RESUMEN
Preventing the protein-protein interaction of the cellular chromatin binding protein Lens Epithelium-Derived Growth Factor (LEDGF) and human immunodeficiency virus (HIV) integrase is an important possible strategy for anti-viral treatment for AIDS. We have used Intracellular Antibody Capture technology to isolate a single VH antibody domain that binds to LEDGF. The crystal structure of the LEDGF-VH complex reveals that the single domain antibody mimics the effect of binding of HIV integrase to LEDGF which is crucial for HIV propagation. CD4-expressing T cell lines were constructed to constitutively express the LEDGF-binding VH and these cells showed interference with HIV viral replication, assayed by virus capsid protein p24 production. Therefore, pre-conditioning cells to express antibody fragments confers effective intracellular immunization for preventing chronic viral replication and can be a way to prevent HIV spread in infected patients. This raises the prospect that intracellular immunization strategies that focus on cellular components of viral integrase protein interactions can be used to combat the problems associated with latent HIV virus re-emergence in patients. New genome editing development, such as using CRISPR/cas9, offer the prospect intracellularly immunized T cells in HIV+ patients.
Asunto(s)
Infecciones por VIH/patología , Integrasa de VIH/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Cristalografía por Rayos X , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/inmunología , Integrasa de VIH/química , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Células Jurkat , Ratones , Simulación de Dinámica Molecular , Unión Proteica , Alineación de Secuencia , Anticuerpos de Dominio Único/química , Técnicas del Sistema de Dos Híbridos , Replicación ViralRESUMEN
HIV-1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV-1 somehow evades detection by the pattern-recognition receptor (PRR) Toll-like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV-1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV-1 trans-infection of CD4+ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV-1 with TLR8+ early endosomes, triggered a pro-inflammatory response, and inhibited trans-infection of CD4+ T cells. Snapin inhibited TLR8 signaling in the absence of HIV-1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV-1 to promote transmission.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , VIH-1/patogenicidad , Interacciones Huésped-Patógeno , Transducción de Señal , Receptor Toll-Like 8/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Linfocitos T CD4-Positivos/virología , Línea Celular , VIH-1/inmunología , HumanosRESUMEN
[This corrects the article on p. 307 in vol. 4, PMID: 24137162.].
RESUMEN
Immune responses at the intestinal mucosa must allow for host protection whilst simultaneously avoiding inappropriate inflammation. Although much work has focused on the innate immune functionality of hematopoietic immune cells, non-hematopoietic cell populations - including epithelial and stromal cells - are now recognized as playing a key role in innate defense at this site. In this study we examined the innate immune capacity of primary human intestinal stromal cells (iSCs). CD90(+) iSCs isolated from human colonic mucosa expressed a wide array of innate immune receptors and functionally responded to stimulation with bacterial ligands. iSCs also sensed infection with live Salmonella typhimurium, rapidly expressing IL-1 family cytokines via a RIPK2/p38MAPK-dependent signaling process. In addition to responding to innate immune triggers, primary iSCs exhibited a capacity for bacterial uptake, phagocytosis, and antigen processing, although to a lesser extent than professional APCs. Thus CD90(+) iSCs represent an abundant population of "non-professional" innate immune effector cells of the human colonic mucosa and likely play an important adjunctive role in host defense and immune regulation at this site.
RESUMEN
NOD2 is an intracellular sensor that contributes to immune defense and inflammation. Here we investigated whether NOD2 mediates its effects through control of microRNAs (miRNAs). miR-29 expression was upregulated in human dendritic cells (DCs) in response to NOD2 signals, and miR-29 regulated the expression of multiple immune mediators. In particular, miR-29 downregulated interleukin-23 (IL-23) by targeting IL-12p40 directly and IL-23p19 indirectly, likely via reduction of ATF2. DSS-induced colitis was worse in miR-29-deficient mice and was associated with elevated IL-23 and T helper 17 signature cytokines in the intestinal mucosa. Crohn's disease (CD) patient DCs expressing NOD2 polymorphisms failed to induce miR-29 upon pattern recognition receptor stimulation and showed enhanced release of IL-12p40 on exposure to adherent invasive E. coli. Therefore, we suggest that loss of miR-29-mediated immunoregulation in CD DCs might contribute to elevated IL-23 in this disease.
