Distribution and activation of T-lymphocyte subsets in tuberculous bovine lymph-node granulomas

The immune response against mycobacterial infections is dependant upon a complex interaction between T lymphocytes and macrophages in the context of the granuloma. For this study, we performed the analysis of 18 stage I or II, and 13 stage III or IV granulomas found in lymph nodes from 8 experimentally and 2 naturally infected cattle. T-cell subpopulations (CD3(+), CD4(+), CD8(+), WC1(+), CD25(+)) were investigated by immunohistochemistry. In the majority of stage I/II lesions, CD8(+) and CD25(+) cells were predominantly found in the lymphocytic outer region of the granuloma, suggesting a possible role for activated CD8(+) cells in the initial attempt to restrain the granuloma growth. CD4(+) T cells appeared equally distributed in the lymphocytic mantle and in the internal areas of the granulomas. WC1(+) cells appeared interspersed among the macrophages. We speculated that this could indicate a role for these 2 subsets in the maintenance and the maturation of the granuloma. In stage III/IV lesions, all of the T-cell subsets investigated appeared interspersed among the mononuclear component of the granulomas. In general terms, there was a higher density of CD8(+) cells compared with CD4(+) cells. However, there was no sense of rimming effect for any of the investigated cell populations.     

Horses with equine recurrent uveitis have an activated CD4+ T‐cell phenotype that can be modulated by mesenchymal stem cells in vitro

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4⁺ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4⁺ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4⁺ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4⁺ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.     

The impact of Trichinella spiralis excretory-secretory products on dendritic cells

Parasitic nematode Trichinella spiralis exert immunomodulatory effect on the host immune response through excretory–secretory products (ES L1) released from the encysted muscle larvae. Rat bone-marrow derived dendritic cells (DCs) stimulated with ES L1 antigens acquire semi-matured status and induce Th2 and regulatory responses in vitro and in vivo. Priming naïve T cells in vitro with ES L1 pulsed DCs caused strong Th2 polarization, accompanied by elevated production of regulatory cytokines IL-10 and TGF-β and no increase in the proportion of CD4⁺CD25⁺Foxp3⁺ among the effector T cell population. In vivo T cell priming resulted in mixed Th1/Th2 cytokine response, with the dominance of the Th2 type and elevated levels of regulatory cytokines. Significant increase in the proportion of CD4⁺CD25⁺Foxp3⁺ cells was found among recipient's spleen cells. We have achieved to create immune status characteristic for the live infection by in vivo application of DCs educated with ES L1 antigens.     

Characterization of immune cell infiltration into canine intracranial meningiomas

Meningiomas are the most common intracranial tumors in dogs. A variety of inflammatory cells have been shown to invade these tumors in people, but little is known about interactions between the immune system and naturally occurring brain tumors in dogs. The purpose of this study was to investigate the presence of a variety of immune cell subsets within canine intracranial meningiomas. Twenty-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry with antibodies specific for CD3, CD79a, CD18, CD11d (αD), CD45RA, forkhead box P3, and Toll-like receptors 4 and 9. Immune cell infiltration was evident in all samples, with a predominance of CD3(+) T cells. Large numbers of CD18(+) microglia and macrophages were noted surrounding and infiltrating the tumors, and a subset of these cells within the tumor appeared to be CD11d(+). Scattered macrophages at the tumor-brain interface were TLR4(+) and TLR9(+). Rare CD79a(+) B cells were noted in only a small subset of tumors. Lesser numbers of lymphocytes that were CD11d(+), CD45RA(+), or FoxP3(+) were noted in a number of the meningiomas. Although the function of these cells is not yet clear, work in other species suggests that evaluation of this immune cell infiltrate may provide important prognostic information and may be useful in the design of novel therapies.     

Effect of supplementation with an electrolyte containing a Bacillus-based direct-fed microbial on immune development in dairy calves

Immune characteristics in 65 calves were evaluated in response to a Bacillus-based direct-fed microbial (DFM) provided in electrolyte scour treatment. Blood samples were analyzed for cell surface markers and α(1)-acid glycoprotein (AGP) concentration. AGP increased in scouring calves given electrolyte containing Bacillus at day 7 post-placement compared to scouring calves administered electrolyte alone and non-scouring calves, enhancing the inflammatory response for pathogen clearance. The Bacillus promotes T cell subsets including greater proportions of activated, mature cells (CD8(-)CD25(+), CD8(-)CD45RO(+), CD8(-)TCR1(+)) in calves given electrolyte containing Bacillus than scouring calves administered electrolyte alone and non-scouring calves. Also, the Bacillus may be alleviating inflammation at day 3 post-placement as the proportion of monocytes and granulocytes lacking L-selectin (CD172a(+)CD62L(-)) was greater in scouring calves given electrolyte compared to the other groups. Electrolyte containing Bacillus administered at the onset of scours influences components of innate and adaptive immune development during and following the scouring event.     

