Figure 3. C12Id⁺ B cells generate strong extrafollicular foci responses.

(A) Immunohistochemistry (left) and double immunofluorescence (right) was done to identify C12Id antibody-expressing cells in MedLN collected from BALB/c mice on day 10 after infection with influenza A/PR8. Immunohistochemistry identifies the accumulation of C12Id⁺ cells (red, arrows) in the medullary cords (M) but not in germinal centers (GC) (Bar = 50 μm). Double immunofluorescence shows double-positive C12Id⁺ (green cytoplasmic staining) and CD138+ positive (red staining and arrows) cells in the medulla (M) of the MedLN (Bar = 20 μm). (B) Shown are pseudocolor FACS plots depicting CD45R (B220) and C12Id-expression on MedLN B cells collected at indicated times after infection, after gating on live (propidium iodide^neg), non-dump (CD3/4/8/F4/80^neg) CD19⁺ CD45R^+/low cells (not shown). Scatter plot shows frequencies of C12Id⁺ (triangle) and C12Id^- (square) cells among CD45R^low lymph node (LN) B cells from BALB/c mice before and after infection (n = 3 - 4). Distal lymph nodes are PLN (inguinal and axillary) from day 7 and 14-infected mice. Each symbol represents data from an individual mouse. Horizontal line indicates the data mean. (C) Analysis of CD138 expression of HA-(A/PR8) specific C12Id⁺ plasmablasts in MedLN of mice infected for 7 days with influenza A/PR8 and treated in vivo with 0.25mg Brefeldin A for 6h prior to tissue collection. Shown are pseudo-color plots from a representative 5-color flow cytometric analysis of CD19⁺ C12Id⁺ B cells, identified by lack of expression for CD3, 4, 8, and F4/80 and expression of CD19 and C12Id (not shown). Virus-specific cells identified by binding to HA showed reduced expression of CD45R (B220) (upper panel, box). A subset of these cells was CD138⁺ (syndecan; lower panel). Number Indicates mean frequencies ± SD CD138⁺ cells among C12Id⁺ HA⁺ B cells from 4 individual mice. (D) Shown are pseudo-color FACS plots for gating of CD19⁺ (left panel) surface and intracytoplasmic HA-A/PR8 binding (s+ icp) (middle panel) cells that express the C12Id (right panel) in MedLN of 7 day influenza virus A/PR8-infected mice. Number in right panel indicates the mean frequency ± SD of C12Id⁺ HA-specific cells analyzed in MedLN of four individual mice.