In this issue of Blood, describe a complex signaling model that explains the mechanism of action of a long-known prognostic marker in chronic lymphocytic leukemia (CLL) and integrates its function with the innate immune system and B-cell receptor signaling.1
Model of mechanism underlying differential effect of innate immune activation on ZAP-70–positive vs ZAP-70–negative CLL. In ZAP-70–positive CLL, activation of TLR9 via CpG induces IgM secretion by a ZAP-70– and Syk-dependent pathway. Robust phosphorylation (P) of Syk results in degradation of the proapoptotic Bim, which is amplified by autocrine stimulation of the B-cell receptor by the secreted IgM. The final effect is CpG-induced CLL cell proliferation and survival. Activation of TLR9 in the absence of ZAP-70 results in cell death. See Figure 7 in the article by Wagner et al that begins on page 436.
Model of mechanism underlying differential effect of innate immune activation on ZAP-70–positive vs ZAP-70–negative CLL. In ZAP-70–positive CLL, activation of TLR9 via CpG induces IgM secretion by a ZAP-70– and Syk-dependent pathway. Robust phosphorylation (P) of Syk results in degradation of the proapoptotic Bim, which is amplified by autocrine stimulation of the B-cell receptor by the secreted IgM. The final effect is CpG-induced CLL cell proliferation and survival. Activation of TLR9 in the absence of ZAP-70 results in cell death. See Figure 7 in the article by Wagner et al that begins on page 436.
Signaling through the B-cell receptor has emerged as a critical pathway in CLL. The aberrant expression of the T-cell receptor ζ-associated protein (ZAP-70) in CLL cells is an unfavorable prognostic marker and is also associated with a more premature B-cell origin, where the variable heavy chain regions of the immunoglobulin genes are intact (ie, unmutated).2 Although aberrant ZAP-70 expression appears to enhance B-cell receptor signaling, the underlying mechanism is unclear and unrelated to its kinase activity.3 Interestingly, unmutated ZAP-70+ CLL cells have previously been noted to have a proliferative response to agonists of the innate immune system receptor Toll-like receptor 9 (TLR9), whereas mutated ZAP-70− CLL cells undergo apoptosis in response to TLR9 agonists, suggesting that the innate immune system contributes to the aggressive phenotype of unmutated ZAP-70+ CLL.4 This response also predicts outcomes in patients.5
Wagner et al report on the mechanism that integrates the innate immune system with the B-cell receptor signaling pathway that underlies the heterogeneity of response that CLL cells have (proliferation vs apoptosis) in response to TLR9 agonists like cytosine guanine dinucleotide (CpG). The authors find that activation of TLR9 via CpG induces immunoglobulin (Ig)M secretion via a Syk- and ZAP-70–dependent pathway. The secretion of IgM in turn activates the B-cell receptor, leading to a survival signal only in ZAP-70+ CLL cells and associated with degradation of the proapoptotic protein Bim (see figure). Blockade of Syk signaling or Btk signaling with small molecule drugs abrogated secretion of IgM and changed the effect of CpG stimulation from prosurvival to proapoptotic in ZAP-70+ CLL cells. This is an important finding in that it identifies a connection between innate immune signaling and B-cell receptor signaling in CLL and the mechanism behind it, and it provides a potential scientific explanation for the observed heterogeneity between ZAP-70+ and ZAP-70− CLL.
In the TCL1 mouse model of CLL, both antigen-dependent and antigen-independent B-cell receptor signals contribute to disease progression.6 The findings by Wagner et al now add a differential signal from innate immune signal (CpG) that leads to different outcomes depending on ZAP-70 expression and Syk activation, but using a pathway that nevertheless depends on B-cell receptor activation (via IgM secretion). This suggests that B-cell receptor signaling has both direct and indirect effects on the pathophysiology of CLL.
The authors first demonstrate the divergent effects of CpG-induced TLR9 activation in ZAP-70+/− and mutated/unmutated CD38+/− CLL cells. The antiapoptotic effect of CpG in ZAP-70+ CLL cells was associated with degradation of Bim. Based on the immune activity of lymphoid cells, the authors hypothesized that CpG activation of TLR9 resulted in secretion of immunologically active proteins; experiments with conditioned media confirmed the differential effect of CpG on ZAP-70+ vs ZAP-70− CLL cells. There was no significant secretion of interleukin (IL)-2, IL-5, IL-21, or BAFF, and no difference in IL-10 secretion. Nevertheless, an unbiased screen for potential soluble factors that could be induced by TLR9 activation revealed that CpG induced the expression of 33 proteins and reduced expression of 9 proteins; of these, IgM emerged as the critical factor that differentiated TLR9 activation–induced survival vs cell death of CLL cells expressing or not expressing ZAP-70. However, IgM alone was necessary but not sufficient for the survival of ZAP-70+ CLL, as addition of purified IgM did not recapitulate the findings. TLR9 activation in ZAP-70–positive CLL cells also resulted in phosphorylation of CD79a and Syk, suggesting that innate immune activation of CLL cells involves ZAP-70, Syk, and autocrine activation of the B-cell receptor via secreted IgM. In this way, ibrutinib-mediated interruption of B-cell receptor signaling blocks the proliferative induction of TLR9 activation in a similar manner to other cytokines and signals from the tumor microenvironment.7 The complexity and positive feedback loops of this pathway also imply that interruption of any part of it, and particularly B-cell receptor signaling and activation of the innate immune system, could have a significant impact on the physiology of CLL.
Conflict-of-interest disclosure: The author declares no competing financial interests.
