Immunity

Published by Elsevier
Online ISSN: 1074-7613
Publications
Article
An important but laborious approach to understanding the concepts underlying T cell lineage commitment is to characterize the cis-acting control elements governing the expression of CD4 and CD8. Previous studies on the CD4 gene have shown that lineage commitment information is directed by the intronic silencer; however, a similarly simple mechanism for controlling CD8 gene expression has not been uncovered. In this issue of Immunity, two groups have investigated the role of putative enhancers in the CD8 locus. The deletion of three different elements in the intergeneic region between CD8 beta and CD8 alpha provides evidence for control at the level of chromatin accessibility.
 
Article
Genetic predisposition to rheumatoid arthritis (RA) is linked to the MHC class II allele HLA-DR4. The charge of the amino acid at DRbeta71 in the peptide-binding site appears to be critical in discriminating DR molecules linked to increased disease susceptibility. We have determined the 2.5 A x-ray structure of the DR4 molecule with the strongest linkage to RA (DRB1*0401) complexed with a human collagen II peptide. Details of a predicted salt bridge between lysine DRbeta71 and aspartic acid at the P4 peptide position suggest how it may participate in both antigen binding and TCR activation. A model is proposed for the DR4 recognition of collagen II (261-273), an antigen immunodominant in human-transgenic mouse models of RA.
 
Article
A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.
 
Article
Mutating the HLA-A*0201 heavy chain from threonine to lysine at position 134 (T134K) results in a molecule that presents exogenous peptide, but cannot present endogenously derived antigen. This is reflected in diminished cell surface expression and altered intracellular trafficking of T134K. The failure of T134K to present endogenous antigen can be overcome by using an ER targeting sequence, suggesting that the antigen presentation defect is restricted to TAP-dependent peptide loading. The ability of T134K to load peptide in a TAP-dependent manner is dramatically reduced compared with HLA-A*0201. By coimmunoprecipitation there is no detectable association of the T134K molecule with the TAP complex. Thus, T134K selectively affects TAP association and peptide loading, suggesting a requirement for the direct interaction of MHC class I heavy chain and the TAP complex for efficient presentation of endogenous antigen.
 
Article
E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.
 
Article
Noninflamed skin venules support constitutive leukocyte rolling. P-selectin controls the rolling frequency, whereas E-selectin dictates rolling velocity (Vroll). Fucosylated selectin ligands are essential for all interactions, as rolling was absent in mice doubly deficient in alpha1,3-fucosyltransferase (FucT)-IV and FucT-VII. The rolling fraction was reduced in FucT-VII-/- animals but normal in FucT-IV-/- mice. However, Vroll was markedly increased in both strains. P-selectin ligands generated by FucT-VII are crucial for initial leukocyte tethering, whereas E-selectin ligands that permit maximum slowing of Vroll require simultaneous expression of FucT-IV and FucT-VII. These results demonstrate a role for FucT-IV in selectin-dependent adhesion and suggest that the endothelial selectins and FucTs have distinct but overlapping functions in the immunosurveillance of the skin.
 
Article
Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.
 
Article
Cytotoxic lymphocytes kill virus-infected target cells and play a critical role in host recovery from viral infections. Granzyme B (GrB) is a cytotoxic lymphocyte granule protease that plays a critical role in mediating cytotoxicity. In these studies, we demonstrate that the adenovirus assembly protein L4--100K (100K) is a GrB substrate that prevents cytotoxic lymphocyte granule-induced apoptosis in infected target cells by potently inhibiting GrB. This inhibition is absolutely dependent on Asp-48 in 100K, found within a classic GrB consensus motif. 100K is the first viral protein described that exclusively targets the GrB pathway. It represents a novel class of viral protease inhibitor, in which an essential, multifunctional viral protein, which is vulnerable to specific proteolysis by GrB, expresses inhibitory function against that protease.
 
Article
NKG2D is known to trigger the natural killer (NK) cell lysis of various tumor and virally infected cells. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles the alpha1 and alpha2 domains of classical MHC molecules without a bound peptide. The lack of alpha3 and beta2m domains is compensated by replacing two hydrophobic patches at the underside of the class I MHC-like beta sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 alpha helices, creating a complementary interface, an asymmetrical subunit orientation, and local conformational adjustments in the receptor. The interface is stabilized primarily by hydrogen bonds and hydrophobic interactions. Unlike the KIR receptors that recognize a conserved HLA region by a lock-and-key mechanism, NKG2D recognizes diverse ligands by an induced-fit mechanism.
 
Article
Cytokine and cytokine receptor gene knockout mice provide powerful experimental systems to characterize the functions of these molecules in resistance to infectious disease. Such mice may also provide unique models of immune deficiency to learn whether manipulation of the immune response can overcome the specific dysfunction. We demonstrate that resistance of IFN gamma gene knockout (GKO-/-) mice to the intracellular bacterium Listeria monocytogenes is severely impaired compared with wild-type mice. However, immunization of GKO-/- mice with an attenuated L. monocytogenes strain generates antigen-specific CD8 T cell responses that can transfer immunity to naive hosts. Furthermore, vaccinated GKO-/- mice themselves exhibit 20,000-fold increased resistance to challenge with virulent L. monocytogenes and this resistance appears to be CD8 T cell mediated. These studies demonstrate that vaccination-induced immunity can overcome the absence of a cytokine that is critical for resistance to acute infection.
 
Article
Interleukin 10 (IL-10) is a dimeric cytokine that plays a central role in suppressing inflammatory responses. These activities are dependent on the interaction of IL-10 with its high-affinity receptor (IL-10R1). This intermediate complex must subsequently recruit the low-affinity IL-10R2 chain before cell signaling can occur. Here we report the 2.9 A crystal structure of IL-10 bound to a soluble form of IL-10R1 (sIL-10R1). The complex consists of two IL-10s and four sIL-10R1 molecules. Several residues in the IL-10/sIL-10R1 interface are conserved in all IL-10 homologs and their receptors. The data suggests that formation of the active IL-10 signaling complex occurs by a novel molecular recognition paradigm where IL-10R1 and IL-10R2 both recognize the same binding site on IL-10.
 
Caspase-11-Deficient Macrophages Allow L. pneumophila Intracellular Replication (A) Wild-type (WT), caspase-11-deficient (Casp4 À/À ), and caspase-1-deficient (Casp1 À/À ) murine BMDMs were infected with L. pneumophila (Leg) and colonyforming units (CFUs) were enumerated at 1, 24, 48, and 72 hr. Data are representative of three independent experiments and presented as means ± SD. Asterisks indicate significant differences from WT macrophages (***p < 0.001). (B) Confocal microscopy of Leg-infected WT or Casp4 À/À BMDMs after 24 hr. Nuclei are stained blue with DAPI and Leg express green florescent protein (GFP). White arrows indicate the sites of Leg. (C and D) WT, Casp1 À/À , and Casp4 À/À BMDMs were nucleofected with plasmid harboring Casp4 (PL-Casp11) or empty vector (PL) for 24 hr. (C) BMDMs were infected with L. pneumophila and CFUs were enumerated at 1, 24, 48, and 72 hr. (D) Samples were lysed and immunoblotted for caspase-11 expression. See also Figure S2.  
Caspase-11 Is Required for the Dynamic Formation of Polymerized Actin around Phagosomes WT, caspase-11-deficient (Casp4 À/À ) (A and B), and Casp1 À/À BMDMs (C) were infected with L. pneumophila (Leg) constitutively expressing GFP. Polymerized actin was stained with rhodamine-phalloidin. (A) The amount of rhodamine-phalloidin within equal areas was quantified by confocal microscopy and expressed as arbitrary units. (B) The percentage of phalloidin-labeled Leg-containing phagosomes was quantified by confocal microscopy. (C) Confocal microscopy showing rhodamine-phalloidin staining (red) around GFP-expressing (green) Leg. Phagosomes containing degraded bacteria are heavily labeled for polymerized actin (white arrowheads). Data in (A) and (B) are representative of three independent experiments and presented as the means ± SD. Asterisks indicate significant differences (*p < 0.05; **p < 0.01; ***p < 0.001). See also Figure S5.  
Article
Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.
 
Article
The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.
 
Article
Mannose-binding protein (MBP), C1q, the recognition component of the classical complement pathway, and pulmonary surfactant protein A (SP-A) are members of a family of molecules containing a collagen-like sequence contiguous with a noncollagen-like sequence, and usually having the properties of a lectin. C1q and SP-A have been shown to enhance monocyte FcR- and CR1-mediated phagocytosis, suggesting that the common structural features of the collagen-like domains may provide a basis for this immunologically important function. Results presented here demonstrate that MBP also enhanced FcR-mediated phagocytosis by both monocytes and macrophages, and stimulated CR1-mediated phagocytosis in human culture-derived macrophages and in phorbol ester-activated monocytes. Furthermore, a monoclonal antibody that recognizes a 126,000 M(r) cell surface protein and inhibits C1q-enhanced phagocytosis, inhibited the MBP-mediated enhancement of phagocytosis. Thus, the receptors that mediate the enhancement of phagocytosis by MBP and C1q share at least one critical functional component, the 126,000 M(r) ClqRP.
 
Article
A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.
 
Article
To further understand the interaction among GATA-3, Stat4, and T-bet in helper T cell development, we first showed that retroviral expression of GATA-3 in developing Th1 cells suppresses Th1 development through downregulation of Stat4 rather through downregulation of the IL-12Rbeta2 chain. Correspondingly, Stat4 levels are greatly suppressed during physiological Th2 development. Then, using cells doubly infected with GFP- and YFP-expressing retroviruses, we showed that retroviral GATA-3 expression in developing Th1 cells does not block Th1 development in cells coexpressing Stat4 but does so in cells coexpressing T-bet. Finally, we showed that retroviral Stat4 expression could facilitate Th2-->Th1 conversion in cells bearing an IL-12Rbeta2 transgene, even in cells lacking T-bet. These findings reassert that Stat4 signaling is a central element of Th1/Th2 development.
 
