Regarding the efficiency of engraftment of huPBLs into SCID/NOD-SCID mice, a wide variety of stimulators of hu- man hematopoiesis have also been tested in efforts to promote the engraftment of lymphocytes into huPBL-SCID/NOD- SCID mice; these stimulators include interleukin 6 (IL-6), IL-4, growth hormone, and chemokines (6, 10, 33, 48). IL-15 was originally isolated from culture supernatants of the simian kidney epithelial cell line CV-1/EBNA (11) and has been shown to have hematopoiesis-promoting effects, including de- velopment and differentiation of natural killer (NK) cells, pro- liferation of T cells, and maturation of B cells (7, 11, 22, 25, 42). A 4-day treatment with IL-15 in serum-free medium alone or synergically with IL-2 enhanced the cytotoxicity of human NK and LAK cells against tumor cells (23, 24). It was also found that IL-15 improved stem cell development in a semi- solid colony assay system (7). Those observations indicate that IL-15 is a strong immune hematopoiesis-promoting cytokine that may be an appropriate candidate for promoting transplan- tation of huPBLs into NOD-SCID mice. In this study, we have found that recombinant human IL-15 (rhIL-15) can be used for reconstitution of human T cells in NOD-SCID mice.
of IL-5 to 0.8 % of control PHA activated cells, and that of IL-13 to 6.2 % of controls (n = 6 for IL-5 and IL-13) (M. Irvine & W. A. Sewell, unpublished observations). How- ever, not all Th2 cytokines are as markedly inhibited by Dex, because IL-4 was only inhibited to 31% of control levels (Table 2). The relative resistance of IL-4 to the inhibitory effects of Dex may explain an unexpected effect of Dex in enhancing the development of Th2 cells ; these findings could be explained by more efficient sup- pression by Dex of IFN- γ than IL-4, leaving sufficient IL-4 to favour differentiation of T cells into Th2 cells.
304 receptor indicates a decrease in AM-sensitivity that appears to distin- 305 guish stimulated T cell phenotype from their unstimulated couterpart. 306 Previous investigations on alternate cell systems have also clearly 307 shown differences in RAMPs and CLR expression depending on the 308 condition cells were exposed to . For example, in calci ﬁ ed VSMC 309 all AM were up-regulated in calci ﬁ ed versus control VSMC , com- 310 pared to the remnant kidneys of rat with mass ablation where the ex- 311 pression of RAMP3 and CLR was lower than that of healthy kidneys 312 , while in an alternative model of renal failure  RAMP2 and 313 CLR were shown to be strongly up-regulated.
A fundamental property of human T cells is to provide lifelong immunity against pathogens lasting several decades (1). The complement of T cells within an individual is heterogeneous and includes naive T cells that develop from thymic precursors, short- lived effector cells, and memory T cells derived from naive T cells following antigen exposure. Maintenance of each of these com- ponents for several decades of life is essential to host defense: memory T cells for defense against previously encountered anti- gens and naive T cells for responses to new antigens. T cell mem- ory is mediated by coordinated action of distinct subsets. Initial pioneering studies classified human memory T cells into effector memory T cells (Tem cells) and central memory T cells (Tcm cells) based on rapid effector function and the expression of lymphoid homing receptors, respectively (2). More recent studies have identified and characterized a subset of memory T cells resident in nonlymphoid tissues (NLT) that mediate regional immune sur- veillance against pathogens (3–6). Tissue-resident memory T cells (Trm cells) in NLTs outnumber memory CD8 + T cells in lymphoid
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The transcription factor kruppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell–mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activ- ity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodula- tory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/ prenylation–dependent pathway. Statins also diminished T cell proliferation and IFN-γ expression. shRNA blockade of KLF2 expression in human T cells increased IFN-γ expression and prevented statin-induced IFN-γ reduction. In a mouse model of myocarditis induced by heart antigen–specific CD8 + T cells, both statin treat-
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Human T cells were enriched from peripheral blood using the T-cell isolation kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer ’ s protocol. Enriched T cells were cultured in RPMI-1640/ 10% heat-inactivated FBS/pen/strep overnight prior to use. On Day -1, 10,000 MSCs or SB623 cells were plated per well of 96-well U-bottom plates. On Day 0 of the culture assay, 100,000 enriched T cells were transferred to each well with a pre-established MSC or SB623 cell monolayer. As an internal control, T cell cultures were maintained in the absence of MSCs or SB623 cells. On Day 7, a sub-optimal dose of 25 ng/ml of phorbol 12- myristate 13-acetate (PMA)/0.5 μM Ionomycin (both from Sigma-Aldrich) was added in the presence of Bre- feldinA (eBioscience, 1:1000) for 6 hours prior to har- vest for intracellular detection of interleukin-10 (IL-10) and interferon gamma (IFN-g). For IL-17 producing TH17 cells, T cells were co-cultured with SB623 cells or MSCs in the presence of IL-23, or in the presence of IL- 23 alone. After sub-optimal activation with PMA/Iono- mycin in the presence of BrefeldinA, the cells were stained with fluorochrome-conjugated antibody against IL-17A (eBioscience) and analyzed by flow cytometry.
