Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis.

Background Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.

Individual Immune-Modulatory Capabilities of MSC-Derived Extracellular Vesicle (EV) Preparations and Recipient-Dependent Responsiveness.

Treatment with extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been suggested as novel therapeutic option in acute inflammation-associated disorders due to their immune-modulatory capacities. As we have previously observed differences in the cytokine profile of independent MSC-EV preparations, functional differences of MSC-EV preparations have to be considered. To evaluate the immune-modulatory capabilities of specific MSC-EV preparations, reliable assays are required to characterize the functionality of MSC-EV preparations prior to administration to a patient. To this end, we established an in vitro assay evaluating the immune-modulatory capacities of MSC-EV preparations. Here, we compared the efficacy of four independent MSC-EV preparations to modulate the induction of T cell differentiation and cytokine production after phorbol 12-myristate 13-acetate (PMA)/Ionomycin stimulation of peripheral blood mononuclear cells (PBMC) derived from six healthy donors. Flow cytometric analyses revealed that the four MSC-EV preparations differentially modulate the expression of surface markers, such as CD45RA, on CD4+ and CD8+ T cells, resulting in shifts in the frequencies of effector and effector memory T cells. Moreover, cytokine profile in T cell subsets was affected in a MSC-EV-specific manner exclusively in CD8+ naïve T cells. Strikingly, hierarchical clustering revealed that the T cell response towards the MSC-EV preparations largely varied among the different PBMC donors. Thus, besides defining functional activity of MSC-EV preparations, it will be crucial to test whether patients intended for treatment with MSC-EV preparations are in principal competent to respond to the envisioned MSC-EV therapy.

Susceptibility of HLA-E*01:03 Allele Carriers to Develop Cytomegalovirus Replication After Living-Donor Kidney Transplantation.

Cytomegalovirus (CMV) causes serious complications among solid organ transplant recipients. We report the positive correlation between the presence of the HLA-E*01:03 allele in living-donor kidney recipients and CMV reactivation during the first year after transplantation. Thus, HLA-E genotyping may help identify CMV replication-prone patients who require individualized patient-based CMV management.

Functional distance between recipient and donor HLA-DPB1 determines nonpermissive mismatches in unrelated HCT.

The role of HLA amino acid (AA) polymorphism for the outcome of hematopoietic cell transplantation (HCT) is controversial, in particular for HLA class II. Here, we investigated this question in nonpermissive HLA-DPB1 T-cell epitope (TCE) mismatches reflected by numerical functional distance (FD) scores, assignable to all HLA-DPB1 alleles based on the combined impact of 12 polymorphic AAs. We calculated the difference in FD scores (ΔFD) of mismatched HLA-DPB1 alleles in patients and their 10/10 HLA-matched unrelated donors of 379 HCTs performed at our center for acute leukemia or myelodysplastic syndrome. Receiver-operator curve-based stratification into 2 ΔFD subgroups showed a significantly higher percentage of nonpermissive TCE mismatches for ΔFD >2.665, compared with ΔFD ≤2.665 (88% vs 25%, P < .0001). In multivariate analysis, ΔFD >2.665 was significantly associated with overall survival (hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.05-1.87; P < .021) and event-free survival (HR, 1.39; 95% CI, 1.05-1.82; P < .021), compared with ΔFD ≤2.665. These associations were stronger than those observed for TCE mismatches. There was a marked but not statistically significant increase in the hazards of relapse and nonrelapse mortality in the high ΔFD subgroup, whereas no differences were observed for acute and chronic graft-versus-host disease. Seven nonconservative AA substitutions in peptide-binding positions had a significantly stronger impact on ΔFD compared with 5 others (P = .0025), demonstrating qualitative differences in the relative impact of AA polymorphism in HLA-DPB1. The novel concept of ΔFD sheds new light onto nonpermissive HLA-DPB1 mismatches in unrelated HCT.

A Valine Mismatch at Position 129 of MICA Is an Independent Predictor of Cytomegalovirus Infection and Acute Kidney Rejection in Simultaneous Pancreas⁻Kidney Transplantation Recipients.

