CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis

A single G protein–coupled receptor (GPCR) can activate multiple signaling cascades based on the binding of different ligands. The biological relevance of this feature in immune regulation has not been evaluated. The chemokine-binding GPCR CXCR3 is preferentially expressed on CD4+ T cells, and canonically binds 3 structurally related chemokines: CXCL9, CXCL10, and CXCL11. Here we have shown that CXCL10/CXCR3 interactions drive effector Th1 polarization via STAT1, STAT4, and STAT5 phosphorylation, while CXCL11/CXCR3 binding induces an immunotolerizing state that is characterized by IL-10^hi (Tr1) and IL-4^hi (Th2) cells, mediated via p70 kinase/mTOR in STAT3- and STAT6-dependent pathways. CXCL11 binds CXCR3 with a higher affinity than CXCL10, suggesting that CXCL11 has the potential to restrain inflammatory autoimmunity. We generated a CXCL11-Ig fusion molecule and evaluated its use in the EAE model of inflammatory autoimmune disease. Administration of CXCL11-Ig during the first episode of relapsing EAE in SJL/J mice not only led to rapid remission, but also prevented subsequent relapse. Using GFP-expressing effector CD4⁺ T cells, we observed that successful therapy was associated with reduced accumulation of these cells at the autoimmune site. Finally, we showed that very low doses of CXCL11 rapidly suppress signs of EAE in C57BL/6 mice lacking functional CXCL11.     

In vitro and in vivo proliferation,differentiation and migration of cardiac endothelial progenitor cells (SCA1+/CD31+ side‐population cells)

To cite this article: Liang SX, Khachigian LM, Ahmadi Z, Yang M, Liu S, Chong BH. In vitro and in vivo proliferation, differentiation and migration of cardiac endothelial progenitor cells (SCA1⁺/CD31⁺ side‐population cells). J Thromb Haemost 2011; 9 : 1628–37. Summary. Background: Side‐population (SP) cells are a select population identified by a capacity to efflux Hoechst dye and are enriched for stem/progenitor cell activity. Previous studies suggested that cardiac SP (CSP) cells could be divided into SCA1⁺/CD31^? and SCA1⁺/CD31⁺ CSP cells. SCA1⁺/CD31^? CSP cells have been shown to be cardiac stem/progenitor cells. However, SCA1⁺/CD31⁺ CSP cells have not been fully characterized. Objective: The aim of the present study was to characterize SCA1⁺/CD31⁺ CSP cells in the adult mouse heart, and investigate their abilities to proliferate, differentiate, vascularize and migrate in vitro and in vivo. Results: Using fluorescence‐activated cell sorting (FACS), RT‐PCR, and assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction (MI), we showed that SCA1⁺/CD31⁺ CSP cells express stem cell and endothelial‐specific genes, and reside in the blood vessels. These cells were able to proliferate, differentiate, migrate and vascularize in vitro and in vivo. After MI, SDF‐1α and CXCR4 were up‐regulated in the damaged myocardium and on SCA1⁺/CD31⁺ CSP cells, respectively. Our results further showed that SDF‐1α induced migration of these cells in vitro. Importantly, we found that SCA1⁺/CD31⁺ CSP cells could migrate into the ischemic region from the non‐ischemic area within the myocardium and form a vascular tube‐like structure after MI. Conclusions: Based on the gene expression profile, localization of SCA1⁺/CD31⁺ CSP cells, and their ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we postulate that SCA1⁺/CD31⁺ CSP cells may represent endothelial progenitor cells in the mouse heart.     

Dimerization of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions

Chemokine receptor CXCR4 (CD184) may play a role in cancer metastasis and is known to form homodimers. However, it is not clear how transmembrane regions (TM) of CXCR4 and receptor homotypic interactions affect the function of CXCR4 in living cells. Using confocal microscopy and flow cytometric analysis, we showed that high levels of CXCR4 are present in the cytoplasm, accompanied by lower expression on the cell surface in CXCR4 transfectants, tumor cells, and normal peripheral blood lymphocytes. CXCR4 homodimers were detected in tumor cells, both on the cell surface membrane and in the cytoplasm using fluorescence resonance energy transfer and photobleaching fluorescence resonance energy transfer to measure energy transfer between CXCR4-CFP and CXCR4-YFP constructs. Disruption of lipid rafts by depletion of cholesterol with methyl-beta-cyclodextrin reduced the interaction between CXCR4 molecules and inhibited malignant cell migration to CXCL12/SDF-1alpha. A synthetic peptide of TM4 of CXCR4 reduced energy transfer between molecules of CXCR4, inhibited CXCL12-induced actin polymerization, and blocked chemotaxis of malignant cells. TM4 also inhibited migration of normal monocytes toward CXCL12. Reduction of CXCR4 energy transfer by the TM4 peptide and methyl-beta-cyclodextrin indicates that interactions between CXCR4s may play important roles in cell migration and suggests that cell surface and intracellular receptor dimers are appropriate targets for control of tumor cell spread. Targeting chemokine receptor oligomerization and signal transduction for the treatment of cancer, HIV-1 infections, and other CXCR4 mediated inflammatory conditions warrants further investigation.     

CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow

Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR⁺ chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2^–/– and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2^–/– neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR⁺ chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF–induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.     

Chemokine receptor patterns in lymphocytes mirror metastatic spreading in melanoma

Melanoma prognosis is dictated by tumor-infiltrating lymphocytes, the migratory and functional behavior of which is guided by chemokine or cytokine gradients. Here, we retrospectively analyzed the expression patterns of 9 homing receptors (CCR/CXCR) in naive and memory CD4⁺ and CD8⁺ T lymphocytes in 57 patients with metastatic melanoma (MMel) with various sites of metastases to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Homing receptor expression on lymphocytes strongly correlated with MMel dissemination. Loss of CCR6 or CXCR3, but not cutaneous lymphocyte antigen (CLA), on circulating T cell subsets was associated with skin or lymph node metastases, loss of CXCR4, CXCR5, and CCR9 corresponded with lung involvement, and a rise in CCR10 or CD103 was associated with widespread dissemination. High frequencies of CD8⁺CCR9⁺ naive T cells correlated with prolonged OS, while neutralizing the CCR9/CCL25 axis in mice stimulated tumor progression. The expansion of CLA-expressing effector memory CD8⁺ T cells in response to a single administration of CTLA4 blockade predicted disease control at 3 months in 47 patients with MMel. Thus, specific CCR/CXCR expression patterns on circulating T lymphocytes may guide potential diagnostic and therapeutic approaches.     

Specific Interactions between the Viral Coreceptor CXCR4 and the Biguanide-Based Compound NB325 Mediate Inhibition of Human Immunodeficiency Virus Type 1 Infection

The present studies were conducted to better define the mechanism of action of polyethylene hexamethylene biguanide (PEHMB) (designated herein as NB325), which was shown in previous studies to inhibit infection by the human immunodeficiency virus type 1 (HIV-1). Fluorescence-activated flow cytometric analyses of activated human CD4⁺ T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 epitope recognition in the absence of altered recognition of selected CD4 or CD3 epitopes. NB325 also inhibited chemotaxis of CD4⁺ T lymphocytes induced by the CXCR4 ligand CXCL12. However, NB325 did not cause CXCR4 internalization (unlike CXCL12) and did not interfere with CXCL12 binding. Additional flow cytometric analyses using antibodies with distinct specificities for extracellular domains of CXCR4 demonstrated that NB325 specifically interfered with antibody binding to extracellular loop 2 (ECL2). This interaction was confirmed using competitive binding analyses, in which a peptide derived from CXCR4 ECL2 competitively inhibited NB325-mediated reductions in CXCR4 epitope recognition. Collectively, these results demonstrate that the biguanide-based compound NB325 inhibits HIV-1 infection by specifically interacting with the HIV-1 coreceptor CXCR4.     

Platelet‐derived CXCL12 (SDF‐1α): basic mechanisms and clinical implications

Platelets are a major source of CXCL12 (stromal cell‐derived factor ‐1α, SDF‐1α) and store CXCL12 as part of their α–granule secretome. Platelet activation enhances surface expression and release of CXCL12. Platelets and megakaryocytes express CXCR4, the major receptor for CXCL12, and interaction of CXCL12 with CXCR4 regulates megakaryopoiesis and the function of circulating platelets. Platelet‐derived CXCL12 also modulates paracrine mechanisms such as chemotaxis, adhesion, proliferation and differentiation of nucleated cells, including progenitor cells. Platelet‐derived CXCL12 enhances peripheral recruitment of progenitor cells to the sites of vascular and tissue injury both in vitro and in vivo and thereby promotes repair mechanisms. CXCL12 expression on platelets is elevated in patients with acute myocardial infarction, correlates with the number of circulating progenitor cells, is associated with preservation of myocardial function and is an independent predictor of clinical outcome. Administration of recombinant CXCL12 reduces infarct size following transient ischemia in mice. The present review summarizes the role of platelet‐derived CXCL12 in cardiovascular biology and its diagnostic and therapeutic implications.     

