Search

The bacterial molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) has critical roles in both interbacterial communication and interkingdom signaling. The ability of C12 to downregulate production of the key proinflammatory cytokine TNF-α in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that, in contrast to TNF-α suppression, C12 amplifies production of the major anti-inflammatory cytokine IL-10 in LPS-stimulated murine RAW264.7 macrophages, as well as peritoneal macrophages. Furthermore, C12 increased IL-10 mRNA levels and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NF-κB DNA-binding levels and prolonged p38 MAPK phosphorylation in RAW264.7 macrophages, suggesting that increased transcriptional activity of NF-κB and/or p38-activated transcription factors serves to upregulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria downregulate immune responses to flourish and establish a chronic infection.

The processes that regulate T cell memory generation are important for therapeutic design and the immune response to disease. However, what allows a subset of effector T cells to survive the contraction period to become memory cells is incompletely understood. The Bcl-2 family is critical for T cell survival, and Bcl-2 has been proposed to be important for the survival of memory cells. However, previous studies have relied on double-knockout models, potentially skewing the role of Bcl-2, and the use of Bcl-2 as a marker in adoptive transfer experiments, a method required to confirm the memory potential of cell subsets, has not been possible because of the intracellular localization of the protein. In this study, we present a novel Bcl-2 reporter mouse model and, to our knowledge, show for the first time that a distinct subset of effector T cells, and also a subset within the CD127(hi)KLRG1(lo) memory precursor effector cell population, retains high Bcl-2 expression at the peak of the CD8(+) T cell response to Listeria monocytogenes. Furthermore, we show that Bcl-2 correlates with memory potential in adoptive transfer experiments using both total responding CD8(+) T cells and memory precursor effector cells. These results show that even within the memory precursor effector cell population, Bcl-2 confers a survival advantage in a subset of effector CD8(+) T cells that allows differentiation into memory cells and cement Bcl-2 as a critical factor for T cell memory.

Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.

Upon activation, naive CD4(+) T cells differentiate into effector Th cell subsets. The stability and plasticity of effector Th cells have not been well understood. In this study we used an IL-17F-red fluorescent protein reporter mouse to analyze the plasticity of Th17 cells in vitro and in vivo. We found that in vitro generated Th17 cells poorly maintained their differentiation program in vitro and could be reprogrammed into other T cell lineages. Moreover, upon transfer into lymphopenic hosts, Th17 cells rapidly lost their IL-17 expression and were converted into Th1 cells independently of IL-7 signaling. However, Th17 cells maintained their phenotypes well in normal animals, even in the absence of Ag and inflammation. These results, although suggesting the plasticity of Th17 cells, also indicate active maintenance of their program in vivo.

Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26(eYfp) reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.

IL-12p40 is induced in macrophages and dendritic cells (DC) after activation by microbial TLR ligands and cytokines and constitutes a component of IL-12 and IL-23. In an effort to understand the location and kinetics of these cytokines during the course of an immune response, we generated knockin (gene-targeted) mice that express the p40 gene linked via a viral internal ribosome entry site element with fluorescent reporters, eYFP or eGFP. Macrophages and DC from these mice faithfully reported biallelic p40 induction using the fluorescent marker. s.c. inoculation with Listeria monocytogenes or LPS led to a rapid, but transient, accumulation of p40-expressing DC in draining lymph nodes, which could be blocked by the addition of pertussis toxin. In situ analysis also revealed the accumulation of IL-12p40 protein around high endothelial venules located in close proximity to p40-expressing DC. Consistent with the in vivo findings, in vitro-activated DC that expressed p40 migrated to draining lymph nodes and promoted Th1 differentiation more efficiently than DC that did not express p40. Accordingly, these mice provide a valuable tool for tracking critical functions of DC in vivo and should bestow a useful reagent for exploring the effector biology of these cells in models of infectious disease, cancer immunity, and vaccine development.

The expression of IFN-gamma is a hallmark of Th1 cells and CD8(+) effector T cells and is the signature cytokine of type 1 responses. However, it is not known whether T cells are homogeneous in their capacity to produce IFN-gamma, whether this potential varies between tissues, and how it relates to the production of other effector molecules. In the present study we used bicistronic IFN-gamma-enhanced yellow fluorescent protein (IFN-gamma-eYFP) reporter mice (Yeti) and MHC class I tetramers to directly quantify IFN-gamma expression at the single cell level. The eYFP fluorescence of Th1 cells and CD8(+) effector T cells was broadly heterogeneous even before cell division and correlated with both the abundance of IFN-gamma transcripts and the secretion of IFN-gamma upon stimulation. CD4(+) and CD8(+) T cells of influenza-infected mice revealed a similarly heterogeneous IFN-gamma expression, and eYFP(high) cells were only found in the infected lung. Ag-specific T cells were in all examined tissues eYFP(+), but also heterogeneous in their reporter fluorescence, and eYFP(high) cells were also restricted to the infected lung. A similar heterogeneity was observed in Toxoplasma gondii-infected animals, but eYFP(high) cells were restricted to different tissues. Highly eYFP fluorescent cells produced elevated levels of proinflammatory cytokines and chemokines in addition to IFN-gamma, suggesting their coregulated expression as a functional unit in highly differentiated effector T cells.

Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.

NF-kappa B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-kappa B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-kappa B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-kappa B cis elements (cis-NF-kappa B(EGFP)). A gene-targeting approach was used to insert a single copy of a NF-kappa B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-kappa B(EGFP) mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-alpha that was blocked by the NF-kappa B inhibitors BAY 11-7082 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-kappa B(EGFP) promoter. Adenoviral delivery of NF-kappa B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-kappa B(EGFP) mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-kappa B(EGFP) mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-kappa B during normal and diseased states.

To evaluate the anti-HSV-1 mechanisms of murine IFN-beta in ocular infection, mice were transduced with an adenoviral vector expressing murine IFN-beta (Ad:IFN-beta). Ocular transduction with Ad:IFN-beta resulted in enhanced survival following infection with HSV-1. The protective effect was associated with a reduction in 1) viral titer, 2) viral gene expression, 3) IFN-gamma levels, and 4) the percentage of CD8(+) T lymphocyte and NK cell infiltration in infected tissue. Expression of IFN-beta resulted in an elevation of the IFN-induced antiviral gene 2',5'-oligoadenylate synthetase (OAS1a) but not dsRNA-dependent protein kinase R (PKR) in the cornea and trigeminal ganglion (TG). Mice deficient in the downstream effector molecule of the OAS pathway, RNase L, were no more sensitive to ocular HSV-1 compared with wild-type controls in the TG based on measurements of viral titer. However, the efficacy of Ad:IFN-beta was transiently lost in the eyes of RNase L mice. By comparison, PKR-deficient mice were more susceptible to ocular HSV-1 infection, and the antiviral efficacy following transduction with Ad:IFN-beta was significantly diminished in the eye and TG. These results suggest that PKR is central in controlling ocular HSV-1 infection in the absence of exogenous IFN, whereas the OAS pathway appears to respond to exogenous IFN, contributing to the establishment of an antiviral environment in a tissue-restricted manner.

A transgene with 8.4-kb of regulatory sequence from the murine IL-2 gene drives consistent expression of a green fluorescent protein (GFP) reporter gene in all cell types that normally express IL-2. However, quantitative analysis of this expression shows that different T cell subsets within the same mouse show divergent abilities to express the transgene as compared with endogenous IL-2 genes. TCR gamma delta cells, as well as alpha beta TCR-NKT cells, exhibit higher in vivo transgene expression levels than TCR alpha beta cells. This deviates from patterns of normal IL-2 expression and from expression of an IL-2-GFP knock-in. Peripheral TCR gamma delta cells accumulate GFP RNA faster than endogenous IL-2 RNA upon stimulation, whereas TCR alpha beta cells express more IL-2 than GFP RNA. In TCR gamma delta cells, IL-2-producing cells are a subset of the GFP-expressing cells, whereas in TCR alpha beta cells, endogenous IL-2 is more likely to be expressed without GFP. These results are seen in multiple independent transgenic lines and thus reflect functional properties of the transgene sequences, rather than copy number or integration site effects. The high ratio of GFP: endogenous IL-2 gene expression in transgenic TCR gamma delta cells may be explained by subset-specific IL-2 gene regulatory elements mapping outside of the 8.4-kb transgene regulatory sequence, as well as accelerated kinetics of endogenous IL-2 RNA degradation in TCR gamma delta cells. The high levels and percentages of transgene expression in thymic and splenic TCR gamma delta and NKT cells, as well as skin TCR gamma delta-dendritic epidermal T cells, indicate that the IL-2-GFP-transgenic mice may provide valuable tracers for detecting developmental and activation events in these lineages.

