Soluble Human Leukocyte Antigen Research Articles

Abstract 1201: Pervasive HLA disruption fuels cancer evolution

Abstract Disruption of the human leukocyte antigen (HLA) molecules has important implications for immune evasion and tumor evolution. However, although genomic loss of HLA is frequent in non-small cell lung cancer (NSCLC) and in some types of breast cancer, the extent and importance of transcriptomic disruption to HLA presentation, including transcript repression and alternative splicing (AS), remains unclear. To address this, we developed MHC Hammer, a tool to call HLA allele-specific mutations, loss of heterozygosity (LOH), transcriptional repression, and alternative splicing. We first assessed HLA allelic expression and the prevalence of AS in normal tissue. Applying MHC Hammer to 510 normal lung samples and 407 normal breast samples from the Genotype-Tissue Expression cohort, we identified extensive heterogeneity in HLA allelic expression depending on both the allele and tissue type. In addition, HLA allelic AS, occurring in at least 20% of the allele transcripts, was observed in 19% of normal lung and 13% of normal breast samples. Next, we applied MHC Hammer to 413 TRACERx NSCLCs and 743 TCGA breast tumours. While mutations were uncommon, HLA LOH was a frequent event, occurring in 32% of lung adenocarcinomas (LUAD) and 57% of lung squamous cell carcinomas (LUSC). We observed a similar rate of HLA LOH in estrogen receptor positive (ER+) and negative (ER-) tumors (ER+: 14%, ER-: 15%), while in triple negative breast tumours we observed a higher rate of LOH (31%). Due to the heterogeneity in HLA expression observed in the normal samples, to measure tumor specific HLA repression and alternative splicing, we restricted our analysis to tumours with WES, tumor and tumor-adjacent normal RNAseq data. This resulted in a cohort of 88 NSCLCs from the TRACERx study as well as 44 ER+ breast tumours from the TCGA cohort. 61% of LUAD, 75% of LUSC and 35% of ER+ tumours harbored class I HLA transcriptional repression that could not be explained by underlying genomic loss. 27% of LUAD, 7% of LUSC and 55% of ER+ breast tumor patients exhibited no HLA disruption. Tumor specific AS affecting HLA exons 2, 3 or 4, potentially disrupting HLA antigen presentation, was identified in 8% of LUADs, 11% of LUSC and 7% of ER+ breast tumours. Tumor specific AS of exon 5, yielding soluble HLA molecules without a transmembrane domain was identified in 20% of LUAD, 14% of LUSCs and 18% of ER+ breast tumours. LUAD and LUSC tumor regions without HLA LOH or repression were enriched for somatic HLA AS events and alleles with somatic HLA AS had a higher neoantigen burden than those without. We found no relationship between survival and HLA expression or AS in the normal tissue. However, consistent with the importance of HLA dysfunction in tumor evolution, LUADs with low HLA expression in at least one tumor region were associated with shorter disease-free survival, HLA LOH was more common in LUAD’s that metastasized and HLA repression was enriched in LUSC primary regions seeding metastases. Citation Format: Clare Puttick, Thomas P. Jones, Michelle Leung, Oriol Pich, Felipe Galvez Cancino, Jiali Liu, Manuel Varas-Godoy, Andrew Rowan, Carlos Martinez-Ruiz, Robert Bentham, Krijn K. Dijkstra, Rachel Rosenthal, Nnennaya Kanu, Kevin Litchfield, Roberto Salgado, David A. Moore, Peter Van Loo, Mariam Jamal-Hanjani, Sergio A. Quezada, Nicholas McGranahan, Charles Swanton. Pervasive HLA disruption fuels cancer evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1201.

The correlation between soluble human leukocyte antigen (sHLA-G) levels and +3010 polymorphism.

Human leukocyte antigen-G (HLA-G) is classified as non-classical HLA, located in the short arm of chromosome 6 and composed of seven introns and eight exons. The HLA-G gene has a lower frequency polymorphism in the coding area and higher variability at the regulatory 5'- and 3'-untranslated regions linked to HLA-G microRNA regulation. HLA-G molecule is known to have an immunomodulatory and tolerogenic features role. In 199 Saudi individuals, we examined the association between plasma soluble HLA-G (sHLA-G) levels and eight polymorphic different sites, including 14bp ins/del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G single nucleotide polymorphisms (SNPs) in exon 8 in the HLA-G gene. Our results revealed higher frequency for rs17179101C (97%), rs1707T (92%) and rs9380142A (73%) alleles. Greater frequencies for the tested genotypes were observed in 3027C/C (rs17179101) (93%), 14bp (rs1704) ins/del (92%), +3003T/T (rs1707) (85%) and +3035C/T (rs17179108) (79%) SNP genotypes. Moreover, we observed a significant association of sHLA-G with +3010G/C (rs1710) SNP. In conclusion, we showed a significant association between 3010G/C (rs1710) SNP and the sHLA-G level among our sample for Saudi populations. Our findings demonstrated that specific SNP within the HLA-G gene is linked to sHLA-G molecule secretion, suggesting sHLA-G levels may be regulated genetically.

IMBAS-MS Discovers Organ-Specific HLA Peptide Patterns in Plasma

Distinction of non-self from self is the major task of the immune system. Immunopeptidomics studies the peptide repertoire presented by the human leukocyte antigen (HLA) protein, usually on tissues. However, HLA peptides are also bound to plasma soluble HLA (sHLA), but little is known about their origin and potential for biomarker discovery in this readily available biofluid. Currently, immunopeptidomics is hampered by complex workflows and limited sensitivity, typically requiring several mL of plasma. Here, we take advantage of recent improvements in the throughput and sensitivity of mass spectrometry (MS)-based proteomics to develop a highly sensitive, automated, and economical workflow for HLA peptide analysis, termed Immunopeptidomics by Biotinylated Antibodies and Streptavidin (IMBAS). IMBAS-MS quantifies more than 5000 HLA class I peptides from only 200μl of plasma, in just 30min. Our technology revealed that the plasma immunopeptidome of healthy donors is remarkably stable throughout the year and strongly correlated between individuals with overlapping HLA types. Immunopeptides originating from diverse tissues, including the brain, are proportionately represented. We conclude that sHLAs are a promising avenue for immunology and potentially for precision oncology.

Determination of soluble HLA-G serum levels in patients with adenomyosis and uterine fibroids before and after surgery

Adenomyosis is a benign gynaecological disease caused by the growth of endometrial tissue in the myometrium that affects approximately 30 % of child-bearing-age women. We evaluated the levels of soluble human leukocyte antigen G (sHLA-G) in the serum of patients with adenomyosis before and after treatment. Serum samples of 34 patients with adenomyosis and 31 patients with uterine fibroids were collected before and after the operation and were analysed for sHLA-G levels byELISAassay. The preoperative levels of serum sHLA-G in the adenomyosis group (28.05 ± 2.466 ng/ml) were significantly higher than those in the uterine fibroid group (18.53 ± 1.435 ng/ml) (P < 0.05). Serum sHLA-G levels in the adenomyosis group showed a decreasing trend at different time points after surgery (28.05 ± 14.38 ng/ml, 18.41 ± 8.34 ng/ml, and 14.45 ± 5.77 ng/ml). Adenomyosis patients who underwent total hysterectomy (n = 20) had a more significant decrease in sHLA-G levels in the early postoperative period (2 days post-operative) than those who underwent partial hysterectomy (n = 14). These results suggest that immunologic dysfunctions may be detected in patients with adenomyosis.

Serum Level of Soluble Human Leukocyte Antigen G in Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis.

This study aimed to evaluate the serum level of human leukocyte antigen G [HLA-G] in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) compared to healthy controls; moreover, it attempted to assess its relationship with SLE and RA disease activity indices. This descriptive study was conducted on 31 SLE patients (17 cases with a recent diagnosis and 14 cases with a previous diagnosis), 21 RA patients (7 cases with a recent diagnosis and 14 cases with a previous diagnosis), and 18 healthy controls who visited Ghaem Hospital affiliated to Mashhad University of Medical Sciences, Mahhad, Iran. SLE and RA activity indices were measured and recorded. Furthemore, soluble isoforms, including shed HLA-G1 and HLA-G5, were measured in serum samples via the ELISA method. A comparison of the five groups showed no significant differences in the serum level of sHLA-G. However, sHLA-G serum level was significantly higher in SLE and RA patients compared to healthy controls (P<0.05). sHLA-G level showed no correlation with disease duration and activity in SLE and RA patients (P>0.05). However, a strong positive correlation was observed between the serum level of sHLA-G and 24-h urine protein in the previously diagnosed SLE group (r=0.83, P=0.01). It seems that the serum level of sHLA-G is higher in RA and SLE patients compared to healthy controls. Furthermore, a strong correlation was found between sHLA-G serum levels and 24-h urine protein in cases with a previous diagnosis of SLE.

Soluble HLA peptidome: A new resource for cancer biomarkers.

Using circulating molecular biomarkers to screen for cancer and other debilitating disorders in a high-throughput and low-cost fashion is becoming increasingly attractive in medicine. One major limitation of investigating protein biomarkers in body fluids is that only one-fourth of the entire proteome can be routinely detected in these fluids. In contrast, Human Leukocyte Antigen (HLA) presents peptides from the entire proteome on the cell surface. While peptide-HLA complexes are predominantly membrane-bound, a fraction of HLA molecules is released into body fluids which is referred to as soluble HLAs (sHLAs). As such peptides bound by sHLA molecules represent the entire proteome of their cells/tissues of origin and more importantly, recent advances in mass spectrometry-based technologies have allowed for accurate determination of these peptides. In this perspective, we discuss the current understanding of sHLA-peptide complexes in the context of cancer, and their potential as a novel, relatively untapped repertoire for cancer biomarkers. We also review the currently available tools to detect and quantify these circulating biomarkers, and we discuss the challenges and future perspectives of implementing sHLA biomarkers in a clinical setting.

Dynamic Alteration in HLA-E Expression and Soluble HLA-E via Interaction with Natural Killer Cells in Gastric Cancer.

Some reports showed the immune tolerance of soluble human leukocyte antigen E (HLA-E), but the role that soluble HLA-E plays in gastric cancer (GC) is unknown. We aimed to clarify the molecular mechanism and clinical significance of soluble HLA-E in GC. We examined the expression of HLA-E on GC cells and soluble HLA-E under co-culture with natural killer (NK) cells in a time-dependent manner. Changes in NK cell activity were investigated using anti-NK group 2 member A (NKG2A) antibodies in the presence of soluble HLA-E. Expression of soluble HLA-E in the serum of GC patients was determined. Whereas HLA-E expression on GC cells peaked with interferon (IFN)-γ secretion by NK cells in a time-dependent manner, soluble HLA-E was upregulated in conditioned medium. Pre-incubation with anti-NKG2A antibodies increased the activation of NKG2A+ NK cells in the presence of soluble HLA-E. Expression of soluble HLA-E in the serum of GC patients correlated with disease progression. HLA-E expression dynamically changes on GC cells and in conditioned medium. Furthermore, soluble HLA-E can contribute to immune escape in GC cell lines, which may have significance in clinical practice. Moreover, soluble HLA-E may be a potential prognostic biomarker.

Effect of Alginate Microbead Encapsulation of Placental Mesenchymal Stem Cells on Their Immunomodulatory Function.

