RESUMO
Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. SIGNIFICANCE: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.
Assuntos
Neoplasias Colorretais , Memória Imunológica , Animais , Linfócitos T CD8-Positivos , Neoplasias Colorretais/metabolismo , Dopamina/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Receptores de Dopamina D5/metabolismoRESUMO
Human Vδ2 cells are innate-like γδ T effectors performing potent immune surveillance against tumors. The constitutive expression of NKG2A identifies a subset of Vδ2 T cells licensed with an intrinsic hyper-responsiveness against cancer. Indeed, the transcriptomic profiles of NKG2A+ and NKG2A- cells characterize two distinct "intralineages" of Vδ2 T lymphocytes that appear early during development, keep their phenotypes, and show self-renewal capabilities in adult life. The hyper-responsiveness of NKG2A+ Vδ2 T cells is counterbalanced by the inhibitory signaling delivered by human leukocyte antigen E (HLA-E) expressed on malignant cells as a tumor-escape mechanism. However, either masking or knocking out NKG2A restores the capacity of Vδ2 T cells to exert the highest effector functions even against HLA-E+ tumors. This is highly relevant in the clinic, as the different degrees of engagement of the NKG2A-HLA-E checkpoint in hepatocellular carcinoma, glioblastoma, and non-small cell lung cancer directly impact patients' overall survival. These findings open avenues for developing combined cellular and immunologic anticancer therapies.
Assuntos
Citotoxicidade Imunológica , Linfócitos Intraepiteliais/metabolismo , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Idoso , Estudos de Casos e Controles , Proliferação de Células , Autorrenovação Celular , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Lactente , Linfócitos Intraepiteliais/imunologia , Células K562 , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Fenótipo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de SinaisRESUMO
Donor lymphocyte infusion (DLI) is a standard of care for relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Currently it is poorly understood how and when CD8+ αß T cells exert graft-versus-leukemia (GVL) activity after DLI. Also, there is no reliable biomarker to monitor GVL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αß T-cell clones in patients with DLI. In this prospective clinical study of 29 patients, we performed deep T-cell receptor ß (TRB ) sequencing of sorted CD8+ αß T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GVL, longitudinal analyses revealed a preferential expansion of distinct CD8+TRB clones (n = 14). This did not occur in samples of patients without signs of GVL (n = 11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36-month study follow-up. Furthermore, absence of clonal outgrowth of the CD8+TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+TRB diversity at day 15 after DLI (n = 13) had a lower relapse incidence (P = .0040) compared with patients without clonal expansion (n = 6). In conclusion, CD8+TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.
Assuntos
Leucemia Mieloide Aguda , Transfusão de Linfócitos , Linfócitos T CD8-Positivos , Humanos , Imunoterapia Adotiva , Leucemia Mieloide Aguda/terapia , Estudos ProspectivosRESUMO
BACKGROUND: In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear. METHOD: In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs. RESULTS: We discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo. CONCLUSION: Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Leucina/metabolismo , Linfócitos do Interstício Tumoral/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma Experimental/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Ativação Enzimática , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Evasão Tumoral , Microambiente TumoralRESUMO
The majority of genetically modified C57BL/6 mice contain congenic passenger DNA around the targeted gene locus as they were generated from 129-derived embryonic stem cells (ESCs) with subsequent backcrossing to the C57BL/6 genetic background. When studying the role of atypical chemokine receptor 4 (ACKR4) in the immune system, we realized that the two available Ackr4-deficient mouse strains (Ackr4-/- and Ackr4GFP/GFP ) show profoundly different phenotypes: Compared to wild-type and Ackr4GFP/GFP mice, Ackr4-/- mice show a strong accumulation of plasma blasts in mesenteric lymph node and spleen as well as increased B cell proliferation after in vitro activation. This phenotype was maintained after further backcrossing to C57BL/6 mice and was even present in heterozygous Ackr4+/- animals, suggesting that a gene variant on the targeted chromosome might cause this phenotype. Exome sequencing revealed that a region of approximately 20 Mbp around the Ackr4 locus on chromosome 9 still originates from the 129 background based on high variant density observed. In activated Ackr4-/- and Ackr4GFP/GFP B cells, transcripts of genes around the Ackr4 locus were equally deregulated compared to C57BL/6 B cells, whereas increased expression of IL-6 was selectively observed in B cells of Ackr4-/- mice. Because the gene encoding for IL-6 is placed on chromosome 5 these findings suggest that passenger DNA around the Ackr4 locus has an indirect effect on B cell activation and IL-6 production. Results of the present study should not only lead to the reinterpretation of data from earlier studies using Ackr4-/- mice but should remind the scientific community about the limitations of mouse models using mice created by gene-targeting of nonsyngeneic ESCs.
Assuntos
Linfócitos B/imunologia , Cromossomos de Mamíferos/imunologia , Interleucina-6/genética , Linfonodos/imunologia , Ativação Linfocitária , Receptores CCR/genética , Animais , Linfócitos B/citologia , Proliferação de Células , Cruzamentos Genéticos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Feminino , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Homozigoto , Interleucina-6/imunologia , Linfonodos/citologia , Masculino , Mesentério/citologia , Mesentério/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptores CCR/deficiência , Receptores CCR/imunologia , Baço/citologia , Baço/imunologia , Sequenciamento do ExomaRESUMO
IL-17-producing γδ T cells express oligoclonal Vγ4+ and Vγ6+ TCRs, mainly develop in the prenatal thymus, and later persist as long-lived self-renewing cells in all kinds of tissues. However, their exchange between tissues and the mechanisms of their tissue-specific adaptation remain poorly understood. Here, single-cell RNA-seq profiling identifies IL-17-producing Vγ6+ T cells as a highly homogeneous Scart1+ population in contrast to their Scart2+ IL-17-producing Vγ4+ T cell counterparts. Parabiosis demonstrates that Vγ6+ T cells are fairly tissue resident in the thymus, peripheral lymph nodes, and skin. There, Scart1+ Vγ6+ T cells display tissue-specific gene expression signatures in the skin, characterized by steady-state production of the cytokines IL-17A and amphiregulin as well as by high expression of the anti-apoptotic Bcl2a1 protein family. Together, this study demonstrates how Scart1+ Vγ6+ T cells undergo tissue-specific functional adaptation to persist as effector cells in their skin habitat.
Assuntos
Antígenos de Histocompatibilidade Menor/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Superfície Celular/metabolismo , Análise de Célula Única/métodos , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Transcriptoma , Animais , Sobrevivência Celular , Células Cultivadas , Interleucina-17/genética , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/genética , Pele/metabolismo , Pele/patologiaRESUMO
Zika virus (ZIKV) is genetically and biologically related to other Flaviviridae family members and has disseminated to many countries. It is associated with severe consequences, including the abnormal development of the neural system in fetuses and neurological diseases in adults. Therefore, the development of anti-ZIKV drugs is of paramount importance. Screening of generic drugs revealed that several nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, acetaminophen, and lornoxicam, potently inhibited the entry of Zika virus Env/HIV-1-pseudotyped viruses. They also significantly inhibited the replication of wild-type ZIKV both in cell lines and in primary human fetal endothelial cells. Interestingly, the NSAIDs exerted this inhibitory effect by potently reducing the expression of AXL, the entry cofactor of ZIKV. Further studies showed that the NSAIDs downregulated the prostaglandin E2/prostaglandin E receptor 2 (EP2)/cAMP/protein kinase A (PKA) signaling pathway and reduced PKA-dependent CDC37 phosphorylation and the interaction between CDC37 and HSP90, which subsequently facilitated CHIP/ubiquitination/proteasome-mediated AXL degradation. Taken together, our results highlight a new mechanism of action of antiviral agents which may assist in designing a convenient strategy for treating ZIKV-infected patients.IMPORTANCE Zika virus (ZIKV) infection, which causes congenital malformations, including microcephaly and other neurological disorders, has attracted global attention. We observed that several NSAIDs significantly inhibited ZIKV infection. Based on our observations, we propose a novel mechanism of action of antiviral compounds which involves the blockade of virus entry via degradation of the entry cofactor. Furthermore, NSAIDs can be practically used for preventing ZIKV infection in pregnant women, as certain NSAIDs, including ibuprofen and acetaminophen, are considered clinically safe.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células Endoteliais/virologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Zika virus/fisiologia , Células A549 , Animais , Linhagem Celular , Chlorocebus aethiops , Regulação para Baixo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Proteólise , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/virologia , Receptor Tirosina Quinase AxlRESUMO
