Noëlle Doyen

The conserved decanucleotide from the immunoglobulin heavy chain promoter induces a very high transcriptional activity in B-cells when introduced into an heterologous promoter.

A conserved decanucleotide (ATGCAAATNA) is present 45‐60 nucleotides upstream from the transcription startpoint in all immunoglobulin heavy chain promoters (VH promoters). We have introduced this decanucleotide (cd sequence) at a similar position into the upstream flanking sequence of the mouse Renin‐1 gene. This gene is only transcribed in highly specialized tissues, and the fragment used here (‐449 to +30 with respect to the main transcription startpoint) has little promoter activity in fibroblastic or myeloma cell lines, even if coupled to a functional enhancer. In contrast, after insertion of the decanucleotide, this fragment, while still inactive in non‐lymphoid cells, becomes a potent promoter in B‐cells when associated with SV40 or immunoglobulin heavy chain enhancer. In all respects, the engineered fragment behaves like an authentic VH promoter isolated in this laboratory, except that it is even more active in B‐cells. Deletion experiments show that all renin sequences are dispensable for the activity of the chimaeric promoter, except probably for the renin TATA box which defines the precise transcription startpoint. We conclude that the decanucleotide is sufficient to activate a promoter in B‐cells but not in non‐B‐cells, and therefore that no other element 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.

TLR9-Dependent Activation of Dendritic Cells by DNA from Leishmania major Favors Th1 Cell Development and the Resolution of Lesions

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-γ production by CD4+ T cells.

THE TWO ISOFORMS OF MOUSE TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE DIFFER IN BOTH THE ABILITY TO ADD N REGIONS AND SUBCELLULAR LOCALIZATION

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.

Study of the antigenic structure of human serum albumin with monoclonal antibodies

Analysis of the antigenic structure of human serum albumin was undertaken using monoclonal antibodies. Nineteen antibodies were prepared and their specificities were studied using fragments which encompass the whole sequence of the albumin molecule. These antibodies recognized 13 different epitopes which are different from the one previously identified with two other monoclonal antibodies [Doyen et al., Immun. Lett. 3, 365-370 (1981)]. Among those 13 different epitopes, six were overlapping. Four epitopes were located on the N-terminal half of the albumin molecule. One of these required integrity of methionine 87 and the other three were overlapping and located around methionine 123. Eight epitopes were located on the C-terminal half of the albumin. Two of them were within the sequence, 330-422 and 299-496 respectively; the other six appeared to be topographic determinants which were altered or lost in the albumin fragments. A last epitope could not be located on any region of albumin. Four monoclonal antibodies directed against a given portion of the albumin molecule reacted slightly with another part of albumin, thus confirming the existence of an intramolecular cross-reactivity between the different domains of human albumin.

Similar binding properties for a neutralizing anti-tetanus toxoid human monoclonal antibody and its bacterially expressed Fab.

A high-affinity anti-tenanus toxoid (TT) human monoclonal antibody showing neutralizing activity was isolated from a fusion between mouse myeloma and human splenic cells. Fab fragments from this antibody were obtained using a recombinant phage surface-display expression system. The parental antibody and the corresponding Fab had identical immunological activities, including specificity and affinity. These results confirm the feasibility of developing Escherichia coli expression of monoclonal human Fab from hybridoma cells.

Substantial N diversity is generated in T cell receptor α genes at birth despite low levels of terminal deoxynucleotidyl transferase expression in mouse thymus

N region diversity in antigen receptors is a developmentally regulated process in B and T lymphocytes, which correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). To precisely determine the onset of TdT gene activation during T cell differentiation and thymic ontogeny, TdT expression was directly detected at the cellular level by in situ hybridization and TdT function was assessed by analyzing the distribution of N additions in α and β TCR genes at early stages of development. Even though TdT transcripts were undetectable at birth, substantial N additions were observed in VαJα junctions and 3 days later in VβDβJβ junctions, indicating that TdT expression could be induced in immature thymocytes much earlier than expected. Indeed low TdT expression level was found in TN3/4 and DP from fetal day 17, suggesting that the onset of TdT expression occurs simultaneously in both populations and may depend on microenvironmental cues. Moreover significant increase in the proportion of thymocytes expressing high levels of TdT mRNA during the first week after birth without a similar increase in the level of N diversity suggests that TdT expression and TdT function in the generation of N diversity are not strictly correlated.

Analysis of promoter and enhancer cell type specificities and the regulation of immunoglobulin gene expression.

