Fig 2: IL-33 overexpression disrupts GC function affecting an NP-KLH immunization.a Model 1 protocol outline. WT mice were immunized with NP-KLH or PBS (as a control) with ALUM adjuvant, and 7 days later, mice were HDD either with IL-33 or EV. At day 14 (7 days after the HDD), mice were harvested. Created in BioRender. Conde, E. (2025) https://BioRender.com/mfs9v6z. b GC B cells (dump-, B220 + , CD38-, IgD-, CD95 +, Gl7 +) quantification (% of B cells) or counts, NP + memory B cells counts (dump-, B220 +, CD38 +, IgD-, CD43-, NP +) and naïve B cell (dump-, B220 +, CD8 +, IgD +) counts in inguinal LN from model 1. c Model 2 protocol outline. WT mice were first HDD either with IL-33 or EV, and 4 days later immunized with NP-KLH and ALUM as adjuvant. 14 days later, mice were harvested. Created in BioRender. Conde, E. (2025) https://BioRender.com/mfs9v6z. d GC B cell counts (dump-, B220 +, CD38-, CD95 +, Gl7 +), NP+ memory B cells (dump-, B220 +, CD38 +, IgD-, CD43-, NP +) and naïve B cells (dump-, B220 +, IgD +, CD38 +) in inguinal LN from model 2. e CD4 + T cells, CD8 + T cells, Tfh cell (B220-, TCRb +, CD4 +, CD44 +, CD62L-, CXCR5 +, PD1 +) and Tfr cell (B220-, TCRb +, CD4 +, CD44 +, CD62L-, CXCR5 +, PD1 +, Foxp3 +) counts in the spleen from model 2. f Plasma cells (dump-, B220-, CD98 +, SLAMF7 +), NP specific plasma cells and non-NP specific plasma cell counts in the inguinal LN from model 2. g NP2 specific IgG1 and IgM titers, NP27 specific IgG1 and IgM titers, and ratio of NP2/NP27 IgG1 and IgM titers. The NP2/NP27 ratio indicates the overall affinity of the specific antibodies. Results show values from individual mice presented as box-and-whiskers plots where the line in the box indicates the median; the box contours indicate upper and lower quartiles, and the whiskers indicate maximum and minimum values, from n = 5 (EV groups), n = 8 (IL-33 ALUM), n = 9 (IL-33, NP-KLH) (a), n = 6 (EV NP-KH), n = 7 (IL-33 NP-KLH) (a bis), n = 7 (e), n = 8 (EV NP-KLH), n = 7 (IL-33 NP-KLH) (g) mice per group pooled from one representative experiment (b, e) or from n = 13 (IL-33 NP-KLH), n = 15 (EV-NP-KLH) pooled from two independent experiments (d, f). Adjusted P-values calculated using a two-tailed Mann-Whitney U test (for not normally distributed samples) or an Unpaired Student’s t test (for normally distributed samples).

Fig 3: IL-33 overexpression induces a break of peripheral tolerance increasing autoantibodies and worsening disease in NZB/W F1 mice, a mouse model of SLE.a anti- dsDNA Ig titers and b anti-dsDNA IgM and IgG1 titers from WT mice. IL-33 or EV HDD was performed, and mice were harvested 7 days later. In addition, prophylactic treatment with either isotype control, anti-IL-5, anti-CD4, or anti-IL-5 and anti-CD4 treatment was performed following protocol outline from Figs. 2a and 3c. c Protocol outline. NZB/W F1 mice were HDD with a low DNA dose (5ug) of either IL-33 or EV. Mice were followed for survival and bled every 2 weeks. In addition, additional mice were harvested at week 4 and the spleens were collected for analysis. Created in BioRender. Conde, E. (2025) https://BioRender.com/dynqqmc. d Survival curve. e Splenic plasma cell quantification 4 weeks after HDD. f Age-associated B cells (dump-, B220 +, CD11c +, Tbet +, CD23-, Gl7-) and MZ B cells (dump-, B220 +, CD23 +, Cd1d + high, IgM + , IgD-) quantification in the spleen 4 weeks after HDD. g Anti-dsDNA Ig found in circulation before HDD (WT and NZB/W F1 at 6 months old), and anti- dsDNA Ig found in circulation overtime after HDD. h Anti-dsDNA specific IgM and IgG1 titers overtime in NZB/W F1 mice. i ACR (albumin to creatin ratio) measured in urine 4 weeks after HDD. a, b, e, f, g, i Results show values from individual mice presented as box-and-whiskers plots where the line in the box indicates the median; the box contours indicate upper and lower quartiles, and the whiskers indicate maximum and minimum values. One representative experiment from n = 5 (EV), n = 6 (IL-33 anti CD4, IL-33 anti IL-5 anti CD4), n = 7 (IL-33, IL-33 iso) (a), n = 7 (IL-33), n = 8 (EV) (b), n = 8 (IL-33 NZB/W), n = 10 (EV NZB/W) (e, f, i), n = 20 (WT), n = 58 (NZB/W F1) (g) mice. b, e, f, g, i Adjusted P-values calculated using a two-tailed Mann-Whitney U test (for not normally distributed samples) or Unpaired Student’s t test (for normally distributed samples). a Kruskal-Wallis test followed by Dunn’s test was performed on samples that did not pass the normality test. For survival analysis in d, the Log-rank (Mantel-Cox) test was used.