Publications by authors named "Giorgio Napolitani"

31 Publications

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Cell Rep 2021 May;35(6):109101

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.
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http://dx.doi.org/10.1016/j.celrep.2021.109101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131582PMC
May 2021

Vi-Vaccinations Induce Heterogeneous Plasma Cell Responses That Associate With Protection From Typhoid Fever.

Front Immunol 2020 3;11:574057. Epub 2020 Dec 3.

The Oxford Vaccine Group, Department of Paediatrics, University of Oxford, and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.

Vi-polysaccharide conjugate vaccines are efficacious against cases of typhoid fever; however, an absolute correlate of protection is not established. In this study, we investigated the leukocyte response to a Vi-tetanus toxoid conjugate vaccine (Vi-TT) in comparison with a plain polysaccharide vaccine (Vi-PS) in healthy adults subsequently challenged with Typhi. Immunological responses and their association with challenge outcome was assessed by mass cytometry and Vi-ELISpot assay. Immunization induced significant expansion of plasma cells in both vaccines with modest T follicular helper cell responses detectable after Vi-TT only. The Vi-specific IgG and IgM B cell response was considerably greater in magnitude in Vi-TT recipients. Intriguingly, a significant increase in a subset of IgA plasma cells expressing mucosal migratory markers α4β7 and CCR10 was observed in both vaccine groups, suggesting a gut-tropic, mucosal response is induced by Vi-vaccination. The total plasma cell response was significantly associated with protection against typhoid fever in Vi-TT vaccinees but not Vi-PS. IgA plasma cells were not significantly associated with protection for either vaccine, although a trend is seen for Vi-PS. Conversely, the IgA fraction of the plasma cell response was only associated with protection in Vi-TT. In summary, these data indicate that a phenotypically heterogeneous response including both gut-homing and systemic antibody secreting cells may be critical for protection induced by Vi-TT vaccination.
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http://dx.doi.org/10.3389/fimmu.2020.574057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793947PMC
June 2021

Potential CD8+ T Cell Cross-Reactivity Against SARS-CoV-2 Conferred by Other Coronavirus Strains.

Front Immunol 2020 5;11:579480. Epub 2020 Nov 5.

MRC Human Immunology Unit, Medical Research Council (MRC) Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

While individuals infected with coronavirus disease 2019 (COVID-19) manifested a broad range in susceptibility and severity to the disease, the pre-existing immune memory to related pathogens cross-reactive against SARS-CoV-2 can influence the disease outcome in COVID-19. Here, we investigated the potential extent of T cell cross-reactivity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be conferred by other coronaviruses and influenza virus, and generated an map of public and private CD8+ T cell epitopes between coronaviruses. We observed 794 predicted SARS-CoV-2 epitopes of which 52% were private and 48% were public. Ninety-nine percent of the public epitopes were shared with SARS-CoV and 5.4% were shared with either one of four common coronaviruses, 229E, HKU1, NL63, and OC43. Moreover, to assess the potential risk of self-reactivity and/or diminished T cell response for peptides identical or highly similar to the host, we identified predicted epitopes with high sequence similarity with human proteome. Lastly, we compared predicted epitopes from coronaviruses with epitopes from influenza virus deposited in IEDB, and found only a small number of peptides with limited potential for cross-reactivity between the two virus families. We believe our comprehensive profile of private and public epitopes across coronaviruses would facilitate design of vaccines, and provide insights into the presence of pre-existing coronavirus-specific memory CD8+ T cells that may influence immune responses against SARS-CoV-2.
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http://dx.doi.org/10.3389/fimmu.2020.579480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676914PMC
December 2020

Predicting Cross-Reactivity and Antigen Specificity of T Cell Receptors.

Front Immunol 2020 22;11:565096. Epub 2020 Oct 22.

