IFN gamma and IL-4 production by CD4, CD8 and WC1 gamma delta TCR(+) T cells from cattle lymph nodes and blood

The synthesis of IFN gamma and IL-4 by CD4, CD8 and WC1 gamma delta TCR(+) T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFN gamma were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFN gamma synthesis was evident within both CD45RO(+) and CD45RB(+) populations, but IL-4 synthesis was predominantly by cells that were CD45RO(+)/CD45RB(-). Expression of CD62L is not related to functional memory in CD4(+) T cells from cattle and CD62L(+) cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFN gamma and IL-4. The findings indicate that at least for CD4(+) T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1(+) cells produce IFN gamma implies the presence of distinct sub-sets of this gamma delta TCR(+) population cattle and suggests a functional role.     

Bovine γδ T cells: cells with multiple functions and important roles in immunity

The γδ T-cell receptor (TCR)-positive lymphocytes are a major circulating lymphocyte population in cattle, especially in young calves. In contrast, human and mice have low levels of circulating γδ TCR(+) T cells (γδ T cells). The majority of the circulating γδ T cells in ruminants express the workshop cluster 1 (WC1) molecule and are of the phenotype WC1(+) CD2(-) CD4(-) CD8(-). WC1 is a 220000 molecular weight glycoprotein with homology to the scavenger receptor cysteine-rich (SRCR) family, closely related to CD163. The existence of 13 members in the bovine WC1 gene family has recently been demonstrated and although murine and human orthologues to WC1 genes exist, functional gene products have not been identified in species other than ruminants and pigs. Highly diverse TCRδ usage has been reported, with expanded variable genes in cattle compared to humans and mice. Differential γ chain usage is evident between populations of bovine γδ T cells, this may have implications for functionality. There is a growing body of evidence that WC1(+) γδ T cells are important in immune responses to mycobacteria and may have important roles in T cell regulation and antigen presentation. In this review, we will summarize recent observations in γδ T cell biology and the importance of γδ T cells in immune responses to mycobacterial infections in cattle.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

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.     

Characterization of CD34⁺ thymocytes in newborn dogs

Using two-color flow cytometry, we characterized CD34(+) cells in the newborn canine thymus. CD34(+) thymic cells comprised approximately 5% of cells recovered by thymus tissue teasing and both large and small thymocytes have been present in this population, the former being 7-12 times more frequent. All CD34(+) cells expressed the pan-leukocyte antigen CD45. The expression of CD44 profile on the large and small CD34(+) thymocytes differed: almost all large CD34(+) cells were CD44(+), while only 75% of small CD34(+) thymocytes co-expressed the CD44 antigen. We have previously described that CD172α is present on the surface of CD34(+) bone marrow cells in dogs. In the thymus, CD172α was expressed on 5-10% and less than 5% of large and small CD34(+) cells, respectively. Some CD34(+) thymocytes also co-expressed T-lineage-specific markers like CD3, CD4, CD8, TCR1 and TCR2. Their expression increased during the large-to-small thymocyte transition. Based on our findings we suggest that thymocyte progenitors enter their primary differentiation center as large CD34(+), CD44(+), CD45(+) and CD172α(+) cells. T-cell specific markers appear on their surface at early stages of differentiation. As the size of progenitors decreases with terminal primary differentiation, the CD34, CD44, and CD172α surface markers are down-regulated.     

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.     

Flow cytometric analysis of lymphocyte subset kinetics in Bali cattle experimentally infected with Jembrana disease virus

Jembrana disease virus (JDV) is an unusual bovine lentivirus that causes an acute and sometimes fatal disease after a short incubation period in Bali cattle (Bos javanicus). The pathological changes occur primarily in lymphoid tissues, which feature proliferating lymphoblastoid-like cells predominantly throughout parafollicular (T-cell) areas, and atrophy of follicles (B-cell) areas. Five Bali cattle were experimentally infected with JDV and all developed typical clinical signs of Jembrana disease characterised by a transient febrile response, enlargement of superficial lymph nodes and a significant leukopenia. Flow cytometric analysis of PBMC during the acute (febrile) disease phase showed that the reduced number of lymphocytes was due to a significant decrease in both the proportion and absolute numbers of CD4(+) T cells, but not CD8(+) T-cells or CD21(+) B-cells. At the end of the febrile phase, total numbers of both CD8(+) T-cells and CD21(+) B-cells increased significantly, while CD4(+) T-cell numbers remained below normal values, resulting in a significantly reduced CD4(+):CD8(+) ratio. We speculate that the persistent depletion of CD4(+) T cells following JDV infection, through lack of CD4(+) T cell help to B cells, may explain the lack of production of JDV-specific antibodies for several weeks after recovery despite an increase in CD21(+) B cell numbers. Further, our previous data showing that IgG(+) plasma cells are targets for JDV infection, correlated with our current data demonstrating an increase in CD8(+) T cell numbers, supports the suggestion that anti-viral cytotoxic T cell or other cell-mediated immune responses may be critical in the recovery process, although this remains to be formally demonstrated for JDV.     

