Low-density cells isolated from the rat thymus resemble branched cortical macrophages and have a reduced capability of rescuing double-positive thymocytes from apoptosis in the BB-DP rat

Biobreeding-diabetes prone (BB-DP) rats spontaneously develop organ-specific autoimmunity and are severely lymphopenic and particularly deficient in ART2(+) regulatory T cells. A special breed, the so-called BB-diabetic-resistant (DR) rats, are not lymphopenic and do not develop organ-specific autoimmunity. The genetic difference between both strains is the lymphopenia (lyp) gene. Intrathymic tolerance mechanisms are important to prevent autoimmunity, and next to thymus epithelial cells, thymus APC play a prominent part in this tolerance. We here embarked on a study to detect defects in thymus APC of the BB-DP rat and isolated thymus APC using a protocol based on the low-density and nonadherent character of the cells. We used BB-DP, BB-DR, wild-type F344, and F344 rats congenic for the lyp gene-containing region. The isolated thymus, nonadherent, low-density cells appeared to be predominantly ED2(+) branched cortical macrophages and not OX62(+) thymus medullary and cortico-medullary dendritic cells. Functionally, these ED2(+) macrophages were excellent stimulators of T cell proliferation, but it is more important that they rescued double-positive thymocytes from apoptosis. The isolated thymus ED2(+) macrophages of the BB-DP and the F344.lyp/lyp rat exhibited a reduced T cell stimulatory capacity as compared with such cells of nonlymphopenic rats. They had a strongly diminished capability of rescuing thymocytes from apoptosis (also of ART2(+) T cells) and showed a reduced Ian5 expression (as lyp/lyp thymocytes do). Our experiments strongly suggest that branched cortical macrophages play a role in positive selection of T cells in the thymus and point to defects in these cells in BB-DP rats.     

hIan5: the human ortholog to the rat Ian4/Iddm1/lyp is a new member of the Ian family that is overexpressed in B-cell lymphoid malignancies

The family of immune associated nucleotide binding proteins (Ian) is a distinct family of GTP-binding proteins conserved in plants, mice, rats and humans that are associated with immune functions, suggesting involvement in conserved defense mechanisms. Recently, the rat Ian4 (rIan4) was cloned and it appears to be identical to the gene Iddm1/lyp responsible for severe lymphopenia and the development of insulin-dependent diabetes in the BB-DP rat. Here we describe the characterization of a new human member of the Ian family: hIan5. hIan5 is highly homologous to rIan4, has a predicted molecular weight of 35 kDa and contains distinct G motifs of GTP-binding proteins (G-1 to G-4) in the N-terminus. Human Ian5 is anchored to the mitochondria by the hydrophobic COOH-terminal domain. Human Ian5 is highly expressed in lymph node and spleen. Different blood fractions show high hIan5 expression in CD4- and CD8-positive T cells and monocytes, but not in B lymphocytes. In contrast, in B-CLL (chronic lymphocytic leukemia) and mantle cell lymphoma samples, hIan5 mRNA was upregulated. The current data underline the role of hIan5 in T-lymphocyte development and function, and for the first time suggest that upregulation of Ian proteins is associated with B-cell malignancy, possibly by inhibiting apoptosis.     

Genetic dissection of lymphopenia from autoimmunity by introgression of mutated Ian5 gene onto the F344 rat

Peripheral T cell lymphopenia (lyp) in the BioBreeding (BB) rat is linked to a frameshift mutation in Ian5, a member of the Immune Associated Nucleotide (Ian) gene family on rat chromosome 4. This lymphopenia leads to type 1 (insulin-dependent) diabetes mellitus (T1DM) at rates up to 100% when combined with the BB rat MHC RT1 u/u genotype. In order, to better study the lymphopenia phenotype without possible confounding effects of diabetes or other autoimmune disease, we generated congenic F344.lyp rats by introgression of lyp on diabetes-resistant MHC RT1 lv1/lv1 F344 rats. Analysis of thymic CD4 and CD8 T lymphocytes revealed no difference in the percentage of CD4(-)CD8(+)and CD4(+)CD8(-)subsets in lyp/lyp compared to +/+ F344 rats. The same subsets was however dramatically reduced in blood (P=0.005), spleen (P=0.019) and mesenteric lymph nodes (MLN) (P<0.0001). Compared to F344 +/+ rats double positive CD4(+)CD8(+)T cells were increased only in lyp/lyp spleen (P=0.034) while double negative CD4(-)CD8(-)were increased in thymus (P=0.033), spleen (P=0.012), MLN (P<0.0001), and peripheral blood (P<0.0001). There were no signs of inflammatory lesions in organs and tissues in F344.lyp/lyp rats examined at 120 days of age or older. We thus conclude that the lymphopenia phenotype was reconstituted by introgression of lyp on to F344 rats without subsequent development of organ-specific autoimmunity. The congenic F344.lyp rat should prove useful to dissect the mechanisms by which the Ian5 frameshift mutation affects T cell selection, differentiation and maturation without organ-specific autoimmunity.     

Loss of a gimap/ian gene leads to activation of NF-kappaB through a MAPK-dependent pathway

The diabetes-prone biobreeding (BB-DP) rat contains the lyp mutation which results in lymphopenia and promotes the progression of a T cell-mediated autoimmune attack of the pancreas in certain rat strains. This mutation has been mapped to a gene which bears homology to human Gimap5/Ian5 and results in the truncation and loss of activity of this protein. The lymphopenic state induced by the loss of this protein has led to the proposal that Gimap5 has an anti-apoptotic function. Previously we described an additional phenotype of incomplete activation mediated by the loss of Gimap5 function. Here we further characterize this incomplete activation phenotype and map a potential signal transduction pathway leading to activation. We show that CD5 expression on peripheral T cells is elevated in Gimap5 animals, while thymocyte expression remains similar between the two strains. Additionally, we show that NF-kappaB but not NFAT is activated in unstimulated Gimap5 mutant T cells as compared to unstimulated wild type T cells. Mapping this activation to its upstream source we show that activation of NF-kappaB is correlated with an activation of IKK. Using a variety of kinase inhibitors we further map this increase in IKK to an increase in MEK activation. Finally, to counter the possibility that activation is an indirect consequence of the lymphopenic environment, we created bone marrow chimeras in which Gimap5 mutant T cells developed in a normal environment and show that these cells retain their activated phenotype. Together, we interpret these data as demonstrating that the activation caused by loss of Gimap5 is a cell intrinsic phenomenon caused, in part, by a MEK-dependent activation of IKK. This, in turn, would suggest that Gimap5 functions to promote both T cell survival and quiescence and that these pathways are biochemically linked.     

Derivation of non-lymphopenic BB rats with an intercross breeding

Previous studies have suggested that the development of diabetes in the BB rats does not require the expression of T lymphopenia. In order to derive non-lymphopenic diabetic rats and define the relationship between the T cell abnormalities, MHC genotype, and diabetes, we performed a cross between BB/H and diabetes resistant BB/control followed by an intercross of the F1. In the F2, the overall incidence of diabetes and lymphopenia was 30% and 27%, respectively. Lymphopenia was strongly associated with diabetes (p less than 0.001) and was seen in 76% of the diabetic F2's. However, 6 of the diabetic were non-lymphopenic (24%) and 3 of the non-diabetics were lymphopenic (5%). In the non-lymphopenic diabetic animals, all T cell levels were within the normal range, but diabetes occurred at an earlier age than their lymphopenic littermates (p less than 0.001). In contrast to the strong association between the inheritance of lymphopenia and diabetes, no relationship between diabetes and Class I MHC restriction fragment length polymorphisms was found. We conclude: 1) Diabetes and lymphopenia are strongly associated inherited abnormalities in the BB rat and are not associated with Class I RFLP defined genotypes within the RTIu haplotype, 2) Animals in whom diabetes occurs in the absence of lymphopenia can be derived using this breeding approach 3) In our non-lymphopenic rats, diabetes occurred at an earlier age possibly reflecting the restoration of quantitative or qualitative T cell defects found in lymphopenic BB rats.     

Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene

The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB (BBDP) rat is polygenic, dependent upon mutations at several loci. Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of the Iddm1/lyp locus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting that Ian5 may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of the Ian gene family as a whole.     

Aberrancies in the differentiation and maturation of dendritic cells from bone-marrow precursors are linked to various genes on chromosome 4 and other chromosomes of the BB-DP rat

BB-Diabetes Prone (BB-DP) rats, a model for endocrine autoimmune diseases, are severely lymphopenic, especially lacking ART2+ regulatory T cells. BB-Diabetes Resistant (DR) rats are not lymphopenic and do not develop autoimmunity. BB-DP and BB-DR rats only differ at the lymphopenia (lyp) gene (iddm2) on chromosome 4. Since BB-DP rats also show aberrancies in the differentiation of dendritic cells (DC) from bone-marrow precursors, we tested the hypothesis that F344 rats congenic for a BB-DP chromosome 4 region (42.5-93.6Mb; including the lyp gene, but also iddm4) display an in vitro DC differentiation different from normal F344 rats. Here we show that the 42.5-93.6Mb BB-DP chromosome 4 region is linked to an increased DC precursor apoptosis, a low MHC class II expression, a reduced IL-10 production and a reduced T cell stimulatory capacity of DC. From our previous report on DC differentiation defects in BB rats (only differing in iddm2) and the present report, we deduce that the abnormal apoptosis and low MHC class II expression is linked to iddm2. The reduced T cell stimulatory capacity is linked to other genes on chromosome 4 (candidate gene: iddm4). The reduced IL-10 production has a complex linkage pattern.     

Both Gimap5 and the diabetogenic BBDP allele of Gimap5 induce apoptosis in T cells

Gimap5, a member of the GTPase of the immunity-associated protein family (Gimap), regulates T cell survival. A strong indication of this is found in the diabetes-prone BioBreeding rat (BBDP), where a frameshift mutation in Gimap5 results in T-cell lymphopenia. We have investigated the function of human Gimap5 in T cells. We found that reduction of Gimap5 by RNA interference in Jurkat cells did not affect the number of apoptotic cells whereas transient over-expression of Gimap5 resulted in a major increase in the number of apoptotic cells. The same effect of over-expression was found in naive human T cells purified from blood but not in activated human T cells. This suggests that the apoptosis-inducing effect of Gimap5 over-expression is dependent on the activation status of the cells. Since the apoptosis-inducing effect of Gimap5 was contrary to the expected function of Gimap5 based on the phenotype of BBDP rats, we over-expressed rat wt Gimap5 and Gimap5 with the mutation found in BBDP (Gimap5-lyp). Both versions of rat Gimap5 induced apoptosis when expressed in the rat T-cell line C58(NT)D.1.G.OVAR.1, however, Gimap5-lyp greatly exacerbated cell death. Finally, we detected the subcellular localization of Gimap5 to be at the endoplasmic reticulum and by quantitative PCR, we found that endogenous Gimap5 mRNA is up-regulated in activated T cells.     

Quantitative analyses comparing all major spleen cell phenotypes in BB and normal rats: autoimmune imbalance and double negative T cells associated with resistant, prone and diabetic animals.

The BB rat is a model of spontaneous autoimmune diabetes. To characterize quantitatively all known immune cell subsets involved in disease pathogenesis, FACS analysis of spleen cells was performed in diabetes-prone (DP) and acutely diabetic (D) BB rats and compared with diabetes-resistant (DR) BB and normal Wistar-Furth (WF) strains. We observed increased percentages of splenic NK cells in DP and D animals compared with DR rats using an NK-specific monoclonal antibody. We found increased proportions of splenic macrophages in the T-lymphopenic DP and D rats and low macrophage contents in DR spleens compared with WF spleens. We observed that percentages of the CD4-CD8- T cell receptor alpha/beta+ (double-negative) T cell subset were strikingly increased in the lymphopenic DP and D animals, compared with DR animals. We observed increased percentages of activated splenic CD5+ T cells expressing the IL-2 receptor and MHC class II antigen in DP and D rats compared with DR animals. Our studies suggest that (a) splenic NK cells and macrophages quantitatively appear to be involved in the pathogenesis of diabetes; (b) double-negative T cells escape from the T cell depletion process; (c) a marked increase of activated splenic T cells suggests diabetes is associated with general T cell activation processes; and (d) an altered balance among the different immune cell subsets may in part explain the pathogenesis of diabetes, since marked relative changes are observed when comparing the DR strain to the DP strain in both the prediabetic and diabetic stages.     

