An Aged/Autoimmune B-cell Program Defines the Early Transformation of Extranodal Lymphomas

Abstract

A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients.

Significance: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8-11). This article is highlighted in the In This Issue feature, p. 1.

Figure 1.. Myd88 mutations increase the competitive fitness of GCB.

A-B, Flow cytometry (FC) analysis of splenic (A) B-cells or (B) GCB. C, H&E, B220 IHC and PNA IHC in consecutive splenic sections from animals treated as in (A). Scale = 100μm. D-E, GC (D) numbers or (E) individual area, based on PNA staining. Dots represent individual (D) animals or (E) GCs. Results for 5 animals per genotype. F, FC analysis of Myd88^L252P/WT and Myd88^WT/WT relative contribution to B-cells and GCB, based on CD45 allele frequencies. G, FC analysis of splenic GCB. Values represent mean ± SEM. Data reproducible with two repeats. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001, using unpaired (A,B) or paired (F,G) two-tailed Student’s t-test with the two-stage step-up method of Benjamini, Krieger and Yekutieli where applicable; or Mann-Whitney U-test (D,E).

Figure 2.. Myd88 mutations confer a proliferative advantage to GCB.

A, Geometric mean fluorescence intensity (gMFI) of Ki67 in splenic GCB. B, FC analysis of EdU incorporation by GCB. NB illustrate non-proliferating cells. C, FC analysis of Ki67+ GCB. D, FC analysis of Myd88^L252P/WT and Myd88^WT/WT relative contribution to total, DZ or LZ GCB. E, FC analysis of Ki67+ DZ and LZ GCB from (D). F, FC analysis of Ki67 expression in Ki67+ DZ and LZ GCB from (E). G, Hierarchical clustering, based on Euclidean distance, for RNA-Seq samples from sorted GCB. H-I, GSVA analysis for samples in (G), relative to canonical (H) LZ or (I) DZ GCB signatures (GSE38696). Values represent mean ± SEM. P-values calculated using unpaired (A,D) or paired (B,C,E,F) two-tailed Student’s t-test with the two-stage step-up method of Benjamini, Krieger and Yekutieli where applicable.

Figure 3.. Myd88 mutations lower the requirement for T-cell-derived co-stimulatory signals.

A-C, FC analysis of (A) CD4+ or (B) GC T_FH cells. (C) GC T_FH abundance relative to GCB in the same animals. D, Experimental scheme for E-G. E-F, FC analysis of GCB as (E) percentage of B-cells or (F) change between conditions. G, FC analysis of Ki67 expression in GCB from (E). H-I, FC analysis of iGCB with variable (H) CD40 or (I) IL-4 stimulation. J, FC analysis of splenic GCB. K, FC analysis of GCB in non-immunized mice. L, B220 and PNA IHC in consecutive sections from animals treated as in K. Scale = 100μm. M-N, GC (M) numbers or (N) individual area in naive mice. Dots represent individual (M) animals or (N) GCs. Results for 12–18 animals per genotype, from 2 experiments. O, FC analysis of Myd88^L252P/WT and Myd88^WT/WT relative contribution to B-cells and GCB. Values represent mean ± SEM. P-values calculated using unpaired (A-C,F,G,J,K) or paired (E,G,O) two-tailed Student’s t-test; or Mann-Whitney U-test (M,N); or two-way ANOVA with Tukey’s post-test (H,I).

Figure 4.. Myd88 mutations enable an aberrantly increased and pervasive GC output.

A-B, GSEA of (A) MBC or (B) PC signatures (GSE4142), against Myd88^L252P/WT LZ GCB. C-D, FC analysis of CD138+ cells (C) relative or (D) absolute abundance. E, FC analysis of the relative fraction of CD138+ cells per cell division, determined by proliferation dye dilution, in cells from (C). Results for 3 animals per genotype. F, FC analysis of (left) total or (right) YFP+ MBC. G, YFP+ MBC abundance relative to YFP+ GCB in the same animals. H, FC analysis of YFP+ MBC. I-J, FC analysis of (left) total or (right) NP-specific (I) YFP+ GCB or (J) YFP+ MBC. Values represent mean ± SEM. P-values calculated using unpaired (F,G) or paired (H-J) two-tailed Student’s t-test; or two-way ANOVA with Tukey’s post-test (C-E).

Figure 5.. MCD mutations trigger an aged/autoimmune-like program in GCBs.

A, Differentially expressed genes in Myd88^L252P/WT LZ GCB. B, Pathway analysis for genes in (A). C-D, FC analysis of (C) CD11c+ or (D) T-BET+ GCB. E, GSEA of genes with a T-BET binding motif in their promoter (HOCOMOCO v11; >90% match within −5kb:TSS:+2kb), against Myd88^L252P/WT LZ GCB. F, GSEA of T-BET-KO MBC (GSE81189), against Myd88^L252P/WT LZ GCB. G, GSEA of canonical murine AiBC signatures (GSE175365), against Myd88^L252P/WT LZ GCB. H, GSEA of Tbl1xr1 mutant GCB (GSE139059), against Myd88^L252P/WT LZ GCB. I, RNA-Seq-based expression of genes of interest (G.O.I.) in Tbl1xr1 mutant (D370Y/WT) or WT GCB (GSE139059). J, RT-qPCR validation of selected genes from (I), on independent animals. K-L, FC analysis of (K) CD11c+ or (L) T-BET+ GCB in Tbl1xr1^MUT mice. M, GSEA of T-BET-KO MBC (GSE81189), against Tbl1xr1^MUT GCB. N, GSEA of canonical murine AiBC signatures, against Tbl1xr1^MUT GCB. Values represent mean ± SEM. P-values calculated using unpaired (D,J-L) or paired (C) two-tailed Student’s t-test.

Figure 6.. MCD mutations cause a cumulative expansion of AiBC-like MBCs.

A-D, FC analysis of (A, C) CD11b+CD11c+ or (B, D) T-BET+ YFP+ MBC. E, FC analysis of CD11c+ YFP+ MBC. F, FC analysis of (left) total or (right) T-BET+CD11c+ MBC in non-immunized young animals. G, FC analysis of Ki67+ AiBC-like MBC in mice from (F). NB illustrate non-proliferating cells. H, FC analysis of splenic T-BET+CD11c+ MBC in non-immunized mice. Tumor samples are plotted but not considered for statistical analysis. I, FC analysis of CD138+ cells in animals from (H). J, BCR mutation burden in splenic B-cells. Values = mean ± SD. K, Clonality based on productive VDJ combinations. Datasets were down-sampled to a common minimum, to prevent size-based bias. L, FC analysis of T-BET and CD11b expression in activated B-cells from the spleen or extranodal tumor from an animal in (H). Values represent mean ± SEM. P-values calculated using unpaired (A-D,F,G-J) or paired (E) two-tailed Student’s t-test with the two-stage step-up method of Benjamini, Krieger and Yekutieli where applicable; or a Kruskal-Wallis test (J,K).

Figure 7.. T-BET supports the fitness of MYD88-mutant B-cells.

A, Experimental scheme for B-D. ON = Overnight; A.T. = Adoptive transfer. B, FC profiling of donor B-cell contribution to total GCB. C, FC analysis of T-BET+ donor-derived GCB. D, FC analysis of KI67 expression in donor-derived GCB. E, FC analysis of donor B-cell contribution to AiBC-like MBC. F, RNA-Seq-based TBX21 expression in primary specimens from (left) NCI (47) or (right) BCCA (48,49) cohorts. G, uMAP depiction of single-cell RNA-Seq data from EBV/HBV-negative DLBCL tumors (50). H, Relative expression of the ITGAX module among specimens in (G). I, RNA-Seq-based TBX21 expression in primary human DLBCL specimens. J, Representative images and quantification of T-BET IHC in specimens from the BCCA cohort (52). Scale = 20μm. K, Experimental scheme for (L). L, (Left) Penetrance of targeted genomic alterations, at time of cell plating. (Center) Number of detectable clonal outgrows 30 days after plating. (Right) WB-based T-BET expression in clonal outgrows. Representative blots for 2 clones per gRNA. M, Schematic representation of the proposed transformation model. Values represent mean ± SEM. P-values calculated using unpaired two-tailed Student’s t-test (B-F), or one-way ANOVA with Tukey’s post-test (I).