The polymorphic major histocompatibility complex class I chain-related molecule A (MICA) and its soluble form (sMICA) interact with activating receptor natural-killer group 2 member D (NKG2D) on natural-killer (NK) and T cells, thereby modifying immune responses to transplantation and infectious agents (e.g., cytomegalovirus). Two single-nucleotide polymorphisms (SNPs), rs2596538GA in the MICA promoter and rs1051792AG in the coding region (MICA-129Val/Met), influence MICA expression or binding to NKG2D, with MICA-129Met molecules showing higher receptor affinity. To investigate the impact of these SNPs on the occurrence of cytomegalovirus infection or acute rejection (AR) in individuals who underwent simultaneous pancreas⁻kidney transplantation (SPKT), 50 recipient-donor pairs were genotyped, and sMICA levels were measured during the first year post-transplantation. Recipients with a Val-mismatch (recipient Met/Met and donor Val/Met or Val/Val) showed shorter cytomegalovirus infection-free and shorter kidney AR-free survival. Additionally, Val mismatch was an independent predictor of cytomegalovirus infection and kidney AR in the first year post-transplantation. Interestingly, sMICA levels were lower in rs2596538AA and MICA129Met/Met-homozygous recipients. These results provide further evidence that genetic variants of MICA influence sMICA levels, and that Val mismatch at position 129 increases cytomegalovirus infection and kidney AR risk during the first year post-SPKT.

HLA-E allelic genotype correlates with HLA-E plasma levels and predicts early progression in chronic lymphocytic leukemia.

BACKGROUND: Human leukocyte antigen-E (HLA-E) is a nonclassical major histocompatibility complex class I molecule that recently came into sharper focus as a putative marker of advanced tumor stages and disease progression. In solid tumors, increased HLA-E expression as well as elevated soluble HLA-E (sHLA-E) plasma levels are associated with a poor prognosis; however, a role for HLA-E in hematologic malignancies remains to be established. METHODS: The authors analyzed HLA-E alleles and sHLA-E levels in a cohort of 110 individuals with chronic lymphocytic leukemia (CLL). RESULTS: In patients with CLL, levels of sHLA-E increased with advanced disease stage (P = .01) and decreased after therapy (P = .01). Longitudinal follow-up revealed that both HLA-E*01:03 alleles and high levels of sHLA-E were significantly associated with a requirement for early treatment in patients with CLL (P = .027 and P = .023, respectively). In vitro, sHLA-E inhibited degranulation and interferon-γ production by natural killer (NK) cells when cocultivated with tumor cells. Moreover, sHLA-E loaded onto microspheres induced transforming growth factor-ß release by NK cells. Multivariate analysis revealed that the presence of at least 1 HLA-E*01:03 allele was an independent predictor of a requirement for early treatment. CONCLUSIONS: HLA-E alleles and sHLA-E levels may represent novel biomarkers for early disease progression in patients with CLL. Cancer 2017;123:814-23. © 2016 American Cancer Society.

Recipient HLA-G +3142 CC Genotype and Concentrations of Soluble HLA-G Impact on Occurrence of CMV Infection after Living-Donor Kidney Transplantation.

The expression modulation of the immunosuppressive non-classical Human leukocyte antigen-G (HLA-G) molecule and its soluble isoforms is an immune evasion strategy being deployed by cytomegalovirus (CMV). The +3142 C>G single nucleotide polymorphism (SNP) located within the 3' untranslated region (3'UTR) is of crucial importance for the regulation of HLA-G expression. Therefore, we analyzed the influence of the +3142 C>G HLA-G SNP on the occurrence of CMV infection in a cohort of 178 living-donor kidney recipients and their 178 corresponding donors. In addition, soluble HLA-G (sHLA-G) levels were quantified before and after transplantation. The presence of the HLA-G +3142 CC genotype in recipients, but not donors of our cohort as along with elevated sHLA-G levels (≥ 6.1 ng/mL) were associated with higher susceptibility to CMV infection after transplantation. Our results provided evidence that i) HLA-G is implicated in the establishment of CMV after living-donor kidney transplantation and ii) recipient HLA-G +3142 CC genotype and sHLA-G concentration levels could represent important predictive risk markers for CMV infection.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease.

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention.

In vivo identification of an HLA-G complex as ubiquitinated protein circulating in exosomes.

The nonclassical human leukocyte antigen-G (HLA-G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA-G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA-G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA-G by ELISA. Complexed HLA-G was detected in exosomes, which indicates an intracellular origin of these forms. 2D-PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA-G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA-G complexes. Immunoblot analysis of exudates and exosomes with an anti-ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA-G. HLA-G ubiquitination could be reproduced in vitro in HLA-G1-transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA-G forms in vivo that open a new perspective in the study of HLA-G function and analysis.

Expression of NTRK1/TrkA affects immunogenicity of neuroblastoma cells.

