Cytokine profile of liver-infiltrating CD4+ T cells separated from murine primary biliary cirrhosis-like hepatic lesions induced by graft-versus-host reaction

BACKGROUND AND METHODS: We have previously reported that CD4+ T cells induced primary biliary cirrhosis (PBC)-like hepatic lesions in mice with graft-versus-host reaction due to major histocompatibility complex class II disparity. To clarify the relationship between the cytokine profile produced by CD4+ T cells and the formation of hepatic lesions, we sorted CD4+ T cells from the liver by using flow cytometry and examined their cytokine mRNA expression at various times after GVHR induction. We also examined the associated changes in the serum levels of antimitochondrial antibodies (AMA). RESULTS: Histologically, the infiltration of CD4+ T cells around the bile ducts was observed from day 5, and the lesions deteriorated gradually until day 14. On day 14, CD8+, B220+ and Mac-1+ cells, as well as CD4+ T cells were seen around the bile ducts. In the liver-infiltrating CD4+ T cells, the expression level of interferon-gamma (IFN-gamma) mRNA was observed to increase at an early phase (day 3), whereas that of interleukin (IL)-10 mRNA was elevated at a later phase (day 14). The elevation of IFN-gamma mRNA expression at an early phase before the appearance of non-suppurative destructive cholangitis suggests that IFN-gamma may be related to the pathogenesis of PBC in this model. Serum levels of AMA on day 14 were significantly higher than those on day 5. Interleukin-10 was considered to stimulate antibody production, to show an inhibitory effect upon the function of T helper 1 cells, and to inhibit fibrosis. CONCLUSIONS: Interferon-gamma may play an important role in the pathogenesis of this model. Moreover, delayed expression of IL-10 mRNA may control PBC-like hepatic lesions.     

Characterization of hepatitis B virus surface antigen-specific CD4+ T cells in hepatitis B vaccine non-responders

AIM: To study the mechanisms of hepatitis B vaccine non-response, we examined hepatitis B virus surface antigen (HBsAg)-induced proliferation of peripheral blood mononuclear cells (PBMC) obtained from hepatitis B (HB) vaccinees. METHODS: Subsequently, we have examined the features of HBsAg-reactive CD4+ T cells in HB vaccine non-responders (NR). Based on serum antibodies to HBsAg (anti-HBs) titers, we divided these vaccinees into three groups: high responder (HR), middle responder (MR) and non-responder (NR), and examined HBsAg-induced proliferation of their PBMC. RESULTS: We found that the in vitro response of PBMC to stimulation with HBsAg was correlated with their serum anti-HBs titer (mean stimulation index was 10.71 in HR, 4.37 in MR and 1.96 in NR). However, by the deletion of CD8+ T cells, the increased response was observed in two of four NRs. CONCLUSIONS: The present results have also shown that at least four distinct HBsAg-reactive CD4+ clones existed (variable gene of T cell receptor beta (V beta) 17 + clone restricted with HLA-DR locus (DR4), V beta 8 + clone restricted with HLA-DQ locust (DQ7), and both V beta 5.1 + clone and V beta 20 + clone restricted with either DR9 or DQ3) in NRs. The results demonstrated that heterogeneous HBsAg-reactive CD4 clones existed in some HB vaccine NRs.     

Restoration of CD4 T-cell responses to cytomegalovirus is short-lived in severely immunodeficient HIV-infected patients responding to highly active antiretroviral therapy

OBJECTIVES: To define the level of pathogen-specific immune reconstitution persisting over 3 to 5 years of highly active antiretroviral therapy (HAART) in HIV-infected patients who began therapy with CD4 T-cell counts below 50 cells/microL. METHODS: Cytomegalovirus (CMV)-specific T-cell responses were analysed in adult HIV-1-infected patients with nadir CD4 T-cell counts below 50 cells/microL before HAART. CMV-specific CD4 T-cell responses were measured by interferon-gamma enzyme-linked immunospot assay (ELISpot assay), lymphoproliferation and interferon-gamma levels in cell culture supernatants. RESULTS: CD4 T-cell responses to CMV were low in untreated patients and remained low during the first year on HAART, but increased progressively to levels similar to those found in HIV-seronegative CMV-seropositive controls at 3 years. Responses then declined markedly and at 5 years were lower than controls. This could not be explained by changes in CD4 or CD8 T-cell counts or plasma HIV RNA levels. Interferon-gamma and interleukin-5 responses to a mitogen were maintained or elevated. CONCLUSIONS: CMV-specific CD4 T-cell responses were found to decline after 3-5 years on HAART and may provide inadequate long-term protection against CMV disease in patients who are severely immunodeficient prior to treatment.     

