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Class IA phosphatidylinositol 3-kinase (PI3K) catalytic subunits p110? and p110? are targets in cancer therapy expressed at high levels in T lymphocytes. The role of p110? PI3K in normal or pathological immune responses is well established, yet the importance of p110? subunits in T cell-dependent immune responses is not clear. To address this problem, mice with p110? conditionally deleted in CD4+ and CD8+ T lymphocytes (p110??/??T) were used. p110??/??T mice show normal development of T cell subsets, but slightly reduced numbers of CD4+ T cells in the spleen. “In vitro,” TCR/CD3 plus CD28 activation of naive CD4+ and CD8+ p110??/??T T cells showed enhanced effector function, particularly IFN-? secretion, T-bet induction, and Akt, Erk, or P38 activation. Tfh derived from p110??/??T cells also have enhanced responses when compared to normal mice, and IL-2 expanded p110??/??T CD8+ T cells had enhanced levels of LAMP-1 and Granzyme B. By contrast, the expansion of p110??/??T iTreg cells was diminished. Also, p110??/??T mice had enhanced anti-keyhole limpet hemocyanin (KLH) IFN-?, or IL-4 responses and IgG1 and IgG2b anti-KLH antibodies, using CFA or Alum as adjuvant, respectively. When compared to WT mice, p110??/??T mice inoculated with B16.F10 melanoma showed delayed tumor progression. The percentage of CD8+ T lymphocytes was higher and the percentage of Treg cells lower in the spleen of tumor-bearing p110??/??T mice. Also, IFN-? production in tumor antigen-activated spleen cells was enhanced. Thus, PI3K p110? plays a significant role in antigen activation and differentiation of CD4+ and CD8+ T lymphocytes modulating antitumor immunity.

11 páginas, 8 figuras, 1 tabla -- PAGS nros. 698-708, Background and purpose: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs). Experimental approach: MDDCs derived from healthy and hepatitis C virus (HCV)-infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T-lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase-based reporter methods, and Toll-like receptor (TLR)-blocking antibodies were employed to analyse TLR activity. Key results: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA-DR, chemokines and chemokine receptors, interleukin (IL)-12p70 and IL-10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF-κB and phosphorylation of p38 mitogen-activated protein kinase. AM3 promoted NF-κB activation in a TLR-4-dependent manner, and blocking TLR-4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation-associated markers in MDDCs from HCV-infected patients and increased the proliferation of T lymphocytes induced by these MDDCs. Conclusions and implications: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation, This work was supported in part by Grants from Industrial Farmacéutica Cantabria (IFC), from CIBER-EHD to Dr R Moreno and Dr M López-Cabrera, from the Ministerio de Educación y Ciencia SAF2007-61201 to M L-C and from the Instituto Salud Carlos III CP 03/0020 and the Ministerio de Educación y Ciencia SAF2007-60677 to Dr Pedro Majano

13 p.-4 fig., Class I phosphoinositide 3-kinases (PI3K) are involved in the development of normal and autoimmune responses, including Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for human multiple sclerosis (MS). Here, the role of the ubiquitously expressed class IA PI3K p110α catalytic subunits in EAE has been analyzed using a model of Cre/flox mediated T cell specific deletion of p110α catalytic chain (p110αΔT). Comparison of two month-old (young) and six month-old (mature) p110αΔT mice and their wild type (WT) counterparts indicated loss of spleen CD4+ T cells that increased with age, indicating a role of p110α in their homeostasis. In contrast, CD4+ T regulatory (Treg) cells were enhanced in mature p110αΔT mice when compared to WT mice. Since Myelin Oligodendrocyte Glycoprotein (MOG) peptide-induced EAE is dependent on, or mediated by CD4+ T cells and CD4+ T cell-derived cytokines and controlled by Treg cells, development of EAE in young and mature WT or p110αΔT mice was analyzed. EAE clinical symptoms and disease scores in six month p110αΔT mice were significantly lower than those of mature WT, or young WT and p110αΔT mice. Furthermore, ex vivo antigen activation of lymph node cells from MOG immunized mature p110αΔT mice induced significantly lower levels of IFN-γ and IL-17A than young p110αΔT or young and mature WT mice. Other cytokines including IL-2, IL-10 or TNF-α showed no significant differences between p110αΔT and WT mature mice. Our data show a lower incidence of MOG-induced EAE in mature p110αΔT mice linked to altered T cell homeostasis and lower secretion of inflammatory cytokines., This research was supported by Grants PI13/01809 (to JMR), PI13/02153 and PI16CIII/00012 (to PP) from “Acción Estratégica en Salud, Plan Estatal I+D+i”, Ministerio de Economía, Industria y Competitividad (MINECO, Spain), and by the Associazione Italiana Ricerca sul Cancro (Grant IG20714, AIRC, Milan) and Fondazione Cariplo (2017-0535) (to U.D.).

18 p.-8 fig.-1 tab., The interaction between the T‐lymphocyte costimulatory molecule ICOS and its ligand (ICOS‐L) is needed for efficient immune responses, but expression levels are tightly controlled, as altered expression of ICOS or ICOS‐L may lead to immunodeficiency, or favor autoimmune diseases and tumor growth. Using cells of mouse B cell lymphoma (M12.C3) and melanoma (B16), or hamster CHO cells transfected with various forms of mouse ICOS‐L, and ICOS+ T cell lines, we show that, within minutes, ICOS induces significant downmodulation of surface ICOS‐L that is largely mediated by endocytosis and trans‐endocytosis. So, after interaction with ICOS+ cells, ICOS‐L was found inside permeabilized cells, or in cell lysates, with significant transfer of ICOS from ICOS+ T cells to ICOS‐L‐expressing cells, and simultaneous loss of surface ICOS by the T cells. Data from cells expressing ICOS‐L mutants show that conserved, functionally important residues in the cytoplasmic domain of mouse ICOS‐L (Arg300, Ser307 and Tyr308), or removal of ICOS‐L cytoplasmic tail have minor effect on its internalization. Internalization was dependent on temperature, and was partially dependent on actin polymerization, the GTPase dynamin, protein kinase C, or the integrity of lipid rafts. In fact, a fraction of ICOS‐L was detected in lipid rafts. On the other hand, proteinase inhibitors had negligible effects on early modulation of ICOS‐L from the cell surface. Our data add a new mechanism of control of ICOS‐L expression to the regulation of ICOS‐dependent responses., Supported by Grants PI13/01809 (to J.M.R.), PI13/02153 and PI16CIII/00012 (to P.P.) from “Acción Estratégica en Salud, Plan Estatal I+D+i”, Ministerio de Economía, Industria y Competitividad(MINECO, Spain) and by the Associazione Italiana Ricerca sul Cancro(Grant IG20714, AIRC, Milan), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017-0535) (to U.D.).

11 p.-5 fig., CD28 expression is generally considered to be T lymphocyte specific. We have previously shown CD28 mRNA expression in M-CSF-dependent anti-inflammatory monocytederived macrophages (M-MØ), and now demonstrate that CD28 cell surface expression is higher in M-MØ than in GM-CSF-dependent macrophages, and that macrophage CD28 expression is regulated by MAFB and activin A. In vivo, CD28 was found in tumorassociated macrophages and, to a lower extent, in pro-inflammatory synovial fluid macrophages from rheumatoid arthritis patients. Analysis of mouse macrophages confirmed Cd28 expression in bone-marrow derived M-MØ. Indeed, anti-CD28 antibodies triggered ERK1/2 phosphorylation in mouse M-MØ. At the functional level, Cd28KO M-MØ exhibited a significantly higher capacity to activate the OVA-specific proliferation of OT-II CD4+ T cells than WT M-MØ, as well as enhanced LPS-induced IL-6 production. Besides, the Cd28KO M-MØ transcriptome was significantly different from WT M-MØ regarding the expression IFN response, inflammatory response, and TGF-β signaling related gene sets. Therefore, defective CD28 expression in mouse macrophages associates to changes in gene expression profile, what might contribute to the altered functionality displayed by Cd28KO M-MØ. Thus, CD28 expression appears as a hallmark of anti-inflammatory macrophages and might be a target for immunotherapy., This work was supported by grants PI17/00037 to APK, grants AES-PI13/02153 and AESIPI16CIII/00012 from Acción Estratégica de Salud del Instituto de Salud Carlos III to PP, grant SAF2017-83785-R from Ministerio de Economía y Competitividad to ALC, and Red de Investigación en Enfermedades Reumáticas, RIER RD16/0012/0007 to APK,and cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF).

26 p.-8 fig., Signaling through the inducible costimulator ICOS is required for the homeostasis and function of various immune cell populations, with an outstanding role in the generation and maintenance of germinal centers. Very recently, it has been suggested that the clinical phenotype of ICOS-deficient patients is much broader than initially anticipated and the innate immune response might be also affected. However, the role of the ICOS/ICOS-Ligand axis in the homeostasis and development of innate NK cells is not known, and reports on its participation in NK cell activation are scarce. NK cells may express low levels of ICOS that are markedly enhanced upon activation. We show here that ICOS-deficient (ICOS-KO) mice present low NK cell numbers and defects in the homeostasis of these cells, with delayed maturation and altered expression of the developmental NK cell markers CD122, NK1.1, CD11b or CD27. Our experiments in mixed bone marrow chimera mice indicate that, both, cell-intrinsic defects of ICOS-KO NK and deficiencies in the milieu of these mice contribute to the altered phenotype. ICOS-deficient NK cells show impaired production of IFN-γ and cytotoxicity, and a final outcome of defects in NK cell-mediated effector function during the response to poly(I:C) or vaccinia virus infection in vivo. Interestingly, we show that murine innate cells like IL-2-cultured NK and bone marrow-derived dendritic cells can simultaneously express ICOS and ICOS-Ligand; both molecules are functional in NK intracellular signaling, enhancing early phosphorylation of Akt and Erk, or IFN-γ secretion in IL-2-activated NK cells. Our study shows the functional importance of the ICOS/ICOS-L pair in NK cell homeostasis, differentiation and activity and suggests novel therapeutic targets for NK manipulation., This work was supported by grants from the Acción Estratégica en Salud (Instituto de Salud Carlos III, ISCIII, MINECO, Spain) (PI13/02153 and PI16CIII/00012 to P.P.; PI13/01809 to J.M.R.; and PI14/00049 to BdA); grants from Ministerio de Economía y Competitividad MINECO/FEDER, Spain (SAF2015-70880-R to M.L.G. and SAF2014-58052 to D.L.); and grants from Associazione Italiana per la Ricerca sul Cancro, AIRC, Milan (IG20714) and the Fondazione Amici di Jean, Torino, Italy (to U.D.).