Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 68
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Cell ; 184(15): 3981-3997.e22, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34157301

RESUMO

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.


Assuntos
Retroalimentação Fisiológica , Homeostase/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Interleucina-2/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Comunicação Parácrina , Transdução de Sinais
2.
Nat Immunol ; 24(12): 2121-2134, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37945821

RESUMO

The T cell antigen receptor (TCR) contains ten immunoreceptor tyrosine-based activation motif (ITAM) signaling sequences distributed within six CD3 subunits; however, the reason for such structural complexity and multiplicity is unclear. Here we evaluated the effect of inactivating the three CD3ζ chain ITAMs on TCR signaling and T cell effector responses using a conditional 'switch' mouse model. Unexpectedly, we found that T cells expressing TCRs containing inactivated (non-signaling) CD3ζ ITAMs (6F-CD3ζ) exhibited reduced ability to discriminate between low- and high-affinity ligands, resulting in enhanced signaling and cytokine responses to low-affinity ligands because of a previously undetected inhibitory function of CD3ζ ITAMs. Also, 6F-CD3ζ TCRs were refractory to antagonism, as predicted by a new in silico adaptive kinetic proofreading model that revises the role of ITAM multiplicity in TCR signaling. Finally, T cells expressing 6F-CD3ζ displayed enhanced cytolytic activity against solid tumors expressing low-affinity ligands, identifying a new counterintuitive approach to TCR-mediated cancer immunotherapy.


Assuntos
Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Receptores de Antígenos de Linfócitos T , Animais , Camundongos , Complexo CD3 , Ligantes , Peptídeos , Linfócitos T
3.
Nat Immunol ; 24(9): 1434-1442, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37500886

RESUMO

Cytotoxic T lymphocytes (CTLs) fight intracellular pathogens and cancer by identifying and destroying infected or transformed target cells1. To kill, CTLs form a specialized cytotoxic immune synapse (IS) with a target of interest and then release toxic perforin and granzymes into the interface to elicit programmed cell death2-5. The IS then dissolves, enabling CTLs to search for additional prey and professional phagocytes to clear the corpse6. While the mechanisms governing IS assembly have been studied extensively, far less is known about target cell release. Here, we applied time-lapse imaging to explore the basis for IS dissolution and found that it occurred concomitantly with the cytoskeletal contraction of apoptotic targets. Genetic and pharmacological perturbation of this contraction response indicated that it was both necessary and sufficient for CTL dissociation. We also found that mechanical amplification of apoptotic contractility promoted faster CTL detachment and serial killing. Collectively, these results establish a biophysical basis for IS dissolution and highlight the importance of mechanosensory feedback in the regulation of cell-cell interactions.


Assuntos
Apoptose , Linfócitos T Citotóxicos , Apoptose/genética , Perforina , Granzimas
4.
Cell ; 183(6): 1520-1535.e14, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33157038

RESUMO

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.


Assuntos
COVID-19/metabolismo , SARS-CoV-2/metabolismo , Via Secretória , Liberação de Vírus , Fatores de Ribosilação do ADP/metabolismo , Animais , COVID-19/patologia , Feminino , Células HeLa , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Lisossomos , Camundongos , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Tratamento Farmacológico da COVID-19
6.
Cell ; 160(4): 619-630, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25679758

RESUMO

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.


Assuntos
Vesículas Citoplasmáticas/virologia , Infecções por Enterovirus/transmissão , Enterovirus/fisiologia , Macrófagos/virologia , Vesículas Citoplasmáticas/química , Humanos , Macrófagos/citologia , Fosfatidilserinas , Poliovirus/fisiologia , RNA Viral/metabolismo , Rhinovirus/fisiologia , Replicação Viral
7.
Immunity ; 46(4): 609-620, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28389069

RESUMO

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 µm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors.


Assuntos
Comunicação Celular/imunologia , Citocinas/imunologia , Sistema Imunitário/imunologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Difusão , Citometria de Fluxo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Imuno-Histoquímica , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Imunológicos , Fator de Transcrição STAT5/imunologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
8.
Immunity ; 44(1): 179-193, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26789923

RESUMO

Current approaches to cancer immunotherapy aim to engage the natural T cell response against tumors. One limitation is the elimination of self-antigen-specific T cells from the immune repertoire. Using a system in which precursor frequency can be manipulated in a murine melanoma model, we demonstrated that the clonal abundance of CD4(+) T cells specific for self-tumor antigen positively correlated with antitumor efficacy. At elevated precursor frequencies, intraclonal competition impaired initial activation and overall expansion of the tumor-specific CD4(+) T cell population. However, through clonally derived help, this population acquired a polyfunctional effector phenotype and antitumor immunity was enhanced. Conversely, development of effector function was attenuated at low precursor frequencies due to irreversible T cell exhaustion. Our findings assert that the differential effects of T cell clonal abundance on phenotypic outcome should be considered during the design of adoptive T cell therapies, including use of engineered T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Melanoma Experimental/imunologia , Evasão Tumoral/imunologia , Transferência Adotiva , Animais , Separação Celular , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Mol Cell ; 66(5): 635-647.e7, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28575659

RESUMO

Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. In vivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both in vitro and in vivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways.


