Fig 2: Markers of adaptive immunity in NEC.Intestinal tissue lysates were assayed for gene expression (open bars) by multiplex real-time PCR and protein abundance (filled bars) by multiplex ELISA or flow cytometry assessing mediators of adaptive immune type 3 (a–c), type 2 (d–h), type 1 (i–l) and regulatory (m) polarization. a, d–m One-way ANOVA or ANOVA on ranks P-values: *P < 0.05; **P < 0.01, and ***P < 0.001 for IL-37tg or WT NEC compared to dam-fed mice. #P < 0.05 for IL-37tg NEC compared to WT NEC. &&&P < 0.001 for IL-37tg dam-fed compared to WT dam-fed. a, d, e, h, i Ileal protein abundance of the indicated mediators is depicted as individual values (dots) and means (bars) normalized to total protein (t.p.). n = 3 pups for both WT and IL-37tg dam-fed, 4 for both WT and IL-37tg NEC. b, c Flow cytometric analysis for T cells on the same cells as in Fig. 4 was performed. As in Fig. 4, data are from n = 2–3 independent experiments; n = 4 mice for WT adult, 5 for IL-37tg adult, 4 for WT dam-fed, and 3 each for IL-37tg dam-fed, WT, and IL-37tg NEC. b Representative gating plot for CD4-TCRß+ROR?t+ cells, which originate from the live CD45+ lymphocyte gate; arrow indicates the source of the solid color fill in the graph. c Measurements from individual mice (dots) and means (open bars) of CD4-TCRß+ cell percentages under live CD45+ lymphocytes are shown. One-way ANOVA P-values: §§P < 0.01 for IL-37tg NEC compared to IL-37tg adult mice. ßP < 0.05 for IL-37tg NEC compared to dam-fed (see “Methods” section). Solid color fill represents the mean percentage within each bar that is ROR?t+, i.e., the ROR?t+ fraction among CD4-TCRß+ cells. Statistics for solid bars by one-way ANOVA (not shown in the figure): P < 0.001 for each of the groups of pups compared to adults. f, g, j–m Real-time PCR results were normalized to Hprt1 and the indicated genes are graphed as fold-change relative to the lowest expressed gene (see “Methods” section). Dots indicate data from individual mice, bars indicate means. n = 3 mice for WT dam-fed, 4 for IL-37tg dam-fed, 8 for both WT and IL-37tg NEC.

Fig 3: Developmental and NEC-associated changes in intestinal ILC subtypes.Lamina propria cells were isolated from the small intestine of 3 day-old pups (n = 4 for WT dam-fed and 3 each for IL-37tg dam-fed, WT and IL-37tg NEC from 3 independent experiments) and adult mice (n = 4 for WT and 5 for IL-37tg from 2 independent experiments) before being subjected to flow cytometric analysis. Cells were stained for NKp46, ROR?t, and Tbet to identify ILC1 (a, c) and ILC3 (a, b, d, e) and for KLRG1 and GATA3 to assess ILC2 (f–h). a, d, f Plots show one representative result per group; arrows indicate the source quadrants of the percentage graphs, which are calculated as a percentage under total live CD45+ cells. b, c, e, g, h Percentage of NKp46+ROR?t+ ILC3 (b), ROR?t-NKp46+Tbet+ ILC1 (c), NKp46-ROR?t+Tbet+ ILC3 (e), KLRG1+GATA3+ ILC2 (g), and KLRG1-GATA3+ ILC2 (h) is shown as data from individual mice (dots) and means (bars). One-way ANOVA or ANOVA on ranks P-values (for details, see “Methods” section): *P < 0.05; **P < 0.01 for IL-37tg or WT NEC compared to dam-fed controls. #P < 0.05; ##P < 0.01; ###P < 0.001 for IL-37tg or WT NEC or dam-fed compared to adult mice. &P < 0.05 for adult IL-37tg compared to adult WT.

Fig 4: GAS2 impairment has mild effects on normal hematopoietic cells. (A) A typical photo of genotyping analyses of +/+, flox/flox and flox/flox;Vav-iCre (conditional knockout, CKO) mice was displayed. (B) The Gas2 transcript expression of peripheral blood (PB), bone marrow (BM), and kidney cells from both CKO (n = 8) and flox/flox mice (n = 8) was analyzed by RT-qPCR. (C) The BM cells from 8 weeks old CKO mice (n = 5) and flox/flox mice (n = 5) were analyzed by flow cytometry for the expression of Mac-1, Gr-1, B220, and Ter119. (D) The absolute numbers of Mac-1+, Gr-1+, B220+, and Ter119+ cells from the bone marrow (2 femurs and 2 tibias) of CKO and flox/flox mice were compared. (E) The PB cells from CKO mice (n = 5) and flox/flox mice (n = 5) were analyzed for the expression of CD4 and CD8. The thymus cells from CKO mice (n = 5) and flox/flox mice (n = 5) were analyzed for the expression of CD4 and CD8 as well. (F) The absolute number of CD4+ and CD8+ cells in PB (100 µL) from CKO and flox/flox mice were compared (n = 5). (G) The absolute numbers of CD4+, CD8+, and CD4+CD8+ cells in the thymus from CKO and flox/flox mice were compared (n = 5). (H) A cartoon was illustrated to depict the role of GAS2 in T-cell leukemogenesis. GAS2 interacts with CXCR4 and enhances its stability. This protein complex sustains the expression of NOTCH/c-MYC and promotes the growth and invasion of T-ALL cells. GAS2 or CXCR4 silencing suppresses the expression of NOTCH/c-MYC to blockade T-cell leukemogenesis. Data were represented as the mean ± SEM, and the statistical significance was estimated with Student's t-test (*P < 0.05, ***P < 0.001. n.s., not significant).

Fig 5: SENP6 is a tumor suppressor of B-cell lymphomagenesis and is recurrently deleted in human DLBCL.a Eµ-myc CISs were analyzed using the Reactome database. Color-coded FDR q-value is shown for the top fourteen pathways. b Schematic depiction of A/R/M lymphomas with Senp6 transposon insertion and without Senp6 transposon insertion. c Transposon insertion pattern in Senp6 indicates tumor suppressor function and the number of affected tumors. Only the dominant insertion per tumor is shown. d Senp6 expression in A/R/M lymphomas with Senp6 transposon insertions (n = 10) and A/R/M lymphomas without Senp6 transposon insertion (n = 13). Senp6 expression was normalized to Ubiquitin. Data are presented as mean ± SD. P-value determined by unpaired t-test (two-tailed). e Copy number alterations (CNA) affecting SENP6 in human DLBCL (n = 304). Wildtype, n = 205; 6q14.1:DEL, n = 38; 6q:DEL, n = 61. f SENP6 mRNA levels in DLBCL groups according to their SENP6 copy number status. 6q14.1:DEL absent, n = 122; 6q14.1:DEL present, n = 15; 6q:DEL absent, n = 114; 6q:DEL present, n = 23. The centerline of the box plot is the median. The box extends from the 25th to 75th percentiles. Whisker length is from minimum to maximum. P-value determined by Wilcoxon Rank Sum Test (two-sided). g Left panel: Kaplan–Meier survival curves of mice transplanted with Eµ-myc;Rosa26Cas9 HSPCs transduced with sgRNAs targeting Senp6. Senp6, n = 5; control n = 15; p = 0.0016. P-value determined by log-rank (Mantel–Cox) test. Right panel: Kaplan–Meier survival curves of mice transplanted with Rosa26Cas9 HSPCs transduced with sgRNAs targeting Senp6. Senp6, n = 6; control n = 5. h Representative histological and immunohistochemical analysis of one of three analyzed Eµ-myc;Rosa26;Cas9Senp6-sgRNA HSPC-derived lymphomas for B220 and CD3 expression. The analysis was repeated three times for each cohort with similar results.