Soluble HLA Class 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.

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.

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.

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.

Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery.

Soluble human leukocyte antigen class I (sHLA)-peptide complexes have been suggested to play a role in the modulation of immune responses and in immune evasion of cancer cells. The set of peptides eluted from sHLA molecules could serve as biomarker for the monitoring of patients with cancer or other conditions. Here, we describe an improved sHLA peptidomics methodology resulting in the identification of 1816 to 2761 unique peptide sequences from triplicate analyses of serum or plasma taken from three healthy donors. More than 90% of the identified peptides were 8-11mers and 74% of these sequences were predicted to bind to cognate HLA alleles, confirming the quality of the resulting immunopeptidomes. In comparison to the HLA peptidome of cultured cells, the plasma-derived peptides were predicted to have a higher stability in complex with the cognate HLA molecules and mainly derived from proteins of the plasma membrane or from the extracellular space. The sHLA peptidomes can efficiently be characterized by using the new methodology, thus serving as potential source of biomarkers in various pathological conditions.

Behavior of soluble HLA-A, -B, -C and HLA-G molecules in patients with chronic hepatitis C virus infection undergoing pegylated interferon-α and ribavirin treatment: potential role as markers of response to antiviral therapy.

The serum levels of soluble HLA class I antigens (sHLA-A, -B, -C and sHLA-G) were determined in 40 HCV genotype 1-infected patients before (T 0), after 3, 6, and 12months (T 3, T 6, and T 12) of pegylated-IFN-α plus ribavirin therapy and 6months (T 18) after the end of treatment. Twenty patients were sustained virological responders (SVR), and 20 were non-responders (NR). sHLA-A, -B, -C levels at T 0 were significantly higher in both SVR (mean 10.48μg/ml) and NR (mean 11.87μg/ml) patients as compared to healthy controls (mean 0.34μg/ml, p<0.0001) and HIV-infected subjects (mean 1.22μg/ml, p<0.0001). sHLA-G levels at T 0 were significantly higher in SVR (mean 24.78ng/ml) and NR (mean 24.93ng/ml) patients as compared to healthy controls (mean 10.34ng/ml, p=0.015 and p=0.014, respectively) but were lower as compared to HIV-infected subjects (mean 48.00ng/ml, p<0.0001). The levels of sHLA-A, -B, -C and sHLA-G significantly decreased in SVR from T 0 to T 18 (mean 1.64 and 1.43ng/ml, respectively, p<0.0001) and correlated with HCV-RNA, AST, ALT, γGT, and ALP levels. The determination of soluble HLA class I levels could be proposed as a surrogate marker to discriminate SVR and NR HCV-infected patients during PEG-IFN-α plus ribavirin therapy.

Quantification of HLA class II-specific memory B cells in HLA-sensitized individuals

For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood.By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy-immunized females, we found memory B cell frequencies ranging from 25 to 756spots per 106 B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no significant spot formation was detected.Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients.

Immunomodulation due to plasma or plasma-platelet apheresis donation: Events occurring during donation procedures.

It has been recently shown that during therapeutic apheresis procedure, a large amount of soluble HLA class I molecules settles onto plastic apheresis circuits, inducing sustained TGFβ1 pre/post-transcriptional modulation in activated patients' leukocytes. Reportedly, donors' leukocytes may be exposed to similar immunosuppressing activities during donor apheresis procedures. On this basis, it could be hypothesized that such events can cause immune modulation. It is uncertain which blood cell population is most impacted by these events. This study is focused on the effects on the T lymphocytes. To assess if such events occur, lymphocytes from 20 apheresis donors collected before and after three closely timed plasma and platelet donation procedures were analyzed for sHLA-I mediated immunomodulation. The results confirmed that sHLA-I molecules bind to the apheresis circuit surfaces. Circuits can also transiently activate donors' CD8(+) T lymphocytes, to which sHLA-I molecules can bind, thus modulating short-lasting immune effects, such as transcriptional and post-transcriptional TGFβ1 modulation and soluble Fas ligand release. However, no significant change in relative proportions, absolute number and cell viability of lymphocyte subpopulations was found and no ex vivo immune effect was detectable longer than 14 days after procedure in any cell type in all donors. Short-lived sHLA-I mediated immunomodulation was demonstrable in lymphocytes from every donor as a consequence of apheresis procedures, but no enrolled subject experienced any adverse reaction or showed any sign of immunosuppression during 24 months of follow-up after the donations.