Assuntos
Comunicação Celular , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/metabolismo , Fosfatidilserinas/metabolismo , Linfócitos T/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Biologia Computacional , Simulação por Computador , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-23/imunologia , Interleucina-23/metabolismo , Janus Quinases/metabolismo , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilserinas/imunologia , Fosforilação , Células RAW 264.7 , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/patologia , Fatores de Tempo , Transcrição Gênica , Receptor de Interferon gama
10.
Nat Methods ; 18(10): 1181-1191, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34594031

RESUMO

Cytokines are critical for intercellular communication in human health and disease, but the investigation of cytokine signaling activity has remained challenging due to the short half-lives of cytokines and the complexity/redundancy of cytokine functions. To address these challenges, we developed the Cytokine Signaling Analyzer (CytoSig; https://cytosig.ccr.cancer.gov/ ), providing both a database of target genes modulated by cytokines and a predictive model of cytokine signaling cascades from transcriptomic profiles. We collected 20,591 transcriptome profiles for human cytokine, chemokine and growth factor responses. This atlas of transcriptional patterns induced by cytokines enabled the reliable prediction of signaling activities in distinct cell populations in infectious diseases, chronic inflammation and cancer using bulk and single-cell transcriptomic data. CytoSig revealed previously unidentified roles of many cytokines, such as BMP6 as an anti-inflammatory factor, and identified candidate therapeutic targets in human inflammatory diseases, such as CXCL8 for severe coronavirus disease 2019.


Assuntos
COVID-19/imunologia , Citocinas/metabolismo , Bases de Dados de Proteínas , SARS-CoV-2 , COVID-19/metabolismo , Citocinas/genética , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/fisiologia , Humanos , Transdução de Sinais/fisiologia
11.
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
12.
Nat Immunol ; 12(7): 647-54, 2011 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-21602810

RESUMO

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.


Assuntos
Polaridade Celular/imunologia , Citoesqueleto/enzimologia , Isoenzimas/imunologia , Proteína Quinase C-épsilon/imunologia , Proteína Quinase C/imunologia , Linfócitos T/enzimologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/enzimologia , Células Apresentadoras de Antígenos/imunologia , Citoesqueleto/imunologia , Diglicerídeos/imunologia , Sinapses Imunológicas/enzimologia , Sinapses Imunológicas/imunologia , Camundongos , Camundongos Transgênicos , Proteína Quinase C-theta , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Linfócitos T/citologia , Linfócitos T/imunologia
13.
PLoS Comput Biol ; 18(10): e1010349, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36191000

RESUMO

Data clustering plays a significant role in biomedical sciences, particularly in single-cell data analysis. Researchers use clustering algorithms to group individual cells into populations that can be evaluated across different levels of disease progression, drug response, and other clinical statuses. In many cases, multiple sets of clusters must be generated to assess varying levels of cluster specificity. For example, there are many subtypes of leukocytes (e.g. T cells), whose individual preponderance and phenotype must be assessed for statistical/functional significance. In this report, we introduce a novel hierarchical density clustering algorithm (HAL-x) that uses supervised linkage methods to build a cluster hierarchy on raw single-cell data. With this new approach, HAL-x can quickly predict multiple sets of labels for immense datasets, achieving a considerable improvement in computational efficiency on large datasets compared to existing methods. We also show that cell clusters generated by HAL-x yield near-perfect F1-scores when classifying different clinical statuses based on single-cell profiles. Our hierarchical density clustering algorithm achieves high accuracy in single cell classification in a scalable, tunable and rapid manner.


Assuntos
Algoritmos , Análise de Célula Única , Análise por Conglomerados
14.
Cytometry A ; 95(10): 1075-1084, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31150166

RESUMO

We present a new method to directly quantify the dynamics of differentiation of multiple cellular subsets in unperturbed mice. We combine a pulse-chase protocol of 5-iodo-2'-deoxyuridine (IdU) injections with subsequent analysis by mass cytometry (CyTOF) and mathematical modeling of the IdU dynamics. Measurements by CyTOF allow for a wide range of cells to be analyzed at once, due to the availability of a large staining panel without the complication of fluorescence spillover. These are also compatible with direct detection of integrated iodine signal, with minimal impact on immunophenotyping based on the surface markers. Mathematical modeling beyond a binary classification of surface marker abundance allows for a continuum of cellular states as the cells transition from one state to another. Thus, we present a complete and robust method for directly quantifying differentiation at the systemic level, allowing for system-wide comparisons between different mouse strains and/or experimental conditions. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.


Assuntos
Citometria de Fluxo/métodos , Hematopoese , Idoxuridina/metabolismo , Modelos Teóricos , Animais , Linfócitos B/citologia , Diferenciação Celular , Feminino , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Fenótipo , Fatores de Tempo
15.
Cytometry A ; 93(6): 611-619, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29451717

RESUMO

Recent efforts in systems immunology lead researchers to build quantitative models of cell activation and differentiation. One goal is to account for the distributions of proteins from single-cell measurements by flow cytometry or mass cytometry as readout of biological regulation. In that context, large cell-to-cell variability is often observed in biological quantities. We show here that these readouts, viewed in logarithmic scale may result in two easily-distinguishable modes, while the underlying distribution (in linear scale) is unimodal. We introduce a simple mathematical test to highlight this mismatch. We then dissect the flow of influence of cell-to-cell variability proposing a graphical model which motivates higher-dimensional analysis of the data. Finally we show how acquiring additional biological information can be used to reduce uncertainty introduced by cell-to-cell variability, helping to clarify whether the data is uni- or bimodal. This communication has cautionary implications for manual and automatic gating strategies, as well as clustering and modeling of single-cell measurements. © 2018 International Society for Advancement of Cytometry.


Assuntos
Análise de Dados , Citometria de Fluxo/métodos , Modelos Biológicos , Linfócitos T/fisiologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL
16.
Proc Natl Acad Sci U S A ; 110(10): E888-97, 2013 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-23431198

RESUMO

Early T-cell activation is selected by evolution to discriminate a few foreign peptides rapidly from a vast excess of self-peptides, and it is unclear in quantitative terms how this is possible. We show that a generic proofreading cascade supplemented by a single negative feedback mediated by the Src homology 2 domain phosphatase-1 (SHP-1) accounts quantitatively for early T-cell activation, including the effects of antagonists. Modulation of the negative feedback with SHP-1 concentration explains counterintuitive experimental observations, such as the nonmonotonic behavior of receptor activity on agonist concentration, the digital vs. continuous behavior on certain parameters, and the loss of response for high SHP-1 concentration. New experiments validate predictions on the nontrivial joint dependence on binding time and concentration for the relative effect of two antagonists: We explain why strong antagonists behave as partial agonists at low concentration and predict that the relative effect of antagonists can invert as their concentrations are varied. By focusing on the phenotype, our model quantitatively fits a body of experimental data with minimal variables and parameters.


Assuntos
Ativação Linfocitária/imunologia , Modelos Imunológicos , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Retroalimentação Fisiológica , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Processos Estocásticos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
17.
Proc Natl Acad Sci U S A ; 109(3): 881-6, 2012 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-22223661

RESUMO

The strength of T-cell receptor (TCR) stimulation and subsequent T-cell response depend on a combination of peptide-major histocompatibility complex (pMHC) density and potency. By comparing two different pMHC at doses yielding similar proliferation in vivo, we have highlighted unexpected differences in the qualitative and quantitative effects of TCR ligand. Measurements of cytokine sensitivity and two-photon imaging of T cell-dendritic cell (T-DC) interactions reveal discrimination between comparably weak stimuli resulting from either decreased pMHC potency or pMHC density. In addition, TCR-induced genes in broad gene expression profiles segregate into two groups: one that responds to cumulative TCR signal and another that responds to pMHC quality, independent of quantity. These observations suggest that models of TCR ligand discrimination must account for disparate sensitivity of downstream responses to specific influences of pMHC potency.


Assuntos
Antígenos de Histocompatibilidade/imunologia , Peptídeos/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Comunicação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-2/farmacologia , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Interleucina-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos
18.
Blood ; 119(22): 5182-90, 2012 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-22510877

RESUMO

Human CD34(+) progenitor-derived Langerhans-type dendritic cells (LCs) are more potent stimulators of T-cell immunity against tumor and viral antigens in vitro than are monocyte-derived DCs (moDCs). The exact mechanisms have remained elusive until now, however. LCs synthesize the highest amounts of IL-15R-α mRNA and protein, which binds IL-15 for presentation to responder lymphocytes, thereby signaling the phosphorylation of signal transducer and activator of transcription 5 (pSTAT5). LCs electroporated with Wilms tumor 1 (WT1) mRNA achieve sufficiently sustained presentation of antigenic peptides, which together with IL-15R-α/IL-15, break tolerance against WT1 by stimulating robust autologous, WT1-specific cytolytic T-lymphocytes (CTLs). These CTLs develop from healthy persons after only 7 days' stimulation without exogenous cytokines and lyse MHC-restricted tumor targets, which include primary WT1(+) leukemic blasts. In contrast, moDCs require exogenous rhuIL-15 to phosphorylate STAT5 and attain stimulatory capacity comparable to LCs. LCs therefore provide a more potent costimulatory cytokine milieu for T-cell activation than do moDCs, thus accounting for their superior stimulation of MHC-restricted Ag-specific CTLs without need for exogenous cytokines. These data support the use of mRNA-electroporated LCs, or moDCs supplemented with exogenous rhuIL-15, as vaccines for cancer immunotherapy to break tolerance against self-differentiation antigens shared by tumors.


Assuntos
Apresentação de Antígeno , Tolerância Imunológica , Interleucina-15/imunologia , Células de Langerhans/imunologia , Receptores de Interleucina-15/imunologia , Fator de Transcrição STAT5/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas WT1/imunologia , Crise Blástica/genética , Crise Blástica/imunologia , Crise Blástica/patologia , Crise Blástica/terapia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Feminino , Humanos , Interleucina-15/farmacologia , Células de Langerhans/patologia , Leucemia/genética , Leucemia/imunologia , Leucemia/patologia , Leucemia/terapia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Receptores de Interleucina-15/genética , Fator de Transcrição STAT5/genética , Linfócitos T Citotóxicos/patologia , Proteínas WT1/genética
19.
Integr Biol (Camb) ; 162024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-39074471

RESUMO

Immune responses against cancer are inherently stochastic, with small numbers of individual T cells within a larger ensemble of lymphocytes initiating the molecular cascades that lead to tumor cytotoxicity. A potential source of this intra-tumor variability is the differential ability of immune cells to respond to tumor cells. Classical microwell co-cultures of T cells and tumor cells are inadequate for reliably culturing and analyzing low cell numbers needed to probe this variability, and have failed in recapitulating the heterogeneous small domains observed in tumors. Here we leverage a membrane displacement trap array technology that overcomes limitations of conventional microwell plates for immunodynamic studies. The microfluidic platform supports on-demand formation of dense nanowell cultures under continuous perfusion reflecting the tumor microenvironment, with real-time monitoring of T cell proliferation and activation within each nanowell. The system enables selective ejection of cells for profiling by fluorescence activated cell sorting, allowing observed on-chip variability in immune response to be correlated with off-chip quantification of T cell activation. The technology offers new potential for probing the molecular origins of T cell heterogeneity and identifying specific cell phenotypes responsible for initiating and propagating immune cascades within tumors. Insight Box Variability in T cell activation plays a critical role in the immune response against cancer. New tools are needed to unravel the mechanisms that drive successful anti-tumor immune response, and to support the development of novel immunotherapies utilizing rare T cell phenotypes that promote effective immune surveillance. To this end, we present a microfluidic cell culture platform capable of probing differential T cell activation in an array of nanoliter-scale wells coupled with off-chip cell analysis, enabling a high resolution view of variable immune response within tumor / T cell co-cultures containing cell ensembles orders of magnitude smaller than conventional well plate studies.


Assuntos
Técnicas de Cocultura , Ativação Linfocitária , Linfócitos T , Microambiente Tumoral , Humanos , Linfócitos T/imunologia , Linfócitos T/citologia , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Neoplasias/imunologia , Neoplasias/patologia , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento
20.
Mol Ther Methods Clin Dev ; 32(1): 101171, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38298420

RESUMO

Chimeric antigen receptor T cells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, T cell selection, removes contaminating cell types that can potentially suppress T cell expansion and transduction. While positive selection of CD4/CD8 T cells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients. While negative selection may avoid this drawback, it is virtually absent from good manufacturing practices. Here, we performed both CD4/CD8-positive and -negative clinical scale selections of mononuclear cell apheresis products and generated CD22 CARTs per our ongoing clinical trial (NCT02315612NCT02315612). While the selection process did not yield differences in CART expansion or transduction, positively selected CART exhibited a significantly higher in vitro interferon-γ and IL-2 secretion but a lower in vitro tumor killing rate. Notably, though, CD22 CART generated from both selection protocols efficiently eradicated leukemia in NSG mice, with negatively selected cells exhibiting a significant enrichment in γδ CD22 CART. Thus, our study demonstrates the importance of the initial T cell selection process in clinical CART manufacturing.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA