RESUMO
Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.
Assuntos
Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias/imunologia , Linfócitos T/imunologia , Biomarcadores Tumorais/metabolismo , Antígenos CD8/metabolismo , Quimiocina CXCL13/metabolismo , Cromossomos Humanos Par 9/genética , Estudos de Coortes , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Exoma/genética , Amplificação de Genes , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Análise Multivariada , Mutação/genética , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único/genética , Receptores CCR5/metabolismo , Linfócitos T/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
We tested a newly described molecular memory system, CCR5 signaling, for its role in recovery after stroke and traumatic brain injury (TBI). CCR5 is uniquely expressed in cortical neurons after stroke. Post-stroke neuronal knockdown of CCR5 in pre-motor cortex leads to early recovery of motor control. Recovery is associated with preservation of dendritic spines, new patterns of cortical projections to contralateral pre-motor cortex, and upregulation of CREB and DLK signaling. Administration of a clinically utilized FDA-approved CCR5 antagonist, devised for HIV treatment, produces similar effects on motor recovery post stroke and cognitive decline post TBI. Finally, in a large clinical cohort of stroke patients, carriers for a naturally occurring loss-of-function mutation in CCR5 (CCR5-Δ32) exhibited greater recovery of neurological impairments and cognitive function. In summary, CCR5 is a translational target for neural repair in stroke and TBI and the first reported gene associated with enhanced recovery in human stroke.
Assuntos
Lesões Encefálicas Traumáticas/terapia , Receptores CCR5/metabolismo , Acidente Vascular Cerebral/terapia , Idoso , Idoso de 80 Anos ou mais , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Córtex Motor/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Receptores CCR5/fisiologia , Reabilitação do Acidente Vascular Cerebral/métodosRESUMO
Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.
Assuntos
Regulação da Expressão Gênica , Variação Genética , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1 , RNA Antissenso/genética , RNA Longo não Codificante/genética , Receptores CCR5/genética , Regiões 3' não Traduzidas , Alelos , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Membrana Celular/metabolismo , Genes Reporter , Genótipo , Infecções por HIV/metabolismo , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Grupos Populacionais/genética , Prognóstico , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Carga ViralRESUMO
The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCRâ arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.
Assuntos
Fosfopeptídeos , Receptores CCR5 , Humanos , Fosforilação , beta-Arrestinas/metabolismo , Fosfopeptídeos/metabolismo , Receptores CCR5/metabolismo , Linhagem CelularRESUMO
Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
Assuntos
Eosinófilos/imunologia , Inflamação/imunologia , Monócitos/imunologia , Receptores CCR/imunologia , Animais , Quimiocinas/imunologia , Quimiocinas/metabolismo , Eosinófilos/metabolismo , Perfilação da Expressão Gênica/métodos , Inflamação/genética , Inflamação/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores CCR/genética , Receptores CCR/metabolismo , Receptores CCR1/imunologia , Receptores CCR1/metabolismo , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Receptores CCR3/imunologia , Receptores CCR3/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismoRESUMO
Real-world memories are formed in a particular context and are often not acquired or recalled in isolation1-5. Time is a key variable in the organization of memories, as events that are experienced close in time are more likely to be meaningfully associated, whereas those that are experienced with a longer interval are not1-4. How the brain segregates events that are temporally distinct is unclear. Here we show that a delayed (12-24 h) increase in the expression of C-C chemokine receptor type 5 (CCR5)-an immune receptor that is well known as a co-receptor for HIV infection6,7-after the formation of a contextual memory determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed expression of CCR5 in mouse dorsal CA1 neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dorsal CA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, and therefore closes the temporal window for memory linking. Our findings also show that an age-related increase in the neuronal expression of CCR5 and its ligand CCL5 leads to impairments in memory linking in aged mice, which could be reversed with a Ccr5 knockout and a drug approved by the US Food and Drug Administration (FDA) that inhibits this receptor, a result with clinical implications. Altogether, the findings reported here provide insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.