P084 : THE HLA PROTEIN CHIP – A HIGH PERFORMANCE MICROARRAY PLATFORM FOR ANTI-HLA ANTIBODY DETECTION UTILIZING SOLUBLE HLA

Aim Successful pairing of a transplant recipient with a donor organ depends on accurate prediction of risk factors for immunologic rejection. As such, there is much interest in accurate clinical measurement of HLA Ab specificities in recipient sera. However, it has become apparent that current methodologies are still surrounded by shortcomings. Here we offer a new solution that is based on recent advancements made in the application of microarray technology in clinical diagnostics, to increase the precision and sensitivity of HLA Ab serology. The aim of this study was to demonstrate the coupling of recombinant soluble HLA proteins to a novel microarray technology as a new, simplified, inexpensive and accurate approach to transplant serology. Methods In order to validate and optimize sHLA microarray performance, multiple 12 × 12 microarrays were fabricated by microfluidic spotting of highly purified soluble HLA class I and II molecules under a range of conditions chosen to optimize protein stabilization on the microarray surface. Of particular interest were the evaluation of maximal signal and background intensities, signal uniformity between different HLA alleles and the structural integrity of the HLA proteins subsequent to microfluidic attachment to the microarray surface. Results HLA protein printed in this array system yielded >65,000 MFI with essentially no background signal. Signal uniformity among alleles tested was consistently below a 10% spread. As assessed by structure-specific antibodies HC-10 and W6/32, it was determined that the native dimer configuration predominated subsequent to microarray printing. Accuracy and reproducibility required for a low cost HLA antibody-screening array was validated with over 200 samples, resulting in a high degree of concordance with known monoclonal Ab patterns and established clinical reference sera. Conclusion We have successfully demonstrated that the combination of microarray and soluble HLA technology has resulted in a simple, accurate, and low cost method for high-performance multiplexed HLA Ab detection, which with additional development in progress will be ready to accommodate very high resolution HLA Ab screening, at time of transplantation and as a routine follow-up test to monitor the earliest stages of graft rejection.

CD8low T-cell subpopulation is increased in patients with chronic hepatitis B virus infection

Recent studies suggest that CD8+ T cells with down-regulated CD8 expression (CD3+CD8low T cells) represented as a distinct phenotype of CD8+ T cells are increased and linked to disease severity in some chronically persistent infection, such as chronic HIV and parasite infection. However, the role of CD3+CD8low T cells in the context of chronic HBV infection is poorly understood. In this study, peripheral blood samples of 47 chronic hepatitis B patients and 19 healthy controls were collected and tested for the frequency and phenotype of CD8low T cells. The circulating CD8low T cells were significantly more frequent in the patients compared to those in healthy controls, and the CD8low T cells in the patients expressed less IFN-γ and more mTGF-β1 than those in the controls, suggesting their type-2 polarized and suppressive properties. Meanwhile, the concentrations of plasma soluble HLA class I molecules were found elevated in the patients, and positively associated with the frequencies of CD8low T cells. Furthermore, the CD8low T-cell frequency in the HLA-A2-positive patients (n=21) was found negatively correlated with the T-cell responsiveness against the HBc18–27 peptide, the latter was impaired as revealed by IFN-γ Elispot assay. Our findings suggested that a better understanding of the involvement of CD8low T cells in chronically persistent HBV infection would add to our knowledge of the impaired T-cell response in the patients.

Systemic inhibition of myeloid dendritic cells by circulating HLA class I molecules in HIV-1 infection

BackgroundHIV-1 infection is associated with profound dysfunction of myeloid dendritic cells, for reasons that remain ill-defined. Soluble HLA class I molecules can have important inhibitory effects on T cells and NK cells, but may also contribute to reduced functional properties of professional antigen-presenting cells. Here, we investigated the expression of soluble HLA class I isoforms during HIV-1 infection and assessed their functional impact on antigen-presenting characteristics of dendritic cells.ResultsSoluble HLA class I molecules were highly upregulated in progressive HIV-1 infection as determined by quantitative Western blots. This was associated with strong increases of intracellular expression of HLA class I isoforms in dendritic cells and monocytes. Using mixed lymphocyte reactions, we found that soluble HLA class I molecules effectively inhibited the antigen-presenting properties of dendritic cells, however, there was no significant influence of HLA class I molecules on the cytokine-secretion properties of these cells. The immunomodulatory effects of soluble HLA class I molecules were mediated by interactions with inhibitory myelomonocytic MHC class I receptors from the Leukocyte Immunoglobulin Like Receptor (LILR) family.ConclusionsDuring progressive HIV-1 infection, soluble HLA class I molecules can contribute to systemic immune dysfunction by inhibiting the antigen-presenting properties of myeloid dendritic cells through interactions with inhibitory myelomonocytic HLA class I receptors.