MRC Human Immunology Unit, Medical Research Council (MRC) Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

Adaptive immune recognition is mediated by specific interactions between heterodimeric T cell receptors (TCRs) and their cognate peptide-MHC (pMHC) ligands, and the methods to accurately predict TCR:pMHC interaction would have profound clinical, therapeutic and pharmaceutical applications. Herein, we review recent developments in predicting cross-reactivity and antigen specificity of TCR recognition. We discuss current experimental and computational approaches to investigate cross-reactivity and antigen-specificity of TCRs and highlight how integrating kinetic, biophysical and structural features may offer valuable insights in modeling immunogenicity. We further underscore the close inter-relationship of these two interconnected notions and the need to investigate each in the light of the other for a better understanding of T cell responsiveness for the effective clinical applications.
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http://dx.doi.org/10.3389/fimmu.2020.565096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642207PMC
May 2021

Homologous and heterologous re-challenge with Salmonella Typhi and Salmonella Paratyphi A in a randomised controlled human infection model.

PLoS Negl Trop Dis 2020 10 20;14(10):e0008783. Epub 2020 Oct 20.

Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, United Kingdom.

Enteric fever is a systemic infection caused by Salmonella Typhi or Paratyphi A. In many endemic areas, these serovars co-circulate and can cause multiple infection-episodes in childhood. Prior exposure is thought to confer partial, but incomplete, protection against subsequent attacks of enteric fever. Empirical data to support this hypothesis are limited, and there are few studies describing the occurrence of heterologous-protection between these closely related serovars. We performed a challenge-re-challenge study using a controlled human infection model (CHIM) to investigate the extent of infection-derived immunity to Salmonella Typhi or Paratyphi A infection. We recruited healthy volunteers into two groups: naïve volunteers with no prior exposure to Salmonella Typhi/Paratyphi A and volunteers previously-exposed to Salmonella Typhi or Paratyphi A in earlier CHIM studies. Within each group, participants were randomised 1:1 to oral challenge with either Salmonella Typhi (104 CFU) or Paratyphi A (103 CFU). The primary objective was to compare the attack rate between naïve and previously challenged individuals, defined as the proportion of participants per group meeting the diagnostic criteria of temperature of ≥38°C persisting for ≥12 hours and/or S. Typhi/Paratyphi bacteraemia up to day 14 post challenge. The attack-rate in participants who underwent homologous re-challenge with Salmonella Typhi was reduced compared with challenged naïve controls, although this reduction was not statistically significant (12/27[44%] vs. 12/19[63%]; Relative risk 0.70; 95% CI 0.41-1.21; p = 0.24). Homologous re-challenge with Salmonella Paratyphi A also resulted in a lower attack-rate than was seen in challenged naïve controls (3/12[25%] vs. 10/18[56%]; RR0.45; 95% CI 0.16-1.30; p = 0.14). Evidence of protection was supported by a post hoc analysis in which previous exposure was associated with an approximately 36% and 57% reduced risk of typhoid or paratyphoid disease respectively on re-challenge. Individuals who did not develop enteric fever on primary exposure were significantly more likely to be protected on re-challenge, compared with individuals who developed disease on primary exposure. Heterologous re-challenge with Salmonella Typhi or Salmonella Paratyphi A was not associated with a reduced attack rate following challenge. Within the context of the model, prior exposure was not associated with reduced disease severity, altered microbiological profile or boosting of humoral immune responses. We conclude that prior Salmonella Typhi and Paratyphi A exposure may confer partial but incomplete protection against subsequent infection, but with a comparable clinical and microbiological phenotype. There is no demonstrable cross-protection between these serovars, consistent with the co-circulation of Salmonella Typhi and Paratyphi A. Collectively, these data are consistent with surveillance and modelling studies that indicate multiple infections can occur in high transmission settings, supporting the need for vaccines to reduce the burden of disease in childhood and achieve disease control. Trial registration NCT02192008; clinicaltrials.gov.
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http://dx.doi.org/10.1371/journal.pntd.0008783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598925PMC
October 2020

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

Nat Immunol 2020 11 4;21(11):1336-1345. Epub 2020 Sep 4.

Sequencing and Flow Cytometry Facility, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4 and/or CD8 epitopes, including six immunodominant regions. Six optimized CD8 epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8 T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
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http://dx.doi.org/10.1038/s41590-020-0782-6DOI Listing
November 2020

Evasion of MAIT cell recognition by the African Typhimurium ST313 pathovar that causes invasive disease.

Proc Natl Acad Sci U S A 2020 08 11;117(34):20717-20728. Epub 2020 Aug 11.

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal strains activate MAIT cells. However, Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of Typhimurium ST313 lineage 2.
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http://dx.doi.org/10.1073/pnas.2007472117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456131PMC
August 2020

Single-cell atlas of colonic CD8 T cells in ulcerative colitis.

Nat Med 2020 09 3;26(9):1480-1490. Epub 2020 Aug 3.

Medical Research Council (MRC) Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine (WIMM), John Radcliffe Hospital, University of Oxford, Oxford, UK.

Colonic antigen-experienced lymphocytes such as tissue-resident memory CD8 T cells can respond rapidly to repeated antigen exposure. However, their cellular phenotypes and the mechanisms by which they drive immune regulation and inflammation remain unclear. Here we compiled an unbiased atlas of human colonic CD8 T cells in health and ulcerative colitis (UC) using single-cell transcriptomics with T-cell receptor repertoire analysis and mass cytometry. We reveal extensive heterogeneity in CD8 T-cell composition, including expanded effector and post-effector terminally differentiated CD8 T cells. While UC-associated CD8 effector T cells can trigger tissue destruction and produce tumor necrosis factor (TNF)-α, post-effector cells acquire innate signatures to adopt regulatory functions that may mitigate excessive inflammation. Thus, we identify colonic CD8 T-cell phenotypes in health and UC, define their clonal relationships and characterize terminally differentiated dysfunctional UC CD8 T cells expressing IL-26, which attenuate acute colitis in a humanized IL-26 transgenic mouse model.
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http://dx.doi.org/10.1038/s41591-020-1003-4DOI Listing
September 2020

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

bioRxiv 2020 Jun 8. Epub 2020 Jun 8.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFNγ based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4 and/or CD8 epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8 T cells than spike-specific CD8 T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8 to CD4 T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.
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http://dx.doi.org/10.1101/2020.06.05.134551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302222PMC
June 2020

Single-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-Specific Transcription Factors Determine Cell Fate.

Cell Stem Cell 2019 05 14;24(5):812-820.e5. Epub 2019 Mar 14.

Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON K1H8L6, Canada; Department of Medicine, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H8L6, Canada. Electronic address:

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However, evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here, we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed, as proteins, in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly, upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory, showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates.
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http://dx.doi.org/10.1016/j.stem.2019.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886472PMC
May 2019

Publisher Correction: Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

Nat Immunol 2019 Apr;20(4):514

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41590-019-0357-6DOI Listing
April 2019

Discovery of trehalose phospholipids reveals functional convergence with mycobacteria.

J Exp Med 2019 04 25;216(4):757-771. Epub 2019 Feb 25.

Department of Infectious Diseases and Immunology, School of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Paratyphi and Typhi, we focused on lipids that are enriched in Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by Typhi, lower amounts by other pathogens, and variable amounts by strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.
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http://dx.doi.org/10.1084/jem.20181812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446866PMC
April 2019

Invasive Salmonella exploits divergent immune evasion strategies in infected and bystander dendritic cell subsets.

Nat Commun 2018 11 19;9(1):4883. Epub 2018 Nov 19.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4 T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.
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http://dx.doi.org/10.1038/s41467-018-07329-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242960PMC
November 2018

Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

Nat Immunol 2018 07 20;19(7):742-754. Epub 2018 Jun 20.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4 T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.
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http://dx.doi.org/10.1038/s41590-018-0133-zDOI Listing
July 2018

Generation of a double binary transgenic zebrafish model to study myeloid gene regulation in response to oncogene activation in melanocytes.

Dis Model Mech 2018 04 6;11(4). Epub 2018 Apr 6.

University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford OX3 9DS, United Kingdom

A complex network of inflammatory genes is closely linked to somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. Here, we describe the development of a double binary zebrafish model that enables regulatory programming of the myeloid cells as they respond to oncogene-activated melanocytes to be explored, focussing on the initial phase when cells become the precursors of cancer. A hormone-inducible binary system allows for temporal control of expression of different Ras oncogenes (, and ) in melanocytes, leading to proliferation and changes in morphology of the melanocytes. This model was coupled to binary cell-specific biotagging models allowing biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed as they respond to the earliest precursors of melanoma , revealed an intricate landscape of regulatory factors that may promote progression to melanoma, including Serpinb1l4, Fgf1, Fgf6, Cathepsin H, Galectin 1 and Galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma.
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http://dx.doi.org/10.1242/dmm.030056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963855PMC
April 2018

MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A.

Nat Commun 2018 01 17;9(1):253. Epub 2018 Jan 17.

Medical Research Council (MRC) Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)β clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRβ clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.
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http://dx.doi.org/10.1038/s41467-017-02540-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772558PMC
January 2018

AS03- and MF59-Adjuvanted Influenza Vaccines in Children.

Front Immunol 2017 13;8:1760. Epub 2017 Dec 13.

Oxford Vaccine Group, Department of Paediatrics, University of Oxford, The NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.

Influenza is a major cause of respiratory disease leading to hospitalization in young children. However, seasonal trivalent influenza vaccines (TIVs) have been shown to be ineffective and poorly immunogenic in this population. The development of live-attenuated influenza vaccines and adjuvanted vaccines are important advances in the prevention of influenza in young children. The oil-in-water emulsions MF59 and adjuvant systems 03 (AS03) have been used as adjuvants in both seasonal adjuvanted trivalent influenza vaccines (ATIVs) and pandemic monovalent influenza vaccines. Compared with non-adjuvanted vaccine responses, these vaccines induce a more robust and persistent antibody response for both homologous and heterologous influenza strains in infants and young children. Evidence of a significant improvement in vaccine efficacy with these adjuvanted vaccines resulted in the use of the monovalent (A/H1N1) AS03-adjuvanted vaccine in children in the 2009 influenza pandemic and the licensure of the seasonal MF59 ATIV for children aged 6 months to 2 years in Canada. The mechanism of action of MF59 and AS03 remains unclear. Adjuvants such as MF59 induce proinflammatory cytokines and chemokines, including CXCL10, but independently of type-1 interferon. This proinflammatory response is associated with improved recruitment, activation and maturation of antigen presenting cells at the injection site. In young children MF59 ATIV produced more homogenous and robust transcriptional responses, more similar to adult-like patterns, than did TIV. Early gene signatures characteristic of the innate immune response, which correlated with antibody titers were also identified. Differences were detected when comparing child and adult responses including opposite trends in gene set enrichment at day 3 postvaccination and, unlike adult data, a lack of correlation between magnitude of plasmablast response at day 7 and antibody titers at day 28 in children. These insights show the utility of novel approaches in understanding new adjuvants and their importance for developing improved influenza vaccines for children.
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http://dx.doi.org/10.3389/fimmu.2017.01760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733358PMC
December 2017

Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells.

J Immunol 2017 10 6;199(8):2631-2638. Epub 2017 Sep 6.

Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. It remains unclear whether, in addition to their cytolytic activity that is important in antimicrobial defense, MAIT cells have immune-modulatory functions that could enhance dendritic cell (DC) maturation. In this study, we investigated the molecular mechanisms dictating the interactions between human MAIT cells and DCs and demonstrate that human MAIT cells mature monocyte-derived and primary DCs in an MR1- and CD40L-dependent manner. Furthermore, we show that MAIT cell-derived signals synergize with microbial stimuli to induce secretion of bioactive IL-12 by DCs. Activation of human MAIT cells in whole blood leads to MR1- and cytokine-dependent NK cell transactivation. Our results underscore an important property of MAIT cells, which can be of translational relevance to rapidly orchestrate adaptive immunity through DC maturation.
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http://dx.doi.org/10.4049/jimmunol.1700615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632842PMC
October 2017

Active nuclear transcriptome analysis reveals inflammasome-dependent mechanism for early neutrophil response to Mycobacterium marinum.

Sci Rep 2017 07 26;7(1):6505. Epub 2017 Jul 26.

University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, United Kingdom.

The mechanisms governing neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilise biotagging, a novel genome-wide profiling approach based on cell type-specific in vivo biotinylation in zebrafish to analyse the initial response of neutrophils to Mycobacterium marinum, a close genetic relative of M. tuberculosis used to model tuberculosis. Differential expression analysis following nuclear RNA-seq of neutrophil active transcriptomes reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, NLRC3 ortholog (wu: fb15h11) and il1β. Crispr/Cas9-mediated knockout of caspase b, which acts by proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterial infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial neutrophil response to mycobacterial infection. Further perturbation of the IEGs exposes egr3 as a key transcriptional regulator controlling il1β transcription.
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http://dx.doi.org/10.1038/s41598-017-06099-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529371PMC
July 2017

Human autoreactive T cells recognize CD1b and phospholipids.

Proc Natl Acad Sci U S A 2016 Jan 30;113(2):380-5. Epub 2015 Nov 30.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;

In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids.
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http://dx.doi.org/10.1073/pnas.1520947112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720340PMC
January 2016

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation.

Proc Natl Acad Sci U S A 2013 Dec 18;110(49):E4753-61. Epub 2013 Nov 18.

Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford OX3 9DS, United Kingdom.

Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the off-rate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a "lipid editor," capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity.
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http://dx.doi.org/10.1073/pnas.1310050110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856826PMC
December 2013

Intestinal DC in migrational imprinting of immune cells.

Immunol Cell Biol 2013 Mar 8;91(3):240-9. Epub 2013 Jan 8.

Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia.

Dendritic cells (DCs) have a pivotal role in instructing antigen-specific immune responses, processing and presenting antigens to CD4(+) and CD8(+) T cells and producing factors capable to modulate the quality of T-cell responses. In this review, we will provide an historic overview on the identification of the mechanisms controlling lymphocyte migration into the largest immune organ of the body: the gut, and we will describe how in recent years an unexpected role for DCs has emerged as the architects in programming gut-homing immune cells. Specifically, we will review how intestinal DCs utilize the dietary vitamin A metabolite retinoic acid (RA) to program gut-homing lymphocytes and how intestinal DCs acquire the unique capacity to become RA producers.
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http://dx.doi.org/10.1038/icb.2012.73DOI Listing
March 2013

Differential IL-17 production and mannan recognition contribute to fungal pathogenicity and commensalism.

J Immunol 2010 Apr 12;184(8):4258-68. Epub 2010 Mar 12.

Department of Preclinic and Clinic Pharmacology, University of Firenze, Firenze, Italy.

In this study, we present evidence of differential Th17 responses in human monocyte-derived dendritic cells exposed to the pathogenic Candida albicans or the nonpathogenic Saccharomyces cerevisiae. We use different forms of the microorganisms, cells, hyphae, and spores, as a toolbox to dissect the role of surface mannan in the fungal immune response. In contrast to the S. cerevisiae yeast cell-induced Th1 response, dendritic cells stimulated with spores or C. albicans hyphae induce cellular responses shifted toward Th17 differentiation. The differential recognition of specific mannan structures is the master regulator of the discrimination between harmful and harmless fungi. The switch between spores and yeast is crucial for the commensalism of S. cerevisiae and depends on the use of a different receptor repertoire. Understanding the role of cell wall recognition during infection might lead to understanding the boundaries between safety and pathogenicity.
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http://dx.doi.org/10.4049/jimmunol.0902972DOI Listing
April 2010

Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+ T cells.

Eur J Immunol 2009 May;39(5):1301-12

Institute for Research in Biomedicine, Bellinzona, Switzerland.

The contribution of Th1 and Th17 cells in chronic inflammatory conditions leading to autoimmunity remains highly controversial. In inflamed tissues, production of prostaglandins by COX-2 has been proposed to favor Th17 responses indirectly by increasing IL-23 and blocking IL-12 release from APC. We report here that prostaglandin E2 (PGE2) can directly modulate cytokine production by human memory CD4(+) T cells. TCR triggering in the presence of PGE2 increased IL-17 and reduced IFN-gamma production by freshly isolated memory T cells or T-cell clones. PGE2 triggered the EP2 and EP4 receptors expressed on T cells leading to a rapid increase of retinoic-acid-related orphan receptor-gammat (ROR-gammat) and decrease of T-cell-specific T-box transcription factor 21 (T-bet) mRNA. Moreover, PGE2 promoted the selective enrichment of IL-17-producing cells by differentially modulating the proliferation of memory T-cell subsets in vitro. Taken together our results indicate that T-cell effector function is a direct target for PGE2 modulation and suggest a novel mechanism by which inhibitors of prostaglandin synthesis, such as COX-2 inhibitors, exert their anti-inflammatory effect.
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http://dx.doi.org/10.1002/eji.200838969DOI Listing
May 2009

Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells.

Nat Immunol 2007 Sep 5;8(9):942-9. Epub 2007 Aug 5.

Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland.

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) have been linked to host defense and autoimmune diseases. In mice, the differentiation of T(H)-17 cells requires transforming growth factor-beta and IL-6 and the transcription factor RORgammat. We report here that for human naive CD4(+) T cells, RORgammat expression and T(H)-17 polarization were induced by IL-1beta and enhanced by IL-6 but were suppressed by transforming growth factor-beta and IL-12. Monocytes and conventional dendritic cells, but not monocyte-derived dendritic cells activated by microbial stimuli, efficiently induced T(H)-17 priming, and this function correlated with antigen-presenting cell production of IL-1beta and IL-6 but not IL-12. Our results identify cytokines, antigen-presenting cells and microbial products that promote the polarization of human T(H)-17 cells and emphasize an important difference in the requirements for the differentiation of T(H)-17 cells in humans and mice.
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http://dx.doi.org/10.1038/ni1496DOI Listing
September 2007

Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells.

Nat Immunol 2007 Jun 7;8(6):639-46. Epub 2007 May 7.

Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland.

Interleukin 17 (IL-17)-producing T helper cells (T(H)-17 cells) have been characterized in mice as a distinct subset of effector cells, but their identity and properties in humans remain elusive. We report here that expression of CCR6 and CCR4 together identified human memory CD4+ T cells selectively producing IL-17 and expressing mRNA encoding the human ortholog of mouse RORgammat, a transcription factor, whereas CCR6 and CXCR3 identified T(H)1 cells producing interferon-gamma and T helper cells producing both interferon-gamma and IL-17. Memory T cells specific for Candida albicans were present mainly in the CCR6+CCR4+ T(H)-17 subset, whereas memory T cells specific for Mycobacterium tuberculosis were present in CCR6+CXCR3+ T helper type 1 subset. The elicitation of IL-17 responses correlated with the capacity of C. albicans hyphae to stimulate antigen-presenting cells for the priming of T(H)-17 responses in vitro and for the production of IL-23 but not IL-12. Our results demonstrate that human T(H)-17 cells have distinct migratory capacity and antigenic specificities and establish a link between microbial products, T helper cell differentiation and homing in response to fungal antigens.
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http://dx.doi.org/10.1038/ni1467DOI Listing
June 2007

Duration, combination and timing: the signal integration model of dendritic cell activation.

Trends Immunol 2007 May 2;28(5):227-33. Epub 2007 Apr 2.

Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500 Bellinzona, Switzerland.

The activation of resting dendritic cells (DCs) is a crucial step in the initiation of adaptive immunity because it links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. It is well recognized that a wide variety of microbial products and endogenous signals can trigger DC activation, and that different DC subsets are specialized in inducing different classes of immune responses. In this review, we will focus on how different aspects of DC maturation are regulated not only by the nature of the DC maturation stimuli, but also by their duration, combination and timing, and provide an overview of how different modes of DC activation can affect T cell responses.
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http://dx.doi.org/10.1016/j.it.2007.03.008DOI Listing
May 2007

Simvastatin inhibits the MHC class II pathway of antigen presentation by impairing Ras superfamily GTPases.

Eur J Immunol 2006 Nov;36(11):2885-93

Department of Evolutionary Biology, University of Siena, Siena, Italy.

Statins are widely used hypocholesterolemic drugs that inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting enzyme of the mevalonate pathway whose biosynthetic endproduct is cholesterol. As a result of this activity, statins may perturb the composition of cell membranes, resulting in lipid raft disruption. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and activity of small GTPases. Antigen uptake, processing and presentation involve the interplay of Rab and Rho family GTPases. Furthermore, lipid rafts have been implicated both in antigen internalization by the BCR and in MHC class II clustering at the immunological synapse. Here we have addressed the effects of simvastatin on antigen processing and presentation by human B cells and dendritic cells. The results show that simvastatin potently suppresses tetanus toxoid processing and presentation to CD4+ T cells by HLA-DR by inhibiting protein antigen uptake through both receptor-mediated endocytosis and macropinocytosis. This effect can be largely accounted for by defective prenylation of Rho and Rab GTPases in the absence of any measurable perturbation of lipid rafts. In addition, simvastatin was found to preferentially affect the invariant chain-dependent MHC class II pathway, thereby identifying this route of antigen processing and presentation as a selective target of statins.
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http://dx.doi.org/10.1002/eji.200636567DOI Listing
November 2006

Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells.

Nat Immunol 2005 Aug 3;6(8):769-76. Epub 2005 Jul 3.

Institute for Research in Biomedicine, Oncology Institute of Southern Switzerland, CH-6500, Bellinzona, Switzerland.

Toll-like receptors (TLRs) sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). As pathogens may contain several TLR agonists, we sought to determine whether different TLRs cooperate in DC activation. In human and mouse DCs, TLR3 and TLR4 potently acted in synergy with TLR7, TLR8 and TLR9 in the induction of a selected set of genes. Synergic TLR stimulation increased production of interleukins 12 and 23 and increased the Delta-4/Jagged-1 ratio, leading to DCs with enhanced and sustained T helper type 1-polarizing capacity. Global gene transcriptional analysis showed that TLR synergy 'boosted' only approximately 1% of the transcripts induced by single TLR agonists. These results identify a 'combinatorial code' by which DCs discriminate pathogens and suggest new strategies for promoting T helper type 1 responses.
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http://dx.doi.org/10.1038/ni1223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760217PMC
August 2005

Cyclic AMP modulates the functional plasticity of immature dendritic cells by inhibiting Src-like kinases through protein kinase A-mediated signaling.

J Biol Chem 2004 Jul 27;279(31):32507-14. Epub 2004 May 27.

Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, I-80131 Naples, Italy.

Immature dendritic cells (iDCs) can be instructed to polarize the immune response toward a noninflammatory pathway by mediators that increase the intracellular concentration of cAMP. This phenomenon is associated with the ability of the cyclic nucleoside to inhibit the release of pro-inflammatory cytokines without affecting the differentiation process of the dendritic cells (DCs). Here we investigated the ability of cAMP to modulate the endotoxin signaling by exposing DCs to exogenous 8-bromium-cyclic AMP in the presence or absence of H89, a selective inhibitor of the protein kinase A, one of the major molecular targets of the cyclic nucleoside. cAMP affects the early lipopolysaccharide-induced signaling cascade dissociating the activation of NF-kappa B, p38, and ERK pathways from the stimulation of c-Src and Lyn kinases. This phenomenon was prevented by H89. The pharmacological block of Src-like tyrosine kinases induces comparable results confirming the involvement of this family of enzymes in the mechanism controlling the release of cytokines in human monocyte-derived iDCs. We propose that the cAMP-protein kinase A-dependent pathway regulates the functional plasticity of iDCs by gating the Toll-like receptor signaling at the level of Src-like kinases.
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http://dx.doi.org/10.1074/jbc.M403355200DOI Listing
July 2004