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.     

Individual antigens of cattle. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2).

The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric molecule expressed in a subpopulation of the WC1+ cells and on an additional small population of T lymphocytes. The cell populations recognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor molecules.     

The role of WC1(+) gamma delta T-cells in the delayed-type hypersensitivity (DTH) skin-test reaction of Mycobacterium bovis-infected cattle

Delayed-type hypersensitivity (DTH) skin-testing with mycobacterial antigens is often used as a means of identifying Mycobacterium bovis-infected cattle. Better understanding of the cellular basis underlying the DTH reaction is required if diagnostic methods are to be improved upon. Previous studies have shown that gamma delta T-cells, particularly those bearing the WC1 molecule, are present at an early stage of developing DTH responses and that such cells may modulate the developing immune response immediately following M. bovis-infection. However, their role, if any, in the DTH response remains unclear. In the present study we have used an in vivo model to deplete WC1(+) gamma delta T-cells, from cattle with established M. bovis-infection, prior to skin-testing. Results indicate that, although WC1(+) gamma delta T-cells do infiltrate the skin-test site in normal calves, they do not appear to be essential for the development of DTH skin swelling, as indicated by effective development of skin responses in calves depleted of circulating WC1(+) gamma delta T-cells.     

Canine dendritic cells from peripheral blood and lymph nodes

Canine dendritic cells were prepared from peripheral blood or lymph nodes using a series of steps including fractionation on bovine plasma albumin (BPA), irradiation with 4000 R, incubation for 16–18 hours, and refractionation on BPA. Dendritic cells were recovered in the low density (LD) fraction containing approximately 0.6% of the unfractionated cells. Measured by the incorporation of ³H-thymidine, the response of the high density (HD) cells to neuraminidase-galactose oxidase (NGO) was lower than that of the unfractionated lymph node cells (LNC) but increased in a concentration dependent manner after the addition of a population of cells enriched for dendritic cells (30–70% by morphologic criteria). Cooperation between HD- and LD- cells was not restricted to identity of the major histocompatibility complex. Canine dendritic cells also displayed stimulatory activity higher than unfractionated peripheral blood mononuclear cells (PBMC) in a one way mixed leukocyte culture (MLC). Canine dendritic cells were nonadherent to plastic, were of low density, and remained viable and functional after irradiation. For the first time, canine dendritic cells have been identified in peripheral blood and lymph nodes and have been shown to act as accessory cells in the response of lymphocytes to NGO and as stimulator cells in a MLC.     

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).     

猪IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系的建立

为了解猪体内细胞免疫应答机制,本研究建立猪细胞因子IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系.以五指山小型猪cDNA为模板,扩增IL-2、IL-4、IL-10和IFN-γ的基因保守区,构建相应的重组质粒,并将其作为标准品构建标准曲线.结果显示,各标准曲线的相关系数r值均大于0.990,扩增效率为90%～105%;该检测方法的敏感性较高,IL-4检测下限为1 copies/μL,IL-2、IL-10及IFN-γ检测下限达为102copies/μL;重复性较好,批内重复试验与批间重复试验变异系数均小于5%.本研究为病毒感染后猪体内免疫应答等研究提供方法.     

The presence of CD25 on bovine WC1+ gammadelta T cells is positively correlated with their production of IL-10 and TGF-beta, but not IFN-gamma

WC1+ cells in cattle exhibit both regulatory and effector activities. However, it has not been elucidated whether they are so plastic that both activities co-exist in one cell or there are separate subpopulations of effector and regulatory cells. Since the production of IFN-gamma and IL-10 seems to be related to WC1+ cells' effector and regulatory function, respectively, the main aim of this study was to determine whether those cytokines are produced by separate subpopulations of WC1+, or are co-produced by the same cells. Due to increasingly frequent emphasised role of consumption of IL-2 in the mechanism of suppressor action of mouse CD25+CD4+ T regulatory cells, expression of the receptor's alpha chain for interleukin 2 (CD25) on WC1+ lymphocytes has been evaluated. An average of 5.21% of WC1+ cells obtained from PBMCs of 12-month-old heifers show constitutive expression of the CD25 molecule, with CD25(high)WC1+ and CD25(low)WC1+ cells accounting for 1.05% and 4.10% of WC1+ lymphocytes, respectively. For detection of intracellular cytokine production, PBMCs were stimulated with concanavalin A. Both IFN-gamma(-) and IL-10-producing cells within the CD25(-)WC1+ and CD25+WC1+ subpopulations were mainly separate subpopulations. The average percentage of IFN-gamma(+)IL-10(-), IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ cells among CD25(-)WC1+ lymphocytes was 4.03%, 2.67% and 0.51%, respectively. A positive correlation was observed between the presence of the CD25 molecule on WC1+ lymphocytes and production of IL-10 and TGF-beta, because the average percentage of IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ among CD25+WC1+ lymphocytes was 3 and 4.5 times higher as compared to the corresponding cells in the CD25(-)WC1+ subpopulation, whereas the percentage of IFN-gamma(+)IL-10(-) cells in both the subpopulations was not significantly different. The percentage of TGF-beta+ cells within the CD25+WC1+ subpopulation was 2.72 times as high as that of CD25(-)WC1+ lymphocytes. Therefore, with respect to the production of IFN-gamma, IL-10 and TGF-beta, CD25+WC1+ lymphocytes turn out to have a more suppressor profile than CD25(-)WC1+.     

Distribution of T and B cells in thymus, blood and lymph nodes of the cat.

