The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific facilities. Pre-prints by Institute authors can be viewed on the Institute's bioRxiv channel. We believe that free and open access to the outputs of publicly‐funded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.

Speir JA, Stevens J, Joly E, Butcher GW, Wilson IA Immunology

The rat MHC class Ia molecule RT1-Aa has the unusual capacity to bind long peptides ending in arginine, such as MTF-E, a thirteen-residue, maternally transmitted minor histocompatibility antigen. The antigenic structure of MTF-E was unpredictable due to its extraordinary length and two arginines that could serve as potential anchor residues. The crystal structure of RT1-Aa-MTF-E at 2.55 A shows that both peptide termini are anchored, as in other class I molecules, but the central residues in two independent pMHC complexes adopt completely different bulged conformations based on local environment. The MTF-E epitope is fully exposed within the putative T cell receptor (TCR) footprint. The flexibility demonstrated by the MTF-E structures illustrates how different TCRs may be raised against chemically identical, but structurally dissimilar, pMHC complexes.

+view abstract Immunity , PMID: 11163232 2001

Clayton E, McAdam S, Coadwell J, Chantry D, Turner M Immunology

Phosphatidylinositol 3-kinases are a family of dual specificity lipid/protein kinases. The products of PI3K's, phosphatidylinositol(3,4,5) triphosphate and phosphatidylinositol(3,4) bisphosphate, act as second messengers connecting activated transmembrane receptors to signaling pathways that control gene transcription, proliferation, transformation, programmed cell death, adhesion, migration and vesicular transport. There is evidence that different isoforms of PI3K's activate specific signaling pathways and are thus responsible for integrating cellular responses. The elucidation of the genomic structure of the catalytic subunits is a necessary step for the investigation of the function of PI3K isoforms by inactivation of the gene in vivo. The structural organization of p110alpha, beta, and gamma genes has been previously reported. Here we report the cloning, sequencing, and structural organization of the mouse p110delta gene from a murine 129/Sv genomic library. The p110delta gene consists of 22 exons and spans over 13 kb. Comparison of the genomic structure with that of p110alpha, beta, and gamma demonstrates that the p110delta gene shares its exon structure with p110beta, the most closely related PI3K at the amino acid level.

+view abstract Biochemical and biophysical research communications , PMID: 11162674 2001

F Hannan, PD Evans

The cDNA of a type 1 ADP-ribosylation factor (ARF) from the desert locust, Locusta migratoria was cloned, sequenced and compared to ARF1 genes of other species. The locust ARF1 protein is 100% identical with the ARF1 protein of the fruit fly Drosophila melanogaster even though the DNA sequences are only 79% identical. The significance of this finding in relation to the considerable evolutionary distance between hemimetabolous and holometabolous insects is discussed.

+view abstract Insect molecular biology , PMID: 11122465 2000

Doody GM, Billadeau DD, Clayton E, Hutchings A, Berland R, McAdam S, Leibson PJ, Turner M Immunology

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

+view abstract The EMBO journal , PMID: 11080163 2000

JE Rudling, J Richardson, PD Evans

The agonist-specific coupling properties of the three cloned human alpha(2)-adrenoceptor subtypes have been compared, when expressed at similar levels in Chinese hamster ovary (CHO) cell lines, using noradrenaline and (+/-)-meta-octopamine as agonists. Noradrenaline can couple the receptor to both the inhibition and stimulation of forskolin-stimulated cyclic AMP production in all three receptor subtypes, with the relative strength of the coupling to the pathways varying for each of the receptor subtypes. meta-Octopamine selectively couples the alpha(2A)-adrenoceptor only to the inhibition of forskolin-stimulated cyclic AMP production. However, meta-octopamine couples the alpha(2B)- and alpha(2C)-adrenoceptors to both the inhibition and stimulation of forskolin-stimulated cyclic AMP production. The relative potency of meta-octopamine to noradrenaline varies between the different alpha(2)-adrenoceptor subtypes. The effects of meta-octopamine are around two orders of magnitude less potent than those of noradrenaline on both the alpha(2A)- and alpha(2B)-adrenoceptor subtypes. In contrast, in the case of the alpha(2C)-adrenoceptor, meta-octopamine is only one order of magnitude less potent than noradrenaline in the stimulation of forskolin-stimulated cyclic AMP production and, in addition, is equipotent with noradrenaline in the inhibition of forskolin-stimulated cyclic AMP production and has an increased maximal response. This raises the possibility that meta-octopamine may have physiologically important actions via alpha(2C)-adrenoceptors in vivo. The results show that the modulation of cyclic AMP production occurs in both a subtype- and agonist-specific manner for alpha(2A)-adrenoceptors and in a subtype specific manner for alpha(2B)- and alpha(2C)-adrenoceptors.

+view abstract British journal of pharmacology , PMID: 11053214 2000

Baker M, Gamble J, Tooze R, Higgins D, Yang FT, O'Brien PC, Coleman N, Pingel S, Turner M, Alexander DR Immunology

The CD45 tyrosine phosphatase lowers T-cell antigen receptor signalling thresholds by its positive actions on p56(lck) tyrosine kinase function. We now show that mice expressing active lck(F505) at non-oncogenic levels develop aggressive thymic lymphomas on a CD45(-/-) background. CD45 suppresses the tumorigenic potential of the kinase by dephosphorylation of the Tyr394 autophosphorylation site. In CD45(-/-) thymocytes the kinase is switched to a hyperactive oncogenic state, resulting in increased resistance to apoptosis. Transformation occurs in early CD4(-)CD8(-) thymocytes during the process of TCR-beta chain rearrangement by a recombinase-independent mechanism. Our findings represent the first example in which a tyrosine phosphatase in situ prevents the oncogenic actions of a SRC: family tyrosine kinase.

+view abstract The EMBO journal , PMID: 10970857 2000

Stevens J, Jones RC, Bordoli RS, Trowsdale J, Gaskell SJ, Butcher GW, Joly E Immunology

The rat major histocompatibility complex class Ia allelomorph RT1-A1(c) is a potent ligand for the recently identified inhibitory rLy-49 receptor, STOK-2. With the ultimate objective of studying the interactions of these molecules using structural and functional methods, we undertook a detailed study of its peptide specificity. The study revealed that designing an "ideal peptide" by choosing the most abundant residues in the "binding motif" obtained by pool sequencing does not necessarily yield an optimal binding peptide. For RT1-A1(c), as many as four positions, P2, P4, P5, and P9, were detected as putative anchors. Since this molecule displays a preference for highly hydrophobic peptides, we tested binding of peptides derived from the known leader peptide sequences of other rat histocompatibility complex class I molecules. One such peptide, found to bind well, requiring 1.6 microm peptide to achieve 50% stabilization, was searched for in vivo. Natural RT1-A1(c) binding peptides were purified from rat splenocytes and characterized by mass spectrometry using a combined matrix-assisted laser desorption ionization/time-of-flight and quadrupole time-of-flight approach. Results showed that the signal sequence-derived peptide was not detectable in the purified peptide pool, which was composed of a complex spectrum of peptides. Seven of these self-peptides were successfully sequenced.

+view abstract The Journal of biological chemistry , PMID: 10856297 2000

Bäckman-Petersson E, Butcher GW, Hedlund G Immunology

We have previously shown activation of NK cells via recognition of an allogeneic, non-classical MHC class I molecule, RT1-E(u). In this study we investigated whether a self-MHC class I molecule could protect the allogeneic targets from being recognized and killed by the alloreactive NK (allo NK) cells. NK cells from BN (RT1 n) rats, primed in vivo by immunization with RT1(u)-expressing cells, manifested cytolytic activity against RT1(u)- as well as RT1(u/lv1)-expressing targets, but not against RT1(u/n)-expressing targets. The absence of cytolytic activity against semiallogeneic targets, i.e. targets expressing self-allotypes, was also valid for allo NK cells from alloimmunized F344 (RT1 (lv1)) rats. To analyze the ability of a distinct MHC class I molecule to protect target cells from NK lysis, Rat2 cells transfected with the activating allogeneic MHC class Ib, RT1-E(u) molecule were also transfected with the self-MHC class Ia, RT1-A1(n) molecule. The allo NK cells from BN rats immunized with RT1(u)-expressing cells were cytolytic against Rat2 transfected with the RT1-E(u) molecule. However, the allo NK cells manifested no cytolytic activity against double-transfected Rat2 cells, expressing the RT1-E(u) as well as the RT1-A1(n) molecule. We conclude that expression of a self-MHC class Ia (RT1-A) molecule protects targets from allo NK killing. Furthermore, the NK inhibition via recognition of the self-MHC class Ia molecule dominates over the activation via recognition of the allogeneic MHC class Ib molecule, RT1-E.

+view abstract International immunology , PMID: 10837412 2000

Forsyth IA, Hutchings A, Butcher GW Immunology

A panel of 11 rat monoclonal antibodies (mAbs) has been raised to ovine placental lactogen (PL). By competitive enzyme-linked immunoabsorbent assay (ELISA), confirmed by two-site ELISA, the antibodies were shown to recognize six antigenic determinants on the ovine PL molecule, two of which overlap. One antigenic determinant (designated 1) was shared by other members of the prolactin/growth hormone (GH)/PL family in ruminants, humans and rodents. The binding of (125)I-labelled ovine PL to crude receptor preparations from sheep liver (somatotrophic) or rabbit mammary gland (lactogenic) was inhibited by mAbs recognizing antigenic determinants 2-6. Both types of receptor preparation were affected similarly. In the local in vivo pigeon crop sac assay, mAbs directed against determinants 3 and 6 enhanced the biological activity of ovine PL.

+view abstract The Journal of endocrinology , PMID: 10810307 2000

Colucci F, Guy-Grand D, Wilson A, Turner M, Schweighoffer E, Tybulewicz VL, Di Santo JP Immunology

The Syk protein tyrosine kinase (PTK) is essential for B, but not T or NK, cell development, although certain T cell subsets (i.e., gamma delta T cells of intestine and skin) appear to be dependent on Syk. In this report, we have re-evaluated the role of Syk in T cell development in hematopoietic chimeras generated by using Syk-deficient fetal liver hematopoietic stem cells (FL-HSC). We found that Syk-/- FL-HSC were vastly inferior to wild-type FL-HSC in reconstituting T cell development in recombinant-activating gene 2 (RAG2)-deficient mice, identifying an unexpected and nonredundant role for Syk in this process. This novel function of Syk in T cell development was mapped to the CD44-CD25+ stage. According to previous reports, development of intestinal gamma delta T cells was arrested in Syk-/- -->RAG2-/- chimeras. In striking contrast, when hosts were the newly established alymphoid RAG2 x common cytokine receptor gamma-chain (RAG2/gamma c) mice, Syk-/- chimeras developed intestinal gamma delta T cells as well as other T cell subsets (including alpha beta T cells, NK1.1+ alpha beta T cells, and splenic and thymic gamma delta T cells). However, all Syk-deficient T cell subsets were reduced in number, reaching about 25-50% of controls. These results attest to the utility of chimeric mice generated in a low competitive hematopoietic environment to evaluate more accurately the impact of lethal mutations on lymphoid development. Furthermore, they suggest that Syk intervenes in early T cell development independently of ZAP-70, and demonstrate that Syk is not essential for the intestinal gamma delta T cell lineage to develop.

+view abstract Journal of immunology (Baltimore, Md. : 1950) , PMID: 10799872 2000

R Chalmers, S Sewitz, K Lipkow, P Crellin

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.

+view abstract Journal of bacteriology , PMID: 10781570 2000

Turner M, Schweighoffer E, Colucci F, Di Santo JP, Tybulewicz VL Immunology

The tyrosine kinase SYK plays critical roles in signalling through immune receptors. Gene-targeting studies have identified the cell types that require SYK for development and function, and the receptors that use SYK as well as their downstream signalling effectors. There is also evidence of a role for SYK in non-immune cells and in the maintenance of vascular integrity.

+view abstract Immunology today , PMID: 10689303 2000

Stevens J, Wiesmüller KH, Butcher GW, Joly E Immunology

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

+view abstract International immunology , PMID: 10607753 2000

Oxley D, Bacic A Mass Spectrometry

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, D-Manalpha(1-2)-D-Manalpha(1-6)-D-Manalpha(1-4)-D-GlcN -inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial beta(1-4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

+view abstract Proceedings of the National Academy of Sciences of the United States of America , PMID: 10588691 1999

Petersson E, Holmdahl R, Butcher GW, Hedlund G Immunology

Previous studies have established that NK cells express both inhibitory and activatory receptors. The inhibitory receptors have been shown to recognize major MHC class I molecules, but the physiological ligands for the activatory receptors have been only partly characterized. In this study we investigated whether NK cells could be activated by recognizing specific non-classical MHC class Ib molecules. NK cells from BN (RT1(n)) rats immunized in vivo with MHC-incompatible WF (RT1(u)) cells displayed cytolytic activity specific for product(s) of the MHC class Ib RT1-E(u) / C(u) region. These cells were shown to kill Rat2 fibroblast cells transfected with cDNA for RT1-E(u) but neither untransfected Rat2 nor a transfectant with the class Ia allele, RT1-A(u). Cytolysis of Rat2-RT1-E(u) was inhibited by the anti-RT1-E(u) antibody 70-3-C2. In addition, NK cells cytolytic against PVG (RT1(c)) targets, but not against WF (RT1(u)) or other allogeneic targets were activated after PVG immunization of BN rats. The generation of NK populations cytolytic for target cells of the same haplotype as the immunizing cells, but not for third-party targets, strongly suggests the existence of a selective NK-mediated response in vivo. We conclude that recognition of an allogeneic MHC class Ib RT1-E molecule activates NK cells and the specific cytolytic response could be regarded as adaptive.

+view abstract European journal of immunology , PMID: 10556822 1999

Nakamura MC, Naper C, Niemi EC, Spusta SC, Rolstad B, Butcher GW, Seaman WE, Ryan JC Immunology

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.

+view abstract Journal of immunology (Baltimore, Md. : 1950) , PMID: 10528166 1999

Mee PJ, Turner M, Basson MA, Costello PS, Zamoyska R, Tybulewicz VL Immunology

The T cell repertoire is shaped by positive and negative selection of thymocytes. TCR-mediated signals that determine these selection processes are only partly understood. The CD45 tyrosine phosphatase has been shown to be important for signal transduction through the TCR, but there has been disagreement about whether CD45 is a positive or negative regulator of TCR signaling. Using CD45-deficient mice expressing transgenic TCR, we show that in the absence of CD45 there is a large increase in the thresholds of TCR stimulation required for both positive and negative selection. Our results conclusively demonstrate that in double-positive thymocytes CD45 is a positive regulator of the TCR signals that drive thymic selection events.

+view abstract European journal of immunology , PMID: 10508267 1999

Pingel S, Baker M, Turner M, Holmes N, Alexander DR Immunology

The pre-TCR complex regulates the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes during T cell development. In CD45(-/-) mice there is an accumulation of DN cells, suggesting a possible role for CD45 in pre-TCR signaling. We therefore crossed CD45(-/-) with Rag-1(-/-) mice to investigate the signaling functions of the CD3 complex in DN thymocytes. Remarkably, treatment of Rag-1(-/-)/CD45(-/-) mice with a CD3 mAb caused maturation to the DP stage at only 3% of the level measured in Rag-1(-/-) mice. Furthermore, ligation of the CD3 complex on Rag-1(-/-) /CD45(-/-) thymocytes in vitro induced less tyrosine phosphorylation in specific proteins when compared to Rag-1(-/-) thymocytes. CD45(-/-) mice were also crossed with pLGFA mice expressing a constitutively active form of the lck tyrosine kinase which restored the DN to DP transition to near normal levels. Our results are consistent with a model in which CD45-activated p56(lck) is critical for pre-TCR signal transduction.

+view abstract European journal of immunology , PMID: 10458749 1999

Colucci F, Turner M, Schweighoffer E, Guy-Grand D, Di Bartolo V, Salcedo M, Tybulewicz VL, Di Santo JP Immunology

Syk and ZAP-70 subserve nonredundant functions in B and T lymphopoiesis. In the absence of Syk, B cell development is blocked, while T cell development is arrested in the absence of ZAP-70. The receptors and the signaling molecules required for differentiation of NK cells are poorly characterized. Here we investigate the role of the Syk protein tyrosine kinase in NK cell differentiation. Hemopoietic chimeras were generated by reconstituting alymphoid (B-, T-, NK-) recombinase-activating gene-2 x common cytokine receptor gamma-chain double-mutant mice with Syk-/- fetal liver cells. The phenotypically mature Syk-/- NK cells that developed in this context were fully competent in natural cytotoxicity and in calibrating functional inhibitory receptors for MHC molecules. Syk-deficient NK cells demonstrated reduced levels of Ab-dependent cellular cytotoxicity. Nevertheless, Syk-/- NK cells could signal through NK1. 1 and 2B4 activating receptors and expressed ZAP-70 protein. We conclude that the Syk protein tyrosine kinase is not essential for murine NK cell development, and that compensatory signaling pathways (including those mediated through ZAP-70) may sustain most NK cell functions in the absence of Syk.

+view abstract Journal of immunology (Baltimore, Md. : 1950) , PMID: 10438908 1999

Naper C, Ryan JC, Kirsch R, Butcher GW, Rolstad B, Vaage JT Immunology

We have generated a monoclonal antibody (STOK2) which reacts with an inhibitory MHC receptor on a subset of alloreactive NK cells in the rat. This receptor, termed the STOK2 antigen (Ag), belongs to the Ly-49 family of lectin-like molecules and displays specificity for the classical MHC class I molecule RT1-A1c of PVG rats. Here, we have investigated the influence of the MHC on the selection, phenotype and function of the STOK2+ NK subset in a panel of MHC congenic and intra-MHC recombinant strains. STOK2 receptor density was influenced by the presence of its classical MHC I ligand RT1-A1c, as evidenced by a reduction of STOK2 Ag on the surface of NK cells from RT1-A1c+, as compared with RT1-A1c-, strains. In addition, a role for nonclassical MHC I RT1-C/E/M alleles in the selection of the STOK2 Ag was demonstrated. The relative number of STOK2+ NK cells was fivefold higher in rats expressing the RT1-C/E/M(av1) as compared with those expressing the RT1-C/E/M(u) class Ib haplotype. The STOK2 ligand RT1-A1c inhibited cytotoxicity of STOK2+ NK cells regardless of effector cell MHC haplotype. Allospecificity of STOK2+ NK cells varied markedly with effector cell MHC, however, and suggested that inhibitory MHC I receptors apart from STOK2 were variably co-expressed by these cells. These data provide evidence for the MHC-dependent regulation of the allospecific repertoire within a subset of potentially autoreactive Ly-49+ rat NK cells.

+view abstract European journal of immunology , PMID: 10382768 1999

G Hernández-Hoyos, S Joseph, NG Miller, GW Butcher Immunology

BB rats develop autoimmune diabetes mellitus at a high frequency. A key factor in the development of the disease is an autosomal recessive mutation determining peripheral T cell lymphocytopenia. Previous studies have suggested that the lymphopenia could be caused by increased cell death. Here we demonstrate that the lyp mutation dramatically reduces the in vitro lifespan of the TCRhi single-positive thymocytes and peripheral T cells, without abolishing their capacity to proliferate. The reduced lifespan is due to an increased rate of apoptosis, and is detected in single-positive thymocytes displaying characteristics of cells which have undergone positive selection. The cell death defect does not affect the in vitro lifespan of peripheral B cells. Interestingly, stimulation can rescue peripheral lyp/lyp T cells from immediate cell death. We propose that the lymphopenia mutation prevents the accumulation of a normal T cell pool, including regulatory subsets, without preventing the activation and proliferation of reactive T cells, thereby creating conditions appropriate for the development of uncontrolled autoimmune responses.

+view abstract European journal of immunology , PMID: 10382745 1999

Briddon SJ, Melford SK, Turner M, Tybulewicz V, Watson SP Immunology

We have characterized changes in [Ca2+]i in primary mouse megakaryocytes in response to fibrillar collagen and in response to cross-linking of the collagen receptor, the integrin alpha2beta1. The response to collagen was markedly different from that seen to a triple helical collagen-related peptide (CRP), which signals via the tyrosine kinases p59(fyn) and p72(syk). This peptide binds to the collagen receptor glycoprotein VI (GPVI), but not to the integrin alpha2beta1. Collagen elicited a sustained increase in [Ca2+]i composed primarily of influx of extracellular Ca2+ with some Ca2+ release from internal stores. In contrast to CRP, this response was only partially (approximately 30%) inhibited by the src-family kinase inhibitor PP1 (10 micromol/L) or by microinjection of the tandem SH2 domains of p72(syk). Collagen also caused an increase in [Ca2+]i in megakaryocytes deficient in either p59(fyn) or p72(syk), although the response was reduced by approximately 40% in both cases: Cross-linking of the alpha2 integrin increased [Ca2+]i in these cells exclusively via Ca2+ influx. This response was reduced by approximately 50% after PP1 pretreatment, but was significantly increased in fyn-deficient megakaryocytes. Collagen therefore increases [Ca2+]i in mouse megakaryocytes via multiple receptors, including GPVI, which causes Ca2+ mobilization, and alpha2beta1, which stimulates a substantial influx of extracellular Ca2+.

+view abstract Blood , PMID: 10339492 1999

Law DA, Nannizzi-Alaimo L, Ministri K, Hughes PE, Forsyth J, Turner M, Shattil SJ, Ginsberg MH, Tybulewicz VL, Phillips DR Immunology

Agonists induce inside-out alphaIIbbeta3 signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in alphaIIbbeta3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of alphaIIbbeta3 by adenine diphosphate (ADP) +/- epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in alphaIIbbeta3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits alphaIIbbeta3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.

+view abstract Blood , PMID: 10194444 1999

Costello PS, Walters AE, Mee PJ, Turner M, Reynolds LF, Prisco A, Sarner N, Zamoyska R, Tybulewicz VL Immunology

Vav is a GTP/GDP exchange factor (GEF) for members of the Rho-family of GTPases that is rapidly tyrosine-phosphorylated after engagement of the T cell receptor (TCR), suggesting that it may transduce signals from the receptor. T cells from mice made Vav-deficient by gene targeting (Vav-/-) fail to proliferate in response to TCR stimulation because they fail to secrete IL-2. We now show that this is due at least in part to the failure to initiate IL-2 gene transcription. Furthermore, we analyze TCR-proximal signaling pathways in Vav-/- T cells and show that despite normal activation of the Lck and ZAP-70 tyrosine kinases, the mutant cells have specific defects in TCR-induced intracellular calcium fluxes, in the activation of extracellular signal-regulated mitogen-activated protein kinases and in the activation of the NF-kappaB transcription factor. Finally, we show that the greatly reduced TCR-induced calcium flux of Vav-deficient T cells is an important cause of their proliferative defect, because restoration of the calcium flux with a calcium ionophore reverses the phenotype.

+view abstract Proceedings of the National Academy of Sciences of the United States of America , PMID: 10077632 1999