ApoE genotype does not affect plasma tPA and PAI-1 antigen levels

The presence of one or two apoliprotein E4 (apoE4) alleles constitutes a major risk factor for Alzheimer's disease (AD) and coronary heart disease (CHD). Numerous observations have suggested that misregulation of proteases may be instrumental in both diseases. Tissue-type plasminogen activator (tPA) has been recently demonstrated to play a key role in neuronal plasticity and in experimental neurodegeneration. One receptor for the ApoE protein is the LRP/α2 macroglobulin receptor, which also binds to and endocytoses tPA and plasminogen activator inhibitor I (PAI-1). Here we tested whether the apoE genotype has an influence on the plasma levels of these proteins. We demonstrate that there is no difference in plasma levels of tPA- and PAI-1-antigens between middled-aged individuals with one apoE4 allele and those having none. This suggests that the impact of apoE4 on Alzheimer's disease is not the result of altered clearance of tPA or PAI-1 by the LRP receptor. Am. J. Med. Genet. 74:172–175, 1997. © 1997 Wiley-Liss, Inc.     

The association of CTLA-4 and HLA class II autoimmune risk genotype with regulatory T cell marker expression in 5-year-old children

Regulatory T cells (Treg) are involved in the maintenance of peripheral tolerance by suppression of autoreactive lymphocytes that have avoided thymic depletion. The defective function of Treg cells has recently attracted attention in autoimmune diseases such as type 1 diabetes (T1D), rheumatoid arthritis and multiple sclerosis. Susceptibility to these diseases is associated with specific human leucocyte antigen (HLA) class II and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene polymorphisms. This study aimed to investigate the relationship between HLA class II and CTLA +49 A/G polymorphisms associated with susceptibility to T1D and the number and characteristics of Treg cells in children. Samples from 47 5-year-old children who participated in the All Babies in South-east Sweden (ABIS) follow-up study were grouped according to the presence of the T1D risk-associated HLA genotype (DQA1*0501-DQB1*0201, DQA1*0301-DQB1*0302) or neutral HLA genotypes. Lower percentages of CD4+ T cells (P = 0.03) and CD4+ CD25high cells (P = 0.06) expressing intracellular CTLA-4 were detected in samples from children with CTLA-4 +49GG compared to children with the +49AA genotype. Similarly, lower percentages of CD4+ (P = 0.002) and CD4+ CD25high (P = 0.002) cells expressing CTLA-4 were observed in children positive for HLA DQA1*0501-DQB1*0201 and DQA1*0301-DQB1*0302 (P = 0.04 for CD4+ and P = 0.02 for CD4+ CD25high) risk haplotypes when compared to children without these alleles. The percentage of CD25high cells among CD4+ cells was correlated inversely with CTLA-4 mRNA expression in PBMC (r = -0.56, P = 0.03). Decreased levels of CTLA-4 in CD4+ and CD4+ CD25high cells in individuals with CTLA-4 and HLA class II alleles associated with T1D may contribute to the initiation and/or progression of autoimmune response.     

Recipient CTLA-4*CT60-AA genotype is a prognostic factor for acute graft-versus-host disease in hematopoietic stem cell transplantation for thalassemia

Polymorphisms of the cytotoxic T-lymphocyte antigen-4 gene (CTLA-4) have been associated with autoimmune diseases and it has recently been reported that donor genotypes correlate with the outcome of allogeneic hematopoietic stem cell transplantation in leukemia patients. With the aim of confirming this finding in thalassemia patients, we investigated the influence of genotype distribution of 3 CTLA-4 gene polymorphisms in 72 thalassemia patients and their unrelated donors. A significant association was observed for recipient CT60-AA genotype and onset of grade II-IV (63.2% vs 24.5%; p = 0.001) and grade III-IV (36.4% vs 7.6%; p = 0.005) acute graft-versus-host disease (aGVHD). The same association was observed for the 88-base-pair allele of the CTLA-4 (AT)n polymorphism, which was determined to be in complete linkage disequilibrium with the CT60 A allele. Multinomial Cox regression demonstrated that this association was independent of CT60 donor genotypes or other risk factors (p = 0.016; hazard ratio = 2.8). Our data confirm that the genetic variability in CTLA-4 is an important prognostic factor for aGVHD and suggest that some of the risk factors for this complication are generated by recipient cells that persist after the myeloablative conditioning regimen.     

XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

TcR-induced regulated secretion leads to surface expression of CTLA-4 in CD4+CD25+ T cells

In this study we show that CD4+ T cells develop a functional regulated secretory compartment after differentiation into effector cells, as shown by their increased expression and T-cell receptor-induced exocytosis of lysosomal and cytotoxic effector proteins. We tested the hypothesis that activation-induced surface cytotoxic T-lymphocyte-associated antigen (CTLA-4) expression in CD4+CD25+ regulatory T cells occurs via a similar regulated secretory pathway. Fluorescence microscopy showed that internal CTLA-4 in these cells was stored in a vesicular compartment distinct from lysosomal vesicles. Rapid activation-induced CTLA-4 surface expression in mouse CD4+CD25+ T cells is independent of protein synthesis and Rab-27a. When antigen-dependent T-cell-antigen-presenting cell (APC) conjugates were analysed for surface distribution of CD86 on APC, a higher concentration of CD86 molecules was observed in the synapse of APC conjugated to CD4+CD25+ cells than APC conjugated to CD4+CD25- cells. These results demonstrate that fast delivery of mediators by the regulated secretory pathway in CD4+ T cells can be used to perform other functions that are not involved in cytotoxic function but that can influence/regulate other cells.     

中国HIV/AIDS患者T细胞及调节性T细胞CTLA-4表达与疾病进展关系研究

目的 对中国HIV感染者T细胞及凋节性T细胞CTLA-4表达与HIV疾病进展的相关性进行研究,探讨CTLA-4在HIV感染中的作用.方法 选取58名HIV/AIDS患者(长期不进展组、无症状HIV组、AIDS组),应用流式细胞仪胞内染色技术检测T细胞及CD4+CD25+Foxp3+调节性T细胞内CTLA-4表达水平,分析其与CD4+T细胞、病毒载量、淋巴细胞活化凋亡水平的相关性.结果 长期不进展组、无症状HIV组、AIDS组CD4+T细胞内CTLA-4表达水平依次增岛(P<0.05);与CD+T细胞显著负相关(P<0.01),与CD8+T细胞活化(CD38表达)、凋亡水平(CD95表达)及CD4+T细胞凋亡水平显著相关(P<0.05),与病毒载量无显著相关性.长期不进展组、无症状HIV组、AIDS组CD8+T细胞内CTLA-4表达水平差异无统计学意义;与CD4+T细胞、病毒载量、CD4,'+>、CD8+T细胞活化及凋亡水平均无显著相关性.CD4+CD25+Foxp3+T细胞内CTLA-4表达水平长期不进展组明显低于无症状HIV组及AIDS纽(P<0.05);与CD4+T细胞显著负相关(P<0.05);与CD4+、CD8+T淋巴细胞活化(HLA-DR表达)显著相关(P<0.01).结论 中国HIV感染者CD4+T细胞及CD4+CD25+Foxp3+调节性T细胞内CTLA-4表达水平与疾病进展及免疫活化状态显著相关,参与HIV感染免疫平衡的调节.     

PD-1 but not CTLA-4 Blockage Abrogates the Protective Effect of Regulatory T Cells in a Pregnancy Murine Model

Problem  Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect paternal/fetal antigens from rejection by maternal effector cells. Accordingly, the transfer of Treg obtained from BALB/c-mated CBA/J females prevents abortion in DBA/2J-mated animals. The actual mechanism through which Treg mediate their protective effect is still inconclusive. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and Programmed cell death 1 (PD-1) are some of known Treg-associated molecules; however, their role in Treg-mediated fetal protection in murine model has not been investigated.
Method of study  Treg obtained from normal pregnant animals (NP; CBA/J × BALB/c) on day 14 were adoptively transferred into abortion-prone mice (AP; CBA/J × DBA/2J) intravenously on day 2 of pregnancy. An amount of 250 μg of either anti-PD-1 or anti-CTLA-4 mAb were injected intraperitoneally on days 0, 3, 6 and 9 of pregnancy. Controls received Treg + IgG or Treg + PBS. NP or AP treated with PBS served as additional controls.
Results  Blocking PD-1 abrogated the protective effect of Treg, resulting in a higher median abortion rate in comparison with the Treg/isotype-treated control while CTLA-4 blockage did not interfere with the protective effect of Treg. This was associated with a diminished number of vascular endothelial growth factor-A⁺ cells, previously reported as stimulators of lymphocyte extravasation in preterm labor.
Conclusion  Our data suggest PD-1 as an important mediator in Treg-induced fetal protection in the CBA/J × DBA/2J murine model.     

Connective tissue growth factor does not affect transforming growth factor-beta 1-induced Smad3 phosphorylation and T lymphocyte proliferation inhibition

Transforming growth factor-beta 1 (TGF-beta(1)) is a key regulator of immune tolerance. TGF-beta(1) controls T lymphocyte activation and is involved in the immunosuppressive function and generation of regulatory T lymphocytes. Connective tissue growth factor (CTGF) has an essential role in the formation of connective tissue and blood vessels. CTGF expression is induced by TGF-beta(1) in several cell types and CTGF mediates several of the downstream actions of TGF-beta(1). Since little is known about the potential synergy between CTGF and TGF-beta(1) in T lymphocyte biology, the purpose of the present study was to determine whether CTGF can modulate TGF-beta(1)-mediated effects on human CD4+ T lymphocytes. Human recombinant CTGF was expressed in HEK293 cells. rCTGF was biologically active demonstrated by induction of proliferation in the endothelial cell line EA hy 926. rCTGF alone did not potentiate or diminish anti-CD3-induced CD4+ T lymphocyte proliferation and did not activate the Smad signaling pathway in CD4+ T lymphocytes. Furthermore, rCTGF did not attenuate TGF-beta(1)-mediated inhibition of CD4+ T lymphocyte proliferation and TGF-beta(1)-induced Smad signaling in CD4+ T lymphocytes. These results indicate that rCTGF had no detectable effects of its own on human CD4+ T lymphocytes and did not potentiate the effects of low amounts of TGF-beta(1) on human CD4+ T lymphocytes. Overall, these data support the hypothesis that CTGF does not act on CD4+ T lymphocytes.     

CT60 single nucleotide polymorphism of the CTLA-4 gene is associated with susceptibility to Graves' disease in the Taiwanese population

Graves' disease (GD) is an autoimmune disease but the underlying etiology has not been completely elucidated. Genetic susceptibility has been believed to play a major role. Recent studies showed that the CT60 single nucleotide polymorphism (SNP), which is in the 3'-noncoding region of the CTLA-4 gene, is strongly associated with some immune-mediated diseases. The aim of this study was to test for association between GD susceptibility and polymorphisms of CTLA-4 (ie, the CT60 SNP and the exon 1 +49 SNP) in the Taiwanese population. Our results demonstrate significant differences in the frequencies of the genotypes and alleles between 107 GD patients and 101 control subjects in the CT60 and exon 1 +49 SNPs (p <0.05). Significant differences in phenotypes were only found for CT60 SNP (78.4% vs 67.8% between patients and controls; chi2 = 3.93, p = 0.047). Furthermore, we found that the G/G genotype of both CT60 and exon 1 +49 was associated with increased risk for GD (p = 0.022, OR = 1.97). Significant linkage disequilibrium was found between the CT60 SNP and the exon 1 +49 SNP in both GD patients and control subjects (D' = 1.00). Because of tight linkage disequilibrium, a combination of these SNPs enhanced the role of the CTLA-4 gene in GD. The frequency of the disease-susceptible G allele of CT60 was comparable to that in Japanese and higher than in Caucasians. In conclusion, we provide evidence that CT60 SNP is associated with susceptibility to GD in the Taiwanese population.     

CTLA4/CT60 polymorphism is not relevant in susceptibility to autoimmune inflammatory intestinal disorders

The aim of this work was to investigate the possible influence of the recently described CT60 A/G dimorphism of the CTLA4 (cytotoxic T-lymphocyte antigen 4) gene in the susceptibility to two different autoimmune inflammatory intestinal disorders, inflammatory bowel disease (IBD) and celiac disease. We analyzed a case-control cohort composed of 528 Spanish patients with IBD (284 with Crohn disease and 244 with ulcerative colitis) and 454 unrelated healthy individuals, and additionally a group of 90 celiac disease families. CT60 genotyping was performed with a TaqMan 5' allelic discrimination assay. After comparing patients with IBD with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CTLA4/CT60 dimorphism. In addition, by means of familial and case-control analysis, no evidence for a statistically significant association was observed between CTLA4/CT60 and celiac disease susceptibility. Therefore, our results suggest that the CTLA4/CT60 polymorphism does not play a major role in inflammatory intestinal disorders.     

Aging does not affect calcium response to CCL2 and LPS in human monocytes

Monocytes play important roles in anti-microbial and anti-viral responses and chronic inflammatory diseases. Monocytes' functions are altered by aging. We investigated age-changes in calcium (Ca²⁺) response to CCL2 and LPS in human monocytes. CCL2 and LPS induced a slow increase of the cytosolic Ca²⁺ level, with a maximum response at ～360 s and ～300 s, respectively, in monocytes of young and older adults. No difference was observed in the magnitude and in the Ca²⁺ kinetic with both stimuli. Furthermore, store-operated Ca²⁺ entry and plasma membrane expression of ORAI1 showed no difference between both groups. In summary, monocytes from older adults maintained the capacity to mobilize calcium as their counterparts in young adults suggesting that the mechanisms underlying the dysfunctions in monocytes in aging might not involve alterations in Ca²⁺ flow through the plasma membrane.     

Disruption of the gene homologous to mammalian Nramp1 in Mycobacterium tuberculosis does not affect virulence in mice

Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1(+/+) and Nramp1(-/-) congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.     

Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.

The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV-JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV-JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host.     

Proliferative T cell anergy to MIs-1a does not correlate with in vivo tolerance

Intravenous or intraperitoneal priming of MIs-1b mice with cells from MIs-1a donors drastically reduces secondary in vitro proliferative responses to specific stimulation. We show here that: (i) priming leads to blast transformation of essentially all CD4+ T cells bearing V beta 6 receptors in spleen and lymph nodes, and to their marked clonal expansion; (ii) secondary in vivo (or in vitro) challenges have no effect on the state of activation and numbers of V beta 6 CD4 T cells, which, however, migrate to the site of antigenic exposure; (iii) priming results in the differentiation of specific V beta 6 CD4 T cells to effector helper activities, manifested in vivo by marked increases in the numbers of splenic plasma cells, which include terminally differentiated donor MIs-1a B cells; (iv) primed mice show accelerated 'second set' rejection of antigenic cells; and (v) MIs-1b mice, thymectomized as adults before exposure to MIs-1a cells, show immune responses that are equivalent to those of control animals. We conclude that, in this experimental system, proliferative 'anergy' does not correlate with tolerance but with memory, and relates to the determination of class in immune responses.     

The SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrins but does not affect proliferation and survival in lymphohematopoietic cells

To better define the role HIV-related chemokine receptor-chemokine axes play in human hematopoiesis, we investigated the function of the CXCR4 and CCR5 receptors in human myeloid, T- and B-lymphoid cell lines selected for the expression of these receptors (CXCR4(+), CXCR4(+) CCR5(+), and CCR5(+) cell lines). We evaluated the phosphorylation of MAPK p42/44, AKT, and STAT proteins and examined the ability of the ligands for these receptors (stromal-derived factor-1 [SDF-1] and macrophage inflammatory protein-1beta [MIP-1beta]) to influence cell growth, apoptosis, adhesion, and production of vascular endothelial growth factors (VEGF), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in these cell lines. We found that A) SDF-1, after binding to CXCR4, activates multiple signaling pathways and that in comparison with the MIP-1beta-CCR5 axis, plays a privileged role in hematopoiesis; B) SDF-1 activation of the MAPK p42/44 pathway and the PI-3K-AKT axis does not affect proliferation and apoptosis but modulates integrin-mediated adhesion to fibronectin, and C) SDF-1 induces secretion of VEGF, but not of MMPs or TIMPs. Thus the role of SDF-1 relates primarily to the interaction of lymphohematopoietic cells with their microenvironment and does not directly influence their proliferation or survival. We conclude that perturbation of the SDF-1-CXCR4 axis during HIV infection may affect interactions of hematopoietic cells with the hematopoietic microenvironment.     

