Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8α⁺ conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8⁺ T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3⁺ DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8⁺ T cells through XCR1.     

Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans

We used multiparameter flow cytometry to characterize leukocyte immunophenotypes and cytokines in skin and peripheral blood of patients with erythema migrans (EM). Dermal leukocytes and cytokines were assessed in fluids aspirated from epidermal suction blisters raised over EM lesions and skin of uninfected controls. Compared with corresponding peripheral blood, EM infiltrates were enriched for T cells, monocytes/macrophages, and dendritic cells (DCs), contained lower proportions of neutrophils, and were virtually devoid of B cells. Enhanced expression of CD14 and HLA-DR by lesional neutrophils and macrophages indicated that these innate effector cells were highly activated. Staining for CD45RO and CD27 revealed that lesional T lymphocytes were predominantly Ag-experienced cells; furthermore, a subset of circulating T cells also appeared to be neosensitized. Lesional DC subsets, CD11c(+) (monocytoid) and CD11c(-) (plasmacytoid), expressed activation/maturation surface markers. Patients with multiple EM lesions had greater symptom scores and higher serum levels of IFN-alpha, TNF-alpha, and IL-2 than patients with solitary EM. IL-6 and IFN-gamma were the predominant cytokines in EM lesions; however, greater levels of both mediators were detected in blister fluids from patients with isolated EM. Circulating monocytes displayed significant increases in surface expression of Toll-like receptor (TLR)1 and TLR2, while CD11c(+) DCs showed increased expression of TLR2 and TLR4; lesional macrophages and CD11c(+) and CD11c(-) DCs exhibited increases in expression of all three TLRs. These results demonstrate that Borrelia burgdorferi triggers innate and adaptive responses during early Lyme disease and emphasize the interdependence of these two arms of the immune response in the efforts of the host to contain spirochetal infection.     

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.     

小鼠胸腺基质细胞系分泌的趋化因子的分析

利用Boyden小室法和FACS分析法,我们分析了五株小鼠胸腺基质细胞系(MTSC)培养上清液对中性粒细胞,单核巨噬细胞和淋巴细胞的化学趋化因子(Chemokines)活性,及定向迁移的淋巴细胞中B细胞、CD 4~ CD 8~-和CD4~-CD8~ T细胞的比例。结果显示,五株MTSC的培养上清液对上述靶细胞均有不同程度的趋化作用.MTSC细胞分泌趋化因子的情况可分为三类:1.MTEC 1和MTEC 2产生的Chemokine(s)对中性粒细胞和淋巴细胞的趋化作用相对较强;2.MTDC 4分泌的Chemokine(s)主要作用于单核巨噬细胞;3.MTEC 3和MTEC 5分泌的Chemokine(s)对多种类型的靶细胞,包括中性粒细胞、单核巨噬细胞和淋巴细胞,表现的趋化作用没有明显的强弱之分。MTSC-SN对B细胞的趋化活性普遍高于对T细胞的趋化活性,对CD 4~-CK 8~ T细胞的趋化活性高于对CD 4~ CD 8~-T细胞的趋化活性。MTSC-SN中趋化因子的分析,有利于新型chemokines的发现及其生物功能的阐明,并可进一步研究Chemok-ine(s)在T细胞发育中的作用。     

Modulation of dendritic cell differentiation and maturation by decoy receptor 3

Decoy receptor 3 (DcR3), a soluble receptor belonging to the TNFR superfamily, is a receptor for both Fas ligand (FasL) and LIGHT. It has been demonstrated that DcR3 is up-regulated in lung and colon cancers, thus promoting tumor growth by neutralizing the cytotoxic effects of FasL and LIGHT. In this study, we found that DcR3.Fc profoundly modulated dendritic cell differentiation and maturation from CD14(+) monocytes, including the up-regulation of CD86/B7.2, and the down-regulation of CD40, CD54/ICAM-1, CD80/B7.1, CD1a, and HLA-DR. Moreover, DcR3-treated dendritic cells suppressed CD4(+) T cell proliferation in an allogeneic MLR and up-regulated IL-4 secretion of CD4(+)CD45RA(+) T cells. This suggests that DcR3.Fc may act not only as a decoy receptor to FasL and LIGHT, but also as an effector molecule to skew T cell response to the Th2 phenotype.     

