In vitro responsiveness of gammadelta T cells from Mycobacterium bovis-infected cattle to mycobacterial antigens: predominant involvement of WC1(+) cells

Abstract

It is generally accepted that protective immunity against tuberculosis is generated through the cell-mediated immune (CMI) system, and a greater understanding of such responses is required if better vaccines and diagnostic tests are to be developed. gammadelta T cells form a major proportion of the peripheral blood mononuclear cells (PBMC) in the ruminant system and, considering data from other species, may have a significant role in CMI responses in bovine tuberculosis. This study compared the in vitro responses of alphabeta and gammadelta T cells from Mycobacterium bovis-infected and uninfected cattle. The results showed that, following 24 h of culture of PBMC with M. bovis-derived antigens, the majority of gammadelta T cells from infected animals became highly activated (upregulation of interleukin-2R), while a lower proportion of the alphabeta T-cell population showed activation. Similar responses were evident to a lesser degree in uninfected animals. Study of the kinetics of this response showed that gammadelta T cells remained significantly activated for at least 7 days in culture, while activation of alphabeta T cells declined during that period. Subsequent analysis revealed that the majority of activated gammadelta T cells expressed WC1, a 215-kDa surface molecule which is not expressed on human or murine gammadelta T cells. Furthermore, in comparison with what was found for CD4(+) T cells, M. bovis antigen was found to induce strong cellular proliferation but relatively little gamma interferon release by purified WC1(+) gammadelta T cells. Overall, while the role of these cells in protective immunity remains unclear, their highly activated status in response to M. bovis suggests an important role in antimycobacterial immunity, and the ability of gammadelta T cells to influence other immune cell functions remains to be elucidated, particularly in relation to CMI-based diagnostic tests.

FIG. 1

Representative two-color flow-cytometric dot plots of phenotype (CD4⁺ or WC1⁺; FL1) and CD25 (IL-2R) expression (FL2) of PBMC from an individual M. bovis-infected animal following 24 h of culture with PBS (control; a and b) or MBSE (c and d). Percentages of cells with the subset phenotype activated (upper right) were calculated by dividing the percentage of CD4⁺ (or WC1⁺) CD25⁺ cells by the percentage of CD4⁺ (or WC1⁺) cells and multiplying by 100.

FIG. 2

Proportions of activated (CD25⁺) and nonactivated (CD25⁻) CD4⁺, CD8⁺, and WC1⁺ T-cell subsets within the total lymphocyte gated population as determined by FCA of uninfected (a) and M. bovis-infected (b) animals following 24 h of culture with PBS (Cont) or MBSE. Results are means ± standard errors of the means for three uninfected and three infected cattle (group 1). Experiments were repeated on five occasions. Statistical comparisons between PBS control and MBSE cultures for the proportion of activated cells within each T-cell subset are shown (∗∗, P < 0.01; ∗∗∗, P < 0.001.

FIG. 3

Kinetics of αβ and γδ T-cell activation. Shown are percentages of T-cell subsets coexpressing CD25 as determined by FCA at various times after 0 to 168 h of culture with PBS (Cont) or MBSE. Results are means ± standard errors of the means for four M. bovis-infected cattle (group 2). Statistical comparisons between control and MBSE cultures at each time point revealed the first significant differences in activation at 18 (αβ T cells; P < 0.05; a and d) and 24 h (γδ T cells; P < 0.01; b and c). Beyond 24 h, significant activation of CD4⁺ (a) cells was apparent up to 144 h (P < 0.01 or P < 0.001), significant activation of CD8⁺ cells was apparent at 48 h only (P < 0.05) (d), and significant activation of γδ T cells (TCR1⁺ and WC1⁺) was apparent at all time points (P < 0.01 or P < 0.001) (b and c).

FIG. 4

Expression of WC1 on activated and nonactivated TCR1⁺ T cells. Shown are representative flow-cytometric dot plots of TCR1 (FL1) and CD25 (IL-2R) expression (FL2) of PBMC from an M. bovis-infected animal following 24 h of culture with PBS (control) or MBSE and corresponding histograms of WC1 expression (FL3). (a) Control (PBS) CD25 versus TCR1 expression dot plot. (b and c) Histograms showing WC1 expression of R2 and R3 gated cells within the dot plot (a), respectively. (d) Dot plot of CD25 and TCR1 coexpression in response to the MBSE antigen. (e and f) Histograms of WC1 expression by cells of gate R2 (e) and gate R3 (f) within the dot plot (d).

FIG. 5

Proliferation and IFN-γ release by purified CD4⁺ or WC1⁺ T cells and APC only from M. bovis-infected animals in response to MBSE and CF antigens. CD4⁺ and WC1⁺ cells were cultured in the presence of autologous mitomycin C-treated PBMC as APC. APC alone were included as controls. (a) Lymphocyte proliferation expressed as mean net counts per minute in excess of control counts per minute (net counts per minute = antigen counts per minute − control PBS counts per minute). (b) IFN-γ release expressed as ODI (ODI of >2 was considered positive). Results are means ± standard errors of the means for four M. bovis-infected cattle (group 2). Experiments were repeated four times. Results of statistical comparisons between PBS control and MBSE- or CF-stimulated cultures for T-cell proliferation and IFN-γ production are shown (∗, P < 0.05; ∗∗∗, P < 0.001).