Gamma interferon production by bovine gamma delta T cells following stimulation with mycobacterial mycolylarabinogalactan peptidoglycan

Abstract

A large percentage of lymphocytes in the blood of cattle express the gamma delta T-cell receptor, but specific functions for these cells have not yet been clearly defined. There is evidence, however, that human, murine, and bovine gamma delta T cells have a role in the immune response to mycobacteria. This study investigated the ability of bovine gamma delta T cells to expand and produce gamma interferon (IFN-gamma) in response to stimulation with mycobacterial products. Bovine gamma delta T cells, isolated from the peripheral blood of healthy cattle, expanded following in vitro stimulation with live mycobacteria, mycobacterial crude cell wall extract, and Mycobacterium bovis culture filtrate proteins. In addition, purified gamma delta T cells, cocultured with purified monocytes and interleukin-2, consistently produced significant amounts of IFN-gamma in response to mycobacterial cell wall. The IFN-gamma-inducing component of the cell wall was further identified as a proteolytically resistant, non-sodium dodecyl sulfate-soluble component of the mycolylarabinogalactan peptidoglycan.

FIG. 1.

Expansion of bovine γδ T cells. In vitro expansion of bovine γδ T cells was measured following stimulation with live M. bovis and live M. avium subsp. paratuberculosis (A), mycobacterial cell wall (CW) and CFP (B), and ManLAM and IPP (C). Data are presented as the mean percent γδ T cells from four normal animals ± standard error of the mean. Statistical significance was determined by using ANOVA and least significant difference. *, P < 0.05.

FIG. 2.

Bovine γδ T cells produce significant amounts of IFN-γ in response to M. tuberculosis cell wall (M.tbCW) when cocultured with purified monocytes and IL-2. Monocytes and γδ T cells were isolated from the peripheral blood of four animals by using magnetic beads. Monocytes and γδ T cells from each individual animal were incubated with antigens or PBS for 48 h. The supernatants were then tested for the presence of IFN-γ. The data are presented as mean OD at 450 to 650 nm ± standard error for four animals. Significant IFN-γ production was determined by one-way ANOVA with the Tukey posttest. ***, P < 0.001.

FIG. 3.

IFN-γ production by γδ T cells in response to M. bovis cell wall (CW). Magnetic beads were used to isolate monocytes and γδ T cells from the peripheral blood of 10 animals. Monocytes and γδ T cells from each individual animal were incubated with M. bovis or M. tuberculosis cell wall or PBS for 48 h. The supernatants were tested for the presence of IFN-γ. Data are presented as mean OD at 450 to 650 nm ± standard error from two successive experiments with five animals per experiment. Significant IFN-γ production was determined by one-way ANOVA with the Tukey posttest. **, P < 0.01; ***, P < 0.001.

FIG. 4.

Bovine γδ T cells produce IFN-γ following culture with proteolytically digested mycobacterial cell wall (Prot.dig.CW). Monocytes and γδ T cells were isolated from the peripheral blood of 10 animals by using magnetic beads. Monocytes and γδ T cells from each individual animal were incubated with proteinase K-treated M. bovis cell wall, the SDS-soluble fraction of M. bovis cell wall, or PBS for 48 h. Supernatants were then tested for the presence of IFN-γ. Data are presented as mean OD at 450 to 650 nm ± standard error from two successive experiments with five animals per experiment. Significant IFN-γ production was determined by one-way ANOVA with the Tukey posttest. ***, P < 0.001.

FIG. 5.

IFN-γ production by γδ T cells following stimulation with mycobacterial mAGP but not with mycobacterial lipids. Monocytes and γδ T cells were isolated from the peripheral blood of five animals by using magnetic beads. Monocytes and γδ T cells were incubated with M. tuberculosis cell wall (M.tbCW), lipid extract from M. tuberculosis, M. tuberculosis mAGP, or PBS for 48 h. Supernatants were frozen and then tested for the presence of IFN-γ. Data are presented as mean OD at 450 to 650 nm ± standard error for five animals. Significant IFN-γ production was determined by one-way ANOVA with the Tukey posttest. *, P < 0.05.