Analyses of the Kinetics and Phenotype of Multiple Intraclonal CXCR4/CD5 B Cell Subsets Suggest Differences in Life Cycle Transitioning in CLL
Authors: Mazzarello Andrea Nicola , Fitch Mark , Ng Anita , Bhuiya Sabreen , Sharma Esha , Shih Andrew , Cardillo Martina , Allen Steven L , Kolitz Jonathan E , Barrientos Jacqueline , Rai Kanti R , Hellerstein Marc , Chiorazzi Nicholas 
Published: November 2021

Abstract
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease so that defining the dynamic features of the clone and its intraclonal subpopulations are essential to understand disease pathogenesis and to develop novel, effective therapies. For instance, because cell division is linked with new mutations, the ability to preferentially select cells that recently divided allows studying the subpopulation(s) most likely responsible for disease progression and resistance to therapies.
The intraclonal kinetics of CLL B cells have been studied in clonal subgroups defined by reciprocal surface levels of CXCR4 and CD5. In that model, three fractions are identified: recently divided "proliferative" (PF; CXCR4 DimCD5 Bright); "intermediate" (IF; CXCR4 IntCD5 Int) and "resting" (RF; CXCR4 BrightCD5 Dim). Here, we have expanded the examination of subpopulations differing for time since last division ("age").
Unmanipulated CLL cells studied ex vivo from 10 patients who drank 2H 2O for 4 weeks were sorted by the relative densities of CXCR4 and CD5 to isolate the formerly identified PF, IF and RF as well as two fractions not previously characterized, "Double Dim" (DDF: CXCR4 DimCD5 Dim) and "Double Bright" (DBF; CXCR4 BrightCD5 Bright). For each fraction, the amount of deuterium incorporated into cellular DNA in vivo was measured. Consistently, the PF contained significantly higher levels of 2H-labeled DNA and higher calculated cell division rates when compared with the RF and IF. Interestingly, the DDF also contained significantly more 2H-labeled DNA compared to the RF; in contrast, the DBF resembled more closely the RF fraction. The overall 2H-incorporation gradient was: PF&gt;DDF&gt;IF&gt;DBF&gt;RF.
In CLL, BCR signaling is fundamental, with the amount of membrane (m) IgM associating with signaling competence and disease aggressiveness. Additionally, when engaged independently, mIgM and mIgD can lead to different signaling sequelae. Therefore, we analyzed the 5 subpopulations for the densities of mIgM and mIgD. This showed a distribution similar to that of 2H-DNA incorporation: for IgM: PF=DDF&gt;IF=DBF=RF, and for IgD: PF&gt;DDF&gt;IF=DBF&gt;RF.
Accordingly, we next measured 2H-DNA in subpopulations with low, intermediate and high levels of IgM and IgD. This revealed a direct correlation between IG densities and in vivo DNA synthesis, consistent with intraclonal subpopulations with high IGs having divided more recently than those with low IGs.
However, these findings are not in line with cell division being primarily initiated by BCR engagement since that would lower mIgM levels. Therefore, we tested if engagement of TLR9 would affect mIG densities on CLL cells. After stimulation of 32 CLL clones with CpG+IL15, anti-IgM+IL4, anti-IgD+IL4, or anti-IgM-IgD+IL4, there was a significant increase in mIGs only after CpG+IL15 activation; each anti-IG stimulation led to downregulation of mIGs.
Finally, we questioned the subclonal responsiveness to BTK inhibition in vivo. CLL samples taken from the same patients, before and during ibrutinib treatment, displayed intraclonal changes in mIG densities and cell size, the latter a marker of cellular and metabolic activation also linked with CLL in vivo birth rates. Ibrutinib treatment normalized mIgM and mIgD intraclonal densities and lead to an overall cell size decrease with larger, 2H-enriched and higher mIG density cells being more affected (PF&gt;DDF&gt;IF&gt;DBF&gt;RF).
Collectively, these findings suggest that the most recently born cells enter the circulation as the PF from which they transition to either lower CD5 (DDF) or higher CXCR4 (IF and DBF) phenotypes. Each eventually converge as the RF. Moreover, since mIG densities on the more recently divided populations (PF and DDF) are high, the data imply that successful cell division is not solely a consequence of BCR engagement; the involvement of the TLR pathways, concomitantly or in series with BCR signaling, is more consistent with the higher mIG levels. Finally, ibrutinib treatment appears to preferentially target more recently divided cells with high mIG levels.

Disclosures
Allen: Alexion: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees.