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2.
Nat Commun ; 13(1): 3222, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35680882

RESUMO

Cytotoxic lymphocytes fight pathogens and cancer by forming immune synapses with infected or transformed target cells and then secreting cytotoxic perforin and granzyme into the synaptic space, with potent and specific killing achieved by this focused delivery. The mechanisms that establish the precise location of secretory events, however, remain poorly understood. Here we use single cell biophysical measurements, micropatterning, and functional assays to demonstrate that localized mechanotransduction helps define the position of secretory events within the synapse. Ligand-bound integrins, predominantly the αLß2 isoform LFA-1, function as spatial cues to attract lytic granules containing perforin and granzyme and induce their fusion with the plasma membrane for content release. LFA-1 is subjected to pulling forces within secretory domains, and disruption of these forces via depletion of the adaptor molecule talin abrogates cytotoxicity. We thus conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance their cytotoxic power and fidelity.


Assuntos
Antígeno-1 Associado à Função Linfocitária , Mecanotransdução Celular , Citotoxicidade Imunológica , Granzimas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Perforina/metabolismo , Sinapses/metabolismo , Linfócitos T Citotóxicos
3.
Cell Host Microbe ; 30(6): 809-823.e6, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35439436

RESUMO

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism ensuring optimal energy balance. However, the interplay between diet, microbes, and host factors sustaining intestinal oscillations is complex and poorly understood. Here, using a mouse model, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bidirectional manner and does not correlate with the intestinal core circadian clock. High-fat diet is the primary driver of microbial oscillators that impair host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression that secondarily drives abundance and oscillation of key gut microbes. This illustrates transkingdom coordination of biological rhythms primarily influenced by diet and reciprocal sensor-effector signals between host and microbial components, ultimately driving metabolism. Restoring the gut microbiota's capacity to sense dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.


Assuntos
Relógios Circadianos , Microbioma Gastrointestinal , Ritmo Circadiano , Dieta , Dieta Hiperlipídica/efeitos adversos , Metabolismo dos Lipídeos
4.
J Exp Med ; 218(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33988715

RESUMO

HIV-specific CD8+ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4+ T cells from HIV+ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8+ T cell response have a profound impact on the emergence and persistence of escape mutations. This "participant-derived xenograft" model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Xenoenxertos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Células HEK293 , Infecções por HIV/virologia , Xenoenxertos/virologia , Humanos , Imunoterapia/métodos , Interleucina-15/imunologia , Camundongos , Mutação/imunologia , Viremia/imunologia , Viremia/virologia , Replicação Viral/imunologia
5.
J Clin Invest ; 130(5): 2542-2559, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32027622

RESUMO

Curing HIV infection will require the elimination of a reservoir of infected CD4+ T cells that persists despite HIV-specific cytotoxic T cell (CTL) responses. Although viral latency is a critical factor in this persistence, recent evidence also suggests a role for intrinsic resistance of reservoir-harboring cells to CTL killing. This resistance may have contributed to negative outcomes of clinical trials, where pharmacologic latency reversal has thus far failed to drive reductions in HIV reservoirs. Through transcriptional profiling, we herein identified overexpression of the prosurvival factor B cell lymphoma 2 (BCL-2) as a distinguishing feature of CD4+ T cells that survived CTL killing. We show that the inducible HIV reservoir was disproportionately present in BCL-2hi subsets in ex vivo CD4+ T cells. Treatment with the BCL-2 antagonist ABT-199 was not sufficient to drive reductions in ex vivo viral reservoirs when tested either alone or with a latency-reversing agent (LRA). However, the triple combination of strong LRAs, HIV-specific T cells, and a BCL-2 antagonist uniquely enabled the depletion of ex vivo viral reservoirs. Our results provide rationale for novel therapeutic approaches targeting HIV cure and, more generally, suggest consideration of BCL-2 antagonism as a means of enhancing CTL immunotherapy in other settings, such as cancer.


