B淋巴瘤中纯化TIL-T的IL-2Rα基因突变及蛋白表达检测

目的：探讨B淋巴瘤(B—NHL)中肿瘤浸润T细胞(TIL-T)的活化标记IL-2Rα(CD25)的基因突变，肿瘤组织中CD25蛋白表达及ⅡLT的增殖特点。方法：19例B-NHL提纯TIL-T，RT-PCR和SSCP检测其IL-2Rα的基因突变与否；MTT法检测17例TIL-T在低浓度rIL-2刺激下的增殖情况；冰冻切片免疫组化检测29例B-NHL中IL-2Rα蛋白的表达。结果：SSCP显示19例TIL-T的IL-2Rα未发现异常泳动条带；rIL-2刺激下，13／17例(76．5％)TIL-T增殖表现下调；免疫组化显示B-NHL中25／29例(86．2％)CD25表达为( /-)和( )，4／29例(13．8％)CD25表达为( ～ )。结论：TIL-T的IL-2Rα未发现基因异常，但TIL-T表达CD25蛋白数量异常，提示B-T接触活化异常可能是B-NHL形成的重要参与机制。     

老年人T细胞膜型CD28及血清中可溶性CD28的表达及其意义

分析老年人(年龄在60岁以上)外周血T细胞膜型CD28(mCD28)及血清中可溶性CD28(sCD28)的表达水平,探讨该分子在老年人免疫功能改变中的意义。分别收集肝素抗凝及不抗凝的老年人外周血,抗凝血经分离获取单个核细胞,间接免疫荧光及流式细胞仪分析,老年人T细胞mCD28的阳性表达率为男性(41.56%±11.23%)及女性(42.97%±12.02%)。与年轻人[男性(61.51%±17.45%)及女性(62.38%±16.22%)]相比,差别具有显著性(P<0.01)。酶联免疫吸附法(ELISA)检测,老年人血清中sCD28的含量为男性(1.08±0.45)及女性(0.98±0.47)ng/ml,与青年人[男性(0.55±0.16)及女性(0.52±0.13)ng/ml]相比,差别具有显著性(P<0.01)。老年人T细胞mCD28的表达水平下降,而血清中sCD28的含量升高。     

1,25二羟维生素D3对人淋巴细胞免疫功能的调节作用

通过淋巴细胞增殖试验、混合淋巴细胞反应及分析淋巴细胞表面IL-2R 和TfR 表达等研究了1.25-(OH)_2D_3对人淋巴细胞的免疫调节作用。结果表明,1,25-(OH)_2D_3对PHA 诱导的淋巴细胞增殖有抑制作用,IC_(50)为19.1nmol/L 外源性IL-2可部分逆转这种作用;低浓度的1,25-(OH)_2D_3能显著增强MLR 诱导的细胞增殖;在由PHA 和MLR 诱导的淋巴细胞表面IL-2R 和TfR 表达中,对TfR 的表达呈抑制作用,对IL-2R 的表达无影响。     

白细胞介素12提高HBV基因疫苗的免疫应答

目的观察编码IL-12的真核表达载体对HBV基因疫苗诱导Babl/c小鼠免疫应答的影响。方法肌肉注射基因疫苗pCR3.1-S及IL-12的真核表达载体pWRG3169,ELISA法检测小鼠血清抗HBs,4h5iCr释放法检测小鼠脾细胞CTL杀伤活性。结果免疫8w后,pCR3.1-S及共注射IL-12真核表达载体组血清450nmA值分别为0.87±0.1及1.67±0.15。CTL细胞杀伤活性分别为50.5%±6.4%及73.3%±8.8%,两组差异显著,CTL细胞杀伤活性在抗CD4单克隆抗体处理脾细胞悬液后无明显改变,抗CD8单克隆抗体处理后明显降低。结论IL-12的真核表达载体能提高小鼠对DNA疫苗的免疫应答,CTL细胞杀伤活性主要由CD8+执行。基因疫苗可能用于预防及治疗HBV感染。     

溃疡性结肠炎患者PBMCs体外sIL-2R诱生水平的研究

目的探讨sIL-2R在溃疡性结肠炎（UC）中的表达水平。方法采用特异性ELISAsIL-2R检测试剂盒，对25例溃结患者PBMCs体外PHA诱导产生的sIL-2R水平进行测定。结果溃结患者PHA诱导48h的细胞培养上清液中sIL-2R的含量为319.63±164.70U/ml，明显低于正常对照组的451.15±246.93U/ml(P<0.05);而未经PHA诱导的sIL-2R含量，两组无明显差异（UC组263.78±115.05U/ml，对照组236.47±139.10U/ml，P＞0.05）。结论溃结患者体外PHA诱导的sIL-2R水平下降，提示其淋巴细胞的免疫活化功能障碍。     

卵巢癌细胞对CD8+T细胞ζ链表达及分泌Tc1/Tc2型细胞因子的影响

目的：通过研究卵巢癌细胞对CD8+T细胞ζ链表达及分泌Tc1/Tc2型细胞因子影响，探讨其在卵巢癌免疫抑制中的作用。方法:Western印迹方法检测3株卵巢癌细胞（OVCAR3、CAOV3和SKOV3）培养上清液作用后CD8+ T细胞ζ链蛋白的表达；分别采用MTT、逆转录-聚合酶链反应（RT-PCR）方法观察卵巢癌细胞培养上清液对CD8+ T细胞增殖和分泌Tc1/Tc2型细胞因子的影响。结果:（1）与对照组比较，OVCAR3、CAOV3和SKOV3细胞培养上清液均能抑制CD8+ T细胞中ζ链的表达，ζ链蛋白平均吸光度值分别为（0.346±0.004; 0.349±0.007; 0.387±0.011; 0.616±0.013）；（2）3株卵巢癌细胞培养上清液皆抑制CD8+ T细胞增殖，分泌的Tc1细胞因子IFN-γ显著降低，而Tc2细胞因子IL-10显著增高。结论:卵巢癌细胞源性的免疫抑制因子通过下调CD8+ T细胞ζ链的表达，从而抑制其增殖并导致Tc1/Tc2细胞因子分泌失衡，这可能是卵巢癌诱导腹腔免疫抑制的机制之一。     

小鼠接种HBV疫苗引起的特异性细胞免疫反应

目的：研究小鼠免疫接种基因重组乙型肝炎表面抗原疫苗(rHBs)产生的特异性细胞免疫反应。 方法： 40只BALB/c小鼠随机分为0.65、1.25、2.5、5 μg 4组，腹腔分别接种0.65、1.25、2.5、5 μg的rHBs 1次或2次。初次免疫后4周或加强免疫后2周分离小鼠脾T淋巴细胞；分别进行以下实验：实验组用rHBs(10 mg/L)刺激脾T淋巴细胞，对照组用PBS代替rHBs刺激脾T淋巴细胞；3 d后用［3H］掺入法检测脾T淋巴细胞特异性增殖反应，以［3H］掺入的同位素counts·min-1值及刺激指数(SI, 实验组counts·min-1值/对照组counts·min-1值)表示。同时用ELISA方法检测培养液中白细胞介素-2(IL-2)及γ-干扰素(IFN-γ)的浓度。 结果：只接受单次免疫的0.65、1.25、2.5、5 μg组小鼠脾T淋巴细胞特异性增殖反应SI分别为1.55、1.93、2.41、2.811；小鼠脾T淋巴细胞释放的IL-2分别为(5.48±8.88)、(9.28±6.98)、(28.53±14.32)、(64.69±20.88)ng/L，释放的IFN-γ分别为(8.22±8.61)、(9.89±9.34)、(20.27±15.50)、(30.77±22.12)ng/L。接受加强免疫的0.65、1.25、2.5、5 μg组小鼠脾T淋巴细胞特异性增殖反应SI分别为1.61、2.05、3.74、3.62；小鼠脾T淋巴细胞释放的IL-2分别为(5.75±5.04)、(102.53±67.52)、(177.13±91.12)、(332.10±124.31)ng/L，释放的γ-干扰素分别为(3.63±4.42)、(28.33±13.04)、(59.66±25.75)、(80.73±19.30)ng/L。 结论： 小鼠接种rHBs后, 脾T淋巴细胞产生特异性增殖反应，并特异性分泌IL-2、γ-干扰素，反应强度与是否加强免疫及接种的剂量密切相关。     

CD40配体对结肠癌细胞株的体外抑制作用

研究免疫共刺激分子CD40在结肠癌细胞株的表达及重组可溶性人CD40配体(rshCD40L)对结肠癌细胞株的体外抑制作用。采用流式细胞仪分别检测4株结肠癌细胞(HCT116、Caco-2、SW48和COLO-320)CD40分子的表达,同时利用流式细胞仪检测γ干扰素(IFN-γ)对结肠癌细胞株CD40表达的影响。MTT法检测rshCD40L对结肠癌细胞的抑制作用,TUNEL法检测rshCD40L对结肠细胞的促凋亡作用,同时检测联合rshCD40L和IFN-γ抗结肠癌作用。结果流式细胞仪检测有3株结肠癌细胞(HCT116、Caco-2和SW48)表达CD40分子,IFN-γ能增强CD40+结肠癌细胞CD40分子的表达。rshCD40L能明显抑制CD40+结肠癌细胞的增殖作用(抑制率分别为HCT116:33.5%±5.5%;Caco-2:21.5%±3.6%;SW48:30.1%±4.2%),而对CD40-结肠癌细胞株(COLO-320)无明显抑制作用(P<0.05)。rshCD40L能诱导CD40+结肠癌细胞的凋亡作用(凋亡细胞百分比为HCT116:25.6%±4.52%;Caco-2:15.71%±2.27%;SW48:18.0%±3.7%),而对CD40-结肠癌细胞株(COLO-320)无诱导凋亡作用(P<0.05)。另外,IFN-γ能明显增强rshCD40L对结肠癌细胞的抑制增殖和促凋亡作用。重组人CD40配体与IFN-γ联合可显著诱导CD40阳性的结肠癌细胞凋亡,抑制其增殖,具有潜在的抗肿瘤作用。     

R848协同CD3抗体诱导C57BL/6小鼠脾脏淋巴细胞功能改变

目的观察并初步分析1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol(R848)协同CD3抗体诱导C57BL/6小鼠脾脏中淋巴细胞发生的功能改变。方法分离正常C57BL/6小鼠脾脏的淋巴细胞,分别在anti-CD3、R848及anti-CD3+R848的作用下培养72 h。通过MTT法观察淋巴细胞的增殖情况;采用ELISA的方法检测脾脏淋巴细胞的IFN-γ和IL-4的产生情况;使用细胞内细胞因子染色的方法检测CD4+T和CD8+T细胞的IFN-γ和IL-4的产生情况;再用细胞表面分子染色的方法检测R848诱导不同淋巴细胞群活化的情况。结果单独使用R848对淋巴细胞的增殖作用不明显,但anti-CD3+R848可促进淋巴细胞的增殖;R848能辅助anti-CD3明显诱导脾CD4+T和CD8+T细胞分泌IFN-γ和IL-4;R848能够使淋巴细胞活化,且以B细胞亚群为主。结论 R848能够协助CD3抗体促进脾脏淋巴细胞的增殖,分泌IFN-γ和IL-4,其机制与R848能明显促进B淋巴细胞活化有关。     