High levels of the NTRK1/TrkA receptor are expressed in low-stage neuroblastomas, which are characterized by a good patient prognosis and often undergo spontaneous regression. In addition to apoptosis, tumor-immune responses might contribute to this regression. We hypothesized that TrkA expression might enhance the immune response to neuroblastomas. Immunohistochemistry on neuroblastoma tissue microarrays confirmed significantly higher lymphocyte infiltration in low-stage compared with high-stage tumors. Flow cytometry of human SH-SY5Y cells stably transfected with NTRK1/TrkA cDNA revealed significant upregulation of major histocompatibility complex (MHC) class I complexes on TrkA-expressing cells. Corresponding to this upregulation, T cell activity and cytoxicity was enhanced in the presence of SY5Y-TrkA cells or by medium conditioned by them, suggesting the existence of additional soluble factors stimulating the T cell response. Activation of natural killer (NK) cells was only increased in the presence of SY5Y-TrkA conditioned medium (CM) and not in co-culture assays, suggesting a dominant inhibitory effect of upregulated MHC class I as the primary NK cell escape mechanism of TrkA-expressing neuroblastomas. We reanalyzed gene expression data obtained from the cell culture model to identify additional genes involved in the TrkA-mediated modulation of immune responses. Upregulation of selected target genes in SY5Y-TrkA cells was confirmed on transcript and protein levels. However, none of the proteins were detected in medium conditioned by SY5Y-TrkA cells, arguing against these factors as soluble mediators of the TrkA-induced immune response. We here provide evidence that TrkA expression in neuroblastoma leads to an increased immunogenicity that may contribute to a less malignant phenotype and/or spontaneous regression of neuroblastoma cells.

HLA-G expression in human embryonic stem cells and preimplantation embryos.

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.

Programmed death receptor ligand-2 (PD-L2) bearing extracellular vesicles as a new biomarker to identify early triple-negative breast cancer patients at high risk for relapse.

PURPOSE: Based on the tumor-promoting features of extracellular vesicles (EV) and PD-L1/2-bearing EV subpopulations (PD-L1/2EV), we evaluated their potential as surrogate markers for disease progression or eligibility criteria for PD-1 immune checkpoint inhibition (ICI) approaches in early triple-negative breast cancer (TNBC). METHODS: After enrichment of EV from plasma samples of 56 patients before and 50 after chemotherapy (CT), we determined levels of EV particle number and PD-L1/2EV by nanoparticle tracking analysis or ELISA and associated the results with clinical status/outcome and the presence of distinct circulating tumor cells (CTC) subpopulations. RESULTS: Compared to healthy controls, patients had a tenfold higher EV concentration and significantly elevated PD L2EV but not PD L1EV levels. The most important clinical implications were found for PD-L2EV. High PD-L2EV levels were associated with a significantly reduced 3-year progression-free and overall survival (PFS and OS). A loss of PD-L2EV after CT was significantly more prominent in patients achieving pathological complete response (pCR). Increased pre-CT PD-L2EV levels were found in patients having NOTCH1-positive or ERBB3-positive CTC. The presence of ERBB3-positive CTC combined with high pre-CT PD-L2EV resulted in a shorter PFS. CONCLUSION: This study highlights PD L2EV as a promising biomarker for risk assessment of TNBC patients and represents the basic for additional studies introducing PD-L2EV as an eligibility criterion for PD-1 ICI approaches.

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients.

Introduction: Triple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets. Materials and methods: To follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3' UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival. Results: sHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3' UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy response. Conclusion: The results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets.

Peripheral HLA-G/ILT-2 immune checkpoint axis in acute and convalescent COVID-19 patients.

The immunosuppressive non-classical human leukocyte antigen-G (HLA-G) can elicits pro-viral activities by down-modulating immune responses. We analysed soluble forms of HLA-G, IL-6 and IL-10 as well as on immune effector cell expression of HLA-G and its cognate ILT-2 receptor in peripheral blood obtained from hospitalised and convalescent COVID-19 patients. Compared with convalescents (N = 202), circulating soluble HLA-G levels (total and vesicular-bound molecules) were significantly increased in hospitalised patients (N = 93) irrespective of the disease severity. During COVID-19, IL-6 and IL-10 levels were also elevated. Regarding the immune checkpoint expression of HLA-G/ILT-2 on peripheral immune effector cells, the frequencies of membrane-bound HLA-G on CD3+ and CD14+ cells were almost identical in patients during and post COVID-19, while the frequency of ILT-2 receptor on CD3+ and CD14+ cells was increased during acute infection. A multi-parametric correlation analysis of soluble HLA-G forms with IL-6, IL-10, activation markers CD25 and CD154, HLA-G, and ILT-2 expression on immune cells revealed a strong positive correlation of soluble HLA-G forms with membrane-bound HLA-G molecules on CD3+/CD14+ cells only in convalescents. During COVID-19, only vesicular-bound HLA-G were positively correlated with the activation marker CD25 on T cells. Thus, our data suggest that the elevated levels of soluble HLA-G in COVID-19 are due to increased expression in organ tissues other than circulating immune effector cells. The concomitant increased expression of soluble HLA-G and ILT-2 receptor frequencies supports the concept that the immune checkpoint HLA-G/ILT-2 plays a role in the immune-pathogenesis of COVID-19.

