Figure 7. TLR7 and cytokine stimulation induce differentiation of naïve B cells into aNAV, DN cells and PC.

A. TLR7 and IFNγ (left) but not IL-4 (right) induce aNAV, DN2 cells, and PC differentiation from HCD resting naïve (rNAV) B cells in an IL-21 dependent manner. Cells from cultures featuring IFNγ and IL-21 reproduce the in vivo phenotype of aNAV and DN2 cells with CD11c, T-bet, and FCRL5 upregulation (bottom left).

B. IL-21 is required for day 7 PC development within the IFNγ cultures from HCD rNAV cells. Similar results were obtained with SLE B cells (not shown).

C. rNAV cells from both SLE patients and HCD stimulated with TLR7 agonist and cytokines differentiate into PC, DN2 cells, and aNAV B cells with equal efficiency (representative examples; n=5).

D. Kinetics of the development of aNAV and DN2 cells from rNAV cultures from HCD (left). Frequencies of aNAV and DN2 cells in the non-PC fraction of day 7 HCD and SLE cultures (right).

E. Direct differentiation into DN2 cells starting from rNAV or aNAV in 3 day cultures using similar stimulation as before (left and middle). aNAV cells generated significantly more DN2 cells in either HCD (n=1) or SLE (n=3) than rNAV. Day 5 cultures started with aNAV cells (2 HCD and 2 SLE; right) contained significantly higher frequencies of PC (Mann Whitney test).

F. DN2 cell cultures have an equivalent ability to generate PC as SWM and DN1 cell cultures and are superior to rNAV cultures (HCD= open circles; SLE = shaded circles). Representative examples are shown for HCD and SLE (n=4). TLR7 stimulation, tested by excluding R848 was essential for DN2 cell to PC differentiation (tested in SLE only; bottom left). When normalized to cell number the IgG output of DN2 cell cultures were equivalent to SWM and DN1 cell cultures and superior to rNAV in both HCD and SLE cells (bottom right; Welch’s t-test).

G. LIPS analysis of day 7 culture supernatants from two patients with Ro, SmD, and RNP autoantibody titers.