Page 144 - Haematologica3

E.M. Coulter et al.
face and downstream BCR signaling, our data strongly suggest that in CLL B cells, they internalize independently, and thus cannot be associated during endocytosis. In nor- mal B cells a small proportion of sCD79B and sIgM co- internalized, again suggesting minimal association of the two during endocytosis. A plausible explanation for these observations is that BCRs exist in two configurations in CLL (Figure 4); the first in which sIgM and sCD79B are not and do not become associated following ligand bind- ing and a second in which sIgM and sCD79B are already colocalized or are induced to associate following activa- tion. The first configuration could not transduce down- stream signals but, because retention of sIgM at the sur- face requires association with CD79B, would internalize efficiently. Adversely, BCRs in which sIgM and CD79B are associated could initiate signaling, however, the asso- ciation with CD79B would favor retention at the cell sur- face, at least until the subunits undergo phosphorylation- induced dissociation.31 Such a model has already been pro- posed for normal B cells,32 and is thought to favor signaling in response to low-affinity ligands, such as autoantigens of the type recognized by CLL BCRs.33
It is currently believed that BCR activation takes place in LNs and that this, combined with other signals from the microenvironment, leads to tumor proliferation. In other receptor systems, ligand binding is generally accompanied by downregulation of cell-surface receptors,34 and our observation that sIgM levels are actually higher in the LN than PB and in recently proliferated CXCR4dimCD5bright compared to CXCR4dimCD5bright PB CLL cells was therefore unexpected. As recently suggested by others, it is possible that the elevated sIgM levels observed within the LN and CXCR4dimCD5bright subsets are due to IL4-induced upregu- lation of CD79B within lymphoid tissues.16,17
Our findings also shed further light on the mechanism of action of BTKis. As noted by others,13,35 a persistent low-level lymphocytosis is frequently seen in such patients and complete remissions are rare. In the early period following the commencement of BTKi therapy, we observed an increase in sIgM expression in PB CLL cells. Since we have shown that cells in the LNs express higher levels of sIgM than those in the PB, this most likely reflects the previously documented redistribution of the tumor from the former compartment to the later.35 Over the longer term, however, the opposite is the case, with a sig- nificant reduction in sIgM that is accompanied by an increase in BCR internalization efficiency and a reduced capacity to phosphorylate ERK. These findings indicate that long-term BTKi therapy causes the emergence of a population of neoplastic B cells with anergic phenotypic
and functional properties. This may occur either through reprograming of the tumor into a more anergic state or because there is subclonal heterogeneity within the tumor and selection of cells with anergic features by BTKi thera- py. The latter possibility is supported by in vitro studies of leukemic B-cell migration29 and in vivo observations using heavy water or glucose labeling36 that suggest the exis- tence of subclonal heterogeneity at a functional level in CLL.
Finally, since BCR internalization into endosomes is the first event in antigen processing, our data support the the- ory that, under some circumstances, CLL B cells might act as antigen presenting cells. We have recently shown that LN derived CLL B cells express higher levels of costimula- tory molecules, form immune synapses and stimulate an allogeneic mixed lymphocyte reaction more efficiently than those from the PB.29 In addition, elution of peptides from CLL major histocompatibility complex (MHC) class II reveals presentation of a range of autologous peptides,37 with evidence for expansion of cognate T-cell clones in CLL but not normal PB. Furthermore, a number of groups have documented the presence of T cells in CLL patients that are capable of responding to a range of other mole- cules, including Rh antigen,38 tumor idiotype23 immunoglobulin framework,39 or CDR3 motifs40 as well as broader responses against tumor lysates41 or intact leukemic cells.42 It is therefore plausible that the abnormal phenotype, repertoire and function of CLL T cells might be a consequence of excessive and aberrant antigen pres- entation occurring within lymphoid tissues.
In summary, we have shown that, as in normal B-cell anergy, CLL B cells internalize ligands that bind to the BCR more efficiently than normal. This process is uncou- pled from downstream signaling and does not involve association with CD79B. We demonstrate that BTKi ther- apy induces or selects for cells with anergic properties that persist in the long term. Understanding how this occurs will be important in order to optimize the efficacy of this important new class of drugs.
Funding
This work was supported by research funding from Bloodwise (grant number: 15012) and the British Society of Haematology (BSH; grant number: 34721/start up). The authors also acknowledge financial support from the UK Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King’s College Hospital NHS Foundation Trust.
504
References
1. Gobessi S, Laurenti L, Longo PG, et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia. 2009; 23(4):686- 697.
2. Muzio M, Apollonio B, Scielzo C, et al. Constitutive activation of distinct BCR-sig- naling pathways in a subset of CLL patients: a molecular signature of anergy. Blood. 2008;112(1):188-195.
3. ZhuangJ,HawkinsSF,GlennMA,etal.Akt
is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhi- bition. Haematologica. 2010;95(1):110-118.
4. ApollonioB,ScielzoC,BertilaccioMT,etal. Targeting B-cell anergy in chronic lympho- cytic leukemia. Blood. 2013;121(19):3879- 3888, S1-8.
5. Hewamana S, Alghazal S, Lin TT, et al. The NF-kappaB subunit Rel A is associated with in vitro survival and clinical disease progres- sion in chronic lymphocytic leukemia and represents a promising therapeutic target. Blood.2008;111(9):4681-4689.
6. Byrd JC, Harrington B, O'Brien S, et al. Acalabrutinib (ACP-196) in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med. 2016;374(4):323-332.
7. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013;369(1):32-42.
8. Cesano A, Perbellini O, Evensen E, et al. Association between B-cell receptor respon- siveness and disease progression in B-cell chronic lymphocytic leukemia: results from single cell network profiling studies. Haematologica.2013;98(4):626-634.
haematologica | 2018; 103(3)