Relationship between Bone Marrow PD-1 and PD-L1 Expression and the Presence of Osteolytic Bone Disease in Multiple Myeloma Patients
Authors: Costa Federica , Bolzoni Marina , Vescovini Rosanna , Accardi Fabrizio , Dalla Palma Anna Benedetta , De Luca Federica , Marchica Valentina , Toscani Denise , Vicario Emanuela , Storti Paola , Aversa Franco , Giuliani Nicola 
Published: October 2019

Abstract
Alterations of the bone marrow (BM) immune-microenvironment characterize the progression of monoclonal gammopathies and the development of osteolytic bone disease in multiple myeloma (MM). MM patients exhibit immune dysfunctions as impaired dendritic, NK and T cells, whereas the onset of MM osteolytic lesions is associated to an increased prevalence of Th17 cells. Recently, the pathophysiological role of the programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) pathway together with an increase of myeloid derived suppressor cells (MDSCs) in the induction of tumor tolerance and immune evasion has been underlined with a therapeutic relevance. However, unclear data on the expression profile of PD-1/PD-L1 axis in MM patients have been reported and it is not known if this axis could be related with the presence of osteolytic bone disease.
In order to elucidate these aspects, we analyzed a total cohort of 80 consecutives patients with monoclonal gammopathies, including 15 monoclonal gammopathy of undetermined significance (MGUS), 23 smoldering MM (SMM), 21 newly diagnosed MM (MMD) and 21 relapsed/refractory MM (MMR) patients. The presence of bone disease was checked by low-dose computerized tomography (CT) with or without positron emission tomography (PET) scan and by X-rays skeletal survey in 11 MM patients. 87% of MM patients displayed osteolytic lesions. High bone disease (HBD) was defined by the presence of 3 or more osteolytic lesions (62% of our cohort). Patients without bone lesions or with minus of 3 lesions were defined as low bone disease (LBD). BM mononuclear cells were analyzed by flow cytometry, evaluating plasma cells (PCs) (CD138+), monocytes (CD14+) and T cells (total CD3+, CD3+CD4+ and CD3+CD8+). PD-L1 (CD274) expression was evaluated on CD138+ and CD14+ cells, and PD-1 (CD279) on CD3+, CD4+ and CD8+ cells. Lastly, in a subgroup of patients we analysed MDSC populations, including both granulocytic (gMDSCs) (CD11b+HLA-DR-CD14-CD15+) and monocytic MDSCs (mMDSCs), (CD11b+HLA-DR-/lowCD14+CD15-).
The percentage of BM CD3+PD-1+ cells increased from MGUS to MMR patients with a significant trend (p=0.004). Indeed, patients with active MM (MMD and MMR) showed both higher % of CD3+PD-1+ cells (median value: 48.5% vs 40.6%, p=0.001) and PD-1 median fluorescence intensity (MFI) on CD3+ (median value: 18.9 vs 16.7 MFI, p=0.024) as compared to patients with SMM and MGUS. CD4+PD-1+, but not CD8+PD-1+ cells are increased in active MM compared to SMM and MGUS patients (p=0.023). On the other hand, any significant difference was not observed in the PD-L1 expression on both BM CD138+ and CD14+ cells across patient groups. The percentage of BM MDSC populations did not significantly change across the different monoclonal gammopathies (p=0.14); moreover, comparing MM with SMM and MGUS patients, the % of gMDSCs was significantly reduced (median %: 12.5% vs 17%, p=0.04) and the % of mMDSCs was increased (median %: 0.36% vs. 0.24%) without reaching a statistical significance.
Focusing on MM bone disease, we found that osteolytic MM patients displayed higher CD4+/CD8+ ratio compared to non-osteolytic ones (p=0.043). Regarding the PD-1 expression, the % of CD3+PD-1+ cells did not change (p=0.192), whereas the % of CD8+PD-1+ cells was significantly reduced in osteolytic patients compared to non-osteolytic ones (p=0.016). Consistently, among the BM CD8+ cells, a significant decrease of %PD-1+ cells was found in HBD vs LBD MM patients (median value: 46.9% vs 57.4%, p=0.045). Interestingly, when compared to LBD MM patients, HBD MM patients displayed a significant reduction of PD-L1 expression on both BM CD138+ PCs (median MFI value: 13.3 vs 21.6 MFI, p=0.007) and CD14+ cells (median MFI value: 15.4 vs 23.8 MFI, p=0.007). In a multivariate analysis, PD-1+ expression on CD8+ cells and PD-L1 MFI on CD138+ were significant independent factors related to the presence of HBD.
In conclusion, our study indicates that the frequency of PD-1+ T cells increases across the progression of the monoclonal gammopathies. On the other hand, for the first time, we show in MM patients a significant relationship between the presence of extensive osteolytic bone disease and a reduced expression profile of BM PD-1/PD-L1 axis on CD8+ and CD138+ cells. We hypothesize that a less immune-suppressive profile could be related to the development of osteolysis consistent with the negative cross talk existing between PD-1/PD-L1 axis and Th17 cells.

Disclosures
Aversa: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding.