Search

NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab′)2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)−/− or CD28−/− mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L−/− and CD28−/− mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.

Allyl ethers of the tricarbonylchromium(0) complexes of benzylic alcohols undergo highly enantioselective benzylic functionalisation using a chiral base/electrophilic quench sequence; the allyl group is readily removed to reveal a hydroxy group as demonstrated in the syntheses of enantioenriched imidazole alcohols, a triol and a tertiary alcohol. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

The prevalence of allergic diseases has significantly increased in industrialized countries. Allergen-specific immunotherapy (AIT) remains as the only curative treatment. The knowledge about the mechanisms underlying healthy immune responses to allergens, the development of allergic reactions and restoration of appropriate immune responses to allergens has significantly improved over the last decades. It is now well-accepted that the generation and maintenance of functional allergen-specific regulatory T (Treg) cells and regulatory B (Breg) cells are essential for healthy immune responses to environmental proteins and successful AIT. Treg cells comprise different subsets of T cells with suppressive capacity, which control the development and maintenance of allergic diseases by various ways of action. Molecular mechanisms of generation of Treg cells, the identification of novel immunological organs, where this might occur in vivo, such as tonsils, and related epigenetic mechanisms are starting to be deciphered. The key role played by the suppressor cytokines interleukin (IL)-10 and transforming growth factor (TGF)-beta produced by functional Treg cells during the generation of immune tolerance to allergens is now well established. Treg and Breg cells together have a role in suppression of IgE and induction of IgG4 isotype allergen-specific antibodies particularly mediated by IL-10. Other cell types such as subsets of dendritic cells, NK-T cells and natural killer cells producing high levels of IL-10 may also contribute to the generation of healthy immune responses to allergens. In conclusion, better understanding of the immune regulatory mechanisms operating at different stages of allergic diseases will significantly help the development of better diagnostic and predictive biomarkers and therapeutic interventions.

Trabajo presentado en el 248th National Meeting of the American-Chemical-Society (ACS), celebrado en San Francisco, CA (Estados Unidos), del 10 al 14 de agosto de 2014

Deprotonation of the benzylic carbon of a chromium tricarbonyl complex of a benzyl ether followed by reaction with N-fluorobenzenesulfonimide (NFSI) generated a species that reacted with oxygen, sulfur and carbon nucleophiles.

Naturally processed peptides, obtained by acid extraction of tumor cells, contain Ags able to activate specific CTL in vitro. We recently reported that the nonprofessional APC, RMA-S, expressing the B7.1 molecule (RMA-S/B7), pulsed with naturally processed peptides from the nonimmunogenic B16F1 melanoma (B16F1a.e.) primed syngenic CD8+ T cells against the tumor in vitro. Here, we show the rejection of B16F1 melanoma by C57BL/6 mice after immunization with RMA-S/B7 cells pulsed with B16F1a.e. This response is critically dependent on both CD4+ and CD8+ cells, but not on NK cells. However, only CD8+ T cells exert anti-B16F1 cytolitic activity in vitro. Moreover, RMA-S/B7 cells pulsed with B16F1a.e. can be used to prevent the growth of 24-h preestablished melanomas. These results may have important implications for the clinical use of natural peptide fractions of tumor cells as therapeutic cancer vaccines.

Cultured thymic fragments (CTF) from WAG/CPB(RTU and DA/01a (RT1a rats were prepared in the presence or absence of 2′ deoxyguanosine or cyclosporine A, and subsequently transplanted under the kidney capsule of congenitally athymic and euthymic WAG CPB recipients. The rationale of the culture supplements was that these may affect the disappearance of medullary dendritic cells, with subsequent induction of allotolerance. However, the immunohistology of the CTF showed more RTI class II-positive cells than keratin-positive cells, indicative of the maintenance of dendritic cells. Grafts in athymic animals showed the recovery of the original thymic architecture within 6 weeks utter transplantation. The influx of host-derived lymphocytes was accompanied by an influx of dendritic cells in the deculla-like area and macrophages in the cortex. A similar recovery was observed for syngeneic CTF in euthymic recipients. In addition lymphocytic infiltration was seen in the connective tissue surrounding the epithelial areas. Allogeneic grafts in euthymic animals were rejected within 3 weeks after transplantation. This outcome of the transplanted CTF under different conditions was not affected by the supplementation of the thymic culture before transplantation with 2′ deoxyguanosine or cyclosporine A. We conclude that there is no tolerance induction after transplantation in euthymic allogeneic rats of CTF prepared in the presence of 2′ deoxyguanosine. This conclusion is in contrast to date in the mouse, which may be explained by the maintenance of dendrite cells during culture. A chimaeric state of donor-derived epithelium and host-derived dendritic cells is obtained by transplantation of allografts in arthymic rats.

Antigen-pulsed dendritic cells (DCs) are used as natural adjuvants for vaccination, but the factors that influence the efficacy of this treatment are poorly understood. We investigated the parameters that affect the migration of subcutaneously injected mouse-mature DCs to the draining lymph node. We found that the efficiency of DC migration varied with the number of injected DCs and that CCR7+/+ DCs migrating to the draining lymph node, but not CCR7-/- DCs that failed to do so, efficiently induced a rapid increase in lymph node cellularity, which was observed before the onset of T cell proliferation. We also report that DC migration could be increased up to 10-fold by preinjection of inflammatory cytokines that increased the expression of the CCR7 ligand CCL21 in lymphatic endothelial cells. The magnitude and quality of CD4+ T cell response was proportional to the number of antigen-carrying DCs that reached the lymph node and could be boosted up to 40-fold by preinjection of tumor necrosis factor that conditioned the tissue for increased DC migration. These results indicate that DC number and tissue inflammation are critical parameters for DC-based vaccination.

An immunohistochemical study was done for the presence of tyrosine hydroxylase (noradrenergic innervation), neuron-specific protein PGP9.5, and anterior pituitary hormones (beta-subunit of follicle-stimulating hormone, growth hormone, beta-subunit of luteinizing hormone, prolactin, and beta-subunit of thyroid-stimulating hormone) in cultured thymic fragments before and after transplantation in congenitally athymic and euthymic rats. The cultured thymic fragments consisted of epithelial cells and were depleted of lymphocytes. After implantation in syngeneic and allogeneic athymic recipients and in syngeneic euthymic recipients, a recovery of the original architecture was found within 6 weeks; rejection occurred within 3 weeks for allogeneic transplantation in euthymic rats. During culture nerve-like profiles almost disappeared from the tissue, and reappeared simultaneously with the influx of host-derived cells and the restoration of the original thymic architecture. A high immunoreactivity for hormones and PGP9.5 was found in epithelial cells after culture and in the first phase after transplantation. These epithelial cells may represent precursor-epithelial cells, based on their unusual ultrastructure and combined expression of markers that in the normal thymus occur only on subcapsular/medullary epithelium or on cortex epithelium. These data indicate a potential role of the neuroendocrine function of the thymus during restoration of the thymus architecture starting from precursor-like epithelial cells.

Tumor cells undergoing programmed death are an attractive source of tumor-associated antigens, and evidences are available for their therapeutic efficacy in vivo when used either alone or in association with dendritic cells. However, little is known about the specificity of the immune response induced by such antigen formulation. Indeed, activation of specific proteases during apoptosis may influence the cytoplasmic degradation of proteins and the generation of CTL epitopes. We show here that on injection of C57BL/6 mice either with RMA lymphoma cells induced to apoptosis or bone marrow-derived dendritic cells pulsed with apoptotic RMA cells, a specific and protective CTL response is induced, which, however, is not directed against the immunodominant CTL epitope gag(85-93). Lack of in vivo expansion of gag(85-93)-specific CTL in vaccinated mice is attributable to the apoptosis-dependent loss of gag(85-93) in dying tumor cells. Indeed, we found loss of gag(85-93) in RMA, MBL-2, and EL-4G+ lymphoma cells, which share gag(85-93) as an immunodominant CTL epitope, induced to apoptosis by UV irradiation, mitomycin C, doxorubicin, or daunorubicin. This phenomenon appears to be caspase-dependent, because caspase inhibition by N-benzyloxycarbonyl-Val-Ala-asp-fluoromethylketone prevents apoptosis of lymphoma cells and loss of gag(85-93). Therefore, subversion of the epitope hierarchy in apoptotic tumor cells might be relevant in the induction of tumor-specific T-lymphocyte responses.

The immunogenic Friend-Moloney-Rauscher (FMR) virus-induced tumors have been used extensively to clarify the cellular and molecular mechanisms responsible for tumor rejection and to develop immunotherapeutic strategies. We characterize here the trimolecular complex MHC class I-antigenic determinant-T cell receptor involved in the induction of a protective CTL response against the RMA thymoma. This complex is mainly composed by the D(b) molecule interacting with a Rauscher virus antigen (Ag) determinant and the Vbeta5+ T cell receptor. We also show that the chemically induced EL-4 thymoma acquires the susceptibility to recognition by anti-RMA CTLs and the ability to elicit a protective anti-RMA CTL response only upon infection by a virus of the FMR family and that RMA and FMR virus infected EL-4 cells share tumor-associated Ag. The data strongly support the hypothesis that the high immunogenicity of virus-induced or infected tumors is determined by the expression of immunodominant virus-encoded Ag. The demonstration of a different outcome in the immune responses elicited in the presence or in the absence of viral Ag further open the contention of the molecular requirements for immunogenicity and should stimulate a more careful revision of unexpected cross-reactivity among tumors.

51 p.-6 fig.-1 tab.-4 schem.-1 graph. abst., Bacterial resistance to antibiotics makes previously manageable infections again disabling and lethal, highlighting the need for new antibacterial strategies. In this regard, inhibition of the bacterial division process by targeting key protein FtsZ has been recognized as an attractive approach for discovering new antibiotics. Binding of small molecules to the cleft between the N-terminal guanosine triphosphate (GTP)-binding and the C-terminal subdomains allosterically impairs the FtsZ function, eventually inhibiting bacterial division. Nonetheless, the lack of appropriate chemical tools to develop a binding screen against this site has hampered the discovery of FtsZ antibacterial inhibitors. Herein, we describe the first competitive binding assay to identify FtsZ allosteric ligands interacting with the interdomain cleft, based on the use of specific high-affinity fluorescent probes. This novel assay, together with phenotypic profiling and X-ray crystallographic insights, enables the identification and characterization of FtsZ inhibitors of bacterial division aiming at the discovery of more effective antibacterials., Thiswork was supported by grants BFU 2014-51823-R (J.M.A.),SAF2016-78792-R, PID2019-106279RB-I00 (M.L.L.-R.), BFU2017-87387-P (C.F.-T.), and predoctoral FPU fellowshipsfrom MECD (A.E.-P. and M.A.).