P-217: The interaction between myeloma cells and microenvironment via LILRB4 signaling promote myeloma progression

Introduction: LILRB4 is a family member of leukocyte immunoglobulin-like receptors (LILRs), which are known to play critical roles in the regulation of immune tolerance. We have suggested that LILRB4 presented to be an ideal immunotherapy target for MM. In this study, we aimed to investigate the underlying mechanisms by which LILRB4 mediated MM progression remain unknown.

Methods: To underscore the immune alterations under the impact of LILRB4 expression in MM cells, we generated LILRB4-overexpressed immunocompetent MM mouse model. A 32-color spectral flow cytometry panel was implemented for deep immunophenotyping of tumor microenvironment in LILRB4-overexpressed MM mouse model.

Results: We have already suggested that CD38⁺LILRB4⁺ MM cells tended to be initiating immature plasma cells by single-cell RNA sequencing analysis. We observed that the level of LILRB4 was significantly increased in precursor stage of plasma cells (pre-PC, CD38⁺CD138⁺CD19⁺CD56⁺) compared with mature malignant MM cells (MPC, CD38⁺CD138⁺CD19^-), which were both higher than normal PCs (NPC, CD38⁺CD138⁺CD19⁺CD56^-). We hypothesized that LILRB4 might also be a marker for MM-initiating cells. Combining the known MM-initiating marker, CD24, we showed that CD24⁺LILRB4⁺ MM cells presented morphologically less mature than the other MM cells. Further, LILRB4⁺ MM cells could form more and larger cell colonies than LILRB4^- MM cells, indicating the tumor-promoting function of endogenous LILRB4.

LILRB4 is an important immune modulating molecule. LILRB4 has been known to mediate T cell suppression. Here, we cocultured LILRB4-overexpressed or LILRB4-deleted MM cell lines with peripheral blood mononuclear cells (PBMCs) from healthy donors in vitro. We found that overexpression of LILRB4 in MM cells could induce the generation of MDSCs and decrease the infiltration of CD3⁺ T cells. Using KaLwRij-5T33 mouse model, the proportion of MDSCs were increased and the proportion of T cells were decreased in bone marrow from 5T33-LILRB4 engrafted animals. Notabaly, 5T33-LILRB4 engrafted mouse displayed more rapid disease progression compared with 5T33-empty vector transfected (EV) engrafted mice. Furthermore, the conditioned medium of LILRB4-overexpressed MM cells had same effect in inducing MDSCs. Interestingly, when we cocultured MM cells with PBMCs, the expression of LILRB4 were also significantly upregulated in MM cells. These results indicated a feedback loop between immune cells and MM cells via LILRB4-related signaling interaction, which promoted the formation of immunosuppressive microenvironment for MM cells.

Conclusions: We suggested that LILRB4⁺ MM initiating cells could modulate the microenvironment to enhance the maintenance of these cells. Interfering the LILRB4 signal interaction between MM cells and the microenvironment would be advantageous to MM treatment.