GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway.

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

GIMAP6 regulates autophagy, immune competence, and inflammation in mice and humans.

Inborn errors of immunity (IEIs) unveil regulatory pathways of human immunity. We describe a new IEI caused by mutations in the GTPase of the immune-associated protein 6 (GIMAP6) gene in patients with infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Patients and Gimap6-/- mice show defects in autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)-containing lipids. We find that GIMAP6 complexes with GABARAPL2 and GIMAP7 to regulate GTPase activity. Also, GIMAP6 is induced by IFN-γ and plays a critical role in antibacterial immunity. Finally, we observed that Gimap6-/- mice died prematurely from microangiopathic glomerulosclerosis most likely due to GIMAP6 deficiency in kidney endothelial cells.

GIMAP6 is required for T cell maintenance and efficient autophagy in mice.

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.

Survival of mature T cells in the periphery is intrinsically dependent on GIMAP1 in mice.

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 ERT2 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-cells' 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 depolarization 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.

GIMAP1 Is Essential for the Survival of Naive and Activated B Cells In Vivo.

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.

Generation and characterisation of mice deficient in the multi-GTPase domain containing protein, GIMAP8.

BACKGROUND: 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. PRINCIPAL FINDINGS: We show that GIMAP8 is expressed in the very early and late stages of T cell development in the thymus, at late stages during B cell development, and peripheral T and B cells. We find no defects in T or B lymphocyte development in the absence of GIMAP8. A marginal decrease in the number of recirculating bone marrow B cells suggests that GIMAP8 is important for the survival of mature B cells within the bone marrow niche. We also show that deletion of GIMAP8 results in a delay in apoptotic death of mature T cell in vitro in response to dexamethasone or γ-irradiation. However, despite these findings we find that GIMAP8-deficient mice mount normal primary and secondary responses to a T cell dependent antigen. CONCLUSIONS: Despite its unique structure, GIMAP8 is not required for lymphocyte development but appears to have a minor role in maintaining recirculating B cells in the bone marrow niche and a role in regulating apoptosis of mature T cells.

Gimap3 and Gimap5 cooperate to maintain T-cell numbers in the mouse.

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.

The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.

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.

Mice lacking Ly49E show normal NK cell development and provide evidence for probabilistic expression of Ly49E in NK cells and T cells.

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 ß-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 ß-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.

Two complementary rat NK cell subsets, Ly49s3+ and NKR-P1B+, differ in phenotypic characteristics and responsiveness to cytokines.

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.

Putative GTPase GIMAP1 is critical for the development of mature B and T lymphocytes.

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.

Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5-deficient mice.

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.

The autoimmunity-related GIMAP5 GTPase is a lysosome-associated protein.

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.

Expression of GIMAP1, a GTPase of the immunity-associated protein family, is not up-regulated in malaria.

BACKGROUND: 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. METHODS: A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. RESULTS: GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. CONCLUSION: The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

A natural hypomorphic variant of the apoptosis regulator Gimap4/IAN1.

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.

Eosinophilic bowel disease controlled by the BB rat-derived lymphopenia/Gimap5 gene.

BACKGROUND & AIMS: 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. METHODS: Genetically lymphopenic rats and congenic wild-type partners were compared for gross pathologic, histologic, and immunologic parameters, the latter including cytokines and autoantibodies. RESULTS: Genetic analysis demonstrated that homozygosity at the lyp locus was required for disease. All rats developed disease, and the median age at humane killing was approximately 36 weeks. This panintestinal disease showed a conspicuous eosinophilic infiltrate in the submucosa and muscle layers, but the villi were unaffected. Diseased rats showed splenomegaly and massive enlargement of the mesenteric lymph nodes. This pathology resembles human eosinophilic gastroenteritis, and several further features indicate a Th2 basis. The rats developed high serum IgE and made IgG autoantibodies that detected a nonleukocytic cell present in the intestinal wall of all rats (including germ free). CONCLUSIONS: The T-lymphopenic state associated with GIMAP5 deficiency renders rats generally susceptible to T-cell-mediated autoimmunity, but the immunoregulatory bias (Th1/Th2) of any disease depends on other genetic (or environmental) factors. In the present model, we suggest that defective peripheral tolerance to an intestine-specific autoantigen leads to uncontrolled inflammation of the intestinal wall.

Characterization of multiple alleles of the T-cell differentiation marker ART2 (RT6) in inbred and wild rats.

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.

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.

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.