Publications

Title / Authors / Details Open Access Download

GIMAP6 is required for T cell maintenance and efficient autophagy in mice.
Pascall JC, Webb LMC, Eskelinen EL, Innocentin S, Attaf-Bouabdallah N, Butcher GW

The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.

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PloS one, 13, 1932-6203, , 2018

PMID:29718959

Open Access

Multi-tissue DNA methylation age predictor in mouse.
Stubbs TM, Bonder MJ, Stark AK, Krueger F, Bolland D, Butcher G, Chandra T, Clark SJ, Corcoran A, Eckersley-Maslin M, Field L, Frising UC, Gilbert C, Guedes J, Hernando-Herraez I, Houseley J, Kemp F, MacQueen A, Okkenhaug K, Rhoades M, Santbergen MJC, Stebegg M, von Meyenn F, Stegle O, Reik W

DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse.

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Genome biology, 18, 1474-760X, , 2017

PMID:28399939

Open Access

Survival of mature T cells in the periphery is intrinsically dependent on GIMAP1 in mice.
Datta P, Webb LM, Avdo I, Pascall J, Butcher GW

An effective immune system depends upon the survival of mature T cells in the periphery. Members of the GIMAP family of GTPases have been proposed to regulate this homeostasis, supported by the paucity of peripheral T cells in rodents deficient for either GIMAP1 or GIMAP5. It is unclear whether this lack of T cells is a consequence of an ontological defect, causing the thymus to generate and export T cells incapable of surviving in the periphery, or whether (alternatively or additionally) mature T cells intrinsically require GIMAP1 for survival. Using the ER(T2) Cre(+) transgene, we conditionally deleted Gimap1 in C57BL/6 mice and demonstrate that GIMAP1 is intrinsically required for the survival of mature T cells in the periphery. We show that, in contrast to GIMAP5, this requirement is independent of the T cell's activation status. We investigated the nature of the survival defect in GIMAP1-deficient CD4(+) T cells and show that the death occurring after GIMAP1 ablation is accompanied by mitochondrial depolarisation and activation of the extrinsic apoptotic pathway. This study shows that GIMAP1 is critical for maintaining the peripheral T-cell pool in mice and offers a potent target for the treatment of T-cell-mediated diseases. This article is protected by copyright. All rights reserved.

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European journal of immunology, , 1521-4141, , 2016

PMID:27792288

Open Access

GIMAP1 Is Essential for the Survival of Naive and Activated B Cells In Vivo.
Webb LM, Datta P, Bell SE, Kitamura D, Turner M, Butcher GW

An effective immune system depends upon regulation of lymphocyte function and homeostasis. In recent years, members of the GTPases of the immunity associated protein (GIMAP) family were proposed to regulate T cell homeostasis. In contrast, little is known about their function and mode of action in B cells. We used a combination of transgenic mice and in vivo and in vitro techniques to conditionally and electively ablate GIMAP1 in resting and activated peripheral B cells. Our data suggest that GIMAP1 is absolutely essential for the survival of peripheral B cells, irrespective of their activation state. Together with recent data showing increased expression of GIMAP1 in B cell lymphomas, our work points to the possible potential of GIMAP1 as a target for manipulation in a variety of B cell-mediated diseases.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2015

PMID:26621859

Generation and characterisation of mice deficient in the multi-GTPase domain containing protein, GIMAP8.
Webb LM, Pascall JC, Hepburn L, Carter C, Turner M, Butcher GW

GTPases of the immunity-associated protein family (GIMAPs) are predominantly expressed in mature lymphocytes. Studies of rodents deficient in GIMAP1, GIMAP4, or GIMAP5 have demonstrated that these GTPases regulate lymphocyte survival. In contrast to the other family members, GIMAP8 contains three potential GTP-binding domains (G-domains), a highly unusual feature suggesting a novel function for this protein. To examine a role for GIMAP8 in lymphocyte biology we examined GIMAP8 expression during lymphocyte development. We also generated a mouse deficient in GIMAP8 and examined lymphocyte development and function.

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PloS one, 9, 1932-6203, , 2014

PMID:25329815

Open Access

The miR-155-PU.1 axis acts on Pax5 to enable efficient terminal B cell differentiation.
Lu D, Nakagawa R, Lazzaro S, Staudacher P, Abreu-Goodger C, Henley T, Boiani S, Leyland R, Galloway A, Andrews S, Butcher G, Nutt SL, Turner M, Vigorito E

A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell-dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B-T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155-PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.

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The Journal of experimental medicine, 211, 1540-9538, , 2014

PMID:25288398

Open Access

Gimap3 and Gimap5 cooperate to maintain T-cell numbers in the mouse.
Yano K, Carter C, Yoshida N, Abe T, Yamada A, Nitta T, Ishimaru N, Takada K, Butcher GW, Takahama Y

Gimap3 (IAN4) and Gimap5 (IAN5) are highly homologous GTP-binding proteins of the Gimap family. Gimap3 and Gimap5, whose transcripts are abundant in mature lymphocytes, can associate with antiapoptotic Bcl-2 family proteins. While it is established that Gimap5 regulates T-cell survival, the in vivo role of Gimap3 is unclear. Here we report the preparation and characteristics of mouse strains lacking Gimap3 and/or Gimap5. We found that the number of T cells was markedly reduced in mice deficient in both Gimap3 and Gimap5. The defects in T-cell cellularity were more severe in mice lacking both Gimap3 and Gimap5 than in mice lacking only Gimap5. No defects in the cellularity of T cells were detected in mice lacking only Gimap3, whereas bone marrow cells from Gimap3-deficient mice showed reduced T-cell production in a competitive hematopoietic environment. Moreover, retroviral overexpression and short hairpin RNAs-mediated silencing of Gimap3 in bone marrow cells elevated and reduced, respectively, the number of T cells produced in irradiated mice. These results suggest that Gimap3 is a regulator of T-cell numbers in the mouse and that multiple Gimap family proteins cooperate to maintain T-cell survival.

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European journal of immunology, 44, 1521-4141, , 2014

PMID:24510501

The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.
Pascall JC, Rotondo S, Mukadam AS, Oxley D, Webster J, Walker SA, Piron J, Carter C, Ktistakis NT, Butcher GW

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

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PloS one, 8, 1932-6203, , 2013

PMID:24204963

Open Access

The autoimmunity-related GIMAP5 GTPase is a lysosome-associated protein.
VW Wong, AE Saunders, A Hutchings, JC Pascall, C Carter, NA Bright, SA Walker, NT Ktistakis, GW Butcher

A mutation in the rat GIMAP5 gene predisposes for autoimmunity, most famously in the BB rat model of autoimmune type 1 diabetes mellitus. This mutation is associated with severe peripheral T lymphopenia, as is mutation of the same gene in mice, but the mechanism by which GIMAP5 normally protects T cells from death is unknown. GIMAP5 is a putative small GTPase, a class of proteins which often fulfil their functions in the vicinity of cellular membranes. The objective of this study was to determine the normal intracellular location of GIMAP5 in lymphoid cells. Combining studies in rat, mouse and human systems, novel monoclonal antibodies (mAbs) were used to examine the localization of GIMAP5 and the closely-related protein, GIMAP1, in lymphoid cells by means of confocal microscopy and sub-cellular fractionation combined with immunoblotting. Additionally, human Jurkat T cells that inducibly express epitope-tagged GIMAP5 were established and used in electron microscopy (EM). Endogenous GIMAP5 was found to be located in a membraneous compartment/s which was also detected by established markers of lysosomes. GIMAP1, by contrast, was found to be located in the Golgi apparatus. EM studies of the inducible Jurkat T cells also found GIMAP5 in lysosomes and, in addition, in multivesicular bodies. This study establishes that the endogenous location of GIMAP5 is in lysosomes and related compartments and provides a clearer context for hypotheses about its mechanism of action.

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Self/nonself, 1, 3, , 2010

PMID:21487483
DOI: 10.4161/self.1.3.12819

Open Access

Mice lacking Ly49E show normal NK cell development and provide evidence for probabilistic expression of Ly49E in NK cells and T cells.
JG Aust, F Gays, F Hussain, GW Butcher, R Kist, H Peters, CG Brooks

Ly49E is an unusual member of the Ly49 family that is expressed on fetal NK cells, epithelial T cells, and NKT cells, but not on resting adult NK cells. Ly49E(bgeo/bgeo) mice in which the Ly49E gene was disrupted by inserting a β-geo transgene were healthy, fertile, and had normal numbers of NK and T cells in all organs examined. Their NK cells displayed normal expression of Ly49 and other NK cell receptors, killed tumor and MHC class I-deficient cells efficiently, and produced normal levels of IFN-γ. In heterozygous Ly49E(+/bgeo) mice, the proportion of epidermal T cells, NKT cells, and IL-2-activated NK cells that expressed Ly49E was about half that found in wild-type mice. Surprisingly, although splenic T cells rarely expressed Ly49E, IL-2-activated splenic T cells from Ly49E(bgeo/bgeo) mice were as resistant to growth in G418 as NK cells and expressed similar levels of β-geo transcripts, suggesting that disruption of the Ly49E locus had increased its expression in these cells to the same level as that in NK cells. Importantly, however, the proportion of G418-resistant heterozygous Ly49E(+/bgeo) cells that expressed Ly49E from the wild-type allele was similar to that observed in control cells. Collectively, these findings demonstrate that Ly49E is not required for the development or homeostasis of NK and T cell populations or for the acquisition of functional competence in NK cells and provide compelling evidence that Ly49E is expressed in a probabilistic manner in adult NK cells and T cells.

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Journal of immunology (Baltimore, Md. : 1950), 186, 4, , 2011

PMID:21248256
DOI: 10.4049/jimmunol.1003698

Open Access

Two complementary rat NK cell subsets, Ly49s3+ and NKR-P1B+, differ in phenotypic characteristics and responsiveness to cytokines.
L Kveberg, P Jiménez-Royo, C Naper, B Rolstad, GW Butcher, JT Vaage, M Inngjerdingen

Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR-P1B lectin-like receptor. Ly49s3(+) NK cells, but not NKR-P1B(+) NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell-associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR-P1B(+) NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3(+) NK cells was associated with more rapid proliferation in response to IL-2 as compared with NKR-P1B(+) NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3(+) NK cells. Moreover, freshly isolated Ly49s3(+) and NKR-P1B(+) NK cells produce similar amounts of cytokines, and a minor Ly49s3(-)NKR-P1B(-) double-negative NK subset appears to be hyporesponsive based on its significantly lower IFN-gamma production. Collectively, our data demonstrate divergent profiles of NKR-P1B(+) and Ly49s3(+) NK cells, indicating distinct tasks in vivo.

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Journal of leukocyte biology, 88, 1, , 2010

PMID:20395458
DOI: 10.1189/jlb.0110039

Open Access

Putative GTPase GIMAP1 is critical for the development of mature B and T lymphocytes.
A Saunders, LM Webb, ML Janas, A Hutchings, J Pascall, C Carter, N Pugh, G Morgan, M Turner, G Butcher

The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene GIMAP1. Homozygous loss of this allele under the influence of the lymphoid-expressed hCD2-iCre recombinase transgene led to severe (> 85%) deficiency of mature T lymphocytes and, unexpectedly, of mature B lymphocytes. By contrast there was little effect of GIMAP1 deletion on immature lymphocytes in either B or T lineages, although in vitro studies showed a shortening of the survival time of both immature and mature CD4(+) single-positive thymocytes. These findings show a vital requirement for GIMAP1 in mature lymphocyte development/survival and draw attention to the nonredundant roles of members of the GIMAP GTPase family in these processes.

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Blood, 115, 16, , 2010

PMID:20194894
DOI: 10.1182/blood-2009-08-237586

Open Access

Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5-deficient mice.
MJ Barnes, H Aksoylar, P Krebs, T Bourdeau, CN Arnold, Y Xia, K Khovananth, I Engel, S Sovath, K Lampe, E Laws, A Saunders, GW Butcher, M Kronenberg, K Steinbrecher, D Hildeman, HL Grimes, B Beutler, K Hoebe

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

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Journal of immunology (Baltimore, Md. : 1950), 184, 7, , 2010

PMID:20190135
DOI: 10.4049/jimmunol.0903164

Open Access

Expression of GIMAP1, a GTPase of the immunity-associated protein family, is not up-regulated in malaria.
A Saunders, T Lamb, J Pascall, A Hutchings, C Dion, C Carter, L Hepburn, J Langhorne, GW Butcher

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi.

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Malaria journal, 8, , , 2009

PMID:19338674
DOI: 10.1186/1475-2875-8-53

Open Access

MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters. 1990.
Deverson EV, Gow IR, Coadwell WJ, Monaco JJ, Butcher GW, Howard JC

Journal of immunology (Baltimore, Md. : 1950), 180, 0022-1767, , 2008

PMID:18292488

Open Access

A natural hypomorphic variant of the apoptosis regulator Gimap4/IAN1.
C Carter, C Dion, S Schnell, WJ Coadwell, M Graham, L Hepburn, G Morgan, A Hutchings, JC Pascall, H Jacobs, JR Miller, GW Butcher

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a "delayed" apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency ( approximately 30%). This suggests an adaptive significance for this hypomorphic allele.

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Journal of immunology (Baltimore, Md. : 1950), 179, 3, , 2007

PMID:17641045

Open Access

Eosinophilic bowel disease controlled by the BB rat-derived lymphopenia/Gimap5 gene.
L Cousins, M Graham, R Tooze, C Carter, JR Miller, FM Powrie, GG Macpherson, GW Butcher

Many models of autoimmunity are associated with lymphopenia. Most involve a T-helper cell (Th)1-type disease, including the diabetic BioBreeding (BB) rat. To investigate the roles of identified susceptibility loci in disease pathogenesis, we bred PVG-RT1(u), lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1(u)) and iddm2 (lyp, Gimap5(-/-)) diabetes susceptibility loci on the PVG background. Surprisingly, these rats developed a spontaneous, progressive, inflammatory bowel disease. To understand the disease pathogenesis, we undertook investigations at the genetic, histologic, and cellular levels.

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Gastroenterology, 131, 5, , 2006

PMID:17064701
DOI: 10.1053/j.gastro.2006.09.023

Characterization of multiple alleles of the T-cell differentiation marker ART2 (RT6) in inbred and wild rats.
Rothenburg S, Haag F, Koch-Nolte F, Carter C, Graham M, Butcher GW

ART2 (RT6) belongs to the family of mono-ADP-ribosyltransferases (ARTs). ART2 is a T-cell differentiation marker expressed by the majority of mature peripheral T cells in the rat. The two known ART2 allotypes display approximately 95% amino acid identity. We sequenced the ART2 coding regions from 18 inbred rat strains and found two additional alleles, termed Art2 ( a2 ) and Art2 ( b2 ). Monoclonal antibody Gy12/61 specifically reacted with Art2 ( a2 ) but not Art2 ( a1 ) lymph node cells. Expression of ART2 allotypes in Jurkat cells confirmed this specificity. A polymerase chain reaction (PCR) assay using restriction fragment length polymorphisms is described, which allows the easy discrimination of Art2 alleles. All four laboratory rat alleles, as well as an additional sequence variant, were found amongst 18 wild rat DNA samples. PCR analysis confirmed the selective presence of a rodent identifier (ID) element in the Art2 ( a ) but not the Art2 ( b ) alleles in all rats studied. Analysis of Art2 ( a1 ) and Art2 ( b2 ) genes showed greater divergence in coding than in non-coding regions. Together with the finding of a high number of non-synonymous mutations leading mostly to non-conservative amino acid substitutions clustered on the side facing away from the cell surface, this suggests that the Art2 polymorphism has been subject to selection.

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Immunogenetics, 57, 0093-7711, , 2005

PMID:16195877

Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs.
C Dion, C Carter, L Hepburn, WJ Coadwell, G Morgan, M Graham, N Pugh, G Anderson, GW Butcher, JR Miller

Reports suggest that two members of the novel immune-associated nucleotide (Ian) GTPase family, Ian1 and Ian5, play roles in T cell development. We performed real-time PCR analysis of the expression of Ian genes of the rat during T cell maturation, in macrophages and in cell lines. We found that all of the genes were expressed at relatively low levels at the early double-negative thymocyte stage but were expressed more strongly at later cell stages. Our study also revealed the fact that the previously reported Ian9, Ian10 and Ian11 genes are, instead, parts of a single gene for which we retain the name Ian9, potentially encoding a GTPase with a highly unusual triplicated structure. Antisera were developed against both Ian1 and Ian9. We established that Ian9 is produced as an approximately 75-kDa protein in both T cells and thymocytes. We observed that levels of both Ian1 and Ian9 proteins are profoundly reduced in T cells from lymphopenic rats as compared with wild-type rats. It was demonstrated that thymocytes and B cells from lymphopenic rats (Ian5 null) did not show enhanced sensitivity to gamma-irradiation-induced apoptosis.

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International immunology, 17, 9, , 2005

PMID:16103028
DOI: 10.1093/intimm/dxh302

Open Access

Competition for access to the rat major histocompatibility complex class I peptide-loading complex reveals optimization of peptide cargo in the absence of transporter associated with antigen processing (TAP) association.
Ford S, Antoniou A, Butcher GW, Powis SJ

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-Aa, when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-Au molecule from associating with TAP. However, stable RT1-Au molecules are assembled efficiently in competition with RT1-Aa, demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-Aa, which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-Aa, which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-Aa binding. Full optimization of RT1-Aa requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.

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The Journal of biological chemistry, 279, 0021-9258, , 2004

PMID:14764587

Open Access

Formation of HLA-B27 homodimers and their relationship to assembly kinetics.
Antoniou AN, Ford S, Taurog JD, Butcher GW, Powis SJ

The human HLA-B27 class I molecule exhibits a strong association with the inflammatory arthritic disorder ankylosing spondylitis and other related arthropathies. Major histocompatibility complex class I heavy chains normally associate with beta(2)-microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface. However, an unusual characteristic of HLA-B27 is its ability to form heavy chain homodimers through an unpaired cysteine at position 67 in the peptide groove. Homodimers have previously been detected within the ER and at the cell surface, but their mechanism of formation and role in disease remain undefined. Here we demonstrate, in the rat C58 thymoma cell line and in human HeLa cells transfected with HLA-B27, that homodimer formation involves not only cysteine at position 67 but also the conserved structural cysteine at position 164. We also show that homodimer formation can be induced in the non-disease-associated HLA class I allele HLA-A2 by slowing its assembly rate by incubation of cells at 26 degrees C, suggesting that homodimer formation in the endoplasmic reticulum may occur as a result of the slower folding kinetics of HLA-B27. Finally, we report an association between unfolded HLA-B27 molecules and immunoglobulin-binding protein at the cell surface.

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The Journal of biological chemistry, 279, 0021-9258, , 2004

PMID:14684742

Open Access

Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites.
Lau P, Amadou C, Brun H, Rouillon V, McLaren F, Le Rolle AF, Graham M, Butcher GW, Joly E

So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered.

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BMC immunology, 4, 1471-2172, , 2003

PMID:12837137

Open Access

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype.
González-Muñoz AL, Le Rolle AF, Brun H, Hedrich HJ, Wedekind D, Powis SJ, Joly E, Butcher GW

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.

