Journal: bioRxiv

Article Title: “A negative feedback loop mediated by the NR4A family of nuclear hormone receptors restrains expansion of B cells that receive signal one in the absence of signal two”

doi: 10.1101/2020.03.31.017434

Figure Lengend Snippet: A, B . Nr4a1 +/+ and Nr4a1 -/- mice were immunized IP with either 100 μg NP-Ficoll (A) or 100 μg NP-LPS (B) and anti-NP IgG3 titers were determined via ELISA at serial time points. C, D . Nr4a1 +/+ and Nr4a1 -/- mice were immunized IP with 100 μg NP-KLH admixed 1:1 with alum and anti-NP IgG1 were determined via ELISA at serial time points. ELISA plates were coated with either NP-24-BSA (C), or NP-1-RSA (D) in order to detect total and high affinity NP-specific Abs respectively. E-G . Diagram of experimental design (E) for adoptive transfer assays depicted in (F, G). B cells were purified from splenocytes harvested from Nr4a1 +/+ B1-8 Tg CD45.1/2+ and Nr4a1 -/- B1-8 Tg CD45.2+ mice via bench-top negative selection, mixed 1:1, and loaded with CTV. 5×10 6 cells were adoptively transferred into WT or TCRα-/- host mice either 1 day prior to or same day as 100 µg NP-OVA/alum IP immunization, followed by spleen harvest on d3. F . Representative flow plots gated on donor B cells depict CTV dilution among NP-binding B cell under varied conditions. G . Adoptive transfers performed as depicted in (E) except all hosts were immunized on day of transfer (day 0 condition) and a cohort of WT recipients also received 600 μg of anti-CD40L antibody at the time of adoptive transfer. Shown is the ratio of Nr4a1 -/- to Nr4a1+/+ donor B cells among NP+ population normalized to the same ratio in the NP-population. Statistical analyses of these data are depicted in Fig S6B. Data in A-D depict N=4-5 mice /genotype, and data in G depicts N=4 recipient mice per condition. Data in G depict N=4. Data in A, C, D are representative of 2-3 independent experiments. Mean +/- SEM displayed for all graphs. Statistical significance was assessed with student’s t-test with Holm-Sidak (A-D). ****p<0.0001

Article Snippet: Goat anti-mouse IgM F(ab’)2 was from Jackson Immunoresearch; Stimulatory anti-IgD was from MD Biosciences; Murine IL-4 (Peprotech), anti-CD40 (hm40-3 clone; BD Pharmingen), recombinant murine BAFF (R&D), LPS (O26:B6; Sigma), CpG (ODN 1826; InvivoGen), anti-CD40L blocking Ab (clone MR-1, BioXcell).

Techniques: Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Purification, Selection, Binding Assay

Journal: bioRxiv

Article Title: “A negative feedback loop mediated by the NR4A family of nuclear hormone receptors restrains expansion of B cells that receive signal one in the absence of signal two”

doi: 10.1101/2020.03.31.017434

Figure Lengend Snippet: A, B . Schematic of adoptive transfer experimental design. B cells were purified from splenocytes harvested from Nr4a1 +/+ B1-8 Tg CD45.1/2+ and Nr4a1 -/- B1-8 Tg CD45.2+ mice via bench-top negative selection, mixed 1:1, and loaded with CTV. 2×10 6 -3×10 6 B cells were then adoptively transferred +/- OTII splenocytes into either WT or CD40L-/- hosts. Host mice were then immunized IP one day later with 100 µg NP-OVA/alum, followed by spleen harvest on d4. C . Representative flow plots gated on donor B cells depict CTV dilution among NP-binding B cell under varied conditions. D-F . Adoptive transfers into WT hosts were performed as described above (A, B). Mice which excess T cell help (OTII: “HI”) received donor B cells co-transferred with 2×10 6 OTII splenocytes Graphs depict CTV dilution among donor NP-binding B cells (D), expansion of NP-binding donor B cells (E), and ratio of Nr4a1 -/- relative to Nr4a1 +/+ donor NP-binding B cells normalized to the ratio in unimmunized hosts (F). N=5 unimmunized recipients, N=13 hosts that received only donor B cells, and N=6 hosts that received donor B cells mixed with excess OTII. G-I . Adoptive transfers into CD40L-/- hosts were performed as described above (A, B). Mice which received limited T cell help (OTII: “LO”) received donor B cells co-transferred co-transferred with 5×10 4 splenocytes harvested from OTII mice, and mice which received excess T cell help (OTII: “HI”) received donor B cells co-transferred co-transferred with 2×10 5 splenocytes. Graphs depict CTV dilution among donor NP-binding B cells (G), expansion of NP-binding donor B cells (H), and ratio of Nr4a1 -/- relative to Nr4a1 +/+ donor NP-binding B cells normalized to the ratio in unimmunized hosts (I). N=5 unimmunized recipitents, N= 7 hosts that received B1-8 donor B cells only, N=3 hosts that received limited T cell help, and N=9 hosts that received excess OTII T cells. J . Graph depicts correlation between ratio of Nr4a1 -/- relative to Nr4a1 +/+ donor NP-binding B cells (as plotted in F, I above) and CTV dilution of NP-binding donor B cells in individual recipients (as a proxy measure of T cell help). Mean +/- SEM displayed for all graphs. Statistical significance was assessed with one-way ANOVA with Tukey’s (D-G, I) or Sidak (H), and Pearson correlation coefficient (J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: Goat anti-mouse IgM F(ab’)2 was from Jackson Immunoresearch; Stimulatory anti-IgD was from MD Biosciences; Murine IL-4 (Peprotech), anti-CD40 (hm40-3 clone; BD Pharmingen), recombinant murine BAFF (R&D), LPS (O26:B6; Sigma), CpG (ODN 1826; InvivoGen), anti-CD40L blocking Ab (clone MR-1, BioXcell).

Techniques: Adoptive Transfer Assay, Purification, Selection, Binding Assay