Phenotypic and functional changes of GM-CSF differentiated human macrophages following exposure to apoptotic neutrophils.

The engulfment of apoptotic cells by monocytes and unprimed macrophages results in M2 polarization. In the current study, we investigated whether apoptotic cells influence the phenotypic and functional characteristics of GM-CSF-differentiated human macrophages (GM-Mφ). Our results demonstrate that GM-Mφ preincubated with apoptotic neutrophils (GM-MφNeu) show significantly increased expression of CD206 and FasL and decreased capacity to stimulate allogeneic T-cell proliferation thus adopting M2 features. The 27-plex analysis demonstrates the down-regulation of 24 cytokines (including IL-10) in GM-MφNeu cultures. In contrast, apoptotic neutrophils enhance PGE2 synthesis by GM-Mφ, and blocking PGE2 production with indomethacin restores an allostimulatory activity of GM-MφNeu. These data provide evidence that GM-Mφ following exposure to apoptotic cells acquire features of M2 cells. Given the global suppression of cytokine secretion, GM-MφNeu resemble deactivated (M2c) macrophages, and their capacity to inhibit allogeneic T-cell proliferation appears to be mediated by an enhanced synthesis of PGE2 but not IL-10.

Elevated levels of dehydroepiandrosterone as a potential mechanism of dendritic cell impairment during pregnancy.

BACKGROUND: This study aimed to test the hypothesis that immune dysfunction and the increased risk of spontaneous abortion in pregnant women with hyperandrogenia (HA) are caused by the reduced tolerogenic potential of dendritic cells (DCs) that results from elevated levels of dehydroepiandrosterone sulfate (DHEAS). METHODS: The phenotypic and functional properties of monocyte-derived DCs generated from blood monocytes from non-pregnant women, women with a normal pregnancy, or pregnant women with HA, as well as the in vitro effects of DHEAS on DCs in healthy pregnant women were investigated. RESULTS: In a normal pregnancy, DCs were shown to be immature and are characterized by a reduced number of CD83(+) and CD25(+) DCs, the ability to stimulate type 2 T cell responses and to induce T cell apoptosis. By contrast, DCs from pregnant women with HA had a mature phenotype, were able to stimulate both type 1 (IFN-γ) and type 2 (IL-4) T cell responses, and were characterized by lower B7-H1 expression and cytotoxic activity against CD8(+) T cells. The addition of DHEAS to cultures of DCs from healthy pregnant women induced the maturation of DCs and increased their ability to activate type 1 T cell responses. CONCLUSION: Our data demonstrated the reduction in the tolerogenic potential of DCs from pregnant women with HA, and revealed new mechanisms involved in the hormonal regulation of DCs mediated by DHEAS.

Mesenchymal stromal cells improve early lymphocyte recovery and T cell reconstitution after autologous hematopoietic stem cell transplantation in patients with malignant lymphomas.

Mesenchymal stromal cells (MSCs) possess a multi-lineage potential and immunoregulatory activities and provide a great potential in cell-based technologies. However, MSC suppressive activity raises concerns regarding the possible adverse effect of MSCs on the immune recovery. The influence of autologous MSC co-transplantation on recovery of T cell subsets in patients receiving autologous hematopoietic stem cell transplantation (AHSCT) for malignant lymphomas and multiple myeloma were characterized. Co-transplantation of MSCs improved lymphocyte recovery most effectively in patients with low input of hematopoietic stem cells or low absolute lymphocyte count in apheresis product. MSC co-transplantation improved early recovery of both memory and naive T cells with more prominent effect on naive CD4(+) T cells. Patients with MSC co-transplantation showed more effective reconstitution of recent thymic emigrants. These data indicate the positive impact of MSCs on immune reconstitution and note MSC co-transplantation is feasible to optimize the outcomes of AHSCT in malignant lymphoma patients.

Interferon alpha induces generation of semi-mature dendritic cells with high pro-inflammatory and cytotoxic potential.

Dendritic cell-based vaccines are considered as a new and promising immunotherapeutic approach for cancer treatment. However, the choice of optimal protocol of dendritic cell generation in vitro represents the major challenge. Here, we compared phenotype and functional characteristics of human monocyte-derived dendritic cells (DCs) generated in the presence of IL-4/GM-CSF (IL4-DCs) and IFNα/GM-CSF (IFN-DCs). We showed that IFN-DCs displayed semi-mature phenotype and expressed higher level of CD123, TNF-related apoptosis-inducing ligand (TRAIL) and B7-H1 molecules in comparison with IL4-DCs. LPS-stimulated IFN-DCs were characterized by greater production of Th1/pro-inflammatory (IFN-γ, IL-2, IL-1ß, TNF-α, IL-17), Тh2/anti-inflammatory cytokines (IL-10, IL-5), hematopoietic growth factors (G-CSF) and chemokines (MCP-1). These data indicated more pronounced ability of IFN-DCs to induce cellular immune response as well as humoral immune response compared to IL4-DCs. LPS-stimulated IFN-DCs possessed higher direct cytotoxic activity against TRAIL-sensitive tumor cell line Jurkat and similar cytotoxicity against TRAIL-resistant tumor HEp-2 cells. Besides, IFN-DCs and IL4-DCs equally induced apoptosis of activated CD4(+) and CD8(+) T cells. These results suggest that IFN-DCs can be used as potent cell-based curative therapies for individuals with cancer.

Cytotoxic activity of ex-vivo generated IFNα-induced monocyte-derived dendritic cells in brain glioma patients.

Recent studies have revealed that besides the important role in triggering the adoptive antitumor immunity, dendritic cells (DCs) possess direct cytotoxic antitumor activity. Here, we investigated brain glioma patient monocyte-derived DCs generated in the presence of IFNα and GM-CSF (IFN-DCs). These DCs were characterized by reduced cytotoxic activity against TRAIL-resistant HEp-2 cells. The impairment of DC cytotoxic function was observed mainly in high-grade glioma patients and associated with poor survival. The dysfunction of patient DC cytotoxicity was partially restored under in vitro pretreatment of DCs with double-stranded human DNA as well as rIL-2. In contrast to healthy donors, IFN-DCs in a part of high-grade glioma patients also failed to lyse primary autologous or allogeneic glioma cells. Our findings point to possible contribution of DC impairment in tumor pathogenesis in brain glioma and justify the necessity to evaluate and correct DC cytotoxic function when exploring DCs as cancer vaccines in glioma.

Cytotoxic activity of dendritic cells as a possible mechanism of negative regulation of T lymphocytes in pulmonary tuberculosis.

The PD-1/B7-H1-mediated induction of T cell apoptosis/anergy as a possible mechanism of immune response failure was studied in 76 patients with pulmonary tuberculosis (TB) with normal and low-proliferative response to antigens of M. tuberculosis (purified protein derivative (PPD)). It was revealed that dendritic cells (DCs), generated in vitro from patient blood monocytes with GM-CSF + IFN-α, were characterized by increased B7-H1 expression, upproduction of IL-10, and reducing of allostimulatory activity in mixed lymphocyte culture (MLC). Moreover, DCs of patients with TB were able to enhance T cell apoptosis and to block T-cell division in MLC. It was shown that neutralizing anti-PD1 antibodies significantly decreased the proapoptogenic/tolerogenic effect of DCs. Correlation analysis revealed a direct relationship between IL-10 production and level of B7-H1 expression in the general group of investigated patients. It was demonstrated that generation of healthy donor DCs in the presence of IL-10 led to an increase in the number of DCs-expressed B7-H1 molecule, DC proapoptogenic activity, and a decrease in their allostimulatory activity. Obviously, the revealed phenomenon of the PD-1/B7-H1-mediated pro-apoptogenic activity of DCs is clinically significant since the cytotoxic/tolerogenic potential of DCs is more pronounced in patients with PPD anergy.

Increased circulating CD3+ T cells are associated with early relapse following autologous hematopoietic stem cell transplantation in patients with classical Hodgkin lymphoma.

Non-malignant host immune cells are the main substrate in classical Hodgkin lymphoma (HL) microenvironment. Reconstitution of lymphocyte populations following the high-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (auto-HSCT) can support tumor growth in HL patients. We investigated recovery dynamics of circulating CD3+, CD4+, CD8+, CD16+/CD56+, CD19+, CD4+FOXP3+ lymphocytes following auto-HSCT in 79 HL patients and assessed relationship between these populations and the development of early relapse. Studied populations were not statistically significant between patients with high or standard/intermediate risk of relapse. CD3+ T cells at the time of engraftment were increased in patients with the early relapse of HL compared to non-relapsed patients (PU = 0.0028). Area under the curve was 0.76 (р = .0037). In logistic regression models, CD3+ T cell count was associated with early relapse/progression as a trend. These findings elucidate several interactions between early systemic T cell recovery and tumor progression following HDC with auto-HSCT.

Increased circulating CD4+FOXP3+ T cells associate with early relapse following autologous hematopoietic stem cell transplantation in multiple myeloma patients.

We investigated dynamics of CD4+FOXP3+ T cell recovery following the high-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (auto-HSCT) in multiple myeloma (MM) patients. Circulating CD4+FOXP3+ T cells of 79 MM patients were evaluated using flow cytometry before HDC with auto-HSCT, at the day of engraftment, and following 6 and 12 months. Percentage of CD4+FOXP3+ T cells restored rapidly following auto-HSCT, became higher than pre-transplant level at the day of engraftment and then subsequently decreased for a year. CD4+FOXP3+ T cells at the time of engraftment were increased in patients with the relapse or progression of MM during 12 months following auto-HSCT (n=10) compared to non-relapsed patients (n=50): 6.7% (5.3-8.9%) vs 4.9% (2.8-6.6%); PU = 0.026. Area under the curve was 0.72 (95% CI: 0.570-0.878; р=0.026). Circulating CD4+FOXP3+ T cell count was not associated with the percentage of myeloma plasma cells in a bone marrow but depended on its amount in autografts. CONCLUSIONS: Relative count of CD4+FOXP3+ T cells restored rapidly following auto-HSCT (at the day of engraftment), became higher than pre-transplant level and then subsequently decreased for a year. Their excess at the time of engraftment is associated with early relapse.

CD4+ memory T cells retain surface expression of CD31 independently of thymic function in patients with lymphoproliferative disorders following autologous hematopoietic stem-cell transplantation.

High-dose chemotherapy with autologous hematopoietic stem-cell transplantation (AHSCT) causes severe and long-lasting immunodeficiency in patients with lymphoproliferative disorders. The thymus begins to restore the T-cell repertoire approximately from the sixth month post-transplant. We assessed the dynamics of post-transplant recovery of CD4+CD45RA+CD31+ T cells, "recent thymic emigrants" (RTEs), and a poorly described subtype of CD4+CD45RA-CD31+ T cells in 90 patients with lymphoproliferative disorders following high-dose chemotherapy with AHSCT. Relative and absolute counts of CD4+CD31+ naïve and memory T cells were evaluated before AHSCT, at the day of engraftment, and 6- and 12-month post-transplant. The pre-transplant count of CD4+CD45RA+CD31+ T cells was lower than in healthy controls, and did not reach donors' values during the 12-month period. The pre-transplant number of CD4+CD45RA-CD31+ T cells was higher than in healthy controls and was restored rapidly following AHSCT. Post-transplant mediastinal radiotherapy reduced counts of RTEs and elongated recovery period. Non-thymic tissue irradiation did not reduce this subset. The obtained data indicate that homeostatic proliferation may decrease the significance of CD31 expression on CD4+CD45RA+ T cells as a marker of RTEs, and suggest that evaluation of RTEs recovery by flow cytometry requires an accurate gating strategy to exclude CD31+ memory T cells.

Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis.

The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generated in vitro macrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity to M. tuberculosis antigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14(+)CD16(+) expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which were in vitro generated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γ coupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generated in vitro from peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γ production in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response to M. tuberculosis was discussed.

Formation of different abzymes in autoimmune-prone MRL-lpr/lpr mice is associated with changes in colony formation of haematopoietic progenitors.

It was shown that IgGs from the sera of 2-7-month-old control non-autoimmune (CBA x C57BL)F1 and BALB/c mice and 2-3-month-old autoimmune prone MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre-diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre-diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs. The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre-diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre-diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre-diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be considered the earliest statistically significant marker of mouse spontaneous SLE and a further significant increase in their activities correlates with the appearance of SLE visible markers and with an increase in concentrations of anti-DNA Abs and urine protein. However, development of autoimmune (AI)-reactions and the increase in the sera anti-DNA antibodies (Abs) and in the abzyme activities in pregnant and lactating mice do not associate with SLE visible markers and proteinuria. The possible differences in immune system reorganizations during pre-disease, disease, pregnancy and lactation leading to production of different auto-antibodies and abzymes are discussed.

Hematopoietic progenitor colony formation in the immunopathogenesis of the autoimmune disorder in MRL/MpJ-lpr mice.

Lymphocyte proliferation and apoptosis at different stages of the development of the autoimmune disorder in MRL/MpJ-lpr mice was studied. Hematopoietic progenitor colony formation during the course of the disease was characterized. A detectable difference at the level of lymphocyte proliferation, apoptosis, and the relative amount of BFU-E, CFU-GM and CFU-GEMM cell colonies was revealed between healthy young mice and animals spontaneously developing pronounced symptoms of the autoimmune disorder. The quantity of BFU-E and CFU-GEMM colonies was remarkably increases in aged MRL/MpJ-lpr mice even before clinical manifestation of the disease (proteinuria). An elevated number of CFU-GEMM was accompanied by a striking increase in their size. The study of hematopoietic disturbances in autoimmune MRL/MpJ-lpr mice may be very useful for understanding the mechanism of the autoimmune disease development and searching for new strategies of the correction of the autoimmune disorder.