Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation.

BACKGROUND: Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. METHODS: We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. RESULTS: We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. CONCLUSIONS: This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.

Immunomodulatory Drugs Alter the Metabolism and the Extracellular Release of Soluble Mediators by Normal Monocytes.

Immunomodulatory drugs (IMiDs) are used in the treatment of hematological malignancies, especially multiple myeloma. IMiDs have direct anticancer effects but also indirect effects via cancer-supporting stromal cells. Monocytes are a stromal cell subset whose metabolism is modulated by the microenvironment, and they communicate with neighboring cells through extracellular release of soluble mediators. Toll-like receptor 4 (TLR4) is then a common regulator of monocyte metabolism and mediator release. Our aim was to investigate IMiD effects on these two monocyte functions. We compared effects of thalidomide, lenalidomide, and pomalidomide on in vitro cultured normal monocytes. Cells were cultured in medium alone or activated by lipopolysaccharide (LPS), a TLR4 agonist. Metabolism was analyzed by the Seahorse XF 96 cell analyzer. Mediator release was measured as culture supernatant levels. TLR4 was a regulator of both monocyte metabolism and mediator release. All three IMiDs altered monocyte metabolism especially when cells were cultured with LPS; this effect was strongest for lenalidomide that increased glycolysis. Monocytes showed a broad soluble mediator release profile. IMiDs decreased TLR4-induced mediator release; this effect was stronger for pomalidomide than for lenalidomide and especially thalidomide. To conclude, IMiDs can alter the metabolism and cell-cell communication of normal monocytes, and despite their common molecular target these effects differ among various IMiDs.

A Pilot Study of Circulating Monocyte Subsets in Patients Treated with Stem Cell Transplantation for High-Risk Hematological Malignancies.

Background and Objectives: Autologous and allogeneic stem cell transplantation is used in the treatment of high-risk hematological malignancies, and monocytes are probably involved in hematological reconstitution as well as posttransplant immunoregulation. The aim of our study was to investigate the levels of circulating monocyte subsets in allotransplant recipients. Materials and Methods: The levels of the classical, intermediate, and nonclassical monocyte subsets were determined by flow cytometry. Sixteen patients and 18 healthy controls were included, and the levels were analyzed during pretransplant remission (n = 13), early posttransplant during cytopenia (n = 9), and early reconstitution (n = 9). Results: Most patients in remission showed a majority of classical monocytes. The patients showed severe early posttransplant monocytopenia, but the total peripheral blood monocyte counts normalized very early on, and before neutrophil and platelet counts. During the first 7-10 days posttransplant (i.e., during cytopenia) a majority of the circulating monocytes showed a nonclassical phenotype, but later (i.e., 12-28 days posttransplant) the majority showed a classical phenotype. However, the variation range of classical monocytes was wider for patients in remission and during regeneration than for healthy controls. Conclusions: The total peripheral blood monocyte levels normalize at the very early stages and before neutrophil reconstitution after stem cell transplantation, and a dominance of classical monocytes is reached within 2-4 weeks posttransplant.

Circulating monocyte subsets in multiple myeloma patients receiving autologous stem cell transplantation - a study of the preconditioning status and the course until posttransplant reconstitution for a consecutive group of patients.

BACKGROUND: Induction therapy of multiple myeloma patients prior to autologous stem cell transplantation has changed from conventional chemotherapy to treatment based on proteasome inhibitors or immunomodulatory drugs. We used flow cytometry to analyze total monocyte and monocyte subset (classical, intermediate and non-classical monocytes) peripheral blood levels before and following auto-transplantation for a consecutive group of myeloma patients who had received the presently used induction therapy. RESULTS: The patients showed normal total monocyte concentrations after induction/stem cell mobilization, but the concentrations of classical monocytes were increased compared with healthy controls. Melphalan conditioning reduced the levels of total CD14+ as well as classical and non-classical monocytes, whereas intermediate monocytes were not affected. Thus, melphalan has a non-random effect on monocyte subsets. Melphalan had a stronger effect on total and classical monocyte concentrations for those patients who had received induction therapy including immunomodulatory drugs. Total monocytes and monocyte subset concentrations decreased during the period of pancytopenia, but monocyte reconstitution occurred before hematopoietic reconstitution. However, the fractions of various monocyte subsets varied considerably between patients. CONCLUSIONS: The total level of circulating monocytes is normalized early after auto-transplantation for multiple myeloma, but pre- and post-transplant levels of various monocyte subsets show considerable variation between patients.

Standardization of sampling and sample preparation for analysis of human monocyte subsets in peripheral blood.

INTRODUCTION: Monocytes are important for innate immunity and include the classical (CD14brightCD16negative), intermediate (CD14brightCD16dim) and non-classical (CD14dimCD16bright) monocyte subsets. The quantification of these functionally different subsets in peripheral blood may become useful for diagnosis and follow-up in human diseases. The aim of the present study was to investigate how different pre-analytical parameters influence analysis of monocyte subsets in peripheral blood samples. METHODS: We determined relative levels of monocytes and monocyte subsets by flow cytometry of peripheral blood samples derived from healthy individuals. A gating strategy exclusively extracting viable CD14+ monocytes and focusing on the three monocyte subsets was applied. We investigated the effects of (i) various anticoagulants (i.e. Li-Heparin, ACD-A, K2EDTA), (ii) insufficient filling of blood sampling tubes, (iii) cryopreservation. In addition, we analysed expression of the CCR2 chemokine receptor. RESULTS: The relative numbers of CD14+ monocytes depended on the anticoagulant used, whereas the fraction of the three monocyte subsets did not. Insufficient filling of blood sampling tubes altered the relative levels of monocytes out of leukocytes, but not the relative levels of the monocyte subsets. Finally, the fraction of CD14+ monocytes out of isolated peripheral blood mononuclear cells was not significantly altered by cryopreservation, but the relative percentages of monocyte subsets was altered (similar effects for ACD-A and K2EDTA samples) and this was observed in correlation to a decreased CD16 expression. CONCLUDING REMARKS: Analysis of the monocyte subsets (i.e. classical, intermediate, non-classical) in peripheral blood samples requires a careful standardization of peripheral blood sampling and pre-analytic handling of the samples with respect to the anticoagulant used, filling of sample tubes, and cryopreservation of cells prior to analysis.