Gut T cell-independent IgA responses to commensal bacteria require engagement of the TACI receptor on B cells.

The gut mounts secretory immunoglobulin A (SIgA) responses to commensal bacteria through nonredundant T cell-dependent (TD) and T cell-independent (TI) pathways that promote the establishment of mutualistic host-microbiota interactions. SIgAs from the TD pathway target penetrant bacteria, and their induction requires engagement of CD40 on B cells by CD40 ligand on T follicular helper cells. In contrast, SIgAs from the TI pathway bind a larger spectrum of bacteria, but the mechanism underpinning their production remains elusive. Here, we show that the intestinal TI pathway required CD40-independent B cell-activating signals from TACI, a receptor for the innate CD40 ligand-like factors BAFF and APRIL. TACI-induced SIgA responses targeted a fraction of the gut microbiota without shaping its overall composition. Of note, TACI was dispensable for TD induction of IgA in gut-associated lymphoid organs. Thus, BAFF/APRIL signals acting on TACI orchestrate commensal bacteria-specific SIgA responses through an intestinal TI program.

NF-AT activation induced by a CAML-interacting member of the tumor necrosis factor receptor superfamily.

Activation of the nuclear factor of activated T cells transcription factor (NF-AT) is a key event underlying lymphocyte action. The CAML (calcium-modulator and cyclophilin ligand) protein is a coinducer of NF-AT activation when overexpressed in Jurkat T cells. A member of the tumor necrosis factor receptor superfamily was isolated by virtue of its affinity for CAML. Cross-linking of this lymphocyte-specific protein, designated TACI (transmembrane activator and CAML-interactor), on the surface of transfected Jurkat cells with TACI-specific antibodies led to activation of the transcription factors NF-AT, AP-1, and NFkappaB. TACI-induced activation of NF-AT was specifically blocked by a dominant-negative CAML mutant, thus implicating CAML as a signaling intermediate.

Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor.

A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay. This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000. An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000. CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S. cerevisiae and adjacent to the TATA element of the adenovirus major late promoter. A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others.

Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location.

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.

Isolation of a cDNA encoding the chicken homologue of the human calcium-modulating cyclophilin ligand.

In order to identify important regions of the human CAML (calcium-modulating cyclophilin ligand) protein, a cDNA encoding the chicken homologue was isolated using low-stringency hybridization screening of a T-lymphocyte cDNA library. The deduced amino acid sequences of chicken and human CAML show areas of extensive conservation, suggesting an evolutionarily important role in signaling.

CAML regulates Bim-dependent thymocyte death.

Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum protein calcium-modulating cyclophilin ligand (CAML) failed to undergo normal T-cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation, or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.

Calcium signalling in T cells stimulated by a cyclophilin B-binding protein.

The immunosuppressant drug cyclosporin A blocks a calcium-dependent signal from the T-cell receptor (TCR) that normally leads to T-cell activation. When bound to cyclophilin, cyclosporin A binds and inactivates the key signalling intermediate calcineurin. To identify potential cellular homologues of cyclosporin A that might regulate calcium signalling, we have cloned human genes encoding cyclophilin B-binding-proteins using the yeast two-hybrid system. One gene product, when overexpressed in Jurkat T cells, specifically induced transcription from the interleukin-2 enhancer, by activating the T-cell-specific transcription factors NF-AT and NF-IL2A. This protein, termed calcium-signal modulating cyclophilin ligand (CAML), acts downstream of the TCR and upstream of calcineurin by causing an influx of calcium. CAML appears to be a new participant in the calcium-signal transduction pathway, implicating cyclophilin B in calcium signalling, even in the absence of cyclosporin.

A hydrophobic domain of Ca2+-modulating cyclophilin ligand modulates calcium influx signaling in T lymphocytes.

Ca2+-modulating cyclophilin ligand (CAML) was originally described as a cyclophilin B-binding protein whose overexpression in T cells causes a rise in intracellular calcium, thus activating transcription factors responsible for the early immune response. As reported here, structure-function analysis of the CAML gene in Jurkat T cells indicates that two of CAML's putative membrane-spanning domains are necessary and sufficient for the modulation of intracellular calcium. We propose that the hydrophobic C-terminal tail of CAML forms its effector domain, thus implicating the N-terminal hydrophilic domain in a regulatory role. These findings define a novel protein motif that functions in intracellular calcium signaling.

Specific protein binding to far upstream activating sequences in polymerase II promoters.

A binding activity specific for the upstream activating sequence of the GAL1-GAL10 promoter of Saccharomyces cerevisiae has been purified 220-fold on the basis of a nitrocellulose filter-binding assay. The binding activity is enriched in a nuclear preparation and is likely to be the GAL4 gene product. DNase I-protection mapping patterns reveal binding to two 30-base-pair regions at the boundaries of the sequence. A nearly identical mapping pattern is obtained with the coordinately regulated GAL7 promoter. The four 30-base-pair regions of binding in the two promoters are closely homologous, with a core consensus sequence of C-G-CG-TG-C-A-A-C-A-G-T-G-C-T-C-C-G-A-A- GC-G-A-T. A synthetic oligonucleotide with such a sequence competes with the upstream activating sequence in the binding reaction.

Co-localization of calcium-modulating cyclophilin ligand with intracellular calcium pools.

The calcium-modulating cyclophilin ligand (CAML) protein activates Ca2+ influx signaling when overexpressed in Jurkat T cells. Although CAML appears to directly participate in Ca2+-dependent signaling initiated by the transmembrane activator and CAML interactor cell surface receptor, its mechanism of action is unknown. To address this issue, we have determined its membrane topology, subcellular localization, and ability to mobilize intracellular Ca2+ pools. Fractionation of cell extracts on discontinuous sucrose gradients and indirect immunofluorescence indicate that CAML co-localizes with sarcoplasmic/endoplasmic reticulum calcium/ATPase-2 and calreticulin at membrane-bound cytosolic vesicles. Limited trypsin digests indicate that the hydrophilic NH2-terminal domain of CAML is directed toward the cytoplasm. Functionally, CAML overexpression was shown to deplete thapsigargin-sensitive intracellular Ca2+ pools. These data suggest that CAML may initiate Ca2+ signaling through activation of a capacitative Ca2+ influx pathway.

A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription.

Binding sites for the GAL4-positive regulatory protein have been identified upstream of six galactose-inducible genes of Saccharomyces cerevisiae on the basis of (i) protection in DNAse I footprints, (ii) loss of protection when excess GAL4-binding oligonucleotide is added and (iii) homology with a 23-bp dyad-symmetric consensus sequence. Many of the binding sites have been shown to function as upstream activating sequences. The number of binding sites upstream of the various genes ranges from one to four, but a feature is conserved: in cases of multiple sites there is a pair with highest binding affinity located at dyad--dyad distances of 82--87 bp. We suggest that a pair of sites facilitates repression by the GAL80-negative regulatory protein, on the basis of (i) a correlation of a pair of sites (or only one) with full (or only partial) repression and (ii) the introduction of a second site abolishing transcription occurring with one.

Regulation of the T-independent humoral response by TACI.

TACI is a TNFR homolog expressed by mature B lymphocytes that has been implicated in the positive regulation of B cell growth and antibody production, as well as in the development of autoimmune disease. Its biology is complex due to the existence of two ligands, BLyS and APRIL, and a homologous receptor, BCMA, that similarly binds both ligands. To determine its critical biological role, we generated TACI knockout mice. Surprisingly, these mice demonstrated a 2-fold increase in numbers of circulating and splenic B cells, apparently due to increased proliferation rate. Maturation of B cells and T-dependent antibody production was normal, but responses to T-independent type II antigens were almost completely abolished. It appears that TACI provides an essential costimulatory signal for the T-independent humoral response.

Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A.

Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.

Calcium-modulating cyclophilin ligand desensitizes hormone-evoked calcium release.

The Ca(2+)-modulating cyclophilin ligand (CAML) protein causes stimulation of transcription factors via activation of a store-operated Ca(2+) entry pathway. Since CAML is widely expressed in mammalian tissues, it may be an important regulator of Ca(2+) store function. In the present study, we investigated the consequence of CAML overexpression on Ca(2+) signaling using rapid confocal imaging of Fluo3-loaded NIH3T3 fibroblasts. Control and CAML-expressing cells gave concentration-dependent responses to the Ca(2+) mobilizing agonist ATP. CAML expression reduced the sensitivity of the cells so that higher concentrations of ATP were needed to achieve global Ca(2+) waves. The amplitudes of Ca(2+) waves were significantly reduced in CAML expressing cells, consistent with earlier suggestions that CAML causes depletion of internal Ca(2+) stores. With low ATP concentrations, only local Ca(2+) release events were observed. CAML did not affect the characteristics of these local Ca(2+) signals, suggesting that it does not directly affect Ca(2+) release channels.

Molecular cloning and functional characterization of murine transmembrane activator and CAML interactor (TACI) with chromosomal localization in human and mouse.

The human Taci gene (Transmembrane Activator and CAML Interactor) encodes a recently discovered member of the Tumor Necrosis Factor Receptor family. TACI is expressed in B-lymphocytes and may act to regulate humoral immunity. To identify functionally important regions of the protein, we have isolated and characterized the murine homolog of the human Taci cDNA. The proteins display 61.5% similarity and 54.6% identity. Mouse TACI is a type III transmembrane protein, as judged by the lack of a cleaved signal sequence and its N-terminal extracellular exposure. The intracellular domains of the mouse and human proteins share a single, defined region of high sequence conservation (19 of 23 residues identical). This constitutes a novel domain that may play a part in the initiation of signal transduction through TACI. In support of this notion, mouse TACI was found to activate NFAT, NFkB, and AP1 transcription factors in a transient transfection assay. The Taci gene was localized to human Chromosome (Chr) 17p11 by fluorescence in situ hybridization. The murine homolog was localized by intraspecific backcross analysis to the middle of Chr 11, a region that is syntenic to human Chr 17p. This work identifies conserved domains within TACI that may mediate the cellular distribution and signal transduction function of the protein and extend the details of homology between mouse Chr 11 and human 17p.

The gene for calcium-modulating cyclophilin ligand (CAMLG) is located on human chromosome 5q23 and a syntenic region of mouse chromosome 13.

The CAMLG gene encodes a novel cyclophilin B-binding protein called calcium-modulating cyclophilin ligand, which appears to be involved in the regulation of calcium signaling in T lymphocytes and other cells. The murine homolog, Caml, was localized by interspecific backcross analysis to the middle of chromosome 13. By fluorescence in situ hybridization, this gene was localized to human chromosome 5 in a region (q23) known to be syntenic to mouse chromosome 13. These results provide further evidence supporting the extensive homology between human chromosome 5q and mouse chromosome 13. In addition, the results will provide a basis for further evaluation of cytogenetic anomalies that may contribute to inherited defects in calcium signaling or immune system function.

Isolation of mutant T lymphocytes with defects in capacitative calcium entry.

Calcium and calcium-binding proteins play important roles in the signaling cascade leading from the initial engagement of TCRs on T cells to the fully activated state. To undertake a molecular dissection of this cascade, we first isolated a Jurkat T cell line derivative containing the NF-AT promoter element driving transcription of the diphtheria toxin A chain gene (dipA), resulting in rapid cell death. Selecting viable cells that fail to activate NF-AT-dependent transcription, we isolated two independent cell lines possessing defects in capacitative Ca2+ entry. NF-AT-dependent transcription can be restored in these cells by expression of a constitutively active calcineurin, but not overexpression of the Ca2+ regulatory protein CAML, which can normally replace the Ca2+ signal. The defect in these cell lines probably lies between CAML and calcineurin in the T cell activation cascade.