Impact of preformed donor-specific antibodies against HLA class Ⅰ on kidney graft outcomes:Comparative analysis of exclusively anti-Cw vs anti-A and/or-B antibodies

AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.

Anti-human leukocyte antigens and anti-major histocompatibility complex class I-related chain A antibody expression in kidney transplantation during a four-year follow-up

Background Humoral immunity is an important factor for long-term survival of renal allograft. Here we performed a four-year follow-up to explore the clinical significance of monitoring anti-human leukocyte antigens （HLA） and anti-major histocompatibility complex class I-related chain A （MICA） antibody expression after kidney transplantation. Methods We obtained serial serum samples from 84 kidney transplant patients over a four-year period. All patients were followed up at least 6 months after transplantation and had at least two follow-up points. Anti-HLA and anti-MICA antibody titres and serum creatinine （SCr） levels were evaluated at each follow-up. Patients were divided into 4 groups： HLA（＋） MICA（-）, HLA（-）MICA（＋）, HLA（＋）MICA（＋） and HLA（-）MICA（-）. The impact of post-transplant antibody level on kidney allograft function was evaluated. Results Antibodies were detected in 38.1% （32/84） of the renal allograft recipients. HLA, MICA and HLA＋MICA expression was observed in 18.89%, 14.44% and 5.93% of the recipients respectively. The most frequent anti-HLA and anti-MICA specific antibodies identified were All, A24, A29, A32, A33, A80; B7, B13, B37; DR17, DR12, DR18, DR52, DR53, DR1, DR4, DR9, DR51; DQ7, DQ4, DQ8, DQ2, DQ9, DQ5, DQ6 and MICA02, MICA18, MICA19, MICA07, MICA27. As the time after transplantation elapsed, more recipients developed de novo antibody expression. Total 11.91% （10/84） of the recipients had de novo antibody expression during the follow up. The average level of SCr and the percentage of recipients with abnormal allograft function were significantly higher in recipients with anti-HLA and/or anti- MICA antibody expression than those without. The appearance of anti-HLA and anti-MICA antibody expression always preceded the increase in SCr value. Conclusions Anti-HLA and anti-MICA antibody expression has predictive value for early and late allograft dysfunction. The presence of donor specific antibody is detrimental to graft function and graft survival.

组织工程血管脱细胞支架主要组织相容性复合体Ⅰ抗原性的免疫组化观察

目的 :探讨组织工程血管全生物支架的免疫原性的检测方法。 方法 :取猪颈总动脉 ,剥离外膜 ,剪成 2 cm长的血管段 ,随机分为 3组 :对照组、脱细胞组和脱细胞加碱处理组 ,每组 5段。以免疫组化方法检测血管脱细胞处理前后主要组织相容性复合体 (MHC )的变化情况 ,并将检测结果作图像分析。 结果 :对照组血管壁 MHC 高表达 ,脱细胞处理后 MHC 明显减少。图像分析得出阳性颗粒面积与视场面积比 :对照组为 (2 4 .0 9± 6 .0 9) % ,脱细胞组为 (6 .71± 2 .1 8) % ,脱细胞加碱处理组为 (1 .6 2± 0 .76 ) % ,组间比较差别均有显著意义 (P<0 .0 1 )。 结论 :MHC

MHC-Ⅰ类链相关基因A真核表达载体的构建及稳定转染舌鳞癌细胞的实验研究

目的构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系。方法采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真核表达载体pEGFP-N1,构建最终的表达载体pEGFP-N1-MICA,脂质体法转染Tca8113-Tb细胞,G418筛选,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定过表达MICA基因的Tca8113-Tb细胞系,RT-PCR、real time PCR和免疫细胞化学检测MICA在该细胞中的表达。结果通过PCR技术获取了MICA基因并成功克隆入载体,测序鉴定该序列与GenBank中的序列相同。转染的细胞可见绿色荧光蛋白表达,RT-PCR、real time PCR及免疫细胞化学检测到目的基因MICA在转染细胞中为过表达。结论 pEGFP-N1-MICA真核表达载体的成功构建与稳定转染Tca8113-Tb细胞系的建立,为进一步研究该基因的功能奠定了良好的实验基础。

肾移植受者术前主要组织相容性复合物Ⅰ类链相关基因A抗体对移植后早期急性排斥反应的影响

目的探讨肾移植受者术前主要组织相容性复合物Ⅰ类链相关基因A（MICA）抗体对肾移植术后早期急性排斥反应和移植肾功能恢复的影响。方法检测2009年~2010年本院197例未使用免疫抑制剂的肾移植受者术前MICA抗体及其特异性,随访其后接受尸体肾移植手术的139例受者的术后早期急性排斥反应（AR）和移植肾功能。结果 197例肾移植受者术前MICA抗体阳性45例（22.84%）。MICA抗体特异性分析发现有11种抗体,其中出现频率最高的是抗MICA019（占65.7%）,出现频率最低的是抗MICA015（占8.6%）和抗MICA017（占8.6%）,高、低频率抗体间的差异具有显著性（χ2=24.48,P〈0.01）。45例阳性受者中单一特异性抗体18例（占51.4%）,多种特异性抗体17例（占48.6%）。197例受者中139例在术后有39例发生早期AR（占28.1%）;其中,45例术前MICA抗体阳性受者中38例接受移植后有14例发生早期AR（占36.8%）;152例术前MICA抗体阴性受者中有101例接受移植后有25例发生了早期AR（占24.8%）。结论中国人群中最常见的MICA抗体中为抗MICA019,推测MICA019为中国人群中较常见基因是其表现出临床上高频率抗体的原因。

五指山小型猪近交系白细胞抗原Ⅰ类3基因的研究

【目的】为探讨五指山小型猪(WZSP)近交系群体中SLAⅠ3(SLA-3)位点等位基因分布结构及其特性。【方法】利用4对引物,采用RT-PCR扩增了32头WZSPSLA-3基因部分外显子1、外显子2和大部分外显子3序列。根据家系和扩增结果,选择8头个体的PCR产物克隆测序。【结果】获得9个不同的核苷酸序列。与GenBank中所用SLA-3基因的核苷酸序列及其编码的氨基酸序列进行比较分析,确定这9个序列均为SLA-3位点上的新等位基因,但这些等位基因间核苷酸变异很少;氨基酸序列比较发现猪和人之间有很高的保守性。同时,构建了23个SLA-3的等位基因和1个HLA-A等位基因核苷酸序列的系统树。【结论】WZSP在SLA-3位点和其它猪品种有明显的差异,拥有其独特的遗传资源。从分子水平为WZSPSLA-3基因的分子分型及特异单倍体猪的培育和异种器官移植实验用动物的研究提供理论依据。

穿膜序列对MHC-Ⅰ类限制性CTL表位呈递影响的研究

目的 :从时间依赖性抗原呈递动力学的角度研究穿膜序列 (CPP)对 MHC- 类限制性 CTL 表位呈递的影响。 方法 :应用多肽固相合成技术 ,分别合成含 CPP与 H- 2 Kb限制性 CTL 表位 OVA2 57- 2 6 4的融合多肽 ,同时合成该表位 N、C端自然延伸的抗原肽和无关对照肽。采用流式细胞仪 (FACS)分析技术 ,检测抗原呈递细胞 (APC)在不同时相点对上述各抗原肽进行 MHC- 类呈递的情况。结果 :与不含 CPP的抗原肽相比 ,含 CPP的抗原肽中的 CTL 表位更易进入 APC内 MHC- 类呈递途径 ,表现为呈递所需时间明显缩短 (P<0 .0 5 ) ,且同一时相点的呈递强度显著增加 (P<0 .0 5 )。 结论 :在外源性抗原肽中引入穿膜序列可明显促进其 MHC- 类限制性 CTL 表位的呈递效率 。

MHC-Ⅰ类分子抗原递呈与病毒免疫逃逸

阐述主要组织相容性复合体(MHC)Ⅰ类分子递呈抗原过程,分析病毒干扰MHC-Ⅰ类分子抗原递呈所采取的策略,以期揭示病毒的免疫逃逸机制,为病毒疫苗的研制提供一定的思路。

蛋白酶体与MHC-Ⅰ类抗原的加工呈递

蛋白酶体是普遍存在于所有被检测过的细胞中的一种保守的蛋白酶复合物 ,有着广泛的功能。它在 MHC- 类抗原的加工呈递过程中起着非常重要的作用 ,而 MHC- 类抗原的加工呈递效率对肿瘤和胞内感染性疾病的防治特别重要 ,因此 ,本文对蛋白酶体的结构、MHC-

γ-干扰素基因的导入及删除对人肿瘤细胞HLA-Ⅰ、Ⅱ类抗原表达的影响

目的：制备有效的细胞因子基因修饰的肿瘤细胞作为“瘤苗”。方法：用缺陷型逆转录病毒作为载体将人γ－干扰素（ＩＦ－γ）含信号肽ｃＤＮＡ转导入人胃癌细胞株ＭＫＮ、４５，经Ｇ４１８筛选后得－ＩＦＮ－γ基因修饰株ＭＫＮ－４５－ＩＦＮ。ＰＣＲ方法测得有标记基因ＮｅｏＲ及ＩＦＮ－γ基因的存在，但经３个月连续传代培养后，ＰＣＲ仅能检测到ＮｅｏＲ基因，而未检出ＩＦＮ－γ基因、ＨＬＡ－Ⅰ、Ⅱ类单抗间接免疫荧光法流式细胞仪测定了上述相应时期ＩＦＮ－γ基因修饰肿瘤细胞ＨＬＡ－Ｉ、Ⅱ类抗原的表达。结果：随着ＩＦＮ－γ基因在染色体中被删除，ＨＬＡ－Ⅰ、Ⅱ类的表达又恢复至与ＭＫＮ－４５相同的水平。结论：为确保ＩＦＮ－γ基因的作用，应不断检测其基因的存在。

人类主要组织相容抗原Ⅰ类分子在涎腺腺样囊性癌中的表达

目的:探讨人类主要组织相容抗原(MHC)Ⅰ类分子(HLA-Ⅰ,HLA-G)在腺样囊性癌发生中的意义。方法:应用HLA-I、HLA-G特异性单抗,通过免疫组织化学的方法检测42例腺样囊性癌、30例正常涎腺组织内HLA-Ⅰ、HLA-G抗原的表达。结果:正常涎腺组织内HLA-Ⅰ抗原表达阳性,HLA-G抗原基本上不表达。腺样囊性癌内HLA-Ⅰ类抗原的表达显著低于正常涎腺组织(χ2=17.17,P<0.05),同时HLA-G抗原的表达显著高于正常涎腺组织组(χ2=13.18,P<0.05),但二者与腺样囊性癌的临床分型并无明确的关系。结论:HLA-Ⅰ抗原表达降低和HLA-G抗原表达上调可能参与了腺样囊性癌的发生过程。HLA-Ⅰ、HLA-G抗原可作为腺样囊性癌发生过程中的标志物,对判断腺样囊性癌的预后有一定价值。

免疫抑制酸性蛋白与HLA－Ⅰ类分子在胃癌组织中的表达及其关系

本文应用抗ＨＬＡ－Ⅰ类重链单态决定簇和抗免疫抑制酸性蛋白Ⅱ型（ＩＡＰ－２）单克隆抗体，分别对６４例胃癌、癌旁组织和６例正常胃粘膜冰冻和石蜡切片进行了免疫组化分析。结果表明，正常胃粘膜及癌旁上皮细胞ＩＡＰ－２表达阴性，ＨＬＡ－Ⅰ类分子表达阳性。但在胃癌组织Ⅰ类分子表达缺失（４２．２％），ＩＡＰ－２表达阳性（９０．５％）。其中肿瘤分化程度与Ⅰ类分子缺失和ＩＡＰ－２高表达均呈负相关，即分化程度越低，Ⅰ类分子缺失率越高，ＩＡＰ－２表达阳性率亦越高。提示：①ＨＬＡ－Ⅰ类分子表达缺失可能是胃癌细胞逃避宿主免疫监视而得以生长、扩散和转移的机理之一；②ＩＡＰ－２的高表达可能是胃癌细胞引起宿主免疫抑制的重要因素。

HLA-Ⅰ类抗原在喉癌中的表达及意义

目的:研究HLA-Ⅰ类抗原在喉癌组织中的表达及意义。方法:应用免疫组化方法检测HLA-I类抗原在40例喉癌组织及癌旁正常组织中的表达。结果:HLA-Ⅰ类抗原在喉癌组织中表达下调比例60%,缺失比例17.5%。有转移淋巴结组与无转移淋巴结组之间差异存在统计学意义。结论:HLA-Ⅰ类抗原在喉癌组织中表达下调或缺失,可能与淋巴结转移相关。

HLA-Ⅰ类分子与非小细胞肺癌的免疫治疗

通过分析HLA-Ⅰ类分子与肿瘤免疫逃逸的关系及其在非小细胞肺癌癌组织中的表达,来探讨HLA-Ⅰ类分子与与非小细胞肺癌的免疫治疗的关系。结果表明在考虑非小细胞肺癌免疫治疗方案时必须考虑癌组织HLA-Ⅰ分子表达状况,并据此选择合适的免疫治疗方案。

骨肉瘤组织中主要组织相容性复合物Ⅰ类链相关A蛋白和基质金属蛋白酶-9的关系

目的:探讨骨肉瘤组织中主要组织相容性复合物Ⅰ类链相关A(MICA)蛋白和基质金属蛋白酶-9(MMP-9)的关系。方法:用免疫组化检测66例骨肉瘤组织中MICA和MMP-9蛋白的表达情况。结果:MICA和MMP-9蛋白在骨肉瘤组织的表达率分别为51.5%和54.5%,两者在骨肉瘤组织的表达呈正相关(r=0.414,P<0.01)。结论:MICA广泛表达于骨肉瘤组织,其与MMP-9表达可能由相同信号通路调控。

组织相容性Ⅰ类相关链A位点基因多态性在预判肾移植物排斥中的意义

目的研究组织相容性Ⅰ类相关链A位点（MICA）基因多态性和抗MICA特异性抗体在肾移植排斥反应发生中的意义。方法采用免疫磁珠液相芯片技术对40例肾移植患者在移植术前和移植术后1个月、3个月、6个月、1年和2年动态检测抗MICA抗体的特异性和阳性分值的变化,同时采用SSOP方法分析16对肾移植供受者的MICA基因分型,并把MICA基因具有相同抗原表位的特征性分为G1组和G2组。结果在40例肾移植中尸体供肾35例,亲缘活体供肾5例。35例肾移植受者均带肾存活良好,其中33例接受动态随访。移植术前预存抗MICA抗体为12例,其中抗MICA抗体10例、抗HLA-Ⅰ类＋MICA抗体2例;4例分为MICA-G1组、6例分为MICA-G2组、2例共为MICA-G1和G2组。16对供受者MICA等位基因频率分布：在MICA-G1组中频率M﹡002最高,M﹡007次之;MICA-G2组中频率M﹡008最高,M﹡009和M﹡019次之。33例移植术后有4例新生抗MICA抗体,3例新生抗HLA抗体,均为抗供者特异性抗体和抗非供者特异性抗体。移植术前预存抗MICA抗体,在移植术后1～2年的动态随访中其特异性均未改变,而抗体的阳性分值呈现高低的变化。12例移植术前预存抗MICA抗体中的3例和抗体阴性1例移植术后发生急性排斥反应,临床表现为发热,尿量减少,肌酐和肾动脉阻力指数升高。33例动态随访资料显示：无论是MICA基因频率分布特点,还是抗MICA特异性抗体类型与排斥反应的关系,M﹡002、M﹡004、M﹡008、M﹡019和M﹡001基因型最常见,但未见MICA﹡012和MICA﹡006基因型。结论移植术前分析供受者的MICA基因多态性,受者的特异性抗体鉴定和特征性分组;移植术后动态监测抗MICA抗体的变化,是肾移植术后预防急性和慢性移植物排斥的重要靶分子。

Hepatitis C virus and ethanol alter antigen presentation in liver cells

Alcoholic patients have a high incidence of hepatitis C virus （HCV） infection. Alcohol consumption enhances the severity of the HCV disease course and worsens the outcome of chronic hepatitis C. The accumulation of virally infected cells in the liver is related to the HCV- induced inability of the immune system to recognize infected cells and to develop the immune responses. This review covers the effects of HCV proteins and ethanol on major histocompatibility complex （MHC） class Ⅰ- and class Ⅱ-restricted antigen presentation. Here, we discuss the liver which functions as an immune privilege organ; factors, which affect cleavage and loading of antigenic peptides onto MHC class I and class ~I in hepatocytes and dendritic cells, and the modulating effects of ethanol and HCV on antigen presentation by liver cells. Altered antigen presentation in the liver limits the ability ＇of the immune system to clear HCV and infected cells and contributes to disease progression. HCV by itself affects dendritic cell function, switching their cytokine profile to the suppressive phenotype of interleukin-10 （IL-10） and transforming growth factor beta （TGFβ） predominance, preventing cell maturation and allostimulation capacity. The synergistic action of ethanol with HCV results in the suppression of MHC class Ⅱ-restricted antigen presentation. In addition, ethanol metabolism and HCV proteins reduce proteasome function and interferon signaling, thereby suppressing the generation of peptides for MHC class I -restricted antigen presentation. Collectively, ethanol exposure further impairs antigen presentation in HCV-infected liver cells, which may provide a partial explanation for exacerbations and the poor outcome of HCV infection in alcoholics.

Impact of PRRSV on activation and viability of antigen presenting cells

Porcine reproductive and respiratory syndrome(PRRS) is one of the most important diseases of swine industry. The causal agent, PRRS-virus(PRRSV), is able to evade the host immune response and survive in the organism causing transient infections. Despite all scientific efforts, there are still some gaps in the knowledge of the pathogenesis of this disease. Antigen presenting cells(APCs), as initiators of the immune response, are located in the first line of defense against microorganisms, and are responsible for antigen recognition, processing and presentation. Dendritic cells(DCs) are the main type of APC involved in antigen presentation and they are susceptible to PRRSV infection. Thus, PRRSV replication in DCs may trigger off different mechanisms to impair the onset of a host effective immune response against the virus. On the one side, PRRSV may impair the basic functions of DCs by regulating the expression of major histocompatibility complex class Ⅱ and CD80/86. Other strategy followed by the virus is the induction of cell death of APCs by apoptosis, necrosis or both of them. The impairment and/or cell death ofAPCs could lead to a failure in the onset of an efficient immune response, as long as cells could not properly activate T cells. Future aspects to take into account are also discussed in this review.

TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation

Limited clinical application of antibody-drug conjugates(ADCs)targeting tumor associated antigens(TAAs)is usually caused by on-target off-tumor side effect.Tumor-specific mutant antigens(TSMAs)only expressed in tumor cells which are ideal targets for ADCs.In addition,intracellular somatic mutant proteins can be presented on the cell surface by human leukocyte antigen class I(HLA I)molecules forming tumor-specific peptide/HLA I complexes.KRAS G12 V mutation frequently occurred in varied cancer and was verified as a promising target for cancer therapy.In this study,we generated two TCR-mimic antibodydrug conjugates(TCRm-ADCs),2E8-MMAE and 2 A5-MMAE,targeting KRAS G12 V/HLAA*0201 complex,which mediated specific antitumor activity in vitro and in vivo without obvious toxicity.Our findings are the first time validate the strategy of TCRm-ADCs targeting intracellular TSMAs,which improves the safety of antibody-based drugs and provides novel strategy for precision medicine in cancer therapy.

Serum major-histocompatibility-complex class Ⅰ-related chain A antibody detection for the evaluation of graft dysfunction in renal allograft recipients

Background In addition to the well-known antibodies against human leukocyte antigens （HLA）-induced kidney-graft rejection, polymorphic major-histocompatibility-complex （MHC） class Ⅰ-related chain A （MICA） antigens can elicit antibodies and have been suggested to play a role in the antibody-mediated allograft rejection （AMR）. We carded out a prospective study of MICA antibodies in post-renal transplant patients to determine the association between MICA antibodies, C4d staining, histological features, and graft outcome.Methods We tested 52 patients who had biopsy results due to graft dysfunction. The MICA antibodies in concurrent sera were determined by Luminex. All patients were followed up for one year after renal biopsy. The influence of antibody production on the function of graft was analyzed.Results Antibodies against MICA were positive in 15 out of the 52 patients （28.9%）. The presence of MICA antibodies was associated with renal-allograft deterioration. During one-year follow-up, the estimated glomerular filtration rate （eGFR） decreased （24.0±3.4）% among recipients with anti-MICA antibodies. However, among recipients without anti-MICA antibodies, the eGFR has declined only （8.4＋3.0）% （P=0.017）. The association between C4d staining,histological features and MICA antibody production was found no significant difference.Conclusion Besides anti-HLA antibodies, the presence of post-transplant MICA antibody is associated with poor graft outcome and increases the risk of graft failure.