Ligation of CD4 on human blood monocytes triggers macrophage differentiation (158.8)

Abstract Human monocytes/macrophages (M/M) have an important role in innate immune responses and in controlling adaptive immune responses. One unique aspect of human M/M is that they express the CD4 molecule. The role of the CD4 molecule in human monocyte/macrophage function and development is not known. To investigate the role of CD4 expressed on human M/M, human blood monocytes were treated with soluble HLA Class II (sHLA II), a natural CD4 ligand. We determined that sHLA II:CD4 ligation triggered the differentiation of human monocytes into functional mature macrophages. These newly developed macrophages had an increased ability to induce T cell activation, suggesting enhanced immune function following CD4 ligation of these cells. We observed a calcium flux and the modulation of gene and cytokine protein expression following CD4 ligation on peripheral monocytes. This indicates that ligation of CD4 transmits a functional signal in this cell type. We found that intracellular signaling of CD4 in primary monocytes occurs through src family kinases as well as via the MAPK pathway to trigger macrophage differentiation. We show that HLA II on IFN-γ-induced HUVECs can ligate CD4 on monocytes and trigger differentiation of these cells into functional macrophages. In all, our results have identified a new function for the CD4 molecule on human peripheral monocytes in triggering the differentiation of these cells into functional macrophages following their interaction with HLA II.

Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.

Elevated Soluble HLA II Protein Levels in Patients With Alpha-1 Antitrypsin Deficiency With or Without COPD

PURPOSE: Elevated levels of HLA proteins in peripheral blood and other body fluids have been reported in several pathologic conditions that are associated with increased concentrations of white blood cells such as infections, inflammation and lymphoproliferative disorders. In this investigation we measured soluble HLA class II proteins (sHLA II) in peripheral blood of patients with Alpha 1 antitrypsin deficiency (A1ATD) with and without co-existing COPD.

TRANSFUSION PRACTICE: sHLA‐I contaminating molecules as novel mechanism of ex vivo/in vitro transcriptional and posttranscriptional modulation of transforming growth factor‐β1 in CD8+ T lymphocytes and neutrophils after intravenous immunoglobulin treatment

BACKGROUND: Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders. Among others' data, an in vitro increase of intracellular TGF‐β expression when culturing CD4+ T lymphocytes in the presence of IVIG has been reported. As IVIG infusion involves administration of soluble contaminants likewise all hemoderivative preparations, we hypothesized that, besides several other immunomodulatory proposed mechanisms, the clinical effects of IVIG therapy might be, at least partly, due to contaminating soluble HLA Class I (sHLA‐I) molecules capable to exert pleiotropic immunomodulatory effects among which TGF‐β1 modulation.STUDY DESIGN AND METHODS: Ex vivo and in vitro transcriptional and posttranscriptional modulation of TGF‐β1 in CD8+ T lymphocytes and neutrophils after IVIG infusion was analyzed.RESULTS: Ex vivo analysis of cells drawn from 10 enrolled IVIG recipients pointed out a significant increase of TGF‐β1 mRNA and intracellular TGF‐β1 molecules in both leukotypes. In vitro comparable results were obtained incubating CD8+ T lymphocytes and neutrophils from healthy donors with IVIG. The immunodepletion of sHLA‐I and/or soluble Fas ligand (sFasL) abolished TGF‐β1 modulation in both leukotypes. Coculture with human immunoglobulin (Ig)M monoclonal antibody or chimeric IgG (MabThera, Roche), whose manufacturing excludes “contamination,” did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA‐I molecules enhanced TGF‐β1 mRNA in both white blood cells to levels comparable to those obtained with IVIG incubation.CONCLUSION: On the whole, these data lead us to speculate that the ability of IVIG administration to modulate TGF‐β1 might be related to the immunomodulatory activities of sHLA‐I and sFasL molecules on activated CD8+ T lymphocytes and neutrophils.

