International Immunology Advance Access originally published online on September 18, 2007
International Immunology 2007 19(10):1183-1189; doi:10.1093/intimm/dxm089
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Cellular FLIP long isoform transgenic mice overcome inherent Th2-biased immune responses to efficiently resolve Leishmania major infection
1 Laboratory of Molecular Genetics
2 Laboratory of Cellular Immunology, Hellenic Pasteur Institute, 127 Vasilissis Sophias Avenue, 115 21 Athens, Greece
Correspondence to: L. Probert; Email: lesley{at}pasteur.gr
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Abstract |
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c-FLIPL expression in T cells is required for mounting effective T cell responses and can also be critical for effector T cell differentiation, as has recently been shown by a number of in vivo studies in conditional knockout and transgenic mouse systems. Available data supports therefore a novel immunomodulatory role of this anti-apoptotic protein besides its traditionally proposed function in homeostatic maintenance of T cell populations. In this study, the responses to infection with Leishmania major of mice over-expressing FLIPL specifically in the T cell compartment (TgFLIPL) are assessed. Although previous studies have shown that FLIPL drives T cells towards a Th2 differentiation programme in various autoimmune and allergic paradigms, in this study, we show that TgFLIPL are able to overcome this Th2 bias in a dermal L. major infection model to mount a robust Th1 response to pathogen and effectively clear infection. Our results suggest that vaccination protocols designed to enhance FLIPL expression in T cells may be useful for the treatment of autoimmune diseases like multiple sclerosis, without necessarily compromising immune responses towards infectious agents.
Keywords: apoptosis, parasite infection, Th cell differentiation, vaccines
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Introduction |
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CD4+ T helper lymphocytes are responsible for orchestrating appropriate immune responses to a diverse array of antigens ranging from self- to pathogen-derived antigens. Following antigen encounter, naive CD4+ T cells show substantial plasticity and have the potential to differentiate towards distinct effector or regulatory cell lineages depending upon the antigen and concomitant signals from cells of the innate immune system. To date, three types of CD4+ effector T cell populations have been described, namely Th1, Th2 and Th17 (1, 2). Th1 cells produce high levels of IFN-
and tumour necrosis factor (TNF)-
and are involved in the immunity against intracellular pathogens and autoimmunity, Th2 cells produce IL-4, IL-5 and IL-13 and control parasitic infections and allergy and recently described Th17 cells produce high levels of IL-17, IL-17F and IL-6 and are involved in pathogenesis of experimental autoimmune diseases. A major goal in the field of T cell differentiation is to identify the factors responsible for directing different effector T cell responses. Specific activation signals transmitted by the TCR, co-stimulatory signals from members of the CD28 or TNF receptor families and cytokine stimuli, interplay to specify the fate of the naive T cell and to shape the resulting immune response (3). Co-stimulatory molecules, with CD28 being the best characterized, have been shown to play a critical role in the development of Th2 effector cells (4), particularly in the presence of weak TCR signals (5). Several lines of evidence indicate that death receptors (DRs) of the TNF receptor family, further to their role in mediating apoptosis in activated T cells, may also modulate TCR signalling and control T cell differentiation. For example, studies in gene knockout mice have shown that the receptor adaptor Fas-associated death domain (FADD) (6), caspase 8 (7, 8) and c-FLIP (9, 10) are essential for pathways that instruct thymocyte development and T cell maturation and effector function. In addition, the modulation of DR signalling by the over-expression of c-FLIP long isoform in T cells of transgenic mice (TgFLIPL) resulted in diminished Th1 profiles and the preferential expansion of the Th2 pool in several in vivo immune paradigms including experimental autoimmune encephalomyelitis (EAE) (11) and airway hypersensitivity (12).
Infection by the intracellular pathogen Leishmania major in mice represents a prototypic model for studying the mechanisms underlying CD4+ Th effector cell differentiation in vivo. To further understand the role of FLIPL in shaping of T cell immunity, we tested the susceptibility of TgFLIPL (C57BL/6) mice in a dermal L. major infection model and determined their ability to mount a parasite-specific Th1 immune response and to clear infection. Disease resolution in genetically resistant strains (e.g. C57BL/6) correlates with a higher level of activation of Th1 cells that produce IFN-, whereas disease progression in genetically susceptible strains (e.g. BALB/c) is associated with a more prevalent Th2 response (13). Overreaction of homeostatic mechanisms involving IL-10 and regulatory T cells has also been suggested to explain a non-healing form of leishmaniasis in conventionally resistant C57BL/6 mice (14). Here, we show that TgFLIPL (C57BL/6) mice overcome inherent Th2-biased immune responses that are apparent in models of autoimmunity and allergy and develop an adequate Th1 effector cell response to L. major, efficiently clear infection and display memory cell formation comparable to that in wild-type (wt) mice. These results indicate that during L. major infection, the activatory signals delivered to T cells through TCR and from the innate immune environment are capable of overriding the Th2-polarizing effects of T cell-specific FLIPL transgene expression that have been observed in other in vivo autoimmune and allergic immune paradigms.
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Methods |
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Mice
The generation and characterization of TgFLIPL has been described previously (11). All experiments were performed with 6- to 8-week old female mice. The transgenic mouse line was backcrossed at least 12 generations in the C57BL/6 background and in for all procedures wt littermate mice were used as controls. Mice were kept under specific pathogen-free conditions in the experimental animal unit of Hellenic Pasteur Institute. All animal procedures were approved by national authorities and conformed to the European Union guidelines for animal experimentation.
Parasites and antigens
The strain MRHO/SU/59/P of L. major (LV39) was used in all experiments. Stationary phase LV39 promastigotes were isolated by centrifugation. Promastigotes were washed three times in PBS (Flow Laboratories, Meckenheim, Germany), adjusted to 109 promastigotes ml–1 and disrupted by six sequential freeze–thaw cycles in order to prepare soluble promastigote lysate. Total protein determination was carried out using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA).
Infection with L. major
Mice received a subcutaneous (s.c.) immunization in the footpad with 2 x 106 stationary stage promastigotes in 40 µl volume PBS. Footpad swelling was determined thereafter at 1 week intervals by subtracting footpad thickness of the non-immunized footpad from the thickness of the L. major-immunized footpad. Footpad thickness was determined using an electronic caliper gauge (Kori Seiki MFG Ltd, Tokyo, Japan). For secondary infection, mice received an identical challenge with L. major in the opposite footpad from the one that received the primary immunization.
In vitro T cell proliferation assays
Spleens and lymph nodes from L. major-infected mice were aseptically excised and used to prepare single-cell suspensions in RPMI-1640 supplemented with 10% foetal bovine serum (FBS), 10 mM HEPES, 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 100 units ml–1 penicillin and 100 g ml–1 streptomycin (Gibco, Invitrogen Corporation, Paisley, UK). Cell viability was >95% as determined by trypan blue exclusion. Cells were cultured in triplicate in round-bottom 96-well plates (Costar, Cambridge, MA, USA) at a concentration of 1 x 106 cells per ml, for proliferation assays, or 24-well flat-bottom plates at 5 x 106 cells per ml for supernatant collection. Splenocytes were stimulated with crude soluble promastigote lysate at a concentration range of 5–20 µg ml–1 or Con A (Sigma, Munich, Germany) at a range of 2.5–10 µg ml–1. In lymphoproliferation experiments, cultures were incubated in 5% CO2 at 37°C for 96 h and pulsed with 0.2 µCi ml–1 [3H]-thymidine (Amersham Co, Buckinghamshire, UK) during the final 18 h of culture. Cells were harvested and [3H]-thymidine incorporation was assessed by liquid scintillation counting (Wallac, Turku, Finland). Results are expressed as the stimulation index (ratio between radioactivity counts of cells cultured in presence of antigen and cells cultured with medium alone).
Measurement of cytokine production
IL-4 and IFN- ELISA kits (Endogen, Woburn, MA, USA) and a mouse IL-17 ELISA set (R&D Systems, Wiesbaden-Nordenstadt, Germany) were used to measure cytokine secretion from splenocyte and draining lymph node culture supernatants according to the manufacturer’s instructions. The detection limit for IL-4 was 10 pg ml–1 and for IFN- 50 pg ml–1. In addition, the mouse Th1/Th2 cytokine cytometric bead array kit (BD Biosciences, San Jose, CA, USA) was used to measure cytokine levels in culture supernatants according to the manufacturer's instructions. The sensitivity of the assay for the different cytokines is the following: IL-2, 5 pg ml–1; IL-4, 5 pg ml–1; IL-5, 5 pg ml–1; IFN-, 2.5 pg ml–1 and TNF-, 6.3 pg ml–1. For intracellular cytokine staining, draining lymph node cells were isolated from wt and TgFLIPL 4 weeks after footpad infection with L. major. After 3 days in culture with or without a mixture of L. major antigens (used at a concentration of 20 µg ml–1), cells were re-stimulated with phorbol myristate acetate (PMA)/ionomycin (Sigma) for 5 h and brefeldin A (5 µg ml–1, Sigma) was added for the last 3 h of culture. Cells were then washed and fixed with 2% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.5% saponin in PBS/BSA/azide and stained with APC-conjugated anti-CD4 (L3T4) and PE-conjugated anti-IFN- (XMG1.2), anti-IL-10 (JES5-16E3) and anti-IL-4 (11B11) (all antibodies were from BD PharMingen, San Jose, CA, USA). Cytometric analysis was performed using a FACSCalibur and the CellQuest software (BD Biosciences).
