共刺激分子CD86在急性髓性白血病细胞的表达及其临床意义

目的探讨共刺激分子CD80、CD86在急性髓性白血病细胞中的表达及其临床意义。方法采用细胞培养白血病细胞（HL60,U937,NB4及I（562）及流式细胞仪检测共刺激分子CD80、CD86在急性髓性白血病患者骨髓细胞中的表达及其在急性髓性白血病细胞HL-60细胞、U937细胞、NB4细胞和K562细胞的表达。结果CD80在急性髓性白血病骨髓细胞中的表达很低或不表达,CD86在急性髓性白血病骨髓细胞表达[（27．86±19．65）％]高于对照组[（1．21±0．13）％,t=3．55,P〈0．01]。其中CD86在M4型（48．65±21．92）％、M5型（39．25±18．67）％表达较高,但与对照组单核细胞表达（50．204-20．31）％比较差异均无统计学意义（P〉0．05）。CD86在HL-60细胞、U937细胞和NB4细胞的培养24h后表达分别为（30．624-5．35）％、（24．12±5．23）％、（21．25±3．78）％,与培养48h后表达[（29．43±4．67）％、（26．564-6．54）％、（23．21±6．98）％]比较差异均无统计学意义（P〉0．05）。CD80在HL一60细胞、U937细胞和NB4细胞的表达很低或不表达,K562细胞不表达共刺激分子CD80和CD86。结论CD86在HL-60细胞、U937细胞和NB4细胞和急性髓性白血病患者骨髓细胞中表达。     

AIHA/Evans综合征患者外周血CD80、CD86及CD4^+CD25^+T细胞的表达及临床意义

目的探究自身免疫性溶血性贫血(autoimmune hemolytic anemia,AIHA)/Evans综合征患者外周血CD80、CD86及CD4^+CD25^+调节性T细胞的表达水平及其临床意义。方法选取2014年1月-2017年12月经南阳医学高等专科学校第一附属医院诊治的AIHA/Evans综合征患者60例,以是否处于发作期为准分为发作组与缓解组,其中发作组39例,缓解组21例,另同期随机选取28名健康志愿者作为对照组,比较三组外周血的淋巴细胞共刺激因子CD80、CD86及CD4^+CD25^+调节性T细胞的表达水平。结果AIHA/Evans综合征发作组CD80、CD86、CD4^+CD25^+调节性T细胞表达水平分别为(7.21±1.84)%、(6.23±4.42)%、(2.83±1.75)%,发作组CD80、CD86表达水平高于缓解组及对照组,CD4^+CD25^+调节性T细胞表达水平低于缓解组及对照组,差异均具有统计学意义(P<0.05),但缓解组与对照组CD80、CD86及CD4^+CD25^+调节性T细胞表达水平的比较差异无统计学意义。结论AIHA/Evans综合征发作患者外周血CD80、CD86及CD4^+CD25^+调节性T细胞的表达水平明显升高,CD80、CD86及CD4^+CD25^+调节性T细胞可能在AIHA/Evans综合征发病机制中起着重要作用。     

CTLA-4及协同刺激信号与孕早期不明原因反复自然流产的关系

目的:研究孕早期不明原因反复自然流产(URSA)患者外周血、蜕膜组织CTLA-4及相关免疫分子的表达及其在母胎免疫调节中的作用。方法:采集10例正常育龄妇女(正常非孕组)黄体期、12例正常育龄妇女早孕期(正常妊娠组)和23例孕早期不明原因反复流产患者(URSA组)流产时的外周血,同时留取正常育龄妇女及反复流产患者早孕期行人工流产时蜕膜组织,应用流式细胞仪进行分析,比较CTLA-4、CD4+CD25+、CD80、CD86的表达水平。结果:CTLA-4在3组外周血中表达差异无统计学意义(P>0.05),正常妊娠组外周血淋巴细胞CD4+CD25+的表达显著高于URSA组与正常非孕组,差异有统计学意义(P<0.05),URSA组外周血淋巴细胞中CD80、CD86的表达显著高于正常妊娠组及正常非孕组,差异有统计学意义(P<0.05),正常妊娠组蜕膜组织CTLA-4、CD4+CD25+的表达显著高于URSA组,差异有统计学意义(P<0.05),URSA组蜕膜组织CD80、CD86的表达显著高于正常妊娠组(P<0.05)。结论:蜕膜组织淋巴细胞CTLA-4、CD4+CD25+、CD80、CD86的表达情况在母胎界面免疫耐受调控中起重要作用,外周血淋巴细胞CD4+CD25+、CD80、CD86的表达水平与孕早期反复流产有一定的相关性。     

早发型重度子痫前期分娩前后Th1/Th2及CD28~+/CTLA-4~+的相关研究

目的通过研究早发型重度子痫前期患者分娩前后Th1/Th2及CD28~+/CTLA-4~+表达的改变,探讨Th1/Th2及CD28~+/CTLA-4~+表达在早发型重度子痫前期发病中的作用。方法采用流式细胞技术分别测定30例早发型重度子痫前期患者分娩前后CD28~+、CTLA-4~+、CD28~+/CTLA-4~+的表达水平,同时测量各受试者血压及肝肾功能。结果早发型重度子痫前期患者分娩前CD3~+、CD8~+含量低于分娩后,CD4~+含量高于分娩后,差异有统计学意义(均P0.05)。分娩前CD3~+CD4~+CTLA-4~+、CD3~+CD8~+CTLA-4~+高于分娩后,CD3~+CD4~+CD28~+/CD3~+CD4~+CTLA-4~+、CD3~+CD8~+CD28~+/CD3~+CD8~+CTLA-4~+低于分娩后,差异有统计学意义(均P0.05)。CD3~+CD4~+CD28~+/CD3~+CD4~+CTLA-4~+和CD3~+CD8~+CD28~+/CD3~+CD8~+CTLA-4~+无相关性(r=-0.037,P=0.844)。分娩前早发型重度子痫前期组Th1细胞和Th1/Th2比率高于分娩后,差异有统计学意义(P0.05)。结论早发型重度子痫前期患者分娩前后Th1/Th2及CD28~+/CTLA-4~+表达有明显差异,可能说明患者体内Th1/Th2与CD28~+/CTLA-4~+的异常表达与早发型重度子痫前期的发病密切相关。     

协同刺激分子CD28、CTLA-4与子痫前期发病的关系

目的研究协同刺激分子CD28、CTLA-4在子痫前期发病中的作用。方法采集45例正常妊娠妇女(对照组)及63例子痫前期患者(子痫前期组:轻度31例,重度32例)外周血及子宫蜕膜,分离其内淋巴细胞,用流式细胞技术分别检测CD28、CTLA-4在CD3+T淋巴细胞上的表达率。结果与对照组相比,子痫前期组外周血CD3+T淋巴细胞上CD28的表达差异无统计学意义(P0.05);子痫前期组蜕膜组织中CD28较低,差异有统计学意义(P0.05);子痫前期组外周血与蜕膜CTLA-4分子表达均增加,且重度子痫前期组高于轻度子痫前期组(P0.05);子痫前期组外周血与蜕膜CD28/CTLA-4比率均降低,且重度子痫前期组低于轻度子痫前期组,差异有统计学意义(P0.05);外周血与子宫蜕膜的CD28、CTLA-4、CD28/CTLA-4均呈正相关。早发型、晚发型子痫前期两组CD28+、CTLA-4+、CD28+/CTLA-4的差异均无统计学意义(P0.05)。结论子痫前期的发病可能是从蜕膜组织局部的免疫反应开始,协同刺激信号分子CD28/CTLA-4表达异常,高表达的CTLA-4可能是子痫前期发生的重要原因。     

特应性皮炎患儿单核细胞和CD4+T淋巴细胞表面共刺激分子的表达

目的 探讨外周血单核细胞和CD4+T淋巴细胞表面共刺激分子在儿童特应性皮炎(AD)中的致病作用.方法 用免疫荧光标记流式细胞术检测25例AD患儿外周血单核细胞和CD4+T淋巴细胞表面共刺激分子,以25例正常人作为对照.结果 与对照组相比,AD患儿外周血单核细胞表面CD80、CD86和CD83分子表达水平均明显增高,CD4+T淋巴细胞表面CD86分子表达水平明显增高,而CD80和CD83分子表达水平差异无统计学意义.结论 AD患儿外周血中单核细胞可能通过上调共刺激分子CD80、CD86和CD83的表达水平促使免疫细胞活化而导致Th1／Th2免疫应答失衡;CD4+T淋巴细胞表面共刺激分子CD86表达水平升高,可能在免疫应答中起维持和放大作用.     

叶酸对柯萨奇病毒B3诱导乳鼠心肌凋亡保护作用

目的 探讨叶酸对柯萨奇病毒B3诱导的乳鼠心肌细胞凋亡及凋亡相关基因与蛋白表达的影响.方法 取成熟健康雌性Wistar大鼠随机分为4组,分别为对照、叶酸、柯萨奇病毒B3及柯萨奇病毒B3+叶酸组;孕前灌胃给予叶酸2周,孕第5d腹腔注射给予柯萨奇病毒B3,连续5d;取自然分娩乳鼠心肌细胞,切片观察乳鼠心肌损伤;原位末端标记法( tunel)检测乳鼠心肌细胞凋亡;蛋白免疫印迹(western blot)及逆转录聚合酶链反应(RT-PCR)测定乳鼠心肌细胞凋亡相关Bax、Bcl-2、Caspase-3蛋白及基因表达.结果 柯萨奇病毒组乳鼠心肌细胞损伤明显,凋亡细胞增加,加用叶酸后状态改善;病毒组乳鼠心肌细胞Bax、Caspase-3蛋白表达(0.608±0.060,0.951±0.038)及基因表达(0.951 ±0.098,0.960±0.074)高于病毒+叶酸组,病毒组Bcl-2蛋白表达(0.350±0.037)及基因表达(0.423±0.051)低于病毒+叶酸组.结论 怀孕母鼠早期感染柯萨奇病毒可致乳鼠心肌细胞凋亡增加,补充叶酸对乳鼠心肌细胞凋亡具有明显保护作用.     

维生素E及叶酸对高血糖孕鼠子代心肌细胞凋亡的影响

目的探讨维生素E(Ve)及叶酸对高血糖诱导的乳鼠心肌细胞凋亡蛋白caspase-3表达的影响。方法将50只清洁级成年SD孕鼠随机分为5组,分别为正常对照组(A组)、妊娠糖尿病组(B组)、叶酸组(C组)、维生素E组(D组)和Ve+叶酸组(E组)。除对照组外,其余大鼠于妊娠d 1腹腔注射链脲佐菌素(STZ)35 mg/kg制备妊娠糖尿病动物模型,对照组给予等量0.1 mmol/L(p H 4.5)柠檬酸钠—柠檬酸缓冲液。孕d 5,D、E 2组予50 mg/kg维生素E灌胃,C、E组予0.4 mg/kg叶酸灌胃,A、B组予等量的生理盐水灌胃,持续7 d。取自然分娩的乳鼠心肌组织于光镜下观察心肌结构的改变,应用免疫组织化学法检测各组乳鼠心肌细胞caspase-3抗原的表达。蛋白免疫印迹(western-blot)检测各组乳鼠心肌细胞caspase-3的表达。结果 (1)造模前各组血糖值比较差异无统计学意义(P>0.05);造模后,B、C、D、E组血糖值明显高于A组,差异有统计学意义(P<0.01);B、C、D、E 4组间比较差异无统计学意义(P>0.05)。(2)光镜下观察B、C、D、E组乳鼠心肌组织呈现不同程度损伤,表现为细胞坏死、水肿、轮廓不清,心肌纤维排列疏松紊乱、炎性细胞浸润。(3)5组乳鼠心肌细胞凋亡蛋白caspase-3的表达不同,差异有统计学意义(P<0.01)。结论妊娠糖尿病孕鼠对子代心肌细胞凋亡影响明显,孕期补充维生素E、叶酸对乳鼠心肌细胞凋亡有明显改善作用,且叶酸抑制凋亡的作用优于维生素E,同时补充维生素E和叶酸的效果优于单独补充一种。     