Intralesional injection of adenovirus encoding CC chemokine ligand 16 inhibits mammary tumor growth and prevents metastatic-induced death after surgical removal of the treated primary tumor

The CC chemokine ligand (CCL)16 exerts chemotactic activity on human monocytes and lymphocytes. Although no murine homologous has been defined, the TSA mouse adenocarcinoma cells engineered to express human CCL16 are rapidly rejected by syngenic mice. An adenovirus encoding CCL16 (AdCCL16) was generated using a Cre-Lox-based system and was used to determine whether this chemokine might also block pre-existing tumors. Both recombinant and viral CCL16 showed in vitro chemotactic activity for murine CD4(+) and CD8(+) lymphocytes and dendritic cells (DC). AdCCL16, but not the control empty vector, when injected in established nodules significantly delayed tumor growth. Immunohistochemistry revealed accumulation of CD4(+) and CD8(+) T cells and DC in the treated tumors as well as in draining lymph nodes. DC from such lymph nodes stimulated IFN-gamma by a T cell clone specific for the known TSA tumor-associated Ag (TAA), suggesting the tumor origin of these cells. Lymphocytes from the same nodes showed specific CTL activity against TSA tumor cells and their immunodominant TAA peptide. Antitumor activity required CD4, CD8, and IFN-gamma production, as shown using subset-depleted and knockout mice. Despite the robust and rapid immune response triggered by intratumoral injection of AdCCL16, the lesions were not completely rejected; however, the same treatment given before surgical excision of primary lesions prevented metastatic spread and cured 63% of mice bearing the 4T1 mammary adenocarcinoma, which is perhaps the most compelling model of spontaneous metastasis.     

Epstein-Barr virus (EBV)-infected monocytes facilitate dissemination of EBV within the oral mucosal epithelium

Epstein-Barr virus (EBV) causes hairy leukoplakia (HL), a benign lesion of oral epithelium that occurs primarily in the setting of human immunodeficiency virus (HIV)-associated immunodeficiency. However, the mechanisms of EBV infection of oral epithelium are poorly understood. Analysis of HL tissues shows a small number of EBV-positive intraepithelial macrophages and dendritic/Langerhans cells. To investigate a role for these cells in spreading EBV to epithelial cells, we used tongue and buccal explants infected ex vivo with EBV. We showed that EBV first infects submucosal CD14(+) monocytes, which then migrate into the epithelium and spread virus to oral epithelial cells, initiating productive viral infection within the terminally differentiated spinosum and granulosum layers. Incubation of EBV-infected monocytes and oral explants with antibodies to CCR2 receptor and monocyte chemotactic protein 1 prevented entry of monocytes into the epithelium and inhibited EBV infection of keratinocytes. B lymphocytes played little part in the spread of EBV to keratinocytes in our explant model. However, cocultivation of EBV-infected B lymphocytes with uninfected monocytes in vitro showed that EBV may spread from B lymphocytes to monocytes. Circulating EBV-positive monocytes were detected in most HIV-infected individuals, consistent with a model in which EBV may be spread from B lymphocytes to monocytes, which then enter the epithelium and initiate productive viral infection of keratinocytes.     

Characterization of human inducible costimulator ligand expression and function

The inducible costimulator (ICOS) is the newest member of the CD28/CD152 receptor family involved in regulating T cell activation. We constructed a soluble-Ig fusion protein of the extracellular domain of human ICOS and used it as a probe to characterize expression patterns of the ICOS ligand (ICOSL). ICOSIg did not bind to CD80- or CD86-transfected Chinese hamster ovary cell lines, demonstrating that ICOSL is distinct from those ligands identified for CD28/CD152. ICOSIg showed selective binding to monocytic and B cell lines, whereas binding was undetectable on unstimulated monocytes and peripheral blood T and B cells. Expression of ICOSL was induced on monocytes after integrin-dependent plastic adhesion. Pretreatment of monocytes with mAb to the beta2-integrin subunit CD18 decreased adhesion and abolished ICOSL up-regulation but had no effect on CD80/86 (CD152 ligand (CD152L)) expression. Both ICOSL and CD152L were up-regulated on monocytes by IFN-gamma but by distinct signaling pathways. Unlike CD152L expression, ICOSL expression did not change when monocytes were differentiated into dendritic cells (DCs) or after DCs were induced to mature by LPS, TNF-alpha, or CD40 ligation. Addition of ICOSIg to allogeneic MLRs between DCs and T cells reduced T cell proliferative responses but did so less efficiently than CTLA4Ig (CD152Ig) did. Similarly, ICOSIg also blocked Ag-specific T cell proliferation to tetanus toxoid. Thus, ICOSL, like CD80/86, is expressed on activated monocytes and dendritic cells but is regulated differently and delivers distinct signals to T cells that can be specifically inhibited by ICOSIg.     