诱导共刺激分子转基因小鼠类风湿性关节炎中滤泡辅助性T细胞的极化

目的 探讨可诱导共刺激分子(ICOS)转基因小鼠类风湿性关节炎(RA)模型中ICOS信号对滤泡辅助性T细胞(Tfh)极化的影响.方法 1.流式细胞仪分析测定ICOS转基因(ICOS-Trangenic,ICOS-Tg)小鼠及其野生型对照(WT)小鼠脾细胞CD4+T淋巴细胞共刺激分子ICOS在不同时期的表达趋势及特征;2.ELISA检测脾淋巴细胞培养悬液中干扰素(IFN)-γ、IL-21、IL-23、IL-17的水平;结果1.野生型RA小鼠脾CD4+T淋巴细胞表面的ICOS的表达水平从4w到12w为上升趋势(％)(4 W:6.5 ±1.0;7 W:13.2±1.3;12 W:23.5±:2.1);ICOS-Tg小鼠脾CD4+T淋巴细胞表面ICOS的表达三期同样呈上升趋势(％)(4W:8.2±0.9;7W:17.2±1.5;12W:31.6 ±3.0),但与野生型小鼠相比各期均表达上调(均P＜0.05);2.野生型小鼠IFN-γ从4W开始表达上升,7周达峰值后下降,ICOS-Tg小鼠同野生型小鼠相比,趋势相同但表达在各期呈下调趋势,差异有统计学意义(4 W～20 W均P＜0.05);野生型小鼠的IL-21、IL-23及IL-17的表达于4周上升,12周达峰值后下降,ICOS-Tg小鼠同野生型小鼠相比,三者的各期表达趋势相同但呈上调趋势(IL-21和IL-17有统计学意义,P＜0.05;IL-23无统计学意义,P＞0.05).结论 RA小鼠在其致病过程中共刺激信号ICOS呈上调表达;ICOS-Tg小鼠同野生型小鼠相比,其脾淋巴细胞培养上清中IFN-γ呈显著下调趋势;Tfh分化相关的细胞因子IL-21、IL-17则均显著上调表达.Tfh细胞及其作用因子很可能参与了RA的免疫应答,与RA的发生发展有关.     

Age-related changes of the function of T cell subsets: predominant defect of the proliferative response in CD8 positive T cell subset in aged persons

The proportion of CD8 positive cells in the peripheral blood AET-rosette forming T cells from aged persons was significantly reduced than that from young persons. The difference in the proportion between aged and young groups became more significant after proliferative response to a mitogen phytohemagglutinin (PHA) or a specific antigen tuberculin active peptide (TAP). The purified macrophage-deprived T cells (Twp), CD4 (T4) positive cells or CD8 positive cells were prepared from aged or young persons. These cell preparations lost proliferative response to PHA or TAP but showed marked proliferative response to the combined stimulation to 1 microM of ionomycin and 1 nM of phorbol-12-myristate-13-acetate (PMA) at usual culture cell density (2.5 X 10(5)/ml). Proliferative responses of these cell preparations to the combined stimulation were significantly reduced in the aged than those in the young and the magnitude of the difference in the proliferative responses between aged and young groups was more pronounced in CD8 positive cell population than in CD4 positive cell population. Although the cell preparations were relatively independent of exogenous IL-2 for the proliferative response to the combined stimulation of ionomycin and PMA at usual culture cell density, they needed exogenous IL-2 for sustained proliferation at lower culture cell density (5 X 10(3)/ml). These IL-2-dependent proliferative responses to the combined stimulation in the aged were significantly lower than those in the young and again the difference in the proliferative magnitude between aged and young groups was greater in CD8 positive population. The mechanism(s) of age-related change of the proportion and proliferative ability of T subsets were discussed.     

Role of common gamma chain utilizing cytokines for immune reconstitution in HIV infection

Many cytokines that utilize the common gamma (Cγ) chain signaling pathway, viz Interleukin (IL)-2, IL-15, and IL-7 are known to be important for inducing T cell maturation, proliferation, or survival. Untreated chronic HIV infection is associated with profound quantitative and qualitative deficiency of CD4 T cells, which is partially reversed following highly active antiretroviral therapy (HAART). A subset of patients, however, fail to recover CD4 T cells despite virologic suppression. The role of Cγ chain cytokines in influencing immune reconstitution following potent antiretroviral therapy is discussed. Maturation markers (naïve, central memory, effector memory, and effector), cytokine receptors IL-2Rβ, Cγ chain, IL-7Rα, IL-15Rα, and cytokine-induced proliferative responses of T cells in a cohort of HIV-infected pediatric patients and adults classified on the basis of immunologic and virologic response to antiretroviral therapy were examined. The studies indicated that patients had increased percentages of effector memory CD8+ T cells in comparison to healthy volunteers. While patients with partially controlled viremia and poor CD4 T cell reconstitution manifested poor proliferative responses to anti-CD3 or HIV gag antigen stimulation, proliferative responses to Cγ chain utilizing cytokines IL-2, IL-7, and IL-15 were robust. Another Cγ chain utilizing cytokine, IL-21 had no influence on cellular proliferation but enhanced perforin expression in effector CD8 T cells. Thus, cytokine receptor deficiencies may contribute to immune deficiency in HIV-infected patients, and Cγ chain cytokines may play an important role in vivo in immune homeostasis in lymphopenic patients by maintaining the memory subsets of T cells in patients with CD4 T cell deficiency.     