Asunto(s)
Enfermedad de Crohn/inmunología , Células Dendríticas/inmunología , Interleucina-23/metabolismo , MicroARNs/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Factor de Transcripción Activador 2/metabolismo , Animales , Células Cultivadas , Células Dendríticas/metabolismo , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Humanos , Inflamación/inmunología , Subunidad p40 de la Interleucina-12/metabolismo , Mucosa Intestinal/inmunología , Ratones , Ratones Noqueados , MicroARNs/genética , Proteína Adaptadora de Señalización NOD2/genética , Polimorfismo de Nucleótido Simple , Células Th17/inmunologíaRESUMEN
HLA-B27 can form beta-2 microglobulin (beta2m)-associated heterotrimers (HLA-B27) and beta2m-free homodimers (B27(2)). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)-like receptors KIR3DL1 and KIR3DL2 and with Ig-like transcripts LILRB1 and LILRB2. HLA-B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen-derived epitopes bound to KIR3DL1-expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA-B27 inhibited NK cell IFN-gamma production in a peptide-dependent fashion. B27 but not HLA-A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B27(2) bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B27(2) inhibited NK and T cell IFN-gamma production. By contrast with HLA heterotrimers, B27(2) binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B27(2) could be involved in the pathogenesis of spondyloarthritis.
Asunto(s)
Presentación de Antígeno/inmunología , Antígeno HLA-B27/química , Antígeno HLA-B27/metabolismo , Activación de Linfocitos/inmunología , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Western Blotting , Dimerización , Electroforesis en Gel de Poliacrilamida , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Citometría de Flujo , Antígeno HLA-B27/inmunología , Humanos , Células Asesinas Naturales/inmunología , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Receptores KIR , Receptores KIR3DL1 , Receptores KIR3DL2 , Espondiloartritis/inmunología , Linfocitos T/inmunología , TransfecciónRESUMEN
The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno HLA-B27/genética , Proteínas de Unión al ARN , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/biosíntesis , Femenino , Antígeno HLA-B27/fisiología , Humanos , Virus de la Influenza A/inmunología , Interferón gamma/biosíntesis , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de la Nucleocápside , Nucleoproteínas/inmunología , Nucleoproteínas/farmacología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/virología , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/virología , Transgenes/inmunología , Proteínas del Núcleo Viral/inmunología , Proteínas del Núcleo Viral/farmacologíaRESUMEN
OBJECTIVE: HLA-B27 is capable of forming in vitro a heavy-chain homodimer structure lacking beta(2)-microglobulin. We undertook this study to ascertain if patients with spondylarthritis express beta(2)-microglobulin-free HLA-B27 heavy chains in the form of homodimers and receptors for HLA-B27 homodimers. METHODS: Expression of HLA-B27 heavy chains by mononuclear cells was analyzed by fluorescence-activated cell sorter staining, Western blotting with the monoclonal antibody HC-10, and 2-dimensional isoelectric focusing. Fluorescence-labeled tetrameric complexes of HLA-B27 heavy-chain homodimers were constructed in which each dimer comprised one His-tagged heavy chain and one biotinylated heavy chain, and were used to stain patient and control mononuclear cells and transfected cell lines. RESULTS: Patients with spondylarthritis expressed cell-surface HLA-B27 homodimers. Populations of synovial and peripheral blood monocytes, and B and T lymphocytes from patients with spondylarthritis, and controls carried receptors for HLA-B27 homodimers. Experiments with transfected cell lines demonstrated that KIR3DL1 and KIR3DL2, and immunoglobulin-like transcript 4 (ILT4), but not ILT2, are receptors for HLA-B27 homodimers. CONCLUSION: Patients with spondylarthritis express both HLA-B27 heavy-chain homodimers and receptors for HLA-B27 homodimers. This may be of significance with regard to disease pathogenesis.