Although antiviral drugs are available for the treatment of influenza infection, it is an urgent requirement to develop new antiviral drugs regarding the emergence of drug-resistant viruses. The nucleoprotein (NP) is conserved among all influenza A viruses (IAVs) and has no cellular equivalent. Therefore, NP is an ideal target for the development of new IAV inhibitors. In this study, we identified a novel anti-influenza compound, ZBMD-1, from a library of 20,000 compounds using cell-based influenza A infection assays. We found that ZBMD-1 inhibited the replication of H1N1 and H3N2 influenza A virus strains in vitro, with an IC50 ranging from 0.41-1.14 µM. Furthermore, ZBMD-1 inhibited the polymerase activity and specifically impaired the nuclear export of NP. Further investigation indicated that ZBMD-1 binds to the nuclear export signal 3 (NES3) domain and the dimer interface of the NP pocket. ZBMD-1 also protected mice that were challenged with lethal doses of A/PR/8/1934 (H1N1) virus, effectively relieving lung histopathology changes, as well as strongly inhibiting the expression of pro-inflammatory cytokines/chemokines, without inducing toxicity effects in mice. These results suggest that ZBMD-1 is a promising anti-influenza compound which can be further investigated as a useful strategy against IAVs in the future.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas do Core Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A/metabolismo , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Proteínas de Ligação a RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas do Core Viral/metabolismoRESUMO
Human coinfection with a novel H7N9 influenza virus and the 2009 pandemic A(H1N1) influenza virus, H1N1pdm09, has recently been reported in China. Because reassortment can occur during coinfection, it is necessary to clarify the effects of gene reassortment between these two viruses. Among the viral ribonucleoprotein complex (vRNP) genes, only the PA gene of H1N1pdm09 enhances the avian influenza viral polymerase activity. Based on a phylogenetic analysis, we show a special evolutionary feature of the H1N1pdm09 PA gene, which clustered with those of the novel H7N9 virus and related H9N2 viruses, rather than in the outgroup as the H1N1pdm09 genes do on the phylogenetic trees of other vRNP genes. Using a minigenome system of the novel H7N9 virus, we further demonstrate that replacement of its PA gene significantly enhanced its polymerase activity, whereas replacement of the other vRNP genes reduced its polymerase activity. We also show that the residues of PA evolutionarily conserved between H1N1pdm09 and the novel H7N9 virus are associated with attenuated or neutral polymerase activity. The mutations associated with the increased activity of the novel H7N9 polymerase are characteristic of the H1N1pdm09 gene, and are located almost adjacent to the surface of the PA protein. Our results suggest that the novel H7N9 virus has more effective PB1, PB2, and NP genes than H1N1pdm09, and that H1N1pdm09-like PA mutations enhance the novel H7N9 polymerase function.
Assuntos
Aminoácidos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Subtipo H7N9 do Vírus da Influenza A/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Aminoácidos/genética , China , Coinfecção/virologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Ativação Enzimática , Evolução Molecular , Genes Virais , Genoma Viral , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H1N1/genética , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Vírus da Influenza A Subtipo H9N2/enzimologia , Vírus da Influenza A Subtipo H9N2/genética , Influenza Humana/virologia , Mutação , Proteínas do Nucleocapsídeo , Filogenia , Estrutura Terciária de Proteína , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Vírus Reordenados/classificação , Vírus Reordenados/genética , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
UNLABELLED: The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP) and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying that its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor of IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-α, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent of its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex. IMPORTANCE: The interaction between the influenza virus vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for the influenza virus life cycle since it inhibited the viral growth rate. Conversely, importin-α has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells but not in avian cells. MOV10 disrupted the interaction between NP and importin-α, suggesting that MOV10 could also be an important host factor for influenza virus transmission and pathogenicity. Importantly, as an interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.