Purification and characterisation of bovine WC1⁺ ?? T lymphocytes from peripheral blood

Abstract

-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

Enhanced secretion of interferon‐γ by bovine γδ T cells induced by coculture with Mycobacterium bovis‐infected dendritic cells: evidence for reciprocal activating signals

“…Activated human γδ T cells may also present antigen in vitro via major histocompatibility complex class II (MHC II), classically considered a feature of professional antigen‐presenting cells (APCs) 12 . Some studies suggest that γδ T cells express CD28 and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) 13 and that CD86 is involved in their activation 14 . This is supported by the finding that dendritic cells (DCs), which express high levels of CD80/86 upon infection, are more potent stimulators of γδ T cells than macrophages 13,15 .…”

“…Some studies suggest that γδ T cells express CD28 and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) 13 and that CD86 is involved in their activation 14 . This is supported by the finding that dendritic cells (DCs), which express high levels of CD80/86 upon infection, are more potent stimulators of γδ T cells than macrophages 13,15 . However, other studies suggest that bovine γδ cells may use alternative costimulatory pathways, independent of CD28 15,16 .…”

Role of anandamide transporter in regulating calcitonin gene-related peptide production and blood pressure in hypertension

“…Results were expressed as the ratio of a-CGRP or b-CGRP mRNA to GAPDH mRNA, and the value of a-CGRP or b-CGRP expression level in the WKY group was regarded as one. Isolation and treatment of lymphocytes Peripheral lymphocytes were purified by gradient centrifugation, using the density separation medium, Lymphoprep (Sigma, Saint Louis, Missouri, USA), as reported previously [21]. Purified lymphocytes were resuspended in RPMI 1640 medium (Gibco, Carlsbad, California, USA) at a density of 1.5 Â 10 6 cells/ml.…”

Effect of Dietary Administration of Bananas on Immunocytes in F1 Hybrid Calves

“…CD3 + TcR + cells are λ δ-type T-lymphocytes that are commonly observed in the juvenile period [1], and it is known that levels of these lymphocytes are low from birth in infirm calves with low resistance to disease [13]. Fikri et al [5] reported that λ δ-type T-lymphocytes produce IFNλ, which promotes the cellular immune response. It is suggested that the increase in the CD3 + TcR + cells seen in the banana group is participating in the cellular immune response.…”