Increased CD4+ T cell-dependent anti-erythrocyte antibody levels following the onset of parasite egg production in Schistosoma mansoni infected mice

Anaemia has been reported to be a symptom of schistosomiasis mansoni. In other chronic infectious diseases, antired blood cell (RBC) antibodies have been suggested or shown to play a role in anaemia by participating in either complement or macrophage-dependent RBC elimination. To examine whether such a situation could be contributing to the anaemia of schistosomiasis, we examined RBC taken from infected mice for surface-bound antibodies. Our data show that prior to the onset of egg production infected mice have plasma haemoglobin levels that are indistinguishable from age matched controls (AMC). However, consistent with previous reports, following the initiation of egg laying, infected mice have significantly lower haemoglobin levels than AMC. Surface-bound IgM, IgG1 and IgG3 on RBC from infected mice increased markedly after egg laying began. Levels of RBC-associated IgG2b were similar on RBC from infected and normal mice. Antibody production against RBC was Th cell-dependent since it did not occur in mice depleted of CD4⁺ cells. Antibodies eluted from RBC of infected mice bound to isolated membranes of RBC from AMC and to a soluble extract of schistosome eggs. Furthermore, antibodies in serum from mice carrying patent infections bound to the membranes of RBC from normal mice. Taken together, these data suggest that schistosome eggs induce an antibody response which may cross react with a RBC surface antigen.     

Age-dependent accumulation of monoclonal CD4+CD8+ double positive T lymphocytes in the peripheral blood of the elderly

Multicolour flow cytometric analysis enabled the identification of monoclonal B-cell lymphocytosis (MBL), frequently resembling chronic lymphocytic leukaemia, at a rather high frequency in peripheral blood (PB) samples from an elderly population. PB T lymphocytes from 103 otherwise healthy subjects >65 years of age and 51 younger donors (<65 years) were analysed. Besides CD4(+) and CD8(+) single positive (SP) cells, CD4(+)CD8(+) double positive (DP) mature T lymphocytes were present in both series and could be further distinguished into CD4(high)CD8(low) and CD4(low)CD8(high) subsets. An age-dependent increase of both DP T-cell subsets was observed, while SP T cells remained stable throughout life. Flow cytometry and polymerase chain reaction analysis of the TRBV expression profiles showed the presence of a TRBV restriction within CD4(+)CD8(+) DP cells in more than half (53/103; 55.3%) of the individuals >65 years of age, regardless the actual number of DP T cells observed. Clonal expansions were more prominent within the CD4(high)CD8(low) subset, accounting for most circulating DP clones (47/103; 45.6%). A few cases showed more than one (up to three) monoclonal expansion. Clonal CD4(low)CD8(high) DP T-lymphocyte expansions were detected in only 10/103 samples (9.7%) and showed a close phenotypic similarity to the rare T-cell large granular lymphocyte leukaemias. The similarities between DP clones and MBL in the elderly may help to better understand the mechanisms of immunosenescence and their relationships with the development of lymphoproliferative disorders.     

Suppression of diabetes mellitus in the non-obese diabetic (NOD) mouse by an autoreactive (anti-I-Ag7) islet-derived CD4+ T-cell line

Summary The non-obese diabetic (NOD) mouse is a spontaneous model of human insulin-dependent diabetes mellitus. Both CD4⁺ and CD8⁺ T cells infiltrate the pancreatic islets of NOD mice prior to beta-cell destruction. T-cell lines isolated from the islets of NOD mice are tools for studying the pathogenesis of insulin-dependent diabetes mellitus. During attempts to generate such lines we isolated an autoreactive CD4⁺ T-cell line, designated C2, from the insulitis lesion of a 20-week-old female non-diabetic NOD/WEHI mouse. Islet T cells were propagated by the addition of interleukin-2 and reexposure every 2 weeks to whole NOD islets and irradiated NOD spleen cells as antigen presenting cells. C2 cells proliferated up to 100-fold upon exposure to NOD antigen presenting cells but did not respond to whole NOD islets or antigen presenting cells from allogeneic mouse strains. Proliferation of C2 cells to NOD antigen presenting cells was blocked by a monoclonal antibody against the unique class II MHC molecule of NOD, I-A_g7. In response to NOD antigen presenting cells, C2 cells secreted Interferon-, tumour necrosis factor- and interleukin-6 but no detectable interleukin-2, interleukin-4 or interleukin-10, a pattern of cytokine secretion more characteristic of Th₁ CD4 cells. C2 cells displayed significant cytotoxicity in a redirected lysis assay. To explore a possible role for autoreactive T cells in the pathogenesis of autoimmune diabetes, C2 cells were injected i.v. into female NOD mice that had received cyclophosphamide to accelerate development of diabetes. Although not different at day 10, by day 16 the frequency of diabetes (blood glucose>15 mmol/l) in mice given cyclophosphamide and C2 cells was significantly less than in mice given cyclophosphamide alone (p<0.05). These results are consistent with the suppressor function of autoreactive T-cells previously reported and with the concept that autoreactive T cells modulate autoimmune beta-cell destruction in the NOD mouse.     

日本血吸虫慢性感染致CD+4T细胞凋亡的分子基础

目的　立足于全基因水平考察日本血吸虫慢性感染小鼠CD 4T细胞的凋亡相关基因的变化。方法　采用三色流式细胞术结合高密度寡核苷酸芯片 (Affemetrix芯片 ) ,对日本血吸虫感染 13周的小鼠脾脏中的CD 4T细胞进行凋亡观察及基因转录分析 ,获得凋亡相关基因的表达图谱。结果　日本血吸虫慢性感染小鼠CD 4T细胞的凋亡率均明显高于正常小鼠 (P <0 0 1) ;感染小鼠的CD 4T细胞中有 2 5个凋亡相关基因与正常小鼠相比有显著性差异 ,包括凋亡促进基因—生长抑制和DNA损伤 -GADD家族和肿瘤坏死因子α诱导蛋白 3,凋亡抑制基因—IAP等。结论　日本血吸虫慢性感染小鼠CD 4T细胞的凋亡可能是导致宿主免疫下调的原因之一。凋亡通路中存在复杂的凋亡促进基因和抑制基因的相互作用 ,其中肿瘤坏死因子α诱导蛋白 3可能是日本血吸虫感染诱导CD 4T细胞凋亡的特征性因子。     

日本血吸虫虫卵抗原诱导CD4+CD5+T细胞及其细胞因子表达

目的 探讨日本血吸虫虫卵抗原诱导宿主免疫应答下调的机制.方法 6～8周龄雌性队LB/c小鼠分为2组,实验组小鼠口服血吸虫虫卵10 000个以及尾静脉注射可溶性虫卵抗原(SEA)200μg,每周免疫1次,共4次;对照组注射PBS.流式细胞仪检测SEA免疫小鼠CD4+CD25+T细胞数量;与CD4+CD25-T细胞共同培养,检测CD4+CD25+T细胞抑制功能;流式细胞仪检测CD4+CD25+T细胞表达IL-4、IL-10与IFN-γ水平;酶联免疫吸附试验检测静脉血IL-10和转化生长因子-β表达水平.结果 实验组小鼠CD4+CD25+T细胞数量为14.7%,对照组为7.4%;实验组IL-10为29.2 pg/ml,对照组为11.0 pg/ml.与CD4+CD25-T细胞相比,CD4+CD25+T细胞主要合成IL-10及少量IFN-γ.CD4+CD25+T细胞显著抑制CD4+T细胞增殖. 结论 日本血吸虫虫卵抗原可能通过诱导CD4+CD25+T细胞和IL-10下调机体免疫应答.     

Profiles of cytokines produced by CD4-positive T lymphocytes stimulated by anti-CD3 antibody in patients with chronic hepatitis C

Helper T cells (Th) are classified as type 1 (Th1) and type 2 (Th2) according to the cytokines they produce; interferon-γ is produced by Th1, and interleukin-4 by Th2. We counted the circulating CD4-positive Th cells that produce interferon-γ or interleukin-4 with an enzyme-linked immunospot assay. CD4-positive T cells isolated from patients with chronic hepatitis B (n = 10), chronic hepatitis C (n = 16), and healthy subjects (n = 10) were stimulated with anti-CD3 antibody in vitro. The number of interferon-γ-producing Th cells was significantly lower in patients with chronic hepatitis C than in healthy subjects (P = 0.0024), whereas in patients with chronic hepatitis B, the number was similar to that in healthy subjects (P = 0.8530). The number of interleukin-4-producing Th cells was significantly higher in patients with chronic hepatitis C (P = 0.0010) and chronic hepatitis B (P = 0.0089) than in healthy subjects. In chronic hepatitis C, the number of interferon-γ-producing Th cells was increased after incubation of the cells with interferon-α (P = 0.008) or with recombinant interferon-γla (P = 0.024), but not with interferon-β (P = 0.051). The number of interleukin-4-producing Th cells was decreased after incubation with interferon-α (P = 0.0004), with interferon-β (P = 0.003), and with recombinant interferon-γla (P = 0.0004). Changes in the numbers of interferon-γ- or interleukin-4-producing Th cells in vitro were more evident in sustained responders to interferon therapy than in non-responders. These results suggest that Th2 cells are the predominant cell type in chronic hepatitis C, and that their activity may be suppressed by the administration of interferon. (Received May 12, 1997; accepted Dec. 19, 1997)     

