Figure 3.
Kinetics and magnitude of antigen-dependent cellular responses. (A) Representative results of a human IFN-γ/IL-4 dual color ELISPOT assay are presented. Human peripheral blood mononuclear cells (PBMCs, 5 × 105 cells per well) isolated from patients were stimulated with 10 μg/mL of the ScaA or TSA56 antigen in the presence of co-stimulatory anti-CD28 and anti-CD49d monoclonal antibodies at 37 °C for 18 h, and cellular spots were stained with anti-IFN-γ (brown) or IL-4 (blue) specific antibodies. Stim., stimulation; PMA/Iono, Phorbol myristate acetate (20 ng/mL)/ionomycin (1 μg/mL) as a positive control; CNT, PBMCs from the control group; ST, PBMCs from scrub typhus patients. (B) Kinetic responses of IFN-γ secretion by mononuclear cells after stimulation with the ScaA or TSA56 antigen were assessed by an ELISPOT assay as described in A. Cellular responses of PBMCs from 19 control individuals were used as a negative control (CNT), and the positive ELISPOT results (counts over the mean plus 3 × s.d. (dashed line) from 19 control samples) are indicated as black circles. The mean counts of the ELISPOT results are indicated as red lines, and statistical analysis was performed using ANOVA followed by the Newman–Keuls t-test for comparisons of the values among different groups (*P<0.05; ***P<0.001). Y0 (n=15), Y1 (n=18) and Y2 (n=11).