In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25−CD4+ cells

This paper investigates the in vitro effect of dexamethasone on bovine CD25^highCD4⁺, CD25^lowCD4⁺ and CD25⁻CD4⁺ T cells. Only a small percentage of bovine CD25^highCD4⁺ (2–4%) and CD25^lowCD4⁺ (1–2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25⁻CD4⁺ cells, but it increased the relative and absolute numbers of CD25^highCD4⁺ and CD25^lowCD4⁺ lymphocytes, while at the same time reducing the percentage of Foxp3⁺ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25⁺CD4⁺, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4⁺ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25⁻CD4⁺ cells and antiapoptotic effect on CD25^highCD4⁺ and CD25^lowCD4⁺ cells. The results obtained from this study indicate that the involvement of CD4⁺ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25⁻CD4⁺ cells. Secretion of TGF-β and IL-10 by CD4⁺ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10⁺CD4⁺ cells.     

The role of CD4+CD25+ regulatory T cells in viral infections

Many virus infections result in the suppression of one or more functions of the immune system. Multiple mechanisms have been proposed to explain viral-induced immunosuppression, including an imbalance in the cellular Th1/Th2 or cytokine profile, induction of anergy, depletion of effector cells and most recently the activation of CD4+CD25+ regulatory T (T reg) cells. CD4+CD25+ T reg cells are a subset of circulating CD4+ T cells with suppressive properties. CD4+CD25+ T reg cells were first identified in mice as cells capable of maintaining self-tolerance by suppressing autoreactive T cells. This review focuses on interactions between CD4+CD25+ T reg cells and viral pathogens. Most cases in which CD4+CD25+ T reg cells participate in response to infection reported so far involve chronic or persistent viral infections. Examples have been growing recently and include members of different viral families including retroviridae, herpesviridae and picornaviridae. It is currently not known how microbes are recognized by CD4+CD25+ T reg cells and whether exoantigen-specific T reg cells are of the same lineage as self-reacting natural T reg cells or represent peripherally induced counterparts derived from CD4+CD25- T cells. The findings that T reg cells influence the functional immunity during viral infections, however, might indicate that, in some cases, virus-specific T reg cells not only influence immune pathology or prevent pathogen elimination but also can promote a generalized state of immunosuppression in vivo such that the host is more susceptible to secondary infections with other pathogens or has reduced resistance to tumors. Conceivably, the activities of T reg cells might be one of the contributing reasons why it has been difficult so far to produce effective vaccines against some persisting viral infections.     

AA肉鸡血液CD3+、CD4+和CD8+T淋巴细胞的变化规律

采用流式细胞仪检测1、3、5、7、14、21、28、35、42、49日龄AA肉鸡血液中的CD3、CD4、CD8阳性T细胞比例。研究结果表明:1～5日龄T细胞逐渐进入血液参与细胞免疫,7、21日龄注射疫苗起免疫应答作用,28日龄后基本形成稳固的细胞免疫水平。     

In vivo lipopolysaccharide injection alters CD4+CD25+ cell properties in chickens

In chickens, thymic CD4(+)CD25(+) cells are characterized as regulatory T cells. The objectives of this experiment were to study the effects of an in vivo lipopolysaccharide (LPS) injection on the percentage of CD4(+)CD25(+) cells in peripheral organs and the suppressive properties of splenic CD4(+)CD25(+) cells in chickens. Chickens were injected with LPS and CD4(+)CD25(+) cells were analyzed at 1, 2, 3, 5, and 10 d post LPS injection. The LPS injection increased CD4(+)CD25(+) cell percentage approximately 5-fold in the blood at 1 d post LPS injection (P < 0.001), 3-fold in the thymus at 3 d post LPS injection (P = 0.001), and 2.5-fold in the spleen at 2 d post LPS injection (P = 0.001) compared with the no-LPS-injected group. The LPS injection did not alter the CD4(+)CD25(+) cell percentage in the cecal tonsil (P = 0.162), lung (P = 0.098), or bone marrow (P = 0.071) at any time point measured. At 2 d post LPS injection, splenic CD4(+)CD25(+) cells lost their suppressive ability (P < 0.001). At 5 d post LPS injection, splenic CD4(+)CD25(+) cells not only regained their suppressive ability, but also became supersuppressive (P < 0.001). Splenic CD4(+)CD25(+) cells at 5 d post LPS injection produced 5.5-fold more (P = 0.005) IL-10 mRNA than splenic CD4(+)CD25(+) cells at 0 and 2 d post LPS injection. In conclusion, chicken regulatory T cells are differentially activated to facilitate immune response during the early stage of inflammation and to facilitate immune suppression at a later stage of inflammation.     

Differential cytokine gene expression in CD4+ and CD8+ T cell subsets of calves

The distinct patterns of cytokine expression in CD4+ and CD8+ T cells are well understood in mice and humans. However, little information is available about cytokine expression in bovine CD4+ and CD8+ T cells. In this study, mRNA expression of 19 different cytokines was analyzed in CD4+ and CD8+ T cells of calves with or without Concanavalin A (Con A) stimulation. CD4+ and CD8+ T cell populations were enriched to 98% purity by positive selection using magnetic cell sorting (MACS). CD4+ T cells spontaneously expressed the mRNAs of interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-18, IFN-gamma, TNF-alpha, TNF-beta and TGF-beta, and augmented the mRNA expression of IL-10, IFN-gamma and TNF-beta after Con A stimulation. The mRNAs of IL-3, IL-4, IL-5, IL-13 and GM-CSF were newly expressed in Con A-stimulated CD4+ T cells. CD8+ T cells displayed spontaneous mRNA expression of IL-6, IL-18, TNF-alpha, TNF-beta and TGF-beta, and newly expressed the mRNA of IL-2, IL-7, interferon-gamma (IFN-gamma) and GM-CSF after Con A stimulation. It was found that CD4+ T cells expressed the mRNA of 17 cytokines except for IL-12 and IL-15, while CD8+ T cells expressed only the mRNA of 9 cytokines after Con A stimulation. The profile of cytokine mRNA expression was substantially different in the CD4+ and CD8+ T cells of calves, indicating that CD4+ T cells can be distinguished from CD8+ T cells by the cytokine gene expression of IL-1alpha, IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-10 and IL-13. Differential cytokine expression between CD4+ and CD8+ T cells serve to interpret an individual function of T cell subsets in the immune system of calves.     

外周血中CD4+、CD8+T、CD4+CD25+Treg在绵羊接种布鲁氏菌M5-90弱毒疫苗后的动态变化

为研究绵羊接种布鲁氏菌弱毒M5-90株后外周血中CD4+、CD8+T、CD4+CD25+Treg细胞的动态变化规律,本研究选择11只健康绵羊,每10 d免疫一次,共免疫3次,分别在免疫前、免疫后10d、20 d、30 d利用流式细胞术检测外周血中CD4+、CD8+T、CD4+CD25+Treg淋巴细胞亚群.在免疫后的第20 d,CD4+T、CD8+T细胞百分含量达到最高水平(P＜0.05)后均缓慢下降;在第10d,CD4+CD25+Treg细胞缓慢升高,至20 d、30 d均显著升高(p＜0.05);在布鲁氏菌M5-90疫苗免疫应答过程中CD4+CD25+Treg细胞参与了机体的免疫反应调控,对CD4+T、CD8+T淋巴细胞的比例进行调节,并且维持CD4+/CD8+比值稳定,起到平衡Th1/Th2细胞间反应的作用.     

CD4+ and CD8+ T- lymphocytopenia in a filly with Pneumocystis carinii pneumonia

Decreased proportion of CD4+ and CD8+ T lymphocytes in peripheral blood likely contributed to susceptibility to Pneumocystis carinii in a foal. Cytological evaluation of bronchoalveolar lavage was required for identification of the pathogen and serial flow-cytometric analysis of peripheral blood lymphocytes documented transient low expression of CD4+ and CD8+ T lymphocytes. Although immunodeficiency is uncommon, it must be included in the differential diagnosis for patients suffering from chronic or opportunistic infections and may provide an indication for immunostimulant therapy.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells

Feline and primate immunodeficiency viruses (FIVs, SIVs, and HIV) are transmitted via direct contact (e.g. fighting, sexual contact, and mother–offspring transmission). This dynamic likely poses a behavioral barrier to cross-species transmission in the wild. Recently, several host intracellular anti-viral proteins that contribute to species-specificity of primate lentiviruses have been identified revealing adaptive mechanisms that further limit spread of lentiviruses between species. Consistent with these inter-species transmission barriers, phylogenetic evidence supports the prediction that FIV transmission is an exceedingly rare event between free-ranging cat species, though it has occurred occasionally in captive settings. Recently we documented that puma and bobcats in Southern California share an FIV strain, providing an opportunity to evaluate evolution of both viral strains and host intracellular restriction proteins. These studies are facilitated by the availability of the 2× cat genome sequence annotation. In addition, concurrent viral and host genetic analyses have been used to track patterns of migration of the host species and barriers to transmission of the virus within the African lion. These studies illustrate the utility of FIV as a model to discover the variables necessary for establishment and control of lentiviral infections in new species.     

猪圆环病毒2型感染小鼠脾脏内CD4~+CD25~+调节性T细胞的动态变化

为分析猪圆环病毒2型(PCV2)感染小鼠后脾细胞中CD4+CD25+调节性T细胞(Tregs)占CD4+T细胞比例的动态变化,探讨Tregs与PCV2感染的关系,本研究选择清洁级昆明小鼠60只,随机分成实验组和对照组,实验组腹腔接种PCV2,分别在接种后第0 d、5 d、10 d、20 d、30 d和60 d取脾脏制备单细胞悬液,用FITC-CD4和PE-CD25单克隆抗体标记Tregs,采用流式细胞仪检测Tregs占总CD4+T细胞百分比的动态变化。结果表明感染组小鼠脾细胞中Tregs百分比从第5 d开始逐步上升,第20 d达峰值后逐渐下降,感染组Tregs百分比第10 d、20 d、30 d时显著高于对照组(p0.05),但第60 d两组间差异不显著(p0.05)。试验结果证明PCV2感染昆明小鼠后,可在小鼠脾脏内诱导明显的Tregs增殖,这些增殖的细胞可能在PCV2感染中发挥免疫抑制作用。     

Effect of CD4+CD25+ T cell-depletion on acute lethal infection of mice with Trypanosoma congolense

Despite the immense socio-economic repercussions of African trypanosomosis (AT), there is currently no effective control measure against the disease. Characterization of mechanisms governing resistance and/or susceptibility to AT could suggest interventions that might lead to more effective disease control. The present study was designed in an attempt to address the possible role of CD4+CD25+ T cells during an acute lethal infection of mice with Trypanosoma congolense, the causative agent of AT in domestic animals, through selective depletion using anti-CD25 monoclonal antibody. Accordingly, CD4+CD25+ T-cell-depletion resulted in a significant reduction or delay in parasitemia, pathology, and mortality, as compared to controls. The apparent resistance in CD4+CD25+-T-cell-depleted mice correlated with a profound suppression of Th2 cytokines in vitro and in vivo, culminating in a net Th1 cytokine environment. Cumulatively, these findings suggest that CD4+CD25+ T-cell- depletion improves the trypanotolerance of highly susceptible BALB/c mice acutely infected with the lethal T. congolense.     

The Localization Changes of CD4+ and CD8+ T Lymphocytes in Chicken Lung at Different Ages

The aim of this study was to investigate the immune state of chicken lung in different periods.With lung tissue of Hy-line White chicken at different ages,the distribution and quantity changes of CD4⁺ and CD8⁺T lymphocytes in lung were studied using immunohistochemistry staining.The results showed that CD8⁺T lymphocytes appeared firstly in embryonic at 18 d,while CD4⁺T lymphocytes appeared at 1-day-old chicken.At 4-day-old,there were aggregates of lymphocytes at the junction of the primary and secondary bronchi,which formed obvious broncho-associated lymphoid tissue (BALT).CD4⁺T lymphocytes of each age mainly occupied the central area of BALT,while CD8⁺T lymphocytes mainly surrounded the periphery.Since 56 days old,CD8⁺T lymphocytes are distributed in the inner wall of third-order bronchial airway,atrial septum,gas exchange area and interlobular connective tissue,and are distributed throughout the lung.In terms of quantity change,with the growth of daily age,the number of CD4⁺T lymphocytes and CD8⁺T lymphocytes gradually increased,and the number of CD4⁺ T lymphocytes was more than that of CD8⁺ T lymphocytes before 35 days of age,while the number of CD8⁺ T lymphocytes was significantly more than that of CD4⁺ T lymphocytes at the same age thereafter.The results showed that the distribution and number of CD4⁺ and CD8⁺T lymphocytes in the lungs of chickens were correlated with the age,and the changes could reflect that the lungs before the age of 35 days were dominated by humoral immunity,while the lungs after that tended to be cellular immunity.     

CD4+CD25+ regulatory T cells are infected and activated during acute FIV infection

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.     

An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes,CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8β+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT.KP induced an early dose-dependent shift to pro-inflammatory CD172α⁺CD14^+high monocytes and an increase of CD3⁺CD16⁺ natural killer (NK) T cells. KP triggered CD8α and CD8β expression on classical CD4⁻CD8αβ⁺ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4⁺CD8α⁺ Th memory cells (CD4⁺CD8α^+low CD8β^+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4⁺CD8α^+high CD8β^+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.     

传染性支气管炎病毒结构蛋白免疫鸡对CD4+CD8+T淋巴细胞亚群的影响

为了比较传染性支气管炎病毒3种主要结构蛋白免疫鸡后对CD4+、CD8+T淋巴细胞亚群的不同影响.本试验以pVAX1载体为携带工具,制备分别含有IBV主要结构蛋白基因的质粒免疫健康雏鸡,采用流式细胞仪(FACS)对免疫鸡外周血中CD4+、CD8+T淋巴细胞数进行检测.结果显示:各试验组间,携带N蛋白基因的质粒在免疫后3周内CD4+T淋巴细胞数和CD8+T淋巴细胞数均高于其他组,且差异极显著(P＜0.01).由此可见,IBV的N蛋白可明显诱导CD8+T淋巴细胞的CTL免疫作用和CD4+T淋巴细胞的辅助性免疫作用,其细胞免疫原性高于S1蛋白和M蛋白.     

刺五加多糖对雏鸡脾脏中CD4+ 和CD8+T淋巴细胞定位分布的影响

为了研究刺五加多糖（ASPS）对雏鸡脾脏中CD4+和CD8+ T淋巴细胞定位分布的影响,从组织学角度评价ASPS对脾脏的免疫调节作用,试验将1日龄海兰褐公雏饲养至7日龄时选取150只,随机分为3组：空白对照组、ASPS低剂量组（ASPSL）和高剂量组（ASPSH）,每组50只,所有组每天注射1次,连续注射3天。免疫后的第7、14、21和28天分别取其脾脏制作冰冻切片,采用免疫组织化学方法检测CD4+和CD8+ T淋巴细胞的定位分布。结果显示,与空白对照组比较,免疫注射后21天和28天时ASPSL组和ASPSH组CD4+ T淋巴细胞的数量均显著增加（P<0.05）,而且ASPS能够促进红髓中CD4+ T淋巴细胞向动脉周围淋巴鞘迁移,从而使单个动脉周围淋巴鞘面积较对照组明显增加,而ASPS对脾脏中CD8+ T淋巴细胞的数量和分布无明显影响。由此可知,ASPS能够通过影响脾脏中CD4+ T淋巴细胞的定位分布发挥免疫调节作用,这对于进一步揭示ASPS的免疫调节机制具有重要意义。     

CD34+, CD41+ acute megakaryoblastic leukemia in a dog

A clinically normal, 5-year-old intact female German Shepherd dog was presented to the local veterinarian to be spayed. Results of a preoperative CBC included mild nonregenerative anemia, severe thrombocytopenia, and 17% unclassified cells. On cytologic examination of aspirates from the dog's enlarged spleen and peripheral lymph nodes, a population of primitive round cells that occasionally resembled megakaryocytes was observed. A bone marrow aspirate specimen was markedly hypercellular with approximately 65% of marrow cells comprising a homogeneous population of immature hematopoietic cells similar to those found in the spleen, lymph nodes, and peripheral blood. Using immunocytochemical stains with canine-specific antibodies, all neoplastic cells strongly expressed cytoplasmic CD41 and 20-70% of the neoplastic cells expressed CD34 weakly to moderately. Rare (<0.5%) neoplastic cells weakly expressed vWF. The cells were negative for all other markers. Based on these results and the morphology of the neoplastic cells, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. In spite of treatment, results of a CBC performed 1 week later indicated progressive anemia and thrombocytopenia, and the dog was euthanized. To our knowledge, this report documents the first case of canine AMegL diagnosed with both anti-canine CD34 and CD41 antibodies.     

青蒿组方对球虫感染雏鸡血液中CD4^+和CD8^+T淋巴细胞的影响试验

为研究青蒿组方中药对鸡免疫力的影响,试验选取14日龄三黄鸡120只,平均分为4组:感染给药组(1组),感染不给药组(2组),不感染给药组(3组),不感染不给药组(4组).感染组人工接种柔嫩艾美耳球虫孢子化卵囊.接种后第4、7、10 d运用流式细胞仪测定各组鸡血液中CD4^+、CD8^+T淋巴细胞值及二者的比值.结果:1组CD4^+、CD8^+T淋巴细胞值及其比例在接种后第4、7、10 d均高于2组,差异显著(P<0.05);2组CD4^+、CD8^+T淋巴细胞平均比值低于其他3个组.结论:青蒿组方中药可促进鸡血液中CD4^+、CD8^+T淋巴细胞生成,进而增强机体的免疫功能.