implicates the adaptive immune response in atherosclerosis, the main underlying cause of coronary artery disease (CAD), yet specific antigens involved remain to be fully identified. The pathobiology of CAD is influenced by sex with many factors that may be involved in the underlying mechanisms. Given the reported sexual dimorphic nature of immune-inflammatory responses, we investigated the influence of sex on potential CAD self-antigens from acute coronary syndrome (ACS) patients using immune-precipitation of soluble HLA Class-I/peptide complexes and mass spectrometry. Relevance of identified self-antigens to atherosclerosis, the major underlying cause of CAD, was tested in the apoE–/– atherosclerotic mouse model. Soluble HLA Class-I complexes from ACS patients and self-reported controls were immune-precipitated and subjected to elution, denaturation and size-exclusion to obtain HLA-bound peptides. Peptides were then subjected to mass spectrometry and patient-unique self-peptides were grouped as common to both female and male, or unique to either sex. Three peptides common to both female and male patients (COL6A1, CDSN, and SAA2), and 2 peptides each unique to female (COL1A1 and COL5A2) or male (SAA1 and KRT 9) patients were selected and mouse homologs of the peptides were screened for self-reactive immune responses in apoE–/– mice. The screening step revealed potential sex-influenced immune responses which was associated with differential immune profiles. Based on the frequency in patient plasma, COL6A1, COL5A2, and KRT 9 peptides were then tested in immunization studies. Neither COL5A2 nor KRT 9 peptide immunization resulted in significant effects on atherosclerosis compared to controls. On the other hand, female mice immunized with COL6A1 peptide had significantly reduced atherosclerosis whereas male mice had significantly increased atherosclerosis, associated with differential immune profiles. Our study identified potential self-antigens involved in atherosclerosis using the immune peptidome of CAD patients. Altering self-reactive immune responses to COL6A1 in apoE–/– mice resulted in differential effects on atherosclerosis burden with sex as a determinant of outcome.

HLA-F Allele-Specific Peptide Restriction Represents an Exceptional Proteomic Footprint.

Peptide-dependent engagement between human leucocyte antigens class I (HLA-I) molecules and their cognate receptors has been extensively analyzed. HLA-F belongs to the non-classical HLA-Ib molecules with marginal polymorphic nature and tissue restricted distribution. The three common allelic variants HLA-F*01:01/01:03/01:04 are distinguished by polymorphism outside the peptide binding pockets (residue 50, α1 or residue 251, α3) and are therefore not considered relevant for attention. However, peptide selection and presentation undergoes a most elaborated extraction from the whole available proteome. It is known that HLA-F confers a beneficial effect on disease outcome during HIV-1 infections. The interaction with the NK cell receptor initiates an antiviral downstream immune response and lead to delayed disease progression. During the time of HIV infection, HLA-F expression is upregulated, while its interaction with KIR3DS1 is diminished. The non-polymorphic nature of HLA-F facilitates the conclusion that understanding HLA-F peptide selection and presentation is essential to a comprehensive understanding of this dynamic immune response. Utilizing soluble HLA technology we recovered stable pHLA-F*01:01, 01:03 and 01:04 complexes from K562 cells and analyzed the peptides presented. Utilizing a sophisticated LC-MS-method, we analyzed the complete K562 proteome and matched the peptides presented by the respective HLA-F subtypes with detected proteins. All peptides featured a length of 8 to 24 amino acids and are not N-terminally anchored; the C-terminus is preferably anchored by Lys. To comprehend the alteration of the pHLA-F surface we structurally compared HLA-F variants bound to selected peptides. The peptides were selected from the same cellular content; however, no overlap between the proteomic source of F*01:01, 01:03 or 01:04 selected peptides could be observed. Recognizing the balance between HLA-F expression, HLA-F polymorphism and peptide selection will support to understand the role of HLA-F in viral pathogenesis.

Soluble HLA in the Aqueous Humour of Uveal Melanoma Is Associated with Unfavourable Tumour Characteristics.

A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression and intratumoural inflammation. Aqueous humour from 108 UM patients was analysed for the presence of sHLA, using a Luminex assay specific for HLA Class I. Clinical and genetic parameters were compared between sHLA-positive and negative eyes. A qPCR analysis was performed on tumour tissue using a Fluidigm assay. In 19/108 UM-containing eyes, the sHLA level in the aqueous was above the detection limit. Tumours in sHLA-positive eyes were significantly larger, more frequently involved the ciliary body, and more often showed monosomy 3, gain of chromosome 8q and loss of BAP1 staining. Melanoma-related survival was worse in patients with sHLA-positive aqueous humour. sHLA in the aqueous did not represent the tumour’s HLA expression and did not relate to immune cell infiltration in the tumour. We conclude that UM-containing eyes may contain sHLA in the aqueous humour, where it is a prognostically-unfavourable sign and may influence local immune responses.