The proportions of T and B cells in the thymus, blood and lymph nodes were estimated in cats from three months to three years of age. T cells were identified by formation of E rosettes with guinea-pig red blood cells and B cells by the presence of surface immunoglobulin (Ig) as shown by the mixed antiglobulin reaction. Using a double test, it was shown that these methods identify separate, non-overlapping cell populations. The proportions of T cells shown in the thymus ranged from 10-73 per cent, in the blood 5-62 per cent and in the nodes, 5-44 per cent. Cells with surface Ig ranged from 0-10 per cent in the thymus, 26-68 per cent in the blood and 32-18 per cent in the nodes. Two cats with lymphadenopathy had unusually high B cell counts and one cat with depletion of the thymus was deficient in peripheral T cells. Papain treatment reduced or abolished E rosette formation by T cells.     

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.     

Effects of the depletion of CD8(+) T cells and monocytes on the proliferative responses of peripheral blood leucocytes from Trypanosoma evansi-infected sheep

Sheep peripheral blood mononuclear cells and those depleted of CD8(+) T cells and/or monocytes were stimulated with polyclonal mitogens and specific antigens, and analysed by means of cell proliferation assay procedure to examine whether these cell populations are involved in Trypanosoma evansi-induced immunosuppression. The removal of CD8(+) T cells failed to normalize the proliferative responses of peripheral blood mononuclear cells from infected sheep to concanavalin A stimulation while the depletion of monocytes resulted in full and enhanced response, showing that macrophages are mainly responsible for the suppression. Although the depletion of CD8(+) T cells, monocytes or both restored the responses of the cells to lipopolysaccharide stimulation, the responsiveness of the undepleted cells to this mitogen was significantly higher from day 24 post infection (p<0.01). The results were discussed in relation to current known mechanisms of depressed lymphocyte proliferation in tsetse-transmitted African trypanosome infections.     

Comparative distribution of immunoglobulin-containing cells in stomach, intestine and associated lymph nodes of cattle

Immunoglobulin (Ig)-containing cells were investigated immunohistochemically in the stomach, intestine and associated lymph nodes of clinically normal calves, cows and fetuses to examine mucosal immune responses in the forestomach to rumen microbial flora. IgG-containing cells were observed in the mucosal propria of the forestomach of a 32 day-old, a 37 day-old, two 90 day-old calves, and of all 5 cases of cows. However, no IgA- and IgM-containing cells were observed. Further, no Ig-containing cells were detected in the associated lymph nodes of all calves and cows. Various numbers of Ig-containing cells, predominantly IgG, were observed in the mucosal propria of the abomasum, small intestine and cecum, and in the mesenteric lymph nodes of aged calves and all cows. No Ig-containing cells were observed in any tissues of fetuses. The results suggest that the development of mucosal immune responses in the forestomach may be incomplete as compared with that of the intestine.     

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.     

CD8(+) T cell knockout mice are less susceptible to Cowdria ruminantium infection than athymic, CD4(+) T cell knockout, and normal C57BL/6 mice

The role of T cells in immunity to Cowdria ruminantium was investigated by studying the responses to infection of normal, athymic, CD4(+) T cell knock out (KO) and CD8(+) T cell KO C57BL/6 mice. Normal C57BL/6 mice could be immunized by infection and treatment, and immunity was adoptively transferable from immune to naive mice by splenocytes. Following infection, athymic mice died sooner than normal mice (P=0.0017), and could not be immunized by infection and treatment. CD4(+) T cell KO mice were as susceptible to infection as normal mice and could be immunized by infection and treatment. In contrast, CD8(+) T cell KO mice were less susceptible than normal and CD4(+) T cell KO mice and 43% self-cured, while those that died did so after a prolonged incubation period. Antibody responses to C. ruminantium were CD4(+) T cell dependent, because responses were detected in immune normal and CD8(+) T cell KO mice but not in immune CD4(+) KO mice (P=0.005). Since CD8(+) T cell KO mice were less susceptible to infection, and since CD4(+) T cell KO mice could be immunized, it can be concluded that immunity to C. ruminantium can be mediated by both CD4(+) and CD8(+) T cells.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.     

Partial protection and intrathecal invasion of CD8(+) T cells in acute canine distemper virus infection

Initial non-inflammatory demyelination in canine distemper virus infection (CDV) develops against a background of severe immunosuppression and is therefore, thought to be virus-induced. However, recently we found a marked invasion of T cells throughout the central nervous system (CNS) in dogs with acute distemper despite drastic damage to the immune system. In the present study, this apparent paradox was further investigated by immunophenotyping of lymphocytes, following experimental CDV challenge in vaccinated and non-vaccinated dogs. In contrast to CDV infected, unprotected dogs, vaccinated dogs did not become immunosuppressed and exhibited a strong antiviral immune response following challenge with virulent CDV. In unprotected dogs rapid and drastic lymphopenia was initially due to depletion of T cells. In peripheral blood, CD4(+) T cells were more sensitive and depleted earlier and for a longer time than CD8(+) cells which recovered soon. In the cerebrospinal fluid (CSF) we could observe an increase in the T cell to B cell and CD8(+) to CD4(+) ratios. Thus, partial protection of the CD8(+) cell population could explain why part of the immune function in acute distemper is preserved. As found earlier, T cells invaded the CNS parenchyma in these dogs but also in the protected challenged dogs, which did not develop any CNS disease at all. Since markers of T cell activation were upregulated in both groups of animals, this phenomenon could in part be related to non-specific penetration of activated T cells through the blood brain barrier. However, in diseased animals much larger numbers of T cells were found in the CNS than in the protected dogs, suggesting that massive invasion of T cells in the brain requires CDV expression in the CNS.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.