AMD3100干预调节性T细胞迁移机制研究

CXCR4+细胞在CD4+CD25+Foxp3+调节性T细胞亚群中占有一定的比例。趋化因子CXCL12与细胞表面的特异性受体CXCR4相互作用,调节这些细胞的迁移和归巢等生理过程。AMD3100是一种人工合成的大环类拮抗剂,可特异性拮抗CXCR4。本研究采用磁珠亲和细胞分选术纯化BALB/c小鼠脾脏CD4+CD25+调节性T细胞,并采用transwell共培养系统,研究AMD3100对调节性T细胞迁移的影响。研究发现,AMD3100以剂量依赖性模式抑制CXCR4+CD4+CD25+T细胞从transwell培养系统的上室迁移至下室,采用中和抗体阻断CXCR4可观察到相似效应。当AMD3100终浓度为2.0μg/ml时,迁移到下室的CXCR4+细胞占总数的2.2%,显著低于磷酸盐缓冲液对照组(36.2%)(P<0.01)。此外,CD4+CD25+细胞24h迁移率也显著下降(AMD3100处理组和磷酸盐缓冲液对照组分别为3.1%和35.5%,P<0.01)。提示AMD3100可特异性抑制CXCR4+CD4+CD25+细胞迁移。由于CXCR4是CXCL12的特异性受体,这一效应提示AMD3100可削弱CXCL12的作用。此外,由于AMD3100处理后可使CD4+CD25+细胞24h迁移率下降,提示这种拮抗剂有可能短时间内削弱调节性T细胞的功能。     

CXCR4–CXCL12 interaction is important for plasma cell homing and survival in NZB/W mice

Antibody‐secreting cells (ASCs), including short‐lived plasmablasts and long‐lived memory plasma cells (LLPCs), contribute to autoimmune pathology. ASCs, particularly LLPCs, refractory to conventional immunosuppressive drugs pose a major therapeutic challenge. Since stromal cells expressing C‐X‐C motif chemokine‐12 (CXCL12) organize survival niches for LLPCs in the bone marrow, we investigated the effects of CXCL12 and its ligand CXCR4 (C‐X‐C chemokine receptor 4) on ASCs in lupus mice (NZB/W). Fewer adoptively transferred splenic ASCs were retrieved from the bone marrow of recipient immunodeficient Rag1^?/? mice when the ASCs were pretreated with the CXCR4 blocker AMD3100. CXCR4 blockade also significantly reduced anti‐OVA ASCs in the bone marrow after secondary immunization with OVA. In this study, AMD3100 efficiently depleted ASCs, including LLPCs. After two weeks, it decreased the total number of ASCs in the spleen and bone marrow by more than 60%. Combination with the proteasome inhibitor bortezomib significantly enhanced the depletion effect of AMD3100. Continuous long‐term (five‐month) CXCR4 blockade with AMD3100 after effective short‐term LLPCs depletion kept the number of LLPCs in the bone marrow low, delayed proteinuria development and prolonged the survival of the mice. These findings identify the CXCR4‐CXCL12 axis as a potential therapeutic target likely due to its importance for ASC homing and survival.     

Mouse serum factor(s) down-modulate the CD4 and CXCR4 molecules on human T cells conferring resistance to HIV infection in NOG mice

Human cells have developed innate immunity, exploiting several means to block virus infection, and viruses have evolved diverse strategies to resist these. We show here that the human immunodeficiency virus 1 (HIV-1) could neither progressively infect engrafted human leukemic T cells nor repress their growth in NOG mice. However, ED-40515(–) cells infected with HIV-1 before inoculation were found to significantly delay the onset of tumor growth and increased the survival period of NOG mice. ED-40515(–) tumor cells showed resistance to HIV-1 which was apparently correlated with the down-regulation of CD4 and CXCR4 molecules in NOG mice. Serum from three different mouse strains, including NOG, retained a suppressive effect on the CD4 molecule of ED-40515(–) cells in vitro. ED-40515(–) cells obtained from mice re-expressed CD4 and CXCR4 molecules upon in vitro culture and were again successfully infected with HIV-1. These findings indicate that HIV-1 may initially successfully delay or regress tumor growth in NOG mice, but eventually fails to do so because of the evolution of HIV-resistant cells due to a rapid down-modulation of CD4 and CXCR4. Our data also demonstrated that some unknown soluble factor(s) present in mouse serum was responsible for conferring resistance to HIV infection to human T cells.Grant sponsor: supported by grants from the Ministry of Education, Science and Culture and Ministry of Health, Labor and Welfare of Japan.     

