Immunoregulatory effects of Lurbinectedin in chronic lymphocytic leukemia.

Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1ß in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.

Function is Dissociated From Activation-Related Immunophenotype on Phagocytes From Patients With SIRS/Sepsis Syndrome.

Sepsis is a life-threatening condition associated with failure of at least one organ in the presence of infection. Along with SIRS, the acute systemic inflammatory syndrome without documented infection, sepsis represents a main health problem in intensive care units around the world. Hypercytokinemia and overexpression of activation-markers on leukocytes are frequently reported in SIRS/sepsis. Leukocyte functions including antibody mediated-phagocytosis, pathogen recognition, and migration appear to be disabled in SIRS/septic patients. Our aim was to evaluate the so-called activation immunophenotype and functions related to infection contention in phagocytes from patients with sepsis. We analyzed blood samples from 44 patients with SIRS/sepsis and 14 healthy volunteers. CD16, CD69, CD64, CCR7, and TREM-1 levels were determined on the surface of neutrophils and monocytes. Phagosome maturation and p38, STAT3, and STAT5 phosphorylation were evaluated in these phagocytes. As expected, sepsis and SIRS patients had increased serological levels of pro- and anti-inflammatory cytokines. E coli internalization was not increased in monocytes from patients with SIRS/sepsis, despite increased numbers of circulating neutrophils and monocytes (P < 0.05) and overexpression of CD64 and CD69 in neutrophils (P < 0.05), TREM-1 (P < 0.01), CD69 (P < 0.001), and CCR7 (P < 0.05). Moreover, phagosome maturation was decreased in phagocytes from patients with SIRS/sepsis syndrome (P < 0.00001). Furthermore, p38 and STAT-3 phosphorylation elicited by LPS or IL-10 (respectively) was diminished in neutrophils and monocytes from patients (P < 0.05). Our results indicate that "activation markers" may not reflect higher functionality, so a more profound analysis should be made before assuming that the activated immunophenotype means increased phagocyte responses.

NH36 and F3 Antigen-Primed Dendritic Cells Show Preserved Migrating Capabilities and CCR7 Expression and F3 Is Effective in Immunotherapy of Visceral Leishmaniasis.

Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.

Common Variable Immunodeficiency and Circulating TFH.

CD4+ T follicular helper cells (TFH) were assessed in adult patients with common variable immune deficiency (CVID) classified according to the presence of granulomatous disease (GD), autoimmunity (AI), or both GD and AI (Group I) or the absence of AI and GD (Group II). TFH lymphocytes were characterized by expression of CXCR5 and PD-1. TFH were higher (in both absolute number and percentage) in Group I than in Group II CVID patients and normal controls (N). Within CXCR5+CD4+ T cells, the percentage of PD-1 (+) was higher and that of CCR7 (+) was lower in Group I than in Group II and N. The percentages of Treg and TFH reg were similar in both CVID groups and in N. TFH responded to stimulation increasing the expression of the costimulatory molecules CD40L and ICOS as did N. After submitogenic PHA+IL-2 stimulation, intracellular expression of TFH cytokines (IL-10, IL-21) was higher than N in Group I, and IL-4 was higher than N in Group II. These results suggest that TFH are functional in CVID and highlight the association of increased circulating TFH with AI and GD manifestations.

Immunomodulation by Trypanosoma cruzi: toward understanding the association of dendritic cells with infecting TcI and TcII populations.

Dendritic cells (DCs) are major immune components, and depending on how these cells are modulated, the protective host immune response changes drastically. Trypanosoma cruzi is a parasite with high genetic variability and modulates DCs by interfering with their capacity for antigen recognition, migration, and maturation. Despite recent efforts, the association between DCs and T. cruzi I (TcI) and TcII populations is unknown. Herein, it was demonstrated that AQ1.7 and MUTUM TcI strains present low rates of invasion of bone marrow-derived DCs, whereas the 1849 and 2369 TcII strains present higher rates. Whereas the four strains similarly induced the expression of PD-L1, the production and expression of IL-10 and TLR-2, respectively, in DCs were differentially increased. The production of TNF-α, IL-12, IL-6, and CCL2 and the expression of CD40, CD80, MHC-II, CCR5, and CCR7 changed depending on the strain. The 2369 strain yielded the most remarkable results because greater invasion correlated with an increase in the levels of anti-inflammatory molecules IL-10 and PD-L1 but not with a change in the levels of TNF-α, MHC-II, or CD40 molecules. These results suggest that T. cruzi strains belonging to different populations have evolved specific evasion strategies that subvert DCs and consequently the host response.

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.

We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.

Activation of PAF-receptor induces regulatory dendritic cells through PGE2 and IL-10.

Activation of the platelet-activating factor receptor (PAFR) in macrophages is associated with suppressor phenotype. Here, we investigated the PAFR in murine dendritic cells (DC). Bone marrow-derived dendritic cells (BALB/c) were cultured with GM-CSF and maturation was induced by LPS. The PAFR antagonists (WEB2086, WEB2170, PCA4248) and the prostaglandin (PG) synthesis inhibitors (indomethacin, nimesulide and NS-398) were added before LPS. Mature and immature DCs expressed PAFR. LPS increased MHCII, CD40, CD80, CD86, CCR7 and induced IL-10, IL-12, COX-2 and PGE2 expression. IL-10, COX-2 and PGE2 levels were reduced by PAFR antagonists and increased by cPAF. The IL-10 production was independent of PGs. Mature DCs induced antigen-specific lymphocyte proliferation. PAFR antagonists or PG-synthesis inhibitors significantly increased lymphocyte proliferation. It is proposed that PAF has a central role in regulatory DC differentiation through potentiation of IL-10 and PGE2 production.

Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+) T lymphocytes.

BACKGROUND: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells). METHODS: We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100. RESULTS: Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change. CONCLUSION: We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.