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.     

Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens.

Postnatal development of various T-lymphocyte subpopulations expressing CD3, CD8, CD4, and antigen-specific TCR heterodimers alpha beta (TCR2) or gamma delta (TCR1) was investigated in two different inbred chicken strains, SC and TK. The ratios of jejunum T-cells expressing TCR1 to TCR2 in the intraepithelium of SC and TK strains gradually increased after hatching and were 3.40 and 4.28 by 12 weeks in TK and SC chickens respectively. The ratios of TCR1+ to TCR2(+)-cells in intraepithelium and the lamina propria in SC chickens were 0.96 and 1.23 at 8 weeks and 4.29 and 2.15 at 12 weeks, respectively. Jejunum intraepithelial lymphocytes expressing the CD8 antigen increased gradually until 4-6 weeks of age and subsequently declined as chickens aged. CD4(+)-cells represented a minor subpopulation among the intestinal lymphocyte subpopulations. Therefore, the composition of various T-cell subpopulations in the intestine depended upon host age, the regions of the gut examined and host genetic background. These results suggest that changes in T-cell subpopulations in the intestine may reflect age-related maturation of the gut-associated lymphoid tissues.     

Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches

ABSTRACT: Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4+ve, CD8+ve, γ/δ TCR+ve T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4+ve T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4+ve T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4+ve cells) and the subsequent identification of the stimulating components using tandem mass spectrometry.     

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.     

In vitro responses to purified protein derivate of caprine T lymphocytes following vaccination with live strains of Mycobacterium avium subsp paratuberculosis

Live attenuated vaccines provide protection against intestinal lesions in goats infected with Mycobacterium avium subsp. paratuberculosis. To examine the role of different T lymphocyte subsets in the development of this protective immunity, CD4(+), CD8(+) and gamma delta T cell receptor (TCR)(+) cells from peripheral blood of goat kids vaccinated with live attenuated strains of M. a. paratuberculosis were studied. After in vitro stimulation with purified protein derivate, the expression of gamma-interferon (IFN-gamma) and the activation marker interleukin-2 receptor (IL-2R) was analysed by flow cytometry. A depletion experiment was performed, where the phenotypes and IL-2R expression was studied after stimulation of cultures depleted of a T lymphocyte subpopulation. Close to all of the IFN-gamma producing cells were of the CD4(+) subset, while only a small number were CD8(+) cells. The gamma delta TCR(+) cells were highly activated, but did not produce IFN-gamma after in vitro stimulation. Depletion of CD4(+) cells lead to a decrease in the percentage of total gamma delta TCR(+) cells and gamma delta TCR(+)IL2-R(+) cells. Removing the gamma delta TCR(+) cells increased the relative numbers of CD4(+), but not the CD4(+)IL-2R(+) cells. Insight into the in vitro recall responses of T cell subsets from animals vaccinated with live paratuberculosis vaccines is essential in the development of more efficient vaccines.     

Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS)

The composition of peripheral blood leukocyte populations was studied following experimental PCV2-infection in 3-week-old piglets. Four of 10 PCV2-infected piglets developed clinical and pathological symptoms consistent with postweaning multisystemic wasting syndrome (PMWS) between 14 and 21 days post-inoculation (p.i.), and were characterised as PMWS-affected. Only these four PMWS-affected piglets, but neither the non-symptomatic infected nor control animals, developed a clear leukopenia. Kinetic analysis demonstrated a clear loss of both CD21(+) B and CD3(+) T lymphocytes in the PMWS-affected piglets. By CD3/CD4/CD8 triple labelling, the influence of PCV2 infection on all T cell sub-populations was discernible. A loss of CD3(+)CD4(+)CD8(+) memory/activated Th lymphocytes was particularly notable. However, all T lymphocyte sub-populations-CD3(+)CD4(+)CD8(+) memory Th, CD3(+)CD4(+)CD8(-) nai;ve Th, CD3(+)CD4(-)CD8(+) Tc and CD3(+)CD4(-)CD8(-) gammadelta TCR(+) lymphocytes-were susceptible to PCV2 infection-induced lymphopenia. CD3(-)CD4(-)CD8(+) NK cells were also depleted in the PMWS-affected animals, but granulocytes and monocytes were less affected. In conclusion, PCV2 infection induces primarily a lymphopenia, but only in animals which subsequently develop PMWS. The lymphopenia can be identified early p.i., particularly with the B lymphocytes. Memory/activated Th lymphocytes might be affected more than the other T cell sub-populations, but as time progressed a collapse of both T and B cell populations was clear.     