Article
We have characterized a cytokine produced by Th2 cells, designated as IL-25. Infusion of mice with IL-25 induced IL-4, IL-5, and IL-13 gene expression. The induction of these cytokines resulted in Th2-like responses marked by increased serum IgE, IgG(1), and IgA levels, blood eosinophilia, and pathological changes in the lungs and digestive tract that included eosinophilic infiltrates, increased mucus production, and epithelial cell hyperplasia/hypertrophy. In addition, our studies show that IL-25 induces Th2-type cytokine production by accessory cells that are MHC class II(high), CD11c(dull), and lineage(-). These results suggest that IL-25, derived from Th2 T cells, is capable of amplifying allergic type inflammatory responses by its actions on other cell types.
 
Article
We report that Th2 cell cultures generated using T cells or splenocytes from IL-13-deficient mice produce significantly reduced levels of IL-4, IL-5, and IL-10 compared with wild-type. In contrast, IL-4 and IL-5 production by mast cells stimulated in vitro with PMA, ionomycin, or IgE cross-linking are unaffected. In vitro Th2 cell differentiation cannot be rescued by the addition of exogenous factors, but in vivo antigen challenge and administration of IL-13 can increase Th2-like cytokine responses as can infection with the parasitic nematode Nippostrongylus brasiliensis. IL-13-deficient mice also have lower basal levels of serum IgE and biased antigen-specific immunoglobulin responses. Thus, IL-13 is an important regulator of Th2 commitment and may therefore play a central role in atopy and infectious diseases.
 
Article
Functional redundancy is highly prevalent among the Th2 interleukins (IL)-4, IL-5, IL-9, and IL-13. To define the critical functions of these cytokines, we have generated a novel panel of compound Th2 cytokine-deficient mice (from single to quadruple cytokine knockouts). We find that these Th2 cytokines are not essential for fetal survival even during allogeneic pregnancy. Using intestinal parasite infection and a pulmonary granuloma model, we demonstrate cryptic roles for IL-4, IL-5, IL-9, and IL-13 in these responses. Significantly, although IL-5, IL-9, and IL-13 add to the speed and magnitude of the response, a threshold is reached at which IL-4 alone can activate all Th2 effector functions. These mice reveal distinct spatial, temporal, and hierarchical cytokine requirements in immune function.
 
Article
Although IL-4 induces expulsion of the gastrointestinal nematode parasite, Nippostrongylus brasiliensis, from immunodeficient mice, this parasite is expelled normally by IL-4-deficient mice. This apparent paradox is explained by observations that IL-4 receptor alpha chain (IL-4Ralpha)-deficient mice and Stat6-deficient mice fail to expel N. brasiliensis, and a specific antagonist for IL-13, another activator of Stat6 through IL-4Ralpha, prevents worm expulsion. Thus, N. brasiliensis expulsion requires signaling via IL-4Ralpha and Stat6, and IL-13 may be more important than IL-4 as an inducer of the Stat6 signaling that leads to worm expulsion. Additional observations made in the course of these experiments demonstrate that Stat6 signaling is not required for IL-4 enhancement of IgG1 production and actually inhibits IL-4-induction of mucosal mastocytosis.
 
Article
Oxazolone colitis (OC) is an experimental colitis that has a histologic resemblance to human ulcerative colitis. Here we show that IL-13 production is a significant pathologic factor in OC since its neutralization by IL-13Ralpha2-Fc administration prevents colitis. We further show that OC is mediated by NK-T cells since it can be induced neither in mice depleted of NK-T cells nor in mice that cannot present antigen to NK-T cells and mice lacking an NK-T cell-associated TCR. Finally, we show that NK-T cells are the source of the IL-13, since they produce IL-13 upon stimulation by alpha-galactosylceramide, an NK-T cell-specific antigen. These data thus describe a cellular mechanism underlying an experimental colitis that may explain the pathogenesis of ulcerative colitis.
 
Article
BCL-6, a transcriptional repressor frequently translocated in lymphomas, regulates germinal center B cell differentiation and inflammation. DNA microarray screening identified genes repressed by BCL-6, including many lymphocyte activation genes, suggesting that BCL-6 modulates B cell receptor signals. BCL-6 repression of two chemokine genes, MIP-1alpha and IP-10, may also attenuate inflammatory responses. Blimp-1, another BCL-6 target, is important for plasmacytic differentiation. Since BCL-6 expression is silenced in plasma cells, repression of blimp-1 by BCL-6 may control plasmacytic differentiation. Indeed, inhibition of BCL-6 function initiated changes indicative of plasmacytic differentiation, including decreased expression of c-Myc and increased expression of the cell cycle inhibitor p27kip1. These data suggest that malignant transformation by BCL-6 involves inhibition of differentiation and enhanced proliferation.
 
Article
Il4 and Il13, closely linked genes, are expressed monoallelically in TH2 cells. Four different approaches (RNA FISH, cultures from Il13T-Il4/Il13-G4 mice, cultures from heterozygous Il13-Il4 double knockout mice, and a highly selected set of BABL/c*CAST/Ei clones displaying strong Il4 allelic bias) were utilized to study monoallelic expression of Il4 and coexpression of Il4 and Il13 on the same chromosome. There was a random probability for expression of one or two Il4 and one or two Il13 alleles; coexpression of cis and trans Il4 and Il13 alleles was equally probable. Histone H3 acetylation of CNS1, located in the Il13-Il4 intergenic region, was permissive for expression of IL-4 and IL-13 but did not determine the degree of their expression. Thus, monoallelism at the Il4 locus is a complex process; expression is linked to opening CNS1 but probability of expression is controlled at other sites. Based on these probabilities, individual cells randomly express Il4 and Il13 alleles.
 
