A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells.

Gene targeting of the adaptor molecule DAP12 in mice caused abnormal distribution and impaired antigen presentation capacity of dendritic cells (DCs). However, the DAP12-associated receptors expressed on DCs and their functions have not been identified yet. Here we show that the triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor on human monocyte-derived DCs, which is associated with DAP12. TREM-2/DAP12 promotes upregulation of CC chemokine receptor 7, partial DC maturation, and DC survival through activation of protein tyrosine kinases and extracellular signal-regulated kinase. In contrast to Toll-like receptor-mediated signaling, TREM2/DAP12 stimulation is independent of nuclear factor-kappaB and p38 stress-activated protein kinase. This novel DC activation pathway may regulate DC homeostasis and amplify DC responses to pathogens, explaining the phenotype observed in DAP12-deficient mice.

Chemokine production by G protein-coupled receptor activation in a human mast cell line: roles of extracellular signal-regulated kinase and NFAT.

Chemoattractants are thought to be the first mediators generated at sites of bacterial infection. We hypothesized that signaling through G protein-coupled chemoattractant receptors may stimulate cytokine production. To test this hypothesis, a human mast cell line (HMC-1) that normally expresses receptors for complement components C3a and C5a at low levels was stably transfected to express physiologic levels of fMLP receptors. We found that fMLP, but not C3a or C5a, induced macrophage inflammatory protein (MIP)-1ss (CCL4) and monocyte chemoattractant protein-1 (CCL2) mRNA and protein. Although fMLP stimulated both sustained Ca(2+) mobilization and phosphorylation of extracellular signal-regulated kinase (ERK), these responses to C3a or C5a were transient. However, transient expression of C3a receptors in HMC-1 cells rendered the cells responsive to C3a for sustained Ca(2+) mobilization and MIP-1ss production. The fMLP-induced chemokine production was blocked by pertussis toxin, PD98059, and cyclosporin A, which respectively inhibit G(i)alpha activation, mitgen-activated protein kinase kinase-mediated ERK phosphorylation, and calcineurin-mediated activation of NFAT. Furthermore, fMLP, but not C5a, stimulated NFAT activation in HMC-1 cells. These data indicate that chemoattractant receptors induce chemokine production in HMC-1 cells with a selectivity that depends on the level of receptor expression, the length of their signaling time, and the synergistic interaction of multiple signaling pathways, including extracellular signal-regulated kinase phosphorylation, sustained Ca(2+) mobilization and NFAT activation.

Accessibility control of T cell receptor gene rearrangement in developing thymocytes. The TCR alpha/delta locus.

The joining of T cell receptor (TCR) and immunoglobulin (Ig) gene segments through the process of V(D)J recombination occurs in a lineage-specific and developmental-stage-specific way during the early stages of lymphocyte development. Such developmental regulation is thought to be mediated through the control of gene segment accessibility to the recombinase. We have studied the regulation of V(D)J recombination at the TCR alpha/delta locus, because this locus provides a fascinating model in which distinct sets of gene segments are activated at different stages of T cell development. The transcriptional enhancers Edelta and Ealpha have been implicated as critical regulators that, in conjunction with other cis-acting elements, confer region-specific and developmental-stage-specific changes in gene segment accessibility within TCR alpha/delta locus chromatin. Current work suggests that they may do so by functioning as regional modulators of histone acetylation.

A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation.

V(D)J recombination and transcription within the TCR alpha/delta locus are regulated by three characterized cis-acting elements: the TCR delta enhancer (Edelta), TCR alpha enhancer (Ealpha), and T early alpha (TEA) promoter. Analysis of enhancer and promoter occupancy and function in developing thymocytes in vivo indicates Edelta and Ealpha to be developmental-stage-specific enhancers, with Edelta "on" and Ealpha "off" in double-negative III thymocytes and Edelta "off" and Ealpha "on" in double-positive thymocytes. Edelta downregulation reflects a loss of occupancy. Surprisingly, Ealpha and TEA are extensively occupied even prior to activation. TCR delta downregulation in double-positive thymocytes depends on two events, Edelta inactivation and removal of TCR delta from the influence of Ealpha by chromosomal excision.

Developmental regulation of V(D)J recombination at the TCR alpha/delta locus.

The T-cell receptor (TCR) alpha/delta locus includes a large number of V, D, J and C gene segments that are used to produce functional TCR delta and TCR alpha chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T-lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis-acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR alpha/delta locus imparts developmental control to VDJ delta and VJ alpha rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR alpha/delta locus, highly localized and region-specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

Cooperation among multiple transcription factors is required for access to minimal T-cell receptor alpha-enhancer chromatin in vivo.