Infection and determination of tissue parasitism
Parasite burden was determined using a sensitive microtitration assay as published (15). Rabbit blood agar was used to support the growth of parasites. Tissue samples were homogenized through serial passage from 21GA11/2 and 30GA1/2 needles and adjusted to 100 µg ml–1 in RPMI-1640 supplemented with 10% FBS. The number of wells positive for parasite growth was scored using an inverted microscope.
Antibody levels
Serum parasite-specific IgG antibodies and specific IgG isotypes were determined by using a standard sandwich ELISA method. Briefly, microtitre plates (Nunc Maxisorp, Roskilde, Denmark) were coated overnight with 10 µg ml–1 of a mixture of L. major antigens in 0.05 M carbonate–bicarbonate coating buffer (pH 9.6). Uncoated sites were blocked with 1% BSA in PBS. Serum samples from immunized transgenic and control littermate mice were plated and HRP-conjugated rat anti-mouse IgG1 and IgG2a mAbs (BD PharMingen) were used to detect bound antibodies. The enzyme-labelled complexes were detected by a reaction with 0.01% TMB substrate (Sigma) and 0.02% hydrogen peroxide in 0.18 M Na-acetate buffer (pH 4.0). The reaction was stopped with 2N H2SO4 and optical density at 450 nm was measured using a microplate reader (Dynatech MR 5000, Dynatech Laboratories, Chantilly, VA, USA).
Statistical analysis
Data were evaluated using Student's t-test and P < 0.05 was considered significant.
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Results |
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TgFLIPL efficiently resolve infection by L. major
TgFLIPL and non-transgenic littermate control mice, in the C57BL/6 genetic background, were inoculated with 2 x 106 stationary phase L. major promastigotes s.c. in one hind footpad, and subsequently monitored for lesion development by measuring the difference in thickness between injected and non-injected footpads. wt mice developed increasing swelling from the first week post-inoculation with maximum swelling observed between 6 and 10 weeks. Lesions were completely resolved by 17–18 weeks after inoculation (Fig. 1A). TgFLIPL showed a similar clinical course with no significant difference in initiation or resolution phases and overall disease pattern, although a significant increase of swelling was seen at the peak of disease (Fig. 1A). Measurement of tissue parasite burden using a sensitive microtitration assay showed that TgFLIPL had equivalent levels of parasite infestation as wt mice at 3 weeks after primary infection (Fig. 1B). By 17 weeks after L. major inoculation, parasites were cleared from the site of infection, since no L. major promastigotes could be recovered from the footpad of either mouse strain (data not shown). These results show that TgFLIPL efficiently resolve infection by L. major with similar kinetics as wt mice.
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0.05), mildly increased swelling compared with control littermate mice (wt, open squares, n = 16) and clearance of infection follows similar kinetics. Footpad swelling was measured as the difference in footpad thickness between the L. major-inoculated and the PBS-inoculated paws. (B) Tissue parasite burden measured from the footpads of infected wt (n = 3) and TgFLIPL (n = 3) mice, 3 weeks after infection (P = 0.49).TgFLIPL mount a normal memory response to secondary infection with L. major
To examine the memory response of TgFLIPL to L. major infection, TgFLIPL and wt mice were re-infected by s.c. inoculation of 2 x 106 stationary stage L. major promastigotes 25 weeks after the primary infection. Re-infection induced rapid intense footpad swelling in both groups of mice indicating the presence of an efficient and equivalent memory cellular immune response. In both groups of mice, footpad swelling reached its peak during the first week and lesions were resolved by 5 weeks after parasite inoculation (Fig. 2). These results show that TgFLIPL mount a normal memory response to L. major infection.
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Priming and recall responses of lymphocytes to L. major are normal in TgFLIPL
To analyze the immune response of TgFLIPL to L. major, TgFLIPL and wt mice were inoculated with parasite and analyzed for in vitro recall responses to a mixture of antigens prepared from L. major soluble promastigote lysate, as described in Methods. As predicted from the observation of efficient lesion resolution in TgFLIPL, splenocytes and lymph node cells showed normal proliferative responses at 3 weeks (progressive phase of infection) (Fig. 3A) and 17 weeks (resolution phase) (Fig. 3B and C) after infection. Moreover, memory T cell proliferation at 3 weeks after secondary challenge with parasites was also normal in TgFLIPL when compared with wt animals (Fig. 3D). Collectively, these results demonstrate that TgFLIPL respond to L. major infection with a normal lymphocyte proliferation response which effectively limits and clears parasitic infection.
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Antibody responses are normal in TgFLIPL following primary infection but show enhanced IgG1 production following secondary infection with L. major
A combination of Th1 cell-mediated and humoral immune responses is considered important for controlling L. major infection in C57BL/6 mice. To determine whether TgFLIPL are able to develop a normal antibody immune response to L. major that is typical of the C57BL/6 strain, we measured the titers of L. major-specific IgG isotypes in sera sampled from TgFLIPL and wt mice at 3 and 17 weeks after primary infection and at 3 weeks after secondary infection (i.e. at 28 weeks) by ELISA (Fig. 4). During the course of primary infection, both groups of mice produced statistically equivalent amounts of IgG1 and IgG2a. Upon re-infection, however, TgFLIPL produced significantly increased levels of the Th2 priming IgG isotype IgG1 at 3 weeks after re-infection. No significant differences were detected between the groups in the production of IgG2a, the Th1 priming antibody isotype. These results show that TgFLIPL develop a normal Th1 response following primary and secondary infection and an enhanced Th2-driven memory response to secondary infection.
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Cytokine production following infection with L. major or in vivo priming with a mixture of parasite antigens
We next measured the levels of IFN-
produced by splenocytes isolated from TgFLIPL and wt mice after primary and secondary infection with L. major and re-stimulated in vitro with L. major antigens using a sandwich ELISA. Splenocytes from both strains showed similar expression of IFN- at both 3 (Fig. 5A) and 17 (data not shown) weeks time points following primary infection. In reflection of the enhanced IgG1 antibody isotype production profiles, IFN- production in the memory phase after re-infection with L. major showed a trend towards lower levels in the TgFLIPL (Fig. 5B), but this difference did not reach significance levels. IL-4 production by splenocytes from both strains was undetectable at all time points studied by ELISA assay (data not shown). We also measured the production of IL-17 using an ELISA assay. IL-17 secretion was undetectable in culture supernatants of draining lymph node cells isolated from both strains of mice at 4 weeks after primary infection with parasites and re-stimulated for 3 days in vitro with L. major antigen (data not shown).
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To measure the production of cytokines characteristic for Th1/Th2 differentiation in a primary response to L. major antigens, rather than following infection, lymph node cells were isolated from TgFLIPL and wt mice 9 days after in vivo priming with soluble L. major antigen and re-stimulated for 3 days in vitro. Cytokine secretion was measured using a bead-based flow cytometric assay in culture supernatants. TgFLIPL produced equivalent levels of IL-2 to wt mice in response to L. major antigens, and showed a trend towards decreased levels of TNF-
(Fig. 5C) and IFN- (Fig. 5D) production although this difference did not reach high levels of statistical significance. The production of IL-4 and IL-5 in lymph node cells from both mouse strains was below the limit of detection using this assay.
To determine cytokine production specifically by CD4+ T cells, intracellular cytokine staining was performed in draining lymph node cells that were isolated 4 weeks after infection with L. major and were ex vivo re-stimulated for 3 days with a mixture of L. major antigens. Intracellular staining of cytokines in CD4+ T cells showed that IFN- production is not significantly different between the groups of wt and transgenic mice (Fig. 5E and F). Low levels of IL-4 production were also detectable using this method and levels were not significantly different between mouse strains (Fig. 5E and F). We also measured IL-10 production in these cells since it has been described to be the cytokine responsible for immunosuppression and parasite persistence in cutaneous leishmaniasis (16–18). TgFLIPL showed comparable IL-10 production to wt mice (Fig. 5E and 5F).
