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(high)CD4(+), CD25(low)CD4(+) and CD25(-)CD4(+) T cells. Only a small percentage of bovine CD25(high)CD4(+) (2-4%) and CD25(low)CD4(+) (1-2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25(-)CD4(+) cells, but it increased the relative and absolute numbers of CD25(high)CD4(+) and CD25(low)CD4(+) 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(high)CD4(+) and CD25(low)CD4(+) 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.     

Co-infection of porcine dendritic cells with porcine circovirus type 2a (PCV2a) and genotype II porcine reproductive and respiratory syndrome virus (PRRSV) induces CD4(+)CD25(+)FoxP3(+) T cells in vitro

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is the primary causative agent, however co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) is often required to induce the full spectrum of clinical PCVAD. While the specific mechanisms of viral co-infection that lead to clinical disease are not fully understood, immune modulation by the co-infecting viruses likely plays a critical role. We evaluated the ability of dendritic cells (DC) infected with PRRSV, PCV2, or both to induce regulatory T cells (T(regs)) in vitro. DCs infected with PCV2 significantly increased CD4(+)CD25(+)FoxP3(+) T(regs) (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of T(regs) than with PCV2 alone (p<0.05). Cytokine analysis indicated that the induction of T(regs) by co-infected DCs may be dependent on TGF-β and not IL-10. Our data support the immunomodulatory role of PCV2/PRRSV co-infection in the pathogenesis of PCVAD, specifically via T(reg)-mediated immunosuppression.     

Regulatory T cell properties of thymic CD4(+)CD25(+) cells in ducks

Thymic CD4(+)CD25(+) cells from ducks were characterized for mammalian T regulatory cells' suppressive and cytokine production properties. The cross reactivity of anti-chicken CD25 monoclonal antibody with duck CD25 was confirmed by evaluating Concanavalin-A-stimulated CD25 upregulation in splenocytes. CD4(+)CD25(+) cells were detectable in the thymus, spleen, cecal tonsil, and lung (airsacs), but not in the bursa. Duck CD4(+)CD25(+) cells had approximately nine-fold higher IL-10 mRNA, 12-fold higher TGF-β, 16-fold higher CTLA-4, and nine-fold higher LAG-3 mRNA amounts than thymic CD4(+)CD25(-) cells. Thymic CD4(+)CD25(+) cells had no detectable levels of IL-2 mRNA. Duck CD4(+)CD25(+) cells had a three-fold higher IL-10 mRNA amount than chicken CD4(+)CD25(+) cells. Duck CD4(+)CD25(+) cells were anergic in vitro. Duck CD4(+)CD25(+) cells suppressed naive cell proliferation at effector: responder cell ratios above 0.5:1 in both contact-dependent and -independent pathways. It could be concluded that thymic CD4(+)CD25(+) cells in ducks are most likely the counterpart of mammalian T regulatory cells.     

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。     

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.     

Induction of inducible CD4+CD25+Foxp3+ regulatory T lymphocytes by porcine reproductive and respiratory syndrome virus (PRRSV)

Increases in numbers or activities of regulatory T lymphocytes (Tregs) have been linked to the establishments of several persistent infections. It has been previously shown that porcine reproductive and respiratory syndrome virus (PRRSV) can negatively modulate the host immune responses, resulting in persistent infection and secondary immunodeficiency. Recently, the existence of porcine CD4⁺CD25⁺ Tregs has been demonstrated. We investigated the effect of PRRSV on the CD4⁺CD25⁺ Tregs. The CD4⁺CD25⁺Foxp3⁺ T lymphocytes in the peripheral blood mononuclear cells (PBMCs) were identified, using the anti-human anti-Foxp3 monoclonal antibody. In vitro culture of porcine PBMC in the presence of PRRSV, but not classical swine fever virus, significantly increased the numbers of Foxp3⁺ lymphocytes, particularly in the CD4⁺CD25^high subpopulation. The time-course study revealed that PRRSV significantly increased the numbers of viral-specific CD4⁺CD25^highFoxp3⁺ subpopulation in the culture starting from 12 h through the end of the observation period. Consistent to the results obtained by flow cytometry, enhanced Foxp3 gene expression was observed in the PBMC cultured with PRRSV in a time-course manner. The presence of monocyte-derived DC in the co-culture significantly enhanced the induction of CD4⁺CD25⁺ Foxp3⁺ T lymphocytes. The PRRSV-induced CD4⁺CD25^high T lymphocytes exhibited suppressive activity when co-cultured with PHA-activated, autologous peripheral blood leukocytes, indicating the suppressive activity of the PRRSV-specific Tregs. In addition, PRRSV exposure significantly increased the numbers of PRRSV-specific CD4⁺CD25⁺Foxp3⁺ subpopulation in the PBMC of infected pigs at 10 days post-infection. In summary, the results indicated that PRRSV could increase the numbers of viral-specific, inducible regulatory T lymphocytes in the porcine PBMC, both in vitro and in vivo. The findings suggested the novel immunomodulatory mechanism induced by PRRSV.     

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.     

Canine CD4(+)CD8(+) double positive T cells in peripheral blood have features of activated T cells

In dogs a CD4(+)CD8(+) double positive T cell subpopulation exists that has not been phenotypically defined yet. We demonstrate that canine CD4(+)CD8(+) T cells are mature CD1a(-) and TCRαβ(+) T cells. To analyse the activation potential of CD4(+)CD8(+) T cells, PBMC from dogs vaccinated against canine distemper virus (CDV) were re-stimulated with CDV. Upon antigen-specific stimulation, the CD4(+)CD8(+) T cell fraction increases and consists nearly exclusively of proliferated cells. Similarly, other features of activated effector/memory T cells such as up-regulation of CD25 and MHC-II as well as down-regulation of CD62L (L-selectin) were observed in CD4(+)CD8(+) T cells after stimulation. Canine CD4(+)CD8(+) T cells are less abundant, but more heterogeneous than porcine ones, comprising a small proportion expressing the β chain of CD8 in addition to the CD8α chain, like human CD4(+)CD8(+) T cells. In summary, this analysis provides the basis for functional characterisation of the in vivo relevance of CD4(+)CD8(+) T cells in T-cell mediated immunity.     

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.     

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.     

Current knowledge on porcine regulatory T cells

Regulatory T cells (Tregs) are known in humans and mice from last 15 years, and several studies led to a detailed knowledge on their phenotype, functions, and role in various immune reactions. In swine, the existence of Tregs was first demonstrated in 2008 and research is still at the beginning. Nevertheless, basic information regarding phenotype, mechanisms and targets of suppression, as well as implications in transplantation and some diseases are available. Purpose of this review is to give a brief summary of the current knowledge about porcine Tregs.     

Gene expression profiles of CD4/CD8 double‐positive T cells in porcine peripheral blood

A characteristic subset of T cells, known as double positive T cells (DPTC) and expressing both cluster of differentiation 4 (CD4) and CD8, is observed in porcine peripheral blood. Previous studies suggested that DPTC might be memory cells. However, detailed phenotypes and functions of DPTC are yet to be fully elucidated and thus, the relatedness of DPTC with memory phenotypes remains unclear. In this study, DPTC gene expression profiles in peripheral blood were analyzed by DNA microarray in Experiment 1 and compared with those of CD4 single positive T cells (4SPTC) and CD8 single positive T cells (8SPTC). Expressions of IFNG, CCL5, NCK2, CCR2 and ITGB1 were higher than that of 4SPTC and 8SPTC. In contrast, expressions of CCR7 and SELL were lower than that of 4SPTC and 8SPTC. These results suggested that DPTC were either effector T cells or effector memory T cells (T_EM). Next, to determine whether DPTC were effector T cells or T_EM, differences in the response of DPTC and 8SPTC against immunized/primed antigens were compared (Experiment 2). While DPTC showed quick elevation of IL2 and CD25 gene expressions against in vitro stimulation of primed/immunized antigens, 8SPTC did not. These results suggest that at least some DPTC likely belong to T_EM.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Analysis of the repertoire of cattle CD4(+) T cells reactive with bovine viral diarrhoea virus