Age and stress related phenotypical changes in bone marrow CD34+ cells

Objective. Phenotypical changes in the human bone marrow (BM) due to age and stress have not so far been properly addressed in the literature. In the present study, we compared CD34⁺ BM cells between older and young volunteers. The influence of stress on CD34⁺ cell phenotype in older patients was investigated in an age‐matched group with acute myocardial infarction (AMI). Cytokines thought to influence BM CD34⁺ cell homeostasis were also analysed. Material and methods. BM mononuclear cells of 10 older volunteers and of 7 young volunteers (18–25 years), as well as 22 AMI patients, were analysed by flow cytometry for the following markers: CD34, CD38, CD117 (c‐kit) and CD133. Blood samples were analysed for CRP, IL‐6, MCP‐1, IL‐8, MMP‐9, TIMP‐1 and TNFα by ELISA methods. Results. Significantly higher numbers of CD34⁺ CD38^? cells (both absolute and relative) were observed in older volunteers than in young volunteers and AMI patients. Higher numbers of immature progenitors, namely CD34⁺CD38^? cells and CD34⁺CD38^?CD117⁺CD133⁺ cells, were observed among older volunteers compared to the other groups. However, the relative number of CD34⁺ cells lacking CD38 expression or expressing CD133 was higher in the old volunteers and AMI patients. None of the circulating factors investigated correlated with any of the cell population yields. Conclusion. In this study, we found that the absolute and relative numbers of BM CD34⁺CD38^? progenitor cells increase with age. The increment is attenuated in patients with AMI.     

High‐fat diet selectively decreases bone marrow lin−/CD117+ cell population in aging mice through increased ROS production

Bone marrow (BM) stem cells (BMSCs) are an important source for cell therapy. The outcome of cell therapy could be ultimately associated with the number and function of donor BMSCs. The present study was to evaluate the effect of long‐term high‐fat diet (HFD) on the population of BMSCs and the role of reactive oxygen species (ROS) in aging mice. Forty‐week‐old male C57BL/6 mice were fed with HFD for 3 months with regular diet as control. Experiments were repeated when ROS production was reduced in mice treated with N‐acetylcysteine (NAC) or using mice overexpressing antioxidant enzyme network (AON) of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase. BM and blood cells were analyzed with flowcytometry for lineage negative (lin^?) and Sca‐1⁺, or lin^?/CD117⁺, or lin^?/CD133⁺ cells. Lin^?/CD117⁺ cell population was significantly decreased with increased intracellular ROS and apoptosis and decreased proliferation in BM, not in blood, in HFD‐treated mice without change for Sca‐1⁺ or CD133⁺ cell populations in BM or blood. NAC treatment or AON overexpression effectively prevented HFD‐induced intracellular ROS production and reduction of BM lin^?/CD117⁺ population. These data suggested that long‐term HFD selectively decreased BM lin^?/CD117⁺ cell population in aging mice through increased ROS production.     

Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM⁺ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM⁺ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A₄ (LXA₄). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA₄ recapitulated neutrophil regulation in B cell–deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM⁺ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.     

CD133+ cells isolated from various sources and their role in future clinical perspectives

Background. CD133 is a member of a novel family of cell surface glycoproteins. Initially, the expression of CD133 antigen was seen only in the hematopoietic derived CD34⁺ stem cells. At present, CD133 expression is demonstrated in undifferentiated epithelium, different types of tumors and myogenic cells. CD133⁺ neurosphere cells isolated from brain are able to differentiate into both neurons and glial cells. These data suggested that CD133 could be a specific marker for various stem and progenitor cell populations.

Objectives. The main goal would be to describe the role for CD133 as a marker of stem cells able to engraft and differentiate, to form functional non-hematopoietic adult lineages and contribute to disease amelioration via tissue regeneration.

Results/conclusion. In conclusion, since the rise of CD133 antigen as a suitable stem cell marker, the possible use of CD133⁺ stem cells in therapeutic applications has opened a new promising field in the treatment of degenerating diseases. The human circulating cells expressing the CD133 antigen behave as a stem cell population capable of commitment to hematopoietic, endothelial and myogenic lineages. CD133 cell therapy may represent a promising treatment for many diseases.     

Role and implications of the CXCL12/CXCR4/CXCR7 axis in atherosclerosis: still a debate

Atherosclerosis is one of the leading causes of mortality and morbidity worldwide. Chemokines and their receptors are implicated in the pathogenesis of atherosclerosis. CXCL12 is a member of the chemokine family exerting a myriad role in atherosclerosis through its classical CXCR4 and atypical ACKR3 (CXCR7) receptors. The modulatory and regulatory functional spectrum of CXCL12/CXCR4/ACKR3 axis in atherosclerosis spans from proatherogenic, prothrombotic and proinflammatory to atheroprotective, plaque stabilizer and dyslipidemia rectifier. This diverse continuum is executed in a wide range of biological units including endothelial cells (ECs), progenitor cells, macrophages, monocytes, platelets, lymphocytes, neutrophils and vascular smooth muscle cells (VSMCs) through complex heterogeneous and homogenous coupling of CXCR4 and ACKR3 receptors, employing different downstream signalling pathways, which often cross-talk among themselves and with other signalling interactomes. Hence, a better understanding of this structural and functional heterogeneity and complex phenomenon involving CXCL12/CXCR4/ACKR3 axis in atherosclerosis would not only help in formulation of novel therapeutics, but also in elucidation of the CXCL12 ligand and its receptors, as possible diagnostic and prognostic biomarkers.

Key messages

1. The role of CXCL12 per se is proatherogenic in atherosclerosis development and progression.
2. The CXCL12 receptors, CXCR4 and ACKR3 perform both proatherogenic and athero-protective functions in various cell types
3. Due to functional heterogeneity and cross talk of CXCR4 and ACKR3 at receptor level and downstream pathways, regional boosting with specific temporal and spatial modulators of CXCL12, CXCR4 and ACKR3 need to be explored.

     

CXCL12/CXCR4轴通过诱导miR-125b促进肝癌细胞肿瘤干性和5-氟尿嘧啶抵抗的机制

目的探究趋化因子CXCL12/趋化因子受体CXCR4轴通过诱导miR-125b促进肝癌细胞肿瘤干性和5-氟尿嘧啶(5-FU)抵抗的机制。方法选择人肝细胞癌细胞系Huh7,分为7组:对照组(正常培养的肝细胞癌系),CXCR4转染组(CXCR4模拟转染超表达),CXCR4沉默组(CXCR4低表达或者不表达),miR-125b转染组(miR-125b超表达),CXCL12+125b抑制剂组(CXCL12超表达的+抑制miR-125b的表达),5-FU处理组(10μg/L的5-FU处理细胞),5-FU+miR-125b转染组(10μg/L的5-FU处理+miR-125b超表达的细胞)。聚合酶链式反应分析miR-125b mRNA表达;蛋白质印迹法检测E-钙粘蛋白、波形蛋白、CXCR4蛋白、Caspase-3、Bcl-2和Bax的蛋白表达;Transwell分析各组细胞中的细胞迁移、侵袭测定;MTT检测细胞存活力;细胞计数试剂盒8(CCK-8)评估细胞增殖。结果与对照组相比,CXCR4转染组miR-125b mRNA、波形蛋白、CXCR4的蛋白表达显著升高,E黏着蛋白表达显著降低(P<0.05);与CXCR4转染组相比,CXCR4沉默组miR-125b mRNA、波形蛋白、CXCR4的蛋白表达显著降低,E黏着蛋白表达显著升高(P<0.05)。与对照组相比,miR-125b转染组细胞迁移、侵袭数量、细胞活力均显著增加,细胞凋亡率显著降低(P<0.05);与miR-125b转染组相比,CXCL12+125b抑制剂组,细胞迁移、侵袭数量、细胞活均显著减少,细胞凋亡率显著升高(P<0.05)。与对照组相比,5-FU处理组细胞增殖率、Bcl-2表达显著降低,caspase-3、Bax的蛋白表达显著升高(P<0.05),与5-FU处理组相比,5-FU+miR-125b转染组细胞增殖率、Bcl-2表达显著升高,caspase-3、Bax的蛋白表达显著降低(P<0.05)。结论MiR-125b通过激活CXCL12/CXCR4轴而被上调,miR-125b增强CXCR4的表达,这在触发肿瘤侵袭和进展中形成了正反馈回路。这些结果为miR-125b在肝癌进程和化学抗性的发展中的作用提供了新的线索,为肝癌的治疗提供了潜在的治疗靶点。     

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4⁺ Tfh TIL, CD8⁺ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1^hiICOS^int phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67⁺ TIL-B in active germinal centers) and cytotoxic (GZMB⁺CD8⁺ and GZMB⁺CD68⁺ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25⁺CXCR5⁺GARP⁺FOXP3⁺ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β–dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8⁺ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.     

Immunomodulatory effect of human bone marrow‐derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a Th1/Th17‐mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease‐modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)‐MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4⁺ and CD8⁺ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM‐MSCs. The expression of (interleukin) IL‐2, tumor necrosis factor alpha (TNF‐α), and interferon‐gamma (IFN‐γ) was evaluated in naive, central memory, effector memory, and effector CD4⁺ and CD8⁺ T cells, whereas IL‐6, IL‐9, and IL‐17 expression was measured in total CD4⁺ and CD8⁺ T cells. mRNA expression of IL‐4, IL‐10, transforming growth factor beta (TGF‐β), cytotoxic T‐lymphocyte‐associated antigen 4, and/or forkhead box P3 was quantified in fluorescence‐activated cell sorting‐purified CD4⁺ T cells, CD8⁺ T cells, and CD4⁺ Treg. BM‐MSCs inhibited the production of TNF‐α, IL‐17, IL‐6, IL‐2, IFN‐γ, and IL‐9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM‐MSCs increased expression of IL‐10 and TGF‐β by CD4⁺ and CD8⁺ T cells. BM‐MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM‐MSC‐derived immunomodulation affected either naive, effector, and memory T cells.     

Harnessing CXCR4 antagonists in stem cell mobilization,HIV infection,ischemic diseases,and oncology

CXCR4 antagonists (e.g., Plerixafor^TM) have been successfully validated as stem cell mobilizers for peripheral blood stem cell transplantation. Applications of the CXCR4 antagonists have heralded the era of cell‐based therapy and opened a potential therapeutic horizon for many unmet medical needs such as kidney injury, ischemic stroke, cancer, and myocardial infarction. In this review, we first introduce the central role of CXCR4 in diverse cellular signaling pathways and discuss its involvement in several disease progressions. We then highlight the molecular design and optimization strategies for targeting CXCR4 from a large number of case studies, concluding that polyamines are the preferred CXCR4‐binding ligands compared to other structural options, presumably by mimicking the highly positively charged natural ligand CXCL12. These results could be further justified with computer‐aided docking into the CXCR4 crystal structure wherein both major and minor subpockets of the binding cavity are considered functionally important. Finally, from the clinical point of view, CXCR4 antagonists could mobilize hematopoietic stem/progenitor cells with long‐term repopulating capacity to the peripheral blood, promising to replace surgically obtained bone marrow cells as a preferred source for stem cell transplantation.     

深低温冻存的外周血单个核细胞活性氧水平与造血干/祖细胞归巢黏附分子表达之间关系的研究

本研究通过检测经程控降温液氮保存前后的富集造血干/祖细胞的外周血单个核细胞(PBMNC)的活性氧(ROS)水平及造血干/祖细胞归巢分子的表达,分析二者之间的相关性。以冻存PBMNC为实验组,以新分离PBMNC为对照组,通过台盼蓝染色方法检测2组细胞的存活率;通过流式细胞分析法检测2组细胞ROS水平;通过流式细胞分析法检测造血干/祖细胞表面抗原CD34、CD133以及归巢相关分子VLA4、CXCR4、CD44的表达;通过相关系数分析ROS水平与归巢分子表达之间的相关性。结果表明:PBMNC随冻存时间延长存活率逐渐降低,冻存3、6、9、12个月的细胞存活率比冻前显著降低(P<0.05);造血干/祖细胞表面标志性抗原CD34、CD133及归巢相关分子CD44、VLA4、CXCR4表达率随冻存时间的延长有所下降,冷冻保存1年后的表达率比冻存前显著降低(P<0.05);随着冷冻保存时间的延长,细胞内ROS水平逐渐升高,冻存6、9、12个月后细胞内ROS水平比冻存前显著升高(P<0.05)。PBMNC内ROS水平与造血干/祖细胞CD34+VLA4+、CD34+CXCR4+、CD34+CD44+双阳性表达率均呈负相关(相关系数分别为-0.503、-0.457、-0.465)。结论:造血干/祖细胞的归巢黏附分子表达随冻存时间延长而逐渐下降;冻存的PBMNC中的ROS水平与造血干/祖细胞归巢黏附分子表达率成显著负相关;ROS水平增高可能是冻存外周血造血干/祖细胞归巢功能降低的原因之一。     

The Chemokine CXCL16 and Its Receptor, CXCR6, as Markers and Promoters of Inflammation-Associated Cancers

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.