Assuntos
HIV/imunologia , HIV/patogenicidade , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Adulto , Terapia Antirretroviral de Alta Atividade , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Técnicas de Cocultura , Terapia Combinada , Citotoxicidade Imunológica/genética , Reservatórios de Doenças/virologia , Feminino , Perfilação da Expressão Gênica , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Sulfonamidas/farmacologia , Latência Viral/efeitos dos fármacos
6.
Cell Host Microbe ; 17(5): 681-9, 2015 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-25891358

RESUMO

Circadian clocks and metabolism are inextricably intertwined, where central and hepatic circadian clocks coordinate metabolic events in response to light-dark and sleep-wake cycles. We reveal an additional key element involved in maintaining host circadian rhythms, the gut microbiome. Despite persistence of light-dark signals, germ-free mice fed low or high-fat diets exhibit markedly impaired central and hepatic circadian clock gene expression and do not gain weight compared to conventionally raised counterparts. Examination of gut microbiota in conventionally raised mice showed differential diurnal variation in microbial structure and function dependent upon dietary composition. Additionally, specific microbial metabolites induced under low- or high-fat feeding, particularly short-chain fatty acids, but not hydrogen sulfide, directly modulate circadian clock gene expression within hepatocytes. These results underscore the ability of microbially derived metabolites to regulate or modify central and hepatic circadian rhythm and host metabolic function, the latter following intake of a Westernized diet.


Assuntos
Relógios Circadianos , Dieta Hiperlipídica , Disbiose/induzido quimicamente , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Metabolismo dos Lipídeos , Animais , Peso Corporal , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Fígado/patologia , Camundongos , Dados de Sequência Molecular , Obesidade , Análise de Sequência de DNA
7.
Am J Stem Cells ; 1(2): 138-145, 2012 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-23087846

RESUMO

Mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent cells that give rise to stromal cells in the marrow. MSCs have been shown to be immunosuppressive and have become an attractive therapeutic option for the modulation of undesired immune responses. Currently, ex vivo expanded human (h)MSCs are being utilized in clinical trials both in the USA and in Europe to treat a variety of immune disorders. hMSCs need to be harvested, isolated and expanded in culture. This necessary expansion may also result in decrease or loss of the immunomodulatory potential of hMSCs. Ideally, the intrinsic immunomodulatory activity (potency) of an hMSC preparation should be assessed prior to its administration. The goal of the experiments described here was to develop a simple potency assay for the immunomodulatory properties of hMSCs. The immunosuppressive activity of hMSCs conditioned media was tested in enzyme-linked immunosorbent spot assays (ELISpot) and the immunosuppressive activity of the conditioned media was correlated with the concentration of several cytokines present in these conditioned media. The concentration of prostaglandin E(2) in the media correlated with their immunosuppressive activity. The concentration of the other cytokines measured did not correlate with the immunosuppressive activity of the media. The dose-response effect could be replicated by adding PGE(2) to ELISpot assays. Furthermore, the immunosuppressive activity of the conditioned media was inhibitable by a neutralizing anti-PGE(2) antibody. These data suggest that measurement of PGE(2) in media conditioned by hMSCs exposed to inflammatory stimuli could be used as a surrogate measure of their immunosuppressive capacity. These findings need to be confirmed in vitro using different assays of immune function and validated in vivo to determine the level of correlation of these data with efficacy in pre-clinical models of immune disorders.

8.
Stem Cells Int ; 2011: 235176, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21437189

RESUMO

Allogeneic hematopoietic stem cell transplantation is the main curative therapy for many hematologic malignancies. Its potential relies on graft-versus-tumor effects which associate with graft-versus-host disease. Mesenchymal stromal cells (MSCs) possess immunomodulatory properties that make them attractive therapeutic alternatives. We evaluated the in vitro immunosuppressive activity of medium conditioned by human MSCs from 5 donors expanded 13 passages with or without FGF-2. FGF-2 supplementation increased expansion 3,500- and 240,000-fold by passages 7 and 13, respectively. There were no differences in immunosuppressive activity between media conditioned by passage-matched cells expanded under different conditions, but media conditioned by FGF-treated MSCs were superior to population doubling-matched controls. The immunosuppressive activity was maintained in three of the preparations but decreased with expansion in two. The proliferation induced by FGF-2 did not result in loss of immunosuppressive activity. However, because the immunosuppressive activity was not consistently preserved, caution must be exercised to ensure that the activity of the cells is sufficient after extensive expansion.

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