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1.
Proc Natl Acad Sci U S A ; 119(34): e2202144119, 2022 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-35969785

RESUMEN

The metabolic capacity of many cells is tightly regulated and can adapt to changes in metabolic resources according to environmental changes. Tissue-resident memory (TRM) CD8+ T cells are one of the most abundant T cell populations and offer rapid protection against invading pathogens, especially at the epithelia. TRM cells metabolically adapt to their tissue niche, such as the intestinal epithelial barrier. In the small intestine, the types of TRM cells are intraepithelial lymphocytes (IELs), which contain high levels of cytotoxic molecules and express activation markers, suggesting a heightened state of activation. We hypothesize that the tissue environment may determine IEL activity. We show that IEL activation, in line with its semiactive status, is metabolically faster than circulating CD8+ T cells. IEL glycolysis and oxidative phosphorylation (OXPHOS) are interdependently regulated and are dependent on rapid access to metabolites from the environment. IELs are restrained by local availability of metabolites, but, especially, glucose levels determine their activity. Importantly, this enables functional control of intestinal TRM cells by metabolic means within the fragile environment of the intestinal epithelial barrier.


Asunto(s)
Linfocitos T CD8-positivos , Linfocitos Intraepiteliales , Células T de Memoria , Linfocitos T CD8-positivos/citología , Mucosa Intestinal/citología , Intestinos/citología , Linfocitos Intraepiteliales/citología , Activación de Linfocitos , Células T de Memoria/citología , Fosforilación Oxidativa
2.
Cancer Immunol Immunother ; 71(10): 2391-2404, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35195762

RESUMEN

Vδ2+ γδ T cell, one of promising strategies for tumor immunotherapy, recognizes and kills cancer cells in a non-MHC dependent manner. Previously, we pioneeringly proved the clinical safety and efficacy of allogeneic Vδ2+ γδ T cells, in vitro expanded from healthy donors, in the treatment of late-stage cancer patients. Nevertheless, how to profoundly potentiate cytotoxic function of expanded Vδ2+ γδ T cells remains to be further explored. Here, we proposed that 40 °C-Shock could be a simple and reliable approach to in vitro boost the effector function. We found that 40 °C-shock could phosphorylate two MAPK proteins ERK and p38 through HSP70, which facilitated actyl-α-tubulin and actin augments and reorganization, elevated Ki-67 expression and cell surface adhesion, and promoted releases of cytokines IFN-γ, perforin and granzyme B, as well as downregulated LAG3 expression. We also observed 40 °C-shock induced elevations of mitochondrial metabolism. These altogether led to potentiated cytotoxic responses against cancer cells. This proof-of-concept work demonstrated that 40 °C-shock would be probably developed into an effective method to in vitro boost the cytotoxicity of Vδ2+ γδ T cell before applying it in immunotherapy, and provided scientific evidences for the view that fever can activate immune responses of innate immune cells.


Asunto(s)
Proteínas HSP70 de Choque Térmico , Linfocitos Intraepiteliales , Linfocitos T , Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Calor , Humanos , Inmunoterapia , Linfocitos Intraepiteliales/citología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos T/citología , Regulación hacia Arriba
3.
J Immunol ; 208(3): 651-659, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-34996838

RESUMEN

The precursors of TCRαß+CD8αα+ intraepithelial lymphocytes (IEL) arise in the thymus through a complex process of agonist selection. We and others have shown that the proapoptotic protein, Bim, is critical to limit the number of thymic IEL precursors (IELp), as loss of Bim at the CD4+CD8+ double-positive stage of development drastically increases IELp. The factors determining this cell death versus survival decision remain largely unknown. In this study, we used CD4CreBcl2f/f mice to define the role of the antiapoptotic protein Bcl-2 and CD4CreBcl2f/fBimf/f mice to determine the role of Bcl-2 in opposing Bim to promote survival of IELp. First, in wild-type mice, we defined distinct subpopulations within PD-1+CD122+ IELp, based on their expression of Runx3 and α4ß7. Coexpression of α4ß7 and Runx3 marked IELp that were most dependent upon Bcl-2 for survival. Importantly, the additional loss of Bim restored Runx3+α4ß7+ IELp, showing that Bcl-2 antagonizes Bim to enable IELp survival. Further, the loss of thymic IELp in CD4CreBcl2f/f mice also led to a dramatic loss of IEL in the gut, and the additional loss of Bim restored gut IEL. The loss of gut IEL was due to both reduced seeding by IELp from the thymus as well as a requirement for Bcl-2 for peripheral IEL survival. Together, these findings highlight subset-specific and temporal roles for Bcl-2 in driving the survival of TCRαß+CD8αα+ IEL and thymic IELp.


Asunto(s)
Proteína 11 Similar a Bcl2/metabolismo , Linfocitos Intraepiteliales/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Timo/citología , Animales , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Supervivencia Celular/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Linfocitos Intraepiteliales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Células Madre/citología , Timo/inmunología
4.
J Cell Mol Med ; 25(22): 10604-10613, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34687276

RESUMEN

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co-culturing HSC-enriched bone marrow cells (HSC-eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC-eBMCs and iTECs cultured with IL-2 + IL-7 for up to 21 days induced CD45.1+ γδT cells that expressed a broad repertoire of Vγ and Vδ T-cell receptors. Notably, the induced lymphocytes contained few or no αßT cells, NK1.1+ natural killer cells, or B220+ B cells. Adoptive transfer of the induced γδT cells to leukemia-bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.


Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Epiteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Linfocitos Intraepiteliales/metabolismo , Traslado Adoptivo , Animales , Biomarcadores , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Técnicas de Cocultivo , Células Epiteliales/citología , Células Madre Hematopoyéticas/citología , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/citología , Linfocitos Intraepiteliales/citología , Ratones , Ratones Transgénicos , Trasplante Autólogo
5.
Adv Sci (Weinh) ; 8(21): e2102778, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34495570

RESUMEN

Gluten-specific CD4+ T cells being drivers of celiac disease (CeD) are obvious targets for immunotherapy. Little is known about how cell markers harnessed for T-cell-directed therapy can change with time and upon activation in CeD and other autoimmune conditions. In-depth characterization of gluten-specific CD4+ T cells and CeD-associated (CD38+ and CD103+ ) CD8+ and γδ+ T cells in blood of treated CeD patients undergoing a 3 day gluten challenge is reported. The phenotypic profile of gluten-specific cells changes profoundly with gluten exposure and the cells adopt the profile of gluten-specific cells in untreated disease (CD147+ , CD70+ , programmed cell death protein 1 (PD-1)+ , inducible T-cell costimulator (ICOS)+ , CD28+ , CD95+ , CD38+ , and CD161+ ), yet with some markers being unique for day 6 cells (C-X-C chemokine receptor type 6 (CXCR6), CD132, and CD147) and with integrin α4ß7, C-C motif chemokine receptor 9 (CCR9), and CXCR3 being expressed stably at baseline and day 6. Among gluten-specific CD4+ T cells, 52% are CXCR5+ at baseline, perhaps indicative of germinal-center reactions, while on day 6 all are CXCR5- . Strikingly, the phenotypic profile of gluten-specific CD4+ T cells on day 6 largely overlaps with that of CeD-associated (CD38+ and CD103+ ) CD8+ and γδ+ T cells. The antigen-induced shift in phenotype of CD4+ T cells being shared with other disease-associated T cells is relevant for development of T-cell-directed therapies.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/terapia , Glútenes/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Enfermedad Celíaca/inmunología , Glútenes/química , Antígenos HLA-DQ/química , Antígenos HLA-DQ/inmunología , Humanos , Inmunoterapia , Cadenas alfa de Integrinas/metabolismo , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Fenotipo , Multimerización de Proteína
6.
Nat Immunol ; 22(8): 969-982, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34312548

RESUMEN

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Linfocitos Intraepiteliales/citología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Reguladores/citología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/inmunología , Colitis/inmunología , Colitis/prevención & control , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Factores de Transcripción/genética , Transcripción Genética/genética
7.
Hum Pathol ; 115: 112-122, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34181982

RESUMEN

Whilst intraepithelial lymphocytes (IELs) are considered normal within the distal esophageal mucosa, they have an increasingly recognised role in the pathogenesis of reflux esophagitis, and IEL quantification establishes the diagnosis of lymphocytic esophagitis. Knowledge regarding the upper limit of a normal IEL count in health is lacking. We studied 117 non-healthcare seeking adult volunteers from a random community sample (the Kalixanda study) with esophageal biopsies 2 cm above the gastroesophageal junction. Subjects were divided into four groups based on the presence or absence of gastro-esophageal reflux symptoms and/or esophagitis on endoscopy. Asymptomatic subjects with no endoscopic esophagitis were selected as controls, and the cell counts in this group were used to define the upper limit of normal of IELs, eosinophils and neutrophils. The entire sample was used to identify independent predictors of increased cellular counts by logistic regression analysis. None of the healthy controls had an IEL count of more than three per five high power fields (HPF), and therefore this was considered as the upper limit of normal; no controls had eosinophils or neutrophils in esophageal biopsies. Independent predictors of an elevated IEL count were spongiosis on histology (OR 11.17, 95% CI 3.32-37.58, P < 0.01) and current smoking (OR 4.84, 95% CI 1.13-2.71, P = 0.03). A receiver operating characteristics analysis concluded that a threshold of 3 IELs/5HPFs performs best in predicting reflux symptoms when a normal esophageal mucosa is visualized on endoscopy (sensitivity = 100.0%, specificity = 35.2%). The healthy esophageal mucosa does not contain more than three IELs per five HPF in the distal esophagus.


Asunto(s)
Eosinófilos , Mucosa Esofágica/citología , Mucosa Esofágica/patología , Reflujo Gastroesofágico/patología , Linfocitos Intraepiteliales , Neutrófilos , Adulto , Anciano , Eosinófilos/citología , Eosinófilos/patología , Femenino , Humanos , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/patología , Masculino , Persona de Mediana Edad , Neutrófilos/citología , Neutrófilos/patología , Valores de Referencia
8.
Int J Mol Sci ; 22(10)2021 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-34067987

RESUMEN

Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.


Asunto(s)
Antígenos CD/metabolismo , Movimiento Celular , Procesamiento de Imagen Asistido por Computador/métodos , Cadenas alfa de Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Linfocitos Intraepiteliales/metabolismo , Organoides/metabolismo , Linfocitos T/fisiología , Animales , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Mucosa Intestinal/citología , Linfocitos Intraepiteliales/citología , Ratones , Organoides/citología , Análisis Espacio-Temporal
9.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-33806063

RESUMEN

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αß or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αß and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bass Dicentrarchus labrax, to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRß, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ+ cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.


Asunto(s)
Inmunidad Adaptativa , Lubina/genética , Linfocitos Intraepiteliales/citología , Receptores de Antígenos de Linfocitos T/genética , Alimentación Animal , Animales , Lubina/inmunología , Linaje de la Célula , Ensayo de Inmunoadsorción Enzimática , Sistema Inmunológico/inmunología , Inmunoglobulina G , Leucocitos/citología , Tejido Linfoide , Microalgas , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T/inmunología , Timo/inmunología , Distribución Tisular
10.
Eur J Immunol ; 51(5): 1080-1088, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33521937

RESUMEN

TCRαß+ CD8α+ CD8ß- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely ß2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of ß2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.


Asunto(s)
Selección Clonal Mediada por Antígenos , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Linfopoyesis , Células Precursoras de Linfocitos T/inmunología , Células Precursoras de Linfocitos T/metabolismo , Timo/citología , Animales , Biomarcadores , Diferenciación Celular , Genes MHC Clase I , Inmunofenotipificación , Linfocitos Intraepiteliales/citología , Linfopoyesis/genética , Linfopoyesis/inmunología , Ratones , Células Precursoras de Linfocitos T/citología , Timocitos/citología , Timocitos/inmunología , Timocitos/metabolismo
11.
Cell Mol Gastroenterol Hepatol ; 11(5): 1483-1503, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33515805

RESUMEN

BACKGROUND & AIMS: The reason why small intestinal cancer is rarer than colorectal cancer is not clear. We hypothesized that intraepithelial lymphocytes (IELs), which are enriched in the small intestine, are the closest immune cells to epithelial cells, exclude tumor cells via cell-to-cell contact. METHODS: We developed DPE-green fluorescent protein (DPE-GFP) × adenomatous polyposis coli; multiple intestinal neoplasia (APCmin ) mice, which is a T-cell-reporter mouse with spontaneous intestinal tumors. We visualized the dynamics of IELs in the intestinal tumor microenvironment and the interaction between IELs and epithelial cells, and the roles of cell-to-cell contact in anti-intestinal tumor immunity using a novel in vivo live-imaging system and a novel in vitro co-culture system. RESULTS: In the small intestinal tumor microenvironment, T-cell movement was restricted around blood vessels and the frequency of interaction between IELs and epithelial cells was reduced. Genetic deletion of CD103 decreased the frequency of interaction between IELs and epithelial cells, and increased the number of small intestinal tumors. In the co-culture system, wild-type IELs expanded and infiltrated to intestinal tumor organoids from APCmin mice and reduced the viability of them, which was cell-to-cell contact and CD103 dependent. CONCLUSIONS: The abundance of IELs in the small intestine may contribute to a low number of tumors, although this system may not work in the colon because of the sparseness of IELs. Strategies to increase the number of IELs in the colon or enhance cell-to-cell contact between IELs and epithelial cells may be effective for the prevention of intestinal tumors in patients with a high cancer risk.


