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1.
J Immunol ; 200(1): 186-195, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29180487

RESUMO

Plasmacytoid dendritic cells (pDCs) are the major producers of IFN-α, an antiviral cytokine involved in immunomodulation and control of HIV type 1 replication, whereas Toxoplasma gondii is a life-threatening opportunistic infection in AIDS patients. During infection with HIV type 1, human pDCs decrease in circulation and remaining pDC produce lower amounts of IFN-α in response to viral stimulation. In this study, we investigated the impact of coinfection with T. gondii on the innate virus-directed responses of human pDCs. Using intracellular flow cytometry and fluorescence microscopy, we determined that T. gondii invaded but did not induce IFN-α or TNF-α in human pDC. However, T. gondii inhibited IFN-α and TNF-α produced in response to HSV and HIV, thus functionally inactivating pDC. IFN-α production was inhibited only in cells infected by T. gondii, which inhibited neither uptake of GFP-HSV nor localization of TLR9 in CD71+ endosomes, directing us to investigate downstream events. Using imaging flow cytometry, we found that both T. gondii and IL-10 inhibited virus-induced nuclear translocation, but not phosphorylation, of IFN response factor 7. Blockade of IFN response factor 7 nuclear translocation and inhibition of the IFN-α response was partially reversed by a deficiency in the T. gondii-derived ROP16 kinase, known to directly phosphorylate STAT3, a critical mediator of IL-10's anti-inflammatory effects. Taken together, our results indicate that T. gondii suppresses pDC activation by mimicking IL-10's regulatory effects through an ROP16 kinase-dependent mechanism. Our findings further imply a convergent mechanism of inhibition of TLR signaling by T. gondii and IL-10 and suggest potential negative consequences of HIV/T. gondii coinfection.


Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Interleucina-10/metabolismo , Infecções Oportunistas/imunologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/imunologia , Toxoplasmose/imunologia , Diferenciação Celular , Células Cultivadas , Coinfecção , Células Dendríticas/parasitologia , Humanos , Imunidade Inata , Imunomodulação , Fator Regulador 7 de Interferon/metabolismo , Interferon-alfa/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Cytometry B Clin Cytom ; 100(1): 79-91, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33373096

RESUMO

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.


Assuntos
Citometria de Fluxo , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/análise , Linfócitos T/citologia , Humanos , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia
3.
Nat Med ; 24(10): 1504-1506, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30275569

RESUMO

We identified genetic mutations in CD19 and loss of heterozygosity at the time of CD19- relapse to chimeric antigen receptor (CAR) therapy. The mutations are present in the vast majority of resistant tumor cells and are predicted to lead to a truncated protein with a nonfunctional or absent transmembrane domain and consequently to a loss of surface antigen. This irreversible loss of CD19 advocates for an alternative targeting or combination CAR approach.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Antígenos CD19/genética , Antígenos CD19/imunologia , Humanos , Imunoterapia Adotiva , Perda de Heterozigosidade/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/imunologia
4.
J Immunol Methods ; 416: 124-36, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25462536

RESUMO

BACKGROUND: Administration of a biotherapeutic can result in the formation of anti-drug antibodies (ADAs). The resulting ADA can potentially form immune complexes (ICs) with the drug leading to altered pharmacokinetic (PK) profiles and/or adverse events. Furthermore the presence of such complexes may interfere with accurate PK assessment, and/or detection of ADA in immunogenicity assays. Here, we present two assays to detect the presence of drug-ADA immune complexes in cynomolgus monkeys. RESULTS: Serum samples were analyzed for IC formation in vivo. 8/8 tested animals were positive for drug specific IC. Depending on the time point tested 4/8 or 7/8 animals tested positive for ADA during drug dosing. All 8 animals were confirmed positive for ADA during the washout phase, indicating drug interference in the bridging assay. Relative amount of IC over time was determined and its correlation with PK and ADA was then assessed. Multivariate data analysis demonstrates good correlation between signals obtained from the anti-drug and FcγRIIIa based capture assays, although due to its biological characteristic FcγRIIIa based assay captured only a subset of drug specific IC. In one animal IC remained in circulation even when the drug levels decreased below detection limit. CONCLUSION: Results from this study indicate the presence of IC during administration of an immunogenic biotherapeutic. Potential application of these assays includes detection of ADA in an IC during high drug levels. The results on the kinetics of IC formation during ADA response can complement the understanding of PK and ADA profiles. Moreover, the presence of IC indicates possible ADA interference in standard PK assays and potential underestimation of total drug exposure in toxicology studies. In addition this study also highlights the need to understand downstream in vivo consequences of drug-ADA IC as no animals under investigation developed adverse events.


Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Macaca fascicularis/imunologia , Preparações Farmacêuticas/metabolismo , Soro/imunologia , Animais , Formação de Anticorpos/imunologia
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