Fig 2: Effect of insulin therapy on InsB:12-20-reactive Helios- and/or Neuropilin-1 (Nrp-1)-expressing Foxp3+ Tregs. Frequencies of InsB:12-20-reactive cells within the CD4+FoxP3+Nrp1+ T cell population (A, D), CD4+FoxP3+Helios+ T cell population (B, E), and CD4+FoxP3+Helios+Nrp1+ T cell population (C, F) are shown at 15 weeks of age in PLN and pancreas of NOD mice. Female NOD mice received four weekly subcutaneous injections, from 10 until 13 weeks of age, of InsB:8-24 peptide formulated in alum (Ins+alum), InsB:8-24 peptide dissolved in buffer (Ins), alum alone (alum), or left untreated (Untr). Data presented as median with interquartile range; symbols (n = 2-6) represent individual mice. *P = 0.05; Treg, regulatory T cell; NOD, non-obese diabetic; PLN, pancreas-draining lymph nodes.

Fig 3: CCR6 expression is highest in TFR cells amongst follicular T cell populations. (A, B) Representative gating strategy for CCR6+ TFH cells (CD4+B220-CXCR5hiPD-1hiFoxp3-), TFR cells (CD4+B220-CXCR5hiPD-1hiFoxp3+), naïve CD4 T cells (CD4+B220-CD44loFoxp3-) and natural T-regulatory cells (nTreg: CD4+B220-CD44midFoxp3+Nrp-1+) 6 days after SRBC immunization. (C) Geometrical mean fluorescence intensity (gMFI) of CCR6 and, (D) percentage of CCR6+ cells within populations from (A) and (B). (E) Representative gating strategy for TFH (Foxp3-) and TFR cells (Foxp3+) within CCR6+ follicular T cells (CD4+B220-CXCR5hiPD-1hiCCR6+). (F) Ratio of TFH : TFR cells within gating strategies from (A, E). (G) Frequency of CCR6+ TFH and TFR cells at indicated time points after i.p. NP-KLH/Alum immunization. (A–C, E, F) Data representative of two independent experiments, n=4 mice ± SEM, one-way ANOVA with Tukey’s multiple comparisons test, (G) n=4-5 mice per time-point ± SEM, two-way ANOVA with Sidak’s multiple comparison test. NS, Not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 4: Nucleolin as the cell surface receptor for Dp1(A) The binding of 10 µg/mL biotin-labeled DC-targeting peptides to NRP-1 protein was detected using fluorescent-labeled streptavidin.(B) The binding of each 250 µg/mL biotin-labeled DC-targeting peptide to DC2.4 cells in the presence of 90 µg/mL anti-NRP-1 polyclonal antibody or 90 µg/mL isotype control antibody was detected using flow cytometry using fluorescent-labeled streptavidin.(C) The binding of 10 µg/mL biotin-labeled Dp1 or Dp2 to wild-type DC2.4 cells or DC2.4 cells treated with siRNA against NRP-1 were detected using flow cytometry using fluorescent-labeled streptavidin.(D) The membrane fraction of BMDCs was incubated with Dp5-conjugated beads or Dp1-conjugated beads. The bead-bound proteins were eluted using an acid solution and the eluted solution was analyzed using Lumitein staining gel after SDS-PAGE. The arrow indicates the band of nucleolin.(E) The binding of 10 µg/mL biotin-labeled Dp1×3 or Dp2×3 to recombinant nucleolin or OVA was measured using PE-labeled streptavidin.(F) The binding of 0.5 µM biotin-labeled AS1411 to recombinant nucleolin in the presence or absence of 100 µg/mL Dp1×3 or Dp2×3 were measured using PE-labeled streptavidin.(G) The binding of 10 µg/mL biotin-labeled Dp1, Dp2, Dp4, or Dp5 to DC2.4 cells in the presence of 10 µM AS1411 or CRO was detected using flow cytometry using fluorescent-labeled streptavidin.(H) Flow cytometry was used to detect the binding of 10 µg/mL biotin-labeled Dp1 or Dp5 to DC2.4 cells in the presence of 50 µg/mL MS-3 or isotype control antibody using fluorescent-labeled streptavidin.(I) The binding of 50 µg/mL biotin-labeled Dp1 to human CAL-1 cells in the presence of 10 µM AS1411 or CRO was evaluated using flow cytometry using fluorescent-labeled streptavidin.(J) The binding of 50 µg/mL biotin-labeled Dp1 to human CAL-1 cells in the presence of 50 µg/mL MS-3 or isotype control antibody was detected using flow cytometry using fluorescent-labeled streptavidin.(K) Mice were immunized with 1 µg PspA-Dp1×3 plus 5 nmol AS1411 or CRO, and the level of PspA-specific IgG in plasma was determined 20 days after the first immunization using ELISA. (A, C, E–G, and J) n = 4; (B, H, and I) n = 3; (K) n = 5. Data are means ± SD. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001 as indicated by (B, C, and H–K) Student’s t-test and (E–G) Tukey’s test. N.S.: not significant.

Fig 5: Follicular T cell populations migrate ex vivo to CCL20. (A–E) Transwell migration of splenocytes from day 6 SRBC immunized WT or Ccr6 -/- mice to increasing concentrations of CCL20. Cell populations were identified within total migrated splenocytes by flow cytometry and migration index was calculated relative to controls without chemokine. (A) Naïve CD4 T cells: CD4+B220-CXCR5-CD44loFoxp3-, (B) B cells: CD4-B220+, (C) nTregs: CD4+B220-CXCR5-CD44midFoxp3+Nrp-1+, (D) TFH cells: CD4+B220-CXCR5hiPD-1hiCD44hiFoxp3-, and (E) TFR cells: CD4+B220-CXCR5hiPD-1hiCD44hiFoxp3+. Data pooled from two independent experiments, n=3-5 mice/strain ± SEM, two-tailed unpaired Student’s t test between strains at each concentration. *p < 0.05, **p < 0.01.