Breaking T-cell tolerance to fight multiple myeloma.

Objective To deplore the immunoregulatory function changes of mesenchymal stem cells(MSCs)from multiple myeloma(MM)patients and its effects on the pathogenesis of myeloma bone disease.Methods MSCs from MM patients and normal controls were isolated and the immunophenotype was detected.Real-time PCR was performed to detect the expressions of TGF-β1,TGF-β2,TGF-β3,IL-6,IL3,TNF-α,FasL and RANKL of MSCs.Transwell coculture systems were performed between MSCs and T cells.Lymphocyte proliferative assay was employed to detect the effect of MSCs on T cell proliferation.The effect of MSCs on T cell cycle and T cell activation markers CD25 and CD69 expression were analyzed by flow cytometry.Cleaved caspase 3 protein by western blot and hoechst 33258 staining were employed to detect the apotosis of T cells.Influence of T cells on the osteogenesis potential of MSCs were detected by Von kossa stain,real-time PCR and Western blot.Results MSCs from both MM patients and normal controls possessed similar morphology and immunophenotypes.MM derived MSCs exhibited increased expressions of TGF-β1,IL-6,IL-3,TNF-α and RANKL and decreased expression of TGF-β2,TGF-β3 and FasL.The inhibitory effect of MM derived MSCs on T cell proliferative ability was attenuated compared to control MSCs.MSCs from normal controls silence more T cells in Go/G1 phase than those from MM patients.The daupening effect of MM derived MSCs on activation-induced T apoptosis seemed to be enhanced.Expression of T cell activation markers were significantly inhibited by MSCs from normal controls.Both T cells cocultured with MM deprived MSCs and T cells directly from MM patients inhibited osteogenesis potential of MSCs from normal controls.Conclusion MSCs from MM patients showed impaired immunoregulatory capability on T cells.The activated T cells,in turn,inhibited the osteogenesis potential of MSCs.This may participate in the pathogenesis of myeloma bone disease. Key words: Multiple myeloma; Mesenchymal stem cells; T cells; Immunoregulatory capability; Myeloma bone disease

The Role of Macrophage Migration Inhibitory Factor (MIF) in Regulation of T-Cell Activity in Multiple Myeloma

Enhanced Antitumoral Efficacy of ARI0002h CAR-T with CD28-TMD in Normal and Low BCMA Expressing Myeloma Cells

Enhanced T cell suppression is directed toward sensitive circulating B cells in multiple myeloma.

Therapeutic monoclonal antibodies (mAbs) have revolutionized treatment options for many cancers. However, the extension of effective mAb-mediated therapy for multiple myeloma (MM) has been limited thus far by lack of an optimal target. Results of experimental agents targeting a number of potential candidate molecules expressed on the surface of MM tumor cells have been reported (including CD20, CD40, CD56, CD74, CD138, CD200, epidermal growth factor receptor, and beta-2 microglobulin), as have those of mAbs targeting other proteins involved in the MM immunologic microenvironment (interleukin-6, vascular endothelial growth factor, and killer immunoglobulin-like inhibitory receptors). Despite these efforts, the benefit of mAb-based therapy directed at these targets in MM remains incompletely articulated. The most promising target for mAb-mediated MM therapy to date seems to be CS1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), a human membrane glycoprotein and member of the immunoglobulin superfamily. CS1 cell surface expression seems nearly ubiquitous in both primary MM tumor cells as well as virtually all MM cell lines. Soluble CS1 is also detectable in serum of patients with MM and correlates with both disease stage and need for therapy. Furthermore, CS1 is expressed in some leukocyte subsets (eg, natural killer [NK] cells and some T-cell subsets) but not in the majority of healthy tissues or other forms of malignancy. Relatively little is known regarding the function of CS1 in MM cells. The CS1 gene resides on the long arm of chromosome 1 (1q23.11q24.1), and gains of chromosome 1q are frequent alterations in MM tumor cells; thus CS1 overexpression may contribute to the pathobiology of the disease. CS1 has been shown to colocalize with CD138 in uropod membranes on the surface of polarized MM cells, which may promote cell-cell adhesion. Moreover, a direct role for CS1 in MM cell adhesion to, and interaction with, bone marrow stromal cells (BMSCs) has been conclusively demonstrated. This is important for MM pathobiology, because the protective effect of BMSCs comprises a substantial component of therapeutic resistance in this tumor type. Much of what is known about human CS1 signaling, however, is derived from studies in other lymphocyte subsets. In NK cells, for example, CS1 is a self ligand that promotes activation through selective expression of the adaptor protein EAT-2. In fact, CS1 may serve an inhibitory role in T cells that lack EAT-2. CS1 ligation independent of Fc R ligation in NK cells results in signal transduction events that activate several pathways including phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and phospholipase C gamma. Whether EAT-2 is expressed in MM cells remains unknown; similarly, the signal transduction events consequent to CS1 ligation in MM cells are a matter of ongoing investigation. Elotuzumab (formerly HuLuc63), a humanized immunoglobulin G1 anti-CS1 mAb, has been shown to impair MM cell adhesion and exert direct (albeit modest) effects on primary MM cell survival in vitro, either alone or in the presence of BMSCs. Its principal mechanism of action, however, is thought to be antibody-dependent cellular cytotoxicity (ADCC). Here, ADCC is mediated by NK cells through interaction of their Fc RIIIa receptor with the Fc binding region of the therapeutic antibody. Preliminary data suggest that elotuzumab may also directly activate NK cells through direct CS1 ligation. Interestingly, elotuzumab-associated NK-cell cytotoxicity via ADCC is directed specifically against CS1-bearing MM tumor cells and not fraternal CS1-bearing NK cells, as demonstrated in both in vitro and correlative studies performed during clinical investigation. Figure 1 summarizes the known functions of CS1 in MM and apparent

The immunosuppressive microenvironment plays a crucial role in T-cell immunodeficiency in multiple myeloma (MM). Overexpression of T-cell immunosuppressive receptors, including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain-containing-3 (Tim-3), may be related to tumor immunosuppression and poor prognosis, and the malignant bone marrow (BM) microenvironment may contribute to such immunosuppression. The purpose of this study was to analyze the distribution of PD-1+ and/or Tim-3+ T cells in different T-cell subset in patients with MM. The expression of PD-1 and Tim-3 with exhausted (CD244+ and CD57+ ) CD3+ , CD4+ and CD8+ T cells between BM and peripheral blood (PB) from 10 patients with untreated MM was detected by multicolor flow cytometry assay. A significant increase in both PD-1+ CD57+ and Tim-3+ CD57+ CD3+ T cells and PD-1+ Tim-3+ CD3+ T cells was detected in PB from patients with MM compared with 10 healthy individuals (HIs), and the alteration was mostly in the CD8+ T-cell subset. Significant higher percentage of PD-1+ CD3+ T cells was found in BM compared with PB from patients with MM. The level of PD-1+ Tim-3+ CD3+ , CD4+ , and CD8+ T cells was high in BM group compared with PB. Moreover, PD-1+ CD244+ or PD-1+ CD57+ CD3+ T cells, particularly PD-1+ CD244+ and PD-1+ CD57+ CD8+ T cells were significantly higher in BM than in PB. In addition, limited dynamic detection data from three MM cases who achieved complete remission after treatment showed that the numbers of either PD-1+ or PD-1+ Tim-3+ T cells in different T-cell subsets were decreased in both BM and PB. We characterized the distribution of PD-1 and TIM-3 concurrent with exhausted CD3+ , CD4+ and CD8+ T cells between BM and PB from patients with MM. Higher numbers of PD-1+ CD244+ or PD-1+ CD57+ CD3+ T cells in BM from patients with MM may contribute to mediate the BM immunosuppressive microenvironment. Although heterogeneous alterations in Tim-3+ T cells may represent a complex immunosuppressive pattern in MM. Overall, higher levels of PD-1+ CD244+ or PD-1/Tim-3+ CD57+ CD8+ T cells may be a major reason for lower T-cell activation and T-cell immunodeficiency in MM.

CS1-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells and T Cells Enhance In Vitro and In Vivo Anti-Tumor Activity Against Human Multiple Myeloma

