HLA-E has been proposed as an immunomodulatory molecule in various physiological and pathological contexts. However, few data are available on its involvement in bowel oncogenesis. Our aim was to investigate whether HLA-E gene polymorphism and its expression as soluble molecules (sHLA-E) contribute to colorectal cancer (CRC) pathology in the Tunisian population. A total of 240 CRC patients and 254 age/gender-matched healthy controls (CTRLs) were genotyped for HLA-E*01:01/01:03 polymorphism (rs1264457) using sequence-specific-primers (PCR-SSP). sHLA-E levels in plasma samples (50 CRC patients and 97 CTRLs) were measured by Enzyme linked ImmunoSorbent assay (ELISA). Our results revealed lower frequencies of the HLA-E*01:01 allele (OR = 0.70, 95 %CI = [0.55-0.90], p = 0.005) and HLA-E*01:01/01:01 homozygous genotype (OR = 0.55, 95 %CI = [0.36-0.85], p = 0.005) in CRC patients compared to CTRLs. Furthermore, individuals with HLA-E*01:01 are less likely to develop CRC before the age of 50. After stratifying the patients and CTRLs by gender, it became evident that frequencies of HLA-E*01:03 allele (p = 0.001) and HLA-E*01:03/01:03 genotype (p = 0.004) were significantly higher in female patients compared to female CTRLs. However, Kaplan-Meier survival analysis showed that female patients carrying the HLA-E*01:01 (N = 82) displayed a significant shorter 3-year overall survival (HR = 2.37; 95 % CI = [1.15-4.85], p = 0.01) than non-carriers (N = 37). Additionally, sHLA-E levels in patients were significantly (p = 0.006) reduced in females (1.8 ng/ml) compared to males (2.6 ng/ml). No relationship between sHLA-E levels and HLA-E polymorphism was found. Further, there was no significant association between sHLA-E levels and CRC outcome although enriched sHLA-E molecules were able to downregulated T cells mediated IFN-γ production in a dose-independent manner. Overall, these data suggest that in the Tunisian population, HLA-E*01:01/01:03 gene polymorphism and circulating sHLA-E molecules in female CRC patients may be important factors enabling gender-specific strategies for the diagnosis and treatment of this cancer.

Background:HLA-G is a non-classical HLA class I molecule that promotes transplant tolerance. It engages the inhibitory receptor LILRB1 on immune effector cells, suppressing cytotoxic responses and inflammation, while promoting tolerogenic and regulatory immune phenotypes. Polymorphisms in the HLA-G 3' untranslated region (3'UTR) modulate HLA-G expression levels, and LILRB1 promoter variants influence receptor expression. The combined effect on kidney transplant (KTx) rejection has not been systematically studied. Methods:Living donor-recipient pairs undergoing KTx were genotyped for nine variants in the HLA-G 3'UTR region and two single nucleotide polymorphisms (SNPs) in the LILRB1 promoter (PROMO) regions. Haplotypes were arranged for both loci. Clinical endpoints were biopsy-proven T cell-mediated rejection (TCMR) within one year and antibody-mediated rejection (AMR) within five years post-transplant. Results:Donor positivity for HLA-G 3'UTR-1 or UTR-2 or negative for UTR-3 haplotype were associated with a significantly higher risk of TCMR in both univariate or multivariate analyses. Recipients lacking the LILRB1-PROMO CG haplotype also had an increased TCMR risk. The combination of an HLA-G 3'UTR-2 positive donor with a LILRB1-PROMO CG haplotype negative recipient was found to be an independent predictor of TCMR. In contrast, HLA-G 3'UTR variants were not associated with AMR, while the presence of the recipient LILRB1-PROMO CG haplotype emerged as an independent AMR risk factor. Conclusions:Donor HLA-G 3'UTR and recipient LILRB1-PROMO haplotypes define a functional immunogenetic axis that differentially influence TCMR and AMR. These results support the clinical potential of HLA-G/LILRB1 genetic profiling to improve donor selection in living KTx and to guide the development of novel rejection therapies.

Aim: Extracellular vesicles (EVs) hold great promise as emerging biomarkers for a variety of diseases. However, their clinical application is still hindered by complex and time-consuming isolation procedures. A clinically relevant scenario where improved biomarker-based diagnostics are urgently needed is prosthetic joint loosening, which may result from either aseptic inflammation or periprosthetic joint infection (PJI) - two conditions requiring fundamentally different therapeutic approaches. This study investigated whether imaging flow cytometry (IFCM) enables the discrimination between aseptic and septic loosening by profiling EVs directly in minimally processed synovial fluid. Methods: We analyzed synovial fluid from 35 aseptic and 13 septic cases using IFCM to detect surface marker-defined EV subpopulations without prior isolation. Samples were classified based on clinical and microbiological findings. Marker abundance was quantified and analyzed using logistic regression. Results: Septic loosening was associated with significantly increased levels of CD82+ EVs and decreased levels of CD9+ EVs. CD82+ EVs showed a sensitivity of 83.3% and specificity of 90.0%, while CD9+ EVs demonstrated 100% specificity but lower sensitivity (58.8%) for aseptic loosening. CD82+ EV abundance was identified as an independent predictor of septic loosening. Conclusion: IFCM enables rapid and direct detection of diagnostically relevant EVs in native synovial fluid. CD9+ and CD82+ EVs serve as promising biomarkers for distinguishing aseptic from septic endoprosthesis loosening, offering a fast and robust diagnostic tool that may complement current clinical diagnostics and support timely treatment decisions.

Hematopoietic cell transplantation (HCT) represents a potentially curative therapeutic approach for various hematologic and non-hematologic malignancies. Human leukocyte antigen (HLA) matching is still the central selection criterion for HCT donors. Nevertheless, post-transplant complications, in particular graft-versus-host disease (GvHD), relapse of disease and infectious complications, represent a major challenge and contribute significantly to morbidity and mortality. Recently, non-classical HLA class I molecules, especially HLA-E, have gained increasing attention in the context of allogeneic HCT. This review aims to summarize the latest findings on the immunomodulatory role of HLA-E, which serves as a ligand for receptors of the innate and adaptive immune system. In particular, we aim to elucidate how (i) polymorphisms within HLA-E, (ii) the NKG2A/C axis and (iii) the repertoire of peptides presented by HLA-E jointly influence the functionality of immune effector cells. Understanding this intricate network of interactions is crucial as it significantly affects NK and T cell responses and thus clinical outcomes after HCT.

HLA-G, an important immune-checkpoint (IC) molecule that exerts inhibitory signalling on immune effector cells, has been suggested to represent a key player in regulating the immune response to Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2). Since specific single-nucleotide polymorphisms (SNP) in the HLA-G 3'untranslated region (UTR), which arrange as haplotypes, are crucial for the regulation of HLA-G expression, we analysed the contribution of these genetic variants as host factors in SARS-CoV-2 infection during acute and post-acute phases. HLA-G gene polymorphisms in the 3'UTR were investigated by sequencing in an unvaccinated Coronavirus Disease 2019 (COVID-19) cohort during acute SARS-CoV-2 infection (N = 505) and in the post-acute phase (N = 253). The HLA-G 3'UTR haplotype known as UTR-3 (p = 0.002) and the variant rs17875408 (also known as +3422) T variant (p = 0.004) are independent prognostic risk factors for fatal COVID-19. The +3422T variant (p = 0.006) predicted also the early loss of neutralising SARS-CoV-2 antibodies. In addition, the HLA-G 3'UTR haplotype UTR-7 (p = 0.023) emerged as an independent prognostic factor for increased susceptibility to Long-COVID symptoms after SARS-CoV-2 infection. Our study highlights that due to the variability of the 3'UTR genetic background, HLA-G has the potential to contribute to the progression of SARS-CoV-2 infection, extending to the development of Long-COVID symptoms, despite the likely alterations in the microenvironment and associated HLA-G-specific regulatory elements over the course of the disease. By spotlighting HLA-G, the importance of the genetic background of IC and their pivotal role in modulating immune responses during and after COVID-19 are emphasised.

IntroductionTriple 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 methodsTo 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.ResultssHLA-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 responseConclusionThe 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.

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.

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.

Objectives Human leukocyte antigen (HLA)-G expression has been correlated with disease status and cancer patients'outcome. In this study, we aimed to investigate the expression levels of both free soluble molecules (sHLA-G) and via extracellular vesicles as a membrane anchored molecule (HLA-GEV) in ascites from epithelial ovarian carcinoma (EOC) patients. We also explored their correlation with CA-125 tumor marker levels and histological subtypes. Methods sHLA-G and HLA-GEV levels were measured using Enzyme-Linked Immunosorbent Assay (ELISA) method in 30 ascites from EOC patients. Results Secreted HLA-G was detected in most of the ascites of EOC patients (sHLA-G 93%; HLA-GEV70%) with increased levels (sHLA-G: 6.46 ng/ml and HLA-GEV: 3.62ng/ml). Secreted HLA-G levels were highly increased in patients with serous ovarian carcinoma versus other subtypes (sHLA-G : 6.94ng/ml vs 4.62ng/ml, and HLA-GEV: 4.20ng/ml vs 1.45ng/ml, respectively). Interestingly, sHLA-G level was increased in EOC patients with high CA-125 tumor marker levels (CA125 ≥35U/ml: 7.20ng/ml vs CA-125 <35U/ml: 3.51 ng/ml). Similarly, HLA-GEV level was increased in EOC patients with high CA-125 tumor marker levels (CA125 ≥35U/ml: 4.16ng/ml vs CA-125 <35U/ml: 1.89ng/ml). Conclusions Our preliminary data established that both sHLA-G and HLA-GEV may provide an interesting new opportunity as tumor markers to evaluate patients with suspected ovarian cancer. Further studies still needed to consolidate our finding and clearly establish secreted HLA-G as new biomarkers for monitoring the disease.

Despite major improvements in diagnostics and therapy in early as well as in locally advanced breast cancer (LABC), metastatic relapse occurs in about 20% of patients, often explained by early micro-metastatic spread into bone marrow by disseminated tumor cells (DTC). Although neoadjuvant chemotherapy (NACT) has been a successful tool to improve overall survival (OS), there is growing evidence that various environmental factors like the non-classical human leukocyte antigen-G (HLA-G) promotes cancer invasiveness and metastatic progression. HLA-G expression is associated with regulatory elements targeting certain single-nucleotide polymorphisms (SNP) in the HLA-G 3’ untranslated region (UTR), which arrange as haplotypes. Here, we systematically evaluated the impact of HLA-G 3’UTR polymorphisms on disease status, on the presence of DTC, on soluble HLA-G levels, and on therapy and disease outcome in non-metastatic LABC patients. Although haplotype frequencies were similar in patients (n = 142) and controls (n = 204), univariate analysis revealed that the UTR-7 haplotype was related to patients with low tumor burden, whereas UTR-4 was associated with tumor sizes >T1. Furthermore, UTR-4 was associated with the presence of DTC, but UTR-3 and UTR-7 were related to absence of DTC. Additionally, increased levels of soluble HLA-G molecules were found in patients carrying UTR-7. Regarding therapy and disease outcome, univariate and multivariate analysis highlighted UTR-1 or UTR-2 as a prognostic parameter indicative for a beneficial course of disease in terms of complete response towards NACT or progression-free survival (PFS). At variance, UTR-4 was an independent risk factor for a reduced OS besides already known parameters. Taken into account the most common HLA-G 3’UTR haplotypes (UTR-1–UTR-7, UTR-18), deduction of the UTR-1/2/4 haplotypes to specific SNPs revealed that the +3003C variant, unique for UTR-4, seemed to favor a detrimental disease outcome, while the +3187G and +3196G variants, unique for UTR-1 or UTR-2, were prognostic parameters for a beneficial course of disease. In conclusion, these data suggest that the HLA-G 3’UTR variants +3003C, +3187G, and +3196G are promising candidates for the prediction of therapy and disease outcome in LABC patients.