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA-I) molecules can regulate survival of NK cells and their anti-tumor killing activity. Herein, we have analysed whether interaction of sHLA-I with CD8 and/or different isoforms of killer Ig-like receptors (KIR) induced secretion of transforming growth factor (TGF)-beta1. CD8+KIR- NK cell clones secreted TGF-beta1 upon the interaction of sHLA-I with CD8 molecule. sHLA-Cw4 or sHLA-Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF-beta1 production elicited through CD8. On the other hand, sHLA-Cw4 or sHLA-Cw3 alleles induced secretion of TGF-beta1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF-beta1 strongly reduced NK cell-mediated tumor cell lysis and production of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Also, TGF-beta1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL-2-dependent surface upregulation of some of these receptors was prevented by TGF-beta1. Furthermore, TGF-beta1 hampered IL-2-induced NK cell proliferation but not IL-2-mediated rescue from apoptosis of NK cells. Depletion of TGF-beta1 restored all the NK cell-mediated functional activities analysed. Taken together these findings suggest that sHLA-I antigens may downregulate the NK cell-mediated innate response by inducing TGF-beta1 release.

TRALI Pathophysiology.

Abstract SCI-48Transfusion related acute lung injury (TRALI) is clinically defined as the new onset of acute lung injury within 6 hours of a transfusion. In TRALI a transfusion activates neutrophils leading to pulmonary leukostasis, endothelial damage, capillary leak and pulmonary edema. A number of elements (bioactive lipids, sCD40L, and leukocyte antibodies) found in blood products can active neutrophils and are risk factors for TRALI. Bioactive lipids and sCD40L accumulate in both stored red cell and platelet components. Leukocyte antibodies are most often found in blood components collected from women alloimmunized during pregnancy. A number of animal and in vitro models have shown that neutrophils activated by leukocyte antibodies cause pulmonary leukostatsis, endothelial damage and capillary leak. Far more blood components contain these TRALI factors than cause TRALI suggesting that additional patient, clinical, or blood component factors are required for the development of TRALI. For example, neutrophil-specific antibodies cause reactions in, at most, 25% of transfusion recipients. Animal models and in vitro studies have found that blood component factors are more likely to induce lung injury if the neutrophils and/or pulmonary endothelial cells are primed or activated. Endothelial cells can be primed by endotoxin and neutrophils can be primed by bioactive lipids and sCD40L. The priming of neutrophils and /or endothelium results in the tight adhesion of neutrophils to endothelial cells and enhances endothelial cell injury Between neutrophil and HLA Class I and II antibodies, neutrophil antibodies are most potent at initiating TRALI and HLA Class I antibodies are least potent. HLA Class I antigens are expressed by platelets, lymphocytes, monocytes and soluble HLA Class I antigens are present in plasma. These sources of Class I antigens likely compete with neutrophils for transfused Class I antibodies and may render them less effective at initiating TRALI than neutrophil-specific or HLA Class II antibodies. Some evidence suggests that HLA Class I antibodies induce TRALI by binding to pulmonary endothelium and activating neutrophils through their Fc portion. Class II antibodies may initiate TRALI by binding to monocytes and stimulating cytokine release. In summary, TRALI is the result of patient and blood component factors which lead to neutrophil activation and endothelial cell damage and capillary leak. Most cases likely require the confluence of multiple factors to form a “perfect inflammatory storm” which leads to significant lung injury. DisclosuresNo relevant conflicts of interest to declare.

281-P: Purification and characterization of soluble HLA class II

281-P: Purification and characterization of soluble HLA class II

Behavior of non-classical soluble HLA class G antigens in human immunodeficiency virus 1-infected patients before and after HAART: Comparison with classical soluble HLA-A, -B, -C antigens and potential role in immune-reconstitution

We have reported that serum level of soluble HLA-A, -B, -C (sHLA-A,-B,-C) antigens is elevated in HIV-infected subjects and decreases after antiretroviral therapy (HAART). In this study, we measured the levels of soluble HLA-G (sHLA-G) antigens in a cohort of HIV-infected patients before and during HAART. sHLA-G and sHLA-A, -B, -C levels were significantly elevated in HIV-infected subjects as compared with controls before antiretroviral treatment and significantly decreased after 36 months of HAART. sHLA-G levels were correlated with sHLA-A, -B, -C levels, the decrease of plasma HIV-RNA level, the increase of CD4+ T-lymphocyte number and the decrease of CD8+ T-lymphocyte number. These results suggest that the measurement of sHLA-G and sHLA-A, -B, -C antigen serum levels might represent a useful surrogate marker to monitor virological response and immune reconstitution in HIV-positive subjects undergoing HAART treatment.