Article Figures & Data
Figures
- Figure 1.
Germinal center hyperplasia and increased plasma cell pools in MBC animals. A, Schematic illustrations of the employed alleles. Exons 2 to 6 of the endogenous Myd88 locus were flanked by loxP sites (triangles). Downstream of the second loxP site, a second set of the exons 2 to 6 was inserted, harboring the L252P point mutation (asterisk). Read-through is prevented by a strong polyadenylation signal (“pA”). Human BCL2 cDNA expression is controlled by a CAGGs promoter and prevented by a lox-stop-lox cassette. GFP expression is coupled to BCL2 expression by an internal ribosomal entry site (IRES). The construct is a knock-in into the Rosa26 locus. Both alleles have been previously published (20). The Cd19Cre allele is a knock-in into the Cd19 locus and has been previously published (85). B, Exemplary axial MR images of 30-week-old animals. Spleens are outlined. C, Spleen volumes of WT (n = 5), MC (n = 5), BC (n = 7), and MBC (n ≥ 7) mice were quantified from MR images. D, IHC stainings for B220, PNA, and CD3 of splenic sections of 30-week-old WT, MC, BC, and MBC animals. E, The germinal center (GC) structures stained by PNA in splenic sections of 30-week-old WT (n = 8), MC (n = 4), BC (n = 5), and MBC animals (n = 5) were quantified. F, Splenocytes of 30-week-old WT (n ≥ 7), MC (n = 8), BC (n = 8), and MBC (n ≥ 7) were analyzed by flow cytometry, and the relative amounts of different B-cell developmental stages were quantified. Early PB, early plasmablasts; late PB/PC, late plasmablasts/plasma cells. G, Serum protein electrophoresis was performed with serum of 30-week-old WT, MC, BC, and MBC animals (n = 6 per genotype). H, Serum immunoglobulin levels of 30-week-old WT, MC, BC, and MBC animals (n ≥ 4 per genotype) were measured by ELISA. *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001; Welch unpaired two-tailed t test.
- Figure 2.
MBC animals show exaggerated immune responses to self- and foreign antigen. A, Self-reactive antibodies of the IgM isotype were visualized by a Kallestad HEp-2 assay adapted to the murine system, and mean fluorescence intensities (MFI) were quantified (n = 6 per genotype, two exemplary cases per genotype are shown). B, Quantification of the observed staining patterns. C, Autoreactive IgG immunoglobulins were visualized by an adapted Kallestad HEp-2 assay, and mean fluorescence intensity (MFI) values were quantified (WT, n = 7; MC, n = 8; BC, n = 8; MBC, n = 9; two exemplary cases per genotype are shown). D, Quantification of the observed staining patterns. E and F, WT, MC, BC, and MBC animals (n = 3) were immunized i.p. with either NP-Ficoll (50 μg) or NP-CGG (50 μg) at day 0, and the NP-specific IgM and IgG levels in the sera of animals were measured at days 0, 4, 7, 10, 21, and 40 after immunization by ELISA. Envelopes represent SEM. *, P ≤ 0.05 and **, P ≤ 0.01; Welch unpaired two-tailed t test.
- Figure 3.
Myd88 p.L252P mutation enhances the formation of the My-T–BCR supercomplex. A, Exemplary images of PLA assays for MYD88 proximity with IRAK4, IRAK1, BTK, and IgM, respectively. Blue, DAPI; red, PLA signal. Scale bar, 10 μm. B, Quantification of the data shown in A. Each dot represents the average number of PLA signals per cell and experimental well. The mean and SEM of at least three independent experiments are depicted. Significant differences between samples were calculated by Welch unpaired t test. C, Lysates were generated from CD43-depleted splenocytes from 10-week-old MC and WT. Immunoblots were generated for the indicated targets. Blots were quantified using ImageJ.
- Figure 4.
MBC animals develop ABC-DLBCL–like tumors. A, Survival curves of WT (n = 10), MC (n = 104, median 101.9 weeks), BC (n = 74, median 68.7 weeks), and MBC animals (n = 107, median 42.3 weeks). B, Quantification of the terminal phenotype of MC (n = 15), BC (n = 12) and MBC animals (n = 30). C, Exemplary illustration of hematoxylin and eosin (H/E), B220, CD138, and Ki67 stainings of MC, BC, and MBC tumors. D, BCR sequencing–based clonality analysis of WT spleens and two MBC primary tumors (“M552 tumor” and “M108 tumor”) and derived cell lines. Each circle represents a unique BCR sequence, with the circle area representing the clone size. Clones differing in one base are connected by lines to clusters. Clusters consisting of ≥10% of reads are highlighted by color, and the exact percentages are given. E, Summary of the clonalities observed in MC (n = 3), BC (n = 5), and MBC lesions (n = 25) compared with the polyclonal scenario observed in WT spleens (n = 3). F, Comparison of the IHC phenotype of two primary tumors and transplanted tumors. For transplantation, stable cell lines derived from the primary tumors M108 and M552 were injected intraperitoneally into Rag1−/− recipients. G, GSEA for ABC- and GCB-DLBCL signatures (11) on MBC (n = 4) and KBC (ref. 43; n = 6) tumors. H, Whole-exome sequencing was performed on 17 MBC tumors. Identified mutated genes (see also Supplementary Table S1) were plotted for the mutation frequencies of the orthologous human genes in two published DLBCL whole-exome sequencing datasets (9, 11). I, Cytokine levels in the sera of lymphoma-bearing MBC animals (n = 15) were measured and compared with WT levels (n = 7). Solid lines represent the mean and envelope the SD. Cytokines with significant differences between MBC and WT are highlighted in red. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001. A, Log-rank test. G and I, Welch unpaired two-tailed t test adjusted for multiple comparisons. Scale bars, 50 μm.
- Figure 5.
MBC tumors are responsive to BCL2 inhibition by venetoclax. A–C, Murine MBC and MYC cell lines as well as human ABC and GCB DLBCL cell lines were treated with increasing doses of ABT-199 (0–300 nmol/L), LY-2409881 (0–15 μmol/L), or ND-2158 (0–5 μmol/L), and cell viability was measured after 96 hours by CellTiter-Glo assay. The mean of a minimum of three independent experiments is shown, with each experiment consisting of three technical replicates. Error bars represent the SEM. D, BH3 profiling (56) of MBC, ABC, MYC, and GCB-DLBCL cell lines. Cells were exposed to either BH3 peptides or small-molecule inhibitors for 1 hour. The coloring indicates the fraction of Cytochrome C releasing (i.e., apoptotic) cells after exposure measured by flow cytometry. E–G, Apoptosis was measured by flow cytometric analysis of the Annexin V/PI double-positive population 48 hours after treatment of the cell lines M191, M108, M552, MYC-14, RYS-202, U2932, RI1, SUDHL10, and OCILY7 with 300 nmol/L ABT-199, 5 μmol/L ND-2158, or 5 μmol/L LY-2409881. H, 107 M108 cells were injected i.p. into Rag1−/− recipients. Two weeks after transplantation, animals were treated with ABT-199 (200 mg/kg daily, oral gavage), ND-2158 (150 mg/kg, i.p., daily), or LY-2409881 (100 mg/kg, i.p., daily) or were left untreated, and survival after transplantation was recorded. *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001. E–G, Welch unpaired two-tailed t test adjusted for multiple comparisons. H, Log-rank test.
- Figure 6.
α–PD-1 treatment is an effective strategy for MBC tumors. A, Expression levels of the indicated genes were compared between ABC (n = 310)- and GCB (n = 328)-DLBCL cases in a previously published RNA-seq dataset (11). B, Thirty-eight human DLBCL samples were categorized into the ABC and GCB subtypes employing the Hans algorithm (101), and PD-L1 was stained immunohistochemically. Grades 0 and 1 were classified as negative/low expression and 2 to 5 as medium/high expression. C, Expression of PD-L1 in primary central nervous system lymphoma samples was analyzed by IHC. MBC animals were monitored for lymphoma development by MRI, and upon tumor detection, treatment was initiated with either ABT-199 (200 mg/kg daily by oral gavage for 3 weeks), α−PD-1 antibody (250 μg twice weekly for 8 weeks), or a combination of both. Exemplary MRI scans 3 weeks after treatment initiation illustrated in D and E show best tumor volume change within 8 weeks of untreated (n = 4), ABT-199–treated (n = 6), α–PD-1–treated (n = 5), or combination-treated (n = 7) MBC. F, Survival after tumor detection of untreated (n = 4), ABT-199–treated (n = 6), α–PD-1–treated (n = 5), or combination-treated (n = 7) MBC animals. G, Timescale of sample collection from α–PD-1–treated MBC tumors. H, Mass cytometric analysis of untreated and α–PD-1–treated tumors. Cells were gated for CD45+DNA+ (not illustrated) and CD3+CD4+ as well as CD3+CD8+ events were selected for further analysis. The adjusted P value and log2 fold change between α–PD-1 (n = 4) and untreated samples (n = 14) for each marker (individually for the CD4+ and CD8+ populations) are depicted in I, and significant markers are highlighted. J, CD3+/CD4+ and CD3+/CD8+ population sizes are given as percentages of the DNA+/CD45+ population. K, Differentially expressed markers in the CD3+/CD4+ and CD3+/CD8+ populations. *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.0001. A, I, and K, Welch unpaired two-tailed t test. B, Fisher exact test. F, Log-rank test. Scale bars, 50 μm.
Additional Files
Supplementary Data
- Table S1 - WES Mutations Table
- Table S2 - Pharmacological Screen IC50 values
- Table S3 - BCR sequencing
- Supplementary Data - Figures S1 - S8
Volume 2, Issue 1
