A Proliferation Inducing Ligand (APRIL) Directly Impacts Immune Regulatory Subsets to Regulate Suppressive Multiple Myeloma Bone Marrow Microenvironment

A proliferation inducing ligand (APRIL), a paracrine factor produced by multiple bone accessory cells in the bone marrow (BM) niche, promotes myeloma cell progression in vivo predominantly through MM-specific receptor B cell maturation antigen (BCMA) (Blood. 2016;127:3225). Here, we investigate whether APRIL directly acts on small non-MM immune subpopulations to influence suppressive BM microenvironment and further define molecular mechanisms regulating these processes. Using flow cytometric analysis, transmembrane activator and calcium modulator (TACI), the other APRIL receptor less frequently expressed and at lower levels than BCMA on MM cell membrane, is expressed at increased levels on CD4+CD25high vs CD4+CD25- T cells from MM patients (n=10). In contrast, BCMA is confirmed undetected in T cells. Specifically, mean fluorescence intensity (MFI) of TACI is appreciably and consistently higher (> 6-fold) on CD4+CD25highFoxp3+/high regulatory T cells (Treg) of BM and peripheral blood (PB) compartments, when compared with conventional T cells (Tcon) (CD4+CD25-) from the same individual (n=9, p<0.02). TACI levels are strongly linked to levels of Foxp3 and IL-10, with highest expression in the IL10+Foxp3+ subset within Foxp3+ Tregs of paired BM and PB samples from MM patients (n=17). Significantly, TACI mRNA is also elevated (>10-fold) in purified Tregs vs autologous Tcon (n=12, p<0.01), associated with upregulated immune inhibitory genes related to Treg, i.e., Foxp3, CTLA4, IL-10, CD38, but not SLAMF7. As expected, BCMA is undetected in any T cell subsets. Importantly, APRIL preferentially induces expression of key growth (CCND1, CCND2) and survival (BCL2, BCL2L1/Bcl-xL) genes in Treg than paired Tcon, confirming elevated TACI levels in Treg vs autologous Tcon (n=4). APRIL more potently stimulates proliferation of Treg than Tcon, as evidenced by > 3-fold increases in CFSE-dilution fractions, [³H]thymidine incorporation, and luminescent-based cell viability assays (n=3). In the presence of Treg, APRIL further suppresses proliferation of autologous Tcon (n=3) stimulated by anti-CD3/CD28 beads in a dose-dependent manner. These results indicate that APRIL further augments suppressive effects of Treg on autologous Tcon in the ex vivo co-cultures. MM-induced Treg (iTreg) can be generated in co-cultures of MM cells with PB mononuclear cells or Tcon, reflecting increased Tregs linked to disease progression. APRIL further augments generation of iTreg in CD4+ and CD8+ T subsets by multiple MM cell lines co-incubated with CD3-enriched T cells or Tcon. Furthermore, osteoclasts, a major physiological source of APRIL in the BM, significantly enhance generation of iTregs induced by multiple MM cell lines. In contrast, APRIL by itself cannot convert Tcon into iTreg, validating a lack of direct impact due to the absence of TACI in paired Tcon. APRIL preferentially induces anti-inflammatory cytokines IL-10 and immuno-suppressive protein PD-L1 in TACI^high Treg vs paired TACI^low/- Tcon. These results further support a suppressive role of APRIL on Tcon via Treg-mediated secretion of immune-inhibitory cytokines. Finally, APRIL upregulates CD19+CD24^highCD38^high B regulatory cells (Breg) which interact with MM cells in the BM to confer an immunosuppressive microenvironment. Importantly, APRIL-promoted Breg is associated with IL-10 upregulation, which is blocked by a blocking APRIL monoclonal antibody. Taken together, we are the first to characterize significantly high TACI expression that is functional in immune checkpoint Treg and Breg subsets which do not express BCMA. Importantly, we identify additional biological functions of APRIL in regulating immunosuppression via specific TACI- but not BCMA-mediated signaling cascades in the MM BM milieu, further supporting targeting this mechanism-based immunotherapeutic pathway to simultaneously target MM cells and overcome immunosuppression.