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The data presented in this report raise several interesting ques- tions. Presently, we do not fully understand the specific signaling events downstream of mTOR that control EC alloimmunoge- nicity. In the cell, mTOR exists in two distinct signaling com- plexes, known as mTORC1 and mTORC2, which have differing upstream inputs and downstream outputs (77). Our data show that disruption of mTORC1 signaling (by raptor knockdown) caused changes in EC surface molecules similar to those induced by rapamycin, and such ECs poorly stimulated allogeneic T cells. In addition, we have found that inhibition of both mTORC1 and mTORC2 signaling reduces the ability of ECs to secrete IL-6 in response to TNF. We are currently investigating whether any of the known mTORC1 and mTORC2 targets are involved in mediating these effects. Additionally, because blockade of PD-1 ligands only partially reduced the ability of rapa-ECs to preferen- tially activate Tregs, it is likely that there are other mechanisms behind this observation. Although ligation of GITR on Tregs by GITR ligand was initially thought to abrogate their suppressive activity (78), recent studies suggest that GITR ligand may act to maintain and expand Tregs (79–81). Additionally, the interaction between OX40 and OX40 ligand has been shown to be important in Treg cell development in the gut (82), and an OX40 agonist enhanced Treg proliferation (83). Given that both GITR ligand and OX40 ligand are highly expressed on rapa-ECs, we are explor- ing whether either of these molecules contributes to the ability of rapa-ECs to activate Tregs.
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Immunoselection of CD34-positive cells tranduced with a thy-1.2-bearing vector and reconstitution of irradiated Thy/Liv implants of SCID-hu mice. We undertook a different strategy that enabled selection by FACS to enrich transduced and transgene-expressing cell populations. The luc gene was re- placed with the murine thy-1.2 gene, which we previously showed to be effective for monitoring of gene expression in individual cells by flow cytometry (32). One additional step was introduced into the reconstitution protocol whereby, following transduction of CD34-positive cells, the cells were purified by FACS on the basis of Thy-1.2 positivity and CD34 surface expression (Fig. 2A). In this manner, Thy/Liv implants of SCID-hu mice should be reconstituted with CD34-positive cells that are highly enriched for the transgene. At 4 to 6 weeks after introduction of CD34-positive cells into Thy/Liv implants, the levels of vector DNA ranged from 0.1 to 8.5 copies of thy-1.2 per donor-derived thymocyte (Table 1 and Fig. 2B). We confirmed Thy-1.2 expression in human cells by demonstrating a distinct population of cells positive for both Thy-1.2 and CD45, a marker for human lymphocytes (Fig. 2A). When nor- malized to the percentage of donor cells, calculated by PCR for human Y chromosome versus b -globin sequences, the propor- tion of cells expressing Thy-1.2 ranged from 4.4 to 71% (Table 1). In other experiments, we compared reconstitution and ex- pression of an unfractionated CD34-positive cell population with the same cells sorted into Thy-1.2-positive and Thy-1.2- negative subpopulations. As expected, we detected little or no thy-1.2 expression in thymocytes derived from reconstitution of
We thank J.J. Fournié and Y. Poquet for providing mycobacterial antigens; H. Fleury, I. Pellegrin, and M.-S. Goni-Urriza for viro- logical tests and DNA sequencing; E. Trannoy and C. Caillet for varicella-zoster virus–infected fibroblast extracts; C. Charnay, I. Gonzalez, and T.-M. Jourdier for free CMV preparation; and the nursing staff of the Service de Transplantation Rénale for excel- lent collaboration. Special thanks are also addressed to all the patients who, by their consent, allowed this study to be per- formed. This work was supported by the Ligue Nationale et Régionale contre le Cancer, the Association pour la Recherche contre le Cancer, the Conseil Régional d’Aquitaine, and the Délé- gation à la Recherche Clinique d’Aquitaine, and by institutional grants from the Institut National de la Santé et de la Recherche Médicale. J. Déchanet is the recipient of grants from the Associa- tion pour la Recherche contre le Cancer, the Fondation pour la Recherche Médicale, and the Ligue Nationale contre le Cancer.
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induced or regulated by normal or abnormal T-cell function. For this purpose we have investigated the cellular requirements for immune responses in vitro to trinitrophenyl- conjugated peripheral blood mononuclear cells. The responding cell was characterized as a T cell on the basis of rosetting with sheep erythrocytes. T-cell recognition of hapten in proliferative responses depended upon presentation of antigen in an appropriate stimulator- cell context. Neither autologous hapten-modified erythrocytes nor T cells restimulated
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patients significantly reduced CD148 activity in CD4 + T cells. SF is an oxidizing environment that also contains high concentrations of cytokines such as TNF, therefore we investigated the effects of these agents on CD148 activity. As expected, hydrogen peroxide (as an oxidising agent) inhibited CD148 activity, presumably by oxidizing the essential Cys1240 in the catalytic site of the mole- cule . However, we also found that TNF increased CD148 activity in CD4 + T cells. Thus, despite an increase in CD148 in isolated synovial T cells (Figure 6B, C), and high levels of TNF that, in isolation, would increase CD148 activity, we conclude that SF contains additional factors that counteract this to result in an overall inhibitory effect on CD148 activity. It is possible that SF contains other cytokines that suppress CD148 suppression, or other factors that maintain an oxidising environment. Since CD148 positively regulates T cell function we therefore expect that overall suppression of CD148 PTP activity in rheumatoid joints would sup- press T cell activation. This may contribute to the observed lack of responsiveness of synovial T cells [49,50]. Whilst immunocytochemistry appears to show macrophage expression of CD148 in tissue joints (Figure 3), macrophages isolated from SF show a reduced expression of CD148 (Figure 6D). This is additional evi- dence that SF contains factors that influence CD148 expression in macrophages as well as in T cells as dis- cussed previously.
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Long noncoding RNAs (lncRNAs) are regulatory RNA molecules that are involved in various biological processes. In the immune system, the lncRNAs play important roles in development, differentiation, survival, cell fate determination, proliferation and activation of immune cells. Lymphocytes are the main players of the adaptive immunity and CD3+ T cells acts as a master regulator for the immune responses. These cells following activation by antigens and co-stimulatory signals are differentiated into various effector T cell subsets, including CD4 and CD8 T cells. These heterogeneous populations can be distinguished based on molecular surface markers and subsets of these markers can be used to denote various stages of T lymphocyte differentiation, notwithstanding the CD3 + T cells phenotypes are markedly influenced by
The present results imply that Pontiac fever is due to infec- tion rather than exposure to dead bacteria or bacterial toxins. Several pieces of evidence support this line. In guinea pigs, which are highly susceptible to L. pneumophila, aerogenic ex- posure to dead organisms of the species caused no signs of disease (1). Moreover, strains of L. pneumophila causing Pon- tiac fever did not differ from those causing Legionnaires⬘ dis- ease (12), and pneumonic and nonpneumonic forms of legion- ellosis have been described in patients with a common-source exposure (15). Finally, urine samples from patients with Pon- tiac fever have shown the presence of Legionella antigen (13). The function of V␥9V␦2 T cells in host response to intra- cellular infections remains