In multiple myeloma (MM), the content of T lymphocytes expressing “checkpoint” molecules PD-1, TIM-3, LAG-3, etc. is increased. Regulatory T cells (Treg) can suppress antitumor immune response and play a sufficient role in MM pathogenesis. Like effector T lymphocytes, some Tregs express checkpoint receptors PD-1, TIM-3, etc., however, the biological meaning of such expression, as well as the consequences of blockade of these receptors, are not clear. The significance of type I regulatory T cells (Tr1), which produce the immunosuppressive cytokine interleukin-10, in MM also remains unexplored. The purpose of this work was to study the content of PD-1- and TIM-3-expressing Tregs and Tr1 in patients with MM. The study included 36 patients with MM and 24 matched healthy donors. The content of CD4+CD25hiCD127-FoxP3+Tregs and IL-10-producing CD4+IL-10+Tr1 populations expressing PD-1 and TIM-3 was assessed in peripheral blood (PB) and bone marrow (BM) by flow cytometry. The relative content of circulating CD4+CD25hiCD127-FoxP3+Tregs and IL-10-producing CD4+IL-10+Tr1 was significantly higher in MM patients compared to healthy donors. A higher relative content of IL-10-producing T lymphocytes was noted compared to Treg. The relative content of Tregs and Tr1 in BM samples did not differ significantly from PB values. The proportion of Tregs expressing PD-1 and TIM-3 in patients with MM did not differ significantly from the values in healthy donors. The content of PD-1- and TIM-3-positive CD4+IL-10+T cells was significantly higher in PB samples from MM patients compared to donors.IL-10-producing CD4+T cells constitute a significant proportion of T lymphocytes in the PB and BM of patients with MM and may play an important role in the pathogenesis of MM. Their content exceeds that of CD4+CD25hiCD127-FoxP3+Treg. A relatively small number of Tregs express the checkpoint receptors PD-1 and TIM-3, no different from donors. The proportion of PD-1-/TIM-3-positive cells is ~20% of CD4+IL-10+T cells and significantly exceeds the values of healthy individuals.

T cells expressing checkpoint receptors PD-1, TIM-3 etc., are potential targets for monoclonal antibody immunotherapy in multiple myeloma (MM). However, checkpoint expressing T cell compartment includes different subsets, and their dysregulation following anti-checkpoint therapy can lead to the development of adverse events.The aim of this study was to evaluate activation markers – homeostatic cytokine receptors and transcription factors expressed by PD-1and TIM-3-positive T cells.Material and Methods. Relative counts of circulating PD-1and/or TIM-3-positive and negative T cells expressing common ɣ-chain cytokine receptors CD25, CD122, CD127, phosphorylated STAT5, and transcription factor EOMES associated with T cell exhaustion were studied using flow cytometry in 17 healthy donors, 22 MM patients with remission and 7 MM patients with progressive disease.Results. T cells expressing PD-1 and/or TIM-3 inhibitory checkpoint receptors in MM patients consisted of CD25+EOMESactivated cells, CD4+CD25+CD127-FOXP3+ regulatory T cells (Treg), CD4+CD25-EOMES+ dysfunctional cells. CD25+ T cells from healthy donors and MM patients, regardless of the expression of the studied checkpoint receptors, were EOMES-negative. No such association was found for CD122 and CD127 cytokine receptors. EOMES is a marker of T cell exhaustion for CD4+ T cells, but not for CD8+ T cells, in which it is more associated with activation. The proportion of CD4+ Tregs among circulating PD-1+ and TIM-3+ T cells was relatively low. A higher content of cytokine receptors in the population of TIM-3+ T cells may indicate the predominant involvement of TIM-3 in the control of homeostatic proliferation of mature T cells under lymphopenic conditions, while the expression of PD-1 may be more associated with the regulation of activation through T cell receptor. PD-1+ and/or TIM-3+ levels of activated, dysfunctional, and regulatory T cells can potentially be used to predict the safety and efficacy of targeted immunotherapy.

The avoidance of immune surveillance by malignant plasma cells (PCs) in multiple myeloma (MM) is mediated by different mechanisms, among which an induction of T cell exhaustion and expansion of myeloid-derived suppressor cells (MDSCs) appear to play substantial roles, but it is still a lack of data on possible MDSC-mediated induction of T cell exhaustion. The aim of the present work was to evaluate possible relationship between frequencies of MM PCs, MDSCs and phenotypically exhausted PD-1+ and TIM-3+ T cells in bone marrow (BM) samples and peripheral blood (PB) of MM patients at various disease stages. Peripheral blood (n = 88) and BM samples (n = 56) were obtained from MM patients (newly diagnosed (n = 6), patients in remission (n = 71) and with progressive disease (n = 11)). Frequencies of T cells expressing checkpoint receptors PD-1 and TIM-3, polymorphonuclear MDSCs (PMN-MDSCs, Lin-CD14-HLA-DR- CD33+CD15+/CD66b+), monocyte MDSCs (M-MDSCs, CD14+HLA-DRlow/-), early MDSCs (E-MDSCs, Lin-HLA-DR-CD33+CD15-/CD66b-), and MM PCs (CD45dimCD38+CD138+CD56+CD19-CD117+CD27- CD81-) were assessed with flow cytometry. Circulating and BM-resident PD-1+/TIM-3+T cell subsets, BM E-MDSCs, as soon as MM PCs and serum beta2-microglobulin (B2-M) levels were gradually increased in patients at different stages. Despite that, there were no associations between the markers of tumor load and the studied cell subsets. In patients in remission, BM PMN-MDSCs negatively correlated with CD4+T cells, CD4+PD-1+ and CD8+TIM-3+T cell subsets; there were positive correlations between BM E-MDSCs and CD4+PD-1+TIM-3+ cells and PB M-MDSCs and CD8+PD-1+ and (as a trend) CD8+TIM-3+T cells. We found no associations for the samples of patients at diagnosis and with progression. We can conclude that a possible mutual influence of malignant PCs, MDSCs and PD-1+/TIM-3+T cells is nonlinear, especially during a manifest tumor growth at diagnosis and progression. The detected negative correlations between resident PMN- MDSCs and T cell subsets might be associated with MDSC suppressive function, affecting both predominantly activated PD-1+ cells and exhausted TIM-3+ subsets. The positive correlations between BM E-MDSCs and CD4+PD-1+TIM-3+ cell subset and circulating M-MDSCs and PD-1+ and TIM-3+ CD8+T cells might confirm an ability of MDSCs to induce T cell exhaustion.

