B cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.


Supplementary Materials and Methods
Animal Models. TCL1 transgenic mice were crossed with IgM knockout mice to obtain the following genotypes: IgM +/+ TCL1 +/tg , IgM +/-TCL1 +/tg , IgM -/-TCL1 +/tg (1-3). The control genotypes included IgM +/+ , IgM +/and IgM -/-. Mice were euthanized using CO 2 at 6, 7, and 8 months of age when B cell clonal expansion are usually obvious. Homozygous strains (IgM +/+ TCL1 +/tg , IgM -/-TCL1 +/tg , IgM +/+ and IgM -/-) were followed and euthanized also at later time points (> 12 months), when most mice exhibit CLL like disease. At sacrifice, spleens were harvested, and single cell suspensions made by homogenizing the organ, filtering through a 45 µm filter. To study bone marrow (BM) cells, femurs and tibiae were removed and crushed in a mortar with containing PBS + 2% FBS, filtered through a 45 µm mesh to remove particulate debris and to obtain single cell suspensions. Blood (20-50 µl) was collected with a Heparin-wetted (Heparin-Natrium 25000 I.E. Ratiopharm, #N68542.04) needle from the tail vein for flow cytometric analysis. Red blood cells (RBCs) in the cell suspensions from all organs were lysed by resuspending the cell pellet in 1 ml of Qiagen RB-lysis solution (#158904) and incubating the sample for 5 min at RT. RBC lysis of blood was done by adding 1 ml of Qiagen RB-lysis solution directly to whole blood, followed by an incubation step for 5 min at RT. This was done twice. RBC lysed single cell suspensions were used for further analyses, such as flow cytometry. Animal experiments were performed according to institutional ethical allowance and in compliance with the guidelines of the German law, license no. 1288, regional board Tübingen, Germany.

cells in 100µL FACS
Buffer. Thereafter, IgM and IgD MFIs of CD19 + CD5 + CLL B cells were normalized over their respective isotype controls and used to calculate the median numbers of ABCs. The ABC equivalent was translated to the number of BCR as described (4). CLL B cells were stained in parallel with polyclonal goat anti-human IgM PE (cat. # 2020-09; Southern Biotech) and antihuman IgD PE (cat. # 2030-09; Southern Biotech) with a saturation point of 2.5 µg/mL for 2x10 6 cells in 100µL FACS Buffer. IgM and IgD MFIs of CD19 + CD5 + CLL cells were normalized over their respective isotype controls and plotted versus their corresponding known ABCs revealing a comparable linear translation for both isotypes.

Use of Triple Knock-Out (TKO) cells to test autonomous and ligand-mediated BCR
signaling. TKO cells were transduced with retrovirus as described (5,6). Briefly, IG H and L chains were expressed using the biomolecular fluorescence complementation vector system (5).
CLL-derived BCRs were amplified from patient samples. Human IGHV-IGHD-IGHJ rearrangements were fused to the µ or δ human constant regions by PCR, whereas the complete human L chains were used. For virus production, Phoenix cells were transfected using FuGeneHD according to the manufacturer's protocol. Supernatants were collected 48 h after transfection and used for transduction of TKO cells (5,6).
Measurement of intracellular calcium mobilization was performed as described (7). Briefly, Glycolysis stress kit was used for: a) Glycolysis = ECAR after glucose minus ECAR after 2-DG; b) Glycolytic Capacity = ECAR after oligomycin minus ECAR after 2-DG; c) Glycolytic Reserve = Glycolytic Capacity minus Glycolysis. OCR and ECAR were normalized by 0% for the raw value of 0 and 100% as the last raw value of each dataset (or first, whichever was larger). Results are presented as fractions.
Cell preparation and staining for Imaging Flow-Cytometry. CLL PBMCs were isolated by density gradient centrifugation with Ficoll-Paque Plus (GE Healthcare) and used after thawing frozen aliquots that had been cryopreserved in liquid nitrogen in 10% DMSO and 90% FCS. To avoid membrane BCR changes, all procedures were carried out on ice and PBS with 0.02% NaN 3 and 1% FCS (Staining Buffer). 100 µl of 2x10 7 cells/ml were aliquoted in V-shaped 96 well plate and incubated for 30 min with murine anti-human mAbs and/or goat F(ab') 2 anti-human as per manufacturer concentrations. Cells were subsequently centrifuged at 300g for 10 min, washed 3 times with 200 µl staining buffer and suspended with 100 µl BD Cytofix solution (BD Bioscience) for 1h before acquisition started.
Imaging Flow-Cytometry Acquisition and Analysis. Cells from the patient cohort that consumed 2 H 2 O were automatically imaged in flow using an Amnis ImageStream X MKII (EMD Millipore) at 60x magnification. The ImageStream X captures simultaneously up to 12 images/cell through 12 channels (ch1-12) divided between 2 cameras (6 channels/camera, Ch1-6 for camera 1 and Ch7-12 for camera 2). The channels correspond to brightfield (one per camera, Ch1 and Ch9 respectively), side scatter (SSC), and up to 9 different fluorescence emission spectra. A halogen light source was used for the brightfield images and 4 lasers at 488nm, 405nm, 561nm and 642nm were used for fluorochrome excitation. For this study, up to 7 channels were used as follow: Ch1  OCR and ECAR were normalized defining 0% as the raw value of 0 and 100% as the last raw value of each dataset (or first, whichever was larger) and presented as fractions. Dashed lines separate values before or after each specific injection. subgroups were defined as the 50% of cases with highest IgM or IgD MFI from the total population. OCR and ECAR were normalized defining 0% as the raw value of 0 and 100% as the last raw value of each dataset (or first, whichever was larger) and presented as fractions. The Pearson correlation coefficient was used for statistical analyses.   before (Pre-T, orange) and during (light blue) ibrutinib treatment, and CLL1803 (BR 1.42%) before (Pre-T, yellow) and during (dark blue) ibrutinib treatment. Limits and medians of cell size are shown. The same curves are duplicated at top and bottom to improve graphic display of data.

B. Differences in MCS (left) and percentage of smallest (middle) and largest (right) CLL B-cell
subpopulations for CLL Int BR group 0.35-0.65% (n = 6) and CLL High BR group 0.80 -1.42% (n = 5), before (Pre-T) and during treatment. C. OCR and D. ECAR measurements before and during treatment with ibrutinib for the two groups based on BR. CLL Int BR 0.35 -0.65% (n = 5) and CLL High BR 0.80 -1.42% (n = 4). A One-Way ANOVA with Tukey test was used for statistical analysis. Tables   Table S1. Characteristics of the cohort of randomly selected CLL patients studied for IGHV mutation status, TTFT, and ZAP70 association with membrane IgM and IgD levels. for which individual cases were employed (*) or not available (NA), imaging flow cytometry (IFC), extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Table S3. Characteristics of the BCRs derived from human CLLs and TCL1 mice analyzed for autonomous and ligand-initiated BCR signaling using TKO cells.