Scheme 1. Schematic of BSA/TPA-Erdn mediated ICD immunotherapy for MM treatment.

Summary High expression of the inhibitory receptor programmed cell death ligand 1 (PD‐L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD‐L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD‐1)/PD‐L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD‐L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD‐1/PD‐L1 pathway in BMSC‐mediated regulation of CD8+ T cells. The data showed that the expression of PD‐L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81 ± 1·61 versus 2·78± 0·70%; P < 0·001). Furthermore, the PD‐1 expression on CD8+ T cells with NDMM patients was significantly higher than that in normal controls (43·22 ± 2·98 versus 20·71 ± 1·08%; P < 0·001). However, there was no significant difference in PD‐1 expression of CD4+ T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co‐culture assays revealed that BMSCs significantly suppressed CD8+ T cell function. However, the PD‐L1 inhibitor effectively reversed BMSC‐mediated suppression in CD8+ T cells. We also found that the combination of PD‐L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+ T cells through the PD‐1/PD‐L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells to promote the immune escape of MM.

Objective: As an important negative regulatory factor of immunological cells, Tim3 plays a regulating role in tumor immune microenvironment. The purpose of this study was to investigate the expression of Tim3 on MM cells and its effect on the proliferation and apoptosis of MM cells, as well as its potential mechanism. Methods: In this study, the expression of Tim3 was detected on myeloma cells (CD38 + CD138 + cells) of bone marrow by flow cytometry (FCM) from 167 patients with MM and 51 healthy donors as controls and making correlation analysis with related clinical indexes. In vitro, MM cell lines (RPMI-8226 and U266) were treated with Tim3 knockdown alone, bortezomib alone and combination of Tim3 knock-down and bortezomib, then cell proliferation, cell apoptosis and downstream signaling pathway were detected by CCK-8, FCM, RT-PCR and western blot. Results: The expression of Tim3 on myeloma cells in MM patients was significantly higher than normal control group and positively correlated with b 2 microglobulin, creatine, and plasma cells of bone marrow, negatively correlated with hemoglobin and red blood cells. In vitro, we validated the high expression of Tim3 in RPMI-8226 and U266 cell lines. After Tim3 knock-down, the cell proliferation was inhibited and cell apoptosis was induced, the relative mRNA and protein expression of Tim3 and NF-kB signal pathway (PI3K, AKT, mTOR, NF-kB) were significantly decreased. Also, the cell proliferation was inhibited, cell apoptosis was increased, the relative mRNA and protein expression of NF-kB were decreased significantly in combination group than bortezomib or Tim3 knock-down group. Conclusions: The high expression of Tim3 on MM cells is associated with progression of MM patients. Tim3 maybe regulate the proliferation of MM cells via NF-kB signal pathway. Down-regulation of Tim3 expression can inhibit proliferation and induce apoptosis of MM cells, also has an additive inhibitory effect of bortezomib on NF-kB signaling pathway, then inhibit proliferation and induce apoptosis. Therefore, Tim3 may be a potential target for the treatment of MM.

Noncoding RNA (ncRNA) is involved in the occurrence, development, metastasis, and drug resistance of tumors and involves a variety of biological functions. In addition, miRNA can regulate proliferation and migration and even regulate epigenetics to promote the development of multiple myeloma (MM). However, the mechanism of ncRNA involved in MM is still unclear, and there are many unknown ncRNAs to be explored. This research is aimed at discovering the unknown lncRNA in MM through high-throughput sequencing and to study the mechanism and role of competitive endogenous RNA (ceRNA) involved in the pathogenesis of MM for the development of novel molecular markers and potential new targeted drugs. We screened out 262 new lncRNAs with statistical differences by RNA sequencing and selected the lncRNA MSTRG.29039.1 according to the expression and function of lncRNAs and their target genes in MM. We verified that MSTRG.29039.1 and its target gene OSMR were highly expressed in MM. After knockdown of MSTRG.29039.1 in MM cell lines, the expression of OSMR was decreased, and the expression of hsa-miR-12119 was upregulated which can also promote cell apoptosis and inhibit proliferation. Then, we knocked down hsa-miR-12119 and MSTRG.29039.1, we found that apoptosis of MM cells was reduced, and cell proliferation was increased compared with just knocking down hsa-miR-12119. We further verified the direct binding relationship between MSTRG.29039.1 and OSMR by the dual-luciferase reporter assay system. Thus, MSTRG.29039.1 can competitively bind with miRNA to counteract the inhibitory effect of miRNA on OSMR, which regulates cell proliferation and apoptosis through the JAK2/STAT3 pathway. In a conclusion, lncRNA MSTRG.29039.1 could promote proliferation by sponging hsa-miR-12119 via the JAK2/STAT3 pathway in multiple myeloma. This may be a molecular marker and a potential therapeutic target for MM.

Background: Multiple myeloma (MM) is a clonal disorder characterized by the proliferation of plasma cells and their accumulation within the bone marrow (BM). The flow cytometric analysis is an essential method for the hematological diseases because of high sensitivity.Aims: This study evaluates the indication role of malignant plasmacytes (PCs) in BM detected by flow cytometry for the risk stratification of MM.Methods: Whole BM samples from 92 newly diagnosed MM patients were included in the study. We collected 10 6 cells each sample by flow cytometry. Then we analyzed the correlation between the malignant PCs in BM and the characteristics of patients.Results: In this study, patients were stratified according to different baseline characteristics and the median ratio of the malignant PCs were compared. The significant statistical differences (p < 0.05) were: Hb < 100 g/L versus ≥100 g/L; β2-microglobulin <3.5 mg/dL versus 3.5-5.5 mg/dL versus >5.5 mg/dL; LDH > 250 U/L versus LDH 250 U/L; ISS I versus ISS II versus ISS III; R-ISS I versus II versus III. The detailed data are showed in Table 2. The significant correlations were observed between the malignant PCs in BM and (Figure 1): plasma cell of biopsy, hemoglobin, β2-microglobulin, lactate dehydrogenase (LDH), creatinine. "Double hit" or "triple hit" are defined as containing any two or three of the high risk cytogenetic abnormalities (t(4;14), t(14;16), t(14;20); del17q; TP53 mutation; 1q21 gain) by mSMAR. "Double or triple hit" had independently unfavorable significance for overall survival. As expected, the malignant PCs of "double or triple hit" group is significantly higher than the group B (one high risk genetic factor) and the group A (normal cytogenetic) (p < 0.0001 and p < 0.019). Conclusion:Multiparametric flow cytometry is a highly sensitive method to identify and quantify malignant PCs. And the ratio of malignant PCs detected by MFC showed strongly correlation with the severity of the pathology of MM. Malignant PCs in BM detected by flow cytometry could be regarded as a predictor for the risk MengYue Tian and ZhaoYun Liu contributed equally to this study.