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1.
Blood ; 140(5): 451-463, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35605184

RESUMO

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393).


Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Animais , Antígenos CD19 , Síndrome da Liberação de Citocina , Citocinas , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Recidiva , Linfócitos T
2.
Cytotherapy ; 24(8): 767-773, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35597752

RESUMO

BACKGROUND AIMS: Selective immune pressure contributes to relapse due to target antigen downregulation in patients treated with anti-CD19 chimeric antigen receptor (CAR) T cells. Bispecific lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T cells may prevent progression/relapse due to antigen escape. Highly polyfunctional T cells within a CAR T-cell product have been associated with response in single-antigen-targeted anti-CD19 CAR T cells. METHODS: The authors performed a single-cell proteomic analysis to assess polyfunctional cells in our LV20.19 CAR T-cell product. Analysis was limited to those treated at a fixed dose of 2.5 × 106 cells/kg (n = 16). Unused pre-infusion CAR T cells were thawed, sorted into CD4/CD8 subsets and stimulated with K562 cells transduced to express CD19 or CD20. Single-cell production of 32 individual analytes was measured and polyfunctionality and polyfunctional strength index (PSI) were calculated. RESULTS: Fifteen patients had adequate leftover cells for analysis upon stimulation with CD19, and nine patients had adequate leftover cells for analysis upon stimulation with CD20. For LV20.19 CAR T cells, PSI was 866-1109 and polyfunctionality was 40-45%, which were higher than previously reported values for other CAR T-cell products. CONCLUSIONS: Stimulation with either CD19 or CD20 antigens resulted in similar levels of analyte activation, suggesting that this product may have efficacy in CD19- patient populations.


Assuntos
Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Antígenos CD19/uso terapêutico , Antígenos CD20/uso terapêutico , Humanos , Imunoterapia Adotiva/métodos , Recidiva Local de Neoplasia , Proteômica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T
3.
Mol Ther ; 29(9): 2691-2706, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33974997

RESUMO

Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor ß receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-ß on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.


Assuntos
Linfoma de Burkitt/terapia , Imunoterapia Adotiva/métodos , NF-kappa B/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição AP-1/genética , Animais , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Regiões Promotoras Genéticas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Gastroenterology ; 158(8): 2250-2265.e20, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32060001

RESUMO

BACKGROUND AND AIMS: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. METHODS: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. RESULTS: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active ß-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. CONCLUSIONS: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.


Assuntos
Carcinoma Hepatocelular/terapia , Glipicanas/metabolismo , Imunoterapia Adotiva , Neoplasias Hepáticas/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/transplante , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Glipicanas/genética , Glipicanas/imunologia , Granzimas/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Perforina/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Tumoral , Microambiente Tumoral , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Lipid Res ; 59(5): 854-863, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29559522

RESUMO

Apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells and macrophages. In zebrafish and mice, AIBP-mediated regulation of cholesterol levels in the plasma membrane of endothelial cells controls angiogenesis. The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. Here, we show that Apoa1bp-/-Ldlr-/- mice fed a high-cholesterol, high-fat diet had exacerbated weight gain, liver steatosis, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and larger atherosclerotic lesions compared with Ldlr-/- mice. Feeding Apoa1bp-/-Ldlr-/- mice a high-cholesterol, normal-fat diet did not result in significant differences in lipid levels or size of atherosclerotic lesions from Ldlr-/- mice. Conversely, adeno-associated virus-mediated overexpression of AIBP reduced hyperlipidemia and atherosclerosis in high-cholesterol, high-fat diet-fed Ldlr-/- mice. Injections of recombinant AIBP reduced aortic inflammation in Ldlr-/- mice fed a short high-cholesterol, high-fat diet. Conditional overexpression of AIBP in zebrafish also reduced diet-induced vascular lipid accumulation. In experiments with isolated macrophages, AIBP facilitated cholesterol efflux to HDL, reduced lipid rafts content, and inhibited inflammatory responses to lipopolysaccharide.jlr Our data demonstrate that AIBP confers protection against diet-induced metabolic abnormalities and atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , Síndrome Metabólica/metabolismo , Fosfoproteínas/metabolismo , Animais , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/deficiência , Racemases e Epimerases , Receptores de LDL/deficiência , Receptores de LDL/metabolismo
6.
Cytotherapy ; 20(3): 394-406, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29287970

RESUMO

BACKGROUND AIMS: Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. METHODS: The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. RESULTS: Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DISCUSSION: The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.