Common variations in the IL4R gene affect splicing and influence natural expression of the soluble isoform

We previously found the soluble interleukin 4 receptor (sIL4R) to be differently expressed in allergic asthma patients compared to healthy individuals. Here we present data demonstrating the involvement of the sequence variations, c.912-1003A > G, c.912-833T > C, c. 912-630A > G, and c.912-577A > G, in the expressional regulation of IL4R splice variants. By using an IL4R minigene construct, genomic DNA and mRNA from asthma patients and nonasthmatic individuals, we analyzed the function of four highly-linked SNPs, flanking the alternatively-spliced exon in the IL4R gene. Results from the minigene assay showed that the form containing the minor alleles significantly decreased the expression of the soluble IL4R (exon 8+) variant, a decrease that could only be seen in the major construct after increasing amounts of either the splicing factor SRp20, or YT521-B. Analysis of mRNA expression in our human material confirmed the results, demonstrating lower expression of the sIL4R in patients and controls carrying the minor alleles. Together these results show sequence variations as a possible way of altering alternative splicing selection of IL4R in vivo.     

Lack of association between CTLA-4 and PDCD1 polymorphisms and acute rejection in German liver transplant recipients

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell-death 1 (PDCD1) are two genes encoding coinhibitory immunoreceptors that are involved in regulation transplant rejection and tolerance induction. Thus, CTLA-4 and PDCD1 may be good candidate genes to evaluate in liver transplant rejection. In this retrospective study, we investigated whether four functional single nucleotide polymorphisms (SNP) of the CTLA-4 gene and PDCD1 gene were associated with susceptibility to liver transplant rejection. The SNPs −1772T > C (rs733618), −1661A > G (rs4553808) of the CTLA-4 gene, and the SNPs 7146G > A (rs11568821), 7209C > T (rs41386349) of the PDCD1 gene were genotyped by polymerase chain reaction allele specific restriction enzyme analysis (PCR-ASRA) in 100 liver recipients with acute rejection, 104 liver transplant recipients without acute rejection and 100 healthy control individuals. For the selected SNPs we did not detect any significant difference in genotypic and allelic frequencies between liver transplant recipients with and without acute rejection. In conclusion, our results suggest that the tested SNPs may not be associated with susceptibility to acute liver transplant rejection in a Caucasian population.     

Constitutive expression of interleukin 4 in vivo does not lead to the development of T helper 2 type CD8+ T cells secreting interleukin 4 or interleukin 5.

Interleukin 4 (IL-4) has been shown to commit CD8+ T cells to a T helper (Th) 2 functional phenotype in vitro. To study the effects of IL-4 on CD8+ T cell development in vivo we analysed the CD8+ T cell phenotype in mice constitutively expressing IL-4. Purified CD8+ T cells from uninfected or flu infected IL-4 transgenic (tg) animals produced no detectable IL-4 or IL-5 after in vitro stimulation on anti-CD3 coated plates. However, CD8+ T cells from IL-4 tg mice could be converted into IL-4 and IL-5 producers in vitro in the presence of exogenous added IL-4, showing that these cells were still responsive to IL-4. IL-4 tg mice also showed a delay in influenza virus clearance from the lung, which was probably due to the observed reduction of total CD8+ T cell numbers in the IL-4 tg animals since IL-4 tg CD8+ T cells showed normal levels of influenza-specific cytotoxicity in comparison to controls. Taken together these results suggest that CD8+ T cells are not necessarily switched to a Th2 phenotype by the presence of IL-4 and that some other factor(s) may be important in the switch process of CD8+ T cells in vivo, since the addition of IL-4 during CD8+ T cell activation in vitro leads to Th2 type CD8+ T cells secreting IL-4 and IL-5.