Fig. 3: Autoantibody derived from the initiating clone is insufficient to break tolerance, even under permissive conditions.

A Experimental setup to evaluate the potential of the autoreactive antibody from the initiating clone (Idiotype, Id) to drive a break in tolerance in a setting deficient in T follicular regulatory cells, compared to no antibody (not injected), a pool of total normal murine IgG (mIgG), or the anti-idiotype (αId). Frequencies of B cells B, CD4 C, CD8 D T cells, GC B cells E and plasmablasts/plasma cells F in IngLN, MesLN and spleen across the four groups. G Schematic representation of assays and nomenclature for identification of Id+ (564Igi) B cells (top left), anti-idiotypic B cells (top right), idiotype antibodies (564 C11) in sera (bottom left), and anti-idiotypic antibodies (9D11) in sera (bottom right). H Idiotype B cell frequency out of total B cells across IngLN, MesLN and spleen in the four groups. I Anti-Idiotype B cell frequency out of total B cells across IngLN, MesLN and spleen in the four groups. Idiotype J and anti-idiotype K antibodies in sera at 0, 14, and 35 days. L Experimental setup to evaluate the potential of the autoreactive antibody from the initiating clone (Idiotype, Id) to drive a break in tolerance during bone marrow reconstitution, compared to no antibody (not injected). 1p = 1 part. Frequencies of B cells M, CD4 N, CD8 O T cells, GC B cells P, and plasmablasts/plasma cells Q in IngLN, MesLN and spleen across the two groups. R Idiotype antibodies (564 C11) in sera of mice presented in panels M-Q. For A–K, n = 3 mice for mIgG, Id and αId, and n = 2 mice for No Ab. For L–R, n = 4 (No Ab) and 6 (Id) mice. Bars and error bars signify mean±SD in all panels. Two-way ANOVA with Tukey’s post-test was used for comparisons of data in panels B-F and H-K. Two-way ANOVA with Šidák’s post-test was used for M–P and R, and unpaired, two-tailed t-test with Welch’s correction was used for statistical comparisons of data in Q. ns = p > 0.05.