Alterations in the expression of interleukin-2R subunits by activated T cells from elderly humans are uncoupled from aberrancies in G1/S progression.

Activated T cells from elderly humans are known to often display a decline in interleukin-2 (IL-2) production. However, the possible effects of aging on the expression of IL-2 receptor (IL-2R) subunits by human T cells are more controversial and less well characterized. In the present investigation, the surface expression of IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma subunits on resting and activated T cells from 15 sets of elderly and young humans was evaluated. The results showed no significant differences in the average expression of IL-2Ralpha, IL2Rbeta, and IL-2Rgamma on resting T cells from elderly and young subjects, with values of 10% or less. Similarly, no significant differences were found in the mean levels of IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma on T cells from elderly and young subjects stimulated with anti-Ig cross-linked anti-CD3 (monoclonal antibody [mAb] OKT3), phorbol myristate acetate (PMA), anti-CD3 and PMA, or 1% phytohemagglutinin (PHA) plus PMA. Analyses of the expression of IL-2R on activated T cells from elderly people revealed a marked heterogeneity in IL2R levels irrespective of the stimuli. Other experiments showed that the age-related alterations in surface expression of IL-2Ralpha were not correlated to changes in the release of soluble IL-2Ralpha. Age-related changes in IL-2R expression on activated T cells from individual donors were not coupled to the ability of the T cells to undergo G(1)/S progression. Collectively, these observations suggest that activated T cells from elderly people exhibit substantial heterogeneity in the expression of IL-2R subunits and that alterations in IL-2R expression may be distinct from intrinsic defects in G(1)/S progression and proliferative responses.     

APC-dependent impairment of T cell proliferation in aging: role of CD28- and IL-12/IL-15-mediated signaling

The age-related impairment of phytohaemagglutinin (PHA)-triggered peripheral blood mononuclear cell (PBMC) proliferation was paralleled by an expansion of CD28 (-) T lymphocytes with a poor capacity to undergo lectin-induced blastogenesis. However, both CD28 (-) and CD28 (+) T cells isolated from aged individuals exhibited a significant reduction of proliferative response to PHA in comparison with young controls, this implies that the CD28-mediated signaling is not the only defective pathway in the elderly. Thus, PBMC or T cell subsets plus monocytes from aged donors were stimulated with PHA and assayed for the production of, or the response to cytokines known to regulate T cell functions. Results can be so summarized: (i). interleukin (IL)-2 as well as IL-10 release was unaffected by age; (ii). in both groups of subjects, IL-15 concentrations were similar to those spontaneously released by PBMC; (iii). surprisingly, IL-12 p70 and IL-12 p40 production by PBMC was markedly increased in the aged group; (iv) in spite of this finding and of the experimental outcome that IFN-gamma synthesis was almost completely dependent on IL-12. PBMC from old individuals did not release higher amounts of IFN-gamma in comparison with young controls; (v). moreover, only a slight increase in IFN-gamma production was observed in PBMC cultures from the aged group as a result of IL-12 and/or IL-15 costimulation; (vi) at the same time, even though IL-12 as well as IL-15 were necessary for an efficient T cell proliferation, the addition of exceeding doses of cytokines proved to be ineffective in enhancing the proliferative outcome of PBMC or of both CD28 (+) and CD28 (-) T cells in the aged group. Taken together, the data outline the role of CD28 and IL-12/IL-15 signaling impairment in T cell proliferative deficiency during senescence.     

Qualitative and Quantitative Analysis of T Lymphocytes During Normal Human Pregnancy

PROBLEM : Human reproduction involves contact between cells which are allogeneic to one another, however the fetus not only survives but thrives. METHODS : Aspects of T-cell-mediated immunity during normal human pregnancy were studied. PBMNCs of pregnant and nonpregnant women were stimulated with PHA and cytomegalovirus antigens (CMV). The capacity of stimulated cells to proliferate, to produce IL-2 and IFN-γ, to express IL-2 receptor (IL2R1) and the effect of rIL2 on the proliferation rate of lymphocytes were examined. FACS was utilized for T-cell subset comparisons. RESULTS : The proliferation rate, IL-2, and IFN-γ synthesis were all significantly impaired at suboptimal concentration of PHA throughout pregnancy. Exogenous rIL-2 corrected this depression of cell-mediated immunity (CMI). At optimal concentration of PHA, proliferation rate and production of IFN-γ and IL-2 were all decreased. Exogenous rIL-2 corrected these deficits only in the third trimester. Third trimester pregnant women demonstrated a significant depression of proliferation as well as IL-2 and IFN-γ production after CMV stimulation, which was partially corrected by exogenous rIL-2. FACS analysis suggested that after stimulation by CMV and optimal concentration of PHA, T cells were activated and both CD4+ and CD8+ lymphoblasts expressed normal density of IL-2R1. With suboptimal PHA, the number of activated CD4+ and CD4+IL2R1+ cells were diminished and CD4+ and CD8+ T lymphoblasts expressed lower number of IL2R1. CONCLUSIONS : CD4 T helper (Thl) cell function is down regulated progressively during the three trimesters of pregnancy without changes in the quantity of T cell subsets.     

尘螨诱导新生儿脐血单个核细胞ICOS与转录因子表达的研究

为了解尘螨(HDM)抗原对新生儿脐血单个核细胞(CBMC)及成人外周血单个核细胞(PBMC)CD3+ICOS+细胞阳性率、转录因子T-bet、GATA-3、Foxp3 mRNA表达水平以及培养上清中IL-4、IL-10、IFN-γ表达水平的影响。用流式细胞术,检测新生儿CBMC、成人PBMC体外经PHA和/或HDM抗原刺激前、后CD3+ICOS+细胞阳性率;用RT-PCR法检测细胞体外经PHA和/或HDM抗原刺激前、后T-bet、GATA-3以及Foxp3 mRNA表达水平;用ELISA法,检测细胞在体外经PHA和/或HDM刺激前、后培养上清液中IL-4、IL-10、IFN-γ表达水平。结果表明,高剂量HDM抗原显著下调CBMC经PHA刺激后的CD3+ICOS+阳性率(P<0.05),同时也显著上调PHA刺激前其T-bet mRNA表达(P<0.05),而显著下调该类细胞经PHA刺激后的Foxp3 mRNA的表达(P<0.05)。也显著增加其PHA刺激前的IFN-γ分泌(P<0.01),减少其PHA刺激后IL-10分泌(P<0.05)。高剂量HDM抗原对CBMC的作用强于PBMC,可下调CD3+ICOS+阳性率,显著上调PHA刺激前CBMC T-bet mRNA表达,增加PHA刺激前IFN-γ的分泌,减少PHA刺激后IL-10分泌,提示早期接触高剂量HDM抗原对机体免疫功能的影响较强,可促进新生儿Th1样反应,高剂量HDM抗原可能通过作用于下调Foxp3 mRNA的表达而下调CD4+CD25+调节性T细胞的功能。     

