抗人CD86单链抗体的基因克隆、表达及结合活性分析

目的 构建并在CHO细胞中表达抗人CD86单链抗体(CD86-scFv),研究该抗体与抗原的结合特性及介导的生物学功能.方法 从分泌鼠源CD86抗体的杂交瘤细胞中克隆出VH和VL基因,构建含有前导肽L-VH-Linker-VL抗体编码基因的表达载体并转染CHO细胞.经筛选高分泌株并扩大培养后收集上清进行纯化.流式细胞术分析CD86-scFv对不同细胞膜型CD86分子的识别及与鼠源亲本抗体1D1的竞争抑制效应.MTT法分析CD86-scFv与Raji细胞表达的CD86结合后对细胞生长的影响.结果 获得了稳定表达抗人CD86-scFv的CHO细胞株及相应抗体.CD86-scFv与CD86基因转染细胞株L929-CD86、天然表达CD86分子的人B系淋巴瘤细胞Raji和Daudi细胞的阳性结合率分别为67.0％、72.3％和80.5％.CD86-scFv对鼠源亲本抗体1D1具有竞争结合能力,将CD86-scFv(终浓度20μg/mL)加入Raji细胞共同培养72 h时的细胞生长抑制率为28.3％.结论 成功构建了稳定分泌抗人CD86-scFv的CHO细胞株(命名为SA-IV).该抗体能够识别CD86分子并具有良好的生物学活性.     

抗人CD80单链抗体的构建、表达及与抗原结合的特性分析

目的:构建及表达抗人CD80单链抗体(ScFv),并初步研究其生物学功能。方法:采用RT-PCR法从分泌鼠抗人CD80mAb的杂交瘤细胞株(4E5)中克隆VH和VL基因。用重叠延伸拼接PCR方法构建具有前导肽的L-VH-Linker-VL单链抗体基因,并亚克隆至pIRES2-EGFP表达载体,脂质体法转染中华仓鼠卵巢细胞(CHO),G418加压筛选。纯化抗CD80-ScFv,并分析其对膜型CD80的识别。竞争抑制实验分析抗CD80-ScFv与相应抗原的结合能力。MTT法体外分析抗CD80-ScFv对CD80介导的共刺激信号的阻断作用。结果:构建的抗CD80-ScFv基因全长为828 bp,经测序含有信号肽和连接肽。实验获得稳定表达细胞株SA-Ⅱ,其培养上清与L929-CD80细胞的阳性结合率达98%以上。抗体纯化后产率约为15.12 mg/L,其能够识别Raji和Daudi细胞天然表达的CD80分子,结合率分别为96.6%和95.0%。抗CD80-ScFv对鼠源亲本抗体4E5与抗原结合的竞争抑制率达98.67%,并能阻断CD80介导的共刺激信号转导,抑制PBMC的增殖,增殖率下降43.48%。结论:成功建立了抗CD80-ScFv CHO细胞的表达株(命名为SA-Ⅱ),该抗体能够特异识别细胞表面膜型CD80分子并介导相应的生物学功能。     

抗人CD86双价抗体的表达及与抗原的结合特性

目的:用中国仓鼠卵巢(CHO)细胞表达抗人CD86双价抗体(CD86-diabody),鉴定该抗体对表达CD86分子肿瘤细胞的识别及介导的生物学效应.方法:从分泌抗人CD86小鼠源抗体的杂交瘤1D1中克隆抗体重、轻链可变区基因VH及VL.SOE-PCR构建VH-(GGGGS)-VL双价抗体基因,整合到真核表达载体plRES2-EGFP中,脂质体法转染CHO细胞,FCM筛选G418加压培养的阳性克隆.IMAC纯化并定量抗体.免疫荧光法分析抗体对人B系淋巴瘤细胞株Raji及Daudi膜型CD86分子的阳性结合率,将抗体加入到Raji细胞的培养体系中,培养72 h,MTT法分析抗体对细胞体外增殖的影响.结果:获得了稳定分泌抗人CD86-diabody的CHO细胞株.培养上清中抗体纯品的得率为5.24 mg/L.抗体与Raji及Daudi细胞的阳性结合率分别为77.2％及70.6％.经抗体孵育72 h,Raji细胞体外增殖抑制率为37％.结论:成功制备了抗人CD86-diabody,可特异性结合细胞膜型CD86分子,并对表达该分子的肿瘤细胞体外增殖具有抑制效应.     