Objective To investigate T-cell receptor(TCR)ζchain gene expression level in peripheral blood T cells from patients with multiple myeloma(MM),thereby to estimate the feature of T cells activation status.Methods Real-time PCR with SYBR Green I technique Was used for detecting TCR ζchain expression level in peripheral blood mononuelear cells(PBMC)of 24 cages with MM and 24 normal individuals.β2-microglobulin(β2M)gene expression was used as an endogenous reference.Relative changes in TCRζchain expression level were analyzed by the 2-△α×100%method between patients with MM and normal individuals.Results Compared with normal individuals,TCRζchain gene expression was obviously down regulated in PBMC from patients with MM(P=0.019).The expression level of TCR ζchain gene is not significantly age-associated in MM patients[(1.83±1.72)%,(3.46±2.75)%](P=0.525).Conclusion This is the first description in the expression feature of TCRζchain gene in MM patients.TCRζchain expression Was decreased in most of MM patients,which might be related to celhar immunodeficiency. Key words: Multiple myeloma; Genes,T-cell receptorζ; Real-time PCR

Tigit: The Potential Mechanisms of Relapse in Multiple Myeloma Following Anti-BCMA CAR-T Therapy and a Promising Target for Improving CAR-T Efficacy

Multiple myeloma (MM) is a clonal B cell malignant disease that is characterized by the proliferation of plasma cells in the bone marrow, leading to bone destruction, hematopoietic failure and producing M protein. In recent years, the remission rate and survival of MM patients have been significantly improved with the application of new drugs. But most patients still experience relapse, progression or drug resistance. It′s urgent to seek new treatment strategies of MM. Chimeric antigen receptor-modified T cell (CAR-T) immunotherapy is one of the important biotherapy techniques in the field of tumor immunotherapy in recent years. CAR-T can kill MM cells by specifically identifying target antigens and provide new therapy for patients with MM. This article intends to expound the relevant targets, existing problems and the measures to improve the effectiveness of CAR-T in treatment of MM. Key words: Multiple myeloma; Immunotherapy, adoptive; Molecular targeted therapy; Clinical protocols; Chimeric antigen receptor T cell immunotherapy

Exploring LAG-3 Expression in Multiple Myeloma Patients Following Autologous Stem Cell Transplant

The Peripheral Blood Stem Cell Immunome Demonstrates Abnormal Immune Effector Cells Early in the Disease Course of Multiple Myeloma and Contributes to Inferior Outcomes and Secondary Myeloid Neoplasms

T-cell subsets defined by monoclonal antibodies (OKT3, OKT4, and OKT8) were analyzed in 117 patients with monoclonal gammopathies--69 multiple myeloma (MM) (30 untreated and 39 treated), 14 Waldenström's macroglobulinaemia (WM), and 34 essential monoclonal gammopathy (EMG) patients. The percentage and absolute numbers of total T-lymphocytes (E+, OKT3+ cells) were within the normal range in all groups except for the treated MM patients, in which a decrease in the absolute number could be observed. The percentages of OKT4+ cells were significantly lower in MM (35 +/- 1.7) than in EMG patients (43 +/- 2) and controls (50 +/- 2). In contrast, OKT8 cells correspondingly increased in MM (38 +/- 1.6) compared with EMG patients (29 +/- 1) and controls (27 +/- 1). The OKT4/OKT8 ratio was lower in MM than that in EMG patients and controls (p less than 0.01) and was shown to be one of the four most significant variables in a linear discriminant analysis used to distinguish between MM and EMG groups. The MM patients in clinical stage III as well as Bence-Jones myeloma patients showed a more pronounced OKT4/OKT8 imbalance. The treatment did not influence the percent distribution of T-cell subpopulations. The patients with WM exhibit an alteration in the distribution of the T-cell subsets similar to the MM patients with a T4/T8 ratio of 1.1 +/- 0.1. This imbalance was more pronounced in WM patients with monoclonal B-lymphocytes in peripheral blood (leukaemic phase of WM). The functional significance of the altered T-cell subsets in MM and WM patients remains to be established, though it is probable that such an imbalance plays an important role in regulating these B-cell proliferations.

Objective To investigate the expression and clinical effect of myeloid-derived suppressor cells(MDSCs)in peripheral blood of patients with multiple myeloma(MM). Methods From October 2015 to June 2017, 40 newly treated MM patients in the Hematology Department of Lishui people′s hospital were selected as the MM group, 27 males and 13 females, aged(58±8)years and ranging from 48 to 76 years.In addition, 40 healthy people were selected as the control group, including 25 males and 15 females, aged(56±11)years, ranging from 45 to 74 years.In MM group, the first-line treatment was used after admission.The expression of MDSCs, TAM and Tregs were detected by flow cytometry.The level of IL-6 was detected by ELISA. Results Before treatment, MDSCs[(8.40±2.15)%], TAM[(6.33±1.34)%], Tregs[(9.25±3.68)%], IL-6[(13.40±0.80)pg/ml]in MM group were higher than those in healthy physical examination group[(0.47±0.26)%, (1.80±0.89)%, (2.42±3.31)%, (2.45±0.24)pg/ml], the difference was statistically significant(P<0.05). After treatment, MDSCs[(0.94±0.41)%], TAM[(2.64±1.12)%], Tregs[(1.92±1.51)%], IL-6[(5.62±0.56)pg/ml]in MM group were significantly lower than those before treatment(P<0.05). Conclusion MDSCs expressed regularly in different stages of MM, which was consistent with the expression trend of TAM and Tregs. Key words: Multiple myeloma; Myeloid-derived suppressor cells; Regulatory T Cells; Tumor-associated macrophages

