Figure 4.

Proliferative and immunoglobulin-secreting capacities of IgT⁺ and IgM⁺ B cells in response to microbial stimulation. (a) Proliferation of IgT⁺ and IgM⁺ B cells among trout head kidney leukocytes left unstimulated (Control) or stimulated for 1, 3 or 7 d with V. anguillarum bacterin (Vibrio), presented as frequency of cells positive for the thymidine analog EdU (5-ethynyl-2′-deoxyuridine) in the IgT⁺ or IgM⁺ B cell subset (n = 10–12 fish). (b) IgM and IgT in supernatants of cells treated as in a, assessed by immunoblot and densitometric analysis and presented relative to values on day 0 (n = 6 fish). (c) Immunoblot analysis of supernatants of cells treated as in a, probed with IgT- and IgM-specific antibodies (n = 6 fish). (d) Flow cytometry of IgT⁺ and IgM⁺ head kidney leukocytes on day 7 in the presence or absence of V. anguillarum bacterin; circles outline the predominant small or large B cell populations. FSC, forward scatter; SSC, side scatter. (e) IgM and IgT in supernatants of head kidney leukocytes collected 7 d after intraperitoneal injection of a mixture of lipopolysaccharide and V. anguillarum bacterin (n = 6 fish), sorted into large and small IgT⁺ and IgM⁺ cells and cultured for 2 d, assessed by immunoblot and densitometric analysis and presented relative to values at day 0. P values, unpaired Student’s t-test. Data are representative of three independent experiments (mean and s.e.m. in a,b,e).