In vitro detection of antigen specific T lymphocyte subsets in cattle

Mitogen and specific antigen activation of bovine CD3+, CD4+, CD8+, and gamma delta T cell receptor+ T cells and B lymphocytes was examined. Lymphocyte activation was detected by measuring increased surface expression of activation markers (Interleukin-2 receptor (IL2r) and major histocompatibility complex class II molecules (MHC II)). Two color flow cytometry was used to simultaneously identify lymphocyte subsets and detect activation marker expression. For mitogen stimulated lymphocyte subsets, IL2r and MHC II expression was compared to subset proliferation as measured directly by flow cytometric analysis of the fluorescent cell membrane stain PKH2. Stimulation by the mitogens Concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and anti-CD3 monoclonal antibody (MAb) caused increased expression of IL2r and MHC II as well as proliferation of CD4+ and CD8+ T cells. CD3+ T cells increased IL2r and MHC II expression in response to all four mitogens, and proliferated in response to Con A, PHA, and anti-CD3 MAb. Gamma delta T cells responded to all four mitogens with increased IL2r and MHC II expression, but only Con A and PHA caused detectable proliferation of gamma delta T cells. B lymphocytes generally responded to all four mitogens with increased IL2r and MHC II expression, but only Con A induced measurable B cell proliferation. Peripheral blood mononuclear cells (PBMC) of cattle hyperimmunized with bovine viral diarrhea virus (BVDV) were incubated in vitro for six days with or without live BVDV. CD3+, CD4+, CD8+, and gamma delta T cells from hyperimmunized cattle became activated and increased IL2r expression when incubated with BVDV as compared to cells incubated without virus. Lymphocytes from BVDV negative control cattle did not increase IL2r expression when incubated in vitro with BVDV. PBMC of cattle immunized with a vaccine containing modified-live bovine herpesvirus 1 (BHV1) were incubated in vitro for six days with or without heat-inactivated BHV1. CD3+, CD4+, and gamma delta T cells were activated and increased expression of IL2r when incubated with BHV1 antigen as compared to cells incubated without virus. CD8+ T cells of vaccinated animals did not increase IL2r expression when incubated with inactivated BHV1. Lymphocytes from non-vaccinated BHV1-negative animals did not increase IL2r expression when incubated with inactivated BHV1