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.     

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4⁺CD25⁺Foxp3⁺ T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3⁺CD4⁺ cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4⁺ cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4⁺ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4⁺ and CD25⁺ T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4⁺ cells in the CD4⁺ T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4⁺CTLA-4⁺ T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.     

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.     

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.     

Membrane markers of the immune cells in swine: an update

Besides their breeding value, swine are increasingly used as biomedical models. As reported in three international swine clusters of differentiation (CD) workshops and in the animal homologue section of the last workshop for the determination of human leukocyte differentiation antigens (HLDA 8), characterisation of leukocyte surface antigens by monoclonal antibodies and other molecular studies have determined the cell lineages and blood leukocyte subsets implicated in the immune response, including cell adhesion molecules involved in cell trafficking. This review focusses on the current state of knowledge of porcine leukocyte differentiation and major histocompatibility complex (SLA) molecules. Examples of porcine particularities such as the double-positive T lymphocytes with the phenotype CD(4+)CD8(low) and CD(4-)CD8(low) alphabeta T cell subsets and the persistence of SLA class II after T-lymphocyte activation are illustrated, as well as the shared characteristics of the Artiodactyla group, such as the high proportion of gammadelta TcR (T cell receptor) T cells in blood and other lymphoid tissues. Furthermore, discrepancies between swine and humans, such as CD16 expression on dendritic cells and CD11b (wCD11R1) tissue distribution are outlined. The rapidly growing information should facilitate manipulation of the swine immune system towards improving disease control, and open new avenues for biomedical research using the pig as a model.     

PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-na?ve regulatory T cells to proliferate

In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)⁺ and N-protein⁻ moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein⁺ moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein⁻ moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3⁺ regulatory T cells present within CD4⁺ T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4⁺ T cells showed differences in regard to proliferation and frequency of Foxp3⁺ T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.     

Monoclonal antibodies putatively recognising activation and differentiation antigens.

In the activation/maturation section, 46 monoclonal antibodies (mAbs) were analysed using freshly isolated as well as mitogen activated and recall antigen re-stimulated cells. A total of 10 internal standards as well as 6 antibodies with established reactivity for human cells, reported to cross-react with porcine leukocytes, were included in the panel. The standard antibodies were anti-CD25, CD44, CD45, SLA II, SWC1, SWC2, SWC7 and SWC8 reagents. The test panel contained antibodies with putative reactivity to CD25, SLA II and other mAbs directed against ill-defined targets. Single and double colour surface staining was performed in the attempt to group the mAbs tested into clusters of differentiation. Five new anti-class II reagents, two directed to SLA-DQ and three to SLA-DR, could be added to the previously established ones. One new anti-CD25 as well as two new antibodies with SWC7 and SWC8 specificities, respectively, could also be added to the previously established ones. The identity of the two latter antibodies was also confirmed in other sections of this workshop (B-cell section for SWC7 antibodies and myeloid section for the SWC8 antibodies). The antibody JM2F12, in our hands, has shown strong similarities to the cross-reactive anti human-CD49f reagent. No other clusters were identified, as all remaining antibodies behaved in a different way on different target leukocyte populations. The second purpose of the section was fulfilled: interesting staining profiles of several antibodies on differentiating lymphocytes were recorded and are discussed here.     

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.     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

Subpopulations of equine blood lymphocytes expressing regulatory T cell markers

Several distinct T lymphocyte subpopulations with immunoregulatory activity have been described in a number of mammalian species. This study performed a phenotypic analysis of cells expressing regulatory T cell (Treg) markers in the peripheral blood of a cohort of 18 horses aged 6 months to 23 years, using antibodies to both intracellular and cell surface markers, including Forkhead box P3 (FOXP3), CD4, CD8, CD25, interferon gamma (IFNγ) and interleukin 10 (IL-10). In peripheral blood, a mean of 2.2 ± 0.2% CD4+ and 0.5 ± 0.1% CD8+ lymphocytes expressed FOXP3. The mean percentage of CD4+FOXP3+ cells was found to be significantly decreased in horses 15 years and older (1.5%) as compared to horses 6 years and younger (2.7%), but did not differ between females and males and ponies and horses. Activation of peripheral blood mononuclear cells by pokeweed mitogen resulted in induction of CD25 and FOXP3 expression by CD4+ cells, with peak expression noted after 48 and 72 h in culture respectively. Activated CD4+FOXP3+ cells expressed IFNγ (35% of FOXP3+ cells) or IL-10 (9% FOXP3+ cells). Cell sorting was performed to determine FOXP3 expression by CD4(+)CD25(-), CD4(+)CD25(dim) and CD4(+)CD25(high) subpopulations. Immediately following sorting, the percentage of CD4+FOXP3+ cells was higher within the CD4(+)CD25(high) population (22.7-26.3%) compared with the CD4(+)CD25(dim) (17% cells) but was similar within the CD4(+)CD25(dim) and CD4(+)CD25(high) cells after resting in IL-2 (9-14%). Fewer than 2% of cells in the CD4(+)CD25(-) population expressed FOXP3. These results demonstrate heterogeneity in equine lymphocyte subsets that express molecules associated with regulatory T cells. CD4+FOXP3+ cells are likely to represent natural Tregs, with CD4+FOXP3+IL-10+ cells representing either activated natural Tregs or inducible Tregs, and CD4+FOXP3+IFNγ+ cells likely to represent activated Th1 cells.     

猪圆环病毒2型衣壳蛋白优势B细胞表位重组蛋白免疫原性研究

猪圆环病毒2型(PCV-2)衣壳蛋白(Cap)是研制基因工程疫苗和血清抗体检测方法的主要候选抗原。为筛选免疫原性强的Cap抗原表位,将定位在Cap上的6个B细胞线性表位的编码基因序列重新组合,命名为R1234、R1235、R1236、R2345、R2346和R3456,然后克隆至pET32a原核表达载体,转化到大肠埃希菌中进行诱导表达。通过对表达和纯化条件的优化,最终获得6个多抗原表位串联体的重组蛋白。Western blot和间接ELISA结果显示,6组重组表位蛋白均能被PCV-2阳性血清特异性识别,具有良好的反应原性。将其与完整Cap分别免疫Balb/c小鼠,检测免疫后血清中特异性抗体和细胞因子水平以及脾脏淋巴细胞中CD4^+和CD8^+T淋巴细胞的比例。结果表明,R2346组合刺激机体产生的特异性抗体和细胞因子IFN-γ、IL-4的水平明显高于其他组,脾脏淋巴细胞中CD4^+和CD8^+T淋巴细胞含量与对照组相比显著增加(P<0.05),说明该重组蛋白能够刺激体液免疫和细胞免疫反应。因此,筛选获得的R2346重组表位蛋白可以作为研制PCV-2多表位基因工程疫苗和血清抗体诊断方法的候选抗原,用于PCV-2的预防和控制工作中。     

猪繁殖与呼吸综合征病毒N蛋白间接ELISA方法的建立

为研制特异性敏感性较好的猪繁殖与呼吸综合征病毒(PRRSV)抗体检测方法,更好地实现PRRSV的流行病学监测和诊断,将PRRSV GDr180毒株N蛋白基因序列克隆到p ET32a(+)载体中,在大肠杆菌BL21(DE3)细胞内实现了高效表达,表达形式为可溶性表达,通过Western blot证明表达产物与PRRSV GDr180株阳性血清具有很好的反应原性和特异性。将大肠杆菌表达的N蛋白经过超声、离心、过柱纯化后,作为间接ELISA包被抗原检测血清中的PRRSV抗体,通过对各参数和试剂的优化建立了能检测PRRSV血清抗体的间接ELISA检测方法;对方法的特异性和重复性以及与同类成品试剂盒间的应用效果对比进行了试验。结果表明,研究建立的ELISA抗体检测方法可用于检测猪血清中PRRSV抗体、监测猪繁殖与呼吸综合征的流行情况和评价相关疫苗的免疫效果。     

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix ‘w' which will lead to ‘wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.     

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.