坐骨神经损伤后长链非编码RNA的聚类分析及长链非编码RNA MX1对大鼠雪旺细胞迁移、增殖能力的影响

目的 探讨坐骨神经损伤后长链非编码RNA（LncRNA）差异表达的基因,以及LncRNA MX1对大鼠雪旺细胞迁移、增殖能力的影响。方法 选取10周龄无特定病原体级雄性SD大鼠24只,随机分为0 d（T0）、3 d（T1）、7 d（T2）和14 d（T3）4组,每组6只,建立坐骨神经损伤动物模型。分别于模型建立后第0、3、7、14天取相应组大鼠坐骨神经损伤处的残端组织提取RNA,制备RNA基因芯片进行Heatmap聚类分析,筛选出各个时间点发生差异表达的基因,并选择其中差异表达显著的基因LncRNA MX1。培养iCell-r030大鼠雪旺细胞,分为对照组、Control siRNA组（siRNA组）和LncRNA MX1 siRNA组（siRNA-MX1组）,使用相应试剂进行转染。转染后采用实时荧光定量聚合酶链反应（qRT-PCR）检测3组雪旺细胞的LncRNA MX1mRNA表达情况,采用Transwell小室检测雪旺细胞的迁移能力,采用5-乙炔基-2'-脱氧尿苷（EDU）实验检测雪旺细胞的增殖能力。结果 各组大鼠均造模成功,实验过程中无大鼠死亡。大鼠损伤坐骨神经组织LncRNA差异基因Heatmap聚类分析的热图显示,与T0组比：T1组共3 066个LncRNA基因发生差异性表达,其中1 634个LncRNA基因表达上调,1 432个LncRNA基因表达下调;T2组共2 498个LncRNA基因发生差异性表达,其中1 634个LncRNA基因表达上调,864个LncRNA基因表达下调;T3组3 567个LncRNA基因发生差异性表达,其中有1 643个LncRNA基因表达上调,1 924个 LncRNA基因表达下调。各组差异表达的LncRNA基因中LncRNA MX1的差异倍数数值较大,差异表达显著。大鼠雪旺细胞qRT-PCR结果显示,转染LncRNA MX1 siRNA后,siRNA-MX1组LncRNA MX1的相对表达量为1.0±0.2,低于对照组的2.3±0.2和siRNA组的2.2±0.2,差异有统计学意义（F=78.47,P<0.001）;而对照组和siRNA组组间差异无统计学意义（P>0.05）。迁移、增殖试验结果显示,siRNA-MX1组细胞迁移数量为（24.1±4.2）个、EDU阳性细胞与DAPI阳性细胞的比率为27.5%±2.8%,低于对照组的（50.3±7.8）个、44.1%±7.2%和siRNA组的（49.2±6.2）个、41.8%±7.0%,差异均有统计学意义（F=93.15、121.26,P值均<0.001）;而对照组和siRNA组间差异均无统计学意义（P值均>0.05）。结论 大鼠坐骨神经损伤后LncRNA MX1差异表达显著,下调LncRNA MX1在大鼠雪旺细胞中的表达可显著降低细胞的迁移、增殖能力。     

Signaling lymphocytic activation molecule (SLAM) is differentially expressed in human Th1 and Th2 cells

We have used a real-time quantitative RT-PCR technique (TaqMan, PE Biosystems) to identify genes that are differentially expressed by human polarised CD4(+) T cell subsets (Th1 or Th2). The goal was to test the feasibility of the detection method in profiling the expression of a set of marker genes important for Th1 and Th2 differentiation. We demonstrate that in polarised human Th1 cells signaling lymphocytic activation molecule (SLAM), a member of the immunoglobulin superfamily, is expressed at 7-25-fold higher levels than in Th2 cells. Along with SLAM, expression of the IL-12 receptor chain beta 2 (IL-12R beta 2) and the IFN-gamma receptor chain beta (IFN-gamma R beta) proved to be useful molecular markers indicating the state of T cell polarisation, as previously reported. Treatment with IL-12 increased SLAM mRNA expression in T cells by 3-4-fold, whereas a number of other cytokines including PDGF-BB, IFN-alpha A, IFN-alpha A/D, IFN-beta, IFN-gamma or IL-9 had no effect. Stimulating T cells by co-ligating CD3 and CD28 increased SLAM protein surface expression in both Th1 and Th2 cells. In conclusion, real-time RT-PCR detection was found to be an accurate, sensitive and highly reproducible method for fast profiling of mRNA expression in Th1 and Th2 cell subsets.     

Toll-Like Receptor Expression and Function in Subsets of Human γδ T Lymphocytes

Two subsets of human γδ T cells can be identified by T cell receptor (TCR) V gene usage. Vδ2Vγ9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vδ1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-γ (IFN-γ) production in peripheral blood γδ T cells and in Vδ2Vγ9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated γδ T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vδ1 and Vδ2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vδ1 and Vδ2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-γ and chemokine secretion in TCR-activated Vδ1 and Vδ2 subsets, although the levels of IFN-γ secreted by Vδ1 T cells were much lower than those produced by Vδ2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vδ1 and Vδ2 T cells and underscore the intrinsically different capacity for IFN-γ secretion of Vδ1 versus Vδ2 T cells.     

Low-affinity ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts.

In the absence of thymic emigration, the peripheral T cell pool is maintained by division of mature lymphocytes. We have examined the molecular interactions required for peripheral CD8+ T cell expansion in lymphopenic mice without conventional antigenic stimulation. Expansion of CD8+ T cells in lymphopenic hosts was found to be peptide specific. An antagonist peptide known to serve as a ligand for positive selection of these T cells promoted expansion; however, a control peptide that binds the same class I molecule did not. Surprisingly, the cells undergoing proliferation in lymphopenic hosts did not mature to cytotoxic effectors and displayed a partially activated surface phenotype. These data suggest that division of T cells in the periphery of lymphopenic hosts requires specific recognition of self-peptide/MHC complexes, similar to the signal for thymocyte maturation.     

敲除ARID1A对人肝癌细胞系PLC/PRF/5基因表达调控的影响

目的探讨肝癌细胞系PLC/PRF/5中敲除ARID1A后对基因表达调控的影响。方法利用CRISPR-Cas9基因编辑技术敲除PLC/PRF/5细胞中ARID1A。然后分别对PLC/PRF/5 ARID1A野生型与敲除型细胞进行全转录组测序分析(RNA-seq),并用DESeq2鉴定ARID1A敲除后差异表达基因。最后,使用Metascape数据库对差异表达基因进行GO功能富集分析。结果成功构建PLC/PRF/5 ARID1A敲除细胞系。利用RNA-seq共筛选出978个差异表达基因,包括480个表达上调差异基因和498个表达下调差异基因。GO功能富集分析显示差异表达基因主要集中在细胞黏附、激素水平调节、激素代谢和MAP激酶活性调节等生物学过程。结论PLC/PRF/5肝癌细胞中,ARID1A可调控大量基因的表达,为进一步研究ARID1A在肝癌发生发展中的作用机制提供了新的线索。     

HIV‐1 susceptibility of transgenic rat‐derived primary macrophage/T cells and a T cell line that express human receptors,CyclinT1 and CRM1 genes

We developed transgenic (Tg) rats that express human CD4, CCR5, CXCR4, CyclinT1, and CRM1 genes. Tg rat macrophages were efficiently infected with HIV‐1 and supported production of infectious progeny virus. By contrast, both rat primary CD4⁺ T cells and established T cell lines expressing human CD4, CCR5, CyclinT1, and CRM1 genes were infected inefficiently, but this was ameliorated by inhibition of cyclophilin A. The infectivity of rat T cell‐derived virus was lower than that of human T cell‐derived virus.     

Tumorigenicity of adenovirus-transformed cells: region E1A of adenovirus 12 confers resistance to natural killer cells

Sensitivity of a library of rat cells transformed in vitro with viable recombinant adenoviruses to natural killer (NK) cells and allogeneic cytotoxic T cells has been studied and correlated with their oncogenic potential in syngeneic rats. All cell lines transformed with the sub370-12E1AB virus (containing E1A and E1B regions of Ad12) and with the sub370-12E1A virions (containing the E1A region of Ad12 and the E1B region of Ad5) showed a high degree of resistance to NK cells. The cell lines transformed with the sub370-12E1B virus (containing the E1A region of Ad5 and the E1B region of Ad12) were highly sensitive to NK cytotoxicity. While all cell lines transformed with virions containing the E1A genes of Ad5 expressed high levels of class I MHC antigen, only three of eight cell lines containing the E1A region of Ad12 showed detectable levels by flow cytometric analysis after staining with specific antibodies. All cell lines containing E1A genes of Ad5 were killed by in vitro generated allogeneic cytolytic T cells. Only three of eight cell lines containing the E1A region of Ad12 were killed by such CTLs; the level of cytotoxicity, however, did not reach that seen with the cells containing the E1A genes of Ad5. All cell lines containing the E1A and E1B genes of Ad12 were highly tumorigenic. Only two of four cell lines transformed with virus containing the E1A genes of Ad12 and E1B region of Ad5 were tumorigenic. The efficiency of tumor induction was low and the latent period was long confirming the importance of the E1B region. None of the cell lines transformed with virus containing the E1A region of Ad5 and the E1B genes of Ad12 were tumorigenic, reflecting their high degree of sensitivity to both natural and induced cellular immunity. Expression of the E1A region of Ad12 in transformed cells modulates not only the level of class I MHC antigens, but also confers resistance to NK cell cytotoxicity.