Nature Immunology

Published by Springer Nature

Online ISSN: 1529-2916


Print ISSN: 1529-2908


La(s)t but not least
  • Article

June 2011


36 Reads

Bernard Malissen


The T cell antigen receptor is functionally coupled to many kinases and adaptor proteins. Analysis of the spatiotemporal organization of the T cell antigen receptor signaling cascade suggests that adaptor-containing intracellular vesicles are essential for proper signal propagation.

Gene-environment interactions in chronic inflammatory disease. Nat Immunol 12(4):273-277, Epub 2011/03/23
  • Literature Review
  • Full-text available

April 2011


482 Reads






Chronic inflammatory diseases represent a major challenge for both clinical research and patient care, and evidence indicates that these disorders develop as a result of complex gene-environment interactions. Better understanding of their cause-and-effect relationship is the basis for emerging proposals for therapy and prevention.

Figure 3: Kinetics of B. abortus replication.Monolayers of HeLa cells (a,b) or C57BL/6 mouse peritoneal macrophages (c,d) were inoculated with a standardized bacterial suspension of B. abortus 2308 virulent strain (open bars), cgs- BvI129 mutant (filled bars) or CG-treated cgs- BvI129 (gray bars). After washing and gentamicin treatment, the 'fold increase' in brucella (as colony-forming units per ml; a,c) and the percentage of infected cells (b,d) were determined (time, horizontal axes). Values are averages s.e.m. for triplicate samples.
Figure 4: Kinetics of cathepsin D, Lamp1 and calreticulin acquisition by BCVs.HeLa cells were infected with B. abortus strains: 2308 virulent strain (open squares), cgs- BvI129 mutant (open circles) and CG-treated cgs- BvI129 (filled circles). Colocalization percentages for cathepsin D (a), Lamp1 (b) and calreticulin (c) were obtained from confocal microscopy images. Data represent mean values from three independent experiments; bars indicate averages of the absolute deviations of data points from their means. In all experiments, 100 vacuoles of each strain were analyzed for each time point.
Figure 7: Accumulation of flotillin-1 in the vicinity of BCVs.HeLa cells were infected for 20 min with the 2308 virulent strain, cgs- BvI129 mutant or CG-treated cgs- BvI129. Cells were washed and were incubated further in cultures supplemented with gentamicin. Cells were then fixed and were immunostained for brucella (green) and flotilin-1 (red). Scale bars, 20 m. Arrows indicate BCV area. Representative of three independent experiments.
Figure 8: CG and MCD prevent lysosome fusion independently of the VirB type IV secretion system.HeLa cells were infected as described in with brucella left untreated or treated with 1 mM CG (+ CG) or 0.5 mM MCD (+ CD). (a) Kinetics of acquisition of cathepsin D by BCVs. (b) Intracellular, gentamicin-protected bacteria in cells lysed 24 h after inoculation. The 'fold increase' was calculated as the ratio of the intracellular colony-forming units at 24 h and 1 h after inoculation. Values represent the means s.e.m. of three independent experiments. (c) Percentage of BCVs positive for cathepsin D in HeLa cells at 24 h after infection with wild-type bacteria, cgs BvI129 mutant (cgs-), the brucella virB- polar mutant (VirBp), the brucella virB- nonpolar mutant (VirBnp), the double brucella mutant, polar virB-;cgs- BvI129 (VirBp-;cgs-) or the double brucella mutant, nonpolar virB-;cgs- BvI129 (VirBnp-;cgs-). In all experiments, 100 vacuoles of each strain were analyzed for each time point. (d) Distribution of cathepsin D in cells infected with polar or nonpolar virB-;cgs- BvI129 and treated with 1 mM CG. Fixed cells were immunostained for brucella (green) and cathepsin D (red). Arrows indicate BCV area. Representative of three independent experiments. Scale bars, 10 m (main images) or 1 m (enlarged images, bottom row).
Cyclic beta-1,2-glucan is a Brucella virulence factor required for intracellular survival

July 2005


476 Reads

Pathogenic brucella bacteria have developed strategies to persist for prolonged periods of time in host cells, avoiding innate immune responses. Here we show that the cyclic beta-1,2-glucans (CbetaG) synthesized by brucella is important for circumventing host cell defenses. CbetaG acted in lipid rafts found on host cell membranes. CbetaG-deficient mutants failed to prevent phagosome-lysosome fusion and could not replicate. However, when treated with purified CbetaG or synthetic methyl-beta-cyclodextrin, the mutants were able to control vacuole maturation by avoiding lysosome fusion, and this allowed intracellular brucella to survive and reach the endoplasmic reticulum. Fusion between the endoplasmic reticulum and the brucella-containing vacuole depended on the brucella virulence type IV secretion system but not on CbetaG. Brucella CbetaG is thus a virulence factor that interacts with lipid rafts and contributes to pathogen survival.

Figure 1: Development of Itpkb-/- B lymphocytes.Flow cytometry of wild-type (WT) and Itpkb-/- bone marrow and spleen B lymphocytes, stained with various antibodies. (a) Total numbers of each B lymphocyte subset in the bone marrow (BM). Populations defined according to the Hardy gating scheme40: pre-pro-B, B220+CD43+BP-1-HSA-; pro-B, B220+CD43+BP-1-HSA+; early pre-B, B220+CD43+BP-1+HSA+; late pre-B, B220+CD43-IgM-IgD-; immature (Imm) pre-B, B220+CD43-IgM+IgD-; and recirculating (Recirc) B cells, B220+CD43-IgM+IgD+. *, P < 0.05. (b) Total numbers of each B lymphocyte (B220+) subset in the spleen. T1, IgM+IgD-CD21-CD23-; T2, IgM+IgD+CD21+CD23+; follicular mature (FM), IgMloIgD+CD21mid; CD138+, IgMloIgD+CD138+; and marginal zone (MZ), IgM+IgD+CD21+CD23-. P < 0.01 for all populations except CD138+ cells, which were not significantly different. (c) Gating schemes; numbers in plots indicate percentages of each gated population. (d) Expression of the pre–plasma lymphocyte marker CD138 by B lymphocytes. Numbers in quadrants indicate percent IgM+ or IgD+ cells (right quadrants) or IgM- or IgD- cells (left quadrants) with (top quadrants) or without (bottom quadrants) CD138 expression. (e) Fraction of mature B lymphocytes in the follicular mature, CD138 or marginal zone subsets. (f) Quantification of IgM on immature (B220+IgMhiIgD-) and mature (B220+IgMloIgD+) B lymphocytes. Below, mean fluorescence intensity (MFI) of IgM staining. (g) Total numbers of peritoneal B-1 B cells among peritoneal lymphocytes counted and stained with antibodies specific for IgM, B220, CD43, CD23 and CD5; B-1 lymphocytes were gated as IgM+B220+CD43+CD23-, and CD5 expression distinguished B-1a (CD5+) and B-1b (CD5-) lymphocytes. Data are representative of at least five independent experiments.
Figure 4: Impaired T cell–independent antibody responses to TNP-Ficoll immunization in Itpkb-/- mice.ELISA of TNP-specific IgM antibody responses (a) and TNP-specific IgG3 antibody responses (b) on days 0 and 7 after TNP-Ficoll immunization of wild-type mice (n = 7) and Itpkb-/- mice (n = 4). Data represent two independent experiments.
Corrigendum: Production of Ins(1,3,4,5)P4 mediated by the kinase Itpkb inhibits store-operated calcium channels and regulates B cell selection and activation

June 2007


224 Reads

Antigen receptor-mediated production of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in lymphocytes triggers the release of Ca2+ from intracellular stores; this release of Ca2+ results in the opening of store-operated Ca2+ channels in the plasma membrane. Here we report that mice lacking Ins(1,4,5)P3 3-kinase B (Itpkb), which converts Ins(1,4,5)P3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), had impaired B lymphocyte development and defective immunoglobulin G3 antibody responses to a T lymphocyte-independent antigen. Itpkb-deficient B lymphocytes had the phenotypic and functional features of tolerant B lymphocytes and showed enhanced activity of store-operated Ca2+ channels after B lymphocyte receptor stimulation, which was reversed by the provision of exogenous Ins(1,3,4,5)P4. Our data identify Itpkb and its product Ins(1,3,4,5)P4 as inhibitors of store-operated Ca2+ channels and crucial regulators of B cell selection and activation.

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development

December 2003


112 Reads

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.

Neutra, M.R., Mantis, N.J. & Kraehenbuhl, J.-P. Collaboration of epithelial cells with organized mucosal lymphoid tissues. Nat. Immunol. 2, 1004-1009

December 2001


104 Reads

Immune surveillance of mucosal surfaces requires the delivery of intact macromolecules and microorganisms across epithelial barriers to organized mucosal lymphoid tissues. Transport, processing and presentation of foreign antigens, as well as local induction and clonal expansion of antigen-specific effector lymphocytes, involves a close collaboration between organized lymphoid tissues and the specialized follicle-associated epithelium. M cells in the follicle-associated epithelium transport foreign macromolecules and microorganisms to antigen-presenting cells within and under the epithelial barrier. Determination of the earliest cellular interactions that occur in and under the follicle-associated epithelium could greatly facilitate the design of effective mucosal vaccines in the future.

Gajewski TF, Schreiber H, Fu YXInnate and adaptive immune cells in the tumor microenvironment. Nat Immunol 14(10): 1014-1022

October 2013


308 Reads

Most tumor cells express antigens that can mediate recognition by host CD8(+) T cells. Cancers that are detected clinically must have evaded antitumor immune responses to grow progressively. Recent work has suggested two broad categories of tumor escape based on cellular and molecular characteristics of the tumor microenvironment. One major subset shows a T cell-inflamed phenotype consisting of infiltrating T cells, a broad chemokine profile and a type I interferon signature indicative of innate immune activation. These tumors appear to resist immune attack through the dominant inhibitory effects of immune system-suppressive pathways. The other major phenotype lacks this T cell-inflamed phenotype and appears to resist immune attack through immune system exclusion or ignorance. These two major phenotypes of tumor microenvironment may require distinct immunotherapeutic interventions for maximal therapeutic effect.

Spilianakis, C.G. & Flavell, R.A. Long-range intrachromosomal interactions in the T helper type 2 cytokine locus. Nat. Immunol. 5, 1017-1027

November 2004


165 Reads

The T helper type 2 (T(H)2) locus control region is important in the regulation of the genes encoding the cytokines interleukins 4, 5 and 13. Using the chromosome conformation capture technique, we found that in T cells, natural killer cells, B cells and fibroblasts, the promoters for the genes encoding T(H)2 cytokines are located in close spatial proximity, forming an initial chromatin core configuration. In CD4(+) T cells and natural killer cells, but not B cells and fibroblasts, the T(H)2 locus control region participates in this configuration. The transcription factors GATA3 and STAT6 are essential for the establishment and/or maintenance of these interactions. Intrachromosomal interactions in the T(H)2 cytokine locus may form the basis for the coordinated transcriptional regulation of cytokine-encoding genes by the T(H)2 locus control region.

Figure 1: IL-12 acts locally in a paracrine manner.(a) Repression of subcutaneous tumors in wild-type (WT) mice given subcutaneous injection of 2 × 105 B16.F10, B16–IL-12 or B16 cells (n = 6 mice per group). (b) Repression of tumors in wild-type mice given subcutaneous injection of a mixture of B16 cells and B16–IL-12 cells at a ratio of 1:1, 2:1 or 10:1 (n = 5 mice per group). (c) Repression of tumors in wild-type or Il12rb2−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 6 mice per group). (d) Tumor repression in wild-type mice given subcutaneous injection of 2 × 105 B16 cells and treated intraperitoneally (i.p.) with PBS or recombinant IL-12 (rIL-12) on days 1, 3, 5 and 9 after tumor injection (n = 3 mice per group). (e) Tumor repression in wild-type mice given subcutaneous injection of 2 × 105 B16 cells in the right abdomen and 2 × 105 B16–IL-12 cells in the left contralateral abdomen (n = 6 mice per group). (f) Tumor repression in wild-type mice given subcutaneous injection of 2 × 105 B16 cells and intratumoral (i.t.) injection of PBS or recombinant IL-12 on days 7, 9, 12, 14, 16 and 19 after tumor injection (n = 3 mice per group). Additional data, Supplementary  NS, not significant. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of four (a), three (c,e) or two (b,d,f) experiments (mean and s.e.m.).
Figure 2: IL-12 elicits the recruitment of leukocytes into the tumor mass.(a) Immunohistochemistry of frozen tumor sections obtained from wild-type mice 3 weeks after challenge with B16 or B16–IL-12 cells and stained with anti-CD4 (CD4), anti-CD8 (CD8) and anti–asialo GM1 (NK). Scale bar, 50 μm. (b) Cytofluorometry of tumor-invading leukocytes in mice treated as in a, assessed in the entire tumor mass after exclusion of cellular debris, dead cells and duplets, and presented as tumor-infiltrating CD45+ leukocytes relative to CD45− melanoma cells. Each symbol represents an individual mouse; long horizontal lines indicate the mean (short lines, s.e.m.). (c–i) Cytofluorometry of tumor-invading leukocytes in mice treated as in a, presented as the frequency of infiltrating CD8+ cells (c), CD4+ cells (d), CD11c+ cells (e), CD11b+ (f), NK1.1+ cells (g), 1A8+ cells (h) and CD19+ cells (i), gated on CD45+ cells, in B16 tumors relative to that in B16–IL-12 tumors. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of two experiments with at least four mice per group (mean and s.e.m. in b–i).
Figure 3: IL-12-mediated repression of subcutaneous tumor acts independently of T cells, B cells, NKT cells and cNK cells.(a) Tumor repression in wild-type and Rag1−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 6 mice per group). (b) Tumor repression in Rag1−/− and anti-NK1.1 (α-NK1.1)-treated Rag1−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 3 mice per group; additional data, Supplementary ). (c) Tumor repression in Rag1−/− and anti-GM1 (α-GM1)-treated Rag1−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 5 mice per group; additional data, Supplementary ). (d) Tumor repression in wild-type and Il15ra−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 6 mice per group). (e) Lung metastasis in Rag1−/− mice given intravenous injection of 1 × 105 B16 or B16–IL-12 cells (n = 6 mice per group), followed by depletion of NK cells with anti-NK1.1 (three times per week); metastases were counted after 21 d (additional data, Supplementary ). *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of four (a) or two (b–e) experiments (mean and s.e.m.).
Eisenring M, vom Berg J, Kristiansen G, Saller E, Becher BIL-12 initiates tumor rejection via lymphoid tissue-inducer cells bearing the natural cytotoxicity receptor NKp46. Nat Immunol 11: 1030-1038

October 2010


397 Reads

The potent tumoricidal activity of interleukin 12 (IL-12) is thought to be mediated by the activation and polarization of natural killer (NK) cells and T helper type 1 (T(H)1) cells, respectively. By systematic analysis of the IL-12-induced immune response to subcutaneous melanoma (B16), we found that tumor suppression was mediated independently of T lymphocytes or NK cells. IL-12 initiated local antitumor immunity by stimulating a subset of NKp46(+) lymphoid tissue-inducer (LTi) cells dependent on the transcription factor RORγt. The presence of these NKp46(+) LTi cells induced upregulation of adhesion molecules in the tumor vasculature and resulted in more leukocyte invasion. Thus, this innate cell type is responsive to IL-12 and is a powerful mediator of tumor suppression.

Figure 2: Expression of pancreatic autoantigens in mTECs versus thymic or splenic DCs.Expression of three pancreatic autoantigens in purified mTECs, thymic DCs and splenic myeloid and lymphoid DCs was assessed by RT-PCR. A fivefold cDNA dilution-series from the indicated populations were used for amplification.
Figure 3: Protein expression of tissue-antigens by mTECs.Protein expression of (a,b) PLP (c,d) insulin and (e,f) P1A by purified mTECs. Control staining included (a) secondary antibody alone for PLP (c) specific blockade by free insulin for insulin and (e) preimmune serum for P1A. mTEC protein expression was heterogenous. Bar, 20 m.
Figure 5: Promiscuous gene expression during ontogeny.Thymic expression of five tissue-specific genes was monitored during ontogeny. Expression of all genes tested was detectable at embryonic day 15 (E15) and was fully maintained into late adulthood. Due to different mTEC contents in the different preparations (note that prenatally unseparated thymus and postnatally purified mTEC were analyzed) approximately 30-fold more embryonic thymus cDNA was used in the RT-PCRs. mTEC expression in 8-week-old mice was arbitrarily defined as 1.0.
Figure 6: Promiscuous expression of a fetal tissue antigen.Pre- and postnatal expression of -fetoprotein was compared in hepatocytes and thymi. Each sample was appropriately diluted to determine the relative signal strength within the linear amplification range.
Derbinski J, Schulte A, Kyewski B, Klein L.. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat Immunol 2: 1032-1039

December 2001


644 Reads

Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity. Here we identified medullary thymic epithelial cells as being a unique cell type that expresses a diverse range of tissue-specific antigens. We found that this promiscuous gene expression was a cell-autonomous property of medullary epithelial cells and was maintained during the entire period of thymic T cell output. It may facilitate tolerance induction to self-antigens that would otherwise be temporally or spatially secluded from the immune system. However, the array of promiscuously expressed self-antigens appeared random rather than selected and was not confined to secluded self-antigens.

Jensen, PE. Recent advances in antigen processing and presentation. Nat Immunol 8: 1041-1048

November 2007


70 Reads

Heterogeneous intracellular pathways and biochemical mechanisms are responsible for generating the glycoprotein complexes of peptide and major histocompatibility complex that are displayed on the surfaces of antigen-presenting cells for recognition by T lymphocytes. These pathways have a profound influence on the specificity of adaptive immunity and tolerance, as well as the context and consequences of antigen recognition by T cells in the thymus and periphery. The field of antigen processing and presentation has continued to advance since the publication of a focus issue on the topic in Nature Immunology in July 2004. Progress has been made on many fronts, including advances in understanding how proteases, accessory molecules and intracellular pathways influence peptide loading and antigen presentation in various cell types.

Jun, J.E. & Goodnow, C.C. Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling. Nat. Immunol. 4, 1057-1064

December 2003


30 Reads

Lymphocyte antigen receptors are responsible for inducing the opposite responses of immunity or tolerance. How the correct polarity of antigen receptor signaling is encoded has been an enduring enigma. Here we summarize recent advances defining key scaffolding molecules, CARMA1 (also known as CARD11) and the Cbl family of ubiquitin ligases, required for either immunogenic or tolerogenic signaling by antigen receptors. These scaffolding proteins may determine the polarity of response to antigen by promoting assembly around antigen receptors of competing multiprotein signal complexes: immunosomes versus tolerosomes. Each of the factors that influence immunogenicity or tolerogenicity--stage of lymphocyte differentiation, concurrent engagement of inhibitory or costimulatory receptors, extent of receptor crosslinking, and prior antigen experience--may be integrated in lymphocytes through their capacity to influence the probability of assembling immunosomes versus tolerosomes.

Figure 7: MCMV infection altered the secretion of IL-12 and IL-2 by DCs.(a) Concentrations of IL-12 secreted after LPS treatment or MCMV infection of D1 cells. LPS (10 g/ml) or MCMV (high MOI, >3 PFU/cell) were added on day 0 and cells cultured for the indicated times. In all cases cells were plated into fresh medium 48 h before collection of supernatants for analysis. (b) Concentration of IL-12 secreted by splenic DCs collected from control or MCMV-infected mice (days 1, 2 or 4 after infection) and cultured in the presence of LPS for 18 h. (c) IL-2 secreted by MCMV-infected D1 cells treated with LPS for various periods of time. The concentration of IL-2 secreted by control and uninfected LPS-treated D1 is shown for comparison. (d) Concentration of IL-2 secreted by splenic DCs after LPS treatment. DCs were collected from control or MCMV-infected mice (days 1 or 2 after infection) and cultured in the presence of LPS for 6 or 18 h before collection of supernatants for cytokine assays.
Figure 8: MCMV infection of DCs impaired their allostimulatory capacity.The allostimulatory capacity of D1 cells infected with MCMV for (a) 2 or (b) 4 days and (c) that of DCs purified from the spleens of mice infected with MCMV for 4 days. D1 cells or purified splenic DCs activated with LPS (10 g/ml for 48 h) were used as controls. For the D1 (H2b, I-Ab) experiments, splenocytes from BALB/c (H2d, I-Ad) mice were used as the allogeneic responders and splenocytes from C57BL/6 mice were used as the syngeneic controls. Conversely, in experiments where the stimulators were DCs purified from BALB/c mice, splenocytes from C57BL/6 mice were used as the allogeneic responders and splenocytes from BALB/c mice were used as the syngeneic controls. APC, antigen-presenting cell.
Andrews, D.M., Andoniou, C.E., Granucci, F., Ricciardi-Castagnoli, P. & Degli-Esposti, M.A. Infection of dendritic cells by murine cytomegalovirus induces functional paralysis. Nat. Immunol. 2, 1077-1084

December 2001


130 Reads

Cytomegalovirus (CMV), measles and HIV are the main human pathogens known to induce immunosuppression. Unlike measles and HIV, and despite the availability of a well studied animal model, little is known about the mechanisms that control CMV-induced immunosuppression. We hypothesized that dendritic cells (DCs), which are crucial in generating and maintaining immune responses, represent a target for CMV and that the transient, but profound, immunosuppression that accompanies CMV infection results from viral interference with DC functions. Here we show that DCs were permissive to murine CMV infection. In addition, DC infection prevented delivery of the signals required for T cell activation. Thus, CMV-mediated impairment of DC function may be crucial for virally induced immunosuppression and interleukin 2 is implicated as a key factor.

Murre C.. Helix-loop-helix proteins and lymphocyte development. Nat Immunol 6: 1079-1086

December 2005


43 Reads

Helix-loop-helix (HLH) proteins are transcriptional regulators that control a wide variety of developmental pathways in both invertebrate and vertebrate organisms. Results obtained in the past decade have shown that HLH proteins also contribute to the development of lymphoid lineages. A subset of HLH proteins, the 'E proteins', seems to be particularly important for proper lymphoid development. Members of the E protein family include E12, E47, E2-2 and HEB. The E proteins contribute to B lineage- and T lineage-specific gene expression programs, regulate lymphocyte survival and cellular proliferation, activate the rearrangement of antigen receptor genes and control progression through critical developmental checkpoints. This review discusses HLH proteins in lymphocyte development and homeostasis.

Figure 1. miR-10a is highly expressed in nT reg cells and is induced by RA and TGF-β. (a) miR-10a expression was evaluated by isolating total RNA from different T cell subsets and assessing expression by quantitative RT-PCR. (b-g) Naïve CD4 + T cells were stimulated with antiCD3 and anti-CD28 antibodies and miR-10a expression was determined in cells cultured in (b) medium alone, IL-2 (50 U/ml) or TGF-β (20 ng/ml), (c) with varying concentrations of TGF-β, (d) varying concentrations of ATRA with TGF-β (20 ng/ml, solid circles) or antiTGF-β antibody (solid squares), or (e) anti-TGF-β antibody, TGF-β (20 ng/ml) or LE540 (pan-RAR antagonist, 1 μM). (f) Expression of RARα was assessed by quantitative RTPCR after stimulating cells with anti-CD3 and anti-CD28 antibodies and varying concentrations of TGF-β. (g) miR-10a levels were determined in isolated naïve CD4 + T cells that were stimulated with anti-CD3 and-CD28 antibodies in the presence of TGF-β alone or with ATRA (1 μM), AM580 (RARα agonist, 1 μM) or A7980 (RARγ agonist, 1 μM) as indicated. All experiments were performed in triplicate. Statistical significance was determined by t-test. * indicates p < 0.05, ** indicates p < 0.01. Similar results were obtained in two (a, c, e, f, g) or three (b, d) independent experiments.
Figure 4. Retinoic acid has biphasic effects on T H 17 differentiation. (a-d) Naïve CD4 + T cells cultured under conditions to generate T H 17 cells (IL-6, TGF-β and anti-IFN-γ antibody) or (IL-6, TGF-β, anti-IFN-γ antibody and anti-IL-2 antibody) and iT reg cells (IL-2, TGF-β and anti-IFN-γ antibody) along with varying concentrations of ATRA and analyzed for expression of IL-17A and Foxp3 by flow cytometry. The effect of ATRA on the proportion of Foxp3 expressing-(a) and IL-17A producing-(b) cells over a wide range of doses. Representative results from three independent experiments are shown in the left panel (a and b). Each data point represents the mean ± SEM (n ≧ 3). A representative experiment is shown in the middle panels. Pooled data from five (iT reg and T H 17 (anti-IL-2) conditions) or seven (T H 17 condition) independent experiments are shown in the right panels. P values were determined by paired sample t-test. (c, d) The effects of ATRA and varying concentrations of TGF-β on IL-17A production. (c) Isolated naïve CD4 + T cells cultured under conditions (IL-6, anti-IFN-γ antibody and anti-IL-2 antibody) to generate T H 17 cells with varying concentrations of ATRA and TGF-β. * indicates P < 0.05 for all conditions. (d) The increase over basal expression represents a ratio determined by the difference in the percentage of IL-17A producing cells in the presence and absence of ATRA (10 nM) for each concentration of TGF-β divided by the percentage of IL-17A-producing cells generated without ATRA. P values were determined by t-test (c, d). Each data point represents the mean ± SEM (n ≧ 3) and similar results were obtained in two independent experiments.
Figure 6. miR-10a constrains T H 17 differentiation in the presence of RA. (a) Naïve CD4 + T cells were transduced with a retroviral construct expressing a miR-10a-5p "sponge" target sequence (miR10a-5pT) or a scrambled sequence (control) and cultured under T H 17 conditions with anti-IL-2 antibody and ATRA (1.5 nM). (b, c) Naïve CD4 + T cells were transduced with a retroviral construct expressing miR-10a or control vector under T H 17 conditions and anti-IL-2 with (b) or without ATRA (1.5 nM) (c). Cells were analyzed for Foxp3 and IL-17A expressions by flow cytometry. A representative experiment is depicted in the left panel and pooled data from multiple independent experiments are shown in the right panels. Statistical significance was determined by paired sample t-test.
Figure 7. Ncor2 and Bcl-6 regulate IL-17A in a T-bet-dependent manner. (a, b) Naïve CD4 + T cells from wild-type mice (a) or Tbx21 −/− mice (b) were transduced with retroviral vectors encoding shRNAs for Ncor2 (iNcor2), Bcl-6 (iBcl6) or a control vector and cultured under T H 17 conditions with anti-IL-2 antibody + ATRA (1.5 nM). IL-17A and Foxp3 expression was assessed by flow cytometry. Representative contour plots are shown on the left and pooled data (n = 4) from this experiment are shown on the right. (c, d) Naïve CD4 + T cells were cultured under T H 17 conditions and anti-IL-2 antibody with or without ATRA at the indicated concentrations. Cells were analyzed for T-bet expression by immunoblotting (c) and flow cytometry (d). Experiments were repeated twice (a, b) or three (c, d) times with similar results.
Figure 8: Ncor2 and Bcl-6 regulate IL-17A in a T-bet-dependent manner. (a,b) Expression of IL-17A and Foxp3 by naive CD4+ T cells obtained from wild-type (WT) mice (a) or T-bet-deficient (Tbx21−/−) mice (b) and transduced with retroviral vector encoding short hairpin RNA specific for Ncor2 (iNcor2) or Bcl-6 (iBcl6) or a control vector and cultured under TH17 conditions with anti-IL-2 plus 1.5 nM ATRA, assessed by flow cytometry (left). Right, frequency of IL-17A+ cells; each symbol represents a T cell culture from a mouse, and small horizontal lines indicate the mean of quadruplicates. (c,d) T-bet expression in naive CD4+ T cells cultured under TH17 conditions and anti-IL-2 without ATRA (0 nM) or with 100 nM ATRA (c) or various concentrations of ATRA (d), assessed by immunoblot analysis (c) or flow cytometry (d). Bottom (c), quantification of T-bet relative to actin (by densitometry). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of two (a,b) or three (c,d) experiments with similar results (error bars, s.e.m.).
TGF-?? and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and constrains the plasticity of helper T cells

April 2012


232 Reads

Distinct CD4(+) T cell subsets are critical for host defense and immunoregulation. Although these subsets can act as terminally differentiated lineages, they have been increasingly noted to demonstrated plasticity. MicroRNAs are factors that control T cell stability and plasticity. Here we report that naturally occurring regulatory T cells (T(reg) cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-β (TGF-β) in inducible T(reg) cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, miR-10a attenuated the phenotypic conversion of inducible T(reg) cells into follicular helper T cells. We also found that miR-10a limited differentiation into the T(H)17 subset of helper T cells and therefore represents a factor that can fine-tune the plasticity and fate of helper T cells.

Cyster, J.G. B cell follicles and antigen encounters of the third kind. Nat. Immunol. 11, 989-996

November 2010


146 Reads

Defining where and in what form lymphocytes encounter antigen is fundamental to understanding how immune responses occur. Although knowledge of the recognition of antigen by CD4(+) and CD8(+) T cells has advanced greatly, understanding of the dynamics of B cell-antigen encounters has lagged. With the application of advanced imaging approaches, encounters of this third kind are now being brought into focus. Multiple processes facilitate these encounters, from the filtering functions of lymphoid tissues and migration paths of B cells to the antigen-presenting properties of macrophages and follicular dendritic cells. This Review will discuss how these factors work together in the lymph node to ensure efficient and persistent exposure of B cells to diverse forms of antigen and thus effective triggering of the humoral response.