elusive. In the samples obtained here 14 to 16 days after the onset of legionellosis, a majority of the cells produced TNF-␣ and IFN-␥ after being exposed for 4 h to phorbol myristate acetate. One month later, the cytokine expression capability of the V␥9V␦2 T cells was decreased, TNF-␣ expression in particular. These data may reflect an initial cytokine response important in the activation of bacte- ricidal macrophages, followed by down regulation or modula- tion of the inflammatory response. A similar change has been observed in tularemia (24). In vitro studies suggest that ␥␦ T cells are subjected to a fine-tuned combination of stimulation and inhibition by their interaction with different membrane receptors (26, 31). One of the receptors involved in down regulation is CD94, a type C lectin. In vitro data suggest that CD94 expression is increased on V␥9V␦2 T cells as a result of antigen stimulation (3). We found that its expression increased at 5 to 7 weeks after onset of disease, compared with 4 to 6 days after onset, i.e., concomitantly with the decrease in cytokine expression. In fact, experimental work on intracellular infec- tions has suggested a dual role for ␥␦ T cells. After contribut- ing to an early inflammatory response by cytokine expression and activation of macrophages, ␥␦ T cells are suggested to participate in termination of the immune response (4).
Immunological tolerance has developed to allow organisms to differentiate between self and nonself. T cell tolerance is achieved primarily through the elimi- nation of potentially autoreactive clones in the thymus through a mechanism known as negative selection (1, 2). The clones that escape central tolerance in the thy- mus are rendered anergic in the periphery upon encounter with an antigen under suboptimal condi- tions (2). In spite of these two mechanisms for main- taining tolerance, autoreactive T cells can be readily detected in normal individuals. Recently, a regulatory T (T R ) cell population has been identified and shown
Background: The FOXP3 gene is the master regulator for T regulatory cells and is under tight DNA methylation control at the Treg specific demethylated region (TSDR) in its first intron. This said, methylation of its promoter region, the significance of which is unknown, has also been associated with various immune-related disease states such as asthma, food allergy, auto-immunity and cancer. Here, we used induced T regulatory cells (iTreg) as a target cell population to identify candidate hypomethylated CpG sites in the FOXP3 gene promoter to design a DNA methyla- tion quantitative assay for this region.
Next, we examined the effects of EGFR inhibitors in transformed-human esophageal epithelial cells. Here, we used two cell lines, T-Epi and T-Mes, which are estab- lished transformed-human esophageal epithelial cells [19, 20]. As shown in Fig. 2a, T-Epi cells were round as seen in epithelial cells and T-Mes cells had a spindle-like morphology as seen in mesenchymal cells. To characterize these cells as either epithelial or mesenchymal pheno- types, we examined the expression levels of E-cadherin (epithelial marker) and vimentin (mesenchymal marker). Consistent with their morphology, T-Epi cells showed high expression of E-cadherin and low expression of vimentin, whereas T-Mes cells showed the reverse (Fig. 2b). Accordingly, T-Epi cells could be catego- rized as epithelial-like esophageal cells, and T-Mes cells as mesenchymal-like esophageal cells. When these cells were treated with erlotinib or cetuximab for 72 h, cell-cell contact was observed in T-Epi cells but not T-Mes cells (Fig. 2a). This result indicates that the effects of EGFR inhibition on epithelial- and mesenchymal-like esophageal cells might be different.
