PMC4378302_01
Histological analysis of CSF-470 vaccination site biopsy from patient #1. Hematoxylin-Eosin stained sections were examined by optical microscopy (Olympus BX40, Tokyo, Japan); pictures were acquired with Olympus Digital Camera DP72 and analyzed with Image J software. (A) Low magnification image of the granulomatous nodule distinguished a fibrosis zone (I), a highly vascularized zone (II), and a brisk-infiltrated with inflammatory cells zone (III). (B) Zone (II), in detail, showing lymphocytes and polymorphonuclear cells. (C) Zone (III) showing dense nested structures with a polynuclear cell (inset). (D) Ziehl-Neelsen staining revealed absence of BCG bacilli in the vaccine site. (E) A positive control for bacilli staining is shown (bowel tuberculosis). Bars = 2 mm (A); 50 mum (B-E).
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F1
The s.c. nodule was located 2 mm below the epidermis (Figure 1A) and three distinct areas of fibrosis, high vascularization, and brisk lymphoid infiltration could be distinguished (Figure 1AI-III). Lymphocytes and polymorphonuclear cells were distributed around vessels (Figure 1B). BCG, were observed in the highly infiltrated zone (Figure 1C). BCG was rapidly cleared from the site, since no remaining bacilli were detected by Ziehl-Nielsen staining at the vaccination site (Figures 1D,E).
Immune profiling and MART-1 distribution in CSF-470 vaccination site biopsy from patient #1. Formalin-fixed, paraffin-embedded tissue sections were stained with appropriate antibodies, amplified with avidin-biotin-peroxidase (ABC) system (Vectastain, Vector Labs), and revealed with 3,3'-diaminobenzidine. Brisk CD8+ and CD20+ lymphocyte infiltration was observed in zones (II, III), with scarce CD4+ or Foxp3+ infiltration (A-D). Expression of MART-1 Ag was observed throughout the inoculation site (E). CD11c+ cells were observed in dense nested structures (F). Quantification of the immune infiltrate (n  cells/mm2 tissue surface, mean +- SD, two determinations) (G). Bars = 50 mum. Antibodies: CD8 (clone C8/144, Dako, CA, USA), CD20 (clone L26, Dako), CD4 (clone 1F6, Novocastra, Wetzlar, Germany), Foxp3 (clone 236A/E7, Abcam, MA, USA), MART-1 (clone A103, Dako), CD11c (clone EP1347Y, Abcam).
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F2
Immune profiling analysis revealed brisk CD8+ and CD20+ lymphocyte infiltration, non-brisk CD4+, and scarce Foxp3+ T cells infiltration (Figures 2A-D,G). MART-1 antigen (Ag), derived from the vaccine, was found throughout the inflammatory focus (Figure 2E). Brisk infiltration of CD11c+ Ag-presenting cells (APCs) was also observed; these cells were either dispersed in the dermis or assembled in multiple dense nested structures (Figures 2F,G).
Analysis of CD11c+ Ag presenting cells in CSF-470 vaccination site biopsy from patient #1. Tissue sections were stained with appropriate antibodies, revealed with fluorophore-conjugated secondary antibodies, and examined by confocal microscopy (LSM 5 Zeiss Pascal, Oberkochen, Germany). Pictures were acquired with Zeiss LSM Image software and analyzed with Image J software. Both nested (A) and dispersed (B) CD11c+ cells showed some proliferating Ki-67+ cells. Most CD11c+ APCs incorporated CM Ag MART-1 [(C-D), inset]. CD8+ lymphocytes were observed surrounding APCs nests, with some T cells interacting with nested (E) and dispersed CD11c+ cells (F). Quantification of Ki-67+ and MART-1+ cells in nested and dispersed CD11c+ cells (mean +- SD, three high power fields, two determinations) (G). Bars = 20 mum. Antibodies: Ki-67 (clone MIB-1, Dako); MART-1-AF647 [clone 2A9 ]. Fluorophore-conjugated secondary antibodies (Jackson Immunoresearch).
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F3
Further analysis revealed that some CD11c+ APCs, both dispersed and nested, proliferated (Figures 3A,B,G), most had phagocytosed MART-1 Ag (Figures 3C,D,G), and some were surrounded by CD8+ lymphocytes (Figures 3E,F), suggesting local Ag presentation.