CLL and its precursor, monoclonal B-cell lymphocytosis (MBL), are associated with immune deficits. In MBL, the higher percentages of normal B cells (NBC) provide a unique opportunity to study B-cell defects. We compared transcriptomes of NBC from healthy donors (HD) and people with high-count MBL (hcMBL) with mutated (M-MBL) and unmutated (U-MBL) IGHV, and with low-count MBL (lcMBL). We also explored B-cell subset distribution and BCR repertoires.

PBMC were collected from 21 hcMBL (12 M-MBL, 9 U-MBL), 7 lcMBL (1 M-MBL, the rest unknown), and 13 HD. 111 sorted B-cell samples were obtained: 21 hcMBL clones (CD20LowCD5+Igκ+/Igλ+), 36 NBC-hcMBL (30 CD20+CD5⁻; 6 CD20+CD5⁺), 12 NBC-lcMBL (CD20+CD5⁻), and 42 NBC-HD (25 CD20+CD5⁻; 17 CD20+CD5⁺). RNA was sequenced using SMART-Seqv4 on HiSeq. Differentially expressed genes (DEG) were analyzed with DESeq2 (Padj<0.05, |FC|≥1.5). Transcriptome variation was assessed by PCA and enriched pathways by IPA. Flow cytometry validated DEG in 6 hcMBL and 8 HD. B-cell subset distribution was analyzed in 14 hcMBL (10 M-MBL, 4 U-MBL) and 9 HD using FACSymphony. BCR repertoire analysis was performed on NBCs of 4 HD, 7 hcMBL (4 M-MBL, 3 U-MBL), and 1 M-lcMBL patient, using the ImmunoRead platform and a custom pipeline built upon the Immcantation framework. Leukemic IGHV-D-J reads were removed.

PCA clearly separated NBC-HD, NBC-lcMBL, NBC-hcMBL, and hcMBL clones, forming a gradient with NBC-HD and hcMBL clones at opposite extremes, and NBC-lcMBL samples occupying an intermediate position between NBC-HD and NBC-hcMBL. Within hcMBL, CD5⁺NBC and NBC-U-MBL clustered closer to hcMBL clones. Comparing CD5⁻NBC-M-hcMBL to NBC-HD revealed 8,380 DEG, with most pathways, including B-cell activation (20/21, 95%), inhibited. Mismatch and base-excision repair (MMR, BER), typically found in follicular B-cell responses, were activated. CD5⁻NBC-U-hcMBL vs. NBC-HD exhibited 12,793 DEG and widespread upregulation of activation pathways (23/26, 88%). Comparing CD5-NBC-U-hcMBL and NBC-M-hcMBL revealed fewer DEG (1,316), consistent with the groups being hcMBL, although the former showed activated pathways (cytokine storm, NET, S-phase and cyclophilin signaling, and RIPK1-mediated necrosis), typical for heightened inflammation and extrafollicular responses.

Phenotypically, NBC-hcMBL had reduced naïve B cells, and increased memory, ABC and DN B cells compared to NBC-HD (P<0.05). NBC-M-hcMBL exhibited increased DN1 cells (P=0.046), considered switched memory precursors, and expanded switched memory B cells (P=0.008), whereas NBC-U-hcMBL displayed increased unswitched memory B cells (P=0.018). NBC-U-MBL also exhibited higher DN2/3 cells which are precursors to antibody-secreting cells compared to NBC-HD (P=0.003) and NBC-M-hcMBL (P=0.024).

BCR analysis of NBCs revealed significant differences (P<0.05) in clonal space occupancy (CSO) among groups. NBC-HD exhibited a polyclonal repertoire with low clonal dominance; expanded clones (0.5-1% CSO) were rare (1.4%) and hyperexpanded clones (>1% CSO) absent. NBC-U-hcMBL showed increased expanded and hyperexpanded clones (5% each). NBC-M-hcMBL had the most restricted repertoire, with 16.3% hyperexpanded clones and the highest SHM rates, significantly surpassing NBC-U-hcMBL and NBC-HD (P<0.001). The sole M-lcMBL case analyzed had a clearly distinct BCR repertoire, with a high proportion of hyperexpanded clones (45% CSO) and unique V gene usage.

Altogether, our results demonstrate a global B-cell defect both in lcMBL and hcMBL, being markedly more pronounced in the latter. Nevertheless, the transcriptomic differences (NBC from lcMBL being much closer to HD), together with the different immunogenetic pressures revealed by BCR analysis point toward separate pathogenic drivers in lcMBL and hcMBL. In hcMBL, the transcriptome defects were demonstrated in CD5⁺ and CD5⁻ NBC subsets and in M- and U- MBL patients. NBC from M-hcMBL appear to follow a follicular B-cell maturation pathway (BER and MMR signatures, switched memory and DN1 B cells, and a more restricted and hypermutated BCR repertoire). Conversely, NBC from U-hcMBL show a gene signature of extensive inflammation compatible with extrafollicular maturation, supported by unswitched memory and DN2/3 B-cell subsets and reduced SHM rates. These findings suggest that different antigenic challenges push hcMBL NBCs into separate developmental paths that correlate with IGHV-mutation status.

GB was supported by the Lymphoma Research Foundation.

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2025
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