PD-1 Blockade Reinvigorates Bone Marrow CD8⁺ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients.

To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8⁺ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8⁺ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β.

First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8⁺ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8⁺ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1⁺CD8⁺ T cells had a higher percentage of Eomes^hiT-bet^lo cells than PD-1^-CD8⁺ T cells. These data demonstrate that PD-1-expressing CD8⁺ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8⁺ T cells specific to myeloma antigens NY-ESO-1 and HM1.24.

Next, we investigated proliferation and cytokine production of BM CD8⁺ T cells from MM patients. BM CD8⁺ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8⁺ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8⁺ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8⁺ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1_157-165 and HM1.24_22-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8⁺ T cells from MM patients.

It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients.

We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8⁺ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8⁺ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8⁺ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8⁺ T cells from HLA-A2⁺ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8⁺ T cells from MM patients in the presence of TGF-β inhibitors.

Taken together, BM CD8⁺ T cells and myeloma antigen-specific CD8⁺ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8⁺ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8⁺ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM.