Fig. 4.

Functionality and antigen-specificity of granuloma infiltrating cells (GICs). GICs were isolated as described in the Material and Methods section and analysed alongside PBMCs isolated from blood drawn on the same day from the same individual. (a) GICs were rested overnight after isolation and the IFNγ response towards the three vaccinated peptides (ADV-Hex, FLU-NCAP and EBV-GP350; Table 1) was determined by IFNγ ELISpot assay. 50,000 cells were seeded per well. HIV-A*01, HIV-B*08 and Fil-A peptides served as the relevant negative controls (rearranged wells). Ex vivo phenotype of GICs is provided as Additional File 8: Fig. S3. (b) PBMCs and GICs were harvested from the ELISpot plate (see panel A) and stained with ADV-Hex APC- and FLU-NCAP-PE- multimers. Percentages of CD8⁺ multimer-positive and multimer-negative cells within CD4^neg are indicated. (c) GICs were stimulated and expanded in vitro using anti-CD3 mAb and IL-2. The cells were then re-stimulated with the indicated peptides or with an equal volume of 10% DMSO for 12 h and the indicated secreted cytokines and surface CD107a expression (degranulation) were quantified by flow cytometry (% of functional cells are given after subtraction of marker-positive cells in the DMSO control well)