IgM and IgD Differential CLL BCRs Organization and Interaction with Co-Receptors before and during Ibrutinib Therapy

Studies of the B cell receptor (BCR) on normal B lymphocytes indicate that membrane IgM and IgD (respectively mIgM and mIgD) are organized in independent protein islands containing different BCR co-receptors that then reorganize as B cells transition from resting (CD19 and CD20 associated with IgD) to activated (CD19 and CD20 associated with IgM). In this way the signaling balance between IgM and IgD regulates B cell fate. Since BCR signaling is a key factor for chronic lymphocytic leukemia (CLL), as evinced by the efficacy of the signaling inhibitory drug ibrutinib, we decided to evaluate the membrane organization of the BCR-complex on primary CLL cells in an isotype dependent fashion.

We analyzed 4 U-CLL and 4 M-CLL samples, taken before and during (4-6 weeks of) ibrutinib treatment, by imaging flow cytometry, to evaluate the tendency of mIgM or mIgD to appear in high density regions (clusters). CLL cells displayed more IgM- than IgD-containing clusters, and the spot diameters for mIgM clusters were twice as broad as those for mIgD. However, while mIgD density correlated with the number of clusters, mIgM density did not. Taken together, these data indicate that mIgM and mIgD are organized differently on CLL cell membranes.

Next, we assessed the association of activating (CD19, CD20) and inhibitory (CD22, Siglec10) co-receptors with the two mIG isotypes. These findings were then correlated with in vivo birth rates measured after patients drank ²H₂O. Co-localization of CD19 and CD20 with mIgM directly correlated with increased proliferation in vivo . Conversely, those patients whose CLL B cells had more CD22 co-localized with IgM than IgD had lower in vivo birth rates. Notably, there was no such correlation for co-localization of Siglec10. Thus, there was differential co-localization of IgM with CD19 and CD20 versus with CD22 and these correlated respectively positively or negatively with CLL cell growth in vivo .

The mIGs of CLL cells can spontaneously self-associate and transmit a signal to the leukemic cells. This property could influence membrane organization of the two isotypes, either directly by self-binding or indirectly by repetitive signaling and subsequent cytoskeleton rearrangement. To test this, we expressed 13 different CLL BCRs in triple knockout (TKO) cells as IgMs or IgDs while maintaining their original IGHVDJ and IGLVJ structure. For BCR receptors expressed as IgM, Ca⁺⁺ mobilization occurred spontaneously, without the need for an external ligand ("autonomous signaling"). In contrast, BCR receptors expressed as IgD required ligand crosslinking for signaling. Therefore, while both mIgM and mIgD BCRs are capable of signaling upon antigen engagement, only mIgM BCRs naturally self-associate and transduce a signal. This implies a constitutive, self-reorganization of the BCR clusters and co-receptor localization that differs from normal resting B cells and primes CLL activation. The self-binding strength and the quality and reorganization capacities of the induced signaling likely lead to different cellular consequences.

Finally, we studied patients undergoing ibrutinib treatment. Ibrutinib significantly upregulated the sm density of IgM and downregulated IgD and CD20; however, there was little change for CD19, CD22 and Siglec10. Ibrutinib also led to even more and bigger IgM and less and smaller IgD clusters. In addition, CD22 no longer co-localized with IgM, while the level of association with IgD did not change. Finally, co-localization of CD19 and CD20 with the IgM and IgD BCRs was not significantly altered indicating: i) their organization is not dependent on Btk signaling; ii) Localization changes take place in subpopulations, but the clonal median is invariant; or iii) Changes occur at levels below our detection method.

In conclusion, IgM and IgD are organized differently on CLL B cells. IgM forms bigger and more clusters than IgD, and higher CLL birth rates are found for cells with more CD19 and CD20 in proximity to IgM than IgD. Also, autonomous signaling occurs only through IgM. Thus, CLL clones exhibit a membrane organization like that of an activated B cell. Conversely, increased association of CD22 with IgM correlates with slower birth rates and a less activated cell status. Ibrutinib treatment disregulates BCR isotype organization increasing IgM cluster size and number, decreasing those with IgD and changing the relative proximity of co-receptors such as CD22.