Article
Macrophage mannose receptor (MMR) is an important component of the innate immune system implicated in host defense against microbial infections such as candidiasis and in antigen presentation. We demonstrate here that the MMR expression is induced in mouse peritoneal macrophages following exposure to PPARgamma ligands or to interleukine-13 (IL-13) via a PPARgamma signaling pathway. Ligand activation of the PPARgamma in macrophages promotes uptake, killing of Candida albicans, and reactive oxygen intermediates production triggered by the yeasts through MMR overexpression. We also show that MMR induction by IL-13 via PPARgamma is dependent on phopholipase A2 activation and that IL-13 induces 15d-PGJ2 production and nuclear localization. These results reveal a novel signaling pathway controlling the MMR surface expression and suggest that endogenous PPARgamma ligand produced by phospholipase A2 activation may be an important regulator of MMR expression by IL-13.
 
Article
To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and Stat5 in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2R beta and IL-7R that can serve as docking sites for Stat proteins. IL-13 Induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.
 
LPS-Induced Downregulation of AChE In Vivo and Ex Vivo Suggests Involvement of miR-132 Predicted to Target AChE mRNA (A) QRT-PCR for AChE mRNA normalized to GAPDH in splenocytes of FVB-N mice 3-24 hr after LPS. Bars indicate SD, n = 3 or more per bar. (B) AChE protein in splenocytes, 24 hr after LPS, normalized to actin (n = 3 per group). Antibodies used to target AChE are described in Experimental Procedures. (C) AChE's hydrolytic activity in splenocytes of FVB-N mice 24 hr after LPS. **p < 0.01; bars, SEM (n = 3 per group). (D-F) FVB-N mice serum titers of IL-6 (D), IL-1b (E), and AChE activity (F) 3 hr (cytokines) and 24 hr (AChE activity) after LPS treatment. Bars indicate SD from triplicates (n = 3 per group). (G) Scatter plot of representative spotted miRNA array in primary human macrophages. Each black spot represents a particular miRNA, with those above the dashed lines upregulated. Spots representing miRNAs 132 and 182* are red and blue, respectively. (H) QRT-PCR for miRs-132, 182*, and 181a (a non-LPS-responding miRNA serving as control) in human macrophages. Bars indicate SD from triplicates. (I) QRT-PCR for miR-132 in bone marrow (BM) or spleen of LPS-injected FVBN mice compared to controls (n = 3 per group). Bars indicate SD; *p = 0.04. 
Counteracting miR-132 In Vivo by an LNA-Anti-miR Oligo Elevates Serum AChE Activity (A) Anti-miR-132 and scrambled control oligonucleotides (Sigma) had fulllength phosphorothioate backbones and >50% LNA bases (in red). (B) Quantification of QRT-PCR for miR-132 and miR-182* in bone marrow (BM) and spleen of FVB-N mice injected i.v. with 3.3 mg anti-miR-132 or scrambled (scr) oligo daily for 3 days and sacrificed 24 hr after last injection. Bars indicate SD; *p = 0.04 (n = 4 per group). (C) Quantification of AChE catalytic activity in intestine, sera, and bone marrow (BM) of mice as in (B). Averages in red. *p = 0.04. Anti-miR132-injected mice (n = 7) represented by squares, triangles, and diamonds; scrambled (scr) oligo-injected or nontreated mice represented by white symbols (n = 8 or 6). (D) Immunoblot for AChE in intestine of anti-132-treated and control mice as in (B). Recombinant AChE used as positive control (C). b-Tubulin was used as loading control (n = 6 per group). (E) Spleen miR-132 levels are inversely associated with serum AChE activity. Inset: R value (n = 20). 
miR-132 Regulates AChE Activity in Cultured Cells (A) QRT-PCR amplification plot for miR-132 in mice brain, CHO cells, and mouse spleen. (B) Quantification of AChE activity in CHO cells transfected (n = 3) with equal amounts of AChE expression vector with native 3 0-UTR or UTR with pointmutated miR-132 binding site or GFP expression vector that served as control for transfection efficiency. Bars indicate SD; **p = 0.001 (n = 3). (C) Scheme of the pre-miR-132 overexpression vector, with sequences for pre-(in black) and mature miR-132 (in red). (D) AChE activity of bone-marrow-derived macrophages infected with lentivirus overexpressing pre-miR-132 or a scrambled sequence. **p = 0.004, Mann-Whitney U test. 
Mice Overexpressing 3 0-UTR Null AChE Show Deregulated, ACh-Refractory Immune Responses (A) 3 0-UTR null AChE sequence in the TGR mice. (B) Working hypothesis: inflammation-induced miRs control AChE activity to enable the anti-inflammatory cholinergic reflex (Metz and Tracey, 2005); the suggested mechanism is disrupted in TgR mice. (C) TgR mice show significantly higher intestinal AChE activity compared to FVB-N wild-type mice. **p = 0.002 (U test, n = 8). Bars indicate SEM. (D) Immunoblot for AChE in intestine of TgR (n = 4) and control FVB-N (n = 4) mice. Anti-132-treated mice (as in Figure 2) showed elevated AChE expression comparable to TgR transgenics. b-Tubulin was used as loading control. (E-G) Intestinal explants from TgR mice display higher basal expression of cytokines, IL1, and IL6 (C; *p = 0.002, **p = 10 À5 ), and IL10 (D; *p = 0.02) compared to FVB/N controls that is quenchable by nicotine. n = 8, bars indicate SEM. (H) Rectal temperatures in FVB-N and TgR mice after i.p. injection of 2 mg/kg LPS. *p < 0.03; bars indicate SEM. (I) QRT-PCR for miR-132 in bone marrow (BM) and brain hypothalamus of TgR mice, normalized to FVB-N controls. Bars indicate SEM; *p = 0.046. (J-L) Cytokine (IL-6, IL-12, TNF-a) amounts in bone marrow macrophages from FVB-N (WT) and TgR mice, naive and treated with LPS, with or without ACh. Bars indicate SD. ***p = 0.0005, *p = 0.03 (n = 3 per group). 
Article
MicroRNAs (miRNAs) contribute to both neuronal and immune cell fate, but their involvement in intertissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induced leukocyte overexpression of the AChE-targeting miR-132. Injected locked nucleic acid (LNA)-modified anti-miR-132 oligonucleotide depleted miR-132 amounts while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3'UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice overexpressing 3'UTR null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 upregulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening avenues for study and therapeutic manipulations of the neuro-immune dialog.
 
