The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.

T cells infiltrating inflammatory sites are usually of the activated/memory type. The precise mechanism for the positioning of these cells within tissues is unclear. Adhesion molecules certainly play a role; however, the intricate control of cell migration appears to be mediated by numerous chemokines and their receptors. Particularly important chemokines for activated/memory T cells are the CXCR3 ligands IP-10 and Mig and the CCR5 ligands RANTES, macrophage inflammatory protein-1alpha, and macrophage inflammatory protein-1beta. We raised anti-CXCR3 mAbs and were able to detect high levels of CXCR3 expression on activated T cells. Surprisingly, a proportion of circulating blood T cells, B cells, and natural killer cells also expressed CXCR3. CCR5 showed a similar expression pattern as CXCR3, but was expressed on fewer circulating T cells. Blood T cells expressing CXCR3 (and CCR5) were mostly CD45RO+, and generally expressed high levels of beta1 integrins. This phenotype resembled that of T cells infiltrating inflammatory lesions. Immunostaining of T cells in rheumatoid arthritis synovial fluid confirmed that virtually all such T cells expressed CXCR3 and approximately 80% expressed CCR5, representing high enrichment over levels of CXCR3+ and CCR5+ T cells in blood, 35 and 15%, respectively. Analysis by immunohistochemistry of various inflamed tissues gave comparable findings in that virtually all T cells within the lesions expressed CXCR3, particularly in perivascular regions, whereas far fewer T cells within normal lymph nodes expressed CXCR3 or CCR5. These results demonstrate that the chemokine receptor CXCR3 and CCR5 are markers for T cells associated with certain inflammatory reactions, particularly TH-1 type reactions. Moreover, CXCR3 and CCR5 appear to identify subsets of T cells in blood with a predilection for homing to these sites.     

Regulation of pulmonary fibrosis by chemokine receptor CXCR3

CXC chemokine receptor 3 (CXCR3) is the receptor for the IFN-gamma-inducible C-X-C chemokines MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. CXCR3 is expressed on activated immune cells and proliferating endothelial cells. The role of CXCR3 in fibroproliferation has not been investigated. We examined the role of CXCR3 in pulmonary injury and repair in vivo. CXCR3-deficient mice demonstrated increased mortality with progressive interstitial fibrosis relative to WT mice. Increased fibrosis occurred without increased inflammatory cell recruitment. CXCR3 deficiency resulted in both a reduced early burst of IFN-gamma production and decreased expression of CXCL10 after lung injury. We identified a relative deficiency in lung NK cells in the unchallenged CXCR3-deficient lung and demonstrated production of IFN-gamma by WT lung NK cells in vivo following lung injury. The fibrotic phenotype in the CXCR3-deficient mice was significantly reversed following administration of exogenous IFN-gamma or restoration of endogenous IFN-gamma production by adoptive transfer of WT lymph node and spleen cells. Finally, pretreatment of WT mice with IFN-gamma-neutralizing Ab's enhanced fibrosis following lung injury. These data demonstrate a nonredundant role for CXCR3 in limiting tissue fibroproliferation and suggest that this effect may be mediated, in part, by the innate production of IFN-gamma following lung injury.     

趋化因子SDF-1及其受体对卵巢癌细胞增殖及迁移的影响

目的:探讨趋化因子受体CXCR4在人卵巢癌细胞中的表达及SDF-1/CXCR4系统与卵巢癌细胞恶性表现的关系.方法:采用RT-PCR检测卵巢癌细胞系SW626中CXCR4的表达,MTT法检测SW626细胞增殖能力的变化,通过体外微孔隔离室板(Transwell)检测SDF-1对SW626细胞迁移及CXCR4的封闭对其迁移的影响.结果:CXCR4 mRNA在SW626细胞呈阳性表达,抗CXCR4单克隆抗体(20 μg/mL)对SW626细胞增殖有明显的抑制作用(P＜0.05),SDF-1可促进SW626细胞的迁移,CXCR4的封闭能抑制SDF-1对SW626迁移的这种促进作用(P＜0.05).结论:SDF-1及其受体CXCR4的相互作用可能在卵巢癌细胞增殖和迁移中发挥着重要作用,干扰SDF-1/CXCR4的相互作用可能成为治疗卵巢癌的一个有意义的靶点和策略.     

Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells

In addition to their role as regulators of leukocyte migration and activation, chemokines and their receptors also function in angiogenesis, growth regulation, and HIV-1 pathogenesis--effects that involve the action of chemokines on nonhematopoietic cells. To determine whether chemokine receptors are expressed in human colonic epithelium, HT-29 cells were examined by RT-PCR for the expression of the chemokine receptors for lymphotactin, fractalkine, CCR1-10, and CXCR1-5. The only receptor consistently detected was CXCR4 (fusin/LESTR), although HT-29 cells did not express mRNA for its ligand, stromal cell-derived factor (SDF-1alpha). Flow cytometric analysis with anti-CXCR4 antibody indicated that the CXCR4 protein was expressed on the surface of roughly half of HT-29 cells. CXCR4 was also expressed in colonic epithelial cells in vivo as shown by immunohistochemistry on biopsies from normal and inflamed human colonic mucosa. The mRNA for SDF-1alpha and other CC and CXC chemokines was present in normal colonic biopsies. The CXCR4 receptor in HT-29 cells was functionally coupled, as demonstrated by the elevation in [Ca2+]i, which occurred in response to 25 nM SDF-1alpha and by the SDF-1alpha-induced upregulation of ICAM-1 mRNA. Sodium butyrate downregulated CXCR4 expression and induced differentiation of HT-29 cells, suggesting a role for CXCR4 in maintenance and renewal of the colonic epithelium. This receptor, which also serves as a coreceptor for HIV, may mediate viral infection of colonic epithelial cells.     

Selective modification of antigen-specific T cells by RNA electroporation

It has been observed that the efficient transfection of T cells by RNA electroporation requires prior activation of T cells with mitogens or by anti-CD3 antibody stimulation. We hypothesized that this requirement for T cell activation could be leveraged to express marker genes within activated T cells responding to antigen-pulsed dendritic cells and allow for the selective enrichment and modification of antigen-specific T cells. Using electroporation of mRNA encoding green fluorescent protein as a marker gene, we demonstrate that RNA electroporation can efficiently allow for the separation of cytomegalovirus-specific CD8+ and CD4+ T cells from bulk culture responding to cytomegalovirus pp65 antigen-pulsed dendritic cells. Furthermore, we demonstrate that cytomegalovirus-specific T cells can be functionally modified by RNA transfection of the C-X-C chemokine receptor, CXCR2, to migrate efficiently toward a variety of CXCR2-specific chemokines in vitro and in vivo. These studies demonstrate the utility of RNA transfection as a simple method by which to purify and selectively modify the function of antigen-specific T cells for use in adoptive immunotherapy, and importantly provide evidence that transient expression of proteins by RNA transfection is an efficient means of modulating the in vivo function of activated T cells.     

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation

Discovering dominant epitopes for T cells, particularly CD4⁺ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2–expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4⁺ T cells in the lung. We discovered lung-resident CD4⁺ T cells that expressed a disease-specific public CDR3β T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLA-DP2–CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4⁺ T cells specific for the HLA-DP2–CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation.     

趋化因子受体CXCR7在人早孕期滋养细胞和蜕膜基质细胞的表达

目的 研究趋化因子受体CXCR7在人早孕期母-胎界面的表达特征.方法 收集人早孕期绒毛与蜕膜组织,免疫组织化学技术分析趋化因子受体CXCR7及CXCR4在母-胎界面的表达情况;分别分离培养人早孕期滋养细胞及蜕膜基质细胞,免疫细胞化学技术分析CXCR7和CXCR4在两种细胞的表达.结果 人早孕期绒毛与蜕膜组织及体外分离培养的人早孕期滋养细胞、蜕膜基质细胞均可观察到特异性CXCR7和CXCR4阳性染色,但两种细胞CXCR7染色强度（平均光密度值）均略低于CXCR4,差异有统计学意义（P＜0.05）.结论 人早孕期滋养细胞及蜕膜基质细胞共表达趋化因子受体CXCR7和CXCR4,提示CXCR7可能在母-胎免疫微环境形成中也起重要作用.     

Ablation of tumor-derived stem cells transplanted to the central nervous system by genetic modification of embryonic stem cells with a suicide gene

Embryonic stem cell (ESC)-based therapies open new possibilities as regenerative medicine for the treatment of human disease, but the presence of small numbers of undifferentiated ESCs within the transplant could lead to the development of tumors. The safety of ESC transplants would be enhanced if uncontrolled cell growth could be suppressed, using external stimuli. A lentiviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) and green fluorescent protein (GFP) genes was used to genetically modify murine ESCs (HSVtk+GFP+ ESCs). In the presence of ganciclovir (GCV), 100% of HSVtk+GFP+ ESCs were killed in vitro, and 100% of flank tumors derived from HSVtk+GFP+ ESCs were eliminated. When CNS tumors were produced by the HSVtk+GFP+ ESCs, the tumor mass was completely eliminated on GCV treatment for 1 week. After GCV treatment for 3 weeks, histologic analysis showed no residual tumor cells and TaqMan realtime polymerase chain reaction analysis showed no genomic HSVtk copies or HSVtk mRNA. These data demonstrate that it is possible to use ex vivo gene transfer to modify ESCs with conditional genetic elements that can be activated in vivo to control undifferentiated ESC outgrowth and to eliminate transduced ESCs that have escaped growth control after ESC-mediated therapy to the CNS.     

