Ex Vivo Evidence for the Combination of Immune Checkpoint Inhibition with TGF-β Blockade to Enhance Anti-Tumor T Cell Responses in Multiple Myeloma

Immunosuppressive milieu of multiple myeloma is associated with various cellular and non-cellular factors that foster immune escape leading to tumor progression. While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti-tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunophenotypic and functional characteristics of tumor antigen-specific T cells in patients with multiple myeloma. In addition, using direct ex vivo experimental techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti-myeloma responses of the tumor-specific T cells.

We first tried to define and characterize CD8⁺ T cell population capable of specifically recognizing and reacting to myeloma cells in bone marrow of myeloma patients using MHC multimer technique. In selected patients, we could successfully define CD8⁺ T cell population specifically recognizing the HLA-A*0201-restricted epitopes (either "SLLMWITQC" or "LLLGIGILV"), included in myeloma tumor antigens NY-ESO-1 and HM1.24. The vast majority of myeloma-antigen specific CD8⁺ T cells expressed high level of PD-1 and also co-expressed other types T cell inhibitory receptors. More strikingly, PD-1⁺ myeloma-specific CD8⁺ T cells had a distinct pattern of transcriptional factor expression, high level of Eomes and low level of T-bet (Eomes^hiT-bet^lo), indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 binding might not be enough to reinvigorate their anti-myeloma activity.

Consistent with the immunophenotypes of myeloma-specific CD8⁺ T cells, malignant plasma cells in bone marrow of myeloma patients, defined as CD14^-CD19^-CD138⁺CS1⁺CD56^hi, also expressed PD-L1 abundantly, compared to normal plasma cells, suggesting that PD-1/PD-L1 axis plays a major role in making myeloma-recognizing T cells unresponsive to TCR stimulation. Interestingly, in addition to tumor cells, various types of immune cells comprising myeloma microenvironment also highly express PD-L1. Indeed, in response to ex vivo TCR stimulation with anti-CD3, CD8⁺ T cells from myeloma bone marrow showed lower proliferation and reduced production of anti-tumor effector cytokines (INF-γ and TNF-α), compared to marrow-infiltrating CD8⁺ T cells of diffuse large B cell lymphoma and Hodgkin lymphoma patients with extensive bone marrow involvement. However, even in the presence of anti-PD-1, myeloma-specific responses of marrow-infiltrating CD8⁺ T cells was only modestly improved in terms of proliferation and cytokine production.

Next, we investigated whether blocking TGF-β signaling in combination with PD-1/PD-L1 axis blockade could restore the function of marrow-infiltrating CD8⁺ T cells of myeloma patients, since TGF-β produced by clonal plasma cells and bone marrow stromal cells is critical in immunosuppressive microenvironment of multiple myeloma. To this end, we found that combination of TGF-β signaling blockade (either anti-TGF-β1 or Galunisertib, a small molecule inhibitor of TGF-β receptor I) with anti-PD-1 significantly increased the frequencies of IFN-γ- and/or TNF-α-producing CD8⁺ T cells in response to ex vivo TCR stimulation, compared to a single PD-1 or a single TGF-β blockade. Likewise, myeloma antigen-specific proliferation of CD8⁺ T cells was significantly enhanced with addition of TGF-β signaling blockade.

Taken together, although PD-1/PD-L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD-1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell-intrinsic and -extrinsic factors. Our results provide an ex vivo evidence of incorporating TGF- β signaling blockade to immune checkpoint inhibition to enhance anti-tumor T cell responses in multiple myeloma