B-Cell Anergy Underlies Indolent Clinical Behavior Of CLL Stereotyped Subset #4

Mounting evidence suggests that specific modalities of B-cell receptor (BcR) and Toll-like receptor (TLR) collaboration and/or regulation may exist in chronic lymphocytic leukemia (CLL), eventually impacting on the behavior of the malignant clones. CLL patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BcRs, SS4), the largest subset within mutated CLL, are characterized clinically by early age at diagnosis and indolent disease course and genetically by few lesions, with deletion of chromosome 13q representing the exclusive recurrent aberration. Examination of the IG sequences of SS4 clonotypic BcRs allows for parallels to be drawn with pathogenic anti-DNA antibodies since both carry long positively charged VH CDR3s. However, SS4 IGs also display distinctive somatic hypermutation (SHM) patterns, the most notable concerning the obligatory introduction of negatively charged residues in either the heavy or the light chains or both, alluding to editing of an autoreactive progenitor. Moreover, SS4 BcRs continue to acquire SHMs within their IG genes overtime, implying antigen selection in the clonal evolution of CLL SS4, albeit without a negative clinical impact. Altogether, these findings suggest that CLL SS4 cells may exist in an anergic state showing attenuated responses to selecting (auto)antigenic elements. To test this hypothesis, after BcR engagement in vitro we analysed basal phosphorylated ERK (pERK) levels and calcium mobilization, both known to be features of B-cell anergic state. Nine of 10 SS4 cases expressed high pERK levels and were unable to increase intracellular Ca²⁺ upon BcR crosslinking; moreover, stimulation through the BcR had no effect on pERK levels. Primary cell culture for 50min caused a statistically significant reduction of pERK, while culture for 6-24 hours restored the ability of the cells to respond to BcR activation by inducing ERK phosphorylation. Furthermore, analysis of serial samples of 4 SS4 cases over a period spanning 5-7 years revealed consistently positive pERK expression. On these grounds, we conclude that SS4 CLL clones are anergic through the BcR possibly because of chronic (auto)antigen activation. To determine if this profile is SS4-biased or linked to the usage of the IGHV4-34 gene, we also performed similar experiments in 6 cases using this gene in mutated non-stereotyped rearrangements of the MD isotype and found a more heterogeneous pattern: 3/4 cases were pERK negative, while 3/6 cases showed Ca²⁺ fluxes after BcR crosslinking. Given that SS4 cells are responsive to TLR1/2 ligands, we next investigated if TLR signals can modulate BcR anergy. To this end, SS4 CLL cells pre-stimulated through TLR1/2 with Pam3CSK4 for 50min were then subjected to BcR crosslinking for 10min. TLR1/2 stimulation up-regulated pERK levels, confirming our previous finding that CLL SS4 cells are responsive to TLR1/2 signalling. Notably, BcR crosslinking in TLR1/2-pretreated cells induced even higher pERK levels, indicating that TLR1/2 triggering restores BcR functionality, likely breaching the anergic state. We have previously demonstrated that TLR1/2 stimulation induces up-regulation of the miR-17∼92 cluster eventually leading to down-regulation of critical BcR and/or TLR signaling molecules (i.e. MAP3K1, MAP2K3, MAPK8, CASP8, IKBKB) which are potential targets of these miRNAs. In order to experimentally validate the predicted mRNA-miRNA interactions, we performed 3’ UTR luciferase reporter assay experiments in HeLa cells and documented 5 novel interactions between miR-17∼92 cluster members and MAP3K1, MAP2K3 and MAPK8. To ensure that these interactions are valid also in CLL, we performed transfection experiments with miRNA mimics in negatively selected CLL B cells and obtained identical results. In conclusion, we demonstrate that SS4 CLL cells are anergic through the BcR and that stimulation through TLR1/2 may break B-cell anergy. Moreover, we propose a regulatory mechanism whereby TLR1/2 signals induce miR-17∼92 cluster up-regulation, leading to down-regulation of MAP3K1, MAP2K3 and MAPK8 and eventual modulation of ERK phosphorylation, a key to the anergic state. On a broader scale, the detailed molecular and biochemical characterization of immune signaling in SS4, a prototype for clinically indolent, good prognosis CLL, is also relevant for the advancement of therapeutic strategies incorporating immune signaling inhibitors.