Asunto(s)
Antígenos CD/fisiología , Comunicación Celular , Cadenas alfa de Integrinas/fisiología , Mucosa Intestinal/inmunología , Neoplasias Intestinales/prevención & control , Intestino Delgado/inmunología , Linfocitos Intraepiteliales/inmunología , Microambiente Tumoral , Animales , Técnicas de Cocultivo , Femenino , Mucosa Intestinal/citología , Neoplasias Intestinales/inmunología , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Intestino Delgado/patología , Linfocitos Intraepiteliales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organoides/inmunología , Organoides/patología
12.
Cell Mol Life Sci ; 78(2): 573-580, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32803399

RESUMEN

Epidermal resident γδ T cells, or dendritic epidermal T cells (DETCs) in mice, are a unique and conserved population of γδ T cells enriched in the epidermis, where they serve as the regulators of immune responses and sense skin injury. Despite the great advances in the understanding of the development, homeostasis, and function of DETCs in the past decades, the origin and the underlying molecular mechanisms remain elusive. Here, we reviewed the recent research progress on DETCs, including their origin and homeostasis in the skin, especially at transcriptional and epigenetic levels, and discuss the involvement of DETCs in skin diseases.


Asunto(s)
Epidermis/inmunología , Linfocitos Intraepiteliales/inmunología , Enfermedades de la Piel/inmunología , Piel/inmunología , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epigénesis Genética , Humanos , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/metabolismo , Ratones , Piel/citología , Piel/metabolismo , Enfermedades de la Piel/genética , Timo/citología , Timo/inmunología , Timo/metabolismo , Saco Vitelino/citología , Saco Vitelino/inmunología , Saco Vitelino/metabolismo
13.
Mucosal Immunol ; 14(1): 68-79, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32483197

RESUMEN

Thymocytes bearing αß T cell receptors (TCRαß) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Selección Clonal Mediada por Antígenos , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Timo/inmunología , Timo/metabolismo , Linfocitos T CD8-positivos/citología , Microambiente Celular , Selección Clonal Mediada por Antígenos/genética , Selección Clonal Mediada por Antígenos/inmunología , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/inmunología , Linfocitos Intraepiteliales/citología , Péptidos/inmunología , Timo/citología
14.
Nanotheranostics ; 4(2): 71-82, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32190534

RESUMEN

Vγ9Vδ2 T cell immunotherapy has been shown to be effective in delaying tumour growth in both pre-clinical and clinical studies. It has been pointed out the importance of the ability of cells to accumulate within tumours and the association with therapeutic efficacy in clinical studies of adoptive T cell transfer. We have previously reported that alendronate liposomes (L-ALD) increase the efficacy of this therapy after localised or systemic injection of γδ T cells in mice, inoculated with ovarian, melanoma, pancreatic or experimental lung metastasis tumour models, respectively. This study aimed to examine the organ biodistribution and tumour uptake of human γδ T cells in subcutaneous (SC), intraperitoneal (IP) or experimental metastatic lung tumours, established in NOD-SCID gamma (NSG) mice using the melanoma cell line A375Pß6.luc. pre-injected with L-ALD. Overall, small variations in blood profiles and organ biodistribution of γδ T cells among the different tumour models were observed. Exceptionally, IP-tumour and experimental metastatic lung-tumour bearing mice pre-injected with L-ALD showed a significant decrease in liver accumulation, and highest uptake of γδ T cells in lungs and tumour-bearing lungs, respectively. Lower γδ T cell count was found in the SC and IP tumours.


