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1.
Nat Immunol ; 24(5): 841-854, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36928412

RESUMO

Regulatory T (Treg) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that Treg cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. Treg cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented TH1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted TH2-like polarization (increased expression of GATA-3, CCR4 and IL4). TH1-like Treg cells limited CD8+ T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how Treg cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.


Assuntos
Interferon gama , Linfócitos T Reguladores , Interferon gama/metabolismo , Citocinas/metabolismo , Linfócitos T CD8-Positivos , Antivirais/metabolismo , Células Th1
2.
Nat Immunol ; 23(5): 757-767, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35437325

RESUMO

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4+ and CD8+ T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor-TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies.


Assuntos
Linfócitos T CD8-Positivos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Antígenos CD/imunologia , Complexo CD3/imunologia , Antígenos CD8/metabolismo , Antígenos de Histocompatibilidade Classe II , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteína do Gene 3 de Ativação de Linfócitos
3.
Nat Immunol ; 21(9): 1010-1021, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661362

RESUMO

Robust CD8+ T cell memory is essential for long-term protective immunity but is often compromised in cancer, where T cell exhaustion leads to loss of memory precursors. Immunotherapy via checkpoint blockade may not effectively reverse this defect, potentially underlying disease relapse. Here we report that mice with a CD8+ T cell-restricted neuropilin-1 (NRP1) deletion exhibited substantially enhanced protection from tumor rechallenge and sensitivity to anti-PD1 immunotherapy, despite unchanged primary tumor growth. Mechanistically, NRP1 cell-intrinsically limited the self-renewal of the CD44+PD1+TCF1+TIM3- progenitor exhausted T cells, which was associated with their reduced ability to induce c-Jun/AP-1 expression on T cell receptor restimulation, a mechanism that may contribute to terminal T cell exhaustion at the cost of memory differentiation in wild-type tumor-bearing hosts. These data indicate that blockade of NRP1, a unique 'immune memory checkpoint', may promote the development of long-lived tumor-specific Tmem that are essential for durable antitumor immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Checkpoint Imunológico/metabolismo , Melanoma Experimental/imunologia , Neuropilina-1/metabolismo , Células Precursoras de Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Humanos , Proteínas de Checkpoint Imunológico/genética , Tolerância Imunológica , Imunidade , Memória Imunológica , Camundongos , Camundongos Knockout , Neuropilina-1/genética , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais
4.
Immunity ; 54(10): 2209-2217.e6, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34551314

RESUMO

CD4+ T cells share common developmental pathways with CD8+ T cells, and upon maturation, CD4+ T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3+ T regulatory cells. We developed a tamoxifen-inducible ThPOKCreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3+ cells. Breeding these mice resulted in a CD4conviCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4+ Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4+ Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8IiCreERT2.GFP mouse that enables inducible targeting of CD8+ T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4+ T cells and CD4+ Tconv cells.


Assuntos
Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Edição de Genes/métodos , Integrases/genética , Alelos , Animais , Linfócitos T CD8-Positivos/citologia , Linhagem Celular , Camundongos
5.
Immunity ; 51(2): 381-397.e6, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31350177

RESUMO

Regulatory T (Treg) cells are crucial for immune homeostasis, but they also contribute to tumor immune evasion by promoting a suppressive tumor microenvironment (TME). Mice with Treg cell-restricted Neuropilin-1 deficiency show tumor resistance while maintaining peripheral immune homeostasis, thereby providing a controlled system to interrogate the impact of intratumoral Treg cells on the TME. Using this and other genetic models, we showed that Treg cells shaped the transcriptional landscape across multiple tumor-infiltrating immune cell types. Treg cells suppressed CD8+ T cell secretion of interferon-γ (IFNγ), which would otherwise block the activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated fatty acid synthesis in immunosuppressive (M2-like) tumor-associated macrophages (TAMs). Thus, Treg cells indirectly but selectively sustained M2-like TAM metabolic fitness, mitochondrial integrity, and survival. SREBP1 inhibition augmented the efficacy of immune checkpoint blockade, suggesting that targeting Treg cells or their modulation of lipid metabolism in M2-like TAMs could improve cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Macrófagos/metabolismo , Melanoma/imunologia , Neoplasias Experimentais/imunologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Carcinogênese , Diferenciação Celular , Ácidos Graxos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Evasão da Resposta Imune , Interferon gama/metabolismo , Macrófagos/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropilina-1/genética , Células Th2/imunologia , Microambiente Tumoral
6.
J Immunol ; 213(8): 1115-1124, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39240167