鼠抗人CD86 单克隆抗体的制备及对肿瘤细胞生长与迁移的影响

目的:制备鼠抗人CD86分子的单克隆抗体(m Ab),分析其对天然表达CD86分子的恶性肿瘤细胞株的生长与迁移的影响。方法:采用小鼠腹水诱生法制备CD86 m Ab,Protein A亲和层析法纯化抗体。采用流式细胞术(FCM)检测抗体对细胞膜型CD86分子的识别。以Raji、Daudi和8266细胞为观察对象,加入CD86 m Ab共同孵育,用FCM检测抗体对细胞凋亡的诱导作用,MTT检测抗体对细胞增殖的影响,Transwell~(TM)研究抗体对细胞迁移的影响。结果:本实验获得的CD86 m Ab能很好地识别细胞膜型分子,其与Raji、Daudi和8266细胞的结合率分别为96. 5%、99. 0%和83. 9%。FCM结果显示,共同孵育24 h,当抗体浓度为20μg/ml时,上述细胞的凋亡率分别为16%、18%及14%,均明显高于同型对照组(P0. 05)。MTT结果显示,共同孵育48 h,20μg/ml抗体对细胞的增殖己可产生抑制作用,40μg/ml抗体对上述细胞增殖的抑制效果更明显(P0. 05),抑制率依次为23%、25%及26%。TranswellTM结果显示,共同孵育48 h,20μg/ml抗体组的Raji、Daudi和8266细胞的迁移率分别为26%、23%和24%,均明显低于同型对照组(P0. 05)。结论:本实验制备的抗体能很好地识别细胞膜型分子,其对表达CD86分子的肿瘤细胞的增殖及迁移具有抑制作用,同时可诱导肿瘤细胞发生凋亡,提示该抗体对表达相应抗原分子的肿瘤具有一定的抗瘤效应。     

CD80人-鼠嵌合抗体对B淋巴瘤细胞株Raji与Daudi的生长抑制及杀伤作用

目的:研究CD80人-鼠嵌合抗体(命名为ch-4E5)对天然高表达CD80分子的人恶性B淋巴瘤细胞株Raji与Daudi的生长抑制及杀伤作用.方法:采用免疫荧光及流式细胞术(FCM)分析ch-4E5对Raji及Daudi细胞膜型CD80分子的识别;将ch-4E5分别加入到Raji及Daudi中共培养, 终浓度为10 mg/L.在培养的第0、 4、 10、 16、 24及48 h, 采用FCM检测细胞表面Fas及FasL的表达;培养至72 h, MTT法检测细胞的增殖;以人外周血PBMC为效应细胞, Raji与Daudi为靶细胞, 效靶比为20∶ 1, 加入ch-4E5, 终浓度为10 mg/L.培养至24 h, MTT法分析ADCC效应.结果:ch-4E5与Raji及Daudi的阳性结合率分别为98.6%和96.4%.ch-4E5分别与Raji及Daudi共培养至4 h, Raji细胞表面FasL的表达开始上调, 16 h的阳性表达率为16.8%, 与人IgG1对照组1.5%比较明显升高(P<0.01);Fas的表达从10 h起逐渐增高, 24 h达高峰, 阳性表达率为15.6%, 与人IgG1对照组4.5%比较具有统计学意义(P<0.01).Daudi的FasL及Fas的变化趋势与Raji一致.ch-4E5分别与Raji及Daudi共培养至72 h, 细胞增殖的抑制率分别为34.60%和32.64%(P<0.01);ch-4E5介导PBMC对Raji及Daudi的杀伤率分别为55.61%及54.42%(P<0.01).结论:CD80人-鼠嵌合抗体能够通过其Fab段及Fc段发挥双重的抗肿瘤效应.     

