Fig 2: GD2+ NXS2 neuroblastoma growth after allogeneic HSCT and hu14.18-IL2. (A) Lethally irradiated CD45.2+ B6AJF1 mice (H-2b x a) were transplanted with either CD3e cell depleted (T cell depleted) or CD3e depleted BM replenished with 2.5 x 103 T cells (T cell replete) from congenic CD45.1+ B6 mice (H-2b) on Day +0. On Day +10, 2 x 106 NXS2 tumor cells were inoculated into the right flank. On days +14-16, PBS (No IC) or hu14.18-IL-2 (IC) was administered and (B) tumor growth was monitored by using a digital caliper as well as (C) clinical GVHD scores. N=5 mice/group. ** p = 0.01.

Fig 3: Loss of miR-146a causes spontaneous accumulation of Tfh and GC B cells.(a–d) Flow cytometric contour plots and dot plots showing percentages and absolute cell number of follicular T cells gated as CXCR5high PD-1high cells (a,b) or BCL-6high PD-1high cells (c,d). (e) Gating strategy for Tfh (CXCR5high PD-1high Foxp3-) and Tfr (CXCR5high PD-1high Foxp3+) cells and dot plots showing their percentages. (f) Percentages of Th1 (IFN-?+), Th2 (IL-4+), Th17 (IL-17+), Treg (Foxp3+) and Tfh (CXCR5high PD-1high Foxp3-) cells among total CD4+ cells. (g,h) Flow cytometric contour plots and dot plots showing percentages and absolute cell numbers of GC B cells. Contour plots shown in a,c,e are gated on CD4+ cells, those in g are gated on B220+ cells. Unimmunized miR-146a-deficient (-/-) and -sufficient (+/+) mice were analyzed. Each symbol represents one mouse and the horizontal bars represent the median values. Data are representative of at least three independent experiments. Statistical significance was determined using Mann–Whitney U-test. NS, not significant, *P<0.05, **P<0.01.

Fig 4: Contribution of IL-6 signalling to the Tfh and GC B-cell accumulation in miR-146a-deficient mice.(a–c) Similar expression of Il6 mRNA between miR-146a-deficient (-/-) and -sufficient (+/+) cells in SRBC-immunized Ly5a+.Mir146a+/+: Ly5b+.Mir146a-/- mixed bone marrow chimeras analyzed 14 weeks post reconstitution. (a) Flow cytometric contour plots showing the gating strategy used to isolate the different cell subsets. (b) Relative amounts of Il6 mRNA in GC B (B220+ GL-7+ Fas+), non-GC B (B220+ GL-7- Fas-), Tfh (CD4+ CXCR5high PD-1high), and CD11b+ cells from Ly5a+ and Ly5b+ cells analyzed directly after isolation. ND=not detectable. The expression of Il6 was normalized to ß-actin. (c) Relative amounts of Il6 mRNA in LPS-stimulated CD11b+ cells from the same set of chimeric mice as in b. (d,e) No increased expression of IL-6 in miR-146a-deficient follicular dendritic cells. (d) Flow cytometric contour plots showing the gating strategy used to isolate follicular dendritic cells. (e) Relative amounts of Il6 mRNA in gp38+ and gp38- FDCs (CD45- CD31- CD35+) from Mir146a+/+ or Mir146a-/- mice. (f) Histograms and dot plots showing the amounts of STAT3 phosphorylation in naïve and effector CD4+ T cells following acute stimulation with recombinant mouse IL-6. Data are representative of at least two independent experiments. Heights of bar graph in b,c,e represent mean and error bar represents s.d. Each symbol in f represents one mouse and the horizontal bars represent the median values. Statistical significance was determined using unpaired Student’s t-test (e) or Mann–Whitney U-test (f). NS=not significant, *P<0.05, **P<0.01.

Fig 5: Cell-autonomous accumulation of miR-146a-deficient Tfh cells.(a) Flow cytometric contour plots showing the gating strategy for follicular T cells in 50:50 mixed bone marrow chimeras gated on all CD4+ (top), Ly5a+ CD4+ (middle) and Ly5b+ CD4+ (bottom) cells. (b) Percentages of Tfh, Tfr, non-follicular effector and non-follicular Tregs in the mixed chimeras. (c,d) Flow cytometric contour plots and dot plot showing the percentages of GC B cells in the mixed chimeras. Contour plots in c are gated on Ly5a+ B220+ and Ly5b+ B220+ cells as indicated. Connecting lines between percentages of Ly5a+ and Ly5b+ in b and d indicate that they are from the same chimeric mouse. Chimeras were analyzed 16 weeks post reconstitution. (e) Percentages of OVA-specific (Vß5+) Tfh cells and total GC B cells in Cd28-/- recipient mice 7 days following adoptive transfer. Data are representative of three independent experiments. (f,g) Adoptive transfer of purified CD4+ T cells and B cells from Mir146a+/+ or Mir146a-/- donor mice into Rag1-/- recipients according to the strategy shown in f. The recipient mice were immunized with sheep red blood cells 14 days after transfer and they were killed 7 days post immunization. (g) Percentages of Tfh and GC B cells in Rag1-/- recipient mice following adoptive transfer and SRBC immunization. Each symbol represents one mouse and the horizontal bars represent the median values. Statistical significance was determined using a paired Student’s t-test (b,d) or Mann–Whitney U-test (e,g). NS, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.