Genetic modification of T cells for immunotherapy

Adoptive transfer of antigen-specific T cells is a promising approach for preventing progressive viral infections in immunosuppressed hosts. By contrast, effective T-cell therapy of malignant disease has proven to be much more difficult to achieve. This, in part, reflects the difficulty of isolating high avidity T cells specific for tumor-associated antigens, many of which are self-antigens that have induced some level of tolerance in the host. Even when tumor-reactive T cells can be isolated, the ability of these cells to survive in vivo and traffic to tumor sites is often impaired. Additionally, most tumors employ multiple mechanisms to escape T-cell recognition, including interference in antigen presentation, secretion of inhibitory factors and recruitment of regulatory or immunosuppressive cells. The genetic modification of T cells prior to transfer provides a potential means to overcome many of these obstacles and enhance the efficacy of T-cell therapy. This review article discusses the rationale for genetic modification of T cells, the critical steps involved in gene transfer, and potential advantages and disadvantages of strategies that are now being examined to engineer improved effector T cells for the treatment of human infectious and malignant disease.     

H-2-restricted helper factor secreted by clone hybridoma cells

Biological and serological characteristics of a helper factor secreted by cloned hybridoma cells was described. The factor is carrier specific and contains determinants shared with immunoglobulin VH bu does not react with V kappa- or V lambda-specific antibodies. Presence of four H-2I-controlled antigenic specificities, Ia.ml, Ia.m2, Ia.17, and Ia.m7, was detected. Hence, it is possible that both A beta and E alpha loci may be involved in its control. Helper effect could be obtained only toward B cell sources that shared the H-2K and I-A antigens with the hybridoma cells. Similarly, the factor was absorbed only by spleen cells syngeneic in I-A. Previous studies have demonstrated that this clone binds antigen in an H-2-restricted manner. It follows that H-2-restricted helper cells produce H-2-restricted helper factors. Hence, they support the view that specific T cell factors may represent secreted T cell receptors.     

Genetic modification of T cells for immunotherapy

Adoptive transfer of antigen-specific T cells is a promising approach for preventing progressive viral infections in immunosuppressed hosts. By contrast, effective T-cell therapy of malignant disease has proven to be much more difficult to achieve. This, in part, reflects the difficulty of isolating high avidity T cells specific for tumor-associated antigens, many of which are self-antigens that have induced some level of tolerance in the host. Even when tumor-reactive T cells can be isolated, the ability of these cells to survive in vivo and traffic to tumor sites is often impaired. Additionally, most tumors employ multiple mechanisms to escape T-cell recognition, including interference in antigen presentation, secretion of inhibitory factors and recruitment of regulatory or immunosuppressive cells. The genetic modification of T cells prior to transfer provides a potential means to overcome many of these obstacles and enhance the efficacy of T-cell therapy. This review article discusses the rationale for genetic modification of T cells, the critical steps involved in gene transfer, and potential advantages and disadvantages of strategies that are now being examined to engineer improved effector T cells for the treatment of human infectious and malignant disease.     

Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of α4β7 and CCR9 by Peyer’s patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid–dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103⁺ MLN DCs, up-regulate the gut-homing integrin α4β7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.Because efficient trafficking of immune cells to the gastrointestinal (GI) tract is critical for host defense against pathogenic challenge, studying cellular recruitment pathways to the GI tract is the key to developing novel vaccines against mucosally transmitted diseases, including HIV-1 infection.Naive T cells acquire the capacity to migrate to extra-lymphoid tissues once activated by their cognate antigen (Butcher et al., 1999; von Andrian and Mackay, 2000). These antigen-experienced effector T cells migrate preferentially to the tissues where they first encountered the antigen (Kantele et al., 1999; Campbell and Butcher, 2002). For example, early observations demonstrated that cells activated in the GI tract home back to the intestinal effector sites (Cahill et al., 1977; Hall et al., 1977). Integrin α4β7 and chemokine receptor 9 (CCR9) are among the best studied gut-specific homing molecules (Berlin et al., 1995; Zabel et al., 1999). The α4β7 ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates recruitment of T cells to the intestinal lamina propria (Berlin et al., 1995), and the CCR9 ligand TECK, expressed by small intestinal epithelial cells, recruits T cells to the small bowel (Zabel et al., 1999).DCs are well recognized as the initiators of the adaptive immune response (Steinman and Cohn, 1973), as well as mediators of tolerance to self-antigens in steady-state conditions (Hawiger et al., 2001). Additionally, there is increasing evidence regarding the role played by DCs as conductors of immunological traffic to the skin and the GI tract (Johansson-Lindbom et al., 2003, 2005; Mora et al., 2003, 2005; Sigmundsdottir et al., 2007). DCs can imprint T cells to migrate to the tissue in which the T cells were originally activated. For example, gut-associated DCs induce the gut-homing receptors α4β7 and CCR9 on T cells upon activation (Johansson-Lindbom et al., 2003; Mora et al., 2003; Stagg et al., 2002).RA is necessary and sufficient to induce gut-homing receptors on T cells (Iwata et al., 2004). The main pathway of RA biosynthesis in vivo is dependent on the intracellular oxidative metabolism of retinol (Napoli, 1999; Duester, 2000), catalyzed by a family of alcohol dehydrogenases including retinal dehydrogenase (RALDH), a class I aldehyde dehydrogenase that mediates the irreversible oxidation of retinal to RA. RA in turn is thought to induce RALDH-2 in a positive feedback loop (Yokota et al., 2009; Hammerschmidt et al., 2011; Villablanca et al., 2011) and RA levels correlate with the ability of the intestinal DCs to induce gut-tropic T cells. Vitamin A is introduced via dietary or biliary sources (Jaensson-Gyllenbäck et al., 2011). Among the cellular sources of RA in the intestinal mucosa are DCs (Iwata et al., 2004), stromal cells (Hammerschmidt et al., 2008; Molenaar et al., 2009), intestinal epithelial cells (Bhat, 1998; Lampen et al., 2000), and intestinal macrophages (Denning et al., 2007), with the DCs likely playing a key role in the induction of gut-homing phenotype on T cells. Among the intestinal DCs, the CD103⁺ DC subsets express high levels of RALDH-2 and are capable of generating high levels of RA (Johansson-Lindbom et al., 2005; Coombes et al., 2007; Sun et al., 2007; Jaensson et al., 2008). In contrast, the CD103⁻CD11b⁺CX3CR1⁺ macrophage-like population in the intestinal lamina propria expresses RALDH-1 and not RALDH-2 and exhibits a lower RA-producing capacity (Schulz et al., 2009; Denning et al., 2011), and therefore a decreased capacity to induce gut-homing potential on T cells (Jaensson et al., 2008).Collectively, a paradigm has emerged wherein only the intestinal CD103⁺ DCs, which are capable of metabolizing vitamin A, can induce GI-specific homing on T cells (Jaensson et al., 2008). This paradigm however, is difficult to reconcile with reports of GI T cell responses after i.n. delivery of antigens that do not directly target the GI lymphoid tissue. For example, mice infected i.n. with influenza virus show no activated virus-specific CD8⁺ T cells in the mesenteric LNs (MLNs) or Peyer’s patches (PPs), yet flu-specific CD8⁺ cells within the lung-associated tissues express α4β7 and memory CD8⁺ T cells are established within the small intestinal epithelium (Masopust et al., 2010). Similarly, a recent study demonstrates that i.n. challenge with H1N1 influenza results in the accumulation of T_H17 cells within the small intestinal lamina propria (SILP; Esplugues et al., 2011). Furthermore, Ciabattini et al. (2011) have demonstrated that after i.n. immunization, antigen-specific T cells are generated in the mediastinal LN and migrate to the MLN in an α4β7- and CD62L-dependent manner. Additionally, it is known that lungs harbor prominent extrahepatic stores of vitamin A (Okabe et al., 1984; Dirami et al., 2004). Its metabolite, RA, plays an important role in pulmonary alveolar development (Dirami et al., 2004) and has a putative therapeutic role in emphysema (Massaro and Massaro, 1997). Finally, although RA production has been considered to be the forte of gut-resident DCs, other DC populations also express RALDH, particularly lung-resident DCs that express RALDH-2 (Heng and Painter, 2008; Guilliams et al., 2010). However, the ability of lung DCs to induce GI-specific T cell homing has not yet been reported.All of the aforementioned factors led us to hypothesize that lung DCs would up-regulate the expression of gut-homing molecules integrin α4β7 and CCR9 on T cells, which in turn would license the migration of T cells to the GI tract. Our hypothesis was made credible by the concept of a common mucosal immunological system proposed by Bienenstock et al. (1978) more than 30 years ago. Indeed, there is increasing appreciation of the mucosal immune system as an integrated network of tissues, cells, and effector molecules, although the cellular factors that link different mucosal compartments are not well understood (Gill et al., 2010).In this study, we show that lung DCs can imprint expression of the gut-homing integrin α4β7 and CCR9 on co-cultured T cells in vitro and on adoptively transferred cells in vivo, licensing T cells to migrate to the GI lamina propria and confer protective immunity against intestinal pathogens. We define a new pathway of DC-mediated mucosal cross talk and challenge the existing dogma that only GI-resident DCs can recruit antigen-specific T cells back to the gut.     

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.