Selective gene expression and activation-dependent regulation of vasoactive intestinal peptide receptor type 1 and type 2 in human T cells

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4(+) T cells and CD8(+) T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4(+) T cells than in CD8(+) T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4(+) T cells and CD8(+) T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4(+) T cell subpopulations is highly correlated with T cell activation.     

Human leptin enhances activation and proliferation of human circulating T lymphocytes

Leptin is an adipocyte-secreted hormone that centrally regulates weight control. However, leptin receptor is expressed not only in the central nervous system, but also in other systems such as reproductive and hematopoietic tissues. Human leptin has previously been shown to enhance cytokine production by murine peritoneal macrophages and human circulating monocytes. In this paper we have assessed the presence of leptin receptors in peripheral human T lymphocytes and we have studied their functional role. Both CD4(+) and CD8(+) T lymphocytes express leptin receptors. Moreover, we show that human leptin dose-dependently enhances proliferation and activation of human circulating T lymphocytes when they are costimulated by PHA or Con A. Leptin alone was not able to activate T lymphocytes. To confirm a direct effect of leptin on T lymphocytes, monocytes were extracted by adhesion to culture flasks. The early activation surface marker CD69 was then induced in both CD4(+) and CD8(+) T lymphocytes after 8 h stimulation with PHA or Con A. Leptin dose-dependently enhanced stimulated CD69 expression. Moreover, leptin dose-dependently enhanced the expression of the late activation markers CD25 and CD71 in both CD4(+) and CD8(+) T lymphocytes after 48 h stimulation with PHA or Con A. Finally, we have found that leptin modulates CD4(+) T lymphocyte activation toward Th1 phenotype by stimulating the synthesis of IL-2 and IFN-gamma. These results demonstrate the presence of the leptin receptor in human circulating CD4(+) and CD8(+) T lymphocytes and a functional role of leptin as a modulator (enhancer) of lymphocyte stimulation with a shift toward Th1 cytokine-production profile. This function of leptin may have some relevance in the pathophysiology of immunologic alterations related to obesity.     

Expression profile of FcgammaRIIb on leukocytes and its dysregulation in systemic lupus erythematosus

FcgammaRIIb (CD32B, Online Mendelian Inheritance in Man 604590), an IgG FcR with a tyrosine-based inhibitory motif, plays a critical role in the balance of tolerance and autoimmunity in murine models. However, the high degree of homology between FcgammaRIIb and FcgammaRIIa in humans and the lack of specific Abs to differentiate them have hampered study of the normal expression profile of FcgammaRIIb and its potential dysregulation in autoimmune diseases such as systemic lupus erythematosus (SLE). Using our newly developed anti-FcgammaRIIb mAb 4F5 which does not react with FcgammaRIIa, we found that FcgammaRIIb is expressed on the cell surface of circulating B lymphocytes, monocytes, neutrophils, myeloid dendritic cells (DCs), and at very low levels on plasmacytoid DCs from some donors. Normal donors with the less frequent 2B.4 promoter haplotype have higher FcgammaRIIb expression on monocytes, neutrophils, and myeloid DCs similar to that reported for B lymphocytes, indicating that FcgammaRIIb expression on both myeloid and lymphoid cells is regulated by the naturally occurring regulatory single nucleotide polymorphisms in the FCGR2B promoter. FcgammaRIIb expression in normal controls is up-regulated on memory B lymphocytes compared with naive B lymphocytes. In contrast, in active SLE, FcgammaRIIb is significantly down-regulated on both memory and plasma B lymphocytes compared with naive and memory/plasma B lymphocytes from normals. Similar down-regulation of FcgammaRIIb on myeloid-lineage cells in SLE was not seen. Our studies demonstrate the constitutive regulation of FcgammaRIIb by natural gene polymorphisms and the acquired dysregulation in SLE autoimmunity, which may identify opportunities for using this receptor as a therapeutic target.     

Eosinophils and monocytes produce pulmonary and activation-regulated chemokine,which activates cultured monocytes/macrophages

Pulmonary and activation-regulated chemokine (PARC/CCL18) belongs to the family of CC chemokines and shares 61% sequence identity with monocyte inflammatory protein (MIP)-1alpha. Produced by dendritic cells and macrophages primarily in the lung, PARC is known to be chemotactic for T cells. Because PARC's biological function is largely unknown, we screened various leukocyte populations for PARC expression and for response to PARC, with the idea that the cellular source may link PARC to disease states in which it may be involved. Here we report that eosinophils obtained from individuals with mild eosinophilia express PARC as assessed by RT-PCR on eosinophil RNA. The eosinophil preparations were free of monocytes, a known source of PARC, and no RT-PCR product was obtained from neutrophils. Furthermore, PARC protein was detected by ELISA in the supernatants of eosinophils from seven of nine donors and in higher concentration in the supernatants of monocytes on day 1 of culture. Purified recombinant PARC activated human monocytes/macrophages kept in culture for 3-4 days but not freshly isolated monocytes. The threshold dose for Ca(2+) mobilization as determined fluorometrically in indo 1-AM-labeled monocytes was 5 nM; maximal response was reached with approximately 50 nM PARC. PARC was chemotactic for these cultured monocytes and caused actin polymerization determined by FITC-phalloidin binding and fluorescence-activated cell sorting analysis. In contrast, PARC activated neither neutrophils nor eosinophils. Eosinophil production of PARC, its chemotactic effect on monocytes and lymphocytes, and PARC's previously described localization to the lung suggest that this chemokine might play a role in pulmonary leukocyte trafficking.     

Acquisition of antigen-presenting functions by neutrophils isolated from mice with chronic colitis

Active episodes of the inflammatory bowel diseases are associated with the infiltration of large numbers of myeloid cells including neutrophils, monocytes, and macrophages. The objective of this study was to systematically characterize and define the different populations of myeloid cells generated in a mouse model of chronic gut inflammation. Using the T cell transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic cytokines (IL-17 and G-CSF), increased production of neutrophils and monocytes, and infiltration of large numbers of myeloid cells into the mesenteric lymph nodes (MLNs) and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1(+)Ly6C(high)Gr-1(low/neg) cells (monocytes), Mac-1(+)Ly6C(int)Gr-1(+) cells (neutrophils), and Mac-1(+)Ly6C(low/neg)Gr-1(low/neg) leukocytes (macrophages, dendritic cells, and eosinophils). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared with neutrophils obtained from the blood, the MLNs, and the spleen of colitic mice. Furthermore, we found that colonic neutrophils had acquired APC function that enabled these granulocytes to induce proliferation of OVA-specific CD4(+) T cells in an Ag- and MHC class II-dependent manner. Finally, we observed a synergistic increase in proinflammatory cytokine and chemokine production following coculture of T cells with neutrophils in vitro. Taken together, our data suggest that extravasated neutrophils acquire APC function within the inflamed bowel where they may perpetuate chronic gut inflammation by inducing T cell activation and proliferation as well as by enhancing production of proinflammatory mediators.     

Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens

In the present study we evaluated the role of IFN-alpha in the generation of dendritic cells (IFN-DCs) with priming activity on CD8(+) T lymphocytes directed against human tumor Ags. A 3-day treatment of monocytes, obtained as adherent PBMCs from HLA-A*0201(+) healthy donors, with IFN-alpha and GM-CSF led to the differentiation of DCs displaying a semimature phenotype, but promptly inducing CD8(+) T cell responses after one in vitro sensitization with peptides derived from melanoma (gp100(209-217) and MART-1/Melan-A(27-35)) and adenocarcinoma (CEA(605-613)) Ags. However, these features were lost when IFN-DCs were generated from immunosorted CD14(+) monocytes. The ability of adherent PBMCs to differentiate into IFN-DCs expressing higher levels of costimulatory molecules and exerting efficient T cell priming capacity was associated with the presence of contaminating NK cells, which underwent phenotypic and functional activation upon IFN-alpha treatment. NK cell boost appeared to be mediated by both direct and indirect (i.e., mediated by IFN-DCs) mechanisms. Experiments performed to prove the role of contaminating NK cells in DC differentiation showed that IFN-DCs generated in the absence of NK were phenotypically less mature and could not efficiently prime antitumor CD8(+) lymphocytes. Reciprocally, IFN-DCs raised from immunosorted CD14(+) monocytes regained their T cell priming activity when NK cells were added to the culture before IFN-alpha and GM-CSF treatment. Together, our data suggest that the ability of IFN-DCs to efficiently prime anti-tumor CD8(+) T lymphocytes relied mostly on the positive cross-talk occurring between DCs and NK cells upon stimulation with IFN-alpha.     

Tumor necrosis factor-alpha regulates the expression of inducible costimulator receptor ligand on CD34(+) progenitor cells during differentiation into antigen presenting cells

The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34(+) cells in bone marrow. We found that CD34(bright) cells regardless of their myeloid commitment were ICOSL(-), whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34(+) hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor alpha (TNF-alpha) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34(+) cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-alpha and was regulated via NF-kappa B as revealed by use of inhibitors specific for I kappa B alpha phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-alpha stimulated CD34(+) cells was strongly inhibited by ICOSIg fusion proteins or by NF-kappa B inhibition. Thus, TNF-alpha-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34(+) hematopoietic progenitor cells.