Differential cytokine expression in T cell subsets from bovine peripheral blood

Although distinct cytokine expression in T cell subsets is well understood in mice and humans, limited information is available on bovine T cell subsets. In the present study, we analyzed the mRNA expression of 10 kinds of cytokines and CD25 expression in CD4⁺, CD8⁺, WC1⁺ and WC1^-γδ T cell subsets in bovine peripheral blood by Concanavalin A (Con A) stimulation. CD25 expression was significantly increased in CD4⁺, CD8⁺ and WC1⁺γδ T cells, but not in WC1^-γδ T cells by Con A stimulation. In CD4⁺ T cells, the mRNAs of Interleukin (IL)-2, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β and transforming growth factor (TGF)-β were expressed in control cultures, and IL-3, IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) were newly expressed when the cells were stimulated with Con A. CD8⁺ T cells expressed the mRNAs of IL-6, TNF-α, TNF-β and TGF-β in control cultures, and newly expressed those of IL-2, IFN-γ and GM-CSF, but did not express those of IL-3, IL-4 or IL-10 after Con A stimulation. The cytokine expression profile of WC1⁺γδ T cells was similar to that of CD8⁺ T cells. However, WC1^-γδ T cells did not express any cytokine mRNA except TGF-â mRNA. These results will contribute to elucidate the participation of T cell subsets in immune responses against infectious disease in cattle.     

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.     

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.     

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.     

Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ gammadelta TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor alpha chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of gammadelta T cells from birth to approximately 5 months of age. However, the absolute numbers of gammadelta T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of gammadelta T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute gammadelta T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.     

Kinetics of IL-2 receptor expression on lymphocyte subsets from goats infected with Mycobacterium avium subsp. paratuberculosis after specific in vitro stimulation

Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.     

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+.     

Molecular cloning and expression of feline CD28 and CTLA-4 cDNA

Feline CD28 and CTLA-4 (CD152) cDNA were cloned from Con-A stimulated feline peripheral blood mononuclear cells (PBMC) by rapid amplification of cDNA end-PCR (RACE-PCR). Both CD28 and CTLA-4 proteins belong to the immunoglobulin superfamily (Ig SF) and are composed of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. The open reading frame (ORF) of CD28 cDNA encoded a predicted protein of 221 amino acids and that of CTLA-4 cDNA encoded a predicted protein of 223 amino acids. The B7 ligands binding motif MYPPPY hexamer was found on the extracellular Ig V-like domains of both receptors and phosphatidylinositol 3-kinase (PI 3-kinase) binding motifs pYMNM for CD28 and pYVKM for CTLA-4 were identified in the cytoplasmic domains. Comparisons of amino acid sequences of feline proteins with known sequences of other species indicated that rabbit CD28 and CTLA-4 were most closely related and mouse molecules were the least conserved with feline molecules. Comparison of each domain of both molecules with that of other animals showed that the cytoplasmic domain of CTLA-4 was 100% conserved and that of CD28 was the most conserved domain. The cloned CD28 and CTLA-4 cDNA could be expressed in transfected mammalian cells. Expression of feline CD28 and CTLA-4 mRNA in freshly isolated feline PBMC was demonstrated by RT-PCR. Stimulation of PBMC with Con-A similarly increased the expression of both CD28 and CTLA-4 mRNA.     

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.     

干扰素α对小鼠Tfh细胞分化的调控作用

通过体外试验观察干扰素-α(IFN-α)对滤泡辅助性T细胞(Tfh)分化及功能的影响。无菌分离小鼠脾细胞,磁珠分选CD4^+T细胞,加入抗CD3单克隆抗体、抗CD28单克隆抗体,重组IL-21、IL-6进行刺激,同时加入重组IFN-α(设不加IFN-α的阴性对照)进行培养,分别在培养后的1、3、5d,流式细胞术检测Tfh细胞(CD4^+CXCR5^+)和CD4^+CXCR5^+PD-1^+细胞的比例;ELISA检测细胞上清中Tfh细胞功能性细胞因子IL-4、IL-21的水平;real-time PCR检测培养3d后Tfh细胞相关转录因子STAT1、Bcl-6以及细胞因子IL-21mRNA的表达水平。结果表明,与阴性组相比,IFN-α能明显促进Tfh细胞的分化及Tfh细胞中PD-1的表达(P<0.05),能提高Tfh细胞功能性细胞因子IL-4、IL-21的分泌水平(P<0.05);IFN-α能正向调控Tfh细胞相关转录因子STAT1、Bcl-6以及细胞因子IL-21mRNA的表达水平(P<0.05)。说明IFN-α可通过正向调控STAT1、Bcl-6的表达,促进Tfh细胞的分化和细胞因子的分泌。     

Expression of adhesion molecules on milk and blood lymphocytes from periparturient dairy cattle with Johne's disease

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.