CD80鼠-人嵌合抗体的CHO细胞表达及体外生物学功能的初步研究

目的:CHO细胞表达抗人CD80鼠-人嵌合抗体ch4E5,并研究其在体外阻断CD80介导的共刺激信号转导的生物学功能.方法:构建含有嵌合重、轻链基因的表达载体pIRES/ch4E5;表达载体先转染293T细胞,FCM检测到ch4E5抗体的瞬时表达后,表达载体再转染CHO细胞,构建稳定表达细胞株CHO-ch4E5;Protein G亲和层析法从CHO-ch4E5细胞无血清培养上清中纯化ch4E5抗体,FCM检测ch4E5抗体对膜型CD80的识别;以丝裂霉素处理的正常人外周血单核细胞PBMCs为刺激细胞,以异体正常人外周血淋巴细胞PBLs为反应细胞,用MTT法分析ch4E5抗体的阻断作用.结果:ch4E5抗体在293T细胞的瞬时表达于48小时达到高峰,上清与L929-B7-1细胞结合率为96.0%;CHO-ch4E5细胞持续表达ch4E5抗体,其培养上清与L929-B7-1细胞结合率为95.7%;3天培养上清中,ch4E5抗体的产率约为5.56 mg/L;ch4E5抗体和L929-B7-1、Daudi、Sub-T细胞结合率分别为94.1%、25.7%、23.5%;ch4E5抗体可以阻断CD80介导的共刺激信号转导,抑制异体PBLs的体外增殖.结论:在CHO细胞中成功表达了抗人CD80鼠-人嵌合抗体ch4E5,在体外该嵌合抗体具有亲本抗体阻断CD80介导的共刺激信号的生物学活性.     

小鼠抗人B7-1(CD80)单克隆抗体的制备及其对肿瘤细胞的抑制作用

目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B7-1的识别。以天然表达B7-1的Daudi细胞、Raji细胞及8266细胞为观察对象,分别加入(5、10、20、40)μg/m L B7-1 mAb处理,采用噻唑蓝(MTT)法检测mAb对肿瘤细胞体外增殖的影响,TranswellTM法分析mAb对肿瘤细胞体外迁移的影响,流式细胞术分析mAb对肿瘤细胞凋亡的诱导作用。结果成功制备1株能稳定分泌小鼠抗人B7-1 mAb的杂交瘤,命名为5G10。经流式细胞术分析,该抗体与Daudi、Raji、8266、U266、BEL-7402及MCF-7肿瘤细胞的阳性结合率分别为(96.30±2.12)%、(95.70±1.79)%、(96.80±2.48)%、(23.20±2.35)%、(1.68±0.35)%、(0.55±0.04)%;与对照组相比,20μg/m L 5G10 mAb明显抑制Daudi细胞、Raji细胞及8266细胞的增殖,肿瘤细胞迁移率明显降低,细胞凋亡率明显增加。结论小鼠抗人B7-1 mAb能特异性识别和结合不同肿瘤细胞膜表面的B7-1,并能显著抑制天然表达B7-1的肿瘤细胞的体外生长、迁移并促进其凋亡。     

5株鼠抗人CD28单克隆抗体的研制及生物学特性的研究

目的：制备鼠抗人CD28分子功能性单克隆抗体，研究其对T细胞活化、增殖及信号转导等方面的生物学效应。方法：以天然高表达CD28分子的人多发性骨髓瘤细胞株U266和小鼠淋巴瘤细胞转人CD28基因细胞株CD28-T分别作为免疫原和检测细胞株，采用B淋巴细胞杂交瘤技术进行单抗的研制；以快速定性试纸法鉴定单抗亚类；腹水诱生法和免疫亲和层析法进行单抗的制备和纯化；经间接免疫荧光法分析单抗对不同细胞膜表面CD28分子的识别；采用竞争抑制法分析单抗识别的抗原位点；利用^3H-TdR掺入法分析单抗对PBTC的刺激效应和免疫荧光法分析PBTC活化前后的表型变化。结果：成功获得5株鼠抗人CD28功能性单克隆抗体，分别命名为2D5、2F5、3136、3F8和8G8，其中2D5为IgG2a亚类，其余均为IgG1亚类；流式细胞仪分析结果显示，5株单抗均能良好识别CD28-T、U266、XGI和Jurkat细胞表面的CD28分子；竞争抑制试验表明，2D5和8G8能完全阻断标准抗人CD28单抗与U266膜CD28分子的结合，其余3株为部分阻断；^3H-TdR掺入法实验结果表明，单抗8G8联合激发型CD3单抗能明显促进PBTC的增殖，刺激指数为7．4，活化细胞CD4、CD25、ICOS、4IBB及OX40分子的表达上调。结论：5株单抗均为抗人CD28单克隆抗体，具有重要的基础研究及潜在的临床应用价值。     