P078 Immune complexes formed by circulating soluble HLA antigen elicit autoantibodies to human leukocyte antigen: A pilot study comparing SLE, RA, and GWI

P078 Immune complexes formed by circulating soluble HLA antigen elicit autoantibodies to human leukocyte antigen: A pilot study comparing SLE, RA, and GWI

Releasing the concept of HLA-allele specific peptide anchors in viral infections: A non-canonical naturally presented human cytomegalovirus-derived HLA-A*24:02 restricted peptide drives exquisite immunogenicity.

T‐cell receptors possess the unique ability to survey and respond to their permanently modified ligands, self HLA‐I molecules bound to non‐self peptides of various origin. This highly specific immune function is impaired following hematopoietic stem cell transplantation (HSCT) for a timespan of several months needed for the maturation of T‐cells. Especially, the progression of HCMV disease in immunocompromised patients induces life‐threatening situations. Therefore, the need for a new immune system that delivers vital and potent CD8+ T‐cells carrying TCRs that recognize even one human cytomegalovirus (HCMV) peptide/HLA molecule and clear the viral infection long term becomes obvious. The transcription and translation of HCMV proteins in the lytic cycle is a precisely regulated cascade of processes, therefore, it is a highly sensitive challenge to adjust the exact time point of HCMV‐peptide recruitment over self‐peptides. We utilized soluble HLA technology in HCMV‐infected fibroblasts and sequenced naturally sHLA‐A*24:02 presented HCMV‐derived peptides. One peptide of 14 AAs length derived from the IE2 antigen induced the strongest T‐cell responses; this peptide can be detected with a low ranking score in general peptide prediction databanks. These results highlight the need for elaborate and HLA‐allele specific peptide selection.

Studies of cell surface and soluble HLA class I expression in pancreatic beta cells exposed to interferons or Poly I:C

HLA class I (HLA-I) molecules play a crucial role in cell-mediated immunity by presenting peptide antigens to cytotoxic CD8+T cells. A pathological hallmark of Type 1 diabetes (T1D) is the hyperexpression of HLA-I in pancreatic islets that contain residual insulin producing beta cells. The expression of HLA-I can be induced following exposure to interferons (IFN). Previous studies have implicated enteroviruses (EV) as major players in triggering an autoimmune response against beta cells and it seems likely that the hyperexpression of HLA-I contributes to the recognition and targeting of beta cells by CD8+T cells. Whilst HLA-I molecules are expressed on the surface of nucleated cells, there is increasing evidence that HLA-I can also be found in a soluble form in the plasma, including in patients with viral infections. Intriguingly, soluble HLA-I (sHLA-I) levels are significantly elevated in the serum of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and T1D, yet the function of sHLA-I is still unclear. In this project, we are investigating the impact of interferons and poly I:C, a viral dsRNA mimetic, on the expression of surface and soluble HLA-I in the human pancreatic beta cell line EndoC-βH1, the human pancreatic ductal cell line PANC1 and in HeLa cells. We show that surface HLA-I is upregulated in response to these stimuli in each of the cell lines, with greatest magnitude in EndoC-βH1. We also show for the first time that release of sHLA-I is significantly increased in response to IFNγ in EndoC-βH1 cell line.

P164 Repetitive in-vitro exposure to DFP affects MHC class I and class II antigen expression