Myeloid-derived suppressor cells (MDSCs) play an important role in the immune response regulation in many pathologies, primarily in malignant tumors, but their role in the hematopoietic stem cell engraftment and the hematopoietic recovery after high-dose chemotherapy and autologous stem cell transplantation remains practically unexplored. This study is aimed at studying the correlation between the number of MDSC subpopulations and blood parameters at the stage of hematopoietic recovery after high-dose chemotherapy and autologous hematopoietic stem cell transplantation in patients with multiple myeloma (MM). Circulating MDSCs were assessed at the stage of leukopenia recovery (absolute leukocyte count in peripheral blood (PB) > 1 x 109/L) by flow cytometry. The number of transplanted CD34+CD45+ hematopoietic stem cells was 4.38 x 106/kg (IQR (3.1—5.6) x 106/kg). The duration of recovery from leukopenia varied from 8 to 18 days (Me 12 days). The number of MDSCs at the engraftment was not associated with the number of CD34+ cells/kg in the graft. The relative number of monocytic MDSCs (M-MDSCs, CD14+HLA-DRlow/-) directly correlated with the number of monocytes at the stage of recovery from leukopenia (R = 0.417, p = 0.002). Granulocytic MDSCs (PMN-MDSCs, Lin-HLA-DR-CD33+CD66b+) were characterized by an inverse correlation with the number of monocytes (R = -0.493, p = 0.0003) while the association with the absolute number of neutrophils was weak (R = 0.273, p = 0.048). The number of lymphocytes at the stage of recovery from leukopenia had an inverse correlation with PMN-MDSCs (R = -0.347, p = 0.014) and did not correlate with M-MDSCs. When analyzing the duration of leukopenia, an inverse correlation with this indicator was revealed for the percentage and absolute number of M-MDSCs (R = -0.347, p = 0.018 and R = -0.469, p = 0.0008, respectively). Multiple regression analysis showed dependence of the lymphopenia duration on the proportion of circulating M-MDSCs (p = 0.014) and the number of transplanted CD34+ cells/kg (p = 0.032). According to the data of multivariate analysis of variance, the number of transplanted CD34+ cells/kg and the number of M-MDSCs were significant factors for the duration of leukopenia. At the same time, such clinical parameters as the depth of response and minimal residual disease status before high-dose chemotherapy and hematopoietic stem cell transplantation, as well as the MM stage, did not affect the duration of hematopoietic recovery. Thus, the obtained results indicate the association of a higher number of M-MDSCs with a shorter duration of leukopenia after high-dose chemotherapy with autologous stem cell transplantation and indicate a positive role of M-MDSCs in hematopoietic recovery in the early post-transplant period in patients with MM.

We studied suppressor potential of myeloid-derived suppressor cells (MDSC) in multiple myeloma patients, including before and after mobilization of hematopoietic stem cells (HSC), by evaluating the expression of arginase-1 (Arg1), indolamine-2,3-dioxygenase (IDO), and PD-L1 in MDSC subsets. The study included 20 multiple myeloma patients in remission, 5 patients with progression, as well as 10 sex-and age-matched healthy donors. The expression of Arg1, IDO, and PD-L1 in circulating granulocytic MDSC (G-MDSC, Lin–HLA-DR–CD33+CD66b+), monocytic MDSC (M-MDSC, CD14+HLA-DRlow/–), and early-stage MDSC (E-MDSC, Lin–HLA-DR–CD33+CD66b–) was evaluated by flow cytometry. Multiple myeloma patients in remission were characterized by reduced expression of Arg1 in M-MDSC in comparison with donors. The expression of Arg1 in M-MDSC depended on the number of induction therapy lines performed and was significantly lower in patients who received ⩾2 lines and responded with remission. Patients with multiple myeloma progression (resistant to therapy) showed significantly increased expression of Arg1 and PD-L1 in M-MDSC, as well as increased expression of Arg1 in E-MDSC. After G-CSF-induced mobilization of HSC, the content of circulating Arg1-expressing M-MDSC increased significantly. Considering the presence of MDSC in apheresis products, MDSC suppressive activity is discussed as a factor affecting the outcomes of autologous HSC transplantation in multiple myeloma patients.