To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor alpha enhancer (Ealpha), we analyzed enhancer occupancy in thymocytes of transgenic mice in vivo by genomic footprinting. We found that the formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5' of the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type Ealpha, all sites are unoccupied on versions of Ealpha with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB. Our data indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single Ealpha binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable binding site occupancy as well as the activation of transcription and V(D)J recombination.

Enhancer control of local accessibility to V(D)J recombinase.

We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.

Regulation of T cell receptor delta gene rearrangement by c-Myb.

Developmental activation of VDJ recombination at the T cell receptor (TCR) delta locus is controlled by an intronic transcriptional enhancer (E delta). Transcriptional activation by E delta is dependent on c-Myb. To determine whether c-Myb plays a role in the activation of TCR-delta gene rearrangement, we compared VDJ recombination in transgenic mice carrying two versions of a human TCR-delta gene minilocus recombination substrate. One includes a wild-type E delta, whereas the other carries an E delta with a mutation that abrogates c-Myb binding. We demonstrate that an intact Myb binding site is necessary for efficient rearrangement of the minilocus substrate, suggesting that c-Myb plays a crucial role in activating VDJ recombination at the endogenous TCR-delta locus.

Transcriptional regulation of the human T cell receptor delta gene.

T cell receptor delta gene expression is regulated by a T cell-specific transcriptional enhancer located within the J delta 3-C delta intron. An essential element of the enhancer was localized to a small 30 bp segment denoted delta E3. Two specific factors, CBF/PEBP2 and c-Myb, bind to adjacent sites within delta E3 and cooperate functionally to mediate transcriptional activation. These factors are likely to play essential roles in the developmental activation of the TCR delta gene in vivo.

c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer.

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor delta gene. We have previously shown that a 30-bp element, denoted delta E3, acts as the minimal TCR delta enhancer and that within delta E3, adjacent and precisely spaced binding sites for core-binding factor (CBF/PEBP2) and c-Myb are essential for transcriptional activity. These data suggested that CBF/PEBP2 and c-Myb synergize to mediate transcriptional activity but did not establish the molecular basis for synergy. In this study, we have examined in detail the binding of CBF/PEBP2 and c-Myb to delta E3. We found that CBF/PEBP2 and c-Myb could simultaneously occupy the core site and one of two overlapping Myb sites within delta E3. However, equilibrium binding and kinetic dissociation experiments suggest that the two factors bind to delta E3 independently, rather than cooperatively. This was found to be true by using isoforms of these factors present in extracts of transfected COS-7 cells, as well as the natural factors present in nuclear extracts of the Jurkat T-cell line. We further showed that CBF/PEBP2 and c-Myb provide unique transactivation functions, since the core-Myb combination cannot be substituted by dimerized core or Myb sites. We propose that spatially precise synergy between CBF/PEBP2 and c-Myb may result from the ability of the two factors to form a composite surface that makes unique and stereospecific contacts with one or more additional components of the transcriptional machinery.

Trypanosoma cruzi: identification of a membrane cysteine proteinase linked through a GPI anchor.

The biochemical and functional properties of T. cruzi GP50/55, a novel glycosylphosphatidylinositol (GPI)-anchored membrane antigen have been investigated. A 50-52-kDa thiol proteinase activity could be immunoprecipitated with monoclonal antibodies (mAb) directed against GP50/55 (mAb C10), different from the one reactive with mAbs against lysosomal cysteine proteinase GP57/51. Furthermore, the mAb C10-reactive proteinase corresponded to the GPI-anchored surface antigen since the proteolytic and antigenic activity partitioned to the aqueous phase after Triton X114 phase separation of phosphatidylinositol specific phospholipase C (PI-PLC)-treated parasites. Of several proteins immunoprecipitated by a polyclonal anti-lysosomal cysteine proteinase, an mAb to GP57/51 recognized a 60-kDa protein, whereas mAb C10 recognized antigens ranging between 52 and 50 kDa. The GP50/55 antigen detected by mAb C10 is expressed on the parasite surface whereas the GP57/51 antigen is mainly intracellular. The internal peptide sequence obtained from purified GP50/55 showed that it is more homologous to the prototype of the cysteine proteinases superfamily, papain, than to the two T. cruzi lysosomal cysteine proteinases so far described. Our data indicate that the T. cruzi GP50/55 is a novel GPI-anchored cysteine proteinase and may represent another isoform of this heterogeneous group of proteinases.

Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors.

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene. The 30-bp minimal enhancer element denoted delta E3 carries a core sequence (TGTGGTTT) that binds a T-cell-specific factor, and that is necessary but not sufficient for transcriptional activation. Here we demonstrate that the transcription factor c-Myb regulates TCR delta enhancer activity through a binding site in delta E3 that is adjacent to the core site. Both v-Myb and c-Myb bind specifically to delta E3. The Myb site is necessary for enhancer activity, because a mutation that eliminates Myb binding abolishes transcriptional activation by the delta E3 element and by the 370-bp TCR delta enhancer. Transfection of cells with a c-Myb expression construct upregulates delta E3 enhancer activity, whereas treatment of cells with an antisense c-myb oligonucleotide inhibits delta E3 enhancer activity. Since intact Myb and core sites are both required for delta E3 function, our data argue that c-Myb and core binding factors must cooperate to mediate transcriptional activation through delta E3. Efficient cooperation depends on the relative positioning of the Myb and core sites, since only one of two overlapping Myb sites within delta E3 is functional and alterations of the distance between this site and the core site disrupt enhancer activity. Cooperative regulation by c-Myb and core-binding factors is likely to play an important role in the control of gene expression during T-cell development.

GP 50/55, a membrane antigen of Trypanosoma cruzi involved in autoimmunity and immunosuppression.

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and the induction of autoantibodies crossreacting with host cells and tissues. We described here a GPI-linked 50/55 kDa antigen (GP50/55) present on the T. cruzi membrane, but not in the membrane of other parasites of the family Trypanosomatidae. We have obtained several monoclonal antibodies which specifically recognize this molecule. One of these GP50/55-specific mAbs (C10) crossreacts with a 28 kDa antigen expressed on the membrane of activated mouse and human T and B lymphocytes, after "in vitro" activation with mitogens, phorbol esters, or antigen, and on several murine T and B lymphocyte cell lines. Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from T. cruzi-infected mice or Chagasic patients but not from uninfected mice or control patients contain antibodies which recognize a similar p28 antigen and also suppress the proliferation of human T lymphocytes. These results suggest a possible role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers.

We have previously shown that the delta E3 site is an essential element for transcriptional activation by the human T-cell receptor (TCR) delta enhancer and identified two factors, NF-delta E3A and NF-delta E3C, that bound to overlapping core (TGTGGTTT) and E-box motifs within delta E3. In this study, we show that protein binding to the core motif is necessary but not sufficient for transcriptional activation by the delta E3 element. In contrast, protein binding to the E-box motif does not contribute significantly to enhancer activity. A similar core motif present within the enhancers of T-cell-tropic murine retroviruses has been shown to contribute to transcriptional activity of the viral long terminal repeat in T lymphocytes and to viral T-cell tropism. We therefore determined the relationship between the nuclear factors that bind to the TCR delta and Moloney murine leukemia virus core motifs. On the basis of electrophoretic mobility shift binding and competition studies, biochemical analysis of affinity-labeled DNA-binding proteins, and the binding of a purified core binding factor, the proteins that bound to the TCR delta core site were indistinguishable from those that bound to the murine leukemia virus core site. These data argue that DNA-binding proteins that interact with the core site of murine leukemia virus long terminal repeats and contribute to viral T-cell tropism also play an essential role in the T-cell-specific expression of cellular genes.

A Trypanosoma cruzi membrane protein shares an epitope with a lymphocyte activation antigen and induces crossreactive antibodies.

Chagas' disease results from the infection of the protozoan parasite Trypanosoma cruzi and affects several million people in South America. Several alterations of the immune response have been described in this disease, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal stimulation with the generation of autoantibodies crossreacting with host cells and tissues. We have obtained monoclonal antibodies (mAbs) from T. cruzi-infected mice that recognized a 50/55-kD antigen (GP50/55) on the T. cruzi membrane, but not in other parasites of the family Trypanosomatidae. One of these GP50/55-specific mAbs (C10) crossreacts with a 28-kD antigen (p28) expressed on the membrane of greater than 85% of activated mouse T and B lymphocytes, after in vitro activation with concanavalin A, Salmonella typhosa lipopolysaccharide, phorbol dibutyrate ester, or antigen, and on several murine T and B lymphocyte cell lines. Human T and B lymphocytes also express upon activation with phytohemagglutinin or Staphylococcus aureus Cowan I (SAC) a similar antigen recognized by mAb C10, although in a lower proportion of cells (30-40%). Furthermore, this mAb was able to suppress mouse and human T and B cell proliferation to any of those stimuli. In addition, sera from chagasic patients and T. cruzi-infected mice, but not from control patients or littermates, contain antibodies that recognize a similar p28 antigen on B lymphocytes. Furthermore, the immunoglobulin fractions of some chagasic sera also suppress the proliferation of human T lymphocytes. These results suggest a possible pathological role of autoantibodies as an alternative mechanism for T. cruzi-associated immunosuppression.

Characterization of a glycosyl-phosphatidylinositol-anchored membrane protein from Trypanosoma cruzi.

Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.