The immunosuppressive molecule HLA-G and its clinical implications.

Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) class I molecule that, through interaction with its receptors, exerts important tolerogenic functions. Its main physiological expression occurs in placenta where it seems to participate in the maternal tolerance toward the fetus. HLA-G has been studied as a marker of pregnancy complications such as abortion or pre-eclapmsia. Although HLA-G is not expressed in most adult tissues, its ectopic expression has been observed in some diseases such as viral infections, autoimmune disorders, and especially cancer. HLA-G neo-expression in cancer is associated with the capability of tumor cells to evade the immune control. In this review, we will summarize HLA-G biology and how it participates in these physiopathological processes. Special attention will be paid to its role as a diagnostic tool and also as a therapeutic target.

Genetic, immunological and clinical risk factors for biliary strictures following liver transplantation.

BACKGROUND: Biliary strictures after liver transplantation (LT) are a major cause of morbidity and reduced graft survival. AIMS: The purpose of this study was to investigate genetic, immunological and clinical risk factors for the occurrence of post-LT ischaemic type biliary lesions (ITBLs) and biliary anastomotic strictures (AS). METHODS: Clinical and laboratory data, chemokine receptor (CCR) genotypes, chemotactic cytokines and anti-major-histocompatibility complex antibodies in serum were investigated in 162 LT patients. RESULTS: In the univariate analysis, older donor and recipient age, partial LT, high peak aspartate aminotransaminase (AST) levels and CC chemokine receptor 5 delta32 loss-of-function mutation (CCR5Δ32) were associated with ITBL, whereas LT for acute liver failure (ALF), ABO-compatible non-identical LT, presence of donor-specific anti-human leucocyte antigen (HLA) class II antibodies and fractalkine receptor (CX3CR1)-249II allele were associated with AS. In the multivariate analysis, CCR5Δ32 was an independent risk factor for ITBL, whereas LT for ALF, ABO-compatible non-identical LT, and CX3CR1-249II allele remained predictive for AS. Serum levels of interferon-gamma and interleukin (IL)-6 as well as IL-10 were significantly increased in patients with biliary strictures. CONCLUSION: Specific chemokine receptor polymorphisms of the recipient are associated with development of post-LT biliary strictures. Altered cytokine profile may contribute to enhanced fibrotic tissue remodelling and biliary stricture formation. Screening of anti-HLA antibodies might be useful for early identification of at-risk patients who could benefit from closer surveillance and tailored immunosuppressive regimen. Our findings may have relevance for prediction and management of post-LT biliary strictures.

The importance of HLA-G expression in embryos, trophoblast cells, and embryonic stem cells.

The nonclassical HLA-G molecule is a trophoblast-specific molecule present in almost every pregnancy. It differs from classical HLA class I molecules by the low degree of allelic variants and the high diversity of protein structures. HLA-G is reported to be a tolerogenic molecule that acts on cells of both innate and adaptive immunity. At the maternal-fetal interface HLA-G seems to be responsible largely for the reprogramming of local maternal immune response. This review will focus on the HLA-G gene expression profile in pregnancy, in preimplantation embryos, and in human embryonic stem cells with emphasis on the structural diversity of the HLA-G protein and its potential functional and diagnostic implications.

Effect of HLA-G5 Immune Checkpoint Molecule on the Expression of ILT-2, CD27, and CD38 in Splenic B cells.