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Discussion |
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Using a cutaneous L. major infection model in conventionally resistant C57BL/6 mice, we have further investigated the role of T cell-specific FLIPL transgene over-expression in the specification of Th fate. In comparison to previous findings, where down modulation of Th1-mediated immune function and Th2 skewing of T cell responses resulted in the suppression of myelin oligodendrocyte glycoprotein 35–55 peptide (MOG35–55)-induced EAE (11) and exacerbation of ovalbumin peptide-induced asthma (12), we show here that in the L. major infection model, TgFLIPL exhibited a robust Th1 adaptive immune response to the pathogen, which is typical of the C57BL/6 strain, and cleared pathogen as effectively as normal mice. Furthermore, even though memory responses induced by secondary infection with the same parasite were characterized by reduced Th1-polarized immunity, as indicated by significantly increased IgG1 production, mice were still able to clear re-infection normally. These observations confirm that FLIPL plays an important role in shaping T cell responses to a wide range of antigens, now including those derived from the parasite L. major, and has functional consequences in several autoimmune and allergic immune paradigms. They also suggest that during an infection, for example by L. major parasites, additional signals are delivered to T cells by the innate immune environment that overrides the otherwise Th2-polarizing effects of T cell-specific FLIPL transgene expression.
It has become clear that apoptosis-related mediators, such as caspase 8 and FLIPL, play important roles in the control of thymocyte development and the maturation and effector function of mature T cells (6–9, 19). Inactivation of flip selectively in the T cell lineage of conditional FLIP knockout mice enhanced the sensitivity of CD4+ and CD8+ single-positive thymocytes to TCR/CD3 and Fas-induced apoptosis resulting in severely reduced numbers of mature T cells in the periphery (9, 10). Further, the over-expression of FLIPL in T cells of transgenic mice resulted in increased CD3- and antigen-induced proliferation (20). We have seen that TgFLIPL (C57BL/6) T cells are capable of differentiating towards both the Th1 and Th2 cellular fates, but show a markedly lower production of Th1 cytokines when compared with wt cells when stimulated ex vivo or in vitro with various peptide antigens or polyclonal activators (V. Tseveleki and L. Probert, unpublished data). As mentioned above, in vivo priming and in vitro recall stimulation of TgFLIPL T cells with a wide range of purified antigens resulted in reduced Th1 and enhanced Th2 cytokine responses, and in several disease paradigms, this effect translated into markedly altered clinical signs (11, 12). The mechanism by which FLIPL alters Th differentiation is not known but biochemical studies have shown that FLIPL expression in cell lines promotes nuclear factor-kappaB and extracellular signal-regulated kinase activation (21) and inhibits c-jun N-terminal kinase activation (22). It remains to be determined whether the Th2 skewing effect of FLIPL in T cell pools is due to altered signalling in T cells which affects their differentiation or whether the Th2 population is selectively protected from apoptosis leading to its aberrant expansion. Our finding, that T cell FLIPL does not lead to a Th2 bias when mice are infected with the intracellular parasite L. major, suggests that although it might be important for physiological T cell functioning, for example for tolerance or memory formation, it might not play such a critical role during host defence reactions, where additional infection-specific signals are delivered to T cells and robust Th1 responses are required. However, we cannot exclude the alternative possibility that differences in TCR signal strength delivered by various antigens determine whether or not FLIPL can be involved in shaping T cell responses.
A major pursuit in the field of T cell biology is to apply basic knowledge to the development of improved therapeutics and vaccination strategies for the treatment of autoimmune diseases. Several of the current approaches that involve the prolonged use of immunosuppressants for the treatment of autoimmune diseases have been linked with significant adverse effects such as increased susceptibility to opportunistic infections that can be fatal (23). For example, there are multiple Food and Drug Administration, USA (FDA) warnings associating the use of the TNF- blocking agents with the occurrence of serious infections, including sepsis and disseminated tuberculosis. It is tempting to envision vaccination strategies that would be able to boost FLIPL expression in T cells for the treatment of autoimmune disorders without necessarily compromising host defence mechanisms to pathogens.
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Funding |
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The sixth Framework Programme of the European Union, NeuroproMiSe (LSHM-CT-2005-018637).
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Acknowledgements |
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Funding to pay the Open Access publication charges for this article was provided by the sixth Framework Programme of the European Union, NeuroproMiSe (LSHM-CT-2005-018637).
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Abbreviations |
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| DR, death receptor |
| EAE, experimental autoimmune encephalomyelitis |
| FBS, foetal bovine serum |
| PMA, phorbol myristate acetate |
| s.c., subcutaneous |
| TgFLIPL, CD2-FLIPL transgenic mice |
| TNF, tumour necrosis factor |
| Wt, wild type |
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Notes |
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Transmitting editor: D. Wallach
Received 7 December 2006, accepted 9 July 2007.
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