Objectives: Colorectal cancer (CRC) is a serious gastrointestinal disease. Cancer cells can survive in a microenvironment that includes distinct immune cells, various immune checkpoints including HLA-G, and different immune effectors such as interleukin-6 (IL-6), TNF-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). Thus, the objective of this study was to investigate HLA-G involvement in CRC as a prognostic factor in the Tunisian population, both in its soluble form (sHLA-G) and in its form linked to extracellular vesicles (HLA-G (EV)). Additionally, we examined its association to the secretion of cytokines. Methods: Fifty Tunisian patients, diagnosed with CRC, matched with ninety-eight healthy blood donors (HD) were enrolled in this study. Levels of sHLA-G, HLA-G(EV), IL-6, TNF-alpha, and IFN-gamma were dosed in plasma samples using specific ELISA. For the functional assay, we assessed sHLA-G effects on the production of cytokines by stimulated T lymphocytes. We measured the concentration of IL-6, TNF-alpha, and IFN-gamma produced by activated T lymphocytes pre-stimulated by plasma sHLA-G and HLA-G(EV). Results: Our case-control analysis showed high concentration of both sHLA-G (8.8 [0-63] ng/ml vs. 2.1 [0-63] ng/ml, p < 0.0001) and HLA-G(EV) (0.9 [0-17.8] ng/ml vs. 0.3 [0-31.0] ng/ml, p = 0.018) in CRC patients compared to HD. Concerning cytokines, IL-6 exclusively increased compared to HD (7.1[0.0-104.0] pg/ml vs. 3.6[0.0-58.22] pg/ml, p = 0.005). The evaluation of diagnostic performance using the receiver operating characteristic (ROC) curves and the area under the curve (AUC) showed sHLA-G (AUC = 0.828) as the more adequate biomarker in colorectal carcinoma prediction compared to HLAG(EV) (AUC = 0.615) and IL-6 (AUC = 0.664). We found that sHLA-G is able to decrease IL-6, TNF-alpha, and IFN-gamma production by stimulated T lymphocytes in a concentration-independent manner. Additionally, HLA-G(EV) appears to downregulate solely IL-6 release by stimulated T lymphocytes. Interestingly, HLAG(EV) was related to lymph node infiltration, which represents the most common metastatic route for colorectal cancer via the lymphatic system. Conclusions: We outlined the importance of sHLA-G and HLA-G(EV) as substantial immune biomarkers in CRC. Our findings suggest that HLA-G could be an effective diagnostic tool for colorectal cancer. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of King Saud University.

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.

Objectives Most patients with ovarian carcinoma are diagnosed at an advanced stage, and therapeutic options for these patients still limited. The present study aims to investigate soluble Human leukocyte antigen-E (sHLA-E) and TNF-α inflammatory cytokine expression in ascites of patients with epithelial ovarian carcinoma (EOC). Methods Thirty ascites specimens from EOC patients were collected. We optimized a direct sandwich enzyme-linked immunosorbent assay (ELISA) method to simultaneously determine the total antibody levels of sHLA-E and TNF-α. Results Ascites from EOC patients showed increased sHLA-E levels (Mean: 1403pg/m) and TNF-α levels (Mean: 40,4pg/ml). Interestingly, sHLA-E was positively correlated to TNF-α expression (Spearman r=0.37, p=0.05). Both soluble molecules were decreased in ascites of EOC patients with high CA-125 without significance (CA-125 <35U vs CA125 ≥35U: sHLA-E: 1914 pg/ml vs 1307pg/ml and TNF-a: 154.2pg/ml vs 24.85pg/ml). Conclusions Our preliminary data demonstrated that sHLA-E and TNF-a are increased in EOC patients' ascites but decreased in those with high CA-125 tumor marker. sHLA-E might be highly secreted to decrease local inflammation and inhibit tumor progression. Further studies could shed light on the potential immune tolerance role of HLA-E in EOC.

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.