Project description:The immune system plays a key role in the protective response against oral cancer, however the tumour microenvironment (TME) is able to impair this anti-cancer response by modulating T helper (Th) responses and promoting an anti-inflammatory environment. Regulatory T cells (Tregs) and Th2 effector cells (Teff) have been associated with bad prognosis in oral squamous cell carcinoma (OSCC), however it is unknown the main chemoattractant or immunomodulatory mechanisms associated with the enrichment of these subsets in OSCC. We characterised T helper-like lineages in Teff and Tregs and evaluated the main immunomodulatory changes induced by the TME in OSCC. Our phenotypic data revealed a higher distribution of tumour-infiltrating CCR8+ and Th2-like Treg in OSCC in comparison with non-malignant samples, whereas the percentages of Th1 cells were reduced in cancer. We then analysed the presence of CCR8 ligands and the chemoattractant effect of tumour secretomes, however despite observing higher presence of CCR8 ligand CCL18, we only observed reduced migration of Teff to tumour secretomes. The direct effect of the TME was then analysed by exposing T cell subsets to cancer secretomes and we observed that the TME induced higher expression of CCR8 and immunomodulatory molecules on both subsets. Finally, the proteomic analysis of the TME suggests that these changes were associated with the rapid membrane-associated vitamin D signalling pathway. In summary, the TME from OSCC promotes immunomodulatory changes by regulating CCR8 expression and disbalancing the Th1/Th2 repertoire.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:Naive CD4+ T cells, sorted from PBMCs of grass-fed beef cattle, were stimulated with anti-bovine CD3 under Th2 differentiation conditions with or without Ostertagia ostertagi (OO) protein extract. Th1 differentiation conditions were included for comparison. The effect of recombinant bovine IL-4 (rbIL-4) and weakening TCR signal strength on Th2 differentiation were also tested. Flow cytometry, qPCR and proteomic assay were performed to analyze the differentiated cells.
The majority of differentiated cells expressed IFNgamma (a hallmark cytokine for Th1) and a small percentage of cells expressed both IFNgamma and IL-4 (a hallmark cytokine for Th2), indicating the presence of Th0 cells, as previously reported in cattle. While weakening TCR signal strength reduced Th2 cell expansion, adding rbIL-4 or OO protein extract enhanced Th2 cell expansion but without significant changes in IFNgamma and IL-4 expression. Proteomic data predicted that, as in mice and humans, bovine Th2 differentiation results from three upstream stimulation with CD3, CD28, and IL-4, which were inhibited when OO protein extract was added into the Th2 differentiation media. Importantly, protein profiling indicated that the addition of rbIL-4 enhanced IL-4 signaling but inhibited IL-12 signaling. Soon, we are planning to explore if other cytokines like IL-10 can be applied to optimize this Th2 differentiation in vitro.
Naive bovine CD4+ T cells differentiate into a mixed population containing a high percentage of IFNgamma producing cells and a small percentage of Th0 cells. Furthermore, bovine Th2 differentiation is sensitive to regulation such as by OO or rbIL-4.