日本血吸虫可溶性成虫抗原和虫卵抗原对CD4~+ T淋巴细胞凋亡和细胞周期的影响

目的观察并比较日本血吸虫可溶性成虫抗原(SWA)、可溶性虫卵抗原(SEA)对小鼠CD4+T淋巴细胞凋亡和细胞周期的影响。方法以免疫磁珠分选正常小鼠脾细胞中的CD4+T淋巴细胞后,与羧基荧光素二醋酸盐琥珀酰亚胺酯标记的抗原递呈细胞共培养,并分别以PBS、SWA、SEA刺激。36h后以流式细胞术(FCM)结合caspase-3荧光抗体标记技术检测CD4+T淋巴细胞的凋亡水平;96h后以碘化丙锭染色结合FCM分析CD4+T淋巴细胞周期分布情况。结果凋亡分析结果显示,经SEA和SWA刺激后,外周CD4+T细胞的凋亡百分比分别为(1.24±0.29)%和(1.52±0.38)%,两者差异无统计学意义(P0.05)。细胞周期分析表明,SWA组处于G1期、S期和G2/M期的细胞百分比分别为(78.91±2.98)%、(7.39±0.85%)和(10.69±1.05)%;与之相比,SEA组G1期细胞百分比[(59.42±1.32)%]显著降低,S期[(21.07±0.88)%]和G2/M期[(18.88±1.21)%]显著增加,差异均有统计学意义(P均0.01)。结论SWA、SEA均可诱导CD4+T淋巴细胞凋亡,SEA促进CD4+T淋巴细胞进入分裂周期的作用更明显。     

免疫调节剂SE抗血吸虫性肝病变的CD+4细胞因子调节动态

目的　进一步研究免疫调节剂SE(含SE - 1和SE - 2 )对感染小鼠诱导的抗肝内血吸虫卵肉芽肿调节效应的动态变化和分子机制。方法　BALB/c小鼠 30只 ,均分 3组 ,2组分别用SE - 1和SE - 2皮下免疫共 3次 ,每次间隔 1w ,总剂量 10 5 μg和 16 0 μg/鼠 ,另为对照组。末次免疫后 10d ,3组每鼠攻击感染尾蚴 30± 2条。在感染后 7～ 11w时 ,剖杀小鼠 :取肝组织 ,制成石腊切片 ,观察和计算肉芽肿大小 ;无菌分离脾淋巴细胞 ,进行培养 ,取上清液测定IL - 2、IL - 4和IL - 10含量水平 (pg/ml)。 结果　小鼠感染后 7～ 11wk时 ,实验鼠肉芽肿的平均面积和体积均显著小于感染对照鼠 (P均 <0 0 1) ,实验组之间无显著性差异 (P均 >0 0 5 ) ;在感染后 7w时 ,实验鼠的IL - 2和IL - 4水平均显著高于对照鼠 ;3组均显示相当高水平的IL - 10 ;在感染后 11w时 ,实验鼠的IL - 10水平显著高于对照鼠 (P均 <0 0 5 )。结论　免疫调节剂SE能诱导明显的抗肝内血吸虫卵肉芽肿效应和血吸虫性纤维化作用 ,其分子机制与Th1型 (IL - 2 )和Th2型 (IL - 4和IL - 10 )细胞因子应答的调节动态密切相关。     

Local skin reaction (chancre) induced following inoculation of metacyclic trypanosomes in cattle by tsetse flies is dependent on CD4 T lymphocytes

The first visible response in livestock to the bite of a trypanosome-infected tsetse fly is the formation of a localized skin reaction, also known as a chancre. This is an inflammatory response in the skin associated with swelling and an influx of cells. It is thought to be associated with an acquired immune response to the injected metacyclic trypanosomes. In this study, we examined the role of T lymphocytes in the development of the inflammatory response, by depleting cattle of T cell subpopulations and monitoring the development of chancres. Depletion of CD4 cells, but not CD8 cells, resulted in a significant reduction in chancre formation, confirming that an acquired response mediates the inflammatory response. In addition, it was established that the CD4 T cells mediate the generation of memory for immunity to a homologueous re-challenge. The inflammatory response in the skin did not affect further progress of the infection.