IFN-γ production in T_H cells conjugated with M2-expressing B cells requires specific peptide presentation.

<p>A20 B cells stably expressing M2 or M2Y were pulsed overnight, or not, with different concentrations of OVA peptide (OVAp) and incubated with OVAp-specific CD4⁺ T cells. (A) Extracellular IFN-γ after 20h of incubation. After incubation the supernatant was recovered and analyzed by sandwich ELISA to determine IFN-γ concentration. Supernatants of three independent experiments were tested in duplicate. (B) Upper panel: percentage of T cells producing IFN-γ after 5h of conjugation in the presence of brefeldin A (BFA). Cells were fixed and stained for CD4 with anti-CD4-APC, and for IFN-γ with anti-IFN-γ-PE, and analyzed on a FACS Calibur. Lower panel: IFN-γ mean fluorescence intensity (MFI) of responding T cells. Average of three independent experiments is shown. Statistical significance of the difference between groups was evaluated by a one-tailed unpaired Student’s t test. (C) Representative FACS plots of intracellular IFN-γ production. (D) Representative confocal images of IFN-γ polarization to the contact zone. Prior to incubation B and T cells were labelled with CMFDA (green) and CMAC (blue) live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ (red), and for pTyr (yellow). Images are from one representative experiment out of three and were obtained using a Zeiss LSM 510 META microscope. (E) Quantification of conjugates with IFN-γ polarization per image. Conjugates were evaluated by confocal microscopy based on B-T_H cell contact, and IFN-γ polarization (red). 45 to 55 images were acquired per sample from three independent experiments. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.