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(p<0.001) increases in proliferation were observed when eosinophils had been pulsed by pre-incubation with the allergens HDM (Figure 1A) or TG (Figure 1B) at final concentrations of 1000, 1500 or 2500 IU/ml, compared with medium alone. The strongest responses were induced by HDM or TG at 2500 IU, and this concentration of the allergens was therefore used in all subsequent experiments. Eosinophils pulsed with the control antigen PPD also elicited significant proliferation at the standard concentration of 5 µg/ml. To confirm that responses to each stimulus required the presence of both eosinophils and Th cells, proliferation was compared in cultures containing each cell type alone or together, with or without antigen pulsing (Figure 2). Proliferative responses were significant only when both antigen-pulsed eosinophils and Th cells were added, and, strikingly, these responses were similar in size to those seen when unfractionated PBMC were stimulated with the respective antigen. The ability of Th cells to respond to antigen-pulsed eosinophils was MHC class II dependent, since incubating HDM-pulsed eosinophils with blocking mAb specific for HLA-DR/DP/DQ (26) significantly (P<0.001) reduced T cell proliferation by 77% (Supplemental
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Vγ9Vδ2 T cells are the predominant subset in blood. Their ligand specificities are well defined, and these include pAg, which are metabolites of the pathway of isoprenoid biosynthesis, a pathway essential for cell survival employed by most eukaryotic cells and many pathogens. There are two distinct pathways, the mevalonate pathway and the MEP (Fig. 1.3) pathway. The MEP pathway, which is used by many Gram-positive bacteria, mycobacteria, protozoa and parasites (Morita, et al., 2007), results in production of HMB-PP, the most potent ligand known to uniquely activate Vγ9Vδ2 T cells and which is not produced by human cells. Thus, since it is not found endogenously, HMB-PP specifically plays a role in recognition of foreign pathogens by Vγ9Vδ2 T cells. HMB-PP can be converted to IPP, another Vγ9Vδ2 T cell antigen of much lower potency than its precursor. IPP can also be produced via the mevalonate pathway used by most eukaryotes, and thus can serve as a self-ligand in human cells. However, the levels normally produced in human cells are not sufficient to activate Vγ9Vδ2 T cells. Cell transformation or dysregulation can result in overproduction of rate-limiting enzymes in this pathway, such as HMG-CoA reductase, or inhibition of the enzyme responsible for breaking down IPP, both of which result in increased IPP levels, and ultimately recognition by Vγ9Vδ2 T cells (Thedrez, et al., 2007, Gober, et al., 2003, Morita, et al., 2007, Idrees, et al., 2013, Riganti, et al., 2012). Some bacteria such as Listeria monocytogens and Streptomyces use both pathways (Heuston, et al., 2012).
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For full activation of the T cells, costimulatory sig- nals in addition to TCR cross-linking are required. Costimulation via CD28 on the T cells is a well-estab- lished dominant pathway (19–23). However, recent studies suggest that additional costimulatory path- ways also exist and that they may have roles at differ- ent stages of T-cell activation or on different subsets of T cells or contribute to the development of differ- ent effector functions. For example, signals through inducible costimulator (ICOS) have effects on activat- ed T cells only, and this pathway enhances IL-10 but not IL-2 production (24); costimulation via signaling lymphocyte activation molecule (SLAM/CD150) enhances IFN-γ production and directs memory T cells toward Th0/Th1 responses (25); crosslink of 4-1BB preferentially promotes CD8 cell proliferation (26); lig- ation of OX40 increases Th2 responses (27). In the past 2 years, several laboratories have demonstrated that a TNFR family member, TR2, is constitutively Figure 4
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To evaluate the impact of different sera on CAR T cell function during the cell expansion phase, we first trans- duced OKT3/CD28 stimulated T cells cultured in medium supplemented with 10% FBS with a retroviral vector encoding either a second generation prostate stem cell antigen (PSCA)-targeted CAR or a CD19- specific CAR, each containing the endodomains CD28 and CD3z (P28z and 1928z, respectively). Three days after transduction (transduction efficiency: P28z - 87.8 ± 1.5%; n = 14, 1928z - 90.9 ± 1.5%; n = 9. Additional file 2: Figure S1a), the T cell cultures were split into three con- ditions and cultured in medium supplemented with ei- ther 10% FBS, 10% ABS or 10% HPL (Fig. 1a) and further expanded for an additional week in the presence of IL2. We first evaluated the impact of differential sera exposure on T cell expansion and found that there was no statistical difference between the 3 conditions (Fig. 1b). However, when we examined the phenotypic profile of the expanded cells we found that those cultured in HPL showed a trend towards increased CD4 + T cell numbers (Additional file 2: Figure S1b) as well as a sig- nificantly higher percentage of CCR7 + cells representing naïve-like (T N ) and central memory T cells (T CM ) in
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