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Journal of immunology (Baltimore, Md. : 1950), 171, 0022-1767, , 2003

PMID:12817008

Open Access

Troponin C in different insect muscle types: identification of two isoforms in Lethocerus, Drosophila and Anopheles that are specific to asynchronous flight muscle in the adult insect.
Qiu F, Lakey A, Agianian B, Hutchings A, Butcher GW, Labeit S, Leonard K, Bullard B

The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca(2+) concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca(2+)-binding subunit of troponin, troponin C (TnC), with a single Ca(2+)-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca(2+)-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca(2+)-binding sites. Ca(2+) binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca(2+)-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca(2+) binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.

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The Biochemical journal, 371, 0264-6021, , 2003

PMID:12558500

Open Access

Molecular characterization of the novel rat NK receptor 1C7.
Bäckman-Petersson E, Miller JR, Hollyoake M, Aguado B, Butcher GW

A novel receptor, named 1C7 or NKp30 and involved in natural cytotoxicity, was recently identified. This receptor is encoded by the 1C7 gene, which is located within the class III region of the human MHC, HLA. It is a member of the immunoglobulin gene superfamily (IgSF) and, remarkably, is expressed at the mRNA level as six different splice variants in human. Recent investigations have indicated that the 1c7 gene of the mouse is silenced by in-frame stop codons. In this study, the molecular characterization of the rat 1c7 gene is described. cDNA derived from this gene encode a protein of 192 amino acid residues predicted to contain a single IgV-set domain in the extracellular region and a positively charged residue in the transmembrane region. Expression of the gene was detected in freshly isolated rat Natural Killer (NK) and T splenocytes. Transfection of rat 1C7 into the NK cell line RNK-16 induced cytolytic activity against glioma as well as lymphoma tumor cells. In addition, binding of a r1C7-Fc fusion protein by a panel of target cells correlated with susceptibility to killing by RNK-16-1C7 effector cells. These results indicate that the r1C7 molecule could function as an NK activating receptor as previously reported for the human NKp30 receptor molecule.

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European journal of immunology, 33, 0014-2980, , 2003

PMID:12548565

Crystal structures of two rat MHC class Ia (RT1-A) molecules that are associated differentially with peptide transporter alleles TAP-A and TAP-B.
Rudolph MG, Stevens J, Speir JA, Trowsdale J, Butcher GW, Joly E, Wilson IA

Antigenic peptides are loaded onto class I MHC molecules in the endoplasmic reticulum (ER) by a complex consisting of the MHC class I heavy chain, beta(2)-microglobulin, calreticulin, tapasin, Erp57 (ER60) and the transporter associated with antigen processing (TAP). While most mammalian species transport these peptides into the ER via a single allele of TAP, rats have evolved different TAPs, TAP-A and TAP-B, that are present in different inbred strains. Each TAP delivers a different spectrum of peptides and is associated genetically with distinct subsets of MHC class Ia alleles, but the molecular basis for the conservation (or co-evolution) of the two transporter alleles is unknown. We have determined the crystal structures of a representative of each MHC subset, viz RT1-A(a) and RT1-A1(c), in association with high-affinity nonamer peptides. The structures reveal how the chemical properties of the two different rat MHC F-pockets match those of the corresponding C termini of the peptides, corroborating biochemical data on the rates of peptide-MHC complex assembly. An unusual sequence in RT1-A1(c) leads to a major deviation from the highly conserved beta(3)/alpha(1) loop (residues 40-59) conformation in mouse and human MHC class I structures. This loop change contributes to profound changes in the shape of the A-pocket in the peptide-binding groove and may explain the function of RT1-A1(c) as an inhibitory natural killer cell ligand.

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Journal of molecular biology, 324, 0022-2836, , 2002

PMID:12470953

Rat natural killer cell receptor systems and recognition of MHC class I molecules.
Rolstad B, Naper C, Løvik G, Vaage JT, Ryan JC, Bäckman-Petersson E, Kirsch RD, Butcher GW

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RT1-A1c on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

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Immunological reviews, 181, 0105-2896, , 2001

PMID:11513136

Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach.
Stevens J, Joly E, Trowsdale J, Butcher GW

Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E.

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BMC immunology, 2, 1471-2172, , 2001

PMID:11432755

Open Access

Rat tapasin: cDNA cloning and identification as a component of the class I MHC assembly complex.
Deverson EV, Powis SJ, Morrice NA, Herberg JA, Trowsdale J, Butcher GW

During the assembly of major histocompatibility complex (MHC) class I molecules transient associations are formed with the endoplasmic reticulum resident chaperones calnexin and calreticulin, ERp57 oxidoreductase, and also with tapasin, the latter mediating binding of the class I molecules to the transporter associated with antigen processing (TAP). We report here the isolation of a cDNA encoding rat tapasin from a DA (RT1av1) library. The cDNA encodes a proline-rich (11.3%) polypeptide of 464 residues with a potential ER-retention KK motif at its COOH-terminus, and a predicted molecular mass of 48 kDa. Matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry of peptides derived from in-gel tryptic digestion of a TAP-associated protein match regions of the predicted translation product. A species of the correct molecular mass and predicted pl was also identified in association with radiolabelled immunoprecipitates of the rat TAP complex analysed by two-dimensional gel electrophoresis. This confirms rat tapasin as a component of the rat MHC class I assembly complex.

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Genes and immunity, 2, 1466-4879, , 2001

PMID:11294569

Two different, highly exposed, bulged structures for an unusually long peptide bound to rat MHC class I RT1-Aa.
Speir JA, Stevens J, Joly E, Butcher GW, Wilson IA

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.

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Immunity, 14, 1074-7613, , 2001

PMID:11163232

Open Access

Peptide specificity of RT1-A1(c), an inhibitory rat major histocompatibility complex class I natural killer cell ligand.
Stevens J, Jones RC, Bordoli RS, Trowsdale J, Gaskell SJ, Butcher GW, Joly E

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.

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The Journal of biological chemistry, 275, 0021-9258, , 2000

PMID:10856297

Open Access

Self-MHC class Ia (RT1-A(n)) protects cells co-expressing the activatory allogeneic MHC class Ib molecule (RT1-E(u)) from NK lysis.
Bäckman-Petersson E, Butcher GW, Hedlund G

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.

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International immunology, 12, 0953-8178, , 2000

PMID:10837412

Open Access

A panel of monoclonal antibodies to ovine placental lactogen.
Forsyth IA, Hutchings A, Butcher GW

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.

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The Journal of endocrinology, 165, 0022-0795, , 2000

PMID:10810307

Open Access

Analysis of peptide length preference of the rat MHC class Ia molecule RT1-A(u), by a modified random peptide library approach.
Stevens J, Wiesmüller KH, Butcher GW, Joly E

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.

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International immunology, 12, 0953-8178, , 2000

PMID:10607753

Open Access

Activation and selection of NK cells via recognition of an allogeneic, non-classical MHC class I molecule, RT1-E.
Petersson E, Holmdahl R, Butcher GW, Hedlund G

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.

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European journal of immunology, 29, 0014-2980, , 1999

PMID:10556822

Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor.
Nakamura MC, Naper C, Niemi EC, Spusta SC, Rolstad B, Butcher GW, Seaman WE, Ryan JC

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.

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Journal of immunology (Baltimore, Md. : 1950), 163, 0022-1767, , 1999

PMID:10528166

Open Access

Genes in two major histocompatibility complex class I regions control selection, phenotype, and function of a rat Ly-49 natural killer cell subset.
Naper C, Ryan JC, Kirsch R, Butcher GW, Rolstad B, Vaage JT

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.

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European journal of immunology, 29, 0014-2980, , 1999

PMID:10382768

The lymphopenia mutation of the BB rat causes inappropriate apoptosis of mature thymocytes.
G Hernández-Hoyos, S Joseph, NG Miller, GW Butcher

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.

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European journal of immunology, 29, 6, , 1999

PMID:10382745
DOI: 10.1002/(SICI)1521-4141(199906)29:063.0.CO;2-F