Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1-transgenic mice.

Susceptibility of activated T cells to apoptosis must be tightly regulated to ensure sufficient T cell progeny for an effective response, while allowing a rapid depletion of them at the end of the immune response. We show here that a previously isolated, NF-kappa B/rel target gene IEX-1 (Immediate Early response gene X-1) is highly expressed in T cells at early stages of activation, but declines with a prolonged period of activation time, coincident with an increased susceptibility of T cells to apoptosis during the late phases of an immune response. Transgenic expression of IEX-1 specifically in lymphocytes impaired apoptosis in activated T cells, extended a duration of an effector-phase of a specific immune response, and increased the accumulation of effector/memory-like T cells and the susceptibility to a lupus-like autoimmune disease. Our study demonstrated an antiapoptotic effect of IEX-1 on T cell apoptosis triggered by ligation of Fas and T cell receptor (TCR)/CD3 complex. The ability of extending life expectancy of T effectors, in line with a decrease in its expression following prolonged T cell activation, suggests a key role for IEX-1 in regulating T cell homeostasis during immune responses.

CD26-mediated signaling for T cell activation occurs in lipid rafts through its association with CD45RO.

CD26 is a T cell activation antigen that contains dipeptidyl peptidase IV activity and is known to bind adenosine deaminase. The mechanism by which CD26 costimulation potentiates T cell receptor-mediated T cell activation, leading to subsequent exertion of T cell effector function, is still not clearly defined. In this article, we demonstrate that CD26 localizes into lipid rafts, and targeting of CD26 to rafts is necessary for signaling events through CD26. Importantly, aggregation of CD26 by anti-CD26 mAb crosslinking also causes coaggregation of CD45 into rafts. Moreover, we show that CD26 directly binds to the cytoplasmic domain of CD45. Our results therefore indicate a mechanism whereby CD26 engagement promotes aggregation of lipid rafts and facilitates colocalization of CD45 to T cell receptor signaling molecules p56(Lck), ZAP-70, and TCRzeta, thereby enhancing protein tyrosine phosphorylation of various signaling molecules and subsequent interleukin-2 production.

Molecular cloning of Porimin, a novel cell surface receptor mediating oncotic cell death.

Anti-Porimin (Pro-oncosis receptor inducing membrane injury) mAb mediates oncosis-like cell death in Jurkat cells. Porimin cDNA was isolated from a Jurkat cell cDNA library by COS cell-expression cloning. The 3,337-bp cDNA has an ORF of 567 bp, encoding a type I transmembrane protein of 189 amino acids. The extracellular domain of Porimin contains many O-linked and seven N-linked glycosylation sites that define it as a new member of the mucin family. COS7 and 293 cells transiently transfected with Porimin cDNA were specifically recognized by anti-Porimin Ab in cell staining and immunoblotting experiments. When expressed in Jurkat cells, a His-tagged Porimin cDNA construct resulted in the generation of a specific 110-kDa-size protein that matched the molecular mass of the endogenous Porimin protein. Crosslinking of the Porimin receptor expressed on COS7 transfectants resulted in the loss of cell membrane integrity and cell death as measured by the leakage of intracellular lactate dehydrogenase. Both COS7 and 293 cells expressing transfected Porimin at a relatively high level lost their ability to adhere to culture dishes, suggesting a role for Porimin in cell adhesion. The Porimin gene was mapped to human chromosome 11q22.1 and is composed of four exons spanning 133 kb of genomic DNA.

B cell co-receptors regulating T cell-dependent antibody production in common variable immunodeficiency: CD27 pathway defects identify subsets of severely immuno-compromised patients.

CD27 and CD134 ligand (CD134L) are two B cell co-receptors for T(h) cell activation-induced ligands (i.e. CD70 and CD134) that promote differentiation of B cells into plasma cells and high-rate antibody production respectively. We explored the CD27 pathway and T cell CD134 expression in common variable immunodeficiency (CVID), a disease characterized by a lack of plasma cells and low Ig serum levels. Twelve patients were compared to seven healthy controls. We found a low percentage of circulating CD27(+) B cells in seven patients and B cell CD27 expression was not up-regulated by in vitro activation in two of them. Importantly, the number of circulating CD27(+) B cells was correlated with the severity of the disease--the patients with the lowest CD27(+) B cell counts having the lowest serum Ig concentrations and the lowest total peripheral blood B cell counts. In contrast, CD70 and CD134 were normally expressed on in vitro activated T cells. CD134L was not detected on patient and control B cells in our activation conditions. Functional studies of in vitro Ig production demonstrated an absence of B cell response to CD27 cross-linking, in particular in a patient with normal CD27 expression. Our results indicate that a defect in CD27 expression or function contributes to the pathogenesis of certain severe forms of CVID.

Defective surface expression of attractin on T cells in patients with common variable immunodeficiency (CVID).

The proliferative responses of T lymphocytes of a subset of patients with CVID are abnormally low. This may be due to abnormalities in extracellular interactions or signalling defects downstream from membrane-associated receptors. Demonstrating that the T cell receptor signalling was normal, we observed no abnormal pattern of activation-induced tyrosine phosphorylation in cells from CVID patients. Moreover, the addition of exogenous IL-2 increased the low proliferation to mitogens, thus indicating the integrity of the IL-2R signalling apparatus. Attractin is a rapidly expressed T cell activation antigen involved in forming an association between T cells and monocytes. Twenty-four to 48 h after activation by CD3 cross-linking, attractin expression was not up-regulated on the cells of CVID patients despite normal up-regulation of CD25 and CD26. On control cells, however, attractin expression was up-regulated together with CD25 and CD26. The addition of the purified 175-kD attractin was capable of restoring the proliferative response of peripheral blood mononuclear cells following CD3 X-L in the presence of suboptimal concentrations of rIL-2 (10 and 20 U/ml). The effect was dose-dependent with the maximal effect at a concentration of 500 ng/ml, and present at a concentration as low as 50 ng/ml. Due to the likely role of attractin in cell guidance and amplification of the immune response, our results indicate that the lack of up-regulation of the molecule in patients with CVID may in turn affect any further step of productive immune response. Our finding may also imply a potential therapeutic role for this novel molecule.

A biochemical function for attractin in agouti-induced pigmentation and obesity.

Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.

Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation.

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.

Attractin: a cub-family protease involved in T cell-monocyte/macrophage interactions.

Attractin is a rapidly upregulated membrane-associated molecule on activated T cells. It is a member of the CUB family of extracellular guidance and development proteins, sharing with them a protease activity similar to that of Dipeptidyl peptidase IV (DPPIV/CD26). Most remarkably, and in sharp contrast to CD26, it is released from the T cell and is presumed to be a major source of a soluble serum-circulating attractin. Genomic sequencing reveals that the soluble form is not a proteolytic product of the membrane form, but is in fact the result of alternative splicing. Recent results prove that the loss of murine membrane attractin results in the mahogany mutation with severe repercussions upon skin pigmentation and control of energy metabolism. In each of these latter instances, there is a strong likelihood that attractin is moderating the interaction of cytokines with their respective receptors. We propose that attractin is performing a similar function in the immune system through capture and proteolytic modification of the N-terminals of several cytokines and chemokines. This regulatory activity allows cells to interact and form immunoregulatory clusters and subsequently aids in downregulating chemokine/cytokine activity once a response has been initiated. These two properties are likely to be affected by the balance of membrane-expressed to soluble attractin.

Secreted and membrane attractin result from alternative splicing of the human ATRN gene.

Attractin, initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T lymphocytes, also has been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form. The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' terminal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene. The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.

CD134L engagement enhances human B cell Ig production: CD154/CD40, CD70/CD27, and CD134/CD134L interactions coordinately regulate T cell-dependent B cell responses.

CD134 is a member of the TNFR family expressed on activated T cells, whose ligand, CD134L, is found preferentially on activated B cells. We have previously reported that the CD70/CD27 interaction may be more important in the induction of plasma cell differentiation after the expansion phase induced by the CD154/CD40 interaction has occurred. When CD134-transfected cells were added to PBMCs stimulated with pokeweed mitogen, IgG production was enhanced in a dose-dependent fashion. Addition of CD134-transfected cells to B cells stimulated with Staphylococcus aureus Cowan I strain/IL-2 resulted in little if any enhancement of B cell IgG production and proliferation. We found that while CD134-transfected cells induced no IgG production by themselves, it greatly enhanced IgG production in the presence of CD40 stimulation or T cell cytokines such as IL-4 and IL-10. The addition of CD134-transfected cells showed only a slight increase in the number of plasma cells compared with that in the culture without them, indicating that an increased Ig production rate per cell is responsible for the observed enhancing effect of CD134L engagement rather than increase in plasma cell generation. These results strongly suggest different and sequential roles of the TNF/TNFR family molecules in human T cell-dependent B cell responses through cell-cell contacts and the cytokine network.

Murine Siva-1 and Siva-2, alternate splice forms of the mouse Siva gene, both bind to CD27 but differentially transduce apoptosis.

CD27, a member of the TNFR family known to provide essential co-stimulatory signals for T cell growth and B cell Ig synthesis, can also mediate cell death. Using the CD27 cytoplasmic tail as the bait in yeast two hybrid assay, we previously cloned human Siva, a pro-apoptotic molecule. Here we report the characterization of the mouse Siva gene as a 4 kb sequence containing 4 exons and 3 introns. RT-PCR has revealed the presence of two forms of mouse Siva mRNA, the longer full length form Siva-1 and the shorter Siva-2 lacking the sequence coded by exon 2. Immunoblotting with anti-Siva (human) antibodies clearly demonstrate the presence of both Siva-1 and Siva-2. Cotransfection experiments in 293T cells reveal that mouse CD27 receptor can interact with both forms of Siva. Although mouse Siva-1 can trigger apoptosis in Rat-1 cells and in some of the mouse cell lines in transient transfection experiments, similar to the observation made with human Siva, intriguingly its alternate splice form, Siva-2 appears to be much less toxic. It is therefore likely that Siva-2 could regulate the function of Siva-1.

Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.

DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.

CD26/dipeptidyl peptidase IV differentially regulates the chemotaxis of T cells and monocytes toward RANTES: possible mechanism for the switch from innate to acquired immune response.

CD26, a 110 kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) enzyme activity and plays an important role in T cell co-stimulation. In the present study, the function of CD26/DPPIV in transendothelial migration was examined using beta-chemokines as chemoattractants. When soluble recombinant CD26 (sCD26/DPPIV+) was added to the transendothelial chemotaxis system, chemotactic migration of T cells toward RANTES was significantly enhanced. Addition of sCD26 to 50 ng/ml of RANTES enhanced the migratory response by a factor of two compared to RANTES alone, whereas mutant soluble CD26 (mCD26), lacking the DPPIV enzyme activity, had no enhancing effect on RANTES-induced T cell migration. In the process of analyzing the mechanisms of the enhancement of T cell migration by sCD26, we showed that RANTES was cleaved by sCD26 under physiologic conditions at the precise site characteristic of its enzyme specificity. However, synthesized RANTES which lacks two N-terminal amino acids showed a chemotactic activity equivalent to full-length RANTES on T cells. Furthermore, addition of sCD26 showed enhancement of T cell migration induced by both forms of RANTES. In contrast to T cells, the truncated RANTES is inactive in chemotaxis of purified monocytes and supplement of sCD26 but not mCD26 reduced the migratory response of monocytes to RANTES. These results suggest that CD26/DPPIV differentially regulate the chemotactic response of T cells and monocytes to RANTES.

The mouse mahogany locus encodes a transmembrane form of human attractin.

Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.

Immune regulatory loops determine productive interactions within human T lymphocyte-dendritic cell clusters.

Help for the induction of cytolytic T lymphocytes is mediated by dendritic cells (DC) that are conditioned by CD40 signaling. We identified tumor necrosis factor family member CD27L/CD70, which is expressed by cytolytic T lymphocytes on interaction with DC to control CD154 (CD40L) up-regulation on CD45RA+ helper T cells for subsequent DC stimulation. The results show that the initiation of a cytolytic immune response is determined by regulatory circuits, requiring simultaneous activation and differentiation of all cells involved in T lymphocyte-DC cluster formation.

APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model.

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.

Attractin (DPPT-L), a member of the CUB family of cell adhesion and guidance proteins, is secreted by activated human T lymphocytes and modulates immune cell interactions.

Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. We have cloned attractin and find that, as in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common gamma cytokine chain. Except for the latter two domains, the overall structure shares high homology with the Caenorhabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.

IEX-1L, an apoptosis inhibitor involved in NF-kappaB-mediated cell survival.

Transcription factors of the nuclear factor-kappaB/rel (NF-kappaB) family may be important in cell survival by regulating unidentified, anti-apoptotic genes. One such gene that protects cells from apoptosis induced by Fas or tumor necrosis factor type alpha (TNF), IEX-1L, is described here. Its transcription induced by TNF was decreased in cells with defective NF-kappaB activation, rendering them sensitive to TNF-induced apoptosis, which was abolished by transfection with IEX-1L. In support, overexpression of antisense IEX-1L partially blocked TNF-induced expression of IEX-1L and sensitized normal cells to killing. This study demonstrates a key role of IEX-1L in cellular resistance to TNF-induced apoptosis.

Correlation of the epitopes defined by anti-CD26 mAbs and CD26 function.

To clarify the different anti-CD26 mAbs corresponding different functions of CD26, the correlation of the epitopes defined by anti-CD26 mAbs and the functions of CD26 have been studied. Using truncated, human-rat CD26 swap mutants and cross-blocking studies, 13 anti-CD26 mAbs were divided into 5 separate groups. These 5 epitopes were localized between the 1-247th, 248-358th, 359-449th (closer to the 359th amino acid), 450-577th and 359 653th amino acid regions. MAbs against two of these five epitopes, the 248-358th and 359-449th amino acid regions, were associated with inducing modulation of CD26 and T-cell costimulation through the CD3 pathway. Furthermore, mAbs against one of these epitopes, the 359-449th amino acid region, appeared to encompass the ADA binding domain. Analysing the avidity of each mAb to the CD26 molecule using DPPIV enzymatic activity as an indicator, we found that the function of CD26 had little correlation with the avidity of anti-CD26 mAbs, suggesting that distinct epitopes defined by anti-CD26 mAbs appeared to be associated with different functions of CD26. These results will be very useful in the further definition of the functional domains of CD26.