PARP-1/PARP-2 double deficiency in mouse T cells results in faulty immune responses and T lymphomas.

The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.

Antagonistic effect of TNF-alpha and insulin on uncoupling protein 2 (UCP-2) expression and vascular damage.

BACKGROUND: It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels. METHODS: We analyzed the effect of insulin or oleic acid in presence or not of TNF-α on UCP-2 expression in murine endothelial and vascular smooth muscle cells. At this step, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE-/- mice under Western type diet for 2, 6, 12 or 18 weeks, BATIRKO mice under high-fat diet for 16 weeks and 52-week-old BATIRKO mice with o without anti-TNF-α antibody pre-treatment. RESULTS: Firstly, we found that TNF-α pre-treatment reduced UCP-2 expression induced by insulin in vascular cells. Secondly, we observed a progressive reduction of UCP-2 levels together with an increase of lipid depots and lesion area in aorta from ApoE-/- mice. In vivo, we also observed that moderate hyperinsulinemic obese BATIRKO mice have lower TNF-α and ROS levels and increased UCP-2 expression levels within the aorta, lower lipid accumulation, vascular dysfunction and macrovascular damage. We also observed that the anti-TNF-α antibody pre-treatment impaired the loss of UCP-2 expression within the aorta and relieved vascular damage observed in 52-week-old BATIRKO mice. Finally, we observed that the pretreatment with iNOS inhibitor prevented UCP-2 reduction induced by TNF-α in vascular cells. Moreover, iNOS levels are augmented in aorta from mice with lower UCP-2 levels and higher TNF-α levels. CONCLUSIONS: Our data suggest that moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.

Human MR1 expression on the cell surface is acid sensitive, proteasome independent and increases after culturing at 26°C.

Mucosal-associated invariant T (MAIT) cells are a population of non-conventional T-lymphocytes which are restricted by the MHC-related 1 (MR1) molecule. MR1 is a non-classical member of the MHC class I family of proteins, it is unknown if MR1 presents any kind of antigens to MAIT cells. In the present manuscript we describe that detection of MR1 on the cell surface by conformation-dependent monoclonal antibodies is enhanced upon culture the cells at 26°C; we also show that detection of MR1 on the cell surface is lost after treating the cells at pH 3.3 as in the case of classical MHC class I molecules. Finally, the re-expression of MR1 on the cell surface is independent of proteasome. Taken together these results strongly suggest that MR1 needs to bind proteasome-independent ligands in order to properly reach the cell surface.

The MHC-related protein 1 (MR1) is expressed by a subpopulation of CD38+, IgA+ cells in the human intestinal mucosa.

MHC-related 1 (MR1) molecule is a non-classical member of the MHC class I family of proteins. The sequence homology between classical MHC class I molecules and MR1 is very high, although the MR1 gene is not polymorphic and is highly conserved between species. MR1 is the restriction molecule of a sub-population of T lymphocytes, which are CD4-,CD8- and display conserved TCR alpha chain. The function of these cells is currently unknown, but they are believed to have regulatory properties similar to those of the CD1d restricted NKT cells. The MR1 gene is ubiquitously transcribed; however it is unknown what types of cells express the MR1 protein "in vivo". In the present work we analyzed the expression of the MR1 protein using specific antisera and monoclonal antibodies in different human cell lines, in primary cells and in mucosal tissues. We found some lymphoid cell lines that express MR1 on the cell surface but at levels much lower than the MR1 transfected cell lines. In addition, we observed that expression of MR1 in the mucosa is restricted to a subpopulation of plasma cells or plasmablasts, CD38+ or CD138+ and IgA+, located in the human intestinal mucosa. This suggests a function for MR1 in the development of IgA producing plasma cells.

A role for DNA polymerase mu in the emerging DJH rearrangements of the postgastrulation mouse embryo.

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ(H) joints and compare them with those present in later life. The embryo DJ(H) joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase mu (Polmu), which was widely expressed in the early ontogeny, as shown by analysis of Polmu(-/-) embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polmu also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polmu participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.

Immature human dendritic cells infected with Leishmania infantum are resistant to NK-mediated cytolysis but are efficiently recognized by NKT cells.

Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-gamma. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania.

Expression of human CD1d molecules protects target cells from NK cell-mediated cytolysis.

The cytotoxic activity of NK cells can be inhibited by classical and nonclassical MHC molecules. The CD1 system is formed by a family of glycoproteins that are related to classical MHC. CD1a, b, and c molecules present lipids or glycolipids to T cells and are involved in defense against microbial infections, especially mycobacteria. It has been shown recently that these molecules can inhibit target cell lysis by human NK cells. It has also been shown that mouse CD1d molecules can protect cells from NK cell-mediated cytotoxicity. In the present study, we describe how human CD1d, orthologous to murine CD1 molecules, can inhibit NK cell-mediated cytolysis. We have expressed CD1d in the HLA class I-deficient cell lines L721.221 and C1R. The inhibitory effect is observed when effector NK cells from different donors are used, as well as in different cell lines with NK activity. The inhibitory effect was reversed by incubating the target cells with a mAb specific for human CD1d. Incubation of target cells with the ligands for CD1d, alpha-galactosylceramide (alpha-GalCer), and beta-GalCer abolishes the protective effect of CD1d in our in vitro killing assays. Staining the effector cells using CD1d tetramers loaded with alpha-GalCer was negative, suggesting that the putative inhibitory receptor does not recognize CD1d molecules loaded with alpha-GalCer.