Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
The Journal of Immunology, 2010 GILT Accelerates Autoimmunity to the Melanoma Antigen Tyrosinase-Related Protein 1 Xu Zhenjie 2011,08,31 1. Background GILT ( Gamma-IFN inducible lysosomal thiol reductase) is expressed in APCs, where it localizes to MHC class II-loading compartments. GILT can facilitates the generation of MHC class II-restricted epitopes from disulfide bond containing Ags. Melanocyte differentiation Ags are melanosomal integral membrane proteins involved in melanin pigment synthesis. These Ags contain a dileucine-based sorting signal that targets them to the endosomal system where they can be processed for MHC class II-restricted presentation. However, it is not known how GILT would influence the development of immune responses to these Ags in vivo. 2. Materials 95-10: an I-Ab-restricted T cell hybridoma recognizing murine TRP1 Cell lines B16.F10: Mel (Melanoma line) PVD: SCC (squamous cell carcinoma) line C57BL/6 : wild-type mice GILT-/- mice mice TRP1tg mice TRP1Bw RAG-/- TRP1tg mice 3. Methods and results GILT is required for efficient MHC class II-restricted processing of a TRP1 epitope in vitro. GILT accelerates the onset of vitiligo in TRP1-specific TCR transgenic mice. Increased percentage of TRP1-specific transgenic T cells in GILTdeficient mice. Effector memory cells are increased in the presence of GILT and following development of vitiligo. GILT facilitated class II-restricted processing of TRP1, thereby enhancing T cell activation and accelerating vitiligo. 3.1. GILT is required for efficient MHC class II-restricted processing of TRP1 B16 cells Wild-type mice freeze-thaw splenic B cells Stained by CD45R and I-Ab lysates Immunoblotted with anti-TRP1 mAb control GILT-/- mice Flow cytometry Coculture with B16 cells lysates and 95-10 cells Measuring IL-2 production by ELISA Positive control, TRP1 peptide Negative control, SCC lysates There was a statistically significant difference between the ability of wild-type and GILT-/- B cells to present the TRP1 epitope. Wild-type mice GILT-/- mice DCs Flow cytometry Stained by CD11c and I-Ab Coculture with B16 cells lysates and 95-10 cells Measuring IL-2 production by ELISA control There was a statistically significant difference between the ability of wild-type and GILT-/- DCs to present the TRP1 epitope. 3. 2. GILT accelerates the onset of vitiligo mediated by TRP1-specific CD4+ T cells 3.2.1 To determine whether GILT would impact the development of a TRP1-specific autoimmune response in vivo, 3.2.2 To demonstrate that GILT in APCs is necessary for the accelerated onset of vitiligo, Wild type (n=127) TRP1tg mice GILT+/- (n = 56) GILT-/- (n =76) A A Transfer CD4+ into TRP1specific T cells RAG-/- mice GILT-/- RAG-/mice GILT in APCs increases the severity and accelerates the onset of vitiligo. 3. 3. Increased percentage of TRP1-specific transgenic T cells in GILTdeficient mice CD4+ TRP1specific T cells Transfer into Total cell numbers isolated from thymi, spleens, and lymph nodes of the GILTpositive and -negative strains were identical. CD4:CD8 T cell ratio were observed in the thymus, spleen, or lymph node. RAG-/- mice GILT-/- RAG-/- mice Transgenic T cells were identified by Vβ14 and CD4 staining. The percentage of transgenic T cells in GILT-/-TRP1tg mice increased. To exclude the absence of GILT in T cells as the cause of increased T cells, we evaluated the percentage of nontransgenic T cells. ? TCR was expressed in both GILT-/-TRP1tg mice and TRP1tg mice. The influence of T regulatory (Treg) cell was exclude. The expansion of T cells was limited to the TRP1-specific CD4+Vb14+ T cells. 3.4 Effector memory cells are increased in the presence of GILT and following development of vitiligo. GILT-/-TRP1tg mice and TRP1tg mice CD4+Vβ14+ T cells CD62L-CD44+ (effector memory) CD62L+CD44+ (central memory) CD62L+CD44- (naive) GILT facilitated class II-restricted processing of TRP1, thereby enhancing T cell activation and accelerating vitiligo.