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1.
Leukemia ; 35(12): 3466-3481, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34035409

RESUMO

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.


Assuntos
Antígenos CD7/genética , Complexo CD3/genética , Imunoterapia Adotiva/métodos , Leucemia de Células T/terapia , Linfócitos T/imunologia , Animais , Antígenos CD7/química , Antígenos CD7/metabolismo , Complexo CD3/antagonistas & inibidores , Complexo CD3/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Edição de Genes , Humanos , Leucemia de Células T/imunologia , Leucemia de Células T/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Elife ; 92020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33300875

RESUMO

HIV-1 must replicate in cells that are equipped to defend themselves from infection through intracellular innate immune systems. HIV-1 evades innate immune sensing through encapsidated DNA synthesis and encodes accessory genes that antagonize specific antiviral effectors. Here, we show that both particle associated, and expressed HIV-1 Vpr, antagonize the stimulatory effect of a variety of pathogen associated molecular patterns by inhibiting IRF3 and NF-κB nuclear transport. Phosphorylation of IRF3 at S396, but not S386, was also inhibited. We propose that, rather than promoting HIV-1 nuclear import, Vpr interacts with karyopherins to disturb their import of IRF3 and NF-κB to promote replication in macrophages. Concordantly, we demonstrate Vpr-dependent rescue of HIV-1 replication in human macrophages from inhibition by cGAMP, the product of activated cGAS. We propose a model that unifies Vpr manipulation of nuclear import and inhibition of innate immune activation to promote HIV-1 replication and transmission.


Assuntos
Infecções por HIV/imunologia , Evasão da Resposta Imune/fisiologia , Imunidade Inata/imunologia , Replicação Viral/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/imunologia , Transporte Ativo do Núcleo Celular/fisiologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Carioferinas/imunologia , Carioferinas/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
3.
Mol Ther Methods Clin Dev ; 17: 209-219, 2020 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31970199

RESUMO

Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-ß also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture.

4.
JCI Insight ; 3(13)2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29997304

RESUMO

T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of T-on-T cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3 (3CAR). Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells, and this has provided a route to generation of 3CAR, CD3-specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ε CAR derived from OKT3, either before or after TALEN-mediated disruption of the endogenous TCRαß/CD3 complex. Only transduction after disrupting assembly of TCRαß/CD3 yielded viable 3CAR T cells, and these cultures were found to undergo self-enrichment for 3CAR+TCR-CD3- T cells without any further processing. Specific cytotoxicity against CD3ε was demonstrated against primary T cells and against childhood T cell acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent antileukemic effects in a human/murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a bridging strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.


Assuntos
Complexo CD3/imunologia , Imunoterapia , Leucemia/tratamento farmacológico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/genética , Engenharia Celular , Linhagem Celular Tumoral , Feminino , Terapia Genética , Humanos , Camundongos , Muromonab-CD3 , Receptores de Antígenos Quiméricos/genética , Transdução Genética
5.
Nature ; 503(7476): 402-405, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24196705

RESUMO

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.


Assuntos
HIV-1/imunologia , Evasão da Resposta Imune , Imunidade Inata/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Ciclofilinas/metabolismo , Ciclosporina/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Macrófagos/citologia , Macrófagos/patologia , Chaperonas Moleculares/metabolismo , Monócitos/citologia , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores de Reconhecimento de Padrão , Internalização do Vírus , Replicação Viral/imunologia , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
6.
J Virol ; 87(5): 2882-94, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23269792

RESUMO

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as "molecular BL" (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.


Assuntos
Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Transcriptoma , Latência Viral/genética , Antígenos Virais/biossíntese , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/classificação , Humanos , Fatores Reguladores de Interferon/biossíntese , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/biossíntese , Regulação para Cima , Proteínas Virais/biossíntese
7.
PLoS Pathog ; 7(12): e1002439, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22174692

RESUMO

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.


Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/virologia , Ciclofilina A/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Replicação Viral , Transporte Ativo do Núcleo Celular/fisiologia , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/fisiologia
8.
Immunology ; 127(3): 429-41, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19538250

RESUMO

Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy.


Assuntos
Células Dendríticas/imunologia , Leucemia Mieloide Aguda/imunologia , Apresentação de Antígeno/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Regulação para Baixo/imunologia , Expressão Gênica/imunologia , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Receptores de Lipopolissacarídeos/análise , Família Multigênica/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica/imunologia , Células Tumorais Cultivadas
9.
Br J Haematol ; 138(3): 281-90, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17614817

RESUMO

Dendritic cells (DCs) are key antigen-presenting cells (APCs), which link innate and adaptive immunity, ultimately activating antigen-specific T cells. This review examines the relationship between the acute and chronic myeloid leukaemias and cells with DC properties. DCs are non-dividing terminally differentiated cells, and ex vivo leukaemic cells or cell lines show little similarity to DCs. However, many leukaemias differentiate further in response to defined stimuli, and retain a degree of lineage plasticity. Therefore, several studies have explored the response of leukaemic cells to the in vitro regimens used to differentiate ex vivo primary DCs. Recent data suggest that the most 'dendritic-like' cells can be derived from more undifferentiated myeloid leukaemias, such as the myelomonocytic Mutz-3 cell line. These findings have important implications for understanding the developmental origins of DCs, for harnessing the APC properties of this class of tumour to stimulate the therapeutic anti-tumour immunity, and for developing useful models for the study of human DC physiology and pathology. There is a strong rationale for further exploration of this class of tumour and its relationship to the normal DC.


Assuntos
Células Dendríticas/patologia , Leucemia Mieloide/patologia , Doença Aguda , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Doença Crônica , Humanos , Imunidade Inata , Imunoterapia Adotiva
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