抗人CD86人-鼠嵌合抗体的构建及在CHO细胞的表达

从分泌鼠抗人CD86单克隆抗体的杂交瘤细胞株(克隆号:1D1)中抽提总RNA,采用简并引物,经RT-PCR扩增单抗VH和VL的DNA编码区基因。通过SMART-PCR扩增VH和VL基因相应的信号肽序列。采用基因重组技术,将单抗VH、VL基因及其相应信号肽序列,与人IgG1的CH基因、Cκ链基因进行拼接,构建人-鼠嵌合抗体基因的表达质粒pIRES/1D1。转染293T细胞进行瞬时表达,采用流式细胞术分析,转染上清与L929-CD86基因转染细胞的阳性结合率为97.6%。既而转染CHO细胞,经G418筛选,获取稳定分泌嵌合抗体的基因转染细胞株(CHO-ch1D1)。收集无血清培养基的培养上清,采用Protein G亲和层析柱分离纯化,经Lowr法定量。从上清中获取的嵌合抗体蛋白的得率为3.066 mg/L。经进一步间接免疫荧光及流式细胞术分析,纯化嵌合抗体与L929-CD86基因转染细胞,及Daudi细胞膜型CD86分子的阳性结合率分别为98.5%和95.7%。将嵌合抗体(终浓度为5μg/ml)加入到Daudi细胞的培养体系中,经显微镜观察及MTT法分析,嵌合抗体对Daudi细胞的生长具有抑制作用(P<0.05)。本研究获取的嵌合抗体在CD86分子的基础和应用研究中具有重要的价值。     

抗人B7-1及B7-2双特异性抗体的构建、表达及与抗原结合的特性分析

目的:构建及表达抗人B7-1及B7-2双特异性抗体(anti-B7-1/B7-2-Bs Ab),并分析其与相应抗原结合的特性。方法:采用PCR的方法分别从重组质粒中克隆出抗人B7-1及B7-2单链抗体基因,将其克隆入p IRES2-EGFP表达载体,并用6×His为纯化的标签。用脂质体法将两个基因转染入中华仓鼠卵巢细胞(CHO),经G418加压筛选高表达株,扩大培养并收集上清纯化。分析抗体对膜型B7-1及B7-2的识别及与其相应抗原的结合能力。以天然高表达B7-1及B7-2分子的Raji细胞用丝裂霉素处理后作为APC,人PBMC为效应细胞,分析抗体通过阻断B7-CD28共刺激信号对PBMC增殖的影响。结果:获得了稳定表达抗人B7-1/B7-2-Bs Ab的CHO细胞株及相应的抗体。该抗体与B7-1及B7-2基因转染细胞株L929-B7-1及L929-B7-2的阳性结合率分别为99.5%及62.0%。Anti-B7-1/B7-2-Bs Ab对抗人B7-1及B7-2单链抗体的竞争结合率分别为0.4%及32.4%。Anti-B7-1/B7-2-Bs Ab通过阻断B7-CD28共刺激信号使PBMC的增殖效应仅为阴性对照组的59.7%。结论:成功构建了稳定分泌抗人B7-1/B7-2-Bs Ab的CHO细胞株(命名为SA-VI)。该抗体可以识别B7-1及B7-2分子并具有良好的生物学活性。     

CD80 and CD86 polymorphisms in populations of various ancestries: 5 new CD80 promoter alleles

CD80 and CD86 are closely linked genes on chromosome 3 that code for glycoproteins of the immunoglobulin superfamily, expressed on the surface of antigen-presenting cells. These costimulatory molecules play essential roles for stimulation and inhibition of T cells through binding to CD28 and CTLA-4 receptors. In this study, CD80 promoter and CD86 exon 8 polymorphisms were analyzed to investigate the genetic diversity and microevolution of the 2 genes. We genotyped 1,124 individuals, including Brazilians of predominantly European, mixed African and European, and Japanese ancestry, 5 Amerindian populations, and an African sample. All variants were observed in Africans, which suggests their origin in Africa before the human migrations out of that continent. Five new CD80 promoter alleles were identified and confirmed by cloning and sequencing, and promoter 2 is most likely the ancestral allele. Nucleotide -79 is monomorphic in 4 Amerindian populations, where the presence of the -79 G allele is probably the result of gene flow from non-Amerindians.     