Article
The brain-immune axis continues to fascinate. In this issue of Immunity, Shaked et al. (2009) describe how miR-132 mediates an anti-inflammatory effect via the targeting of acetylcholinesterase, leading to an increase in the neurotransmitter acetylcholine.
 
Article
T cell activation induces functional changes in cell shape and cytoskeletal architecture. To facilitate the collection of dynamic, high-resolution images of activated T cells, we plated T cells on coverslips coated with antibodies to the T cell receptor (TCR). Using these images, we were able to quantitate the morphological responses of individual cells over time. Here, we show that TCR engagement triggers the formation and expansion of contacts bounded by continuously remodeled actin-rich rings. These processes are associated with the extension of lamellipodia and require actin polymerization, tyrosine kinase activation, cytoplasmic calcium increases, and LAT, an important hematopoietic adaptor. In addition, the maintenance of the resulting contact requires sustained calcium influxes, an intact microtubule cytoskeleton, and functional LAT.
 
Article
Complex genetic disorders have begun to yield to sequential positional cloning initiatives. Both in humans and in mice, given that multiple susceptibility loci can be established, it is generally possible to dissect phenotypes one gene at a time. The first step has been taken in Crohn's disease. But even simple genetic disorders pose problems of interpretation once they are solved by positional methods. How does huntingtin create its phenotype? How do mutations of superoxide dismutase create amyotrophic lateral sclerosis? How do mutations of NRAMP enhance susceptibility to mycobacterial infection? When numerous mutations are responsible for a phenotype, the riddle is more challenging still. NOD2 has become the eighth protein known to have strong ties to cell death pathways and to autoimmunity. Mutations of NOD2 are responsible for a discrete autoimmune disease. But as to how they create the phenotype, much remains to be learned.
 
Article
Tumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP(∗) isoform of C/EBPβ transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPβ LAP(∗) by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.
 
Article
The flk2 receptor tyrosine kinase has been implicated in hematopoietic development. Mice deficient in flk2 were generated. Mutants developed into healthy adults with normal mature hematopoietic populations. However, they possessed specific deficiencies in primitive B lymphoid progenitors. Bone marrow transplantation experiments revealed a further deficiency in T cell and myeloid reconstitution by mutant stem cells. Mice deficient for both c-kit and flk2 exhibited a more severe phenotype characterized by large overall decreases in hematopoietic cell numbers, further reductions in the relative frequencies of lymphoid progenitors, and a postnatal lethality. Taken together, the data suggest that flk2 plays a role both in multipotent stem cells and in lymphoid differentiation.
 
Article
Invariant Valpha14i NKT (iNKT) cells are a specialized subset of T lymphocytes with regulatory functions. They coexpress TCRalphabeta and natural killer cell markers. They differentiate through interaction of their Valpha14-Jalpha18 invariant TCRalpha chains with CD1d expressed on double-positive (DP) thymocytes. Although their development has been shown to be thymus dependent, their developmental pathway has not been definitively established. By using genetic analyses, we show here that all iNKT cells are selected from a pool of DP thymocytes. Their development is absolutely dependent on Runx1 and ROR(gamma)t, transcription factors that influence, but are not required for, development of conventional T cells. Our results indicate that even though CD1d binding DP thymocytes have yet to be observed, Valpha14-Jalpha18 rearrangement in these cells is required for development of iNKT cells.
 
Article
The bare lymphocyte syndrome (BLS) is characterized by the absence of MHC class II transcription and humoral- and cellular-mediated immune responses to foreign antigens. Three of the four BLS genetic complementation groups have defects in the activity of the MHC class II transcription factor RFX. We have purified the RFX complex and sequenced its three subunits. The sequence of the smallest subunit describes a novel gene, termed RFX-B. RFX-B complements the predominant BLS complementation group (group B) and was found to be mutant in cell lines from this BLS group. The protein has no known DNA-binding domain but does contain three ankyrin repeats that are likely to be important in protein-protein interactions.
 
Article
microRNA-155 (miR-155) is expressed by cells of the immune system after activation and has been shown to be required for antibody production after vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and -independent antigens. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high-affinity IgG1 antibodies. Gene-expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function, many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155-mediated inhibition. When Pu.1 is overexpressed in wild-type B cells, fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155-deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.
 
Article
B lymphocytes perform somatic hypermutation and class-switch recombination (CSR) of the immunoglobulin locus to generate an antibody repertoire diverse in both affinity and function. These somatic diversification processes are catalyzed by activation-induced cytidine deaminase (AID), a potent DNA mutator whose expression and function are highly regulated. Here we show that AID was regulated posttranscriptionally by a lymphocyte-specific microRNA, miR-155. We found that miR-155 was upregulated in murine B lymphocytes undergoing CSR and that it targeted a conserved site in the 3'-untranslated region of the mRNA encoding AID. Disruption of this target site in vivo resulted in quantitative and temporal deregulation of AID expression, along with functional consequences for CSR and affinity maturation. Thus, miR-155, which has recently been shown to play important roles in regulating the germinal-center reaction, does so in part by directly downmodulating AID expression.
 
Article
Chronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a(-/-) mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155(fl/fl) mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.
 
Article
MiR-155 is encoded within a region known as bic, B cell integration cluster, identified originally as a frequent integration site for avian leucosis virus (Lagos-Quintana et al., 2002 and Tam et al., 1997). An important role for miR-155 in B cell malignancies was strongly suggested by the observations that miR-155 is elevated in certain B cell lymphomas (Eis et al., 2005) and that transgenic expression of miR-155 in B cells causes pre-B cell lymphomas in mice (Costinean et al., 2006). Furthermore, the expression pattern of miR-155 in normal B cells suggests a role in differentiation of normal activated B cells because it is induced upon B cell receptor (BCR) ligation of Ramos cells (van den Berg et al., 2003).
 
Article
MicroRNAs (miRNAs) regulate the function of several immune cells, but their role in promoting CD8(+) T cell immunity remains unknown. Here we report that miRNA-155 is required for CD8(+) T cell responses to both virus and cancer. In the absence of miRNA-155, accumulation of effector CD8(+) T cells was severely reduced during acute and chronic viral infections and control of virus replication was impaired. Similarly, Mir155(-/-) CD8(+) T cells were ineffective at controlling tumor growth, whereas miRNA-155 overexpression enhanced the antitumor response. miRNA-155 deficiency resulted in accumulation of suppressor of cytokine signaling-1 (SOCS-1) causing defective cytokine signaling through STAT5. Consistently, enforced expression of SOCS-1 in CD8(+) T cells phenocopied the miRNA-155 deficiency, whereas SOCS-1 silencing augmented tumor destruction. These findings identify miRNA-155 and its target SOCS-1 as key regulators of effector CD8(+) T cells that can be modulated to potentiate immunotherapies for infectious diseases and cancer.
 
Article
Mammalian noncoding microRNAs (miRNAs) are a class of gene regulators that have been linked to immune system function. Here, we have investigated the role of miR-155 during an autoimmune inflammatory disease. Consistent with a positive role for miR-155 in mediating inflammatory responses, Mir155(-/-) mice were highly resistant to experimental autoimmune encephalomyelitis (EAE). miR-155 functions in the hematopoietic compartment to promote the development of inflammatory T cells including the T helper 17 (Th17) cell and Th1 cell subsets. Furthermore, the major contribution of miR-155 to EAE was CD4(+) T cell intrinsic, whereas miR-155 was also required for optimum dendritic cell production of cytokines that promoted Th17 cell formation. Our study shows that one aspect of miR-155 function is the promotion of T cell-dependent tissue inflammation, suggesting that miR-155 might be a promising therapeutic target for the treatment of autoimmune disorders.
 
Article
MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3'-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda(155) mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID.
 
Article
We report intriguing aspects of the contribution of IL-15Ralpha to IL-15 functions. Consistent with high-affinity interactions between IL-15 and IL-15Ralpha, these two molecules form stable complexes on the cell surface of activated monocytes. The formation of IL-15/IL-15Ralpha complexes on cell surfaces induces a trans-endosomal recycling of IL-15 leading to the persistence of surface-bound IL-15 due to the constant reappearance of IL-15 on plasma membranes. This complex contributes to the long survival of T cells expressing IL-15Ralpha after IL-15 withdrawal. Finally, these complexes on activated monocytes present IL-15 in trans to target cells such as CD8(+) T cells that express only IL-2/15Rbeta and gammac upon cell-cell interaction.
 
Article
We examined the relationship between cell death and tolerance induction following antigen injection into the anterior chamber of the eye. Our data show that when inflammatory cells undergo apoptosis following infection with HSV-1, tolerance to the virus was observed. In contrast, when cell death was absent due to defects in Fas or FasL, immune tolerance was not observed. Further studies revealed that cell death and tolerance required that the lymphoid cells be Fas+ and the eye be FasL+. Additionally, we show that while Fas/FasL-mediated apoptosis occurred in the eye, it was apoptotic cell death that was critical for tolerance induction. Our results further demonstrate immune privilege is not a passive process involving physical barriers, but is an active process that employs an important natural mechanism to induce cell death and immune tolerance.
 
Article
The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.
 
Article
Several tumor antigens are recognized by autologous cytolytic T lymphocytes (CTL) on human melanoma MZ2-MEL. Some of them are encoded by genes MAGE-1 and MAGE-3, which are not expressed in normal tissues except in testis. Here, we report the identification of a new gene that codes for another of these antigens. This gene, named BAGE, codes for a putative protein of 43 aa and seems to belong to a family of several genes. The antigen recognized by the autologous CTL consists of BAGE-encoded peptide AARAVFLAL bound to an HLA-Cw 1601 molecule. Gene BAGE is expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas, and 6% of non-small cell lung carcinomas. Like the MAGE genes, it is silent in normal tissues with the exception of testis. Because of its tumor-specific expression, the BAGE-encoded antigen may prove useful for cancer immunotherapy.
 
Article
Phenotypic plasticity of T helper 17 (Th17) cells suggests instability of chromatin structure of key genes of this lineage. We identified epigenetic modifications across the clustered Il17a and Il17f and the Ifng loci before and after differential IL-12 or TGF-beta cytokine signaling, which induce divergent fates of Th17 cell precursors. We found that Th17 cell precursors had substantial remodeling of the Ifng locus, but underwent critical additional modifications to enable high expression when stimulated by IL-12. Permissive modifications across the Il17a-Il17f locus were amplified by TGF-beta signaling in Th17 cells, but were rapidly reversed downstream of IL-12-induced silencing of the Rorc gene by the transcription factors STAT4 and T-bet. These findings reveal substantial chromatin instability of key transcription factor and cytokine genes of Th17 cells and support a model of Th17 cell lineage plasticity in which cell-extrinsic factors modulate Th17 cell fates through differential effects on the epigenetic status of Th17 cell lineage factors.
 
Article
Gammadelta T cells are an innate source of interleukin-17 (IL-17), preceding the development of the adaptive T helper 17 (Th17) cell response. Here we show that IL-17-producing T cell receptor gammadelta (TCRgammadelta) T cells share characteristic features with Th17 cells, such as expression of chemokine receptor 6 (CCR6), retinoid orphan receptor (RORgammat), aryl hydrocarbon receptor (AhR), and IL-23 receptor. AhR expression in gammadelta T cells was essential for the production of IL-22 but not for optimal IL-17 production. In contrast to Th17 cells, CCR6(+)IL-17-producing gammadelta T cells, but not other gammadelta T cells, express Toll-like receptors TLR1 and TLR2, as well as dectin-1, but not TLR4 and could directly interact with certain pathogens. This process was amplified by IL-23 and resulted in expansion, increased IL-17 production, and recruitment of neutrophils. Thus, innate receptor expression linked with IL-17 production characterizes TCRgammadelta T cells as an efficient first line of defense that can orchestrate an inflammatory response to pathogen-derived as well as environmental signals long before Th17 cells have sensed bacterial invasion.
 
Article
The immune system has evolved to protect an organism against a wide variety of infectious agents, including viruses, fungi, bacteria, and multicellular parasites. These pathogens employ many different strategies to survive and multiply in the host, which can lead directly or indirectly to pathology because they interfere with homeostasis or function of affected cells and tissues. However, the immune system is quite adept in facing and handling the threats imposed by these pathogens, using an array of specialized cells and effector mechanisms. In this issue of Immunity, Lin et al. (2009) describe a unique way in which the immune system uses the strengths of two T cell subsets to eliminate the intracellular pathogen Francisella tularensis.
 
