In this study, the ability of purified bovine gammadelta T cells in vitro to be activated by superantigens (SAg) was investigated. Freshly isolated WC1(+) gammadelta T cells, in the presence of autologous glutaraldehyde-fixed or gamma-irradiated antigen presenting cells (APC) and IL-2, were incubated with staphylococcal enterotoxins A and B (SEA and SEB), and toxic shock syndrome toxin-1 (TSST-1). Both a proliferative response and the expression of particular T cell receptor genes of the gamma variable (TCR Vgamma) repertoire family were induced. Genes encoding TCR Vgamma1 and TCR Vgamma2 family, but not TCR Vgamma5 were detected. The cells also expressed cytokine transcripts, namely, those of IL-12, IFN-gamma and TNF-alpha, but not IL-2, IL-4, IL-6, IL-7 and IL-10. The activation and proliferation of freshly isolated gammadelta T cells by non-processed antigens required two signals, one originating from the APC and a second dependent on exogenous IL-2. Our results show that purified bovine WC1(+) gammadelta T cells could be driven to proliferate and to express a particular TCRVgamma profile in response to superantigen activation. The possible implication of cytokines expressed by bovine gammadelta T cells in immunopathogenesis is discussed.
-In order to isolate and characterise resting WC1 + γδ T cells from cattle, we developed a protocol for purifying these cells by negative selection from peripheral blood. The purification method included five steps: separation of mononuclear cells on lymphoprep, depletion of monocytes by adherence to plasma-coated gelatin, enriching T cells on a nylon wool column, depleting CD2 + T cells by sheep red blood cells (SRBC), and finally depleting CD4 + and CD8 + T cells by the magnetic cell sorting technique (MACS). This procedure proved efficient and reproducible, and the purity of the isolated WC1 + γδ T cells was more than 97% as analysed by flow cytometry (FACS). Cytokines and costimulatory molecules mRNA expression was assessed by the reverse transcriptase polymerase chain reaction (RT-PCR) technique in freshly isolated resting WC1 + T cells. We found that purified uncultured WC1 + T cells express TNF-α, CD28, CTLA-4 and IL-2Rα mRNA transcripts but do not express those for IL-2, IL-4, IL-6, IL-10 and IFN-γ. The expression of CD28 and CTLA-4 transcripts on bovine WC1 + T cells indicates that these genes are evolutionarily conserved.antigen / cytokine mRNA expression / γδ T lymphocyte Résumé -Purification et caractérisation des cellules bovines T γδ WC1 + du sang périphérique. Afin de purifier et de caractériser les cellules T γδ du type WC1 + d'origine bovine, nous avons déve-loppé un protocole de purification par sélection négative de ces cellules contenues dans le sang péri-phérique. La méthode de purification comporte cinq étapes, à savoir : la séparation des cellules mononucléés sur lymphoprep, la déplétion des monocytes par adhérence sur de la gélatine couverte de plasma, l'enrichissement des cellules T sur colonne de nylon, la déplétion des cellules T du type CD2 + par formation des rosettes avec les globules rouge du mouton, et enfin la déplétion des cellules T CD4 + et CD8 + par la technique de « Magnetic cell sorting » (MACS). Le procédé s'est révélé Vet. Res. 31 (2000) 229-239 229
We have previously shown that the proliferation of freshly isolated bovine WC1gd T cells to superantigens (SAgs) including staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) or toxic shock syndrome type-1 (TSST-1) required the presence of antigen-presenting cells (APC) and the addition of exogenous interleukin (IL)-2. The costimulatory activity provided by molecules expressed on professional APC for the proliferation of gd T cells has not been addressed hitherto. In the present study, we investigated the ability of two selected APC populations, the dendritic cells (DCs) highly expressing CD80 and CD86 molecules (CD80 high CD86 high ) and the monocytes expressing the same molecules at a rather low level (CD80 low CD86 low ), to stimulate the proliferation of purified bovine WC1 gd T cells to SAgs. DCs were more efficient than monocytes in inducing gd T-cell proliferation, and this response was dependent on exogenous IL-2 in both presentation modes. Stimulating gd T cells with gradual doses of SAgs or concanavalin A (ConA) resulted in similar dose-dependent reaction profiles suggesting a minimal role of the major histocompatibility complex (MHC). However, significant proliferation was already obtained with the starting doses in the presence of DC compared with monocytes, and higher proliferation was reached with DC at optimal doses. Finally, the addition of monoclonal antibody (MoAb) anti-CD86 markedly inhibited SAgs-and ConA-mediated proliferation, whereas MoAb anti-CD80 had no effect. The combination of both anti-CD80 and anti-CD86, however, suppressed this response. These results suggest that bovine gd T-cell proliferation response requires indubitably CD86 costimulation. The role of CD80 molecule seems less clear.
Using commercially available antibodies and toxoids as templates, an ELISA, immuno-quantitative PCR (iqPCR), and multiplex immuno-PCR (iPCR) were developed for detection of Clostridium botulinum neurotoxins A and B. The obtained sensitivities for ELISA ranged from 1 ng/ml in PBS + 1% BSA to 15 and 10 ng/ml in skimmed milk for botulinum neurotoxins (BoNT)/A and BoNT/B, respectively. In semi-fat milk, the limit of detection (LOD) for both toxoids was 30 ng/ml. Quantitative immuno-PCR (iqPCR) had an LOD of 4.5-9 pg/reaction for BoNT/A in both PBS and semi-fat milk, while this was 18.5-37 pg/reaction for BoNT/B in PBS + 1% BSA and semi-fat milk, respectively. The sensitivities of ELISA and iqPCR were improved to 0.5 ng/ml and 3.75 pg/ml (0.2 pg/reaction) in semi-fat milk, respectively, when toxoid of BoNT/A was substituted with actual toxin. Multiplex iPCR with both toxoids run in the same reaction was able to distinguish presence/absence of tested BoNT/A and BoNT/B at 25 pg/reaction. The tested system offers a realistic alternative with much better sensitivity to the standard mouse assay
NKG2D is an activating lectin-like receptor that initiates natural killer (NK) cell responses against transformed tumor cells expressing its ligands, i.e., molecules related to major histocompatibility complex (MHC) class I molecules. NKG2D lacks signaling elements in its cytoplasmic domain and can deliver stimulatory signals only in association with transmembrane adaptor proteins DAP10 or DAP12. The complementary DNAs (cDNAs) encoding the bovine homologues of NKG2D and the adaptor proteins DAP10 and DAP12 were cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from resting bovine peripheral blood mononuclear cells (PBMC) and sequenced. Comparison with human, pig, and mouse sequences showed that bovine NKG2D is most similar to pig NKG2D and short mouse NKG2D (NKG2D-S). Similar to its human, mouse, and pig homologues, the cDNA for bovine DAP10 codes for a phosphatidyl-inositol-3 (PI-3) kinase-binding site (YxxM) in its cytoplasmic region. Finally, similar to its human, mouse, and pig homologues, the cDNA encoding bovine DAP12 demonstrates one tyrosine-based activated motif (ITAM) in its cytoplasmic domain. Bovine NKG2D cell surface expression was analyzed by flow cytometry on HEK 293 cells transiently transfected with cDNA expression vectors encoding COOH-terminal polyhistidine-tagged NKG2D and NH(2)-terminal Flag-tagged DAP10 and DAP12. Confirming previous findings for short mouse NKG2D-S, bovine NKG2D immunoreceptor could associate with either DAP10 or DAP12 adaptor protein for its cell surface expression.
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