Assuntos
Antígenos CD19/metabolismo , Técnicas Citológicas/instrumentação , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Centros Médicos Acadêmicos , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos CD20/genética , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Linfócitos B/imunologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Técnicas Citológicas/métodos , Humanos , Imunofenotipagem , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Transdução Genética
7.
Am J Physiol Endocrinol Metab ; 306(12): E1378-87, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24760988

RESUMO

Incomplete ß-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete ß-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 µM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.


Assuntos
Carnitina/análogos & derivados , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos , Macrófagos/imunologia , Receptores de Reconhecimento de Padrão/agonistas , Animais , Carnitina/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Indução Enzimática , Inativação Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/agonistas , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Ácidos Mirísticos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo
8.
J Immunol ; 188(6): 2894-904, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22331068

RESUMO

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-α, -ß, and -γ peaked 1 d postinfection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335(+), TCR-ß(+) cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti-IL-13 neutralizing Ab attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are mediated, at least in part, by IL-13.


Assuntos
Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/patologia , Hipersensibilidade Respiratória/virologia , Mucosa Respiratória/patologia , Animais , Animais Recém-Nascidos , Separação Celular , Citocinas/biossíntese , Citocinas/imunologia , Citometria de Fluxo , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Picornaviridae/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Mucosa Respiratória/imunologia
9.
J Immunother Cancer ; 12(4)2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38609317

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain variable fragment (scFv)-R12 targeting domain failed to induce durable remissions, in part due to the immunosuppressive tumor microenvironment (TME). Herein, we describe the development of an improved ROR1-CAR with a novel, fully human scFv9 targeting domain, and augmented with TGFßRIIDN armor protective against a major TME factor, transforming growth factor beta (TGFß). METHODS: CAR T cells were generated by lentiviral transduction of enriched CD4+ and CD8+ T cells, and the novel scFv9-based ROR1-CAR-1 was compared with the clinically-characterized ROR1-R12-scFv-based CAR-2 in vitro and in vivo. RESULTS: CAR-1 T cells exhibited greater CAR surface density than CAR-2 when normalized for %CAR+, and produced more interferon (IFN)-γ tumor necrosis factor (TNF)-α and interleukin (IL)-2 in response to hematologic (Jeko-1, RPMI-8226) and solid (OVCAR-3, Capan-2, NCI-H226) tumor cell lines in vitro. In vivo, CAR-1 and CAR-2 both cleared hematologic Jeko-1 lymphoma xenografts, however only CAR-1 fully rejected ovarian solid OVCAR-3 tumors, concordantly with greater expansion of CD8+ and CD4+CAR T cells, and enrichment for central and effector memory phenotype. When equipped with TGFß-protective armor TGFßRIIDN, CAR-1 T cells resisted TGFß-mediated pSmad2/3 phosphorylation, as compared with CAR-1 alone. When co-cultured with ROR-1+ AsPC-1 pancreatic cancer line in the presence of TGFß1, armored CAR-1 demonstrated improved recovery of killing function, IFN-γ, TNF-α and IL-2 secretion. In mouse AsPC-1 pancreatic tumor xenografts overexpressing TGFß1, armored CAR-1, in contrast to CAR-1 alone, achieved complete tumor remissions, and yielded accelerated expansion of CAR+ T cells, diminished circulating active TGFß1, and no apparent toxicity or weight loss. Unexpectedly, in AsPC-1 xenografts without TGFß overexpression, TGFß1 production was specifically induced by ROR-1-CAR T cells interaction with ROR-1 positive tumor cells, and the TGFßRIIDN armor conferred accelerated tumor clearance. CONCLUSIONS: The novel fully human TGFßRIIDN-armored ROR1-CAR-1 T cells are highly potent against ROR1-positive tumors, and withstand the inhibitory effects of TGFß in solid TME. Moreover, TGFß1 induction represents a novel, CAR-induced checkpoint in the solid TME, which can be circumvented by co-expressing the TGßRIIDN armor on T cells.


Assuntos
Neoplasias Ovarianas , Neoplasias Pancreáticas , Humanos , Animais , Feminino , Camundongos , Apoptose , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Fator de Crescimento Transformador beta , Microambiente Tumoral , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética
10.
Am J Physiol Lung Cell Mol Physiol ; 304(3): L162-9, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23204071

RESUMO

Human rhinovirus (HRV) infections lead to exacerbations of lower airways disease in asthmatic patients but not in healthy individuals. However, underlying mechanisms remain to be completely elucidated. We hypothesized that the Th2-driven allergic environment enhances HRV-induced CC chemokine production, leading to asthma exacerbations. Ovalbumin (OVA)-sensitized and -challenged mice inoculated with HRV showed significant increases in the expression of lung CC chemokine ligand (CCL)-2/monocyte chemotactic protein (MCP)-1, CCL4/macrophage inflammatory protein (MIP)-1ß, CCL7/MCP-3, CCL19/MIP-3ß, and CCL20/MIP3α compared with mice treated with OVA alone. Inhibition of CCL2 with neutralizing antibody significantly attenuated HRV-induced airways inflammation and hyperresponsiveness in OVA-treated mice. Immunohistochemical stains showed colocalization of CCL2 with HRV in epithelial cells and CD68-positive macrophages, and flow cytometry showed increased CCL2(+), CD11b(+) cells in the lungs of OVA-treated, HRV-infected mice. Compared with lung macrophages from naïve mice, macrophages from OVA-exposed mice expressed significantly more CCL2 in response to HRV infection ex vivo. Pretreatment of mouse lung macrophages and BEAS-2B human bronchial epithelial cells with interleukin (IL)-4 and IL-13 increased HRV-induced CCL2 expression, and mouse lung macrophages from IL-4 receptor knockout mice showed reduced CCL2 expression in response to HRV, suggesting that exposure to these Th2 cytokines plays a role in the altered HRV response. Finally, bronchoalveolar macrophages from children with asthma elaborated more CCL2 upon ex vivo exposure to HRV than cells from nonasthmatic patients. We conclude that CCL2 production by epithelial cells and macrophages contributes to HRV-induced airway hyperresponsiveness and inflammation in a mouse model of allergic airways disease and may play a role in HRV-induced asthma exacerbations.


Assuntos
Quimiocina CCL2/imunologia , Células Epiteliais/imunologia , Hipersensibilidade/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Rhinovirus/fisiologia , Animais , Anticorpos Neutralizantes/farmacologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Quimiocina CCL19/genética , Quimiocina CCL19/imunologia , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiocina CCL20/genética , Quimiocina CCL20/imunologia , Quimiocina CCL4/genética , Quimiocina CCL4/imunologia , Quimiocina CCL7/genética , Quimiocina CCL7/imunologia , Criança , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Expressão Gênica , Humanos , Hipersensibilidade/patologia , Hipersensibilidade/virologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Pulmão/patologia , Pulmão/virologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Ovalbumina , Rhinovirus/patogenicidade , Células Th2/imunologia
11.
PLoS Pathog ; 7(5): e1002070, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21637773

RESUMO

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.


Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/virologia , RNA Helicases DEAD-box/fisiologia , Inflamação/fisiopatologia , Inflamação/virologia , Rhinovirus/fisiologia , Transdução de Sinais/fisiologia , Receptor 3 Toll-Like/fisiologia , Animais , Hiper-Reatividade Brônquica/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferons/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Pulmão/virologia , Pneumopatias/metabolismo , Pneumopatias/fisiopatologia , Pneumopatias/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/fisiopatologia , Infecções por Picornaviridae/virologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Rhinovirus/isolamento & purificação
12.
Cell Rep Med ; 4(10): 101212, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37774704

RESUMO

Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.


Assuntos
Receptores de Antígenos Quiméricos , Rabdomiossarcoma , Animais , Criança , Humanos , Camundongos , Linhagem Celular Tumoral , Imunoterapia Adotiva , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Antígenos Quiméricos/genética , Rabdomiossarcoma/tratamento farmacológico
13.
J Lipid Res ; 53(9): 2002-13, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22766885

RESUMO

Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88(-/-) macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.


Assuntos
Ácidos Graxos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Animais , Linhagem Celular , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/agonistas , Transcriptoma/efeitos dos fármacos
14.
J Immunol ; 185(4): 2525-35, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20644177

RESUMO

Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-gamma. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.


Assuntos
Quimiocina CCL11/imunologia , Macrófagos Alveolares/imunologia , Ovalbumina/imunologia , Infecções por Picornaviridae/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Eosinófilos/patologia , Células HeLa , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Infecções por Picornaviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/imunologia
15.
Front Immunol ; 13: 832645, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35222421

RESUMO

CAR T-cell therapies targeting the B-cell maturation antigen eliminate tumors in relapsed/refractory multiple myeloma patients, however durable remissions remain difficult to attain. Transforming growth factor beta (TGF-ß) is a multifunctional cytokine abundantly expressed in the multiple myeloma bone marrow niche, where it promotes an immunosuppressive tumor microenvironment. We hypothesized that BCMA CAR T-cells armored to resist the suppressive effects of TGF-ß will provide an advantage in treating multiple myeloma. The armored B2ARM CAR T cells, co-expressing BCMA targeting CAR with TGF-ß dominant-negative receptor II, were generated by lentiviral transduction of primary human CD4+ and CD8+ T cells. The B2ARM CAR T cells eliminated MM.1S multiple myeloma targets in long-term cytotoxicity assays, even under TGF-ß-high conditions, whereas cytotoxic function of the non-armored B2 CAR -T cells was inhibited by TGF-ß. Concordantly, after long-term exposure to targets in the presence of TGF-ß, the B2ARM CAR T cells were enriched for Granzyme B, CD107a, Ki67 and polyfunctional cells T-cells (double or triple-positive for IFN-γ, IL-2 and/or TNF-α), as determined by flow cytometry. In addition, the B2ARM CAR T-cells, but not the conventional B2 CAR T-cells, resisted the TGF-ß-mediated suppression of activation (CD25), exhaustion (PD-1, LAG3), and differentiation to T effectors (CD45RA+ CD45RO-CD62L-). In NSG mice bearing RPMI-8226 tumors overexpressing TGF-ß, the B2ARM CAR mediated 100% tumor rejection and survival, superior infiltration of tumors on day 7 post CAR T treatment (%CD3+CAR+), and greater expression of IFN-γ, TNF-α, Ki67, Granzyme B, and PD-1, as compared to tumor-infiltrating non-armored B2 CAR T-cells. In NSG RPMI-8226 xenograft model in which tumors were additionally supplemented with TGF-ß injections on days -1 through 11 of CAR T treatment, the B2ARM CAR T cells rejected tumors faster than the non-armored B2 CARs, and showed greater numbers of CD3+ and CD3+CAR+, central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) T cells in the peripheral blood 18 days after treatment. In summary, the armored B2ARM CAR T cells mediate superior persistence, proliferation, multi-functionality, effector differentiation and anti-tumor function in pre-clinical models of multiple myeloma, while abrogating TGF-ß-mediated suppression.


Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Animais , Antígeno de Maturação de Linfócitos B , Granzimas , Humanos , Antígeno Ki-67 , Camundongos , Receptor de Morte Celular Programada 1 , Receptores de Antígenos Quiméricos/genética , Fator de Crescimento Transformador beta , Microambiente Tumoral , Fator de Necrose Tumoral alfa
16.
Blood Cancer J ; 12(4): 61, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35418180

RESUMO

Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced, AML remains a therapeutic challenge with insufficient cure rates. In the last years, immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced, which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However, the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression, unimpeded proliferation, and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore, CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects.


Assuntos
Células Matadoras Naturais , Leucemia Mieloide Aguda , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Hematopoese , Humanos , Imunoterapia Adotiva , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Camundongos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética
17.
Commun Med (Lond) ; 2(1): 49, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35603278

RESUMO

Background: With the rising number of chimeric antigen receptor (CAR) T cell treated patients, it is increasingly important to understand the treatment's impact on patient-reported outcomes (PROs) and, ideally, identify biomarkers of central nervous system (CNS) adverse effects. Methods: The purpose of this exploratory study was to assess short-term PROs and serum kynurenine metabolites for associated neurotoxicity among patients treated in an anti-CD20, anti-CD19 (LV20.19) CAR T cell phase I clinical trial (NCT03019055). Fifteen CAR T treated patients from the parent trial provided serum samples and self-report surveys 15 days before and 14, 28, and 90 days after treatment. Results: Blood kynurenine concentrations increased over time in patients with evidence of neurotoxicity (p = 0.004) and were increased in self-reported depression (r = 0.52, p = 0.002). Depression improved after CAR T infusion (p = 0.035). Elevated 3-hydroxyanthranilic acid (3HAA) concentrations prior to cell infusion were also predictive of neurotoxicity onset (p = 0.031), suggesting it is a biomarker of neurotoxicity following CAR T cell therapy. Conclusions: Elevated levels of kynurenine pathway metabolites among CAR T cell recipients are associated with depressed mood and neurotoxicity. Findings from this exploratory study are preliminary and warrant validation in a larger cohort.


This study examined the impact of chimeric antigen receptor (CAR) T cell therapy­a therapy that gets immune cells to fight cancer by changing them in the lab to find and destroy cancer cells­on blood markers associated with depression, anxiety, pain, fatigue, and poor sleep. Fifteen CAR T cell patients provided blood samples and completed surveys before and three timepoints after treatment. We found that the amount of kynurenine, a normal blood constituent, and related molecules was higher in patients who experienced significant CAR T cell side effects on the brain and in patients reporting more depression. These results identify the excessive elevation of blood constituents related to the mood that may also be associated with depression and brain dysfunction following CAR T. These blood constituents could potentially be used as markers and targeted with interventions to prevent brain dysfunction.

18.
J Immunol ; 183(11): 6989-97, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19890046

RESUMO

Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-beta (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-beta, IFN-lambda1, IFN-lambda2/3, IRF7, RIG-I, MDA5, 10-kDa IFN-gamma-inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-beta, IFN-lambda1, IFN-lambda2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.


Assuntos
RNA Helicases DEAD-box/imunologia , Células Epiteliais/imunologia , Infecções por Picornaviridae/imunologia , Mucosa Respiratória/imunologia , Receptor 3 Toll-Like/imunologia , Western Blotting , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , Células Epiteliais/virologia , Expressão Gênica , Humanos , Helicase IFIH1 Induzida por Interferon , RNA de Cadeia Dupla/imunologia , RNA Interferente Pequeno , Receptores Imunológicos , Receptores de Reconhecimento de Padrão/biossíntese , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Mucosa Respiratória/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/imunologia , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/biossíntese , Receptor 3 Toll-Like/genética
19.
Am J Respir Crit Care Med ; 182(3): 332-40, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20395558