RESUMO

EBV-induced gene 3 (Ebi3) is a ß subunit within the IL-12 cytokine family that canonically binds to α subunits p19, p28, or p35 to form the heterodimeric cytokines IL-39, IL-27, and IL-35, respectively. In the last decade, the binding partners for Ebi3 have continued to expand to include IL-6 and the other IL-12 family ß subunit p40, revealing the possibility that Ebi3 may be able to bind to other cytokines and have distinct functions. We first explored this possibility utilizing an in vivo mouse model of regulatory T cell-restricted deletions of the subunits composing the cytokine IL-35, p35, and Ebi3, and we observed a differential impact on CD8+ T cell inhibitory receptor expression despite comparable reduction in tumor growth. We then screened the ability of Ebi3 to bind to different cytokines with varying structural resemblance to the IL-12 family α subunits. These in vitro screens revealed extracellular binding of Ebi3 to both IFN-γ and IL-10. Ebi3 bound to IFN-γ and IL-10 abrogated signal transduction and downstream functions of both cytokines. Lastly, we validated that extracellular complex formation after mixing native proteins resulted in loss of function. These data suggest that secreted partnerless Ebi3 may bind to cytokines within the extracellular microenvironment and act as a cytokine sink, further expanding the potential immunological impact of Ebi3.


Assuntos
Interferon gama , Interleucina-10 , Antígenos de Histocompatibilidade Menor , Animais , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Ligação Proteica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Transdução de Sinais/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Interleucinas/metabolismo , Interleucinas/imunologia , Receptores de Citocinas
7.
Nat Immunol ; 14(3): 262-70, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23377202

RESUMO

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.


Assuntos
Complexo CD3/imunologia , Citocinas/biossíntese , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/metabolismo , Linhagem Celular , Proliferação de Células , Células HEK293 , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo
8.
Nat Immunol ; 13(3): 290-9, 2012 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-22306691

RESUMO

Interleukin 35 (IL-35) belongs to the IL-12 family of heterodimeric cytokines but has a distinct functional profile. IL-35 suppresses T cell proliferation and converts naive T cells into IL-35-producing induced regulatory T cells (iTr35 cells). Here we found that IL-35 signaled through a unique heterodimer of receptor chains IL-12Rß2 and gp130 or homodimers of each chain. Conventional T cells were sensitive to IL-35-mediated suppression in the absence of one receptor chain but not both receptor chains, whereas signaling through both chains was required for IL-35 expression and conversion into iTr35 cells. Signaling through the IL-35 receptor required the transcription factors STAT1 and STAT4, which formed a unique heterodimer that bound to distinct sites in the promoters of the genes encoding the IL-12 subunits p35 and Ebi3. This unconventional mode of signaling, distinct from that of other members of the IL-12 family, may broaden the spectrum and specificity of IL-35-mediated suppression.


Assuntos
Receptores de Interleucina-1/imunologia , Receptores de Interleucina/imunologia , Transdução de Sinais , Animais , Receptor gp130 de Citocina/imunologia , Interleucinas/imunologia , Camundongos , Camundongos Knockout , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/deficiência , Receptores de Interleucina/metabolismo , Receptores de Interleucina-1/química , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-12/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT4/imunologia
9.
J Immunol ; 209(8): 1586-1594, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36104110

RESUMO

Lymphocyte activation gene 3 protein (LAG3; CD223) is an inhibitory receptor that is highly upregulated on exhausted T cells in tumors and chronic viral infection. Consequently, LAG3 is now a major immunotherapeutic target for the treatment of cancer, and many mAbs against human (h) LAG3 (hLAG3) have been generated to block its inhibitory activity. However, little or no information is available on the epitopes they recognize. We selected a panel of seven therapeutic mAbs from the patent literature for detailed characterization. These mAbs were expressed as Fab or single-chain variable fragments and shown to bind hLAG3 with nanomolar affinities, as measured by biolayer interferometry. Using competitive binding assays, we found that the seven mAbs recognize four distinct epitopes on hLAG3. To localize the epitopes, we carried out epitope mapping using chimeras between hLAG3 and mouse LAG3. All seven mAbs are directed against the first Ig-like domain (D1) of hLAG3, despite their different origins. Three mAbs almost exclusively target a unique 30-residue loop of D1 that forms at least part of the putative binding site for MHC class II, whereas four mainly recognize D1 determinants outside this loop. However, because all the mAbs block binding of hLAG3 to MHC class II, each of the epitopes they recognize must at least partially overlap the MHC class II binding site.