Introduction . Myeloid-derived suppressor cells (MDSCs) play an important role in restriction of the immune response and are associated with a poor prognosis in cancer. Mobilization of hematopoietic stem cells (HSCs) before high-dose chemotherapy (HCT) with autologous HSC transplantation (auto-HSCT) is accompanied by a signifcant increase in MDSC counts in peripheral blood and apheresis product of multiple myeloma (MM) patients. However, quantitative changes of these cells at the post-transplant and their role at the immune recovery remain unexplored. The study was aimed to analyze the dynamics of circulating MDSC counts and the expression of suppressor molecule arginase-1 in patients with MM in the frst 12 months after HCT and auto-HSCT and evaluate association between MDSCs and transplantation outcomes. Material and Methods . The study included 44 MM patients who underwent HCT and auto-HSCT. The relative number of granulocytic MDSCs (G-MDSCs), monocytic MDSCs (M-MDSCs), and early-stage MDSCs (E-MDSCs), as well as the expression of arginase-1 in each of MDSC subsets was evaluated by fow cytometry in patient peripheral blood samples. Results. At the engraftment (day +12 – +16, leukocytes >1×10 9 /l), M-MDSC relative count was increased (p U =0.038), as well as the relative (p U =0.003) and absolute (p U ˂ 0.0001) counts of G-MDSCs, decreasing after 6 months down to pre-transplant values (рU=0.007, рU=0.024 and рU=0.02, respectively) and remaining at the same level at the 12-month follow-up period. The absolute count of E-MDSCs by the time of the engraftment decreased transiently (p U =0.004 vs before HCT), gradually recovering by 12-month follow-up (p U =0.032 vs day +12 – +16). The remission within 12 months in the group with G-MDSCs˂0.17 % at the engraftment was observed in 67 ± 11 % of patients, with G-MDSCs >0.17 % – in 94 ± 6 % of patients (p=0.049). During the 12-month post-transplant, the number of M-MDSCs expressing arginase-1 has been increasing, with a tendency to lower values at the engraftment in patients with early MM relapse (p U =0.09). Conclusion . The association of early MM relapse after auto-HSCT with the lower count of G-MDSCs and the lower count of arginase-1 + M-MDSCs at the engraftment suggests that MDSCs is involved in the restriction of homeostatic proliferation as a factor for more effective immune recovery.

Introduction . High-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (auto-HSCT) is the standard of treatment for multiple myeloma (MM) patients. The post-transplant period appears to be promising for targeted anti-checkpoint therapy in MM. Aim — to study the dynamics and functional properties of T-cells expressing inhibitory checkpoint molecules PD-1 and TIM-3 in patients with MM under conditions of lymphopenia after HDC with auto-HSCT. Methods . The study included 40 patients with MM who underwent HDC with auto-HSCT. The counts of PD-1- and TIM3-positive CD8 + and CD4 + T-cells and their functional activity on the intracellular expression of Ki-67, production of granzyme B, and interferon-γ were assessed by fl ow cytometry. Results . Relative counts of patient PD-1 + and TIM-3 + subsets of CD8 + and CD4 + T-cells obtained from bone marrow samples were higher compared to peripheral blood. CD8 + PD-1 + and CD4 + PD-1 + T-cells of MM patients had a pronounced cytotoxic and cytokine-producing potential. The functional activity of CD8 + TIM-3 + and CD4 + TIM-3 + T-cells was signifi cantly reduced compared with TIM-3-negative subsets. Low functional activity was also detected in populations of CD8 + and CD4 + T-lympho cytes, co-expressing PD-1 and TIM-3. The frequencies of T-cells expressing PD-1 and TIM-3 increased signifi cantly on the engraftment day after auto-HSCT. The proliferative activity of PD-1 + and TIM-3 + CD4 + and CD8 + T-cells and the cytotoxic potential of PD-1 + and TIM-3 + CD8 + T-cells were also signifi cantly increased compared to the data prior auto-HSCT. Conclusions . PD-1-positive T-cells in MM patients are related to activated or “early dysfunctional” but not exhausted subsets, while T-cells exhaustion is more analogous with CD8 + TIM-3 + and CD4 + TIM-3 + T-cells, as well as with subsets co-expressing PD-1 and TIM-3. To identify the state of T-cells exhaustion, it is necessary to evaluate T-cells subsets co-expressing PD-1, TIM-3, and other ICMs, and/or to study their functional properties. In the early post-transplant period, the proportion of Tcells expressing PD-1 and TIM-3 increases due to an increase in their proliferative potential.

Introduction. Multiple myeloma (MM) is a B-cell malignancy with clonal expansion of plasma cells in bone marrow. Highdose chemotherapy with autologous haematopoietic stem cell transplantation is among main consolidation therapies in MM. Myeloid-derived suppressor cells (MDSCs) are immature myeloid-accompanying cells able to suppress the immune response. The administration of granulocyte colony stimulating factor (G-CSF) to mobilise haematopoietic stem cells (HSCs) increases the MDSC count in peripheral blood (PB).Aim — to study MDSC subsets in PB of remission MM patients and their incidence dynamics at HSC mobilisation.Methods. The study surveyed 35 MM patients prior to and after HSC mobilisation. The counts of granulocytic (G-MDSCs; Lin–HLA-DR–CD33+ CD66b+), monocytic (М-MDSCs; CD14+ HLA-DRlow/–) and early MDSCs (E-MDSCs; Lin–HLA-DR– CD33+ CD66b–) were estimated in flow cytometry.Results. Remission MM patients differed from healthy donors in higher relative counts of G-MDSCs (Lin–HLA-DR– CD33+ CD66b+) and increased relative and absolute counts of М-MDSCs (CD14+ HLA-DRlow/–). М-MDSCs significantly outnumbered G-MDSCs. MDSC subset counts were elevated in complete response (CR) and very good partial response (VGPR), as well as in partial response (PR). Higher relative MDSC counts were associated with greater pretreatment (2–3 lines of chemotherapy). After HSC mobilisation with cyclophosphamide 2–4 g/m2 + G-CSF (filgrastim 5 μg/kg/day), the median relative E-MDSC and M-MDSC counts increased by 2.3 and 2.0 times, respectively, while the relative G-MDSC count raised 46-fold perturbing the MDSC subset balance.Conclusion. Remission MM patients had the increased relative G-MDSC and both relative and absolute M-MDSC counts compared to donors. A greater patient pretreatment was associated with higher relative G-MDSC counts. Treatment response (CR/VGPR vs. PR) was not coupled with MDSC count variation. The G-CSF-induced HSC mobilisation entailed a significant expansion of all three MDSC subsets in PB.

We have investigated the dynamics of post-transplant recovery of CD4(+)FOXP3(+) T cells in patients with multiple myeloma (MM). The relationship between CD4(+)FOXP3(+)T cell counts and clinical outcomes of MM patients after high-dose chemotherapy and autologous hematopoietic stem cell transplantation (AHSCT) also has been evaluated. Fifty-nine patients were included in the study. The content of circulating CD4(+)FOXP3(+)T cells has been assessed with the use of flow cytometry before AHSCT, at the day of engraftment, and 6 and 12 months later. After AHSCT the relative count of CD4(+)FOXP3(+)T cells in MM patients restored rapidly, it became higher than initial level at the day of engraftment and then subsequently decreased for a 1 year to values in healthy donors. A relative count of CD4(+)FOXP3(+)T cells at the time of engraftment increased in patients with relapsed disease during 12 months after AHSCT. Receiver operating characteristic (ROC) analysis showed that evaluation of CD4(+)FOXP3(+)T cells at the day of engraftment allowed to predict the early relapse of MM after AHSCT.

Recent research revealed dendritic cells (DCs) to have direct antitumor cytotoxic activity and to inhibit the growth and proliferation of tumor cells in vitro. The aim of the present study was to investigate the association between the cytotoxic activity of dendritic cells generated in the presence of interferon alpha (IFN-DCs) and TNFα expression by IFN-DCs in patients with malignant lymphomas. It was shown that IFN-DCs of malignant lymphoma patients possessed low cytotoxic activity against tumor cell line HEp-2 associated with low expression of transmembrane TNFα (tmTNFα) and high level of soluble TNFα (sTNFα) secretion. Reduced DC cytotoxic activity and low tmTNFα expression on DC surface were observed mainly in Hodgkin’s lymphoma patients. In contrast, IFN-DCs of patients with non-Hodgkin lymphoma were endowed with the ability to lysis of HEp-2 cells and tmTNFα molecule expression was similar to that in IFN-DCs from healthy donors. It was determined that the increase of expression of tmTNFα molecule on lymphoma patient IFN-DCs induced by the addition of TNFα-converting enzyme inhibitor into IFN-DC cultures was associated with the enhancement of IFN-DC cytotoxic activity against HEp-2 cells.

Recent research revealed dendritic cells (DCs) to have direct antitumor cytotoxic activity and to inhibit the growth and proliferation of tumor cells in vitro . The aim of the present study was to investigate the association between the cytotoxic activity of dendritic cells generated in the presence of interferon alpha (IFN-DCs) and TNFα expression by IFN-DCs in patients with malignant lymphomas. It was shown that IFN-DCs of malignant lymphoma patients possessed low cytotoxic activity against tumor cell line HEp-2 associated with low expression of transmembrane TNFα (tmTNFα) and high level of soluble TNFα (sTNFα) secretion. Reduced DC cytotoxic activity and low tmTNFα expression on DC surface were observed mainly in Hodgkin’s lymphoma patients. In contrast, IFN-DCs of patients with non-Hodgkin lymphoma were endowed with the ability to lysis of HEp-2 cells and tmTNFα molecule expression was similar to that in IFN-DCs from healthy donors. It was determined that the increase of expression of tmTNFα molecule on lymphoma patient IFN-DCs induced by the addition of TNFα-converting enzyme inhibitor into IFN-DC cultures was associated with the enhancement of IFN-DC cytotoxic activity against HEp-2 cells.

We have evaluated the dynamics of post-transplant recovery of CD4(+)CD45RA(+)CD31(+)T cells and CD4(+)CD45RA(-)CD31(+)T-cells in patients with lymphoproliferative diseases after high-dose chemotherapy with autologous stem cell transplantation (auto-HSCT). 87 patients were included in the study. The content of circulating CD4(+)CD31(+) naive and memory T-cells has been assessed with the use of flow cytometry before auto-HSCT, at the day of engraftment, and in 6 and 12 months. Relative amount of CD4(+)CD45RA(-)CD31(+)T-cells in patients was elevated in comparison with healthy controls, restored rapidly following auto-HSCT and reached initially high level at the day of engraftment. Post-transplant mediastinal radiotherapy significantly reduced counts of CD4(+)CD45RA(+)CD31(+)

The parameters reflecting the activity of the thymus in patients with hematological malignancies were studied and their relationship with the clinical factors was analyzed. The study was carried out in 42 patients with lymphoproliferative diseases. The counts of CD4(+)CD45RA(+)CD31(+) T-cells were evaluated by flow cytometry and the content of TREC molecules in CD4(+) cells was evaluated by PCR. The patients were characterized by a pronounced reduction of the counts of CD4(+)CD45RA(+)CD31(+) cells and of TREC molecules in CD4(+) T-cells and increased counts of CD4(+) memory T-cells expressing CD31 molecule. Inverse correlation between TREC content and number of first-line chemotherapy courses and age-associated reduction of their content were detected. No relationship between CD4(+)CD45RA(+)CD31(+) cells counts and clinical factors was detected. In lymphopenia the most sensitive parameter of thymic activity was TREC in CD4+ T-cells. There was negative correlation between TREC and patients age, between TREC and previous therapy. Homeostatic proliferation reduced the significance of evaluation of CD4(+)CD45RA(+)CD31(+) cells as a marker of "recent migrants from the thymus".

Cell subtypes and cytokine profile of apheresis products collected from lymphoma and acute leucosis patients were analyzed. It was shown, that acute leucosis patients' grafts contained higher relative numbers of naïve T-cells, CD4+CD25high T-cells, T-lymphocytes (non-significant trend) and lower counts of granulocytes. Significant increase of relative numbers of dividing CD34+ cells (in S, G2/M phases of the cell cycle) was demonstrated, in acute leucosis patients' grafts. In lymphoma grafts the levels of CD34+ cells in G0/G1 phases were found, to be increased. Cells isolated from grafts of acute leucosis patients characterized by higher levels of proinflammatory cytokines production, such as IL-1, IL-6, MIP-1β, TNF-α, IL-8, IFN-γ (the last two ones— non-significant trend) and cytokines, essential for humoral immune response (IL-4 and — in trend — IL-10). The existing differences didn't influence on effectiveness of early lymphocyte recovery.