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2.
Nat Biotechnol ; 2021 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-34873326

RESUMO

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.

4.
Cell ; 184(24): 5869-5885.e25, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34758294

RESUMO

RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

5.
J Interferon Cytokine Res ; 41(10): 355-359, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34672799
6.
Nat Commun ; 12(1): 5577, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552066

RESUMO

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfoma Anaplásico de Células Grandes/genética , Receptores de Interleucina-2/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoconjugados/farmacologia , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Antígeno Ki-1/genética , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Receptores de Interleucina-2/imunologia , Receptores de Interleucina-2/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nature ; 597(7877): 544-548, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34526724

RESUMO

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.

8.
FEBS J ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543517

RESUMO

Interleukin-10 (IL-10) is an immunomodulatory cytokine that plays important roles in terminating inflammatory responses and preventing tissue damage resulting from autoimmunity. Although these anti-inflammatory actions have led to considerable clinical interest, efforts to exploit IL-10 therapeutically have been hindered by the highly pleiotropic nature of IL-10 and its ability to elicit proinflammatory effects in vivo. In this structural snapshot, we review the recent cryo-EM structure of the IL-10 receptor signaling complex, highlighting its unique structural features, insights into the mechanism of receptor sharing by the IL-10 cytokine family, and the implications for manipulating IL-10 signaling therapeutically.

9.
Immunity ; 54(7): 1405-1416.e7, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34216564

RESUMO

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Fatores Imunológicos/imunologia , Receptores Acoplados a Proteínas G/imunologia , Proteínas Virais/imunologia , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Quimiocinas/imunologia , Microscopia Crioeletrônica/métodos , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Humanos , Ligação Proteica/imunologia , Células Sf9 , Transdução de Sinais/imunologia
10.
Nat Commun ; 12(1): 4357, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272386

RESUMO

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.


Assuntos
Citomegalovirus/metabolismo , Receptores de Quimiocinas/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Domínio Único/química , Esferoides Celulares/efeitos dos fármacos , Proteínas Virais/imunologia , Quimiocina CX3CL1/metabolismo , Cromatografia Líquida , Citomegalovirus/química , Células HEK293 , Humanos , Ligantes , Conformação Molecular , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Espectrometria de Massas em Tandem , beta-Arrestinas/metabolismo
11.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
12.
Elife ; 102021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34003116

RESUMO

Interleukin-2 is a pleiotropic cytokine that mediates both pro- and anti-inflammatory functions. Immune cells naturally differ in their sensitivity to IL-2 due to cell type and activation state-dependent expression of receptors and signaling pathway components. To probe differences in IL-2 signaling across cell types, we used structure-based design to create and profile a series of IL-2 variants with the capacity to titrate maximum signal strength in fine increments. One of these partial agonists, IL-2-REH, specifically expanded Foxp3+ regulatory T cells with reduced activity on CD8+ T cells due to cell type-intrinsic differences in IL-2 signaling. IL-2-REH elicited cell type-dependent differences in gene expression and provided mixed therapeutic results: showing benefit in the in vivo mouse dextran sulfate sodium (DSS) model of colitis, but no therapeutic efficacy in a transfer colitis model. Our findings show that cytokine partial agonists can be used to calibrate intrinsic differences in response thresholds across responding cell types to narrow pleiotropic actions, which may be generalizable to other cytokine and growth factor systems.


Assuntos
Interleucina-2/agonistas , Interleucina-2/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Colite/induzido quimicamente , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL
13.
Immunity ; 54(4): 660-672.e9, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33852830