We have analysed the properties of IgH promoter (VH) and enhancer (Ig) regions which were used to drive the expression of the chloramphenicol acetyl transferase (CAT) gene (cat) in recombinant plasmids. We observe little synergistic effect between the VH promoter and Ig enhancer on cat gene expression in our constructs. Replacing the VH promoter by the thymidine kinase (TK) promoter does not affect the enhancer-mediated B-cell-specific expression of the cat gene. However, replacement of the VH promoter by the mouse renin gene promoter, which is not normally expressed in B cells, completely abolishes cat gene expression in cells of this lineage. When the Ig enhancer is replaced by the SV40 enhancer (SV), CAT activity is restricted to B cells. The VH promoter is as efficient as the TK promoter in a preB cell line. Extending the size of the VH promoter fragment to include sequences between 126 to 639 bp upstream from the transcription start point results in an eight-fold decrease in CAT activity. In this situation, the tissue specificity of the promoter cat fusion is maintained. Among the various combinations tested here, the association of the TK promoter and the Ig enhancer expresses the cat gene most efficiently. The implications of these observations are discussed.

TLR–CD40 Cross-Talk in Anti-Leishmanial Immune Response

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and activate innate immune cells to induce cytokines and co-stimulatory molecules such as CD40 and to enhance antigen presentation to T cells (1) that, upon activation, can either eliminate or support the pathogen (2). Herein, we propose that this duality in TLR functions results from their cross-talk with CD40. While all TLRs enhance CD40 expression, CD40 augments the expression of only TLR9 (3). As both CD40 and TLR9 induce expression of IL-12, a cytokine that induces the IFN-γ secreting Th1 cell differentiation (4), the CD40–TLR9 cross-regulation implies a positive feedback loop. By contrast, TLR1–TLR2 heterodimer down-regulates TLR9 expression (5) and antagonizes the development of Th1 response but favors the differentiation of regulatory T (T-reg) cells (Pandey et al., unpublished observation). Low CD40 expression levels in dendritic cells also promote T-reg cell differentiation (6). This duality can emerge from the sharing of signaling molecules. CD40 induces TRAF6-mediated, ERK-1/2-dependent IL-10 (7), which can inhibit the TLR-induced p38-MAPK activation and IL-12 production, antagonizing Th1 development. CD40-induced TRAF3-dependent p38-MAPK activation (7) can synergize with the TLR-activated p38-MAPK-dependent IL-12 production and Th1 differentiation. Using Leishmania infection, we show that the TLR–CD40 cross-talk can induce contrasting anti-leishmanial immune responses. Leishmania, a protozoan parasite, lives in macrophages. Leishmania expresses lipophosphoglycan (LPG), proteoglycans, flagellin, and profilin for possible recognition by the host cell-expressed TLRs. Recognition of the Leishmania-expressed PAMPs results in differential immune responses, which can either reduce or exacerbate Leishmania infection. As TLRs modulate the expression of CD40, a co-stimulatory molecule whose expression levels modulate anti-leishmanial T cell responses, we propose that TLR–CD40 cross-talk significantly regulate the outcome of an anti-leishmanial immune response.

Evidence That the Long Murine Terminal Deoxynucleotidyltransferase Isoform Plays No Role in the Control of V(D)J Junctional Diversity

Two TdT isoforms have been found in the mouse. The short isoform is known to add N regions to gene segment junctions during V(D)J recombination, but the role of the long (TdTL) isoform is controversial. We have shown that TdTL, although endowed with terminal transferase activity, is thermally unstable and unable to add N regions in vivo. In this study, we demonstrate that TdTL is devoid of 3′-5′ exonuclease activity, and provide an analysis of nucleotide deletion and addition patterns in large series of V(D)J coding joins, arguing against a role of TdTL in the control of junctional diversity in Igs and TCRs.

A mouse renin promoter containing the conserved decanucleotide element binds the same B-cell factors as an authentic immunoglobulin heavy chain promoter

A mouse renin‐1 gene promoter fragment, normally inactive in B‐cells, becomes a potent promoter in these cells after insertion of the highly conserved decanucleotide (dc/cd sequence) of immunoglobulin heavy and light chain promoters [(1987) EMBO J. 6, 1685–1690]. We observe retarded complexes of the same electrophoretic mobility when the cd‐containing renin promoter fragment or an authentic immunoglobulin heavy chain promoter fragment is incubated with a nuclear extract from myeloma cells, suggesting that the renin promoter is activated due to its acquired ability to bind a B‐cell‐specific positive factor. No retarded complexes are observed with the original renin promoter fragment thus questioning the presence of a repressor as an explanation for its lack of activity in B‐cells.