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1.
Methods Mol Biol ; 2779: 287-303, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38526791

RESUMO

The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Perfilação da Expressão Gênica/métodos , Epitopos , Separação Celular , Anticorpos , Análise de Célula Única/métodos
2.
Cytometry A ; 101(1): 27-44, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34390166

RESUMO

T-cell activation is a key step in the amplification of an immune response. Over the course of an immune response, cells may be chronically stimulated, with some proportion becoming exhausted; an enormous number of molecules are involved in this process. There remain a number of questions about the process, namely: (1) what degree of heterogeneity and plasticity do T-cells exhibit during stimulation? (2) how many unique cell states define chronic stimulation? and (3) what markers discriminate activated from exhausted cells? We addressed these questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study -with unprecedented depth-how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, activation downregulated naïve-cell markers and upregulated effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation, defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10); expression of these markers could be associated with the emergence of short-lived cell types. Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data reveal the high heterogeneity and plasticity of chronically stimulated T cells. Our study demonstrates the power of a single-cell multiomic approach to comprehensively characterize T-cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures during cell stimulation.


Assuntos
Linfócitos T CD8-Positivos , Ativação Linfocitária , Humanos , Imunofenotipagem , Cinética , Análise de Célula Única
3.
STAR Protoc ; 2(4): 100893, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34712996

RESUMO

The dual interrogation of the transcriptome and proteome with single-cell resolution provides exquisite insights into immune mechanisms in health and disease. Here, we describe a cutting-edge protocol wherein we combine Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), a technique utilizing antibody-oligonucleotide conjugates (AOCs), with fluorescence-activated cell sorting to enrich rare cell populations. Our protocol incorporates co-staining of cells with both fluorescent antibodies and AOCs simultaneously for subsequent input into the cell sorting and CITE-Seq pipeline. For complete details on the use and execution of this protocol, please refer to Mair et al. (2020).


Assuntos
Citometria de Fluxo/métodos , Imunofluorescência/métodos , Perfilação da Expressão Gênica/métodos , Leucócitos Mononucleares , Análise de Célula Única/métodos , Epitopos/química , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia
4.
Annu Rev Anal Chem (Palo Alto Calif) ; 12(1): 411-430, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-30699035

RESUMO

Thousands of transcripts and proteins confer function and discriminate cell types in the body. Using high-parameter technologies, we can now measure many of these markers at once, and multiple platforms are now capable of analysis on a cell-by-cell basis. Three high-parameter single-cell technologies have particular potential for discovering new biomarkers, revealing disease mechanisms, and increasing our fundamental understanding of cell biology. We review these three platforms (high-parameter flow cytometry, mass cytometry, and a new class of technologies called integrated molecular cytometry platforms) in this article. We describe the underlying hardware and instrumentation, the reagents involved, and the limitations and advantages of each platform. We also highlight the emerging field of high-parameter single-cell data analysis, providing an accessible overview of the data analysis process and choice of tools.


Assuntos
Citometria de Fluxo , Análise de Célula Única , Citometria de Fluxo/instrumentação , Humanos , Análise de Célula Única/instrumentação
5.
Virology ; 394(1): 91-8, 2009 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-19744690

RESUMO

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vírus da Imunodeficiência Símia/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Reações Cruzadas , Anticorpos Anti-HIV/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Ressonância de Plasmônio de Superfície
6.
Proc Natl Acad Sci U S A ; 104(37): 14700-5, 2007 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-17804805

RESUMO

The R-Spondin (RSpo) family of secreted proteins act as potent activators of the Wnt/beta-catenin signaling pathway. We have previously shown that RSpo proteins can induce proliferative effects on the gastrointestinal epithelium in mice. Here we provide a mechanism whereby RSpo1 regulates cellular responsiveness to Wnt ligands by modulating the cell-surface levels of the coreceptor LRP6. We show that RSpo1 activity critically depends on the presence of canonical Wnt ligands and LRP6. Although RSpo1 does not directly activate LRP6, it interferes with DKK1/Kremen-mediated internalization of LRP6 through an interaction with Kremen, resulting in increased LRP6 levels on the cell surface. Our results support a model in which RSpo1 relieves the inhibition DKK1 imposes on the Wnt pathway.


Assuntos
Proteínas Relacionadas a Receptor de LDL/antagonistas & inibidores , Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem Celular , Drosophila/citologia , Drosophila/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/citologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Fosforilação , Testes de Precipitina , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Trombospondinas/genética , Transfecção , beta Catenina/genética , beta Catenina/metabolismo
7.
Br J Haematol ; 137(4): 307-18, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17456053

RESUMO

NTB-A is a CD2-related cell surface protein expressed primarily on lymphoid cells including B-lymphocytes from chronic lymphocytic leukaemia (CLL) and lymphoma patients. We have generated a series of monoclonal antibodies (mAbs) against NTB-A and assessed their therapeutic potential for CLL. Selective mAbs to NTB-A were further tested in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL patients. While lower levels of NTB-A were detected in T and natural killer (NK) cells, CDC activity was demonstrated primarily in B cells isolated from CLL patients and B lymphoma cell lines. Knockdown of NTB-A by small interfering RNA in target cells results in lower cytotoxicity, demonstrating the specificity of the mAbs. Furthermore, anti NTB-A mAbs demonstrated anti-tumour activity against CA46 human lymphoma xenografts in nude mice and against systemically disseminated Raji human lymphoma cells in severe combined immunodeficient mice. Taken together, these results demonstrate NTB-A as a potential new target for immunotherapy of leukaemia and lymphomas.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Imunização Passiva/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Reações Antígeno-Anticorpo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo , Humanos , Hibridomas , Leucemia Linfocítica Crônica de Células B/imunologia , Ativação Linfocitária , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Interferência de RNA , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transplante Heterólogo
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