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4(+) T cells into CD4(+) Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP(+) cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP(+) cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4(+) T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.

The MHC class I-related receptor, FcRn, plays a central role in regulating the serum levels of IgG. FcRn is expressed in endothelial cells, suggesting that these cells may be involved in maintaining IgG levels. We have used live cell imaging of FcRn-green fluorescent protein transfected human endothelial cells to analyze the intracellular events that control IgG homeostasis. We show that segregation of FcRn-IgG complexes from unbound IgG occurs in the sorting endosome. FcRn or FcRn-IgG complexes are gradually depleted from sorting endosomes to ultimately generate multivesicular bodies whose contents are destined for lysosomal degradation. In addition, the pathways taken by FcRn and the transferrin receptor overlap, despite distinct mechanisms of ligand uptake. The studies provide a dynamic view of the trafficking of FcRn and its ligand and have relevance to understanding how FcRn functions to maintain IgG homeostasis.

Tissue repair often occurs in organs damaged by an inflammatory response. Inflammatory stimuli induce a rapid and massive release of inflammatory cells including neutrophils from the bone marrow. Recently, many studies suggested that bone marrow cells have the potential to differentiate into a variety of cell types. However, whether inflammatory stimuli induce release of bone marrow-derived progenitor cells (BMPCs), or how much impact the suppression of BMPCs has on the injured organ is not clear. Here we show that LPS, a component of Gram-negative bacterial cell walls, in the lung airways, induces a rapid mobilization of BMPCs into the circulation in mice. BMPCs accumulate within the inflammatory site and differentiate to become endothelial and epithelial cells. Moreover, the suppression of BMPCs by sublethal irradiation before intrapulmonary LPS leads to disruption of tissue structure and emphysema-like changes. Reconstitution of the bone marrow prevents these changes. These data suggest that BMPCs are important and required for lung repair after LPS-induced lung injury.

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.

Small intestinal cryptopatches (CP) are the major anatomic site for extrathymic differentiation by precursors destined to become intestinal intraepithelial T lymphocytes (IEL). We found that mice deficient in CCR6 exhibited a 2.7-fold increase in the number of alphabeta TCR IEL, but little or no expansion of gammadelta TCR IEL. Among the alphabeta TCR IEL subsets, the CD4- CD8alphaalpha+ and CD4+ CD8alphaalpha+ subsets were preferentially expanded in CCR6 null mice. Because some CD8alphaalpha+ IEL can arise through extrathymic differentiation in CP, we investigated CCR6 expression by T lymphocyte precursors undergoing extrathymic differentiation in intestinal CP. In sections of CP, 50-60% of c-kit+ precursors were CCR6+. CD11c(+) cells concentrated at the periphery of CP did not express CCR6. A subset of c-kit+, Lin- cells in lamina propria suspensions was CCR6+, but CCR6 was absent from c-kit+ precursors in bone marrow. CCR6 was absent from the vast majority of mature IEL. CCR6 is present on lymphocyte precursors in cryptopatches, expressed transiently during extrathymic IEL development, and is required for homeostatic regulation of intestinal IEL.

CD4(+)Vbeta5(+) T cells enter one of two tolerance pathways after recognizing a peripherally expressed superantigen encoded by an endogenous retrovirus. One pathway leads to deletion, while the other, termed TCR revision, results in cellular rescue upon expression of an alternate TCR that no longer recognizes the tolerogen. TCR revision requires the rearrangement of novel TCR beta-chain genes and depends on recombinase-activating gene (RAG) expression in peripheral T cells. In line with recent findings that RAG(+) splenic B cells are immature cells that have maintained RAG expression, it has been hypothesized that TCR revision is limited to recent thymic emigrants that have maintained RAG expression and TCR loci in a recombination-permissive configuration. Using mice in which the expression of green fluorescent protein is driven by the RAG2 promoter, we now show that in vitro stimulation can drive reporter expression in noncycling, mature, peripheral CD4(+) T cells. In addition, thymectomized Vbeta5 transgenic RAG reporter mice are used to demonstrate that TCR revision can target peripheral T cells up to 2 mo after thymectomy. Both sets of experiments strongly suggest that reinduction of RAG genes triggers TCR revision. Approximately 3% of CD4(+)Vbeta5(+) T cells in thymectomized Vbeta5 transgenic reporter mice have undergone TCR revision within the previous 4-5 days. TCR revision can also occur in Vbeta5(+) T cells from nontransgenic mice, illustrating the relevance of this novel tolerance mechanism in unmanipulated animals.

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8alpha specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.

Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-kappaB-dependent mechanisms, which can be blocked by double-stranded "decoy" oligodeoxyribonucleotides (ODNs) containing binding sites for NF-kappaB. Herein, we describe the combined use of NF-kappaB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-kappaB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.

Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent protein fusion construct (VEcadGFP) that mimics the native molecule, we visualized alterations in endothelial junctional structure in real time during transmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant transmigration occurring exclusively at the cell borders (paracellularly). Surprisingly, transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4-6 microm in size were formed after a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contributing to a junction. Gaps rapidly resealed within 5 min after leukocyte transmigration. Migrating leukocytes appeared to push aside VEcadGFP in the plane of the junction, and this displaced material subsequently diffused back to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time during transmigration.

Intravital microscopy allows detailed analysis of leukocyte trafficking in vivo, but fails to identify the nature of leukocytes investigated. Here, we describe the development of a CD2-enhanced green fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte trafficking during inflammation in vivo. A CD2-EGFP plasmid construct including the CD2 promoter, the EGFP transgene, and the CD2 locus control region was injected into B6CBA/F1 pronuclei. EGFP+ offspring were backcrossed into C57BL/6 mice for six generations. Flow cytometry demonstrated that all peripheral blood EGFP+ cells were positive for CD2 and negative for the granulocyte Ag Ly 6-G (GR-1). EGFP(high) cells stained positive for CD2, CD3, CD8, TCR beta-chain, and NK1.1 but did not express the B cell and monocyte markers CD45RA, CD19, and CD11b. In vitro stimulation assays revealed no difference in lymphocyte proliferation and IL-2 secretion between EGFP+ and EGFP- mice. Intravital microscopy of untreated or TNF-alpha-treated cremaster muscle venules showed EGFP+ cells in vivo, but these cells did not roll or adhere to the vessel wall. In cremaster muscle venules treated with both TNF-alpha and IFN-gamma, EGFP(high) cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 microm/s) than that of neutrophils (10 microm/s). Blocking alpha4 integrin with a mAb increased rolling velocity to 24 microm/s. These findings show that CD8+ T cells roll in TNF-alpha/IFN-gamma-pretreated vessels in vivo via an alpha4 integrin-dependent pathway.

Dendritic cells (DCs) play a pivotal role in the development of anti-viral CD8(+) CTL responses. This is straightforward if they are directly infected with virus, but is less clear in response to viruses that cannot productively infect DCS: Human CMV (HCMV) shows strain-specific cell tropism: fibroblast (Fb)-adapted laboratory strains (AD169) and recent clinical isolates do not infect DCs, whereas endothelial cell-adapted strains (TB40/E) result in productive lytic DC infection. However, we show here that uninfected DCs induce CD8(+) T cell cytotoxicity and IFN-gamma production against HCMV pp65 and immediate early 1 Ags following in vitro coculture with HCMV-AD169-infected Fbs, regardless of the HLA type of these FBS: CD8(+) T cell stimulation was inhibited by pretreatment of DCs with cytochalasin B or brefeldin A, indicating a phagosome/endosome to cytosol pathway. HCMV-infected Fbs were not apoptotic as measured by annexin V binding, and induction of apoptosis of infected Fbs in vitro did not augment CTL induction by DCs, suggesting a mechanism other than apoptosis in the initiation of cross-presentation. Furthermore, HCMV-infected Fbs provided a maturation signal for immature DCs during coculture, as evidenced by increased CD83 and HLA class II expression. Cross-presentation of HCMV Ags by host DCs enables these professional APCs to bypass some of the evasion mechanisms HCMV has developed to avoid T cell recognition. It may also serve to explain the presence of immediate early 1 Ag-specific CTLs in the face of pp65-induced inhibition of Ag presentation at the level of the infected cell.

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.

Although the promoter/enhancer of the IL-2 gene mediates inducible reporter gene expression in vitro, it cannot drive consistent expression in transgenic mice. The location and existence of any regulatory elements that could open the IL-2 locus in vivo have remained unknown, preventing analysis of IL-2 regulation in developmental contexts. In this study, we report the identification of such a regulatory region, marked by novel DNase-hypersensitive sites upstream of the murine IL-2 promoter in unstimulated and stimulated T cells. Inclusion of most of these sites in an 8.4-kb IL-2 promoter green fluorescent protein transgene gives locus control region-like activity. Expression is efficient, tissue specific, and position independent. This transgene is expressed not only in peripheral T cells, but also in immature thymocytes and thymocytes undergoing positive selection, in agreement with endogenous IL-2 expression. In contrast, a 2-kb promoter green fluorescent protein transgene, lacking the new hypersensitive sites, is expressed in only a few founder lines, and expression is dysregulated in CD8(+) cells. Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells.

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 10(4). Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.

TGF-beta is an important immunomodulatory cytokine that can inhibit differentiation of effector T cells. In this report, we address the molecular mechanisms through which TGF-beta inhibits differentiation of CD4(+) cells into Th type 2 cells. We demonstrate that TGF-beta inhibits GATA-3 expression in developing Th cells. We also show that inhibition of GATA-3 expression by TGF-beta is a major mechanism of inhibition of Th2 differentiation by TGF-beta as ectopic expression of GATA-3 in developing T cells overcomes the ability of TGF-beta to inhibit Th2 differentiation. TGF-beta likely inhibits GATA-3 expression at the transcriptional level and does so without interfering with IL-4 signaling.

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-gamma, but they remained CD1a- and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.

Chemokines play necessary and important roles in regulating the trafficking of lymphocytes to intra- or interlymphoid tissues as well as to sites of inflammation. The complex migratory patterns of lymphoid lineage cells is governed by subset-specific expression of chemokine receptors and their access to specific ligands. Several chemokine receptors and chemokine receptor-like orphan receptors also serve, in conjunction with CD4, as coreceptors for infection by human and simian immunodeficiency viruses (HIV and SIV). Here we show that the expression pattern of Bonzo/STRL33, an orphan SIV/HIV coreceptor, is highly restricted to the memory subset of T cells and is up-regulated upon stimulation of these cells with IL-2 or IL-15. Both the pattern and the regulation of Bonzo expression closely paralleled that of CC family chemokine receptors CCR5 or CCR6 and inversely correlated with CXCR4 expression. However, in striking contrast to CCR5, Bonzo expression was not down-modulated by PMA or mitogen stimulation of T cells. Targeted replacement of the Bonzo gene with a gene encoding green fluorescent protein in mice revealed that the expression and cytokine regulation of mouse Bonzo are comparable to those of its human counterpart. The similar expression and regulation patterns of Bonzo and the HIV coreceptor CCR5 may have implications for understanding the role of HIV/SIV receptors in viral evolution and pathogenesis.

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.