In its vertebrate host, Leishmania encounters cells that express TLRs. Using genetically resistant C57BL/6 mice deficient in either TLR2, 4, or 9, we show in this study that only TLR9-deficient mice are more susceptible to infection with Leishmania major. TLR9-deficient mice resolved their lesions and controlled parasites growth with much lower efficiency than wild-type C57BL/6 mice. The absence of TLR9 also transiently inhibited the development of curative Th1 response. In an attempt to analyze the possible basis for such aberrant response in TLR9(-/-) mice, we have studied the importance of TLR9 for the activation of dendritic cells (DCs) by L. major. Results show that DCs in the draining lymph nodes are activated following infection with L. major. Furthermore, bone marrow-derived DCs as well as DCs freshly isolated from the spleen of C57BL/6 mice can be activated by either heat-killed or live L. major in vitro. In sharp contrast, L. major failed to activate DCs from TLR9(-/-) mice. Noteworthily, activation of DCs was abolished either following treatment of the parasites with DNase or after acidification of the endosomal compartment of DCs by chloroquine, pinpointing the DNA of L. major as the possible ligand of TLR9 leading to the activation of DCs. Results showed that DNA purified from L. major was indeed capable of activating DCs in a strictly TLR9-dependent manner. Moreover we showed that the L. major DNA-induced TLR9 signaling in DCs condition these cells to promote IFN-gamma production by CD4(+) T cells.

Communicated by P.Chambon is needed to account for the B-cell specificity of the VH promoter. In addition, our results suggest that the lack of activity of the renin promoter in non cognate cells is not due to the binding of a repressor.

Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy‐terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site‐specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3′ end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half‐life of the TdTL protein in these cells is 2‐fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.

The somatic diversity immunglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Ragl and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Ragl and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types ofjunctional diversity observed during lymphoid development.Immunoglobulin (Ig) and T-cell receptor (TCR) gene assembly is achieved through site-specific recombination events, from separately encoded variable (V), in some cases diversity (D), and junction (J) gene segments. Much of the immunoglobulin and TCR diversity is generated by the combinatorial rearrangement of a large number of V, D, and J gene segments (for a review see refs. 1 and 2).Recombination signal sequences (RSSs) situated adjacent to each gene segment provide the targets for recombination. RSSs are composed of a palindromic heptamer and an (A+T)-rich nonamer separated by a spacer of 12 or 23 base pairs (bp) (3). Rearrangement only occurs between RSSs with spacers of different length. RSSs are sufficient to target rearrangement of artificial substrates containing no other antigen receptor sequences (4-7).Two main types of joints are formed during the recombination process: coding joints created by the juxtaposition of the gene segments and reciprocal joints by contiguous RSSs (8). Whereas the heptamers in the reciprocal joints are generallyjoined back to back without nucleotide insertions or deletions, the coding joints are subjected to extensive processing (8). The junctions formed during rearrangement constitute another source of diversity. Several nucleotides can be deleted and two types of insertions can be found. Random nucleotide additions, resulting in N-region insertions, are thought to be introduced by terminal deoxynucleotidyltransferase (TdT) (9-11). P-nucleotide insertions represent the inverted repeat of the adjacent coding sequence. Their addition has been proposed to be a compulsory step of the recombination mechanism (12).Genomic DNA transfection experiments have allowed the isolation of two recombination activating genes, Ragl and Rag2 (13-15). Together these genes are able to confer sitespecific recombination activity to NIH 3T3 fibroblasts. Although it is not formally excluded that they activate other genes that would be responsible for the recombination activity, Ragl...

The physiologic role played by plasmacytoid dendritic cells (pDCs) in the induction of innate responses and inflammation in response to pathogen signaling is not well understood. Here, we describe a new mouse model lacking pDCs and establish that pDCs are essential for the in vivo induction of NK-cell activity in response to Toll-like receptor 9 (TLR9) triggering. Furthermore, we provide the first evidence that pDCs are critical for the systemic production of a wide variety of chemokines in response to TLR9 activation. Consequently, we observed a profound alteration in monocyte, macrophage, neutrophil, and NK-cell recruitment at the site of inflammation in the absence of pDCs in response to CpG-Dotap and stimulation by microbial pathogens, such as Leishmania major, Escherichia coli, and Mycobacterium bovis. This study, which is based on the development of a constitutively pDC-deficient mouse model, highlights the pivotal role played by pDCs in the induction of innate immune responses and inflammation after TLR9 triggering. (Blood. 2012;120(1): 90-99) IntroductionPlasmacytoid DCs (pDCs) are characterized by their ability to contribute to antiviral innate immunity by producing type I IFNs on stimulation. 1 These cells display a CD11c low B220 ϩ Ly6C ϩ CD45RA ϩ phenotype and also express markers, such as CD317 (BST-2) 2 and SiglecH. 3 Their Toll-like receptor (TLR) expression pattern is limited to TLR7 and TLR9, which recognize viral single-stranded RNA and unmethylated DNA, respectively. The constitutive expression of IFN regulatory factor 7 enables pDCs to rapidly produce high levels of type I IFNs after TLR stimulation. After activation via TLR7 or TLR9 signaling, pDCs produce cytokines, such as IL-12, IL-6, and TNF-␣, and chemokines, including CCL3, CCL4, CCL5, CXCL9, and CXCL10, in addition to type I IFNs. 4 NK cells exhibit potent cytotoxic activity against infected or tumor cells and are efficient producers of several cytokines and chemokines. 5 NK-cell activation is controlled by the recognition of ligands expressed on the surface of target cells. However, NK cells require additional signals for activation, including type I IFNs and IL-12. 6 Because of their ability to produce these cytokines, pDCs may play an important role in stimulating and inducing NK-cell responses. Indeed, pDCs can promote murine cytomegalovirus clearance by NK cells through TLR9 interaction. 7 Furthermore, NK cells express the chemokine receptors CCR5 and CXCR3, which interact with the chemokines produced by activated pDCs, 8 suggesting that pDCs may also influence their recruitment. In addition to NK cells, immature conventional DCs (cDCs), monocytes, macrophages, polymorphonuclear basophiles (PMBs) and eosinophils (PMEs) also respond to type I IFNs 9 and to CCL3, CCL4, and CCL5, 8 suggesting that pDCs participate in the activation and recruitment of inflammatory cells.To directly assess the physiologic role of pDCs in innate immunity, it is crucial to analyze these responses in vivo in the absence of pDCs. pDCs could be immunodeple...