Evelyne Mougneau

A subset of dendritic cells induces CD4+ T cells to produce IFN-γ by an IL-12–independent but CD70-dependent mechanism in vivo

Interferon (IFN)-γ, a cytokine critical for resistance to infection and tumors, is produced by CD4+ helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12–independent pathway whereby DCs induce IFN-γ–secreting T helper (Th)1 CD4+ T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-γ in vivo but required IL-12, not CD70. Isolated CD205+ DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205+ DC function in vivo. Detection of the IL-12–independent IFN-γ pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205+ DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.

Tolerance to Islet Antigens and Prevention from Diabetes Induced by Limited Apoptosis of Pancreatic β Cells

Crosspresentation of self-antigens by antigen-presenting cells is critical for the induction of peripheral tolerance. As apoptosis facilitates the entry of antigens into the crosspresentation pathway, we sought to prevent the development of autoimmune diabetes by inducing pancreatic beta cell apoptosis before disease onset. Accordingly, young nonobese diabetic (NOD) mice injected with a single low dose of streptozotocin (SZ), a drug cytotoxic for beta cells, exhibited impaired T cell responses to islet antigens and were protected from spontaneous diabetes. Furthermore, beta cell apoptosis was necessary for protection since SZ did not protect RIP-CrmA transgenic NOD mice in which beta cells expressed the caspase inhibitor CrmA. Our results support a model in which apoptosis of pancreatic beta cells induces the development of regulatory cells leading to the tolerization of self-reactive T cells and protection from diabetes.

Listeria monocytogenes as a Short-Lived Delivery System for the Induction of Type 1 Cell-Mediated Immunity against the p36/LACK Antigen of Leishmania major

ABSTRACT Listeria monocytogenes has been used as an experimental live vector for the induction of CD8-mediated immune responses in various viral and tumoral experimental models. Susceptibility of BALB/c mice to Leishmania major infection has been correlated to the preferential development of Th2 CD4 T cells through an early production of interleukin 4 (IL-4) by a restricted population of CD4 T cells which react to a single parasite antigen, LACK (stands forLeishmania homologue of receptors for activated C kinase). Experimental vaccination with LACK can redirect the differentiation of CD4+ T cells towards the Th1 pathway if LACK is coadministrated with IL-12. As IL-12 is known to be induced by L. monocytogenes, we have tested the ability of a recombinant attenuated actA mutant L. monocytogenes strain expressing LACK to induce the development of LACK-specific Th1 cells in both B10.D2 and BALB/c mice, which are resistant and susceptible toL. major, respectively. After a single injection of LACK-expressing L. monocytogenes, IL-12/p40 transcripts showed a rapid burst, and peaks of gamma interferon (IFN-γ)-secreting LACK-specific Th1 cells were detected around day 5 in the spleens and livers of mice of both strains. These primed IFN-γ-secreting LACK-reactive T cells were not detected ex vivo after day 7 of immunization but could be recruited and detected 15 days later in the draining lymph node after an L. major footpad challenge. Although immunization of BALB/c mice with LACK-expressing L. monocytogenes did not change the course of the infection withL. major, immunized B10.D2 mice exhibited significantly smaller lesions than nonimmunized controls. Thus, our results demonstrate that, in addition of its recognized use for the induction of effector CD8 T cells, L. monocytogenes can also be used as a live recombinant vector to favor the development of potentially protective IFN-γ-secreting Th1 CD4 T lymphocytes.

Antigen Presentation by Dendritic Cells In Vivo

Although dendritic cells (DCs) are known to be critical for inducing T cell immunity in immunized or infected individuals, it was recently proposed that DCs are also essential to silence potentially pathogenic self-reactive T cells that have escaped negative selection in the thymus ([1][1], [2][2

Direct Visualization of Peptide/MHC Complexes at the Surface and in the Intracellular Compartments of Cells Infected In Vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.

c-myc and Functionally Related Oncogenes Induce Both High Rates of Sister Chromatid Exchange and Abnormal Karyotypes in Rat Fibroblasts

Activated myc genes are found in a broad variety of spontaneous malignancies in rodents and humans as well: Burkitt Lymphoma (Klein and Klein 1985; Croce et al. 1984; Dalla-Favera et al. 1985; this volume), neuroblastoma (Schwab 1985), small cell lung carcinoma (Nau et al. 1985), mouse plasmacytoma (Klein and Klein 1985; this volume) and rat immunocytoma (Pear, this volume).

Integration sites and sequence arrangement of SV40 DNA in a homogeneous series of transformed rat fibroblast lines.

The state and organization of viral DNA sequences present in independently isolated rat cell lines transformed with SV40 were investigated using restriction-enzyme cleavage of the cellular DNA and blot hybridization with a viral probe. The transformed lines were established under conditions as identical as possible, except for a limited number of variables (multiplicity of infection, physiological state of the cells after infection and procedures used for selecting the transformed derivatives). They were characterized after a limited number of generations in culture. Two distinct types of organization were found: covalently integrated viral genomes were present either as single inserts or as head-to-tail oligomeric structures. The latter was observed among transformants derived from cells maintained after infection under growth-inhibiting conditions (suspension in agarose medium, confluency on a solid substrate). Single inserts were observed only among cell lines isolated after an initial period of active growth. Recurrent patterns of hybridizaton were observed in independently isolated lines, indicating that the sites of the integrative recombinations were close enough, both in the viral and the cellular sequences, not to be distinguished at the level of sensitivity of the technique (more than +/- 100 bp). Among cell lines with multiple integration sites, only part of the inserts were found in several instances to be identical to inserts observed in other transformed lines.

Frontline: Self‐peptides that bind with low affinity to the diabetes‐associated I‐Ag7 molecule readily induce T cell tolerance in non‐obese diabetic mice

Although non‐obese diabetic (NOD) mice spontaneously develop T cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self‐Ag. Furthermore, as autoimmunity has been postulated to result from T cell responses directed toward self‐peptides that bind with low affinity to NOD I‐Ag7 MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic β cells. We identified LACK peptides that were the targets of T cells in LACK‐immunized NOD mice while binding to I‐Ag7 with low affinity. While CD4+ T cells from NOD mice secreted IFN‐γ, IL‐4, IL‐5 and IL‐10 in response to LACK, those from LACK‐expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK‐expressing Tg mice exhibited self‐reactive CD4+ T cells with impaired proliferation capabilities. Hence, even self‐peptides that bind to I‐Ag7 with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that T cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation

Vascotto et al. 2007. J. Cell Biol. doi:10.1083/jcb.200611147 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200611147%26rft_id%253Dinfo%253Apmid%252F17389233%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

Cooperative Interactions Involving Cellular and Viral Oncogenes in the Development of Malignant Tumors

A group of cellular and viral oncogenes including the plt gene of polyoma virus (large T protein), the E1A genes of adenoviruses and rearranged forms of the c-myc oncogene cooperate with oncogenes of a second class, including the cellular ras oncogenes and the pmt gene of polyoma virus in the oncogenic transformation fat embryo fibroblasts (1). The myc, plt and E1A genes induce by themselves an early stage of transformation (“immortalization”). The properties of cells at this stage are of interest, since they may constitute a useful experimental model for the study of a very early stage of tumor progression. Transfer of the genes did not result in the immediate appearance of a tumorigenic potential in vivo, and, in fact most of the properties of cells grown in vitroremained similar to those of the normal controls. In primary rat embryo fibroblast cultures, the only phenotypes consistently associated with the expression of these genes in cell culture were a change in the hormonal growth, indicated by a decrease in their requirement in serum factors, and the ability of long term growth (2). When rearranged myc genes were transferred into cells of an established line (FR3T3) together with the neor drug-resistance gene, selection in G418 medium produced cell lines which, upon successive generations in culture, exhibited a characteristic high frequency of spontaneous transformation and acquisition of a tumorigenic potential in syngeneic animals. Fluctuation assays indicated a wide distribution of frequencies between parallel sub-cultures, indicative of the random occurrence of secondary genetic events leading to tumorigenesis (3).