Authors
Gonzalo Blanco, Joy Yan, Anita Ng, Andrew Shih, Jacqueline Aldridge, Anna Puiggros, Lindybeth Sarmiento, Jonathan Kolitz, Steven Allen, Kanti Rai, Douglas Stewart, Blanca Espinet, Gerald Marti, Neil Caporaso, Marcelo Navarrete, Nicholas Chiorazzi.
Background
People with CLL and monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, exhibit immune dysfunction. Since in MBL there are more normal B cells (NBC), it provides an opportunity to study B-cell defects.
Aims
Compare the transcriptomes of NBC from healthy donors (NBC-HD) with those from MBL patients (NBC-MBL), including CD5⁻ and CD5⁺ subsets of IGHV-mutated (M-MBL) and unmutated (U-MBL) patients; Analyze B-cell subset distribution in the NBC of MBL and HD.
Methods
PBMCs were collected from 18 high-count MBL (hcMBL) patients (12 M-MBL, 6 U-MBL) and 13 HD, and 99 FACS-purified fractions were obtained: 21 MBL clones (CD20LowCD5⁺Igκ⁺/Igλ⁺), 36 NBC-MBL (30 CD20BrightCD5⁻; 6 CD20BrightCD5⁺), and 42 NBC-HD (25 CD20BrightCD5⁻; 17 CD20BrightCD5⁺). RNA from these was sequenced using SMART-Seq v4 Ultra-Low-Input and HiSeq platform. DESeq2 was used to analyze RNA-seq data. Differentially expressed genes (DEGs) were defined by Padj < 0.05 and |FC|≥1.5. PCA was used to assess transcriptome variance, and IPA to identify enriched pathways. Protein expression of DEGs was validated by flow cytometry in 6 hcMBL and 8 HD. For dissection of B-cell subset types, PBMCs from 14 hcMBL (10 M-MBL and 4 U-MBL) and 9 HD were studied. Data were acquired on a FACSymphony.
Results
PCA of all B-cell fractions distinctly separated NBC-HD, NBC-MBL, and MBL clones. Within NBC-MBL, CD5⁺ NBC and NBC-U-MBL clustered closer to MBL clones.
Comparing CD5⁻ NBC-M-MBL to NBC-HD revealed 8,380 DEGs. IPA predicted inhibition of 95% of Summary Graph pathways (20/21), including B-cell activation and proliferation. Canonical pathway analysis indicated upregulation (Z-score≥2) of mismatch repair (MMR) and base-excision repair (BER), typically found in NBC undergoing follicular responses when CSR and SHM occur.
CD5⁻ NBC-U-MBL vs. NBC-HD showed 12,793 DEGs, a larger difference, consistent with PCA showing NBC-U-MBL farther from NBC-HD. IPA predicted activation of 88% of Summary Graph pathways (23/26); these included Cell cycle progression and Migration. Inflammation-related Canonical pathways such as Pathogen-induced cytokine storm, IL-8, Chemokine, and IFNG signaling, were all exclusively upregulated in NBC-U-MBL, indicating augmented inflammation specific to this group.
Comparing NBC-U-MBL and NBC-M-MBL showed the fewest differences among comparisons (1,316 DEGs), consistent with both groups being NBC-MBL. Nevertheless, IPA identified six significantly upregulated (Z-score≥2) Canonical pathways in NBC-U-MBL: Cytokine storm, NET signaling, S-phase, Cyclophilin signaling, RIPK1-mediated necrosis, and Fibrin clot dissolution. Note that NETosis can occur in B cells (Bukhari 2023). These pathways converge in the immune response, connecting pathogen sensing, inflammation (cytokine storm, NETs), cell proliferation (S phase), stress signaling (cyclophilin), and inflammatory cell death (RIPK1). This exclusive activation in NBC-U-MBL could foster rapid, uncontrolled inflammation that typically drives extrafollicular B-cell responses. Flow cytometry analyses of selected proteins validated significant overexpression of 10 DEG measurements.
Phenotypically, NBC-MBL had reduced naïve B cells (IgM⁺IgD⁺CD27⁻) and increased CD27⁺ memory B cells compared to NBC-HD (32.8% vs. 76%, P< 0.0001; 34% vs. 9.1%, P=0.0003). ABC (T-bet⁺CD11c⁺CD21⁻) and DN (IgD⁻CD27⁻) B cells were also elevated among NBC-MBL (0.9% vs. 0.2%, P=0.0492; 11.1% vs. 4.7%, P=0.0001). Subgroup analysis by IGHV mutation status revealed distinct memory B-cell profiles. NBC-M-MBL uniquely showed increased DN1 cells (IgD⁻CD27⁻CD38⁺CD21⁺CD24⁺, 3.3% vs. 1.2%, P=0.0459), which are precursors to switched memory B cells, and expanded switched memory B cells (IgM⁻IgD⁻CD27⁺; 26.4% vs. 5%, P=0.0076). On the contrary, only NBC-U-MBL displayed increased percentages of unswitched memory B cells compared to NBC-HD (IgM⁺⁺IgDLowCD27⁺CD21⁺CD24⁺; 8.6% vs. 1.7%, P=0.0182). NBC-U-MBL also exhibited higher percentages of DN2/3 cells (IgD⁻CD27⁻CD21⁻) compared to NBC-HD (6% vs. 0.7%, P=0.0028) and NBC-M-MBL (6% vs. 2.1%, p=0.0240). DN2 and DN3 cells are precursors to antibody-secreting cells.
Conclusions
NBC-MBL exhibit transcriptomes that lie between NBC-HD and MBL clones, suggesting a global B-cell defect potentially shaped by genetic and/or environmental factors. This defect affects both CD5⁺ and CD5⁻ NBC-MBL and is present in M-MBL and U-MBL. Follicular maturation signatures (BER and MMR) and increased follicular B-cell subsets (switched memory and DN1 cells) characterize CD5⁻ NBC-M-MBL. CD5⁻ NBC-U-MBL exhibit inflammatory pathway activation and extrafollicular unswitched and DN2/3 memory subsets. These results align with IPA Summary Graph pathways showing inhibition in NBC-M-MBL, and activation in NBC-U-MBL, supporting a low/controlled inflammation in M-MBL (typical for follicular responses), and high/uncontrolled inflammation in U-MBL (typical in extrafollicular responses). BCR repertoire analysis to verify follicular versus extrafollicular maturation is ongoing.
Acknowledgments. GB was supported by the Lymphoma Research Foundation.
Keywords : monoclonal B-cell lymphocytosis (MBL), normal B cells, immune deficiency
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