Objective To investigate the expression levels,relevance and clinical significance of forkhead box protein (Foxp3) and interleukin (IL)-6 in multiple myeloma (MM).Methods From August 2011 to February 2013,a total of 41 MM patients in Shantou Central Hospital were collected into this study,as study group (n=41),including newly-diagnosis group (n=34) and disease progression group (n=7).According to the International Staging System (ISS) criteria,the newly-diagnosis group were divided into stage Ⅰ group (n=3),stage Ⅱ group (n=14) and stage Ⅲ group (n=17).After 4 courses of chemotherapy,38 MM patients could evaluate the efficacy.These patients were classified into after chemotherapy group (n =38).According to the efficacy,the after chemotherapy goup were divided into complete response (CR) group (n =7),partial response (PR) and minimal response (MR) group (n=16),no-change (NC) and progression disease (PD) group (n-=15).A total of 16 healthy individuals who received medical examination in the same period were included into control group (n =16).The study protocol was approved by the Ethical Review Board of Investigation in Human at Shantou Central Hospital.Informed consent was obtained from all participants.The blood specimens from the subjects were collected at the clinical laboratory.Expression levels of Foxp3 and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA).The cilinical data of the subjects were retrospectively analyzed:expression levels of Foxp3 and IL-6 were compared among different ISS stages,efficacy and before and after treatment.The correlation and clinical significance of Foxp3 and IL-6 expression levels in MM were analyzed.Results ① Expression levels of Foxp3 were significantly reduced in MM patients compared with in levels of Foxp3 healthy controls irrespective of treatment (P＜ 0.001),and considerably equal to those in the newly-diagnosis group and after chemotherapy group (P＞ 0.05).Expression levels of IL-6 were significantly higher in MM patients compared with those in healthy controls irrespective of treatment (P＜ 0.001),and considerably equal to those in the newly-diagnosis group and the after chemotherapy group (P＞ 0.05).② Expression levels of Foxp3 were reducing along with the increasing of the ISS stages in the newlydiagnosis group,but the difference wasn't statistically significant (P＞0.05).And the difference in pairwise comparison of different stages either wasn't statistically significant (P＞0.05).Expression levels of IL-6 were elevating along with the increasing of the ISS stages (P＜0.05).And there was a significant difference in IL-6 level between stage Ⅱ and Ⅲ group (P=0.009).③After chemotherapy,the expression levels of Foxp3 were reduced in order of CR group,PR+MR group and NC+PD group(P＜0.05).Foxp3 levels were significantly higher in CR group than those in PR+ MR group and NC+ PD group (P=0.012,0.001),and there was no significant difference those in between PR+MR group and NC+PD group (P＞ 0.05).Expression levels of IL-6 were elevated in order of CR group,PR+MR group and NC+PD group.IL-6 levels in NC+ PD group were significantly higher than those in CR group and PR+ MR group (P＜ 0.001),and there were significant difference in IL-6 levels between PR+MR group and CR group (P=0.028).④ There was a negative correlation between expression levels of Foxp3 and IL-6 in the study group before chemotherapy (R=-0.632,P＜0.01).Conclusions Expression levels of Foxp3 were significantly reduced in MM patients.The dynamic variation of Foxp3 levels may reflect patients' condition,and could be an important predictor of the efficacy.Expression level of IL-6 might contribute to early diagnosis,monitoring the chemotherapy efficacy and disease progression of patients with MM.The correlation between Foxp3 and IL-6 levels may reflect the dynamic balance between the tumor and the host immune response in the pathogenesis of MM. Key words: Multiple myeloma; Forkhead transcription factors; Interleukin-6; Prognosis; T-lymphocytes, regulatory