Immunolocalization of CD80 and CD86 in Non-Small Cell Lung Carcinoma: CD80 as a Potent Prognostic Factor

It has been demonstrated that tumor cells express programed cell death protein 1 (PD-L1) to escape T lymphocytes that express programed cell protein 1 (PD-1), and PD-1/PD-L1 immune checkpoint inhibitors have been regarded in lung cancer patients. CD80 and CD86 are members of B7 superfamily which regulates T lymphocyte activation and tolerance. However, immunolocalization of CD80 and CD86 has not been examined in the lung carcinoma tissues and their clinical significance remains unknown. Therefore, to clarify clinical significance of CD80 and CD86, we immunolocalized these in 75 non-small cell lung carcinomas (NSCLC) in this study. Immunoreactivities of CD80 and CD86 were mainly detected in tumor-infiltrating macrophages. Immunohistochemical CD80 status was high in 56% of NSCLC, and it was positively associated with stage, pathological T factor, distant metastasis, histological type and PD-L1 status. Moreover, multivariate analysis turned out that the CD80 status was an independent worse prognostic factor. CD86 status was high in 53% of the cases, but it was not significantly associated with any clinicopathological parameters. These findings suggest that CD80 is a potent worse prognostic factor possibly in association with escape from immune attack in NSCLC.     

Regulation of self-tolerance by CD80/CD86 interactions

Antigen presentation by CD80/CD86-positive ‘professional APCs’ induces T-cell activation, whereas antigen presentation in the absence of sufficient CD80/CD86 costimulation may induce a form of tolerance. Blocking CD80/CD86 costimulation inhibits autoimmune disease progression in a variety of animal models, but whether these effects result from restoration of self-tolerance or temporary disease blockade is still unclear. The individual roles of CD80 and CD86 in autoimmune diseases are complicated by multiple factors in vivo. Data from B7 gene knockout mice further clarify the importance of CD80/CD86 in the regulation of T-cell activation and tolerance.     

CD80/CD28 co-stimulation in human brucellosis

Despite treatment, 10-30% of brucellosis patients develop chronic disease, characterized by atypical clinical picture and/or relapses. A defective T helper 1 (Th1) response and a low [corrected] percentage of CD4(+)/CD25(+) cells have been described in chronic brucellosis patients. CD80/CD28 co-stimulation is critical for an efficient Th1 response and has not been studied previously in human brucellosis. In order to investigate the role of CD80/CD28 co-stimulation, 13 acute brucellosis patients (AB), 22 chronic brucellosis patients (CB, 12/22 relapsing type-CB1 and 10/22 atypical type-CB2), 11 'cured' subjects and 15 healthy volunteers (controls) were studied. The percentage of CD4(+)/CD28(+) T lymphocytes and CD14(+)/CD80(+) monocytes were analysed by flow cytometry both ex vivo and after phytohaemagglutinin (PHA)-stimulation with or without heat-killed Brucella abortus (HkBA). Ex vivo analysis showed no differences between all groups studied. PHA stimulation up-regulated the percentage of CD80(+) monocytes in AB compared to 'cured' subjects and controls (P < 0.001), although the proportion of CD4(+)/CD28(+) cells did not alter. A higher percentage of CD80(+) monocytes was observed in the CB1 subgroup, compared to AB, 'cured' subjects and controls (P = 0.042, < 0.001 and < 0.001, respectively). CB2 was characterized by a lower percentage of CD80(+) monocytes in comparison to CB1 (P = 0.020). HkBA in PHA cultures down-regulated the percentage of CD80(+) monocytes compared to PHA alone in all groups, especially in AB and CB patients (P < 0.001 and P = 0.007, respectively). In conclusion, the diminished percentage of CD4(+)/CD25(+) T cells in CB is not associated with inadequate CD80/CD28 co-stimulation. We speculate that differential frequency of CD80(+) monocytes after PHA stimulation could serve as a qualitative parameter of disease status, related to the different clinical forms of chronic brucellosis.     

