Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV Gag p24 protein

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4+ T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development

DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes

Optimal HIV vaccines should elicit CD8+ T cells specific for HIV proteins presented on MHC class I products, because these T cells contribute to host resistance to viruses. We had previously found that the targeting of antigen to dendritic cells (DCs) in mice efficiently induces CD8+ T cell responses. To extend this finding to humans, we introduced the HIV p24 gag protein into a mAb that targets DEC-205/CD205, an endocytic receptor of DCs. We then assessed cross-presentation, which is the processing of nonreplicating internalized antigen onto MHC class I for recognition by CD8+ T cells. Low doses of αDEC-gag, but not control Ig-gag, stimulated proliferation and IFN-γ production by CD8+ T cells isolated from the blood of HIV-infected donors. αCD205 fusion mAb was more effective for cross-presentation than αD209/DC-SIGN, another abundant DC uptake receptor. Presentation was diverse, because we identified eight different gag peptides that were recognized via DEC-205 in 11 individuals studied consecutively. Our results, based on humans with highly polymorphic MHC products, reveal that DCs and DEC-205 can cross-present several different peptides from a single protein. Because of the consistency in eliciting CD8 + T cell responses, these data support the testing of αDEC-205 fusion mAb as a protein-based vaccine

Interferon regulatory factor 2 protects mice from lethal viral neuroinvasion.

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study, we demonstrate that IFN regulatory factor 2 (IRF2), an ISG and a negative regulator of IFN signaling, influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2-/- mice after peripheral inoculation. Irf2-/- mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2-/- mouse brains despite the presence of peripheral neutralizing antibodies, suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient μMT mice are significantly more susceptible to viral infection, yet WT B cells and serum are unable to rescue the Irf2-/- mice. Collectively, our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections

Immunotherapy with internally inactivated virus loaded dendritic cells boosts cellular immunity but does not affect feline immunodeficiency virus infection course

Immunotherapy of feline immunodeficiency virus (FIV)-infected cats with monocyte-derived dendritic cells (MDCs) loaded with aldrithiol-2 (AT2)-inactivated homologous FIV was performed. Although FIV-specific lymphoproliferative responses were markedly increased, viral loads and CD4+ T cell depletion were unaffected, thus indicating that boosting antiviral cell-mediated immunity may not suffice to modify infection course appreciably

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV. IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV

Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves

Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP+ cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c+ cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8+ TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves

UTILIZZO DELLE CELLULE DENDRITICHE NELL’INDUZIONE DI IMMUNITÁ CELLULARE NELLE INFEZIONI LENTIVIRALI

Vaccini efficaci nella prevenzione di HIV dovrebbero indurre cellule T CD4+ e CD8+ specifiche per le proteine virali presentate in associazione a molecole MHC di classe I e II, in quanto sia le cellule T CD8+ sia le cellule CD4+ contribuiscono alla resistenza ai virus. Le cellule dendritiche (DC) sono cellule presentanti l’antigene (Ag) altamente specializzate in grado di presentare Ag di natura esogena sia alle cellule T CD4+ helper sia alle cellule T CD8+ citotossiche e polarizzare le risposte immune nei due tipi Th1 e Th2. Date le loro uniche funzioni immunoregolatorie, le DC sono state considerate come adiuvanti naturali utili in approcci vaccinali contro il cancro e contro importanti malattie infettive. Nella prima parte di questa tesi, abbiamo valutato l’efficacia di una strategia di targeting dell’Ag alle DC nell’uomo. A questo scopo, abbiamo inserito la proteina gag p24 di HIV all’interno di un anticorpo monoclonale (mAb) specifico per DEC-205, recettore endocitotico espresso sulle DC. Basse dosi del mAb di fusione ?-hDECp24, inducevano proliferazione e produzione di IFN? da parte delle cellule T isolate dal sangue di donatori HIV+. Il mAb di fusione ?-hDECp24 risultava più efficiente nel mediare cross-presentazione rispetto a ?-hDC-SIGNp24, un mAb di fusione specifico per un altro recettore endocitotico delle DC. La presentazione dell’Ag via DEC-205 è risultata diversificata in quanto abbiamo identificato 8 diversi peptidi di gag riconosciuti su differenti aplotipi HLA in 11 pazienti infetti con HIV. Nella seconda parte, descriviamo un protocollo per coltivare DC feline in assenza di proteine esogene per il loro uso in vivo. Abbiamo analizzato quale fosse lo stimolo maturativo più efficace per indurre maturazione delle DC ed abbiamo definito i correlati della maturazione. Secondo il nostro protocollo, le DC feline sono state generate a partire dai PBMC in presenza di IL-4 e GM-CSF e dopo 5 giorni di coltura le cellule sono state maturate con LPS, oppure con TNF? o IFN? umani, oppure con piastrine attivate. Dopo 48 h dall’aggiunta dello stimolo maturativo, è stata analizzata l’espressione dei marcatori di superficie CD14, MHC di classe II e B7.1 in parallelo con la capacità delle DC di catturare l’Ag stimolare cellule T allogeniche in cosiddette mixed leucocyte reactions. I risultati presentati mostrano che le DC feline coltivate in plasma autologo differenziavano e maturavano in presenza di stimoli simili a quelli attualmente in uso per altre specie. Questi risultati consolidano l’uso delle DC così ottenute in approcci vaccinali e/o immunoterapeutici in gatti infetti con il virus dell’immunodeficienza felina (FIV). FIV è un lentivirus che è stato a lungo studiato come modello per HIV. L’infezione sostenuta da FIV nei gatti domestici è molto simile alla sindrome umana (AIDS), causando una progressiva compromissione del sistema immunitario; perciò la sindrome felina (FAIDS) è considerata un valido modello per testare eventuali vaccini contro HIV-1. Lo studio presentato nella terza parte di questa tesi è stato condotto per verificare se le DC autologhe caricate con l’isolato primario FIV-M2 inattivato con alditriolo-2 ed inoculate in vivo fossero capaci di stimolare una risposta immunitaria protettiva contro il virus omologo. L’esito del challenge con FIV è stato monitorato misurando la risposta cellulare e umorale, il carico virale e provirale, quantificando il virus anche mediante isolamento virale dai PBMC e valutando l’andamento del numero delle cellule T CD4+ e CD8+ nel sangue durante l’infezione. I dati mostrano che gli animali vaccinati sono risultati infetti in seguito al challenge similmente agli animali di controllo. Sebbene questi risultati non sembrano supportare l’idea che le DC caricate con l’Ag possano indurre immunità protettiva meglio di altre forme di Ag nel modello FIV, il presente studio evidenzia aspetti importanti che devono essere valutati nel design di un vaccino basato sulle DC, quali la maturazione delle DC, il metodo di caricamento ed il numero delle cellule inoculate. Tali argomenti dovranno essere approfonditi in studi futuri di vaccinazione e/o immunoterapia con DC feline

Unboxing dendritic cells: Tales of multi‐faceted biology and function

Often referred to as the bridge between innate and adaptive immunity, dendritic cells (DCs) are professional antigen‐presenting cells (APCs) that constitute a unique, yet complex cell system. Among other APCs, DCs display the unique property of inducing protective immune responses against invading microbes, or cancer cells, while safeguarding the proper homeostatic equilibrium of the immune system and maintaining self‐tolerance. Unsurprisingly, DCs play a role in many diseases such as autoimmunity, allergy, infectious disease and cancer. This makes them attractive but challenging targets for therapeutics. Since their initial discovery, research and understanding of DC biology have flourished. We now recognize the presence of multiple subsets of DCs distributed across tissues. Recent studies of phenotype and gene expression at the single cell level have identified heterogeneity even within the same DC type, supporting the idea that DCs have evolved to greatly expand the flexibility of the immune system to react appropriately to a wide range of threats. This review is meant to serve as a quick and robust guide to understand the basic divisions of DC subsets and their role in the immune system. Between mice and humans, there are some differences in how these subsets are identified and function, and we will point out specific distinctions as necessary. Throughout the text, we are using both fundamental and therapeutic lens to describe overlaps and distinctions and what this could mean for future research and therapies.Recent studies of phenotype and gene expression at the single cell level have identified DC heterogeneity even within the same type, supporting the idea that DCs have evolved to greatly expand the flexibility of the immune system to react appropriately to a wide range of threats. This review is meant to serve as a quick and robust guide to understand the basic divisions of DC subsets and their role in the immune system. Throughout the text, we are using both fundamental and therapeutic lens to describe overlaps and distinctions and what this could mean for future research and therapies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170887/1/imm13394_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170887/2/imm13394.pd

Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats