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3.04 times more efficient in CLL compared to normal B cells (Figure 1D, P<0.0001). The uptake index varied con- siderably between patients, but was significantly higher in those whose BCRs lacked the ability to mobilize calcium in response to BCR ligation and cases with low CD38 expression (see Online Supplementary Figure S2 and Online Supplementary Table S1).
anergic and signaling competent CLL cells and normal B cells (Figure 2E,F). Thus, although both normal and CLL B cells are capable of internalizing IgM and CD79B follow- ing ligation with cognate agonistic antibody, the two mol- ecules do not cointernalize, and thus cannot be closely associated during membrane trafficking.
Having established that sCD79B is not required for sIgM internalization, we proceeded to assess its role in BCR signaling by measuring the effect of prior sCD79B downregulation on αIgM ERK phosphorylation in CLL cells. As expected, prior sCD79b downregulation reduced, but did not abolish αIgM-induced ERK phosphorylation (Figure 2G). In contrast, depletion of sCD79B was without effect on pHrodo-αIgM uptake (Figure 2H).
In vivo relationship between BCR signaling and internalization
We next investigated the relationship between BCR internalization and signaling in vivo in CLL by studying subsets of PB cells, those from the LN where BCR activa- tion and proliferation is thought to take place and longitu- dinal samples obtained from patients being treated with BTKis ibrutinib and acalabrutinib.
Recently proliferated and quiescent subsets
It has previously been shown that recently proliferated LN emigrants express low levels of CXCR4 and high levels of CD5 (CXCR4dimCD5bright).28 Since proliferation within LNs is linked to BCR signaling, we compared BCR expres- sion and internalization efficiency on CXCR4dimCD5bright and CXCR4brightCD5dim subsets. Since the BCR internaliza- tion assay involves incubation with agonistic αIgM, we first investigated whether 1-hour incubation with pHrodo-αIgM altered the expression of these markers. As expected, CXCR4 expression was reduced and CD5 increased following ligation of BCR, however, over the 1- hour assay period, the magnitude of change was small (CXCR4 mean fold change = 0.93±0.02 and CD5 mean fold change 1.24±0.04, data not shown). Since the MFI of the top and bottom decade of CXCR4 and CD5 expres- sion differed by 11.8±5.4-fold and 8.7±6.7-fold, respec- tively, assay-induced changes could not have changed the composition of these subsets. Since the CXCR4dimCD5bright subset are thought to have recently undergone BCR acti- vation within the LN, we expected to find downregulation of sIgM in this fraction, however, this was not the case and levels were higher than in the CXCR4dimCD5bright sub- set (Figure 3A, P<0.0001). In keeping with post-activation anergy, in the majority of patients (14/21) there was more efficient uptake of pHrodo-αIgM in the CXCR4dimCD5bright subset, however, there was significant variability and, in the whole population, this did not reach statistical signifi- cance (Figure 3B; n=21; P=0.281).
BCR expression and internalization in the PB and LN of CLL patients
Since our results suggested that BCR internalization is influenced by the capacity to initiate downstream signal- ing, which occur within LNs, we went on to directly com- pare sIgM levels and uptake of pHrodo-αIgM by LN CLL cells to those derived from simultaneously obtained PB from the same patients. As we have previously shown,29 LN CLL cells expressed higher levels of CD5 than those derived from the PB, in keeping with BCR activation at these sites (data not shown). As was the case for the recent- ly proliferated CXCR4dimCD5bright subset, sIgM levels were
Prolonged ex vivo incubation has previously been shown to reverse features of anergy, namely, re-expression of sIgM and restoration of BCR responsiveness.4,9 We there- fore examined BCR expression and internalization after 24 hours ex vivo incubation, however, results were heteroge- neous with no significant recovery in sIgM expression (Online Supplementary Figure S3A, P=0.83, n=7) or change in BCR internalization efficiency (Online Supplementary Figure S3B, P=0.88, n=7). It was not possible to assess internalization efficiency at later time points as the assay critically depends on metabolic integrity of the cells, which was not consistently maintained after 24 hours.
Since the pHrodo-αIgM signal reflects both uptake into and retention within acidic endosomes, we also assessed the BCR internalization rate more directly by measuring loss from the cell surface following ligation with an ago- nistic antibody. This showed that all cases of CLL internal- ize their BCRs more rapidly than normal B cells (Figure 1E; P=0.04, 2 minute time point). More subtle differences were observed between anergic and signal competent cases of CLL, with more rapid initial loss from the surface in the former (Figure 1E; P=0.02 at 2 minute time point).
We also measured sIgD expression and the levels of pHrodo-αIgD uptake using the same assays. All CLL B cells internalized pHrodo-αIgD, and pre-incubation with unlabeled αIgD reduced the level of uptake (Online Supplementary Figure S4A; n=18; P=0.001). No significant correlation between pHrodo-αIgD uptake and surface sIgD expression was observed (Online Supplementary Figure S4B) and there was no difference in the pHrodo-αIgD uptake index between CD38high and low, anergic/non anergic and IGHV-mutated and unmutated patients (Online Supplementary Figure S4C). This suggests that uptake and retention mechanisms differ between IgD and IgM; this was not addressed further herein.
Mechanism of dissociation of BCR signaling and internalization
We next investigated the role of sCD79B, a molecule that is closely associated with the BCR and essential for signal transduction, in BCR internalization. As previously reported,27 CLL B cells express lower levels of sCD79B compared to normal B cells, with a particularly reduced expression in anergic compared to signaling competent cases (normal B-cell MFI=2561.2±400.9, anergic CLL MFI=318.2±63.4, signaling competent MFI=397.5±71.1). We also found that sIgM expression correlates with sCD79B both between (Figure 2A) and within a patient with CLL (Figures 2B,C). Despite these findings and the fact that sIgM and sCD79B are known to be associated during BCR signaling, no significant reduction in sCD79B expression occurred following ligation and downregula- tion of sIgM with agonistic antibody in both anergic and signaling competent cases of CLL (Figure 2D). In normal B cells, a slight but significant reduction in sCD79B expres- sion was observed 30 and 60 minutes after BCR ligation with αIgM. Similarly, downregulation of CD79B with an agonistic anti-CD79B had no effect on the level of sIgM in
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