RESUMEN

Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16) E7 oncoprotein under a keratin 14 promoter (K14E7 mice) display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb) led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes) to premalignant skin.

Asunto(s)

Regulación de la Expresión Génica , Papillomavirus Humano 16 , Proteínas E7 de Papillomavirus/metabolismo , Piel/inmunología , Piel/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Recuento de Células , Epitelio/inmunología , Epitelio/metabolismo , Epitelio/patología , Hiperplasia/inmunología , Hiperplasia/metabolismo , Tolerancia Inmunológica , Ratones , Proteínas E7 de Papillomavirus/química , Proteína de Retinoblastoma/metabolismo , Piel/citología , Piel/patología , Linfocitos T/citología , Microambiente Tumoral/inmunología

RESUMEN

We recently demonstrated that CD1d-restricted NKT cells resident in skin can inhibit CD8 T cell-mediated graft rejection of human papillomavirus E7-expressing skin through an IFN-γ-dependent mechanism. In this study, we examined the role of systemically derived NKT cells in regulating the rejection of skin grafts expressing viral proteins. In lymph nodes draining transplanted skin, Ag-specific CD8 T cell proliferation, cytokine production, and cytotoxic activity were impaired by NKT cells. NKT cell suppression was mediated via CD11c(+) dendritic cells. Inhibition of CD8 T cell function did not require Foxp3(+) regulatory T cells or NKT cell-secreted IFN-γ, IL-10, or IL-17. Thus, following skin grafting or immunization with human papillomavirus-E7 oncoprotein, NKT cells reduce the capacity of draining lymph node-resident APCs to cross-present Ag to CD8 T cell precursors, as evidenced by impaired expansion and differentiation to Ag-specific CD8 T effector cells. Therefore, in the context of viral Ag challenge in the skin, systemic NKT cells limit the capacity for effective priming of adaptive immunity.

Asunto(s)

Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Rechazo de Injerto/inmunología , Papillomavirus Humano 16/inmunología , Células T Asesinas Naturales/inmunología , Proteínas E7 de Papillomavirus/inmunología , Trasplante de Piel , Piel/inmunología , Inmunidad Adaptativa , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos Virales/genética , Proliferación Celular , Citocinas/inmunología , Rechazo de Injerto/virología , Papillomavirus Humano 16/genética , Humanos , Ganglios Linfáticos/inmunología , Ratones , Ratones Noqueados , Proteínas E7 de Papillomavirus/genética , Piel/virología , Linfocitos T Reguladores/inmunología , Trasplante Homólogo

RESUMEN

NKT cells can promote or inhibit adaptive immune responses. Cutaneous immunity is tightly regulated by cooperation between innate and adaptive immune processes, but the role of NKT cells in regulating cutaneous immunity is largely unknown. In this study, we show, in a mouse model, that skin-infiltrating CD1d-restricted NKT cells in HPV16-E7 transgenic hyperplastic skin produce IFN-gamma, which can prevent rejection of HPV16-E7-expressing skin grafts. Suppression of graft rejection is associated with the accumulation of CD1d(hi)-expressing CD11c(+)F4/80(hi) myeloid cells in hyperplastic skin. Blockade of CD1d, removal of NKT cells, or local inhibition of IFN-gamma signaling is sufficient to restore immune-mediated graft rejection. Thus, inhibition of NKT cell recruitment or function may enable effective immunity against tumor and viral Ags expressed in epithelial cells.

Asunto(s)

Rechazo de Injerto/prevención & control , Tolerancia Inmunológica , Interferón gamma/biosíntesis , Células T Asesinas Naturales/inmunología , Piel/inmunología , Piel/patología , Animales , Antígenos CD1d/biosíntesis , Antígenos CD1d/genética , Células Cultivadas , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Técnicas de Cocultivo , Rechazo de Injerto/inmunología , Rechazo de Injerto/virología , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Humanos , Hiperplasia , Tolerancia Inmunológica/genética , Interferón gamma/deficiencia , Interferón gamma/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/trasplante , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Piel/metabolismo , Trasplante de Piel/inmunología , Trasplante de Piel/patología , Técnicas de Cultivo de Tejidos

RESUMEN

Sulfate plays an important role in maintaining normal structure and function of tissues, and its content is decreased in certain cancers including lung carcinoma. In this study, we investigated tumor growth in a mouse model of hyposulfatemia (Nas1(-/-)) and compared it to wild-type (Nas1(+/+)) mice. Lung epithelial tumor cells (TC-1 cell line) were injected subcutaneously into male Nas1(-/-) and Nas1(+/+) mice on a mixed 129Sv and C57BL/6 genetic background. Tumor sections were stained with anti-glycosaminoglycan antibodies to assess the distribution of proteoglycans and Gomori's trichrome to detect collagen. After 14 days, tumor weights were markedly increased (by approximately 12-fold) in Nas1(-/-) mice when compared with Nas1(+/+) mice. Histological analyses of tumors revealed increased (by approximately 2.4-fold) vessel content, as well as markedly reduced collagen and immunoreactivity against glycosaminoglycan structural epitopes in the tumors from Nas1(-/-) mice. No significant differences were found for the growth of cultured TC-1 cells supplemented with Nas1(-/-) or Nas1(+/+) serum, as determined by (3)H-thymidine incorporation, implying that the cell culture conditions may not reflect the in vivo situation of enhanced tumor growth. This study has revealed increased tumor growth and an altered extracellular tumor matrix in hyposulfatemic Nas1(-/-) mice. These findings highlight the importance of blood sulfate levels as a possible modulator of tumor growth, and could lead to future cancer studies in humans with altered sulfate homeostasis.

Asunto(s)

Proteínas de Transporte de Catión/fisiología , Neoplasias Experimentales/patología , Sulfatos/metabolismo , Simportadores/fisiología , Animales , Proliferación Celular , Colágeno/análisis , Glicosaminoglicanos/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cotransportador de Sodio-Sulfato

RESUMEN

RNA interference (RNAi) for cancer treatment relies on the ability to directly kill cancer cells via down-regulation of target genes, but issues of delivery and efficacy have limited clinical adoption. Furthermore, current studies using immune-deficient animal models disregard potential interactions with the adaptive immune system. It has previously been observed that certain viral antigens appear to be more rapidly presented to the immune system than normal proteins due to the production of defective ribosomal products by the virus. Given that RNAi could potentially result in the generation of truncated mRNAs, we wondered whether a similar mechanism of immune presentation of a target gene was possible. Here we show that RNAi-cleaved mRNAs can be translated into incomplete protein, and if cleavage was downstream of cytotoxic T cell epitopes, resulted in increased presentation of target protein and the generation of a tumor-protective immune response. We show that mice inoculated with tumor cells treated with such short hairpin RNAs (shRNAs) were protected from subsequent challenge with untreated tumors. However, protection was only found if shRNAs were targeted downstream of the dominant cytotoxic T cell (CTL) epitope. Our work suggests that RNAi can alter immunity to targets and shows that not all tumor cells require direct RNAi exposure for treatment to be effective in vivo, pointing the way to a new class of RNAi-based therapy.

Asunto(s)

Antígenos de Neoplasias/genética , Inmunidad/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , Regulación hacia Arriba/inmunología , Animales , Antígenos de Neoplasias/efectos de los fármacos , Epítopos/genética , Humanos , Hidrólisis , Ratones , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Linfocitos T Citotóxicos/inmunología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The role of thymic versus peripheral epithelium in the regulation of the antigen-specific CD8 T-cell repertoire is still largely unresolved. We generated TCR-beta chain transgenic mice in which an increased frequency of peripheral CD8 T cells recognizes an epitope from a viral oncoprotein (HPV16E7) in the context of H-2D(b) MHC class I. When T cells from these mice developed through the thymus of mice expressing functional E7 protein from a keratin 14 promoter, no major perturbation to transgenic T-cell development in the thymus was observed in these double-transgenic mice. In contrast, peripheral CD8 T-cell responses in the single-transgenic, K14E7 mice, including those unrelated to E7 antigen, are reduced whereas CD4 T-cell responses and antibody production are unchanged in these mice. Peripheral non-responsiveness among CD8 T cells was mediated largely by CD4(+)CD25(+) T cells. This suggested that epithelium expressing HPV16E7 protein induces Treg that specifically down-regulate CD8 T-cell responses in the periphery. This may have important consequences for the treatment of cervical pre-cancers and provides a model for understanding differential suppression of T and B lymphocyte subsets by Treg.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Proteínas Oncogénicas Virales/inmunología , Linfocitos T Reguladores/inmunología , Timo/inmunología , Animales , Epitelio/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Queratina-14/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Linfocitos T Reguladores/metabolismo

RESUMEN

Expansion of high- or low-avidity CD8 T cells in vitro inversely correlates with the concentration of peptide ligand present during culture. In contrast, the selective enrichment of high- or low-avidity T cell populations in vivo using peptide immunisation is not well documented. In our study, a single immunisation with different doses of wild-type peptide or a variant peptide able to stimulate CTL responses cross-reactive with wild-type peptide failed to shift the average avidity of the responding CD8 T cell population specific to either peptide. However, in contrast to homologous prime-boost immunisation, heterologous prime-boost immunisation incorporating high doses of the second immunogen resulted in peptide-specific CD8 T cell populations polarized toward a low average functional avidity. These data suggest that sequential exposure to structurally related viral peptides could impair rather than promote anti-viral immunity by lowering the avidity of the responding CD8 T cell population. This study has implications for improving vaccine strategies against viruses and tumours and enhances our understanding of heterologous immunity during sequential viral infection.

Asunto(s)

Citotoxicidad Inmunológica , Inmunización Secundaria/métodos , Proteínas E7 de Papillomavirus/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Activación de Linfocitos/inmunología , Ratones , Proteínas E7 de Papillomavirus/genética , Péptidos/genética , Péptidos/inmunología