Figure 4
Increase in NCR1+/CD16+ lymphocyte percentage and perforin+ cell percentage in the jejunum during infection. (A) Cells labelled with anti NCR1 and CD16 mAbs and gated on the lymphocyte gate (shown in Figure 1I) were analyzed by flow cytometry in spleen, mesenteric lymph nodes (MLN), jejunal Peyer’s patches (JPP) and jejunum. The quadrants were set according to the labelling obtained with the isotype controls for the same number of cells analyzed. Percentages indicated in black represent the CD16-/NCR1+ and CD16+/NCR1+ lymphocyte percentages of which the non-specific labelling was subtracted. Percentages of CD16+ cells among NCR1+ lymphocytes are indicated in green. Data are from an inoculated lamb at 6 dpi and its control. (B) Individual CD16+ percentages of NCR1+ lymphocytes are indicated for animals 2–6 dpi, control lambs (open symbols), inoculated lambs (filled symbols). The red bars indicate the mean values. (C) The points represent the inoculated/control ratio of NCR1+/CD16+ lymphocytes for each pair of age-matched lambs. (D-E) NCR1 and CD16 double labelled cells were permeabilized for intracellular labelling with an anti-human perforin mAb or isotype control to determine the perforin+ cell percentage in both NCR1+ subpopulations by histogram subtraction at 6 dpi. Control lambs (open symbols), inoculated lambs (filled symbols). (E) The data shown are representative of the lambs whose individual percentages of perforin+ cells are shown in Figure 4D. The red and black line limited histograms correspond to the inoculated lamb (In) and its matched control (Co) respectively, the gray filled histogram to the isotype control of the perforin mAb (iso); the mean fluorescence intensity (MFI) is indicated for each histogram. The comparisons were made with the Mann–Whitney (control/inoculated) and Wilcoxon tests (paired data). Significant difference probability: * p < 0.05, ** p < 0.01, *** p < 0.001. The inoculated/control CD16+/NCR1+ lymphocyte ratio was calculated for each pair of lambs (C); the mean was significantly superior to 1 with confidence interval of 1.4-2.82 for JPPs and 1.48-2.32 for jejunum (p < 0.01).