Increased cytokine production in mononuclear cells of healthy elderly people

The production of cytokines during aging, except interleukin (IL)-2, has been neglected in humans. We measured the in vitro production of IL-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-1β by peripheral mononuclear cells from selected healthy young (mean age 26.8 years) and aged (mean age 80.2 years) subjects. Significant increases of IL-6, TNF-α and IL-1bT levels were found in mitogen-stimulated cultures from aged donors, occurring at 24 to 72 h after stimulation. No significant differences were observed for IFN-γ production. Proliferative capability of cells stimulated with PHA was not impaired in aged subjects. Since the amounts of all cytokines studied were similar in unstimulated cultures from young and aged subjects, and also serum levels of TNF-a did not differ, these data indicate that the cellular machinery for the production of these cytokines is well preserved in aging, and also that cells from old people are able to up-regulate their production in response to appropriate stimuli. The increases in cytokine synthesis were not dependent on changes in the number of monocytes, nor were they related to the significant rise of CD45RO⁺, and the concomitant decrease of CD45RA⁺ occurring in both CD4⁺ and CD8⁺ lymphocytes from aged subjects. The increased production of pro-inflammatory cytokines by stimulated mononuclear cells of healthy aged subjects may be relevant to several aspects of age-associated pathological events, including atherosclerosis, osteoporosis, fibrosis and dementia.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo.

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

PHA-induced IL-12Rβ2 mRNA expression in atopic and non-atopic children

BACKGROUND: IL-12 is a strong inducer of Th1 responses. Stimulation via the CD2 receptor increases IFN-gamma production and enhances the responsiveness of activated T-cells to IL-12, possibly due to an up-regulation of the signal transducing beta(2) chain of the IL-12 receptor (IL-12R beta(2)). Atopic children have a reduced Th1-like immunity and a reduced CD2 expression. Our hypothesis is that atopic individuals have a reduced function of the CD2 pathway, causing reduced responsiveness to IL-12 and decreased IFN-gamma production. OBJECTIVE: The aim was to study the mRNA expression of the IL-12R beta(2) chain, after stimulation via the CD2 pathway in peripheral blood mononuclear cells (PBMC), of atopic and non-atopic children, and to investigate correlations to the production of Th1 and Th2 cytokines. MATERIALS AND METHODS: The study included 23 skin prick test positive, and 9 non-sensitized, 12-year-old children. PBMC were stimulated for 24 h with phytohemagglutinin (PHA) (2 microg/mL), which stimulates T cells through the CD2 pathway. Expression of IL-12R beta(2) mRNA was analysed by quantitative real time PCR and the cytokine production was detected with ELISA. RESULTS: Atopic and non-atopic children had similar baseline expression of IL-12R beta(2) mRNA, whereas PHA-induced IL-12R beta(2) mRNA expression was lower in atopic than in non-atopic children. The PHA-induced IL-12R beta(2) mRNA expression correlated well with the PHA-induced IFN-gamma production and with the IFN-gamma/IL-4 ratio. CONCLUSION: PBMC from atopic children expressed less IL-12R beta(2) mRNA than non-atopic children after stimulation via the CD2 pathway (PHA). This may indicate a reduced capacity to respond to Th1-inducing stimuli in atopic children.     

Lymphocyte proliferative response to PHA and anti-CD3/Ti monoclonal antibodies, T cell surface marker expression, and serum IL-2 receptor levels as biomarkers of age and health

Alteration of T cell surface marker expression with a decrease of CD3 positive cells relative to the number of CD4 and CD8 positive cells, diminished in vitro proliferative response to mitogenic stimuli like PHA and antibodies to the CD3/Ti complex, and increase in serum IL-2 receptor levels, are among the changes in immunologic parameters that have been associated with advanced age. To distinguish between effects of the primary aging process and diseases of aging not known to be directly related to immune function, we investigated these variables in two well characterized populations of elderly donors (greater than 70 years) and a young adult control group (less than 35 years). The first group of older donors reported no evidence of significant chronic or recent acute illness and saw a physician only for routine medical care. The second group was randomly selected from individuals seen in a geriatric medicine clinic for diagnoses that included osteoarthritis and cardiopulmonary disorders. Altered surface marker expression and increased serum IL-2 receptor levels were seen only in the second group. On the other hand, lymphocyte proliferative responses to PHA, Leu 4 (anti-CD3) and a monoclonal antibody to the beta-chain of the T cell antigen receptor (WT31) were significantly decreased in both populations. Because we would expect primary aging to affect even extremely fit individuals of advanced age, we conclude that decrease in T cell proliferative response may represent a biomarker of primary aging in man. The alteration in surface marker expression and increased IL-2R levels in serum appear to be effects secondary to non-immunologic disease rather than aging.