GARP: a key receptor controlling FOXP3 in human regulatory T cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

A new monoclonal antibody (CAL2) detects CALRETICULIN mutations in formalin-fixed and paraffin-embedded bone marrow biopsies.

Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.

Incorporation of nitrotyrosine into alpha-tubulin by recombinant mammalian tubulin-tyrosine ligase.

Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin.

Cloning and genomic organization of the TTL gene on mouse chromosome 2 and human chromosome 2q13.

Tubulin tyrosine ligase (TTL) is a cytosolic enzyme involved in the posttranslational modification of tubulin. In the assembled form microtubules are detyrosinated over time at the C-terminus of alpha-tubulin. After microtubular disassembly TTL restores tyrosine residues back to the detyrosinated tubulin leading to a cycle of detyrosination/tyrosination. Here we report the isolation of the human and mouse TTL cDNA. In comparison with other known TTL sequences, namely bovine, rat and porcine, we found that only porcine TTL deviates in length by having an insertion of two glutamate residues. In mouse and human TTL the genomic coding sequence is composed of seven exons with normal intron/exon boundaries. Using fluorescence in situ hybridization (FISH), we mapped the murine TTL gene to mouse chromosome 2 (MMU2). Human TTL has been located to chromosome 2q13 (HSA2q13). In addition, we found frequently truncated PCR products of hTTL transcripts with aberrant splicing in tumors.

Expression of nerve growth factor and neurotrophin receptors in testicular cells suggest novel roles for neurotrophins outside the nervous system.

The present study was designed to clarify the non-neurotrophic role for neurotrophins in mouse testis. By means of SI nuclease protection assay we could demonstrate that the gene coding for the low-affinity nerve growth factor (NGF) receptor p75NGFR is transiently expressed during germ cell development. Gene expression for p75NGFR was detected in late-meiotic spermatocytes and early spermatids and was found to be co-expressed with trkB and trkC, two tyrosine kinase receptors, commonly regarded as the high-affinity receptors for brain-derived neurotrophic factor and neurotrophin-3. Gene transcripts for the high-affinity NGF receptor trkA were found exclusively in non-germ cells. Isolated Leydig cells, peritubular myoid cells and Sertoli cells, but not germ cells, could be identified as potential testicular NGF sources. Non-germ cells respond after incubation for several days with a sharp induction in NGF synthesis, which is accompanied by a loss of phenotypic expression patterns. The fact that p75NGFR mRNA expression was induced in cultured Sertoli cells and peritubular myoid cells suggests an autocrine mode of NGF action in these cells. Induction of NGF synthesis in cultured Leydig cells could be prevented by the glucocorticoid dexamethasone. Results indicate different roles for the individual neurotrophins in distinct testicular compartments and suggest that these neurotrophins might support testicular functions by signalling between individual cell types in an autocrine and paracrine manner.

Constitutive expression of murine c-FLIPR causes autoimmunity in aged mice.

Death receptor-mediated apoptosis is a key mechanism for the control of immune responses and dysregulation of this pathway may lead to autoimmunity. Cellular FLICE-inhibitory proteins (c-FLIPs) are known as inhibitors of death receptor-mediated apoptosis. The only short murine c-FLIP splice variant is c-FLIPRaji (c-FLIPR). To investigate the functional role of c-FLIPR in the immune system, we used the vavFLIPR mouse model constitutively expressing murine c-FLIPR in all hematopoietic compartments. Lymphocytes from these mice are protected against CD95-mediated apoptosis and activation-induced cell death. Young vavFLIPR mice display normal lymphocyte compartments, but the lymphocyte populations alter with age. We identified reduced levels of T cells and slightly higher levels of B cells in 1-year-old vavFLIPR mice compared with wild-type (WT) littermates. Moreover, both B and T cells from aged vavFLIPR animals show activated phenotypes. Sera from 1-year-old WT and transgenic animals were analysed for anti-nuclear antibodies. Notably, elevated titres of these autoantibodies were detected in vavFLIPR sera. Furthermore, tissue damage in kidneys and lungs from aged vavFLIPR animals was observed, indicating that vavFLIPR mice develop a systemic lupus erythematosus-like phenotype with age. Taken together, these data suggest that c-FLIPR is an important modulator of apoptosis and enforced expression leads to autoimmunity.

MyoD and MEF2A mediate activation and repression of the p75NGFR gene during muscle development.

In an effort to clarify transient expression of the NGF low-affinity receptor p75NGFR during muscle development we have focused on the molecular mechanisms involved in the initiation and cessation of p75NGFR gene expression. Using quiescent C3H10T1/2 fibroblast as a tool, we observed that induction of differentiation competence in MyoD-transfected 10T1/2 fibroblasts was accompanied by the initiation of p75NGFR expression. Moreover, we could show that the bHLH transcription factor MyoD itself is a powerful candidate for transcriptional activation of the p75NGFR gene in muscle precursor cells. By means of MyoD-mutants we have found that both the amino terminus of the MyoD molecule as well as the bHLH-region are essential for transcriptional activity on the p75NGFR promoter. The fact that myocyte enhancer factor MEF2A inactivated MyoD-induced p75NGFR promoter activity strongly suggests that cell-specific regulation of the p75NGFR gene might be strictly dependent on the intracellular composition and balance of the appropriate bHLH-transcription factors and their modulators.

Evidence for the participation of nerve growth factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic program.

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75(neurotrophin receptor) (p75NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion.

Tubulin-tyrosine ligase, a long-lasting enigma.

Tubulins and microtubules are subjected to several post-translational modifications of which the reversible detyrosination/tyrosination of the carboxy-terminal end of most alpha-tubulins has been extensively analysed. This modification cycle involves a specific carboxypeptidase and the activity of the tubulin-tyrosine ligase (TTL). The true physiological function of TTL has so far not been established. This review describes the purification of TTL to homogeneity by biochemical methods, its in vitro properties and the generation of monoclonal antibodies. These mabs not only enabled a very convenient and rapid purification of TTL by immunoaffinity chromatography but also its extensive characterization by protein sequencing, which led to the isolation of the full length cDNA. With this information, gene disruption should be feasible in order to determine the physiological significance of the tyrosination cycle.

Regulation of nerve growth factor and its low-affinity receptor (p75NTR) during myogenic differentiation.

In our preceding report, we have shown that nerve growth factor (NGF) and its low-affinity receptor (p75NTR) are expressed in C2C12 myoblasts and downregulated during myogenic differentiation. Furthermore, NGF affects myogenic differentiation and cell growth via p75NTR and downregulation of p75NTR is essential for myogenic differentiation (Seidl et al., 1998). Here we show that NGF and p75NTR are regulated by mechanisms preceding terminal differentiation in myogenic cells. These mechanisms include cell-density phenomena such as cell-cell contact as well as signaling of basic fibroblast growth factor (FGF-2) and its receptor (FGFR1). Downregulation of NGF and p75NTR occurred as a consequence of increasing cell density, an important trigger for the onset of myogenic differentiation. FGF-2 and FGFR1 were shown to be present in C2C12 cells and exogenous FGF-2 induced NGF and p75NTR expression, implying that FGF/FGFR signaling is an upstream regulator of the NGF/p75NTR system. The fact that FGF-2 could suspend yet not abolish density-induced downregulation indicates that cell-cell contact counteracts the FGF effect and ultimately terminates NGF/p75NTR signaling. This evidence, together with the observation that p75NTR expression is suppressed in muscle progenitors, which constitutively express adenovirus E1A proteins and thus lack the competence of myogenic differentiation, underline the important role for the NGF/p75NTR system in the interplay of multiple factors and biological systems that balance myogenic differentiation at the appropriate spatial and temporal level.