Reconstitution of IL-17-Producing ab but Not gd T Cells in Il17af-/-Hosts by Bone Marrow Transplantation with TcrdH2BeGFP gd Reporter Mice Lethally irradiated Il17af À/À mice were reconstituted with bone marrow from TcrdH2BeGFP mice. Organs were analyzed 8-14 weeks after bone marrow reconstitution. (A) Representative intracellular IL-17A staining of gd T cells in chimeras (right) compared to TcrdH2BeGFP mice (left) derived from indicated organs. Top panels exemplify the parent gates that identify H2BeGFP hi gd T cells. Thymic gd T cells were additionally gated on CD44 + CD25 À cells. Pie charts in left panels show the relative contribution of the indicated Vg chains to all IL-17 + gated gd T cells. (B) Flow cytometry analysis of gated gd T cells in chimeras (right) compared to TcrdH2BeGFP mice (left) for expression of CCR6 and CD27. (C) Frequency of either Vg1 (top)-or Vg4 (bottom)expressing cells among all gd T cells in untreated or chimeric mice. Data are representative of four independent experiments with three to six mice per group. Either representative plots (B) or mean ± SD of four (A) or pooled data from two (C) experiments is shown. Statistically significant differences were tested with the Mann-Whitney test (*p < 0.05; **p < 0.01; ***p < 0.001). See also Figure S2.
Induction of T Cell Development in Adult Indu-Rag1 3 TcrdH2BeGFP Mice Does Not Give Rise to gdT17 Cells, but to IFN-g-Producing gd T Cells (A) Schematic description of cre induction for the recombination of exon 2 of the Rag1 gene. Tamoxifen induction of cre expression rescues Rag1 gene expression in T cells whose development was blocked in the double-negative 3 stage (DN3) in the thymus. (B-E) Indu-Rag1 3 TcrdH2BeGFP mice were induced with tamoxifen for 2 weeks at the age of 710 weeks when analyzed. (B) Frequencies of gd T cells among lymphocytes in peripheral LNs in TcrdH2BeGFP mice, induced or uninduced Indu-Rag1 3 TcrdH2BeGFP mice. (C) Frequencies of different Vg chains among gd T cells in either untreated TcrdH2BeGFP, induced Indu-Rag1 3 TcrdH2BeGFP mice, or bone marrow chimeras. (D) Intestinal intraepithelial lymphocytes (iIELs) were isolated from the small intestines from either TcrdH2BeGFP, induced, or uninduced Indu-Rag1 3 TcrdH2BeGFP mice and stained for Vg7Vd6.3. Contour plots show gated gd T cells detected by their TCRb À H2BeGFP hi expression. (E) Histological analysis of epidermal sheets from ears of either TcrdH2BeGFP, induced, or uninduced Indu-Rag1 3 TcrdH2BeGFP mice stained for CD3. H2BeGFP reporter fluorescence expression is shown in green. (F) Intracellular staining for IL-17A + in gated gd T cells from Indu-Rag1 3 TcrdH2BeGFP mice (lower) compared to TcrdH2BeGFP mice (top). Data are representative from two (C, E), three (D, F), or four (B) independent experiments with 2-3 (D-F) or 3-5 (B) mice per group. (B), (C), and (D) show mean ± SD. See also Figure S4.
IL-17 Induces a Negative Feedback on IL-17-Producing gd T Cells (A and B) Lethally irradiated Il17af À/À mice (Thy1.1 + , recipients) were reconstituted with Thy1.2 + , donor bone marrow from Il17af +/+ (left) or from Il17af À/À (right). After 8-10 weeks, donor Thy1.2 + (A) or residual recipient Thy1.1 + (B) cells were separately analyzed for frequencies of CCR6 + CD27 À or CCR6 À CD27 + gd T cells. (C) Real-time qPCR with primers for Il17rc on ex vivo sorted CCR6 + CD44 hi and CD27 + CCR6 À gd T cells from mixed spleen and peripheral lymph nodes of either C57BL/6 or Il17af À/À. Each dot represents one independent experiment with 15-20 mice per sort. (D) Lethally irradiated Il17af À/À mice were reconstituted with either a 1:1 mix of WT and Il17af À/À bone marrow (middle) or a 1:1 mix of Tcra À/À and Il17af À/À bone marrow (right) and were compared to control animals (left). Intracellular IL-17A staining of gated gd T cells in peripheral lymph nodes (LNs), spleen, lung, and liver. During bone marrow reconstitution, mice were 8-12 weeks old. Error bars indicate SD (A-C). Data are representative from one (A, B) or three (C, D) independent experiments with four to five mice per group.
Article
γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.
 
Article
Toll-like receptors (TLRs) have previously been shown to play critical roles in the activation of innate immunity. Here, we describe that T cell expression of TLR2 regulates T helper 17 (Th17) cell responses. Stimulation with TLR2 agonists promoted Th17 differentiation in vitro and led to more robust proliferation and Th17 cytokine production. Using the experimental autoimmune encephalomyelitis (EAE) model, we found that TLR2 regulated Th17 cell-mediated autoimmunity in vivo and that loss of TLR2 in CD4(+) T cells dramatically ameliorated EAE. This study thus reveals a critical role of a TLR in the direct regulation of adaptive immune response and pathogenesis of autoimmune diseases.
 
Article
Patients with systemic autoimmune diseases show increased incidence of atherosclerosis. However, the contribution of proatherogenic factors to autoimmunity remains unclear. We found that atherogenic mice (herein referred to as LDb mice) exhibited increased serum interleukin-17, which was associated with increased numbers of T helper 17 (Th17) cells in secondary lymphoid organs. The environment within LDb mice was substantially favorable for Th17 cell polarization of autoreactive T cells during homeostatic proliferation, which was considerably inhibited by antibodies directed against oxidized low-density lipoprotein (oxLDL). Moreover, the uptake of oxLDL induced dendritic-cell-mediated Th17 cell polarization by triggering IL-6 production in a process dependent on TLR4, CD36, and MyD88. Furthermore, self-reactive CD4(+) T cells that expanded in the presence of oxLDL induced more profound experimental autoimmune encephalomyelitis. These findings demonstrate that proatherogenic factors promote the polarization and inflammatory function of autoimmune Th17 cells, which could be critical for the pathogenesis of atherosclerosis and other related autoimmune diseases.
 
Top-cited authors
Richard A Flavell
  • Yale University
Robert Kastelein
  • Merck & Co.
J. Fernando Bazan
  • Stanford University
Daniel Gorman
Albert Zlotnik
  • University of California, Irvine