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Measurement of secreted interferon (IFN)-gamma has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-gamma assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-gamma were observed. Intradermal injection resulted in significant increases in secreted IFN-gamma for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-gamma was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-gamma was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-gamma, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.     

Peanut and Amaranthus leucocarpus lectins discriminate between memory and naive/quiescent porcine lymphocytes

Lectins are relevant tools to isolate and characterize different cellular sub-populations. In this work, we used the lectins Arachis hypogaea (Peanut agglutinin, PNA) and Amaranthus leucocarpus (ALL), specific for Galss1, 3GalNAc, to characterize naive and memory lymphocytes from pigs, experimentally infected with the porcine rubulavirus (RvP). Our results showed that both lectins recognized preferentially lymphocytes with the CD4(+)CD8(+) phenotype (P<0.05). The phenotypic analysis of the cells recognized by these lectins indicated that PNA(+) lymphocytes showed higher rate of the CD29 antigen (PNA(+)CD29(high)) than ALL(+) (ALL(+)CD29(low)). The number of PNA(+)CD29(high) lymphocytes increased after 8 weeks of experimental infection with RvP, and most of the ALL(+)CD29(low) cells became CD29(middle). PNA(+) lymphocytes isolated from infected pigs proliferated after stimulation with the RvP, whereas ALL(+) cells did not. In vitro assays indicated that the ALL(+) cells from previously infected pigs diminished from 7.5 +/- 2 to 0.5 +/- 0.3% after RvP stimulation; whereas PNA(+) cells increased from 4 +/- 1 to 42 +/- 2%, whereas no modification in ALL(+) or PNA(+) cellular population was identified in lymphocytes from naive animals after RvP stimulation. Our results suggest that the cellular distribution/organization of the O-glycosydically linked glycans on lymphocytes may correlate with biological functions, and that PNA could be a tool to isolate specifically porcine memory T cell subsets, whereas ALL could be useful to isolate naive/quiescent T lymphocytes.     

Postnatal Development of Lymphocyte Subpopulations in the Intestinal Lymph Nodes in Goats

The incidence and location of CD2⁺, CD4⁺, CD8⁺ and γ/δ T lymphocytes and IgM⁺ B lymphocytes were studied in the intestinal lymph nodes in 1-week, 1-month, 3-month and 7-month-old goats, using monoclonal antibodies and immuno-histochemical methods. The cortical area of the intestinal lymph nodes in 1-week-old animals contains only primary follicles occupied by IgM⁺ B lymphocytes and some CD2⁺CD4⁺ T lymphocytes. In goats older than 1 month, secondary follicles, that increased in number and size with age, were observed; the light zone of the germinal centre was occupied by IgM⁺ lymphocytes and some CD2⁺ and CD4⁺ T lymphocytes. In the other compartments of the lymph nodes, B lymphocytes were scarce, their number increasing with age in the medulla and diminishing in the paracortex. The numerous CD2⁺ T lymphocytes in the interfollicular area increased in number in the paracortical area of the 7-month-old goats, simultaneously with an increase in the MHC II⁺ dendritic cells and the CD4/CD8 ratio, which was greater than 1. The γ/δ T lymphocytes represented a minor subpopulation scattered through the lymph nodes.     

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3⁺CD5⁺CD6⁺ T-cells (−40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8 and CD8β lymphocytes, but was not expressed in γδ T-cells or monocyte/macrophages. The perforin positive CD3⁻ subset was phenotypically homogeneous and defined as CD5⁻CD6⁻CD8β⁻CD16⁺CD11b⁺. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3⁺) could be divided into at least three subsets. The first subset was CD4⁻CD5⁺CD6⁺CD11b⁻CD16⁻ most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8β. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4⁺CD5⁺CD6⁺CD8β⁺CD16⁻CD11b⁻ and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3⁺CD4⁻CD5⁺CD6⁻CD8β^±CD11b⁺CD16⁺, and possessed MHC-unrestricted cytotoxicity and LAK activity.