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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Nat Immunol. 2009 Aug 2;10(9):1008–1017. doi: 10.1038/ni.1753

Figure 2. Nef is sufficient for macrophage-like cells to acquire class switch-inhibiting functions.

Figure 2

(a) Imaging of CellTracker-loaded IgD+ B cells (blue) co-cultured with eGFP-THP-1 or Nef-eGFP-THP-1 macrophage-like cells (Mφ, green) for 24 h. Original magnification, ×5. (b) Time course analysis of eGFP-THP-1 or Nef-eGFP-THP-1 in IgD+ B cells cultured as in a for 48 h. (c-e) QRT-PCR of Iα1-Cα1, Iα2-Cα2, AICDA and Iα-Cμ transcripts from IgD+ B cells cultured as in b, sorted and then incubated with or without CD40L and IL-10 for additional 48 h. Iα1-Cα1, Iα2-Cα2 and AICDA mRNAs were normalized to ACTB mRNA and Iα-Cμ mRNA to Iμ-Cμ mRNA. RE, relative expression. (f) ELISAs of IgG, IgA and IgM proteins from IgD+ B cells cultured as in c-e, except that the activation step was carried out for 7 days. (g) IgG, IgA and IgM proteins from IgD+ B cells incubated with supernatants from eGFP-THP-1- or Nef-eGFP-THP-1 cells in the presence or absence of CD40L and IL-10 for 7 days. (h, i) Immunohistology of HIV-1− and HIV-1+ follicles from systemic lymph nodes and intestinal Peyer's patches stained for IgD (green), AID (red), and Nef (blue or dark gray). DAPI (blue) counterstains nuclei. Original magnification, ×10. Panels a, h and i show 1 of 5 experiments yielding similar results, whereas panels b-g summarize 3 experiments (bars indicate mean s.d.; asterisk is p < 0.05). Scale bar equals 10 μm.