Assuntos
Antígenos CD/imunologia , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais , Mapeamento de Epitopos , Epitopos , Humanos , Camundongos , Anticorpos de Cadeia Única/metabolismo , Linfócitos T , Proteína do Gene 3 de Ativação de Linfócitos
10.
J Immunol ; 199(5): 1555-1560, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28733484

RESUMO

For the αß or γδTCR chains to integrate extracellular stimuli into the appropriate intracellular cellular response, they must use the 10 ITAMs found within the CD3 subunits (CD3γε, CD3δε, and ζζ) of the TCR signaling complex. However, it remains unclear whether each specific ITAM sequence of the individual subunit (γεδζ) is required for thymocyte development or whether any particular CD3 ITAM motif is sufficient. In this article, we show that mice utilizing a single ITAM sequence (γ, ε, δ, ζa, ζb, or ζc) at each of the 10 ITAM locations exhibit a substantial reduction in thymic cellularity and limited CD4-CD8- (double-negative) to CD4+CD8+ (double-positive) maturation because of low TCR expression and signaling. Together, the data suggest that ITAM sequence diversity is required for optimal TCR signal transduction and subsequent T cell maturation.


Assuntos
Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Complexos Multiproteicos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Timo/imunologia , Motivos de Aminoácidos/genética , Animais , Complexo CD3/genética , Diferenciação Celular , Células Cultivadas , Hematopoese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais
11.
J Biol Chem ; 290(32): 19796-805, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26109064

RESUMO

The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αß TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the ß chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant ß (Cß) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9-11 solvent-accessible Cß residues, is relatively small (∼400 Å(2)), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cß, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.


Assuntos
Complexo CD3/química , Subunidades Proteicas/química , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Sequência de Aminoácidos , Complexo CD3/genética , Complexo CD3/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/química , Linfócitos T/imunologia
12.
J Immunol ; 193(1): 258-67, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24899501

RESUMO

The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.


Assuntos
Complexo CD3/imunologia , Membrana Celular/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Timócitos/imunologia , Animais , Complexo CD3/genética , Membrana Celular/genética , Camundongos , Camundongos Knockout , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Timócitos/citologia
13.
Nature ; 450(7169): 566-9, 2007 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-18033300

RESUMO

Regulatory T (T(reg)) cells are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and preventing autoimmunity, for limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease, and for regulating homeostatic lymphocyte expansion. However, they also suppress natural immune responses to parasites and viruses as well as anti-tumour immunity induced by therapeutic vaccines. Although the manipulation of T(reg) function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. Here we demonstrate that Epstein-Barr-virus-induced gene 3 (Ebi3, which encodes IL-27beta) and interleukin-12 alpha (Il12a, which encodes IL-12alpha/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) T(reg) cells but not by resting or activated effector CD4+ T (T(eff)) cells, and that an Ebi3-IL-12alpha heterodimer is constitutively secreted by T(reg) but not T(eff) cells. Both Ebi3 and Il12a messenger RNA are markedly upregulated in T(reg) cells co-cultured with T(eff) cells, thereby boosting Ebi3 and IL-12alpha production in trans. T(reg)-cell restriction of this cytokine occurs because Ebi3 is a downstream target of Foxp3, a transcription factor that is required for T(reg)-cell development and function. Ebi3-/- and Il12a-/- T(reg) cells have significantly reduced regulatory activity in vitro and fail to control homeostatic proliferation and to cure inflammatory bowel disease in vivo. Because these phenotypic characteristics are distinct from those of other IL-12 family members, this novel Ebi3-IL-12alpha heterodimeric cytokine has been designated interleukin-35 (IL-35). Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. Taken together, these data identify IL-35 as a novel inhibitory cytokine that may be specifically produced by T(reg) cells and is required for maximal suppressive activity.


Assuntos
Subunidade p35 da Interleucina-12/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Subunidade p35 da Interleucina-12/deficiência , Subunidade p35 da Interleucina-12/genética , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Receptores de Citocinas/deficiência , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/transplante
14.
Nat Biotechnol ; 22(5): 589-94, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15064769

RESUMO

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.


Assuntos
Vetores Genéticos , Peptídeos/metabolismo , Retroviridae/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Homologia de Sequência de Aminoácidos
15.
Sci Immunol ; 2(9)2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28783703

RESUMO

Inhibitory receptors (IRs) are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (Tconv) cell proliferation, viability, and function. However, the role of IRs on regulatory T cells (Tregs) remains obscure because they could be required for suppressive activity and/or limit Treg function. We evaluated the role of lymphocyte activation gene 3 (LAG3; CD223) on Tregs by generating mice in which LAG3 is absent on the cell surface of Tregs in a murine model of type 1 diabetes. Unexpectedly, mice that lacked LAG3 expression on Tregs exhibited reduced autoimmune diabetes, consistent with enhanced Treg proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3-deficient Tregs was largely comparable, substantial differences between intra-islet Tregs were evident and involved a subset of genes and pathways that promote Treg maintenance and function. Consistent with these observations, Lag3-deficient Tregs outcompeted WT Tregs in the islets but not in the periphery in cotransfer experiments because of enhanced interleukin-2-signal transducer and activator of transcription 5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits Treg proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment, and may contribute to Treg insufficiency in autoimmune disease.

16.
J Immunol Methods ; 271(1-2): 137-51, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12445737

RESUMO

MHC tetramers have proven to be powerful reagents for the analysis of MHC class I-restricted T cells. However, generating similarly reliable reagents for MHC class II-restricted T cells has been elusive. Here we evaluated the utility of MHC class II:gamma2aFc multimers, which contain the MHC class II extracellular domains, with or without recombinantly attached peptides, dimerized via a fos-jun leucine zipper and attached to the hinge of murine IgG2a. We have successfully generated 24 multimers in either myeloma or Drosophila melanogaster S2 cells, with an average yield of 7 mg/L. 'Empty' MHC class II:gamma2aFc multimers were effectively used in peptide binding assays. Similar versions that contained recombinantly attached peptides stimulated T cells in an antigen-specific, MHC-restricted manner, and identified antigen-specific nai;ve and effector T cells by flow cytometry. Furthermore, we have successfully used these reagents to stain T cells generated following viral infection. Thus, MHC class II:gamma2aFc multimers are robust and reliable reagents for the analysis of MHC class II-restricted T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Drosophila melanogaster , Feminino , Citometria de Fluxo , Vírus da Influenza A/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Plasmídeos/imunologia , Proteínas Recombinantes/imunologia , Vírus Sendai/imunologia , Organismos Livres de Patógenos Específicos , Células Tumorais Cultivadas
17.
Cold Spring Harb Protoc ; 2012(2): 199-204, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301656

RESUMO

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.


Assuntos
Expressão Gênica , Vetores Genéticos , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , Genes , Fases de Leitura Aberta , Processamento de Proteína Pós-Traducional , Proteólise , Ribossomos/metabolismo
18.
Cold Spring Harb Protoc ; 2012(2): 251-4, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301657

RESUMO

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos , Peptídeos/genética , Peptídeos/metabolismo , Reação em Cadeia da Polimerase/métodos , Expressão Gênica , Genes , Fases de Leitura Aberta , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteólise , Ribossomos/metabolismo
19.
Cold Spring Harb Protoc ; 2012(2): 255-7, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301658

RESUMO

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.


Assuntos
Engenharia Genética/métodos , Peptídeos/genética , Peptídeos/metabolismo , Proteólise , Western Blotting , Linhagem Celular , Expressão Gênica , Genes , Vetores Genéticos , Humanos , Fases de Leitura Aberta , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Transfecção
20.
EMBO J ; 26(2): 494-504, 2007 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-17245433

RESUMO

Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases approximately 12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCtheta-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3(-/-) T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target.


Assuntos
Antígenos CD/fisiologia , Metaloproteases/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/citologia , Proteínas ADAM/fisiologia , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , Linfócitos T/fisiologia , Proteína do Gene 3 de Ativação de Linfócitos
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