B cell malignancies including chronic lymphocytic leukemia (CLL) and diffuse large B cell lymphoma (DLBCL) are age-associated diseases driven by clonal B cell proliferation. Signaling through B cell antigen receptor (BCRs) is dysregulated in these diseases. In addition to BCRs, chemokine receptors, such as CXCR4 and CXCR5, are used to predict clinical course. The chemokine receptor CXCR4 is expressed at different developmental stages of B cells, serving different homeostatic functions including migration. We previously reported that the cross-talk of CXCR4 and the BCR isotype IgD is supporting survival and activation of mature B cells in mice. In this process, the B cell marker and co-activator CD19 plays a pivotal role. Nevertheless, interaction of BCR with CXCR4 has not been analyzed in detail in B cell malignancies.
In this study, we further elucidated the CXCR4 signaling in mouse as well as human B cell subsets including immature and mature B cells. Consistent with murine B cells, CXCR4 signaling in human B cells from healthy donors remains tightly linked to surface IgD-BCR expression, although CXCR4 is highly expressed in human IgG positive memory B cell compartment. Furthermore, proximity of CXCR4 and IgD in human mature B cells is reminiscent of that of mouse B cells. In contrast, IgD:CXCR4 proximity is skewed towards IgM:CXCR4 in CLL cells. In in vitro assays, unmutated (U)-CLL cells migrate better compared to mutated (M)-CLL. Nevertheless, our analyses reveal a frequent association of IgM:CXCR4 in M-CLL. Taking together our murine and human data, we propose that IgD:CXCR4 association is crucial for CXCR4 signaling in both CLL and healthy B cells.
Apart from CXCR4, mutations within the immunoreceptor tyrosine-based activation motif (ITAM) residues of CD79a and CD79b are frequently associated with B cell malignancies including DLBCL. Knowing the potential role of BCR and its isotypes in CXCR4 induced signaling, we further analyzed the role of CD79a and CD79b. Here, we took advantage of transgenic mice, whose CD79a and CD79b cytoplasmic tails carrying ITAM motifs can be inducibly deleted. Our analysis reveals the indispensable role of the CD79b cytoplasmic tail, whose loss of function causes complete impairment of CXCR4 induced signaling in murine B cells. In contrast, loss of CD79a cytoplasmic tail partially blocks CXCR4 induced signaling, which could be rescued by CD19 co-stimulation. Extending our murine results, we established an in vitro read-out system to test the role of ITAM mutants derived from DLBCLs, as well as DLBCL-derived isotypes for analyzing their impact on CXCR4 signaling.
Taking our findings together, IgD:CXCR4 association is crucial for CXCR4 signaling in CLL and healthy B cells. An increased association of IgM:CXCR4 in M-CLL compared to U-CLL suggests the necessity of IgD:CXCR4 for functional CXCR4 signaling. Furthermore, CD79b is crucial for CXCR4 induced signaling in mature B cells and loss of CD79b function abrogates CXCR4 signaling in mature B cells.
Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.
Asterisk with author names denotes non-ASH members.