CD28/CTLA-4 and CD80/CD86 families

T cell stimulation in the absence of a second, costimulatory signal can lead to anergy or the induction of cell death. CD28 is a major T cell costimulatory receptor, the coengagement of which can prevent anergy and cell death. The CD28 receptor is a member of a complex family of polypeptides that includes at least two receptors and two ligands. Cytotoxic lymphocyte-associated molecule-4 (CTLA-4, CD152) is the second member of the CD28 receptor family. The ligands or counterreceptors for these two proteins are the B7 family members, CD80 (B7-1) and CD86 (B7-2). This article reviews the CD28/CTLA4 and CD80/CD86 families, and outlines the functional outcomes and biochemical signaling pathways recruited after CD28 ligation.     

Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation

CD80 is a costimulatory factor mainly expressed on the surface of activated monocytes, B cells and dendritic cells. In this study, we demonstrate that 24% of healthy individuals have soluble forms of CD80, sCD80, in their serum. The concentration of sCD80 ranged from 0 to 1 mg/l. At the mRNA level, we detected a spliced form s1CD80 (771 bp), in unstimulated monocytes and B cells, while another form named s2CD80 (489 bp) was expressed in activated T cells as well as in freshly isolated and activated monocytes. s1CD80 lacks the transmembrane domain, and the IgC-like domain plus the transmembrane domain are spliced out of s2CD80. We also present data demonstrating that recombinant s1CD80 binds to recombinant CD152-Ig and CD28-Ig. It can also bind to T cells, preferentially to activated T cells. Recombinant sCD80 had immunomodulatory effects shown by its inhibition of the mixed lymphocyte reaction and inhibition of T-cell proliferation. sCD80 in human serum adds a new member to the family of soluble receptors, implying a network of soluble costimulatory factors with functional relevance. The inhibitory effect of the recombinant protein on T-cell activation makes it a possible candidate for treatment of diseases associated with hyperactivated T cells.     

CD80和CD86在肾细胞癌组织中的表达

目的　观察肾细胞癌细胞中共刺激分子CD80 (B7- 1)和CD86(B7- 2 )的表达 ,以了解共刺激途径在肾细胞癌的肿瘤免疫中的作用和意义。方法　以正常肾组织和癌周组织为对照 ,采用免疫组织化学方法观察肾细胞癌组织中CD80 (B7-1)和CD86(B7- 2 )的表达。结果　CD80和CD86在肾细胞癌组织中的表达的阳性率明显低于癌周组织和正常肾组织 (P <0 0 5 ) ,但癌周组织与正常肾组织的CD80和CD86表达的阳性率无显著差别 (P >0 0 5 )。结论　共刺激分子CD80和CD86在肾细胞癌组织中呈低水平表达。肾细胞癌组织低水平表达共刺激分子可能与肿瘤细胞“逃避”机体的免疫攻击有关。     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

Trogocytosis of CD80 and CD86 by induced regulatory T cells

Trogocytosis is a process which involves the transfer of membrane fragments and cell surface proteins between cells. Various types of T cells have been shown to be able to acquire membrane-bound proteins from antigen-presenting cells and their functions can be modulated following trogocytosis. However, it is not known whether induced regulatory T cells (iTregs) can undergo trogocytosis, and if so, what the functional consequences of this process might entail. In this study, we show that iTregs can be generated from CD80^−/−CD86^−/− double knockout (DKO) mice. Using flow cytometry and confocal fluorescence microscopy, we demonstrate that iTregs generated from DKO mice are able to acquire both CD80 and CD86 from mature dendritic cells (mDCs) and that the acquisition of CD86 occurs to a higher extent than that of CD80. Furthermore, we found that after co-incubation with iTregs, dendritic cells (DCs) downregulate their surface expression of CD80 and CD86. The trogocytosis of both CD80 and CD86 occurs in a cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), CD28 and programmed death ligand-1 (PDL1)-independent manner. Importantly, we showed that iTregs that acquired CD86 from mDCs expressed higher activation markers and their ability to suppress naive CD4⁺ T-cell proliferation was enhanced, compared to iTregs that did not acquire CD86. These data demonstrate, for the first time, that iTregs can acquire CD80 and CD86 from mDCs, and the acquisition of CD86 may enhance their suppressive function. These findings provide novel understanding of the interaction between iTregs and DCs, suggesting that trogocytosis may play a significant role in iTreg-mediated immune suppression.