类风湿性关节炎患者外周血T、B淋巴细胞中共刺激分子CD80和CD86的表达

目的探讨共刺激分子CD80和CD86在类风湿性关节炎(RA)患者外周血T、B淋巴细胞中的表达情况。方法运用流式细胞仪检测RA患者外周血T、B淋巴细胞中共刺激分子CD80和CD86的表达,并与正常对照组进行比较。结果 RA患者外周血T淋巴细胞中CD80的表达率明显高于正常对照组,差异有统计学意义(P0.05)。RA缓解组、RA活动组患者外周血T淋巴细胞CD80的表达率均明显高于正常对照组,差异有统计学意义(P0.05);RA缓解期组外周血B淋巴细胞CD86的表达率明显低于正常对照组,差异有统计学意义(P0.05);RA活动期组外周血B淋巴细胞CD86的表达率则明显高于RA缓解组和正常对照组,差异有统计学意义(P0.05)。结论共刺激分子CD80和CD86在RA患者外周血T、B淋巴细胞异常表达,可能在RA发病中起重要作用。     

伯氏疟原虫感染不同年龄小鼠免疫反应特点

目的 了解伯氏疟原虫(Plasmodium berghei NK65,P.b NK65)感染幼年和中年C57BL/6小鼠免疫反应特点.方法 P.b NK65感染3周龄幼年和8月龄中年C57 BL/6小鼠,采用流式细胞分析技术检测不同年龄组小鼠脾细胞悬液中髓样树突状细胞(dendritic cells,DCs)数量,表面表达MHCⅡ类分子和CD86分子的DCs数量以及分泌IgG抗体的B细胞数量;ELISA方法检测脾细胞培养上清中IFN-γ和IL-4水平.结果 3周小鼠DCs数量[(40.08 ±3.54)×105]在感染后5d明显升高,8月龄鼠在感染后5 d[(103.47 ±11.07) ×105]及8 d[(104.04±1.92) ×105]明显升高;3周鼠表面表达MHCⅡ类分子和CD86分子的DCs数量在感染后明显升高,8月龄鼠在感染后8d表达MHCⅡ类分子[(239.40±16.32)×105]和CD86分子[(148.48±14.84)×1o5]数量明显高于3周鼠(P ＜0.05);3周鼠[(251.01 ±34.02) pg/mL]和8月龄鼠[(584.17±54.03) pg/mL] IFN-γ水平在感染后5 d均明显升高,2组比较差异有统计学意义(P＜0.05).结论 3周和8月龄C57BL/6小鼠感染P.b NK65后,其细胞免疫和体液免疫存在明显差异.     

Chronic ethanol ingestion by mice increases expression of CD80 and CD86 by activated macrophages.

Results from previous studies from our laboratory have shown that T cells obtained from the spleens of C57BL/6 mice that consumed ethanol chronically have increased expression of activation markers and increased second signal-independent production of interferon-gamma (IFN-gamma). We now report that in vitro-activated CD11b(+) splenocytes obtained from C57BL/6 and BALB/c mice that consumed ethanol chronically express increased levels of the T cell co-stimulatory molecules CD80 and CD86. CD11b(+) splenocytes encompass at least two populations: the CD11b(+)Gr.1(-) population, which is primarily monocytes-macrophages, and a smaller CD11b(+)Gr.1(+) population, which is in the myelocytic-monocytic cell series and contains precursors of both macrophages and neutrophils. Evaluation of cultures of purified CD11b(+) cells, obtained from mice that consumed ethanol chronically, incubated overnight, showed increased up-regulation of CD80 and CD86 expression on Gr.1(-) mouse splenic macrophages. Results of functional studies of purified CD11b(+) cells have demonstrated that CD11b(+) cells obtained from C57BL/6 mice that were exposed to ethanol chronically secrete higher levels, in comparison with the levels secreted by CD11b(+) cells obtained from control animals, of nitric oxide and several proinflammatory cytokines after stimulation by the oligodeoxynucleotide (ODN) CpG 1826. These findings indicate that CD11b(+) splenocytes are in some way sensitized to activating stimuli by chronic ethanol exposure in vivo. Such cells may contribute to systemic immunodysregulation, including T-cell activation, by providing abnormal second signals to T cells, or through excessive release of cytokines, such as interleukin (IL)-6 or IL-12.     

