Immunization of cattle by infection with Cowdria ruminantium elicits T lymphocytes that recognize autologous, infected endothelial cells and monocytes

Abstract

Peripheral blood mononuclear cells (PBMC) from immune cattle proliferate in the presence of autologous Cowdria ruminantium-infected endothelial cells and monocytes. Endothelial cells required treatment with T-cell growth factors to induce class II major histocompatibility complex expression prior to infection and use as stimulators. Proliferative responses to both infected autologous endothelial cells and monocytes were characterized by expansion of a mixture of CD4+, CD8+, and gammadelta T cells. However, gammadelta T cells dominated following several restimulations. Reverse transcription-PCR analysis of cytokine expression by C. ruminantium-specific T-cell lines and immune PBMC revealed weak interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) transcripts at 3 to 24 h after stimulation. Strong expression of IFN-gamma, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and IL-2 receptor alpha-chain mRNA was detected in T-cell lines 48 h after antigen stimulation. Supernatants from these T-cell cultures contained IFN-gamma protein. Our findings suggest that in immune cattle a C. ruminantium-specific T-cell response is induced and that infected endothelial cells and monocytes may present C. ruminantium antigens to specific T lymphocytes in vivo during infection and thereby play a role in induction of protective immune responses to the pathogen.

FIG. 1

In vitro response of bovine PBMC to autologous C. ruminantium-infected endothelial cells. (A) Responses to whole C. ruminantium EB antigen. MED, medium. (B) Responses to irradiated TCGF-treated C. ruminantium-infected endothelial cells. (C) Responses to glutaraldehyde-fixed TCGF-treated C. ruminantium-infected endothelial cells. Values are mean counts per minute (± standard error) of triplicate cultures.

FIG. 2

In vitro response of bovine PBMC to autologous glutaraldehyde-fixed C. ruminantium-infected monocytes. Values are mean counts per minute (± standard error) of triplicate cultures.

FIG. 3

Kinetic analysis of cytokine profiles of immune PBMC and T-cell lines by RT-PCR. Total RNA was prepared from PBMC stimulated with either autologous C. ruminantium-infected EC or monocytes (MΦ) for 3 and 6 h. Similarly, T-cell lines (TC) stimulated with either autologous C. ruminantium-infected EC or monocytes for 6 and 24 h were used for RNA preparation to evaluate IFN-γ, IL-4, and IL-2 expression. Normal PBMC stimulated with ConA for 6 h (ConA Blasts) served as a positive control. Marker sizes are in base pairs.

FIG. 4

RT-PCR analysis of cytokine mRNA in T-cell lines stimulated for 48 h. (A) Stimulation with autologous C. ruminantium-infected monocytes (MΦ). Lanes 2, 3, and 4 contain primers for G3PDH, IL-1β, and IL-4, while lanes 5, 6, and 7 contain primers for G3PDH, IL-2Rα, IL-10, IL-2, IFN-γ, TNF-α, and TNF-β. (B) Stimulation with autologous C. ruminantium-infected EC. F100 ConA blasts and PCR mixture with no template were used as positive and negative controls, respectively. Marker sizes (M) are in base pairs.