趋化因子受体CXCR4在人肺癌高转移细胞株的表达和意义

目的：以人肺癌高、低转移细胞株95D、95C为研究对象，研究趋化因子受体CXCR4的表达及其在肿瘤细胞体外转移潜能中的作用和意义。方法：采用RT-PCR检测95D、95C细胞CXCR4 mRNA的表达情况；以PMA活化肿瘤细胞，研究CXCR4 mRNA表达水平与细胞活性状态的关系；应用钙离子内流实验验证其表达是否具有功能；通过趋化实验观察CXCR4特异性配件SDF-α和裸鼠组织匀浆液对95D细胞的趋化迁移作用；通过MTT法测定95D细胞对SDF-1α作用的增殖反应。结果：95D细胞功能性地高表达趋化因子受体CXCR4，且其表达水平与细胞活性状态有关；CXCR4特异性配件SDF-1α和裸鼠肺、淋巴结组织匀浆均可在体外趋化95D细胞的迁移，SDF-1α还可促进95D细胞的增殖。结论：95D细胞功能性高表达趋化因子受体CXCR4可能与人肺癌细胞株95D的体外高转移潜能有关。     

Comparison of in vivo and in vitro evolution of CCR5 to CXCR4 coreceptor use of primary human immunodeficiency virus type 1 variants

During the course of at least 50% of HIV-1 subtype B infections, CCR5-using (R5) viruses evolve towards a CXCR4-using phenotype. To gain insight in the transition from CCR5 to CXCR4 coreceptor use, we investigated whether acquisition of CXCR4 use in vitro of R5 viruses from four patients resembled this process in vivo. R5 variants from only one patient acquired CXCR4 use in vitro. These variants had envelopes with higher V3 charge and higher number of potential N-linked glycosylation sites when compared to R5 variants that failed to gain CXCR4 use in vitro. In this patient, acquisition of CXCR4 use in vitro and in vivo was associated with multiple mutational patterns not necessarily involving the V3 region. However, changes at specific V3 positions were prerequisite for persistence of CXCR4-using variants in vivo, suggesting that positive selection targeting the V3 loop is required for emergence of CXCR4-using variants during natural disease course.     

Effect of seasonal exposure to pollen on nonspecific interleukin-4, interleukin-5, and interferon-gamma in vitro release by peripheral blood mononuclear cells from subjects with pollinosis

The immune response to environmental allergens depends on both genetic and environmental factors. Allergen exposure triggers the activation of allergen-specific Th2 cells in allergic patients, as well as increased Th2-type cytokine mRNA expression and eosinophil recruitment. Nevertheless, different patterns of release of cytokines could explain the heterogeneity of atopic response. In our study, 25 patients with pollinosis and 15 healthy donors were selected to characterize their release of Th2 (interleukin [IL]-4 and IL-5) and Thi (interferon-gamma [IFN-y]) cytokines, both during and outside the pollen season. Peripheral blood mononuclear cells from patients and controls were isolated, cultured in the presence of phorbol-12-myristate-13- acetate plus ionomycine, and phytohemagglutinin (PHA), and cytokine release was assessed by titration in the supernatants. Both IL-4 and IL-5 showed higher levels during than outside the pollen season in pollinic patients (P<0.05) after nonspecific stimuli, whereas IFN-y levels were significantly lower during than outside the pollen season only after culture with PHA. Significant differences were not observed in the control group. Our results are consistent with the hypothesis that release of cytokines by peripheral blood mononuclear cells from patients with pollinosis depends on environmental exposure to sensitizing pollens, and that influence can be revealed by in vitro nonspecific stimulation. Nevertheless, the heterogeneity in results suggests that the use of mitogens to assess Thl/Th2 dominance may need careful evaluation.