Immunologic Characterization of Tumor Antigen-Specific T Cells in Multiple Myeloma: Implication in Immune Checkpoint Blockade

Various immune checkpoint inhibitory molecules expressed on tumor-recognizing T cells are a critical component of the immunosuppressive tumor microenvironment. Trials of monoclonal antibody drugs targeting immune checkpoint have achieved noteworthy clinical benefit in many types of solid tumors, including melanoma and non-small cell lung carcinoma, by blocking inhibitory signals and restoring anti-tumor immune response. In contrast, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant single-agent anti-tumor efficacy. To predict and improve the clinical responsiveness to immune checkpoint blocking treatment in multiple myeloma, explicit functional characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunologic characteristics of marrow-infiltrating myeloma antigen-specific T cells, focusing on the expression of T cell inhibitory molecules.

First, we examined the expression of immune checkpoint ligands by malignant plasma cells in bone marrow of newly diagnosed multiple myeloma patients, using multicolor flow cytometry. As a result, we found that malignant plasma cells, defined as CD14^-CD19^-CD138⁺CS1⁺CD56^hi, expressed PD-L1 abundantly. Interestingly, frequency of PD-L1-expressing plasma cells in bone marrow correlated positively with tumor burden and clinical stage. Several immune checkpoint ligands, including PD-L1 and galectin-9, were also expressed in various cellular components of bone marrow other than tumor cells.

Next, we investigated the immunophenotypes of marrow-infiltrating CD8⁺ T cells. Marrow-infiltrating CD8⁺ T cells showed significantly higher PD-1 expression in multiple myeloma patients, compared to control group. In myeloma patients, PD-1⁺ CD8⁺ T cells were more enriched in bone marrow compartment, than in peripheral circulation. PD-1⁺ marrow-infiltrating CD8⁺ T cells co-expressed other types of T cell inhibitory receptors including Tim-3, LAG3 and TIGIT.

In selected patients, we could successfully define CD8⁺ T cell population specifically recognizing the HLA-A2-restricted epitope "LLLGIGILV", included in myeloma tumor antigen HM1.24, using MHC multimer technique. We further characterized the immunophenotypes of myeloma antigen-specific CD8⁺ T cells. Myeloma antigen-specific CD8⁺ T cells were exclusively enriched in effector-memory T cell population and vast majority of them expressed high level of PD-1. They also express other types of T cell inhibitory receptors simultaneously. Strikingly, myeloma antigen-specific CD8⁺ T cells expressed high level of Eomes, indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 axis might not be sufficient to reinvigorate their anti-myeloma activity. When marrow-infiltrating T cells of myeloma patients were ex vivo cultured and stimulated with myeloma antigens, however, their antigen-specific proliferation was significantly restored in the presence of PD-1 blocking in some cases.

Collectively, although PD-1/PD-L1 axis acts as a major component of immunosuppressive microenvironment in multiple myeloma, the clinical efficacy of PD-1 blockades in multiple myeloma might be hampered by (1) heterogeneity and multiplicity in expression patterns of T cell inhibitor receptors and (2) molecular depth of T cell exhaustion. Therefore, for successful rejuvenation of anti-myeloma T cell responses, combination immunotherapeutic approaches are required. Our results provides a basis for utilizing multiple immune checkpoint inhibitors targeting different molecules and incorporating immunomodulatory agents into the immune checkpoint inhibitor in treatment of multiple myeloma.