Figure 1: Binding of DC-SIGN by gp120 is essential for early HIV-1 transcription. (a) Quantitative real-time PCR analysis of Tat-Rev mRNA expression in DCs infected for 6 or 24 h with HIV-1 BaL in the presence (Chx) or absence (DMSO (dimethyl sulfoxide)) of the translation inhibitor cycloheximide. (b) Quantitative real-time PCR analysis of Tat-Rev mRNA expression in DCs infected for 6 h with CCR5-tropic HIV-1 (BaL) in the presence or absence (−) of blocking antibody to (α-) DC-SIGN, CD4 or CCR5. (c) Quantitative real-time PCR analysis of Tat-Rev mRNA expression in DCs infected for 6 h with VSV-G-pseudotyped HIV-1 (VSV-G) in the presence or absence of blocking antibodies as in b and simultaneously stimulated with the DC-SIGN ligands gp120 or ManLAM. (d) HIV-1 integration into DCs infected for 6 h with HIV-1 BaL or VSV-G-pseudotyped HIV-1, determined by Alu-PCR and presented relative to HIV-1 integration in HIV-1 BaL–infected cells, set as 1. (e) Quantitative real-time PCR analysis of Tat-Rev mRNA expression in DCs infected for 6 h with VSV-G-pseudotyped HIV-1 in the presence (XL α-DC-SIGN) or absence (–) of crosslinked anti-DC-SIGN (H-200). Tat-Rev mRNA expression (a–c,e) is presented relative to the expression of GAPDH (glyceraldehyde phosphate dehydrogenase). Data are representative of at least four (a–c) or two (d,e) independent experiments (mean and s.d.).
Gringhuis, SI, van der Vlist, M, van den Berg, LM, den Dunnen, J, Litjens, M and Geijtenbeek, TB. HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells. Nat Immunol 11: 419-426

April 2010


189 Reads

Pattern-recognition receptors (PRRs) elicit antiviral immune responses to human immunodeficiency virus type 1 (HIV-1). Here we show that HIV-1 required signaling by the PRRs Toll-like receptor 8 (TLR8) and DC-SIGN for replication in dendritic cells (DCs). HIV-1 activated the transcription factor NF-kappaB through TLR8 to initiate the transcription of integrated provirus by RNA polymerase II (RNAPII). However, DC-SIGN signaling was required for the generation of full-length viral transcripts. Binding of the HIV-1 envelope glycoprotein gp120 to DC-SIGN induced kinase Raf-1-dependent phosphorylation of the NF-kappaB subunit p65 at Ser276, which recruited the transcription-elongation factor pTEF-b to nascent transcripts. Transcription elongation and generation of full-length viral transcripts was dependent on pTEF-b-mediated phosphorylation of RNAPII at Ser2. Inhibition of either pathway abrogated replication and prevented HIV-1 transmission. Thus, HIV-1 subverts crucial components of the immune system for replication that might be targeted to prevent infection and dissemination.

Ziegler SF, Artis D.Sensing the outside world: TSLP regulates barrier immunity. Nat Immunol 11:289-293

March 2010


109 Reads

Thymic stromal lymphopoietin (TSLP) is an interleukin 7 (IL-7)-like cytokine originally characterized by its ability to promote the activation of B cells and dendritic cells (DCs). Subsequent studies have shown that TSLP promotes T helper type 2 (TH2) cell responses associated with immunity to some helminth parasites and the pathogenesis of many inflammatory diseases, including atopic dermatitis and asthma. This review will focus on recent findings indicating that in addition to influencing B cell and DC function, TSLP can promote TH2 cytokine-associated inflammation by directly promoting the effector functions of CD4+ TH2 cells, basophils and other granulocyte populations while simultaneously limiting the expression of DC-derived proinflammatory cytokines and promoting regulatory T cell responses in peripheral tissues.

Doulatov, S, Notta, F, Eppert, K, Nguyen, LT, Ohashi, PS and Dick, JE. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat Immunol 11: 585-593

July 2010


183 Reads

The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. Here we provide a comprehensive analysis of the human hematopoietic hierarchy by clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow. Human multilymphoid progenitors, identified as a distinct population of Thy-1(neg-lo)CD45RA(+) cells in the CD34(+)CD38(-) stem cell compartment, gave rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells, which indicated that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation.

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