CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测,并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析,为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV）,KM-HN-I303-211,（FLPTAPPNV）,KM-HN-I629-637。（TLLQIIETV）,KM-HN-I87-95（ILNKSIIEV）,KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）;对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低,KM-HN—I203-211（FLPTAPPNV）结合亲和力最高,其余3条肽结合亲和力介于2者之间;稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h,KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间,KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间,KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测,结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析,为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。     

HLA-A*0201与EGFP融合蛋白表达载体的构建及其在K562细胞的表达和定位

目的:构建增强型绿色荧光蛋白(EGFP)与HLA-A^*0201(A^*0201)融合蛋白哺乳动物细胞表达载体,分析其在K562细胞中的表达和亚细胞定位。方法:以RT-PCR方法克隆A^*0201cDNA,构建A^*0201-EGFP融合蛋白表达载体,转染K562细胞,以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果:从两个HLA-A2阳性供者外周血细胞中克隆到A^*0201cDNA编码区全长序列。通过PCR方法在起始位点前加入Kozak序列并删除终止密码,成功构建A^*0201-EGFP融合蛋白表达载体。以该质粒转染K562细胞,5h后A^*0201和EGFP的表达百分率分别为25.12±2.26、27.37±3.59,24h后表达水平无明显提高。表达的融合蛋白主要分布于细胞膜上,胞内分布较少。相反,转染空载体的细胞不表达A^*0201分子,仅表达EGFP,且其在细胞内呈弥散样分布。结论:成功构建A^*0201-EGFP融合蛋白表达载体,并在K562细胞中得到表达,表达产物主要分布于细胞膜表面,提示表达该融合蛋白的K562细胞是潜在的人工抗原提呈细胞。     

Identification of a novel HLA-A allele (A*1115) in the UK National External Quality Assessment Schemes for Histocompatibility and Immunogenetics' Educational Cell Exchange

The novel allele, HLA-A*1115, was identified in an 'Educational Scheme' sample (ED03/03 - from a north-western European Caucasoid blood donor) distributed by the UK National External Quality Assessment Schemes for Histocompatibility and Immunogenetics. ED03/03 was typed by serology, the polymerase chain reaction using sequence-specific primers and sequence-based typing. A*1115 is most similar to A*110101 with a single mismatch (G to C) at constant position 565, leading to a conservative amino acid change from valine (GTG) to leucine (CTG) at codon 165 in the alpha(2) domain. This substitution has not been reported for any other HLA class I allele so far. The HLA-A*1115-bearing haplotype was B*350101; Cw*040101; DRB1*140101; DRB3*020201; DQA1*010401; DQB1*0503; DPA1*0103/07; DPB1*030101. Extensive serological typing indicated that this allele essentially encodes a 'normal' HLA-A11 specificity.     

Risk factors of hepatitis C virus-related liver cirrhosis in young adults: positive family history of liver disease and transporter associated with antigen processing 2(TAP2)*0201 Allele

The aim of this study was to clinically characterize young patients with hepatitis-C-related cirrhosis. We compared 27 patients with liver cirrhosis (Group LC) who were anti-HCV positive, aged 40 years or less at the time of diagnosis, with 323 consecutive patients with HCV-related chronic hepatitis (Group CH) matched for age and gender. Furthermore, Group LC was divided into two arbitrary groups (29-35 years, n = 8 /36-40 years, n = 19), based on the age of patients at the time of diagnosis of liver cirrhosis. Patients' characteristics and family history were investigated, and the frequency of transporter associated with antigen processing 2 (TAP2) was determined. A family history of liver disease was present in 40.7% of Group LC but in 18.0% of Group CH (P < 0.05). The younger the age of diagnosis of cirrhosis in Group LC, the higher the frequency of a positive family history (29-35 years, 87.5%; 36-40 years, 21.1%, P < 0.05). The frequency of TAP2*0201 was significantly higher in young adult patients with HCV-related liver cirrhosis than in HCV carriers with normal ALT (P < 0.05), and tended to be higher than in uninfected normal subjects (P = 0.05). The cumulative survival rate of cirrhosis patients with family history of liver diseases was significantly lower than that of cirrhosis patients without such history (P < 0.05). Our findings suggest that a positive family history of liver disease and TAP2*0201 polymorphism may be risk factors for HCV-related liver cirrhosis in young adults.     

