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Mammary gland mononuclear cells (MGM) were compared with peripheral blood mononuclear cells (PBM) for their ability to proliferate in response to phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), and to produce interleukin-2 (IL-2) when cultured with Con A. MGM proliferative responses to all three mitogens were at a level only just above background. Following removal of adherent cells by passage of the MGM population over Sephadex G10, their ability to proliferate increased but these responses were still significantly lower (P < 0.001) than those of PBM. Mammary secretions from non-lactating cows and colostrum, but not milk, were found to suppress the proliferative responses to PHA and Con A of PBM following a 24-hr preincubation period. There was no difference in IL-2 production in response to Con A by Sephadex G10-treated MGM and PBM, and dry-cow secretions had no effect on IL-2 production. [ABSTRACT FROM AUTHOR]

Dendritic cells were identified in afferent lymph derived by lymphatic cannulation of cattle, stained with monoclonal antibody (mAb) to the bovine workshop cluster 6 (WC6) antigen, which is highly expressed on bovine afferent lymph veiled cells, and sorted with a fluorescence-activated cell sorter. These cells expressed major histocompatibility complex (MHC) class I and II and CD1b but not CD14. They bound human and routine CTLA4-immunoglobulin (CTLA4-Ig) fusion proteins indicating expression of CD80 and or CD86. Dendritic cells induced proliferative responses in allogeneic CD4+ and CD8+ cells sorted from blood but did not induce responses in purified allogeneic WC1+, γ/δ T cells, which are CD2-, CD4-, CD8- and are the major γδ T-cell population in cattle blood, even when interleukin-2 (IL-2) was added to cultures. A WC1-, CD2+ γδ T-cell receptor (TCR)+ population predominates in cattle spleens and proliferation of a T-cell line with this phenotype was not induced by aliogeneic dendritic cells, with or without added IL-2. The observations imply that the ligand for the γδ TCR expressed on the two populations is not present on allogeneic dendritic cells or that the costimulatory molecules expressed on dendritic cells that render them highly effective at stimulating MHC class I- and class II-restricted CD8+ and CD4+ T cells are not recognized by the WC1+ or WC1- γ/δ T cells. Expression of CD28 by the four cell types was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Purified CD4+ and CD8+ cells both produced CD28 transcripts but neither purified WC1+ cells nor the WC1- γδ TCR+ cell line did so. The findings indicate that CD80 and or CD86 are involved in the stimulation of CD4+ and CD8+ αβ TCR+ T cells but not in the stimulation of either of the two γδ TCR+ populations. [ABSTRACT FROM AUTHOR]

The 180000 MW isoform of CD45 (CD45RO) has been identified in cattle with a novel monoclonal antibody (mAb) (IL-A116). This has allowed a more precise analysis of T-cell function in relation to CD45 isoform expression. Within the CD44 and CD8+ T-cell populations, CD45RO+ and CD45RO- subsets were evident. Most CD4+ and CD8- T cells that expressed the CD45RO isoform did not express the 220000 and 205000 MW isoforms recognized by mAb CC76. In contrast, the WCI +, CD2-, CD4-, CD8+, γδ T-cell receptor (TCR)+ T cells in bovine peripheral blood mononuclear cells (PBMC) were all CD45RO+. Monocytes and granulocytes were CD45RO+ but B cells were CD45RO+. Sorting experiments with CD4+ T cells from an immunized calf demonstrated that proliferative responses to ovalbumin (OVA) were entirely within the CD45RO+ subset. Following stimulation with concanavalin A (Con A) the CD45RO- subset of CD4+ T cells produced transcripts for interleukin-2 (IL-2) but not IL-4 or interferon-γ (IFN-γ), while the CD45R0+ subset produced mRNA for IL-2, IL-4 and IFN-γ. Biologically active IL-2 was present in supernatants from both CD45RO+ and CD45RO-, CD4+ T cells, and IFN-γ protein was identified by ELISA in supernatants from the CD45RO+ subset, confirming the production of cytokines implied by polymerase chain reaction (PCR). In contrast, sorting experiments with CD8+ T cells from animals immune to the protozoan parasite Theileriaparva revealed substantial numbers of cytotoxic T-lymphocyte precursors in both the CD45RO+ and CD45RO- subsets. Thus it appears that although all antigenically primed CD4+ T cells remain CD45RO+, and expression of this molecule consequently identifies memory cells within PBMC, antigenically primed CD8+ T cells down-regulate CD45RO expression after activation. [ABSTRACT FROM AUTHOR]

Dendritic cells were identified in afferent lymph derived by lymphatic cannulation of cattle, stained with monoclonal antibody (mAb) to the bovine workshop cluster 6 (WC6) antigen, which is highly expressed on bovine afferent lymph veiled cells, and sorted with a fluorescence-activated cell sorter. These cells expressed major histocompatibility complex (MHC) class I and II and CD1b but not CD14. They bound human and murine CTLA4-immunoglobulin (CTLA4-Ig) fusion proteins indicating expression of CD80 and or CD86. Dendritic cells induced proliferative responses in allogeneic CD4+ and CD8+ cells sorted from blood but did not induce responses in purified allogeneic WC1+, gamma/delta T cells, which are CD2-, CD4-, CD8- and are the major gamma delta T-cell population in cattle blood, even when interleukin-2 (IL-2) was added to cultures. A WC1-, CD2+ gamma delta T-cell receptor (TCR)+ population predominates in cattle spleens and proliferation of a T-cell line with this phenotype was not induced by allogeneic dendritic cells, with or without added IL-2. The observations imply that the ligand for the gamma delta TCR expressed on the two populations is not present on allogeneic dendritic cells or that the costimulatory molecules expressed on dendritic cells that render them highly effective at stimulating MHC class I- and class II-restricted CD8+ and CD4+ T cells are not recognized by the WC1+ or WC1- gamma/delta T cells. Expression of CD28 by the four cell types was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Purified CD4+ and CD8+ cells both produced CD28 transcripts but neither purified WC1+ cells nor the WC1- gamma delta TCR+ cell line did so. The findings indicate that CD80 and or CD86 are involved in the stimulation of CD4+ and CD8+ alpha beta TCR+ T cells but not in the stimulation of either of the two gamma delta TCR+ populations.

The study of bovine cytokines and lymphocyte surface markers has been restricted by the lack of stable, long-lived, easily maintained cell lines. To address this problem, bovine mononuclear leucocytes were fused with the mouse thymoma cell line BW5147 to produce interspecies hybrid cell lines and clones. Hybridomas were produced which contained more than 40 chromosomes and expressed one or more bovine leucocyte surface markers, they grew well and were cloned without the use of exogenous factors. Fusion rates were improved when exogenous factors, including recombinant human interleukin-2 (rhIL-2), were added. Some hybrids secreted immunoregulatory factors able to maintain the growth of either an IL-2-dependent bovine T-cell line (C625) and/or preactivated normal bovine B cells. Anti-human IL-2 partially blocked the T-cell growth factor (TCGF) activity produced by these hybrids. The release of growth factors by the hybrid clones 5/AA6/16 and 5/AA6/19 was augmented by concanavalin A stimulation.