The human leukocyte antigen G (HLA-G) is an immune checkpoint molecule with a complex network of interactions with several inhibitory receptors. Although the effect of HLA-G on T cells and NK cells is well studied, the effect of HLA-G on B cells is still largely elusive. B cells are of particular interest in the context of the HLA-G-ILT-2 interaction because the ILT-2 receptor is constitutively expressed on most B cells, whereas it is only present on some subsets of T and NK cells. To characterize the effect of HLA-G5 molecules on B cells, we studied splenic B cells derived from cytomegalovirus (CMV) sero-positive donors after CMV stimulation with antigens in the presence and absence of soluble HLA-G5. In the presence of HLA-G5, increased expression of the ITIM-bearing Ig-like transcript (ILT-2) was observed on B cells, but its expression was not affected by stimulation with CMV antigens. Moreover, it became evident that HLA-G5 exposure resulted in a decreased expression of CD27 and CD38 and, accordingly, in lower proportions of CD19+CD27+CD38+ and higher proportions of CD19+CD27-CD38- B cells. Taken together, our in vitro findings demonstrate that soluble HLA-G5 suppresses markers of B cell activation, suggesting that HLA-G5 has an impact on splenic B cell differentiation and activation. Based on these results, further investigation regarding the role of HLA-G as a prognostic factor and a potential therapeutic agent with respect to B cell function appears reasonable.

The GNB3 c.825C>T (rs5443) polymorphism and protection against fatal outcome of corona virus disease 2019 (COVID-19).

Background and aims: Albeit several factors which influence the outcome of corona virus disease (COVID-19) are already known, genetic markers which may predict the outcome of the disease in hospitalized patients are still very sparse. Thus, in this study, we aimed to analyze whether the single-nucleotide polymorphism (SNP) rs5443 in the gene GNB3, which was associated with higher T cell responses in previous studies, might be a suitable biomarker to predict T cell responses and the outcome of COVID-19 in a comprehensive German cohort. Methods: We analyzed the influence of demographics, pre-existing disorders, laboratory parameters at the time of hospitalization, and GNB3 rs5443 genotype in a comprehensive cohort (N = 1570) on the outcome of COVID-19. In a sub cohort, we analyzed SARS-CoV-2-specific T cell responses and associated GNB3 rs5443 genotypes. We investigated the influence of all factors on COVID-19 fatality in multivariable analysis. Results: We found a younger patient age, normotension or absence of diabetes mellitus or cardiovascular diseases, normal blood cell counts, and low inflammatory markers at hospital admission were protective factors against fatal course of disease. In addition, the rs5443 TT genotype was significantly associated with protection against COVID-19 fatality (OR: 0.60, 95% CI: 0.40-0.92, p = 0.02). We also observed significantly increased SARS-CoV-2-specific T cell responses in rs5443 TT genotype carriers (p = 0.01). Although we observed a significant association of the factors described previously in univariate analysis, only a younger age of the patients, normal blood cell counts, and the GNB3 rs5443 TT genotype remained independent predictors against COVID-19 fatality in multivariable analysis. Conclusion: Immutable predictors for COVID-19 fatality are relatively rare. In this study we could show that the TT genotype of the SNP rs5443 in the gene GNB3 is associated with protection against COVID-19 fatality. It was as well correlated to higher SARS-CoV-2-specific T cell responses, which could result in a milder course of disease in those patients. Based on those observations we hereby provide a further prognostic biomarker, which might be used in routine diagnostics as a predictive factor for COVID-19 mortality already upon hospitalization.

Low Soluble Programmed Cell Death Protein 1 Levels After Allogeneic Stem Cell Transplantation Predict Moderate or Severe Chronic GvHD and Inferior Overall Survival.

Programmed cell death protein-1 (PD-1) is an inhibitory co-receptor required for regulating immune responsiveness and maintaining immune homeostasis. As PD-1 can be released as bioactive soluble molecule, we investigated the clinical significance of soluble PD-1 (sPD-1) after allogeneic hematopoietic stem cell transplantation (HSCT) regarding graft-versus-host disease (GvHD), relapse, and overall survival (OS) in a mono-centric cohort of 82 patients. Compared to pre-HSCT and to healthy controls, post-HSCT sPD-1 plasma levels were significantly increased during an observation time of three months. Univariate analysis revealed that low sPD-1 plasma levels at month one, two or three post HSCT were associated with acute GvHD grade III-IV, the onset of moderate/severe chronic GvHD (cGvHD) and inferior OS, DFS, and TRM, respectively. No relationship was detected to relapse rates. sPD-1 plasma levels were significantly increased in ATG-treated patients compared to ATG-untreated patients. Multivariate analysis revealed that a low sPD-1 plasma levels status at one or two month(s) after HSCT is an independent indicator for inferior OS, DFS, or TRM. A low sPD-1 plasma levels status at month three post HSCT is predictive for the onset of moderate/severe cGvHD. Thus, our study pinpoints the soluble inhibitory co-receptor PD-1 as a promising candidate molecule for the prediction of clinical HSCT outcome.