Asunto(s)
Alendronato , Inmunoterapia Adoptiva/métodos , Linfocitos Intraepiteliales , Liposomas , Alendronato/administración & dosificación , Alendronato/farmacocinética , Animales , Células Cultivadas , Humanos , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/metabolismo , Liposomas/administración & dosificación , Liposomas/farmacocinética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Distribución Tisular
15.
Methods Mol Biol ; 2121: 141-152, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32147793

RESUMEN

Intestinal intraepithelial lymphocytes (IEL) comprise distinct groups of innate-like and memory T cells that collectively form one of the largest T cell compartments in the body. IEL are located within the intestinal epithelium and are the first immune cells in the gut to interact with the food, microbiota, and pathogens that the gut is continually exposed to. IEL can respond rapidly to external insults to protect the small intestinal epithelium but are also considered regulatory cells that are important to maintain the homeostasis of the gut. However, the mechanisms of IEL activation and their interactions within the epithelium remain largely elusive. Indeed, IEL are not commonly evaluated even in studies of gut immunology, potentially because they are perceived as being difficult to isolate and study. In this protocol, we present a simplified method to isolate IEL from the murine small intestine and provide representative data for flow cytometric analyses of the different IEL subsets. We also outline two procedures for culturing IEL, which can permit functional studies and coculture with epithelial cells. These strategies should make studies of this large but enigmatic T cell compartment more accessible and open up understanding of homeostatic mechanisms in the intestine, and tissue-associated immunity.


Asunto(s)
Citometría de Flujo/métodos , Mucosa Intestinal/citología , Intestino Delgado/citología , Linfocitos Intraepiteliales/citología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/citología , Animales , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Inmunidad Innata , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Linfocitos Intraepiteliales/inmunología , Ratones , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
16.
J Biol Chem ; 295(10): 3239-3246, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-31992596

RESUMEN

The immune co-receptor CD8 molecule (CD8) has two subunits, CD8α and CD8ß, which can assemble into homo or heterodimers. Nonclassical (class-Ib) major histocompatibility complex (MHC) molecules (MHC-Ibs) have recently been identified as ligands for the CD8αα homodimer. This was demonstrated by the observation that histocompatibility 2, Q region locus 10 (H2-Q10) is a high-affinity ligand for CD8αα which also binds the MHC-Ib molecule H2-TL. This suggests that MHC-Ib proteins may be an extended source of CD8αα ligands. Expression of H2-T3/TL and H2-Q10 is restricted to the small intestine and liver, respectively, yet CD8αα-containing lymphocytes are present more broadly. Therefore, here we sought to determine whether murine CD8αα binds only to tissue-specific MHC-Ib molecules or also to ubiquitously expressed MHC-Ib molecules. Using recombinant proteins and surface plasmon resonance-based binding assays, we show that the MHC-Ib family furnishes multiple binding partners for murine CD8αα, including H2-T22 and the CD94/NKG2-A/B-activating NK receptor (NKG2) ligand Qa-1b We also demonstrate a hierarchy among MHC-Ib proteins with respect to CD8αα binding, in which Qa-1b > H2-Q10 > TL. Finally, we provide evidence that Qa-1b is a functional ligand for CD8αα, distinguishing it from its human homologue MHC class I antigen E (HLA-E). These findings provide additional clues as to how CD8αα-expressing cells are controlled in different tissues. They also highlight an unexpected immunological divergence of Qa-1b/HLA-E function, indicating the need for more robust studies of murine MHC-Ib proteins as models for human disease.


Asunto(s)
Antígenos CD8/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/química , Animales , Antígenos CD8/genética , Dimerización , Humanos , Interferón gamma/metabolismo , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Células Jurkat , Ligandos , Ratones , Ratones Endogámicos C57BL , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Resonancia por Plasmón de Superficie , Antígenos HLA-E
17.
Int Immunol ; 32(5): 307-319, 2020 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-31875880

RESUMEN

Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intra-epithelial lymphocytes (IELs), especially CD8αα + IELs (CD8αα IELs). In the intestine, IL-15 is produced by intestinal epithelial cells (IECs), blood vascular endothelial cells (BECs) and hematopoietic cells. However, the precise role of intestinal IL-15 on IELs is still unknown. To address the question, we generated two kinds of IL-15 conditional knockout (IL-15cKO) mice: villin-Cre (Vil-Cre) and Tie2-Cre IL-15cKO mice. IEC-derived IL-15 was specifically deleted in Vil-Cre IL-15cKO mice, whereas IL-15 produced by BECs and hematopoietic cells was deleted in Tie2-Cre IL-15cKO mice. The cell number and frequency of CD8αα IELs and NK IELs were significantly reduced in Vil-Cre IL-15cKO mice. By contrast, CD8αα IELs were unchanged in Tie2-Cre IL-15cKO mice, indicating that IL-15 produced by BECs and hematopoietic cells is dispensable for CD8αα IELs. Expression of an anti-apoptotic factor, Bcl-2, was decreased, whereas Fas expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. Forced expression of Bcl-2 by a Bcl-2 transgene partially restored CD8αα IELs in Vil-Cre IL-15cKO mice, suggesting that some IL-15 signal other than Bcl-2 is required for maintenance of CD8αα IELs. Furthermore, granzyme B production was reduced, whereas PD-1 expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. These results collectively suggested that IEC-derived IL-15 is essential for homeostasis of IELs by promoting their survival and functional maturation.


Asunto(s)
Células Endoteliales/inmunología , Interleucina-15/inmunología , Intestinos/citología , Intestinos/inmunología , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/inmunología , Animales , Femenino , Interleucina-15/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos
18.
Biochem Biophys Res Commun ; 523(2): 328-335, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-31864702

RESUMEN

Intraepithelial lymphocytes (IELs) are very unique in the intestinal immune system. They include γδT cells and CD4-CD8-TCRαß+T cells (double negative: DNT), both of which are specific for the intestine, in addition to CD4+ and CD8+ T cells. IELs exist within the monolayer of the intestinal epithelial cells and dynamically move between lamina propria (LP) and intraepithelial (IE) region. The localization and movement patterns of IEL subsets and the regulatory factors have been unknown. Here, we developed a novel in vitro live imaging system and quantified the motility and morphological changes among subsets of IELs. We identified CD8αα as the key regulatory factor. IELs, especially γδ and DNT cells, showed amoeboid shape and frequent morphological change, while most T cells in MLN or SP showed round shape in vitro. TCR signal, IL-15, gut microbes, CCL25, and integrin αEß7 expression were non-essential for IEL movement in vitro. CD8αα+ cells showed higher motility and larger morphological changes than CD8αα- cells. Adoptive transferred CD8αα+CD4-IELs localized to IE region of recipient NSG mice, while CD8αα-CD4-IELs localized to the LP. Our results showed that the CD8αα/TL signal is essential for the localization of IELs to IE region in vivo. CD8αα/TL may be an effective target to increase the number of IELs, which protects against intestinal infection, allergy, tumorigenesis or inflammation.


Asunto(s)
Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/clasificación , Movimiento Celular/inmunología , Forma de la Célula , Quimiocinas CC/metabolismo , Femenino , Inmunidad Mucosa , Interleucina-15/metabolismo , Intestino Delgado/citología , Intestino Delgado/inmunología , Linfocitos Intraepiteliales/clasificación , Microscopía Intravital , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos
19.
J Vis Exp ; (148)2019 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-31282900

RESUMEN

Intraepithelial lymphocytes expressing γδ T cell receptor (γδ IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the γδ T cell receptor, our understanding of the regulation of γδ IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating γδ IEL function and the responsiveness of these cells to the local microenvironment. Although γδ IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP γδ T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.


Asunto(s)
Intestino Delgado/citología , Linfocitos Intraepiteliales/citología , Microscopía Intravital , Animales , Movimiento Celular , Proteínas Fluorescentes Verdes/metabolismo , Imagenología Tridimensional , Intestino Delgado/inmunología , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo
20.
Cell Rep ; 27(10): 3034-3048.e5, 2019 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-31167146

RESUMEN

Dermal γδT cells play critical roles in skin homeostasis and inflammation. However, the underlying molecular mechanisms by which these cells are activated have not been fully understood. Here, we show that the mechanistic or mammalian target of rapamycin (mTOR) and STAT3 pathways are activated in dermal γδT cells in response to innate stimuli such as interleukin-1ß (IL-1ß) and IL-23. Although both mTOR complex 1 (mTORC1) and mTORC2 are essential for dermal γδT cell proliferation, mTORC2 deficiency leads to decreased dermal γδT17 cells. It appears that mitochondria-mediated oxidative phosphorylation is critical in this process. Notably, although the STAT3 pathway is critical for dermal Vγ4T17 effector function, it is not required for Vγ6T17 cells. Transcription factor IRF-4 activation promotes dermal γδT cell IL-17 production by linking IL-1ß and IL-23 signaling. The absence of mTORC2 in dermal γδT cells, but not STAT3, ameliorates skin inflammation. Taken together, our results demonstrate that the mTOR-STAT3 signaling differentially regulates dermal γδT cell effector function in skin inflammation.


Asunto(s)
Linfocitos Intraepiteliales/metabolismo , Factor de Transcripción STAT3/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Factores Reguladores del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interleucina-17/metabolismo , Linfocitos Intraepiteliales/citología , Diana Mecanicista del Complejo 2 de la Rapamicina/deficiencia , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Psoriasis/diagnóstico , Psoriasis/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Piel/metabolismo , Piel/patología
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