Non PC liposome entrapped promastigote antigens elicit parasite specific CD8+ and CD4+ T-cell immune response and protect hamsters against visceral leishmaniasis

Leishmania donovani promastigote soluble antigens (sLAg) were encapsulated in non-phosphatidylcholine (non-PC) liposomes (escheriosomes) prepared from E. coli lipids. The escheriosome-based vaccine was investigated for its potential to elicit a protective immune response against experimental visceral leishmaniasis. The vaccine administration induced strong humoral as well as cell mediated immune responses both in hamsters and BALB/c mice. Immunization of BALB/c mice with escheriosome entrapped sLAg (EL-sLAg) elicited stronger CD8+ cytotoxic T lymphocyte (CTL) response as compared to sLAg entrapped in egg PC/chol liposome (EPC-sLAg) or sLAg administered with incomplete Freund's adjuvant (IFA-sLAg). EL-sLAg also induced the release of mixed (Th1 and Th2) types of cytokines in the immunized BALB/c mice. In addition, the delivery of sLAg via escheriosomes enhanced the expression of costimulatory signals (CD80 and CD86) as determined in peritoneal macrophages obtained from BALB/c mice. In another set of experiments, the EL-sLAg immunized hamsters were found to be better protected than those immunized with EPC-sLAg. The prophylaxis coincided with increased lymphocyte proliferation as well as high nitric oxide (NO) production by peritoneal macrophages among EL-sLAg immunized hamsters. Escheriosomes thus seem to have potential in delivering the antigen to cytosol of the antigen presenting cells (APCs) and in the development of liposome-based vaccine against leishmaniasis as well as other intracellular infections.     

Identification of murine T-cell epitopes on low-molecular-mass secretory proteins (CFP11, CFP17, and TB18.5) of Mycobacterium tuberculosis

The low-molecular-mass secretory proteins of Mycobacterium tuberculosis have been shown to be major T-cell antigens during infection with the pathogenic bacterium. In this study, we determined murine T-cell epitopes on three low-molecular-mass proteins, CFP11 (Rv2433c), CFP17 (Rv1827), and TB18.5 (Rv0164) using DNA immunization of inbred mice. We analyzed interferon-γ production from immune splenocytes in response to overlapping peptides covering these proteins. We identified two CD8+ T-cell epitopes on CFP11 and CFP17, one in BALB/c mice and the other in C57BL/6 mice, respectively. On TB18.5, we identified a CD8+ T-cell epitope in BALB/c mice and a CD4+ T-cell epitope in C57BL/6 mice. With the aid of computer algorithms, we could identify the minimal CD8+ T-cell epitopes. These T-cell epitopes are feasible for analysis of the role of antigen-specific T cells during M. tuberculosis infection.     

Identification of non-CSP antigens bearing CD8 epitopes in mice immunized with irradiated sporozoites

Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(−/−)]. This protection is mediated exclusively by CD8⁺ T cells [1]. To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8⁺ T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8⁺ T cells secreting interferon-γ (IFN-γ) in splenocytes from CSP-Tg/JhT(−/−) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-γ-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20-100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8⁺ specific T cells were generated.     

CD4~+CD25~+调节性T细胞对狼疮样小鼠的干预性研究

目的分析CD4+CD25+调节性T细胞(regulatory T cells,Tregs)对狼疮样小鼠的干预效应,为系统性红斑狼疮(systemic lupus erythematosus,SLE)的免疫干预提供新的思路。方法采用磁活性标记的细胞分选方法(magnetic activated cell sorting,MACS)分选BALB/c小鼠CD4+CD25+T细胞,应用流式细胞术(flow cytometry,FCM)检测分选细胞纯度。将6~8w♀CB6F1小鼠随机分为3组,包括正常对照组、狼疮鼠模型组(亲代淋巴细胞免疫)、狼疮鼠干预组(亲代淋巴细胞免疫后2w,输入5×106Tregs/鼠)。分别于诱导后2、4、8、12w,采用ELISA法检测ANA和抗-dsD-NA抗体,并观察肾标本的病理学改变。结果BALB/c小鼠脾脏单个核细胞经MACS分选后,CD4+CD25+T细胞纯度达98.5%。经亲代淋巴细胞输注后,CB6F1小鼠出现体重降低、皮肤干涩;ANA和抗ds-DNA抗体上升;以及狼疮肾炎的病理学改变。而输入Tregs对已出现自身抗体的狼疮鼠有明显的逆转效应,CB6F1小鼠于输注后抗-dsDNA即...     

Role of CD80 and CD86 in host immune responses to the recombinant hemagglutinin domain of Porphyromonas gingivalis gingipain and in the adjuvanticity of cholera toxin B and monophosphoryl lipid A

The gingipains of Porphyromonas gingivalis have been implicated in the virulence of this bacterium, and antibodies to the hemagglutinin/adhesin domain (HArep) of the gingipains have been shown to protect against P. gingivalis colonization. However, the cellular mechanisms involved in host responses to HArep have not been elucidated. The purpose of the present study was to determine the functional role of CD80 and CD86 in mediating systemic and mucosal immune responses to the recombinant HArep derived from the gingipain Kgp (Kgp-HArep) after intranasal (i.n.) immunization. We also investigated the effect of the mucosal adjuvants the B subunit of cholera toxin (CTB) and monophosphoryl lipid A (MPL) on the functional role of the costimulatory molecules for the induction of systemic and mucosal responses to Kgp-HArep. The in vivo functional roles of CD80 and CD86 were assessed in C57BL/6 wild-type (wt), CD80(-/-), CD86(-/-) and CD80/CD86(-/-) mice following intranasal immunization with Kgp-HArep with or without adjuvant. Serum IgG and mucosal IgA antibody responses were induced following i.n. immunization of mice with Kgp-HArep, and were potentiated by CTB or MPL. A differential requirement of CD80and/or CD86 was observed for systemic IgG anti-Kgp-HArep responses following the primary and secondary immunization with antigen alone or antigen+adjuvant. Compared to wt and CD80(-/-) mice, CD86(-/-) mice had reduced serum IgG anti-Kgp-HArep responses following the second immunization with antigen alone or antigen+CTB, whereas similar levels of serum IgG anti-Kgp-HArep antibody activity were observed in wt, CD80(-/-) and CD86(-/-) mice immunized with antigen+MPL. Analysis of the serum IgG subclass responses revealed that CD80 influenced both Th1- and Th2-like IgG subclass responses, while CD86 preferentially influenced a Th2-associated IgG subclass response to Kgp-HArep. Mucosal IgA anti-Kgp-HArep responses in saliva and vaginal washes were diminished in CD86(-/-) mice. In vitro stimulation of murine bone marrow-derived dendritic cells with Kgp-HArep, CTB and MPL resulted in an up-regulation of CD80 and especially CD86 expression. Taken together, our results demonstrate that CD80 and CD86 can play distinct as well as redundant roles in mediating a systemic immune response and that CD86 plays a unique role in mediating a mucosal response to Kgp-HArep following immunization via the i.n. route alone or with adjuvant.     

DNA vaccination as a tool to identify subdominant CD8 T cell epitopes

DNA vaccination is highly efficient at inducing CD8⁺ T cell responses in animal models. Here we investigated whether DNA vaccine technology could be exploited to identify subdominant cytotoxic T lymphocytes (CTL) epitopes. Previous studies have shown that the Sendai virus HN protein does not induce a CD8⁺ T cell response in C57BL/6 mice. Thus, we vaccinated C57BL/6 mice with a DNA vaccine encoding Sendai virus hemagglutinin neuraminidase (HN) protein. The data show that this strategy elicited a potent D^b-restricted CD8⁺ CTL response against at least one subdominant HN-derived epitope. These CTL were able to lyse Sendai virus-infected target cells, demonstrating that the epitope was appropriately processed and present at sufficient levels for T cell recognition. However, these cells did not confer protection against lethal challenge with Sendai virus. These data demonstrate the capacity of DNA vaccine to raise CTL responses to subdominant epitopes, but show that such responses may be limited in their efficacy against non-persistent viruses.     

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.     

Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice

The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency.