RESUMEN
BACKGROUND & AIMS: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. METHODS: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. RESULTS: In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. CONCLUSIONS: These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.
Asunto(s)
Permeabilidad de la Membrana Celular/inmunología , Uniones Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Animales , Western Blotting , Modelos Animales de Enfermedad , Inmunohistoquímica , Inmunoprecipitación , Mucosa Intestinal/inmunología , Mucosa Intestinal/parasitología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ocludina , Fosforilación , ARN/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Toxoplasma/aislamiento & purificación , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/metabolismo , Toxoplasmosis Animal/patologíaRESUMEN
Although gammadelta T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual gammadelta T-cell subsets to infection is unknown. Here we show that in the absence of Vgamma1+ T cells, novel subsets of gammadelta T cells, expressing T-cell receptor (TCR)-Vgamma chains that normally define TCRgammadelta+ dendritic epidermal T cells (DETCs) (Vgamma5+), intestinal intraepithelial lymphocytes (iIELs) (Vgamma7+), and lymphocytes associated with the vaginal epithelia (Vgamma6+), are recruited to the spleen in response to bacterial infection in TCR-Vgamma1-/- mice. By comparison of phenotype and structure of TCR-Vgamma chains and/or -Vdelta chains expressed by these novel subsets with those of their epithelium-associated counterparts, the Vgamma6+ T cells elicited in infected Vgamma1-/- mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of Vgamma1+ T cells. By contrast, Vgamma5+ and Vgamma7+ T cells found in infected Vgamma1-/- mice were distinct from Vgamma5+ DETCs and Vgamma7+ iIELs. Functional analyses of the novel gammadelta T-cell subsets identified for infected Vgamma1-/- mice showed that whereas the Vgamma5+ and Vgamma7+ subsets may compensate for the absence of Vgamma1+ T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of Vgamma1+ T cells. Collectively, these findings identify novel subsets of gammadelta T cells, the recruitment and activity of which is under the control of Vgamma1+ T cells.
Asunto(s)
Listeria monocytogenes/patogenicidad , Listeriosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena gamma de los Receptores de Linfocito T , Homeostasis , Humanos , Listeria monocytogenes/inmunología , Listeriosis/microbiología , Activación de Linfocitos , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Análisis de Secuencia de ADN , Bazo/citología , Bazo/inmunologíaRESUMEN
Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.