O,O-Diisopropyl phosphofluoridate also known as Diisopropylfluorophosphate (DFP) a surrogate compound of nerve agents (Cyclosarin, VX, Sarin), is an organophosphate, used in this study to understand the mechanisms of toxicity on T cells, B cells and monocytes. Our aim was to study MHC class I & II antigen expression on T cells, B cells and monocytes after repetitive exposure to sub-lethal doses of DFP in-vitro. This study was conducted as a proof of concept since this compound was used in rodent models to study Gulf War Illness (GWI), which is a chronic multisymptom disorder affecting military veterans and civilian worker that served during 1990–91 Gulf War. We isolated T cells, B cells, and macrophages from 10 healthy donors. Freshly isolated cells were maintained in culture and were exposed to 0.1–100 μM DFP for 3 days at a 24 h interval. HLA typing was performed both by using the PCR-sequence specific primer (SSP) and by serological methods before exposure to DFP. After exposure, the cells were screened for the presence of the specific HLA-B and HLA-DR antigen. We isolated the culture supernatant to study the soluble HLA antigens. HLA typing was done for both A and B allele for T cells and DR for B cells using serology and by PCR-SSP. Once the antigens were delineated, after exposure they were tested for the specific antigen. Doses above 50 μM of DFP showed weak antigen expression. The cell surface antigen appeared weak by serology. The cell supernatant tested positive for HLA antigens, although they were combined with other proteins, it will be interesting to see how these HLA cell surface markers detach from the cell. We have isolated the supernatant for LC–MS analysis. Control cells that were not treated with DFP gave a consistently positive result and the HLA A, B, and DR were same as pre-exposure. There was no detectable sHLA in the supernatant. Exposure to organophosphate-based pesticides can cause immunotoxicity. Although we suspected and were trying to understand the GWI, these results offer a valid clue that there is relevance to HLA antigen expression and to look for soluble HLA antigen in the serum/plasma, as this sHLA, may very well initiate an autoimmune response by triggering inflammatory signals.

HLA-G mediated immune regulation is impaired by a single amino acid exchange in the alpha 2 domain

The trade-off from HLA class I expression to HLA-G expression support the immune evasion of malignant cells. The essential role of the virtually invariant HLA-G in immune tolerance, tumor immunology and its expression frequency in immune privileged tissues is known; however the specific importance of allelic subtypes in immune responses is still not well understood. HLA-G∗01:01, ∗01:03 and ∗01:04 are the most prevalent allelic variants differing at residues 31 and 110, respectively. In cytotoxicity assays applying K562 cells transduced with the HLA-G variants as targets and NK cells as effectors the differential protective potential of HLA-G variants was analyzed. Their peptide profiles were determined utilizing soluble HLA technology. An increased protective potential of HLA-G∗01:04 could be observed. All variants exhibit a unique peptide repertoire with marginal overlap, while G∗01:04 differs in its peptide anchor profile substantially. The functional differences between HLA-G subtypes could be explained by the constraint of the bound peptides, modifying the pHLA-G accessible surface. For the first time a contribution of amino acid alterations within the HLA-G heavy chain for peptide selection and NK cell recognition could be observed. These results will be a step towards understanding immune tolerance and will guide towards personalized immune therapeutic strategies.

Membranal and Blood-Soluble HLA Class II Peptidome Analyses Using Data-Dependent and Independent Acquisition.

The interaction between HLA class II peptide complexes on antigen-presenting cells and CD4+ T cells is of fundamental importance for anticancer and antipathogen immunity as well as for the maintenance of immunological tolerance. To study CD4+ T cell reactivities, detailed knowledge of the presented peptides is necessary. In recent years, dramatic advances in the characterization of membranal and soluble HLA class I peptidomes could be observed. However, the same is not true for HLA class II peptidomes, where only few studies identify more than hundred peptides. Here we describe a MS-based workflow for the characterization of membranal and soluble HLA class II DR and DQ peptidomes. Using this workflow, we identify a total of 8595 and 3727 HLA class II peptides from Maver-1 and DOHH2 cells, respectively. Based on this data, a motif-based binding predictor is developed and compared to NetMHCIIpan 3.1. We then apply the workflow to human plasma, resulting in the identification of between 34 and 152 HLA-DR and between 100 and 180 HLA-DQ peptides, respectively. Finally, we implement a data-independent acquisition workflow to increase reproducibility and sensitivity of HLA class II peptidome characterizations.

OR1 The bound peptides are the determinants for the potential of HLA-G mediated immune regulation

Aim The essential role of HLA-G in immune tolerance and its expression frequency in immune privileged tissues is well known; however the specific importance of allelic subtypes in immune responses remains unclear. 14 allelic variants exist of which HLA-G*01:01, *01:03 and *01:04 are the most prevalent with these variants differing at residues 31 and 110, respectively. Systematic analysis of these subtypes was the aim of this study. Methods In cytotoxicity assays using artificial K562 cells expressing the HLA-G variants and NK cells as effector cells the differential protective potential of HLA-G variants was analyzed. The inhibitory receptors ILT-2 and KIR2DL4 are known HLA-G ligands. To determine binding of HLA-G to these molecules, recombinant sHLA-G molecules were used to block inhibitory NK surface receptors. These experiments were repeated reciprocally by blocking or silencing the respective receptor and analyze the sHLA-G blocking sites. HLA-G*01:01, G*01:03 and G*01:04 restricted peptide profiles were determined utilizing soluble HLA technology and mass spectrometry. Results An increased protective potential of HLA-G*01:04 could be observed, G*01:04 demonstrated a stronger affinity to the inhibitory receptors on NK cells than G*01:01 or G*01:03 which we attribute to their altered peptide motifs. All variants exhibit a unique repertoire with marginal overlap, while G*01:04 differs in its peptide anchor profile substantially and appears more invariant than G*01:01 or G*01:03. Using the identified peptides bound to the G*01:04 binding groove, structural analysis explains the impact of the L110I mismatch on the HLA-G conformation and its differential interaction with NK cell receptors. Conclusions For the first time a contribution of structural alterations within the HLA-G heavy chain for peptide specificity and NK cell engagement could be observed. These results will be a step towards understanding immune tolerance and guide towards immune therapeutic strategies.

P075 Dose response features and parameters to consider in serological evaluation of transplant patients

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.

P098 Characterization and quantitation of soluble HLA molecules in human plasma

Aim The identification of prognostic parameters for the clinical course of disease states is of considerable scientific and therapeutic relevance. Serum levels of soluble HLA (sHLA) class I molecules have been advocated at various occasions possessing such predictive potential. However, lack of quantitation of sHLA levels, unavailability of appropriate assay standards and small population sizes investigated have dominantly contributed to the inability to establish the physiological importance of the presence of sHLA in body fluids and to make accurate disease associations. In this work, we have redesigned the assay approach for sHLA class I detection and introduced optimized assay conditions including a sHLA standard of highest purity and structural integrity. Methods Applying a standardized detection methodology, serum samples were screened from one hundred normal volunteer blood donors with known HLA-phenotypes. In addition, plasmapheresis samples from various patients were utilized for large-scale sHLA purification using a W6/32-affinity column. Eluted probes were further analyzed applying multiple assay strategies. Results Our findings showed an average serum level of sHLA class I in normal individuals of 494 ng/ml [190–1402 ng/ml]. No significant differences in sHLA serum levels were observed between different blood types or between male and female donors. Closer investigation of the sHLA content of several plasmapheresis samples indicated that more than 75% of sHLA is complexed with other proteins and not available in free form. HLA specificity testing using mAb matched the determined allotype of the patients confirming the self-origin of the purified sHLA. Conclusions The design and development of assay methods is crucial to meet analytical performance requirements for intact sHLA quantitation. Extraction of sHLA from human blood is highly feasible and offers a valid approach for the elucidation of the relevance of sHLA-mediated immunoregulation. Analysis of HLA-complexed antibody species or just the simple presence of elevated sHLA amounts can provide a source of information to establish a foundation for unique diagnostic approaches to gain insights into immunopathological processes for transplantation and for diseases such as cancer, autoimmunity, allergy, and viral diseases.

Allostimulatory capacity of conditionally immortalized proximal tubule cell lines for bioartificial kidney application

Novel renal replacement therapies, such as a bioartificial kidney (BAK), are needed to improve current hemodialysis treatment of end-stage renal disease (ESRD) patients. As BAK applications may reveal safety concerns, we assessed the alloimmunization and related safety aspects of readily available conditionally immortalized human proximal tubule epithelial cell (ciPTEC) lines to be used in BAK. Two ciPTEC lines, originally derived from urine and kidney tissue, were characterized for the expression and secretion of relevant molecules involved in alloimmunization and inflammatory responses, such as HLA class-I, HLA-DR, CD40, CD80, CD86, as wells as soluble HLA class I and proinflammatory cytokines (IL-6, IL-8 and TNF-α). A lack of direct immunogenic effect of ciPTEC was shown in co-culture experiments with peripheral blood mononuclear cells (PBMC), after appropriate stimulation of ciPTEC. Tight epithelial cell monolayer formation on polyethersulfone flat membranes was confirmed by zonula occludens-1 (ZO-1) expression in the ciPTEC tight junctions, and by restricted inulin-FITC diffusion. Co-culture with (activated) PBMC did not jeopardize the transepithelial barrier function of ciPTEC. In conclusion, the absence of allostimulatory effects and the stability of ciPTEC monolayers show that these unique cells could represent a safe option for BAK engineering application.