Mismatch on Glutathione S-Transferase T1 Increases the Risk of Graft-versus-Host Disease and Mortality after Allogeneic Stem Cell Transplantation

Several drug-metabolizing enzymes, preferentially expressed in the liver, have the potential to act as minor histocompatibility antigens. In the present study, we analyzed the impact of glutathione S-transferase T1 (GSTT1), glutathione S-transferase M1, glutathione S-transferase P1, and UDP glucuronosyl transferase 2B17 (UGT2B17) disparities on the outcome of 125 patients undergoing allogeneic hematopoietic stem cell transplantation. Grades 2 to 4 acute graft-versus-host disease (aGVHD) developed in 56.2% versus 73.3% of GSTT1-matched versus mismatched patients (P = .048). Remarkably, 8.6% GSTT1-matched patients developed grades 2 to 4 liver aGVHD, compared with 36.8% among GSTT1-mismatched recipients (P < .001). Regarding chronic graft-versus-host disease (cGVHD), 34.8% versus 70.7% matched versus mismatched patients developed overall cGVHD (P = .038) and 16.3% versus 48% developed hepatic cGVHD (P = .006). We also found a strong association between the UGT2B17 mismatch and the risk of severe aGVHD (P = .001), especially with gut involvement (P < .001). Most striking was the influence of the GSTT1 mismatch on nonrelapse mortality (26.8% versus 52.6%, P = .031) and overall survival (62% versus 36.9%, P = .045). In summary, UGT2B17 and GSTT1 mismatch are risk factors for the development of GVHD and the latter also influences on mortality and survival after allogeneic transplantation from HLA-identical donors.     

Direct Measurement of Peptide-Specific CD8+ T Cells Using HLA-A2:Ig Dimer for Monitoring the In Vivo Immune Response to a HER2/neu Vaccine in Breast and Prostate Cancer Patients

HER2/neu is a proto-oncogene and a member of the epidermal growth factor receptor family of proteins that is overexpressed in numerous types of human cancer. We are currently conducting clinical trials with the HER2/neu E75 peptide vaccine in breast and prostate cancer patients. We have evaluated the use of HLA-A2 dimer molecule for the immunological monitoring of cancer patients receiving the E75 peptide vaccine. Peripheral blood samples from patients receiving the vaccine were stained with HLA-A2 dimers containing the vaccine peptide E75 or control peptides and analyzed by flow cytometry. We compared the HLA-A2 dimer assay to standard methods of immunologic monitoring (IFN-gamma release, lymphocyte proliferation, and cytotoxicity). The HLA-A2 dimer assay was also compared with the HLA-A2 tetramer assay. E75 peptide-specific CD8 T cells were detected directly in the peripheral blood of patients by staining with E75-HLA-A2 dimers and CD8 antibodies. T cell cultures generated by repeated stimulations using E75 peptide-pulsed dendritic cells showed increased staining with E75-peptide loaded HLA-A2 dimers. Simultaneously analysis by the dimer assay and standard immunologic assays demonstrated that the dimer-staining assay correlated well with these methods of immunologic monitoring. A direct comparison using E75-specific HLA-A2 tetramers and HLA-A2 dimers for the detection of E75-specific CD8 T cells in peripheral blood showed comparable results with the two assays. Our findings indicate that the HLA-A2 dimer is a powerful new tool for directly quantifying and monitoring immune responses of antigen-specific T cells in peptide vaccine clinical trials.     

