Publications by authors named "Uzi Gileadi"

31 Publications

PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses.

Front Immunol 2021 25;12:641703. Epub 2021 Feb 25.

Department of Tumor Immunology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 . Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.
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http://dx.doi.org/10.3389/fimmu.2021.641703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947615PMC
February 2021

Hepcidin-Mediated Hypoferremia Disrupts Immune Responses to Vaccination and Infection.

Med (N Y) 2021 Feb;2(2):164-179.e12

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

Background: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear.

Methods: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant . We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency.

Findings: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity.

Conclusions: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders.

Funding: Medical Research Council, UK.
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http://dx.doi.org/10.1016/j.medj.2020.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895906PMC
February 2021

Nanovaccine administration route is critical to obtain pertinent iNKt cell help for robust anti-tumor T and B cell responses.

Oncoimmunology 2020 03 17;9(1):1738813. Epub 2020 Mar 17.

Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center & Oncode Institute, Nijmegen, The Netherlands.

Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.
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http://dx.doi.org/10.1080/2162402X.2020.1738813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790498PMC
March 2020

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

MAbs 2020 Jan-Dec;12(1):1801230

Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation . Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the p of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
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http://dx.doi.org/10.1080/19420862.2020.1801230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531564PMC
September 2020

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics.

Sci Adv 2020 Jul 29;6(31):eaba5345. Epub 2020 Jul 29.

Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.
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http://dx.doi.org/10.1126/sciadv.aba5345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439646PMC
July 2020

Impacts of combining anti-PD-L1 immunotherapy and radiotherapy on the tumour immune microenvironment in a murine prostate cancer model.

Br J Cancer 2020 09 9;123(7):1089-1100. Epub 2020 Jul 9.

Department of Oncology, University of Oxford, Oxford, UK.

Background: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1.

Methods: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1.

Results: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8 T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy.

Conclusion: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
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http://dx.doi.org/10.1038/s41416-020-0956-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525450PMC
September 2020

Enhanced Immunogenicity of Mitochondrial-Localized Proteins in Cancer Cells.

Cancer Immunol Res 2020 05 23;8(5):685-697. Epub 2020 Mar 23.

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

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8 T cells . We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0467DOI Listing
May 2020

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Proc Natl Acad Sci U S A 2019 11 5;116(47):23671-23681. Epub 2019 Nov 5.

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

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.
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http://dx.doi.org/10.1073/pnas.1910097116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876220PMC
November 2019

Addendum: Precise tuning of gene expression levels in mammalian cells.

Nat Commun 2019 06 10;10(1):2622. Epub 2019 Jun 10.

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

Following re-sequencing of the miSFIT constructs used in the paper, two of the construct variants inserted into the 3'UTR of PD-1, namely '12C' and '17A, 18G', have been found to contain additional insertions not present in the other construct variants. The data points corresponding to these constructs in Figs. 2c, f and Supplementary Fig. 9 are therefore no longer valid. However the overall conclusion that step-wise control over gene expression levels using the miSFIT constructs remains unaffected by these errors. Updated versions of Fig. 2 and Supplementary Fig. 9 are presented in the accompanying Addendum.
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http://dx.doi.org/10.1038/s41467-019-10615-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557827PMC
June 2019

NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses.

Front Immunol 2019 30;10:958. Epub 2019 Apr 30.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

NOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8 T cells are important for immunity to such pathogens but how NOD2 and TLR2 induce antigen specific CD8 T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation in DCs. PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES), which positively regulates MHC class I peptide loading and immunoproteasome stability. Following NOD2 and TLR2 stimulation, depletion of PI31 or inhibition of TBK1 activity impairs DC cross-presentation and CD8 T cell activation. DCs from Crohn's patients expressing polymorphisms show dysregulated cross-presentation and CD8 T cell responses. Our findings reveal unidentified mechanisms that underlie CD8 T cell responses to bacteria in health and in Crohn's.
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http://dx.doi.org/10.3389/fimmu.2019.00958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503738PMC
September 2020

Precise tuning of gene expression levels in mammalian cells.

Nat Commun 2019 02 18;10(1):818. Epub 2019 Feb 18.

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

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3'UTR, demonstrating that this versatile tool can be used to tune endogenous genes.
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http://dx.doi.org/10.1038/s41467-019-08777-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379387PMC
February 2019

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

Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

Nat Immunol 2017 Jul 15;18(7):762-770. Epub 2017 May 15.

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

Trafficking of tissue dendritic cells (DCs) via lymph is critical for the generation of cellular immune responses in draining lymph nodes (LNs). In the current study we found that DCs docked to the basolateral surface of lymphatic vessels and transited to the lumen through hyaluronan-mediated interactions with the lymph-specific endothelial receptor LYVE-1, in dynamic transmigratory-cup-like structures. Furthermore, we show that targeted deletion of the gene Lyve1, antibody blockade or depletion of the DC hyaluronan coat not only delayed lymphatic trafficking of dermal DCs but also blunted their capacity to prime CD8 T cell responses in skin-draining LNs. Our findings uncovered a previously unknown function for LYVE-1 and show that transit through the lymphatic network is initiated by the recognition of leukocyte-derived hyaluronan.
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http://dx.doi.org/10.1038/ni.3750DOI Listing
July 2017

Nutritional Stress Induced by Tryptophan-Degrading Enzymes Results in ATF4-Dependent Reprogramming of the Amino Acid Transporter Profile in Tumor Cells.

Cancer Res 2016 11 20;76(21):6193-6204. Epub 2016 Sep 20.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

Tryptophan degradation is an immune escape strategy shared by many tumors. However, cancer cells' compensatory mechanisms remain unclear. We demonstrate here that a shortage of tryptophan caused by expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) resulted in ATF4-dependent upregulation of several amino acid transporters, including SLC1A5 and its truncated isoforms, which in turn enhanced tryptophan and glutamine uptake. Importantly, SLC1A5 failed to be upregulated in resting human T cells kept under low tryptophan conditions but was enhanced upon cognate antigen T-cell receptor engagement. Our results highlight key differences in the ability of tumor and T cells to adapt to tryptophan starvation and provide important insights into the poor prognosis of tumors coexpressing IDO and SLC1A5. Cancer Res; 76(21); 6193-204. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096689PMC
November 2016

Induced Disruption of the Iron-Regulatory Hormone Hepcidin Inhibits Acute Inflammatory Hypoferraemia.

J Innate Immun 2016 16;8(5):517-28. Epub 2016 Jul 16.

Department of Biochemistry, Birmingham Heartlands Hospital, Heart of England NHS Foundation Trust, Birmingham, UK.

Withdrawal of iron from serum (hypoferraemia) is a conserved innate immune antimicrobial strategy that can withhold this critical nutrient from invading pathogens, impairing their growth. Hepcidin (Hamp1) is the master regulator of iron and its expression is induced by inflammation. Mice lacking Hamp1 from birth rapidly accumulate iron and are susceptible to infection by blood-dwelling siderophilic bacteria such as Vibrio vulnificus. In order to study the innate immune role of hepcidin against a background of normal iron status, we developed a transgenic mouse model of tamoxifen-sensitive conditional Hamp1 deletion (termed iHamp1-KO mice). These mice attain adulthood with an iron status indistinguishable from littermate controls. Hamp1 disruption and the consequent decline of serum hepcidin concentrations occurred within hours of a single tamoxifen dose. We found that the TLR ligands LPS and Pam3CSK4 and heat-killed Brucella abortus caused an equivalent induction of inflammation in control and iHamp1-KO mice. Pam3CSK4 and B. abortus only caused a drop in serum iron in control mice, while hypoferraemia due to LPS was evident but substantially blunted in iHamp1-KO mice. Our results characterise a powerful new model of rapidly inducible hepcidin disruption, and demonstrate the critical contribution of hepcidin to the hypoferraemia of inflammation.
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http://dx.doi.org/10.1159/000447713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322583PMC
October 2017

Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses.

Oncoimmunology 2016;5(1):e1068493. Epub 2015 Aug 12.

Department of Tumor Immunology, Radboud University Medical Center and Radboud Institute for Molecular Life Sciences , Nijmegen, The Netherlands.

Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here, we compared the efficacy of the invariant NKT (iNKT) cell agonist α-galactosylceramide (α-GalCer) and TLR ligands (R848 and poly I:C) as an adjuvant for the full length ovalbumin (OVA) in PLGA nanoparticles. We observed that OVA+α-GalCer nanoparticles (NP) are superior over OVA+TLR-L NP in generating and stimulating antigen-specific cytotoxic T lymphocytes without the need for CD4 T cell help. Not only a 4-fold higher induction of antigen-specific T cells was observed, but also a more profound IFN-γ secretion was obtained by the addition α-GalCer. Surprisingly, we observed that mixtures of OVA containing NP with α-GalCer were ineffective, demonstrating that co-encapsulation of both α-GalCer and antigen within the same nanoparticle is essential for the observed T cell responses. Moreover, a single immunization with OVA+α-GalCer NP provided substantial protection from tumor formation and even delayed the growth of already established tumors, which coincided with a prominent and enhanced antigen-specific CD8 T cell infiltration. The provided evidence on the advantage of antigen and α-GalCer coencapsulation should be considered in the design of future nanoparticle vaccines for therapeutic purposes.
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http://dx.doi.org/10.1080/2162402X.2015.1068493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760331PMC
August 2015

Non-glycosidic compounds can stimulate both human and mouse iNKT cells.

Eur J Immunol 2016 05 1;46(5):1224-34. Epub 2016 Mar 1.

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

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non-glycosidic CD1d-binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6- or 7-membered ring results in significantly more potent non-glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti-tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α-galactosylceramide (α-GalCer), achieving an enhanced T-cell response at lower concentrations compared with α-GalCer both in vitro, using human iNKT-cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non-glycosidic ThrCer-based analogs that have improved potency in iNKT-cell activation compared with that of α-GalCer, and are clinically relevant iNKT-cell agonists.
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http://dx.doi.org/10.1002/eji.201546114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913735PMC
May 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

Processing of human toll-like receptor 7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes.

Immunity 2013 Oct;39(4):711-21

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

Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.
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http://dx.doi.org/10.1016/j.immuni.2013.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839496PMC
October 2013

DOCK8 is critical for the survival and function of NKT cells.

Blood 2013 Sep 8;122(12):2052-61. Epub 2013 Aug 8.

Medical Research Council Human Immunology Unit, Weatherall Institute for Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom;

Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1(+) NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.
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http://dx.doi.org/10.1182/blood-2013-02-482331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778549PMC
September 2013

Hepcidin regulation by innate immune and infectious stimuli.

Blood 2011 Oct 26;118(15):4129-39. Epub 2011 Aug 26.

Molecular Immunology Group, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6-dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-β-dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.
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http://dx.doi.org/10.1182/blood-2011-04-351957DOI Listing
October 2011

A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus.

PLoS One 2011 Feb 3;6(2):e14646. Epub 2011 Feb 3.

Nuffield Department of Clinical Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK.

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8(+) T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8(+) T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8(+) T cell responses to MCMV - both conventional memory responses and those undergoing long-term expansion or "inflation". We infected LMP7(-/-) and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8(+) T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory "inflating" epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8(+) T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014646PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033404PMC
February 2011

Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells.

J Immunol 2008 May;180(10):6452-6

Tumour Immunology Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.
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http://dx.doi.org/10.4049/jimmunol.180.10.6452DOI Listing
May 2008

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses.

J Virol 2008 Jan 24;82(1):86-95. Epub 2007 Oct 24.

Division of Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 4JF, United Kingdom.

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2(+) cells after intravenous injection and in CD11c(+) dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8(+) T-cell response in HLA-A2 transgenic mice and stimulated a CD4(+) T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A(b). As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.
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http://dx.doi.org/10.1128/JVI.01289-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224357PMC
January 2008

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells.

J Immunol 2007 Mar;178(5):2721-9

Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom.

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.
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http://dx.doi.org/10.4049/jimmunol.178.5.2721DOI Listing
March 2007

Role of immunoproteasomes in cross-presentation.

J Immunol 2006 Jul;177(2):983-90

Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom.

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty(246-254) epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7(-/-)) skin grafts by female LMP7(-/-) mice. Second, we ruled out an altered Uty(246-254)-specific T cell repertoire in LMP7(-/-) female mice and demonstrated efficient Uty(246-254) presentation by re-expressing LMP7 in male LMP7(-/-) cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty(246-254)-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7(-/-) female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.
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http://dx.doi.org/10.4049/jimmunol.177.2.983DOI Listing
July 2006

CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef.

J Immunol 2005 Oct;175(7):4618-26

James Martin 21st Century School and Nuffield Department of Clinical Medicine, The Peter Medawar Building, University of Oxford, Oxford, United Kingdom.

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.
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http://dx.doi.org/10.4049/jimmunol.175.7.4618DOI Listing
October 2005

Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell-mediated immunotherapy.

J Clin Invest 2004 Dec;114(12):1800-11

Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

Activation of invariant CD1d-dependent NK T cells (iNKT cells) in vivo through administration of the glycolipid ligand alpha-galactosylceramide (alpha-GalCer) or the sphingosine-truncated alpha-GalCer analog OCH leads to CD40 signaling as well as the release of soluble molecules including type 1 and gamma interferons that contribute to DC maturation. This process enhances T cell immunity to antigens presented by the DC. The adjuvant activity is further amplified if APCs are stimulated through Toll-like receptor 4, suggesting that iNKT cell signals can amplify maturation induced by microbial stimuli. The adjuvant activity of alpha-GalCer enhances both priming and boosting of CD8(+) T cells to coadministered peptide or protein antigens, including a peptide encoding the clinically relevant, HLA-A2-restricted epitope of the human tumor antigen NY-ESO-1. Importantly, alpha-GalCer was used to induce CD8(+) T cells to antigens delivered orally, despite the fact that this route of administration is normally associated with blunted responses. Only T cell responses induced in the presence of iNKT cell stimulation, whether by the i.v. or oral route, were capable of eradicating established tumors. Together these data highlight the therapeutic potential of iNKT cell ligands in vaccination strategies, particularly "heterologous prime-boost" strategies against tumors, and provide evidence that iNKT cell stimulation may be exploited in the development of oral vaccines.
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http://dx.doi.org/10.1172/JCI22046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC535067PMC
December 2004

The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.

J Immunol Methods 2004 Feb;285(1):25-40

Tumour Immunology Unit, Nuffield Department of Medicine, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK.

Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.
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http://dx.doi.org/10.1016/j.jim.2003.10.017DOI Listing
February 2004

NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells.

J Immunol 2003 Nov;171(10):5140-7

Tumor Immunology Unit, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

Modification in the function of dendritic cells (DC), such as that achieved by microbial stimuli or T cell help, plays a critical role in determining the quality and size of adaptive responses to Ag. NKT cells bearing an invariant TCR (iNKT cells) restricted by nonpolymorphic CD1d molecules may constitute a readily available source of help for DC. We therefore examined T cell responses to i.v. injection of soluble Ag in the presence or the absence of iNKT cell stimulation with the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GalCer). Considerably enhanced CD4(+) and CD8(+) T cell responses were observed when alpha-GalCer was administered at the same time as or close to OVA injection. This enhancement was dependent on the involvement of iNKT cells and CD1d molecules and required CD40 signaling. Studies in IFN-gammaR(-/-) mice indicated that IFN-gamma was not required for the adjuvant effect of alpha-GalCer. Consistent with this result, enhanced T cell responses were observed using OCH, an analog of alpha-GalCer with a truncated sphingosine chain and a reduced capacity to induce IFN-gamma. Splenic DC from alpha-GalCer-treated animals expressed high levels of costimulatory molecules, suggesting maturation in response to iNKT cell activation. Furthermore, studies with cultured DC indicated that potentiation of T cell responses required presentation of specific peptide and alpha-GalCer by the same DC, implying conditioning of DC by iNKT cells. The iNKT-enhanced T cell responses resisted challenge with OVA-expressing tumors, whereas responses induced in the absence of iNKT stimulation did not. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble Ag by modulating DC function.
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http://dx.doi.org/10.4049/jimmunol.171.10.5140DOI Listing
November 2003