Normal B Cells in MBL Are Biased Toward Antigen-Experienced B Cells, with Increased Percentages of DN1 Cells in IGHV-Mutated MBL and ABC/DN2 Cells in IGHV-Unmutated MBL Suggesting Distinct Follicular/Extrafollicular Maturation Pathways

Background. People with the pre-leukemic condition MBL suffer from immune deficiency, a major cause of death in CLL. This immune deficiency affects both the B and T cell lineages, and the causative mechanism(s) are not well defined. We previously determined that the CD5^- normal B cell (NBC) compartment of people with MBL exhibits distinct transcriptomes compared to healthy donors. Moreover, NBC from MBL patients of distinct IGHV mutation status differ.

Hypothesis. The variations in NBC transcriptomes stem from differences in the distribution of NBC subsets, and these cell subsets differ according to the IGHV-mutation status of the MBL cell clone reflecting distinct maturation pathways.

Objectives. Compare the immunophenotypic subsets of NBC (CD20^BrightCD5ˉ) from people with MBL (NBC-MBL) and their M-MBL and U-MBL subtypes (NBC-M-MBL and NBC-U-MBL) to those of healthy donors (NBC-HD) and try to corroborate the findings with the divergent transcriptomic profiles.

Methods. Peripheral blood mononuclear cells from 9 people with clinical MBL (5 M-MBL, 4 U-MBL) and 9 age-matched HD were labeled with antibodies targeting IgM, IgD, CD5, CD11c, CD20, CD21, CD23, CD24, CD27, CD38, CD45RB, CD62L, CD95, CXCR5, and T-bet. FVS510 was used to discriminate viable cells. BD FACSymphony and FlowJo were employed.

Results. NBC-MBL exhibited significantly reduced percentages of Naïve B cells (IgM⁺IgD⁺CD27^-) compared to NBC-HD (29.9% vs. 76%, P<0.0001). This difference was due to a loss in Naïve Resting B cells (IgM⁺IgD⁺CD27^-CD21⁺, 25.7% vs. 72.7%, P<0.0001); Naïve Activated B cells were comparable in the two (IgM⁺IgD⁺CD27^-CD21^-CD23^-, 2% vs. 1.6%, P=0.863). Consistently, CD27⁺ Memory subsets were enriched in NBC-MBL vs. NBC-HD (34.1% vs. 9.1%, P=0.002), with an overrepresentation of Late Memory CD45RB⁺CD95⁺ NBC (11.7% vs. 3.6%, P=0.011). Further discrimination indicated increased percentages of ABC cells in NBC-MBL vs. NBC-HD (Tbet⁺CD11c⁺, 1.8% vs. 0.7%, P=0.015; and Tbet⁺CD11c⁺CD21^-, 0.9% vs. 0.2%, P=0.036) and Double Negative (DN) NBC (IgD^-CD27^-, 12.8% vs. 4.7%, P<0.001). The latter was due to an increase in the DN1 subset (IgD^-CD27^-CD38⁺CD21⁺CD24⁺, 5% vs. 1.3%, P=0.046) as well as in the DN2/DN3 subsets (IgD^-CD27^-CD21^-, 4.6% vs. 0.7%, P=0.011).

When the NBC-MBL were subcategorized based on the IGHV-mutation status of the MBL clone for each person, several interesting restrictions emerged. Increases in late memory NBC (11.7% vs. 3.6%, P=0.012), switched memory NBC (IgM^-IgD^-CD27⁺, 31.8% vs. 5%, P=0.012), and the DN1 memory subset (5.6% vs. 1.3%, P=0.012) were found in NBC-M-MBL. These features suggest the NBC-M-MBL followed a classical, follicular, germinal center maturation pathway. In contrast, NBC-U-MBL showed increased percentages of DN2/DN3 cells (6.1% vs. 0.7%, P=0.003), Tbet⁺CD11c⁺CD21^- ABC cells (1.3% vs. 0.2%, P=0.064) and unswitched memory B cells (IgM⁺⁺IgD^LowCD27⁺CD21⁺CD24⁺, 8.6% vs. 1.7%, P=0.018). DN2/ABC and unswitched memory NBCs often follow an extrafollicular maturation pathway.

Two additional sets of data support distinct follicular and extrafollicular developmental pathways. First, significant protein overexpression of CD62L in NBC-M-MBL compared to NBC-U-MBL and NBC-HD; and CD62L and CXCR5 in NBC-M-MBL vs. NBC-HD. The presence of CXCR5 and CD62L on memory B cells indicates their follicular features. Second, RNA-seq data revealed two unique characteristics of NBC-M-MBL: their transcriptomes were significantly suppressed by IL-10, which can be T cell (Th2) enabled; and the Base Excision Repair (BER) pathway was the most significantly upregulated. BER is required for processing AID lesions needed for class switch recombination and also contributes to SHM.

Conclusions. The NBC compartment in MBL is biased to antigen-experienced B cell subsets compared to HD, disclosing an increase in DN/ABC percentages in MBL. NBC of M-MBL showed features of a classical follicular maturation pathway (increased late memory, switched memory, and DN1 NBCs; overexpression of CD62L and CXCR5; and IL-10/Th2 and BER related transcriptomes). In contrast, NBC of U-MBL displayed characteristics of an extrafollicular maturation pathway (increased DN2/ABC and unswitched memory NBCs). These findings are also consistent with the pre-leukemic B cell clones being derivatives of distinct antigenic drives in M-MBL and U-MBL.

Acknowledgments. GB is supported by the Lymphoma Research Foundation.