In this research we have used different cytokines and progesterone to enhance the immunomodulatory capacity of placental-derived stem cells (PLSCs) prior to their encapsulation. We assessed the effect of microencapsulation of the cells without (control) or after 3-day treatment with interferon gamma (INFγ), interleukin10 (IL-10), or progesterone (P4). Treated PLSCs demonstrated strong immunosuppressive effects on phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMNCs). INFγ treatment resulted in the strongest immune inhibition among the treated groups. The treatments enhanced soluble human leukocyte antigen (sHLAG) secretion compared to control. The IL-10-treated group showed the highest effect on HLAG secretion compared to other groups. Alginate encapsulation of PLSCs did not affect cell viability, or sHLAG secretion. Also, after treatment the encapsulated PLSCs inhibited PHA-activated PBMNCs in the same manner as unencapsulated cells. We studied two groups of encapsulated PLSCs, one without perm-selective poly-L-ornithine (PLO)-coating and the other with PLO-coating, and measured levels of sHLAG secreted. We found no difference in sHLAG secretion between both groups. In summary, our data show that immunomodulatory function of the PLSC is not affected by encapsulation. These findings provide good promise for potential use of encapsulated PLSCs for immunomodulation treatment of disease by stem cell therapy.

Novel Cardiovascular Biomarkers Associated with Increased Cardiovascular Risk in Women With Prior Preeclampsia/HELLP Syndrome: A Narrative Review.

Evidence has shown that women with a history of preeclampsia or haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome have an increased risk of cardiovascular disease later in life. Recommendations for screening, prevention and management after such pregnancies are not yet defined. The identification of promising non-traditional cardiovascular biomarkers might be useful to predict which women are at greatest risk. Many studies are inconsistent and an overview of the most promising biomarkers is currently lacking. This narrative review provides an update of the current literature on circulating cardiovascular biomarkers that may be associated with an increased cardiovascular disease risk in women after previous preeclampsia/HELLP syndrome. Fifty-six studies on 53 biomarkers were included. From the summary of evidence, soluble fms-like tyrosine kinase-1, placental growth factor, interleukin (IL)-6, IL-6/IL-10 ratio, high-sensitivity cardiac troponin I, activin A, soluble human leukocyte antigen G, pregnancy-associated plasma protein A and norepinephrine show potential and are interesting candidate biomarkers to further explore. These biomarkers might be potentially eligible for cardiovascular risk stratification after preeclampsia/HELLP syndrome and may contribute to the development of adequate strategies for prevention of hypertension and adverse events in this population.

Novel nucleic acid aptamer gold (Au)-nanoparticles (AuNPs-AptHLA-G5-1 and AuNPs-AptHLA-G5-2) to detect the soluble human leukocyte antigen G5 subtype (HLA-G5) in liquid samples.

BackgroundThe human leukocyte antigen G5 subtype (HLA-G5) is a major histocompatibility complex (MHC) molecule that is selectively expressed at the maternal-foetal tissue interface and is required for the successful implantation of the in vitro fertilized embryo. It is critical to detect HLA-G5, especially HLA-G5 expression in embryo fluid, during in vitro embryo incubation and culture. However, the specificity and sensitivity of traditional ELISA methods to detect sHLA-G5 are insufficient. This work aimed to explore novel nucleic acid aptamer gold (Au)-nanoparticles to detect soluble HLA-G5 in liquid samples.MethodsSoluble HLA-G5 was obtained using a prokaryotic expression system, and two novel aptamers (HLA-G5-Apt1 and HLA-G5-Apt2) detecting HLA-G5 were screened by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. Small (10 nm) gold nanoparticles (AuNPs) were incubated with AptHLAs to form two novel nucleic acid aptamers: Au-nanoparticles (AuNPs-AptHLA-G5-1 and AuNPs-AptHLA-G5-2).ResultsThe results showed that AptHLA-G5-1 and AptHLA-G5-2 have a high affinity for HLA-G5 and can detect its presence in liquid samples. Using the colorimetric sensing method, AuNPs-AptHLA-G1 had a detection limit as low as 20 ng/mL (recovery range between 98.7% to 102.0%), while AuNPs-AptHLA-G2 had a detection limit as low as 20 ng/mL (recovery range between 98.9% to 103.6%).ConclusionsOur work demonstrates that novel AuNPs are efficient detectors for HLA-G5 and are useful for diagnosis and treatment in the field of obstetrics-gynaecology.

Expression and change of miR-199b-5p, s HLA-G in thyroid carcinoma

ObjectiveThis research aims to investigate the relationship between the expression of microRNA-199b-5p (miR-199b-5p) and soluble human leukocyte antigen G (sHLA-G) in thyroid cancer tissues and its clinicopathological characteristics, as well as its impact on prognosis. MethodsFrozen tissues and serum from 85 patients with thyroid cancer, 27 with thyroid adenoma, 19 with Hashimoto's thyroiditis and 14 with nodular goiter from February 2014 to March 2016 were sampled. The miR-199b-5pmRNA expression in tissues was analyzed by real-time quantitative PCR. Serum HLA-G expression was detected by ELISA, and the relationship between s HLA-G expression and clinicopathological characteristics of thyroid cancer was analyzed. The relationship between 1- and 3-year survival rates of all patients and the expression of both detection indexes was observed. ResultsCompared with normal thyroid specimens, nodular goiter, Hashimoto's thyroiditis, thyroid adenoma and thyroid cancer patients, the relative expression of miR-199b-5pmRNA in thyroid cancer tissues was the lowest, while that of s HLA-G was the highest in serum of patients (P < 0.05). The levels of miR-199b-5pmRNA and serum s HLA-G in tumor tissues were correlated with clinical pathological features such as tumor size, differentiation degree, capsule invasion, lymph node metastasis, etc. (all P < 0.05). The expression of miR-199b-5pmRNA and s HLA-G were negatively correlated. ROC curve identified that miR-199b-5pmRNA and HLA-g had obvious diagnostic value for thyroid cancer patients. Kaplan-Meier survival analysis manifested that the 1- and 3-year survival rates of the miR-199b-5p low expression group in thyroid cancer tissues were lower than the miR-199b-5p high expression group, and the rates of the s HLA-G low expression group were higher than the s HLA-G high expression group. ConclusionThe miR-199b-5p expression in thyroid cancer tissues and HLA-g in serum were related to tumor size, differentiation degree, capsular invasion, lymph node metastasis and other characteristics. MiR-199b-5p may jointly affect the progression of thyroid cancer with s HLA-G.

Cytokines in embryonic secretome as potential markers for embryo selection.

Despite performing certain morphological assessments for selecting the best embryo for transfer, the results have not been satisfactory. Given the global tendency for performing quick and noninvasive tests for embryo selection, great efforts have been made to discover the predictive biomarkers of embryo implantation potential. In recent years, many factors have been detected in embryo culture media as a major source of embryo secretions. Previous studies have evaluated cytokines, miRNAs, extracellular vesicles, and other factors such as leukemia inhibitory factor, colony-stimulating factor, reactive oxygen species, soluble human leukocyte antigen G, amino acids, and apolipoproteins in these media. Given the key role of cytokines in embryo implantation, these factors can be considered promising molecules for predicting the implantation success of assisted reproductive technology (ART). The present study was conducted to review embryo-secreted molecules as potential biomarkers for embryo selection in ART.

The Potential of Soluble Human Leukocyte Antigen Molecules for Early Cancer Detection and Therapeutic Vaccine Design.

Human leukocyte antigen (HLA) molecules are essential for anti-tumor immunity, as they display tumor-derived peptides to drive tumor eradication by cytotoxic T lymphocytes. HLA molecules are primarily studied as peptide-loaded complexes on cell membranes (mHLA) and much less attention is given to their secretion as soluble HLA–peptide complexes (sHLA) into bodily fluids. Yet sHLA levels are altered in various pathologies including cancer, and are thus of high interest as biomarkers. Disconcordance in results across studies, however, hampers interpretation and generalization of the relationship between sHLA levels and cancer presence, thereby impairing its use as a biomarker. Furthermore, the question remains to what extent sHLA complexes exert immunomodulatory effects and whether shifts in sHLA levels contribute to disease or are only a consequence of disease. sHLA complexes can also bear tumor-derived peptides and recent advancements in mass spectrometry now permit closer sHLA peptide cargo analysis. sHLA peptide cargo may represent a “liquid biopsy” that could facilitate the use of sHLA for cancer diagnosis and target identification for therapeutic vaccination. This review aims to outline the contradictory and unexplored aspects of sHLA and to provide direction on how the full potential of sHLA as a quantitative and qualitative biomarker can be exploited.

Endothelial Progenitor Cell CD34+ and CD133+ Concentrations and Soluble HLA-G Concentrations During Pregnancy and in Cord Blood in Undifferentiated Connective Tissue Diseases Compared to Controls.

The objective of this study is to evaluate endothelial progenitor cells (EPCs) CD34+ CD133- and CD34+ CD133+ and soluble HLA-G (sHLA-G) concentrations among undifferentiated connective tissue disease (UCTD) subjects, compared to controls, during pregnancy and in cord blood. This is a case-control study including 29 controls and 29 UCTDs. CD34+ CD133-, CD34+ CD133+, and sHLA-G concentrations were detected in maternal plasma and in cord blood. This study was approved by the Medical-Ethical Committee of our Institution (Current Research Project N. 901-rcr2017i-23 of IRCCS Foundation Policlinico San Matteo of Pavia). Circulating CD34+ CD133- and CD34+ CD133+ counts and sHLA-G (soluble human leucocyte antigen G) concentrations in maternal peripherical blood were higher in UCTD compared to those in controls in first and third trimester of pregnancy and at delivery (p< 0.001). Maternal CD34+ CD133- and CD34+ CD133+ counts were strongly and significantly correlated in UCTD (Spearman's rho = 0.79, p< 0.0001) but not in controls (Spearman's rho = 0.10, p= 0.35). Cord blood CD34+ CD133- and CD34+ CD133+ median counts and median sHLA-G concentrations were higher among UCTD subjects than in controls (p< 0.001). Cord blood CD34+ and CD133+ counts were inversely and significantly correlated with sHLA-G concentrations among UCTDs, but not in controls. Early UCTD is characterized by increased EPC levels in maternal plasma and in cord blood and higher levels of sHLA-G, compared to controls. Data suggest that fetoplacental unit plays an independent role in the EPC response to a systemic autoimmune disease.

Pregnancy and postpartum levels of circulating maternal sHLA-G in preeclampsia

Preeclampsia is a leading cause of maternal and offspring mortality and morbidity, and predicts increased future cardiovascular disease risk. Placental dysfunction and immune system dysregulation are likely key pathophysiological factors. Soluble human leukocyte antigen G (sHLA-G) may dampen the specific immune response towards placental trophoblasts. Previous studies have shown low sHLA-G levels in preeclampsia, but postpartum, levels are unknown. Furthermore, the relationship between sHLA-G and sFlt-1 and PlGF, placental function markers, is unknown. We hypothesized that low maternal sHLA-G during pregnancy would be associated with placental dysfunction, including preeclampsia, gestational hypertension, and dysregulated sFlt-1 and PlGF, and that sHLA-G would remain decreased following preeclampsia.We included 316 pregnant women: 58 with early-onset preeclampsia (<34 weeks’ gestation), 81 with late-onset preeclampsia (≥34 weeks’ gestation), 25 with gestational hypertension, and 152 normotensive controls. Postpartum (1 or 3 years), we included 321 women: 29 with early-onset preeclampsia, 98 with late-onset preeclampsia, 57 with gestational hypertension, and 137 who were normotensive during their index pregnancies.In pregnancy, plasma sHLA-G was significantly lower both in the early- and late-onset preeclampsia groups compared to controls. In women with preeclampsia or gestational hypertension, sHLA-G was inversely correlated with serum sFlt-1. Postpartum, plasma sHLA-G levels were significantly higher in women who had had early-onset preeclampsia compared to controls.Our results support that sHLA-G may be important for placental function. Unexpectedly, sHLA-G was elevated up to 3 years after early-onset preeclampsia, suggesting an excessively activated immune system following this severe preeclampsia form, potentially contributing to future cardiovascular disease risk.

