Article Figures & Data
Figures
- FIG. 1.
Extent of IFN-γ production in an M. bovis-free area. Young cattle between 2 and 18 months of age (n = 54) from three different herds were tested four times for IFN-γ production after stimulation of whole blood with PPD, ESAT-6, and MPB70 for 24 h. The mean response in each animal is shown as dots, while the means for each antigen stimulation are shown as lines. Corrected OD is expressed as [(mean of antigen-stimulated wells − mean control wells)/(test positive control − test negative control)].
- FIG. 2.
IFN-γ responses in 14 young calves during the first 2 months of life. Whole blood was stimulated for 24 h with MPP14, ESAT-6, and MPB70 at a concentration of 2 μg/ml and PPD at a concentration of 10 μg/ml. The responders (n = 7) are animals with an IFN-γ response (corrected OD > 0.3) at at least one time point against any of the purified antigens. Nonresponders (n = 7) never had a corrected OD of >0.3 against any of the antigens. Stimulations were made with (A and B) PPD, (C and D) MPP14, (E and F) ESAT-6, or (G and H) MPB70.
- FIG. 3.
IFN-γ production in PBMC and PBMC depleted of NK cells stimulated with mycobacterial antigens. The samples were stimulated with MPP14 (2 μg/ml), ESAT-6 (2 μg/ml), or PPD (10 μg/ml) for 24 h and the supernatant was assayed for IFN-γ production. The results are the means of cells stimulated in triplicates ± standard error of the mean. The experiment was repeated three times using PBMC from six different animals.
- FIG. 4.
Flow cytometry plots verifying IFN-γ production by NK cells. Whole blood was stimulated with MPP14 or ESAT-6 or left unstimulated for 6 h. Brefeldin A was added to stop protein export and the samples were incubated for another 12 h. PBMC was subsequently purified by density gradient centrifugation. The cells were labeled with monoclonal antibodies against A) the NK cell marker NKp46 and B) CD3, followed by permeabilization and labeling for intracellular IFN-γ; 100,000 lymphocytes were analyzed and gated for NKp46-positive and CD3+ cells, respectively. Numbers in the upper right quadrant indicate the percentages of NK cells or CD3+ cells producing IFN-γ.
- FIG. 5.
Mechanisms for IFN-γ production by NK cells. A) Intracellular flow cytometry staining for IFN-γ and surface markers in IL-2 (100 U) and IL-12 (5 U)-stimulated whole blood (100,000 gated lymphocytes); 4.9% of the NKp46-positive cells produced IFN-γ. B) IFN-γ production in IL-2-activated NK cell cultures after stimulation with IL-2 and different levels of IL-12 with or without neutralization by anti-IL-12. C) Production of IFN-γ by NK cells stimulated with supernatant from antigen-stimulated adherent cells. Supernatants from antigen-stimulated adherent cells were diluted 1:10 and incubated with or without anti-IL-12 for 1 hour before addition of NK cells. The amount of IFN-γ was measured by ELISA after 24 h of incubation.
Tables
- TABLE 1.
Increase in IFN-γ production in NK cell cultures after stimulation with supernatant from antigen-stimulated adherent cells compared to medium alonea
Calf no. Mean IFN-γ production (ng/ml) ± SEM MPP14 ESAT-6 PPD MPB70 1 1.3 ± 0.1* 2.8 ± 0.7* 4.6 ± 0.8 0.4 ± 0.3 2 2.5 ± 0.6* 1.9 ± 0.4* 1.8 ± 0.5 0.0 ± 0.5 3 5.0 ± 0.5* 4.5 ± 0.5 0.8 ± 0.4 2.8 ± 0.5 4 4.8 ± 1.1* 3.0 ± 0.8* 3.8 ± 1.1* 0.6 ± 0.2 ↵ a *, P = 0.05, Wilcoxon rank test, compared to MPB70.