On the use of R1 and R2* for measurement of contrast agent concentration in isolated perfused rat liver

We present experimental MRI protocols at 4.7 T for quantitative determination of the Dotarem distribution volume in isolated perfused rat liver. The procedures involved either constant contrast agent (CA) concentration or bolus administration conditions. R1 and R2* effects of the CA in liver and perfusate were measured using gradient echo fast imaging (GEFI) experiments by varying either the excitation angle or the echo time. CA concentrations in liver and perfusate were also measured after MRI by inductively coupled plasma atomic emission spectroscopy, in order to determine in situ relaxivities in the perfusate (r1=4.2 +/- 0.1 s(-1) mm(-1), r2*=17 +/- 2 s(-1) mm(-1)) and in the liver (r1=7.2 +/- 0.2 s(-1) mm(-1), r2*=99 +/- 5 s(-1) mm(-1)). When CA concentrations were estimated from R1 measurements and r1, the CA distribution volume estimations in liver resulting from bolus (0.31 +/- 0.01) and stationary (0.32 +/- 0.05) experiments were not significantly different. In contrast, after a bolus, CA concentrations derived from R2* and r2* were overestimated in liver and even more in perfusate. However, with R1 and R2* being measured before CA bolus administration, zero echo time signal intensities computed from multiple TE measurements during multiple boli yielded good estimations of R1 and thus correct CA concentrations in liver and in perfusate. Under these conditions, a single multi-echo GEFI acquisition should be sufficient to determine the concentration-time curves. Consequently, this protocol should be appropriate to rapidly estimate the distribution volume in vivo when multiple boli have to be avoided.     

Antigen presentation in SARS-CoV-2 infection: the role of class I HLA and ERAP polymorphisms

Given the highly polymorphic nature of Human Leukocyte Antigen (HLA) molecules, it is not surprising that they function as key regulators of the host immune response to almost all invading pathogens, including SARS-CoV-2, the etiological agent responsible for the recent COVID-19 pandemic. Several correlations have already been established between the expression of a specific HLA allele/haplotype and susceptibility/progression of SARS-CoV-2 infection and new ones are continuously emerging. Protective and harmful HLA variants have been described in both mild and severe forms of the disease, but considering the huge amount of existing variants, the data gathered in such a brief span of time are to some extent confusing and contradictory. The aim of this mini-review is to provide a snap-shot of the main findings so far collected on the HLA-SARS-CoV-2 interaction, so as to partially untangle this intricate yarn. As key factors in the generation of antigenic peptides to be presented by HLA molecules, ERAP1 and ERAP2 role in SARS-CoV-2 infection will be revised as well.     

HLA-inferred extended haplotype disparity level is more relevant than the level of HLA mismatch alone for the patients survival and GvHD in T cell-replate hematopoietic stem cell transplantation from unrelated donor

Serious risks in unrelated hematopoietic stem cell transplantation (HSCT) including graft versus host disease (GvHD) and mortality are associated with HLA disparity between donor and recipient. The increased risks might be dependent on disparity in not-routinely-tested multiple polymorphisms in genetically dense MHC region, being organized in combinations of two extended MHC haplotypes (Ehp). We assessed the clinical role of donor-recipient Ehp disparity levels in N?=?889 patients by the population-based detection of HLA allele phase mismatch. We found increased GvHD incidences and mortality rates with increasing Ehp mismatch level even with the same HLA mismatch level. In multivariate analysis HLA mismatch levels were excluded from models and Ehp disparity level remained independent prognostic factor for high grade acute GvHD (p?=?0.000037, HR?=?10.68, 95%CI 5.50–32.5) and extended chronic GvHD (p?<?0.000001, HR?=?15.51, CI95% 5.36–44.8). In group with single HLA mismatch, patients with double Ehp disparity had worse 5-year overall survival (45% vs. 56%, p?=?0.00065, HR?=?4.05, CI95% 1.69–9.71) and non-relapse mortality (40% vs. 31%, p?=?0.00037, HR?=?5.63, CI95% 2.04–15.5) than patients with single Ehp disparity. We conclude that Ehp-linked factors contribute to the high morbidity and mortality in recipients given HLA-mismatched unrelated transplant and Ehp matching should be considered in clinical HSCT.