RESUMO

RATIONALE: Airway inflammation is a central feature of chronic obstructive pulmonary disease (COPD). COPD exacerbations are often triggered by rhinovirus (RV) infection. OBJECTIVES: We hypothesized that airway epithelial cells from patients with COPD maintain a proinflammatory phenotype compared with control subjects, leading to greater RV responses. METHODS: Cells were isolated from tracheobronchial tissues of 12 patients with COPD and 10 transplant donors. Eight patients with COPD had severe emphysema, three had mild to moderate emphysema, and one had no emphysema. All had moderate to severe airflow obstruction, and six met criteria for chronic bronchitis or had at least one exacerbation the previous year. Cells were grown at air-liquid interface and infected with RV serotype 39. Cytokine and IFN expression was measured by ELISA. Selected genes involved in inflammation, oxidative stress, and proteolysis were assessed by focused gene array and real-time polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Compared with control subjects, cells from patients with COPD demonstrated increased mRNA expression of genes involved in oxidative stress and the response to viral infection, including NOX1, DUOXA2, MMP12, ICAM1, DDX58/RIG-I, STAT1, and STAT2. COPD cells showed elevated baseline and RV-stimulated protein levels of IL-6, IL-8/CXCL8, and growth-related oncogene-alpha/CXCL1. COPD cells demonstrated increased viral titer and copy number after RV infection, despite increased IL-29/IFN-lambda1, IL-28A/IFN-lambda2, and IFN-inducible protein-10/CXCL10 protein levels. Finally, RV-infected COPD cultures showed increased mRNA expression of IL28A/IFNlambda2, IL29/IFNlambda1, IFIH1/MDA5, DDX58/RIG-I, DUOX1, DUOX2, IRF7, STAT1, and STAT2. CONCLUSIONS: Airway epithelial cells from patients with COPD show higher baseline levels of cytokine expression and increased susceptibility to RV infection, despite an increased IFN response.


Assuntos
Citocinas/metabolismo , Células Epiteliais/virologia , Doença Pulmonar Obstrutiva Crônica/patologia , Rhinovirus/imunologia , Idoso , Brônquios/citologia , Estudos de Casos e Controles , Células Cultivadas , Resfriado Comum/imunologia , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , RNA Mensageiro/metabolismo , Traqueia/citologia , Carga Viral
20.
Sci Transl Med ; 13(586)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33762438

RESUMO

A substantial number of patients with leukemia and lymphoma treated with anti-CD19 or anti-CD22 monoCAR-T cell therapy relapse because of antigen loss or down-regulation. We hypothesized that B cell tumor antigen escape may be overcome by a chimeric antigen receptor (CAR) design that simultaneously targets three B cell leukemia antigens. We engineered trispecific duoCAR-T cells with lentiviral vectors encoding two CAR open reading frames that target CD19, CD20, and CD22. The duoCARs were composed of a CAR with a tandem CD19- and CD20-targeting binder, linked by the P2A self-cleaving peptide to a second CAR targeting CD22. Multiple combinations of intracellular T cell signaling motifs were evaluated. The most potent duoCAR architectures included those with ICOS, OX40, or CD27 signaling domains rather than those from CD28 or 4-1BB. We identified four optimal binder and signaling combinations that potently rejected xenografted leukemia and lymphoma tumors in vivo. Moreover, in mice bearing a mixture of B cell lymphoma lines composed of parental triple-positive cells, CD19-negative, CD20-negative, and CD22-negative variants, only the trispecific duoCAR-T cells rapidly and efficiently rejected the tumors. Each of the monoCAR-T cells failed to prevent tumor progression. Analysis of intracellular signaling profiles demonstrates that the distinct signaling of the intracellular domains used may contribute to these differential effects. Multispecific duoCAR-T cells are a promising strategy to prevent antigen loss-mediated relapse or the down-regulation of target antigen in patients with B cell malignancies.


Assuntos
Imunoterapia Adotiva , Linfoma de Células B , Animais , Antígenos CD19 , Linfócitos B , Humanos , Linfoma de Células B/terapia , Camundongos , Receptores de Antígenos de Linfócitos T , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos T
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