Cell-mediated immunity and CD4(+) cells in particular are important for the resolution of acute infection with non-cytopathic bovine viral diarrhoea virus (BVDV). CD4(+) T cells were shown to recognise virus-infected and non-infectious-protein-pulsed APCs, whereas CD8(+) T cells recognised only virus-infected APCs. T cell recognition was strain cross-reactive and MHC-restricted. Using native and recombinant antigens, we identified the structural glycoprotein E2 and the non-structural protein NS3 as dominant CD4(+) T cell determinants. The repertoire of CD4(+) T cell responses to E2 and NS3 was examined using inbred, homozygous cattle and overlapping synthetic peptides. The repertoire was biased toward conserved regions of NS3 and excluded the hypervariable regions of E2. The number of peptides that were recognised varied between animals but patterns could be distinguished in those animals that shared the same DRB3(*) allele. Of particular interest were: (i) a determinant that was recognised in the context of both DRB3(*) alleles (i.e. DRB3(*)2002 and DRB3(*)0701), (ii) two determinants that were juxtaposed to B cell sites, and (iii) a determinant that had structural analogy with a NS3 epitope previously described for the closely related hepatitis C virus. The minimum stimulatory sequence of the latter, NS3(397-414), was located to residues NS3(400-410).     

Phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus

It is well documented that there is a delay in the development of effective immunity to porcine reproductive and respiratory syndrome virus (PRRSV) in infected and vaccinated pigs. This suggests that PRRSV might possess some inherent properties to evade host defense mechanisms during the early stage of infection. Dendritic cells (DCs) play a crucial role in the activation and control of T-cells in response to viral antigens. In this study, we investigated the phenotypic and functional property changes of bone marrow-derived immature DCs (BM-imDCs) that take place after infection by PRRSV. Results showed that BM-imDCs were permissive to PRRSV infection, as productive replication took place in these cells. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 is observed at 48 h following infection. Also at 48 h following PRRSV infection, a significant increase of IL-10 secretion by BM-imDCs was noticed. Results suggest that the inhibited expression of MHC I and the enhanced secretion of IL-10 by BM-imDCs after PRRSV infection might be among the strategies used by the virus to evade the host immune defenses.     

Phenotypic and functional characteristics of bovine T lymphocytes

Monoclonal antibodies have been derived which detect the bovine equivalents of the human pan-T cell marker CD2 and the T lymphocyte subpopulation markers CD4 and CD8. We refer to the bovine analogues as BoT2, BoT4 and BoT8. Monoclonal antibodies have also been derived which detect an antigen(s) with similarities to CD3, although the precise nature of the target molecule(s) in this instance remains to be elucidated. In general there is close similarity between the tissue distributions and, where these have been determined, the molecular masses of the BoT2, BoT4, BoT8 and putative BoT3 entities and their counterparts in other species. BoT2 is expressed on a majority of peripheral blood T lymphocytes and thymocytes and BoT2+ cells are found in both thymic cortex and medulla. In contrast, the putative BoT3 marker is expressed by a minority of thymocytes which are moreover, largely restricted to medulla. Monoclonal antibodies detecting BoT2 determinants have been shown to precipitate 55 kDa molecules. Antibodies to the BoT2 and BoT3 entities have been shown to induce proliferation in peripheral blood mononuclear cells of some cattle, and to be capable of inhibition of antigen-driven proliferative responses and cytolytic function. The BoT4 and BoT8 markers are expressed in a mutually exclusive manner by bovine peripheral blood mononuclear cells but they are coexpressed on a large population of thymocytes. Monoclonal antibodies have been used to precipitate molecules of 52 and 55 kDa in the case of those detecting BoT4 and 34 and 35 kDa in the case of an antibody reactive with a BoT8 determinant. The BoT4 and BoT8 markers have been associated with specificity for, and restriction by, MHC class II and class I molecules respectively.     

CD4+CD25+ T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.