RESUMEN
Low-avidity autoreactive CD8 T cells (CTLs) escape from thymic negative selection, and peripheral tolerance mechanisms are essential for their regulation. We report the role of proinsulin (PI) expression on the development and activation of insulin-specific CTLs in the NOD mouse model of type 1 diabetes. We studied insulin B-chain-specific CTL from different T-cell receptor transgenic mice (G9Cα-/-) expressing normal PI1 and PI2 or altered PI expression levels. In the absence of PI2 (Ins2-/-), CTL in pancreatic lymph nodes (PLNs) were more activated, and male G9Cα-/- mice developed T1D. Furthermore, when the insulin-specific CTLs developed in transgenic mice lacking their specific PI epitope, the CTLs demonstrated increased cytotoxicity and proliferation in vitro and in vivo in the PLNs after adoptive transfer into NOD recipients. Dendritic cell-stimulated proliferation of insulin-specific T cells was reduced in the presence of lymph node stromal cells (LNSCs) from NOD mice but not from mice lacking the PI epitope. Our study shows that LNSCs regulate CTL activation and suggests that exposure to PI in the periphery is very important in maintenance of tolerance of autoreactive T cells. This is relevant for human type 1 diabetes and has implications for the use of antigen-specific therapy in tolerance induction.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Proinsulina/metabolismo , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Diabetes Mellitus Tipo 1/inmunología , Epítopos/genética , Femenino , Citometría de Flujo , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones TransgénicosRESUMEN
Murine adoptive CD8+ T-cell immunotherapy studies require the generation of large numbers of high viability CD8+ cells. Here we report a tissue culture protocol for the reliable expansion of CD8+ T-cells derived from murine spleen to give a 20-fold expansion after 4 days in culture. The cells were transfected with an mRNA GFP construct and transferred into NOD mice. GFP positive cells could be detected 7 days after transfer thus confirming that the cells survive and are functional for up to 1 week.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/trasplante , Técnicas de Cultivo de Célula/métodos , Inmunoterapia Adoptiva/métodos , Animales , Recuento de Células , Células Cultivadas , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NODRESUMEN
Assessments of synaptic density in human brain are often based on measurements of synaptic proteins. Little information is available on their post-mortem stability. We have investigated this by ELISAs of the pre-synaptic proteins syntaxin and synaptophysin, and the post-synaptic protein PSD-95, in rat and human cortex. The rat brains were cooled in situ from 37 to 20 or 4 degrees C over 3 h, and then kept at 20 or 4 degrees C for a further 24-72 h, to simulate post-mortem storage at room temperature or in a mortuary refrigerator. Synaptophysin and PSD-95 levels in rat cerebral cortex were not significantly decreased after 72 h of incubation at 20 degrees C. Syntaxin was stable for 24 h but decreased by 39-44% at 48-72 h. Storage at 4 degrees C resulted in a similar reduction of syntaxin levels over 72 h. In human brain tissue from 160 people aged 24-102 years, post-mortem delay had little effect on synaptic protein levels in superior temporal cortex, but was associated with a decline in PSD-95 and syntaxin in mid-frontal cortex after 24 h. The more robust stability of synaptophysin may be related to its multi-transmembrane structure.
Asunto(s)
Corteza Cerebral/química , Proteínas de la Membrana/análisis , Proteínas del Tejido Nervioso/análisis , Cambios Post Mortem , Sinapsis/química , Sinaptofisina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Homólogo 4 de la Proteína Discs Large , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Proteínas Qa-SNARE , Ratas , Sinapsis/metabolismo , Sinaptofisina/metabolismo , Factores de TiempoRESUMEN
OBJECTIVE: We have previously reported a highly diabetogenic CD8 T-cell clone, G9C8, in the nonobese diabetic (NOD) mouse, specific to low-avidity insulin peptide B15-23, and cells responsive to this antigen are among the earliest islet infiltrates. We aimed to study the selection, activation, and development of the diabetogenic capacity of these insulin-reactive T-cells. RESEARCH DESIGN AND METHODS: We generated a T-cell receptor (TCR) transgenic mouse expressing the cloned TCR Valpha18/Vbeta6 receptor of the G9C8 insulin-reactive CD8 T-cell clone. The mice were crossed to TCRCalpha-/- mice so that the majority of the T-cells expressed the clonotypic TCR, and the phenotype and function of the cells was investigated. RESULTS: There was good selection of CD8 T-cells with a predominance of CD8 single-positive thymocytes, in spite of thymic insulin expression. Peripheral lymph node T-cells had a naïve phenotype (CD44lo, CD62Lhi) and proliferated to insulin B15-23 peptide and to insulin. These cells produced interferon-gamma and tumor necrosis factor-alpha in response to insulin peptide and were cytotoxic to insulin peptide-coated targets. In vivo, the TCR transgenic mice developed insulitis but not spontaneous diabetes. However, the mice developed diabetes on immunization, and the activated transgenic T-cells were able to transfer diabetes to immunodeficient NOD.scid mice. CONCLUSIONS: Autoimmune CD8 T-cells responding to a low-affinity insulin B-chain peptide escape from thymic negative selection and require activation in vivo to cause diabetes.