外周血干细胞采集物来源的巨细胞病毒特异性T细胞的体外培养和扩增

目的建立巨细胞病毒特异性细胞毒性T淋巴细胞(CMV CTL)体外扩增的方法。方法用1μg/mL全长巨细胞病毒pp65(CMV pp65)多肽体外多轮刺激由粒细胞-集落刺激因子(G-CSF)动员的外周血干细胞采集物中分离的单个核细胞(PBMC),同时加入白细胞介素2(IL-2)、 IL-15、 IL-21扩增20 d。在培养第7天,加入丝裂霉素处理的负载CMV pp65抗原肽的自体PBMC,第10天补充经γ射线辐照处理并负载CMV pp65抗原肽的PBMC和CD3(OKT3);对照组为未加入抗原肽进行扩增的PBMC组。采用多色流式细胞术分别对扩增前、扩增后的T淋巴细胞表型及细胞内肿瘤坏死因子α(TNF-α)和γ干扰素(IFN-γ)的分泌水平进行分析, ELISA检测供者血清中CMV IgM/IgG抗体滴度水平。结果培养后可以收集得到(165.26±6.14)×10~6个细胞,其中CD3~+ T细胞占89.21%, CD8~+ T细胞占CD3~+ T细胞的(43.54±28.03)%, CD4~+ T细胞占CD3~+ T细胞(34.23±26.18)%。获得的CD3~+ T细胞以效应记忆性T细胞(T_(EM))为主,且扩增培养后的CD8~+和CD4~+ T_(EM)比例较培养前显著增高。另外,培养后干细胞样记忆性T细胞(T_(SCM))和组织原位记忆T细胞(T_(RM))的比例均较培养前显著增加。在功能实验中,培养后得到的IFN-γ~+的分泌型CMV特异性CD8~+ T细胞群比例较扩增前明显增加, TNF-α~+ CMV特异性CD8~+ T细胞比例呈增长趋势;能够分别分泌IFN-γ和TNF-α的CMV特异性CD4~+ T细胞群比例与培养前无明显变化。此外,供者体内CMV IgG水平与供者年龄呈现正相关,且在该培养体系下, IFN-γ~+和TNF-α~+ CMV特异性T细胞扩增比例与供者年龄呈负相关。结论本研究成功建立了在体外有效的培养和扩增CMV CTL的方法。     

活动期结核病患者外周血NK细胞免疫反应特征分析

为了探讨自然杀伤(nature killer,NK)细胞在抗结核免疫应答中的特征,我们表达并纯化结核分枝杆菌特异性抗原蛋白ESAT-6,体外刺激结核病患者和正常人外周血单个核细胞(PBMC),采用流式细胞术分析不同亚群NK细胞IFN-γ释放,并与T细胞释放IFN-γ水平进行相关性分析。结果显示,经结核特异性抗原ESAT-6刺激后,结核患者来源的PBMC中有2.80%±0.65%的NK细胞可以释放IFN-γ,而正常人NK细胞释放的比例仅为0.09%±0.01%,两者间有显著差异(P0.001);同时CD56highCD16dim NK亚群细胞分泌IFN-γ的水平(6.50%±0.94%)显著高于CD56dimCD16high NK亚群细胞(1.78%±0.38%);比较同一抗原刺激条件下T细胞释放IFN-γ的能力,显示NK细胞释放IFN-γ的能力高于CD4+T和CD8+T细胞,并与释放IFN-γ的CD4+T细胞的比例呈显著正相关。上述研究结果表明,NK细胞在结核特异性抗原ESAT-6刺激下可通过分泌高水平的IFN-γ协同CD4+T细胞在抗结核感染中发挥重要作用,增强NK细胞的功能,以提高结核病患者的免疫应答水平,将为结核病的治疗提供新的策略。     

丙型肝炎病毒非结构区5个细胞毒性T细胞表位的鉴定

目的 采用T细胞表位预测软件结合体外实验鉴定丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位.方法 采用T表位预测软件Rankpep预测HCV特异性CTL表位,选择候选CTL表位加以合成;用候选CTL表位肽分别刺激HCV感染者以及健康志愿者的外周血单个核细胞(PBMC),采用酶联免疫斑点试验(ELISPOT)检测PBMC中肽特异性分泌IFN-γ的斑点形成细胞(spots forming cells,SFC)的水平,采用细胞内细胞因子染色(intracellular cytokine staining,ICS)检测PBMC中肽特异性IFN-γ+CD8+T细胞的水平.结果 用5条候选CTL表位肽[NS3 450(TVPQDAVSR)、NS3 594(GPTPLLYRL)、NS4b 78(SMMAFSAAL)、NS5a 416(SEENVSVVF)和NS5a 367(TVSSALAEL)]分别刺激10个HCV感染者和2个健康者的PBMC后,健康者的PBMC不产生IFN-γ而7个HCV感染者的PBMC产生IFN-γ;HCV感染者的PBMC中肽特异性分泌IFN-γ的细胞的频率为(5-36)SFC/105 PBMC,肽特异性IFN-γ+CD8+T细胞占总CD8+T细胞的百分比为0.02%～0.25%.结论 ELISPOT结果和ICS结果证实5条肽NS3 450、NS3 594、NS4b 78、NSSa 416和NS5a 367为全新的HCV特异性CTL表位.     

人类巨细胞病毒特异性CD8+ T细胞IFN-γ和穿孔素水平的检测

目的研究人巨细胞病毒(hCMV)结构蛋白pp65衍生抗原肽(pp65495-503, NLV)特异性CD8+ T细胞的功能特性.方法将HLA-A2/NLV四聚体染色与细胞内细胞因子或穿孔素染色相结合,以流式细胞仪直接分析NLV特异性CD8+ T细胞内穿孔素的表达水平,或者在NLV抗原肽刺激6 h后分析γ-干扰素(IFN-γ)的表达水平.结果 NLV抗原肽能诱导hCMV特异性CD8+ T细胞分泌IFN-γ,不同供者特异性CD8+ T细胞产生IFN-γ的比率存在较大差异(55.69±17.64) %,当NLV抗原肽质量浓度为10 μg/mL时IFN-γ阳性细胞百分率最高;未经刺激的特异性CD8+ T细胞内表达较高水平的穿孔素(53.90±16.41)%.结论识别单一表位的hCMV特异性CD8+ T细胞合成IFN-γ和穿孔素的能力存在多样性.     

EB病毒转化B淋巴母细胞在汉滩病毒CTL表位研究中的应用

目的:建立EB病毒转化B淋巴母细胞系(B-LCL),作为抗原提呈细胞(APC)提呈抗原肽,刺激短期培养的特异性T细胞活化并分泌IFN-γ,从而应用于T细胞表位鉴定中.方法:用B95-8细胞培养上清中的EB病毒转化肾综合征出血热(HFRS)患者PBMC,建立HFRS患者的B-LCL,以自身BLCL为APC,加载抗原肽后,刺激短期培养的G9L特异的HFRS患者CD8+ T细胞系,应用ELISPOT测定CD8+ T细胞受到抗原肽刺激后产生IFN-γ的能力.结果:加载过抗原肽G9L或V15R的B-LCL可刺激G9L特异的CD8+ T细胞活化并产生IFN-γ,而与G9L无同源序列的115P则不能刺激G9L特异的CD8+ T细胞活化.结论:B-LCL可作为非专职APC有效地将抗原肽提呈给特异性T细胞.     

HBV特异性IFN-γ分泌细胞的体外扩增和功能研究

目的:体外分离扩增HBV多肽特异性IFN-γ分泌细胞,并进行自体扩增,检测其扩增后的多肽特异和IFN-分泌功能。方法:酶联免疫斑点法筛选HBV自然感染献血个体,分离外周血单核细胞,体外刺激并分选IFN-γ分泌细胞,并进行自体细胞体外扩增,流式细胞术检测扩增后细胞的CD4、CD8表型,多肽负载自体淋巴瘤细胞系(lymphoblastoid cell lines,LCLs)细胞检测其多肽特异性和IFN-γ分泌能力。结果:经过4周体外自体扩增,HBV特异性IFN-γ分泌细胞扩增数量达1000多倍,CD4和CD8比例变化不显著,4周扩增后的T淋巴细胞能有效识别HBV特异性多肽并分泌IFN-γ。结论:HBV多肽特异性IFN-γ分泌细胞能在体外有效扩增并保持功能表型不变。     

CD107a/b分子用于评价SARS-CoV/S抗原特异性免疫应答

目的 观察抗原刺激以后CD4+和CD8+T细胞表面CD107a/b分子的表达与效应性细胞因子的产生之间的关系.方法 正常Balb/c小鼠脾细胞经多克隆刺激剂刺激或者SARS-CoV/S DNA疫苗免疫小鼠脾细胞经S抗原多肽刺激以后,使用流式细胞仪检测CD4+和CD8+T细胞表面CD107a/b分子的表达与IFN-γ、INF-α、IL-2等细胞因子的表达之间的关系.结果 经抗原多肽刺激后,抗原特异性CD4+ 和CD8+T细胞表面均表达CD107a/b.约80%的CD8+IFN-γ+细胞同时表达CD107a/b;约25%～40%的CD8+TNF-α+细胞表达CD107a/b;而大部分分泌IL-2的抗原特异性T细胞均不表达CD107a/b.结论 可以通过对抗原特异性T细胞表面CD107a/b分子的检测来鉴定抗原特异性T细胞.同时检测效应性细胞因子,可以提高检测的准确性和灵敏度.     