The Loss of HLA-F/KIR3DS1 Ligation Is Mediated by Hemoglobin Peptides.

The human leukocyte antigen (HLA)-Ib molecule, HLA-F, is known as a CD4+ T-cell protein and mediator of HIV progression. While HLA-Ia molecules do not have the chance to select and present viral peptides for immune recognition due to protein downregulation, HLA-F is upregulated. Post HIV infection, HLA-F loses the affinity to its activating receptor KIR3DS1 on NK cells leading to progression of the HIV infection. Several studies aimed to solve the question of the biophysical interface between HLA ligands and their cognate receptors. It became clear that even an invariant HLA molecule can be structurally modified by the variability of the bound peptide. We recently discovered the ability of HLA-F to select and present peptides and the HLA-F allele-specific peptide selection from the proteomic content using soluble HLA (sHLA) technology and a sophisticated MS method. We established recombinant K562 cells that express membrane-bound HLA-F*01:01, 01:03 or 01:04 complexes. While a recombinant soluble form of KIR3DS1 did not bind to the peptide-HLA-F complexes, acid elution of the peptides resulted in the presentation of HLA-F open conformers, and the binding of the soluble KIR3DS1 receptor increased. We used CD4+/HIV− and CD4+/HIV+ cells and performed an MS proteome analysis. We could detect hemoglobin as significantly upregulated in CD4+ T-cells post HIV infection. The expression of cellular hemoglobin in nonerythroid cells has been described, yet HLA-Ib presentation of hemoglobin-derived peptides is novel. Peptide sequence analysis from HLA-F allelic variants featured hemoglobin peptides as dominant and shared. The reciprocal experiment of binding hemoglobin peptide fractions to the HLA-F open conformers resulted in significantly diminished receptor recognition. These results underpin the molecular involvement of HLA-F and its designated peptide ligand in HIV immune escape.

Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique.

Effective and versatile screening of the peptide ligands capable of selectively binding to diverse receptors is in high demand for the state-of-the-art technologies in life sciences, including probing of specificity of the cell surface receptors and drug development. Complex microenvironment and structure of the surface receptors significantly reduce the possibility to determine their specificity, especially when in vitro conditions are utilized. Previously, we designed a publicly available platform for the ultra-high-throughput screening (uHTS) of the specificity of surface-exposed receptors of the living eukaryotic cells, which was done by consolidating the phage display and flow cytometry techniques. Here, we significantly improved this methodology and designed the fADL-1e-based phage vectors that do not require a helper hyperphage for the virion assembly. The enhanced screening procedure was tested on soluble human leukocyte antigen (HLA) class II molecules and transgenic antigen-specific B cells that express recombinant lymphoid B-cell receptor (BCR). Our data suggest that the improved vector system may be successfully used for the comprehensive search of the receptor ligands in either cell-based or surface-immobilized assays.

Natural soluble human leukocyte antigen class I in donor serum neutralizes donor-specific HLA alloantibodies in recipient serum.

BackgroundHuman leukocyte antigen (HLA) molecules are cell-bound but can be identified in a soluble form. These soluble HLA (sHLA) molecules have an immunomodulatory function. We investigated whether natural sHLA in donor serum can neutralize donor-specific HLA alloantibodies (DSAs) in recipient serum.MethodsNeutralizing effects of donor serum on DSAs in recipient serum were measured using inhibition assay principle of flow cytometric crossmatch (FCXM), performed using sera from 143 kidney transplant recipients and their donors. The adding of donor serum to recipient serum yielded lower mean fluorescence intensity (MFI) ratios (test/control) than when diluent was added [Roswell Park Memorial Institute (RPMI) or third-party serum], which was presumed to be caused by the neutralizing effects of sHLA.ResultsIn the recipient group with class I DSAs alone (N=14), donor serum addition to recipient serum resulted in lower T cell MFI ratios [2.25 (1.31‒32.51)] than those observed on RPMI addition [3.04 (1.33‒125.39), P<0.05]. In the recipient group with class II DSAs alone (N=27), donor serum addition showed no significant difference in B cell MFI ratios [5.03 (1.41‒103.53)] compared to diluent addition: RPMI [4.50 (1.34‒145.98)] or third-party serum [5.08 (1.44‒138.47)], P>0.05 for both.ConclusionUsing inhibition FCXM, we verified that natural sHLA class I in donor serum neutralizes DSAs in recipient serum. However, no neutralizing effects of sHLA class II were revealed in this study. These potentially beneficial effects of sHLA infused via blood-derived products should be considered when desensitizing highly HLA-sensitized patients.

Plasma soluble human leukocyte antigen G predicts the long-term prognosis in patients with colorectal cancer.

BackgroundColorectal cancer (CRC) is the third most common tumor in the world. It is crucial to further screen for indicators that are strictly related to long-term prognosis. Human leukocyte antigen G (HLA-G) is closely involved in tumor development. This study aims to evaluate the long-term predictive value of soluble HLA-G (sHLA-G) in CRC through a double-center retrospective study.MethodsPatients who underwent radical CRC surgery in two hospitals from April 2010 to March 2015 were included. Baseline data of the patients were collected. The patients’ preoperative baseline plasma sHLA-G level was detected. According to whether they survived during five years of follow-up, these patients were divided into two groups. Univariate and multivariate analysis were used to find factors associated with survival. The receiver operation curve (ROC) was performed to analyze the relationship between sHLA-G level and five-year survival, and the cutoff value was obtained. The baseline data and follow-up results of the two groups of patients were compared, and the Kaplan-Meier survival curve was used to analyze the difference in the long-term prognosis of the two groups of patients.ResultsA total of 1,037 cases were included and followed up for more than five years. Three hundred two cases (29.1%) died during the five-year follow-up. The baseline data of the survival group and the death group showed that the baseline level of sHLA-G in the survival group was significantly lower than the death group (41.4±16.9 vs. 64.7±23.2, P<0.05). Univariate and multivariate analyses suggest that age, differentiation, family history, TNM stage, and baseline sHLA-G levels are important factors affecting the survival of CRC patients. The area under ROC of sHLA-G with five-year survival is 0.766, and the cutoff value is 50.8 U/mL. The specificity at this point is 78.1%, and the sensitivity is 72.3%. The Kaplan-Meier survival curve showed that the five-year prognosis of patients in the low-level group was better than that in the high-level group.ConclusionsLower baseline sHLA-G level predicts better prognosis during a five-year follow-up in colorectal cancer patients.

The P2X7 Receptor 489C>T Gain of Function Polymorphism Favors HHV-6A Infection and Associates With Female Idiopathic Infertility.

The P2X7 receptor (P2X7R) is an ATP-gated ion channel known for its proinflammatory activity. Despite its participation in host defense against pathogens, the role played in viral infections, notably those caused by herpes viruses, has been seldom studied. Here we investigated the effect of P2X7R expression on human herpes virus 6 A (HHV-6A) infection of P2X7R-expressing HEK293 cells. We show that functional P2X7R increases while its blockade decreases viral load. Interestingly, HHV-6A infection was enhanced in HEK293 cells transfected with P2X7R cDNA bearing the gain of function 489C>T SNP (rs208294, replacing a histidine for tyrosine at position 155). The P2X7R 489C>T polymorphism correlated with HHV-6A infection also in a cohort of 50 women affected with idiopathic infertility, a condition previously shown to correlate with HHV-6A infection. None of the infertile women infected by HHV-6A was homozygote for 489CC genotype, while on the contrary HHV-6A infection significantly associated with the presence of the rs208294 allele. Levels of soluble human leukocyte antigen G (sHLA-G), a factor promoting embryo implant, measured in uterine flushings negatively correlated with the 489TT genotype and HHV-6A infection, while proinflammatory cytokines interleukins 1α (IL-1α), 1β (IL-1β), and 8 (IL-8) positively correlated with both the 489T allele presence and viral infection. Taken together these data point to the P2X7R as a new therapeutic target to prevent HHV-6A infection and the associated infertility.