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.     

Peripheral T lymphocyte changes in neonatal piglets: Relationship with growth hormone (GH), prolactin (PRL) and cortisol changes

Taking into account the role played by the neuroendocrine network in affecting the early development of the immune response, the present study aims to assess neonatal immunity in piglets by testing peripheral lymphocyte age-related changes in relationship to plasma levels of some relevant immunoregulatory hormones, such as growth hormone (GH), prolactin (PRL) and cortisol. For this purpose, we studied the peripheral lymphocyte age-related changes in relationship to plasma levels of GH, PRL and cortisol in conventional piglets from birth (day 0) to 41 days of age. A significant decrease was observed in the total number of lymphocytes at day 0, with a subsequent constant increment up to 41 days of age. Concomitantly, the number of T cell subsets (mainly CD8(+) cells and double positive CD4(+)CD8(+)) was low at birth, with strong increments between the 19th and 41st days of life. The CD4(+) T cell number subset was less diminished at birth than that of CD8(+), albeit with significant increments in the post-weaning period. Of interest, gammadelta T cells, which are more involved in innate immune efficiency, displayed the same trend as CD8(+) T cells from birth to the 41st day of life. From day 0 up to the 19th day, significant inverse correlations were found between T cell subsets and GH or PRL or cortisol, albeit with more significant inverse correlations with cortisol. The high levels of GH and PRL in the pre-weaning period may be due to the fact that they have to counteract the cortisol-mediated negative effect on lymphocyte production and development. These findings suggest that stress condition occurs at birth with decreases in the immune parameters, in the same way as in human newborns, with a subsequent gradual normalisation and immune development, as shown by decreased cortisol, GH and PRL normalisation and concomitant increments in T cell subsets.     

Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.     

Flow cytometric characterization of T lymphocyte subsets in the peripheral blood of Chinese rhesus macaques: normal range, age- and sex-related differences

Available data on the normal levels of white blood cell populations in healthy rhesus macaques (Macaca mulatta) originated and living in China is scanty. To obtain such data, blood samples from 150 Chinese rhesus macaques were collected and the normal range of white blood cells and their subsets were analyzed according to age and sex by flow cytometry. CBC data showed that the count of total white blood cells and lymphocytes decreased with age. Phenotypic analysis of CD4 and CD8 expression on CD3+ T lymphocytes showed that the percentage of CD4+ T cells (51.4+/-9.6%), CD4-CD8- T cells (8.5+/-4.1%) and the ratio of CD4+ T to CD8+ T cells (1.26+/-0.55) decreased with age; and the percentage of CD8+ T cells (42.0+/-9.7%), CD4+CD8+ T cells (1.3+/-0.9%) and CD3+ lymphocytes (55.3+/-13.3%) increased with age. However, no statistically significant difference was observed between the male and female groups in most parameters in these monkeys except for the percentage of CD4+CD8+ T cells. This study provided basic information about blood cell count and T lymphocyte subsets in Chinese rhesus macaques. It may be useful for comparative studies using Indian and Chinese rhesus macaques.     

Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli

γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In na?ve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in na?ve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.     

Changes in peripheral leukocyte subsets in dairy cows with inflammatory diseases after calving

To clarify the cellular immune system in dairy cows with inflammatory diseases after calving, the leukocyte subsets were examined in Holstein dairy cows. Twenty Holstein cows reared in one herd, were used in this study. Nine cows (Group 1) experienced onset of mastitis or puerperal fever within 2 weeks after calving, and the other eleven cows remained healthy (Group 2) after calving. The numbers of CD3(+), CD4(+) and CD8(+) cells tended to be lower in Group 1 than in Group 2 from the day of calving through week 1. These results suggested that the cows with inflammatory diseases might have experienced a decline in T cells by the day of calving, before the onset of disease.     

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.