RESUMO

Interleukin-22 (IL-22) acts on epithelial cells to promote tissue protection and regeneration, but can also elicit pro-inflammatory effects, contributing to disease pathology. Here, we engineered a high-affinity IL-22 super-agonist that enabled the structure determination of the IL-22-IL-22Rα-IL-10Rß ternary complex to a resolution of 2.6 Å. Using structure-based design, we systematically destabilized the IL-22-IL-10Rß binding interface to create partial agonist analogs that decoupled downstream STAT1 and STAT3 signaling. The extent of STAT bias elicited by a single ligand varied across tissues, ranging from full STAT3-biased agonism to STAT1/3 antagonism, correlating with IL-10Rß expression levels. In vivo, this tissue-selective signaling drove tissue protection in the pancreas and gastrointestinal tract without inducing local or systemic inflammation, thereby uncoupling these opposing effects of IL-22 signaling. Our findings provide insight into the mechanisms underlying the cytokine pleiotropy and illustrate how differential receptor expression levels and STAT response thresholds can be synthetically exploited to endow pleiotropic cytokines with enhanced functional specificity.


Assuntos
Inflamação/metabolismo , Interleucinas/metabolismo , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Células HEK293 , Células HT29 , Células Hep G2 , Humanos , Camundongos Endogâmicos C57BL , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia
14.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855972

RESUMO

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.


Assuntos
Interleucina-2/imunologia , Ativação Linfocitária , Receptores de Interleucina-2/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante , Animais , Interleucina-2/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptores de Interleucina-2/genética , Transdução de Sinais/genética , Linfócitos T Reguladores/citologia
15.
Science ; 371(6535)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33737461

RESUMO

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.


Assuntos
Interleucina-10/química , Interleucina-10/metabolismo , Animais , Sítios de Ligação , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Microscopia Crioeletrônica , Citocinas/metabolismo , Evolução Molecular Direcionada , Humanos , Inflamação , Interleucina-10/agonistas , Subunidade alfa de Receptor de Interleucina-10/química , Subunidade alfa de Receptor de Interleucina-10/metabolismo , Subunidade beta de Receptor de Interleucina-10/química , Subunidade beta de Receptor de Interleucina-10/metabolismo , Ativação de Macrófagos , Camundongos , Modelos Moleculares , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Ligação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Fator de Transcrição STAT3/metabolismo , Sepse/imunologia , Transdução de Sinais
16.
Immunity ; 54(3): 586-602.e8, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33691136

RESUMO

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRß chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/genética , Neoplasias Pulmonares/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Algoritmos , Apresentação do Antígeno , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Reações Cruzadas , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/metabolismo , Humanos , Ligação Proteica , Especificidade do Receptor de Antígeno de Linfócitos T
17.
Cell ; 184(4): 983-999.e24, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606986

RESUMO

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.


Assuntos
Interleucina-12/química , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Imunidade , Interleucina-12/agonistas , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/patologia , Estrutura Quaternária de Proteína , Receptores de Interleucina/ultraestrutura , Receptores de Interleucina-12/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
18.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33384332

RESUMO

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.


Assuntos
Receptores de Trombopoetina/metabolismo , Trombopoetina/metabolismo , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Epitopos/imunologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ligantes , Megacariócitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoetina/imunologia , Receptores de Trombopoetina/fisiologia , Transdução de Sinais/fisiologia , Trombocitemia Essencial/metabolismo , Trombopoetina/fisiologia
19.
Nature ; 588(7839): 670-675, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33238290

RESUMO

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.


Assuntos
COVID-19/virologia , Pulmão/citologia , Modelos Biológicos , Organoides/citologia , Organoides/virologia , SARS-CoV-2/fisiologia , Técnicas de Cultura de Tecidos , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , COVID-19/metabolismo , COVID-19/patologia , Diferenciação Celular , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Células Clonais/virologia , Humanos , Técnicas In Vitro , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/fisiologia , Integrina alfa6/análise , Integrina beta4/análise , Queratina-5/análise , Organoides/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Receptor de TWEAK/análise
20.
Nature ; 586(7831): 779-784, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33087934

RESUMO

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.


Assuntos
Antígenos Comuns de Leucócito/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Reagentes para Ligações Cruzadas , Modelos Animais de Doenças , Progressão da Doença , Feminino , Células HEK293 , Humanos , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/química , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Nivolumabe/farmacologia , Fosforilação , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
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