结核杆菌全菌裂解物和全脂质抗原对人外周血单个核细胞的作用

目的观察结核杆菌全菌裂解物和全脂质抗原对人外周血单个核细胞(PBMC)活化和增殖的作用。方法健康成人PBMC(1×106/ml)分别加结核杆菌全菌裂解物(10μg/ml)或全脂质(10μg/ml)进行刺激,同时给予重组人白介素-2(rhIL-2,50 U/ml)维持细胞增殖,另外设仅加rhIL-2的对照组,培养至第6、9、12天,收集培养扩增细胞,流式细胞术检测T细胞以及不同亚群的增殖情况和IFN-γ的产生。结果 2组抗原均能刺激PBMC中T细胞的增殖和活化,第9天时反应最为明显,与对照组相比,全菌裂解物组CD8+T细胞比例降低而CD4-CD8-T细胞比例明显增高;全脂质组CD4+T细胞以及CD4+CD8+T细胞比例增高,差异有统计学意义(P0.05)。培养至第12天,对分泌IFN-γ的细胞亚群分析,全菌裂解物组与全脂质CD8+T细胞所占比例均高于其相应的rhIL-2对照组(P0.05);全菌裂解物组CD8-T细胞所占比例低于其相应对照组(P0.05),而全脂质组高于其相应对照组(P0.05)。结论结核杆菌全脂质抗原与全菌裂解物一样,能激活特异性免疫反应,具有潜在的疫苗组分可能性。     

八色流式细胞术检测人外周血BCG特异性效应型记忆CD4+T细胞的表型特征

目的:检测BCG刺激后,PPD+正常人外周血中细胞因子产生及其亚群.方法:分离PPD+正常人外周血单个核细胞(PBMC),BCG刺激后检测CD4+和CD8+T细胞细胞因子分泌,并用八色流式细胞术分析BCG特异性T细胞亚群.结果:BCG刺激PBMC后,主要是CD4+T细胞分泌Th1细胞因子(IFN-γ、IL-2和TNF-α),而CD8+T细胞几乎不产生细胞因子.进一步分析分泌细胞因子的细胞亚群,主要是CD4+CD45RO+ CD62L(-)CD27(-)和CD4+ CD45RO+ CD62L(-)CD27+分泌细胞因子.结论:BCG刺激PPD+正常人外周血PBMC后,主要诱导CD4+T细胞分泌细胞因子,且该细胞表现出CD4+CD45RO+ CD62L(-)效应型记忆细胞特征,可能在预防结核感染中发挥重要作用.     

不同种类乙型肝炎表面抗原诱导小鼠早期细胞免疫应答的比较

目的 比较不同表达系统来源的乙型肝炎(乙肝)表面抗原(HBsAg)免疫小鼠诱导早期脾淋巴细胞抗原特异性细胞免疫应答的特点,探讨影响乙肝疫苗保护效果的因素.方法 3种HBsAg(汉逊酵母、CHO细胞和血源)分别皮下接种不同组小鼠(BALB/c,H-2d),每只3μg,于免疫后4d分离脾单个核细胞(MNC),经细胞分选仪(MACs)分选后,获得纯度高于90％的CD4+和CD8+T细胞,应用ELISPOT测定MNC、CD4+和CD8+T细胞体外刺激后所产生的细胞因子IFN-γ、IL-2斑点数( SFC).结果 细胞分选后,汉逊抗原诱导CD8+T细胞分泌IFN-γ的水平和CD4+T细胞分泌IL-2水平均显著高于CHO抗原组(8/10、2/10,P=0.035;6/10、0/10,P=0.005).汉逊抗原组与血源抗原组CD8+T细胞经体外刺激诱导IFN-y全部阳转(10/10),但分泌水平上汉逊抗原组显著高于血源抗原组(t=2.479,P=0.035);汉逊抗原组诱导CD4+T细胞IFN-γ阳转率及分泌水平均显著高于血源抗原组(10/10、4/10,P=0.005;t=3.967,P=0.003).结论 乙肝抗原免疫小鼠4d即可诱导细胞免疫应答,且汉逊酵母抗原早期诱导抗原特异性IFN-γ、IL-2的能力显著高于CHO和血源抗原,与其临床考核母婴传播阻断HBV保护率显著优于CHO和血源乙肝疫苗相一致,为及时接种乙肝疫苗的必要性和高危新生儿选择接种乙肝疫苗的类型提供依据.     

Evaluation of antigen-specific responses using in vitro enriched T cells

Antigen-specific lymphocytes are important in the immune response to viral infection. Peripheral blood mononuclear cells (PBMC) are traditionally used as a source of effector cells in most immunological studies. We described here the use of the bispecific monoclonal antibodies (BSMAB) anti CD3:CD8 (CD3,8) and anti CD3:CD4 (CD3,4B) to expand and selectively enrich CD4+ and CD8+ T cells populations, respectively. The expanded cells demonstrated >90% CD3+CD4+ or CD3+CD8+ by 14 days. We measured HIV- and CMV-specific responses of these subset-enriched T cell and found that sensitivity and specificity is similar or higher when compared to PBMC in various cellular immunology assays (CMI). Vbeta analysis of BSMAB-enriched cells demonstrated comparable repertoire to the parent PBMC. Although both CD45RA(hi) and CD45RO(hi) cell populations were expanded with the BSMAB, selective subset depletion demonstrated that the antigen-specific T cell responses were restricted to the initial CD45RO(hi) memory effector subgroup. In conclusion, BSMAB in vitro enrichment of T cells allows significant expansion of the cell population without loss of specificity. This technique of cell expansion permits studies of T cell subset function in situations where the initial cell source is scarce, and presents an alternative for viable and functional T cells in immunological assays.     

Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15--the Amplispot assay

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-gamma) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.     

38 000 MW antigen-specific major histocompatibility complex class I restricted interferon-gamma-secreting CD8+ T cells in healthy contacts of tuberculosis.

CD8+ T lymphocytes are required to protect mice against Mycobacterium tuberculosis, although in early infection the mechanism appears not to be via perforin or granzyme-mediated lysis of the infected target, and may be via interferon-gamma (IFN-gamma) production. We therefore investigated whether CD8+ T cells specific for the immunoprotective 38 000 MW antigen of M. tuberculosis could be detected in infected humans. Using a recombinant vaccinia virus expressing the 38 000 MW antigen of M. tuberculosis (rV38) and a control vaccinia virus (rVras) we demonstrated that both viruses stimulated IFN-gamma production from freshly isolated peripheral blood mononuclear cells (PBMC) in a 36-hr enzyme-linked immunospot assay. Cell depletion and antibody blockade established that the bulk of the 38 000 MW antigen-specific IFN-gamma response was mediated by CD8+, major histocompatibility complex class I-restricted T cells, whereas the anti-vaccinia virus response was predominantly mediated by CD4+ T cells. In further evaluations PBMC from all seven healthy tuberculosis-exposed contacts had a 38 000 MW antigen-specific IFN-gamma response, whereas seven patients with untreated sputum-positive pulmonary tuberculosis had very low levels of 38 000 antigen-specific IFN-gamma-producing cells. These preliminary observations demonstrate the utility of recombinant vaccinia viruses in restimulating freshly isolated CD4+ and CD8+ T cells. The bias towards a higher frequency of IFN-gamma-producing CD8+ T cells in contacts rather than patients may indicate a protective role for CD8+ cells in human tuberculosis.     

In vitro susceptibility of Thai seronegative donor CD8+ T lymphocytes to human immunodeficiency virus-1 (HIV-1) infection

To determine whether CD8+ T lymphocytes from Thai donor cells are susceptible to HIV-1 infection, undepleted peripheral blood mononuclear cells (PBMC) and CD8-enriched PBMC were infected with HIV-1 Thai subtype B and CRF01_AE (E) primary isolates. Virus kinetics in HIV-1 infection of CD4+ and CD8+ T lymphocytes peaked at day 7 or 10 post infection (pi); the TCID50 used for cell infection was proportional to the level of p24 production in the cultures. We also found that the level of p24 antigen in the supernatants of infected undepleted PBMC was significantly higher than that of infected CD8-enriched PBMC. Interestingly, both single positive T lymphocytes (CD4+ and CD8+ T lymphocytes) as well as double positive CD4+/CD8+ T lymphocytes were infected with HIV-1. The double positive T lymphocytes in PBMC were found only in the presence of both CD4+ and CD8+ T lymphocytes. The majority of p24+/CD4-/CD8- T lymphocytes were HIV-1 infected CD4 down-modulated PBMC. This report provides direct evidence that single positive CD8+ T lymphocytes and double positive CD4+/ CD8+ T lymphocytes from Thai donors can be infected with HIV-1 subtypes B and E in vitro.     

Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques

Standard proliferation assays used for analysis of CD4+ T cell function have significant shortcomings, including limited sensitivity, lack of truly quantitative readouts and significant variability. We have optimized an intracellular cytokine staining (ICS) assay in rhesus macaques which allows us to identify virus-specific CD4+ T cells at the single-cell level with high sensitivity while reducing background staining to a minimum. A variety of parameters were tested to determine the optimal experimental conditions necessary for the detection of antigen-specific CD4+ T cells in macaques. Central to our optimized protocol was the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification which resulted in up to threefold enhancement of the frequency of TNF-alpha-secreting CD4+ T cells following superantigen- or antigen-specific stimulation. The ICS protocol was also optimized with respect to antigen concentration and duration of antigenic stimulation. These modifications resulted in a convenient and highly reproducible assay with intra- and inter-assay variability of less than 10%. Although cryopreservation of PBMC generally led to a 40% to 80% decrease in the frequency of antigen-specific CD4+ T cells detected by ICS using stimulation with viral proteins, the use of overlapping peptide pools minimized the effects of cryopreservation on ICS responses. The use of more sensitive techniques such as ICS permits delineation of antigen-specific cells at the single cell level and should provide new insights into pathogen-specific immune responses in the rhesus macaque model.     

CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays

The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot (ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion.     

Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides

The frequencies of human cytomegalovirus (HCMV) protein-specific CD8 T cells, identified by the presence of intracellular IFN-gamma, were measured by flow cytometry following stimulation of freshly isolated peripheral blood mononuclear cells (PBMC) with comprehensive peptide pools. These pools spanned the entire amino acid sequences of the HCMV pp65 and major immediate early (IE-1) proteins and consisted of 15-amino acid peptides with at least nine overlaps between neighboring peptides. As a result all potential CD8 T cell epitopes contained in these proteins were provided by the complete pools and, therefore, unlike with single epitopes, testing was independent of donor HLA type. Individual stimulating peptides from the same pools were identified in parallel experiments. Thus we found that our results with the complete pools using PBMC from 26 healthy HCMV-seropositive donors were 100% sensitive and specific with respect to predicting the presence of recognized epitopes in the respective proteins. In addition, cells from 15 renal transplant patients were tested with complete pools alone. While our results confirmed our previous contention that HCMV IE-1 is an important CD8 T cell target, the technical improvement we made in order to address this question has clearly wider implications. Similar pools may be applied to examine the role of proteins from other pathogens, in autoimmune disease or following vaccination.     

Stimulated proliferative responses in vertically HIV-infected children on HAART correlate with clinical and immunological markers

The objective of the study was to investigate the relationship between various CD4+ T cell subsets and the ability of peripheral blood mononuclear cells (PBMC) to proliferate to several stimuli in vertically human immunodeficiency virus type 1 (HIV-1)-infected children. We studied 29 HIV-1-infected children on highly active antiretroviral therapy (HAART) (median duration: 12.3 months). T cell subsets were determined by flow cytometry. Plasma viral load (VL) was quantified using a standardized molecular method. Proliferative responses were evaluated by [3H]-thymidine incorporation. Decreased proliferative responses of PBMC to pokeweed mitogen (PWM) were found for HIV-1-infected children in Centers for Disease Control (CDC) clinical categories B and C when compared to the control group (P < 0.05). Similarly, children with < or = 15% CD4+ T cells showed a decrease in proliferative responses to PWM (P < 0.01), anti-CD3 + anti-CD28 (P < 0.01) and phytohaemagglutinin (PHA) (P < 0.05) with respect to the control group and to children with CD4+ T cells > or = 25%. Proliferative responses to PWM, anti-CD3+, anti-CD28 and PHA had a statistically significant positive correlation with CD3+/mm3, CD4+/mm3, % CD4 T cells, CD4/CD8 ratio and the percentage of naive T cell subsets (CD4+CD45RO-HLA-DR-, CD4+ CD45RA+ CD62L+, CD4+ CD45RA+), CD4+ CD62L+ and CD4+ T cells co-expressing CD38+ (CD4+ HLA-DR-CD38+, CD4+ CD38+). Moreover, we found a negative correlation between PBMC proliferative responses and % CD8 T cells, memory, memory-activated and activated CD4+ T cell subsets. Lower proliferative responses to PWM (P < 0.01) and PHA (P < 0.01) were associated with higher VL. Our data show that higher proliferative responses to PWM, anti-CD3 + anti-CD28 and PHA are associated with both non-activated and naive CD4+ T cell subsets in HIV-1-infected children on HAART.     

Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.     

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.