Assuntos
Região CA1 Hipocampal , Memória , Neurônios , Receptores CCR5 , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Memória/fisiologia , Rememoração Mental/fisiologia , Camundongos , Neurônios/metabolismo , Receptores CCR5/deficiência , Receptores CCR5/genética , Receptores CCR5/metabolismo , Fatores de TempoRESUMO
Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
Assuntos
Macrófagos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Mioblastos/citologia , Nicotinamida Fosforribosiltransferase/metabolismo , Nicho de Células-Tronco , Peixe-Zebra/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Humanos , Macrófagos/citologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Fator de Transcrição PAX7/metabolismo , RNA-Seq , Receptores CCR5/genética , Receptores CCR5/metabolismo , Regeneração/fisiologia , Análise de Célula Única , Peixe-Zebra/imunologiaRESUMO
Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.
Assuntos
Linfócitos T CD8-Positivos , Movimento Celular , Leishmaniose Cutânea , Receptores CCR5 , Animais , Receptores CCR5/metabolismo , Receptores CCR5/imunologia , Linfócitos T CD8-Positivos/imunologia , Camundongos , Humanos , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Camundongos Knockout , Camundongos Endogâmicos C57BL , Antagonistas dos Receptores CCR5/farmacologia , Maraviroc/farmacologia , FemininoRESUMO
CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.
Assuntos
Quimiocina CCL5/química , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/imunologia , HIV-1/fisiologia , Modelos Moleculares , Mimetismo Molecular , Receptores CCR5/química , Animais , Antagonistas dos Receptores CCR5/química , Antagonistas dos Receptores CCR5/farmacologia , Quimiocina CCL5/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Cicloexanos/química , Cicloexanos/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/química , Infecções por HIV/tratamento farmacológico , Humanos , Maraviroc , Ligação Proteica , Conformação Proteica , Receptores CCR5/metabolismo , Células Sf9 , Spodoptera , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Cenicriviroc, a dual CCR2/CCR5 antagonist, initially developed as an anti-HIV drug, has shown promising results in nonalcoholic steatohepatitis phase 2 clinical trials. It inhibits the infiltration and activation of CCR2+/CCR5+ monocytes and macrophages to the site of liver injury, preventing liver fibrosis. However, the role of Cenicriviroc in the modulation of helper T cell differentiation and functions remains to be explored. In inflamed colons of Crohn's disease patients, CCR2+ and CCR5+ CD4+ T cells are enriched. Considering the role of CCR2+ and CCR5+ T cells in IBD pathogenesis, we investigated the potential role of Cenicriviroc in colitis. Our in vitro studies revealed that Cenicriviroc inhibits Th1-, Th2-, and Th17-cell differentiation while promoting the generation of type 1 regulatory T cells (Tr1), known for preventing inflammation through induction of IL-10. This study is the first to report that Cenicriviroc promotes Tr1 cell generation by up-regulating the signature of Tr1 cell transcription factors such as c-Maf, Prdm1, Irf-1, Batf, and EGR-2. Cenicriviroc displayed a protective effect in experimental colitis models by preventing body weight loss and intestinal inflammation and preserving epithelial barrier integrity. We show that Cenicriviroc induced IL-10 and inhibited the generation of pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and IL-1ß during colitis. Based on our data, we propose Cenicriviroc as a potential therapeutic in controlling tissue inflammation by inhibiting the generation and functions of effector T cells and promoting the induction of anti-inflammatory Tr1 cells.
