Figure 4

Vaccination with rBCG/rAd35 resulted in induction of IL-7 as well as perforin and granulysin protein in lung and spleen from Mtb-infected animals. (A) Representative immunohistochemical images illustrate IL-7 (upper panel), perforin (middle panel) and granulysin (lower panel) expression in TB lung lesions from the rBCG/rAd35 and BCG/rAd35 vaccine groups as well as the unvaccinated control. Granulomatous areas (gr) with the presence of characteristic giant cells (GC; arrowheads) are shown in the TB lesions. Arrows indicate positive cells (brown), whereas negative cells (blue) were counterstained with hematoxylin. The expression of IL-7, perforin and granulysin were very low in the granulomatous areas. Magnification 125×. (B) Images of IL-7, perforin and granulysin expression in spleen from an rBCG/rAd35-vaccinated animal. These proteins were rarely found in the B-cell areas (Bc) of the spleen. Magnification 125×. Higher magnification (600×) shows cell-associated (high-intensity) and extracellular (low-intensity) expression of IL-7 in spleno-cytes as well as granular and polarized expression of granule-associated effector molecules in splenic lymphocytes. (C) Two-color staining demonstrates high colocalization of perforin (green; Alexa-488) and CD8⁺ T cells (red; Alexa-594) in spleen tissue. White arrows indicate double-positive cells. In situ computerized image analysis was used to assess the median expression (± IQR) of IL-7 (D), perforin (E) and granulysin protein (F) in lung lesions and spleen tissues among the different groups as indicated. The results from n = 5–6 animals/group are presented as percent positive area of the total cell area, and statistical significance of differences in protein expression was determined by a nonparametric Kruskal-Wallis test. (G) The frequency of perforin-positive cells among CD8⁺ T cells was detected by flow cytometric analysis after in vitro IL-7 short-term stimulation of PBMCs from NHPs for 6 h. Representative data from one of three animals are shown. *P < 0.05.