Comparative Density Analyses of B Cell Receptor-Complex Components (Membrane IgM, Membrane IgD, and Stimulatory/Inhibitory Co-Receptors) on Intraclonal Subpopulations of CLL B Cells, before and during Ibrutinib Therapy
Authors: Mazzarello Andrea Nicola , Vergani Stefano , Ferrer Gerardo , Liu Yun , Chen Shih-Shih , Yancopoulos Sophia , Yan Xiao-Jie , Barrientos Jacqueline C. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
B cell receptor signaling is a key factor in chronic lymphocytic leukemia (CLL), evinced by inhibitory drug ibrutinib's efficacy. Studies of normal and CLL B cells indicate surface membrane IgM or IgD engagement has diverse signaling consequences. But little is known about relative amounts of sIg/co-receptor components within intraclonal fractions and how their compositions affect signaling. We studied relative densities of IgM and IgD and Ig-associated stimulatory and inhibitory co-receptors in two subsets based on: [1] relative densities of IgM and IgD and [2] reciprocal expression of CXCR4/CD5 indicating activation state. Samples from 5 U and 5 M-CLL patients, pre and during (4-6 weeks) ibrutinib treatment, were tested by conventional and by imaging flow cytometry using an ISX Mark II providing multiple spectral images of individual cells in a flow setting.
After subfractionating clones for IgM (IgMDim, IgMInt, IgMBright) or IgD (IgDDim, IgDInt, IgDBright) densities, we quantified relative densities of stimulatory/inhibitory molecules on the subsets. A directly proportional change was observed for CD5, CD19, CD20, Siglec10, CD25, HLA-DR, and CD38 as IgM moved from Dim to Bright. Exceptions were CXCR4, which dramatically decreased as IgM density increased, and CD22, that had a constant density in all fractions. Similar changes were seen for IgD except for CD22 which increased in IgDBright density. Again CXCR4 showed the opposite pattern. CD25 and HLADR remained constant within IgD increments. Stimulatory markers CD25 and HLADR changed upward only in the transition to IgMBright. Together these imply signaling through IgM heightens as IgM density increases, but does not through IgD, likely due to increased Siglec 10 and CD22. The reduced amounts of smCXCR4 suggest impaired ability of cells with high smIgM or smIgD to traffic.
We examined subpopulations based on CXCR4 and CD5. IgD and IgM densities increased from resting (RF) CXCR4Bright/CD5Dim to intermediate (INT) CXCR4Int/CD5Int to proliferative (PF) CXCR4Dim/CD5Bright fractions, although the degree of upregulation was more marked for IgD than IgM. All stimulatory/inhibitory coreceptors also increased in density from RF to PF. Interestingly, CD22 and IgD retained a constantdensity from RF to INT but increased considerably at the PF. Although stimulatory molecules CD25, CD38 and HLADR had upward trends, peaking occurred in the PF. Collectively, this implies signaling capacity through smIgM amplifies toward the PF due to higher smIgM density and IgD upregulation.
Finally, we evaluated CLL clones as a whole as well as based on the subsets above for ibrutinib treatment induced changes. Clonally, a density decrement occurred for smIgD and an increment for smIgM. Data for the other molecules fell into 3 categories based on relative density changes: Decreasing: CD5, CD20, CD38, CD25, HLA-DR; Increasing: CXCR4: Invariant: CD22, CD19, Siglec10.
For subfractionating based on smIgs density, ibrutinib drastically reduced density differences for co-receptors and activation markers among IgM or IgD density subpopulations with IgMBright and IgDBright fractions more affected than the Dim and Intermediate density counterparts. Similarly for the CXCR4/CD5 subpopulations, lower differences in relative expression of coreceptors from RF to PF were found. As for IgM and IgD levels, the PF was the most reduced. The composite effect was a reduced slope of change among the original sets of density change categories (IgMDim -> IgMBright; IgDDim -> IgDBright; RF to PF), leading to more phenotypically homogenous subpopulations.
In summary, prior to ibrutinib therapy, both the Ig and most of the associated molecules increase density in subsets marked by increased IgM and IgD densities and marked by decreasing CXCR4/CD5 densities. However, there was a major difference in density correlations for CXCR4 and CD22. CXCR4, levels were lowest on IgMBright and IgDBright cells which differs from published data using anti-IgM beads. For CD22 the highest levels were in IgDBright but not in IgMBright and in CXCR4/CD5 subset with the highest IgD and IgM (CXCR4Dim/CD5Bright). In contrast, ibrutinib treatment led to an overall change in coreceptor molecules altering considerably the density relationships with IgM and IgD. Concomitantly, these culminate in a downregulatory membrane stimulatory environment.

Disclosures
No relevant conflicts of interest to declare.