Assuntos
Antagonistas dos Receptores CCR5 , Diferenciação Celular , Colite , Receptores CCR2 , Receptores CCR5 , Linfócitos T Reguladores , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Camundongos , Receptores CCR2/metabolismo , Receptores CCR2/antagonistas & inibidores , Colite/imunologia , Colite/tratamento farmacológico , Colite/induzido quimicamente , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Antagonistas dos Receptores CCR5/farmacologia , Antagonistas dos Receptores CCR5/uso terapêutico , Receptores CCR5/metabolismo , Humanos , Células Th17/imunologia , Células Th17/efeitos dos fármacos , Sulfóxidos/farmacologia , Camundongos Endogâmicos C57BL , Células Th1/imunologia , Células Th1/efeitos dos fármacos , Interleucina-10/metabolismo , Células Th2/imunologia , ImidazóisRESUMO
CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.
Assuntos
Infecções por HIV , HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Replicação Viral , Animais , Feminino , Masculino , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/patologia , Progressão da Doença , HIV/classificação , HIV/crescimento & desenvolvimento , HIV/patogenicidade , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Intestinos/virologia , Tecido Linfoide/virologia , Macaca mulatta/imunologia , Macaca mulatta/metabolismo , Inoculações Seriadas , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/classificação , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Vírus da Imunodeficiência Símia/patogenicidade , Vírus da Imunodeficiência Símia/fisiologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Carga Viral , Tropismo Viral , Virulência , Receptores CCR5/metabolismoRESUMO
The dense patch of high-mannose-type glycans surrounding the N332 glycan on the HIV envelope glycoprotein (Env) is targeted by multiple broadly neutralizing antibodies (bnAbs). This region is relatively conserved, implying functional importance, the origins of which are not well understood. Here we describe the isolation of new bnAbs targeting this region. Examination of these and previously described antibodies to Env revealed that four different bnAb families targeted the (324)GDIR(327) peptide stretch at the base of the gp120 V3 loop and its nearby glycans. We found that this peptide stretch constitutes part of the CCR5 co-receptor binding site, with the high-mannose patch glycans serving to camouflage it from most antibodies. GDIR-glycan bnAbs, in contrast, bound both (324)GDIR(327) peptide residues and high-mannose patch glycans, which enabled broad reactivity against diverse HIV isolates. Thus, as for the CD4 binding site, bnAb effectiveness relies on circumventing the defenses of a critical functional region on Env.
Assuntos
Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Polissacarídeos/metabolismo , Motivos de Aminoácidos , Antígenos CD4/metabolismo , Mapeamento de Epitopos , Epitopos/metabolismo , Engenharia Genética , Células HEK293 , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Imunidade Humoral , Memória Imunológica , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/imunologia , Ligação Proteica , Receptores CCR5/metabolismoRESUMO
A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression.
Assuntos
Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1 , Transplante de Células-Tronco Hematopoéticas/métodos , Receptores CCR5/química , Receptores CCR5/genética , Linfócitos T CD4-Positivos/imunologia , Citomegalovirus/química , Citomegalovirus/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/complicações , HIV-1/química , HIV-1/imunologia , Doença de Hodgkin/complicações , Doença de Hodgkin/tratamento farmacológico , Humanos , Receptores CCR5/deficiência , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Transplante Homólogo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.
Assuntos
Antígenos CD4/química , Antígenos CD4/ultraestrutura , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/ultraestrutura , Receptores CCR5/química , Receptores CCR5/ultraestrutura , Receptores de HIV/química , Receptores de HIV/ultraestrutura , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Sítios de Ligação , Antígenos CD4/isolamento & purificação , Antígenos CD4/metabolismo , Linhagem Celular , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Proteína gp120 do Envelope de HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/ultraestrutura , Humanos , Ligantes , Maraviroc/química , Maraviroc/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores CCR5/isolamento & purificação , Receptores CCR5/metabolismo , Receptores de HIV/antagonistas & inibidores , Receptores de HIV/metabolismoRESUMO
T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal. Despite the significance of such chemotactic repolarization to the accurate navigation and function of T cells, the precise signaling mechanisms that underlie it remain elusive. Notably, it remained unclear whether the distribution of chemokine receptors on the T cell surface is altered during repolarization. Here, we developed parallel cell-secreted and microfluidics-based chemokine gradient delivery methods and employed both fixed imaging and live lattice light-sheet microscopy to investigate the dynamics of chemokine receptor CCR5 on the surface of primary murine CD8+ T cells. Our findings show that, during constitutive migration, chemokine receptor distribution is largely isotropic on the T cell surface. However, upon exposure to a CCL3 gradient, surface chemokine receptor distributions exhibit a transient bias toward the uropod. The chemokine receptors then progressively redistribute from the uropod to cover the T cell surface uniformly. This study sheds new light on the dynamics of surface chemokine receptor distribution during T cell repolarization, advancing our understanding of the signaling of immune cells in the complex chemokine landscapes they navigate.
Assuntos
Receptores CCR5 , Animais , Camundongos , Receptores CCR5/metabolismo , Membrana Celular/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Camundongos Endogâmicos C57BL , Movimento Celular , Receptores de Quimiocinas/metabolismo , Linfócitos T/metabolismo , Linfócitos T/citologia , Polaridade CelularRESUMO
Inflammatory bowel disease (IBD) is a chronic systemic inflammatory condition regarded as a major risk factor for colitis-associated cancer. However, the underlying mechanisms of IBD remain unclear. First, five GSE data sets available in GEO were used to perform 'batch correction' and Robust Rank Aggregation (RRA) to identify differentially expressed genes (DEGs). Candidate molecules were identified using CytoHubba, and their diagnostic effectiveness was predicted. The CIBERSORT algorithm evaluated the immune cell infiltration in the intestinal epithelial tissues of patients with IBD and controls. Immune cell infiltration in the IBD and control groups was determined using the least absolute shrinkage selection operator algorithm and Cox regression analysis. Finally, a total of 51 DEGs were screened, and nine hub genes were identified using CytoHubba and Cytoscape. GSE87466 and GSE193677 were used as extra data set to validate the expression of the nine hub genes. CD4-naïve T cells, gamma-delta T cells, M1 macrophages and resting dendritic cells (DCs) are the main immune cell infiltrates in patients with IBD. Signal transducer and activator of transcription 1, CCR5 and integrin subunit beta 2 (ITGB2) were significantly upregulated in the IBD mouse model, and suppression of ITGB2 expression alleviated IBD inflammation in mice. Additionally, the expression of ITGB2 was upregulated in IBD-associated colorectal cancer (CRC). The silence of ITGB2 suppressed cell proliferation and tumour growth in vitro and in vivo. ITGB2 resting DCs may provide a therapeutic strategy for IBD, and ITGB2 may be a potential diagnostic marker for IBD-associated CRC.
Assuntos
Biologia Computacional , Doenças Inflamatórias Intestinais , Humanos , Animais , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Biologia Computacional/métodos , Camundongos , Perfilação da Expressão Gênica , Modelos Animais de Doenças , Antígenos CD18/genética , Antígenos CD18/metabolismo , Mapas de Interação de Proteínas , Receptores CCR5/genética , Receptores CCR5/metabolismoRESUMO
This study explores the impact of senescence on autocrine C-C motif chemokine ligand 5 (CCL5) in human endothelial progenitor cell (EPCs), addressing the poorly understood decline in number and function of EPCs during ageing. We examined the effects of replication-induced senescence on CCL5/CCL5 receptor (CCR5) signalling and angiogenic activity of EPCs in vitro and in vivo. We also explored microRNAs controlling CCL5 secretion in senescent EPCs, its impact on EPC angiogenic activity, and validated our findings in humans. CCL5 secretion and CCR5 levels in senescent EPCs were reduced, leading to attenuated angiogenic activity. CCL5 enhanced EPC proliferation via the CCR5/AKT/P70S6K axis and increased vascular endothelial growth factor (VEGF) secretion. Up-regulation of miR-409 in senescent EPCs resulted in decreased CCL5 secretion, inhibiting the angiogenic activity, though these negative effects were counteracted by the addition of CCL5 and VEGF. In a mouse hind limb ischemia model, CCL5 improved the angiogenic activity of senescent EPCs. Analysis involving 62 healthy donors revealed a negative association between CCL5 levels, age and Framingham Risk Score. These findings propose CCL5 as a potential biomarker for detection of EPC senescence and cardiovascular risk assessment, suggesting its therapeutic potential for age-related cardiovascular disorders.
Assuntos
Senescência Celular , Quimiocina CCL5 , Células Progenitoras Endoteliais , MicroRNAs , Neovascularização Fisiológica , Animais , Humanos , Masculino , Camundongos , Angiogênese , Proliferação de Células , Quimiocina CCL5/metabolismo , Quimiocina CCL5/genética , Regulação para Baixo/genética , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/citologia , Isquemia/metabolismo , Isquemia/patologia , Isquemia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Receptores CCR5/metabolismo , Receptores CCR5/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in leukocyte development and inflammation and their status as coreceptors for HIV-1 infection, among other roles. Both receptors form dimers or oligomers of unclear function. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate their dimerization interfaces, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that change how the receptors self-associate, either via specific oligomer assembly or alternative mechanisms of clustering in close proximity. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations predicted from the scan to reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. A mutation in the dimer interface of CXCR4 had increased binding to the ligand CXCL12 and yet diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.
Assuntos
Modelos Moleculares , Mutação , Receptores CCR5 , Receptores CXCR4 , Dimerização , Mutagênese , Receptores CCR5/química , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/química , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Humanos , Linhagem Celular , Estrutura Terciária de ProteínaRESUMO
The HIV-1 envelope glycoprotein (Env) plays crucial role in viral infection by facilitating viral attachment to host cells and inducing fusion of the virus with the host cell membrane. This fusion allows the HIV-1 viral genome to enter the target cell then triggering various stages of the viral life cycle. The native Env directly interacts with the main receptor CD4 and the co-receptor (CCR5 or CXCR4) in human cell membrane then induces membrane fusion. The elucidation of the structure of Env with CD4 and co-receptors in different HIV-1 subtypes is essential for the understanding of the mechanism of virus entry. Here we report the Cryo-EM structure of the CD4-bound HIV-1 heterotrimeric Env from Asia prevalent CRF07_BC CH119 strain. In this structure, the binding of three CD4 molecules with Env induced extensively conformational changes in gp120, resulting in the transformation of the Env from close state to intermediate open state. Additionally, the conformational shift of V1/V2 loops of the heterotrimeric Env allosterically expose the V3 loop and promoting the further interactions with co-receptor CCR5 or CXCR4. These findings not only illustrate the structural complexity and plasticity of HIV-1 Env but also give new insights how the biological trimeric Env initialize the immune recognition and membrane fusion.
Assuntos
Antígenos CD4 , Proteína gp120 do Envelope de HIV , HIV-1 , HIV-1/metabolismo , Humanos , Antígenos CD4/metabolismo , Antígenos CD4/química , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Microscopia Crioeletrônica , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Receptores CCR5/metabolismo , Receptores CCR5/química , Ligação Proteica , Modelos Moleculares , Conformação Proteica , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Multimerização Proteica , Receptores CXCR4/metabolismo , Receptores CXCR4/química , ÁsiaRESUMO
Autoimmune uveitis is a leading cause of severe vision loss, and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq, RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU), revealing that EAU causes broad retinal neuron degeneration and marker downregulation, and that Müller glia may act as antigen-presenting cells. Moreover, EAU immune response is primarily driven by Th1 cells, and results in dramatic upregulation of CC chemokines, especially CCL5, in the EAU retina. Accordingly, overexpression of CCR5, a CCL5 receptor, in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU, by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together, our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.