Ion between the tetramer and TCR transgenic T cells by measuring cytokine production from transgenic cells cultured in the presence of plate-bound tetramer. In keeping with the Th2 phenotype, IL-4, IL-5, IL-9 and IL-13 but not IFN were detected (Figure 6F). The Th17 polarized T cell lines produced less IL-17 with each successive re-stimulation such that by the fourth re-stimulation cytokine production was more in keeping with that of a Th1 cell line (Figure 7A, B). We hypothesized that this shift in cytokine production would be accompanied by a reduction in tetramer binding avidity and longer TCRVCDR3 length as T cells with a IFN producing phenotype clonally expand. In keeping with this, we observed a reduction in tetramer binding avidity between the first and fourth re-stimulations of polarized Th17 lines such that at the fourth re-stimulation, tetramer binding curves for Th1 and Th17 lines overlapped (Figure 7C, D). TCR repertoire analysis at the first and fourth restimulations confirmed that the change in cytokine production and tetramer binding avidity occurred alongside an increase in average TCRVCDR3 length (Figure 7E). The mean TCRVCDR3 lengths for the first and fourth re-stimulations are 10.95 + 0.22 (SE) (n = 37) and 11.39 + 0.21 (SE) (n = 51), respectively, (P = 0.038). The difference in mean CDR3 length between the first and fourth restimulation shows progression to a TCR repertoire with longer CDR3 LY317615 site regions as cells lose their IL-17 producing phenotype and become more Th1-like in their tetramer binding characteristics and cytokine production. In light of previous observations in TCR transgenics, notably the early studies by Hosken and colleagues [41] using the DO11.10 TCR transgenic line, it might be predicted that the Th2 TCR transgenic-skewed polarization may be overcome by altering peptide dose. Certainly, among the many factors that can skew cytokine bias would be peptide dose itself. In the DO11.10 TCR transgenic studies, it was shown that under otherwise equivalent in vitro primary culture of na e cells, mid-range peptide doses favored the generation of moderate IFN responses, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28250575 while either higher or lower doses favored a switch to development of more Th2-like responses, givingReynolds et al. BMC Biology 2014, 12:32 http://www.biomedcentral.com/1741-7007/12/Page 11 ofAH thymidine incorporation ( cpm)B10,C40,IFN- (pg/ml)6,8,000 6,000 4,000 2,000 0 Day 10 Day 28 Day30,000 20,000 10,000Day 10 DayIFN- (pg/ml)5,000 4,000 3,000 2,000 1,000TCRV Littermate*DCD4+CD69- (n = 48)CDR3 regionGDSALYLC NEMAVFLC SHSGFYLC SHSGFYLC SHSGFYLC SHSGFYLC SHSGFYLC SHSGFYLC SHSGFYLC SQTSVYFC SQTSVYFC SQTSVYFC SQTSVYFC SQTSLYFC SQTSLYFC SQTSLYFC NEMAVFLC DDSATYFC DDSATYFC DDSATYFC EDSAVYLC EDSAVYLC EDSAVYLC EDSAVYLC QDSAVYLC QDSAVYLC EDRGLYLC KDRGLYLC EDSAVYLC CASSLYGNNQAPLF CASSIRVPGQLYF CAWKGLGGAETLYF CAWSLGTGANTLYF CAWSPDWGGSAETLYF CAWSRDSSAETLYF CAWSPGNSNERLFF CAWSLRVANTEVFF CAWSLGINQDTQYF CASGDRDRGHEQYF CASGDDGQGSAETLYF CASAPATTNTGQLYF CASGDRDSQNTLYF CASSGGGENTLYF CASSYGQGSGQLYF CASSDAGRANSDYTF CASSKDTSQNTLYF CASSQTGPDTQYF CASSQDRYNYAEQFF CASSPDRYEQYF CASSPLGSYEQYF CASSWGLGEYEQYF CASSHRDRGTEVFF CASSPLGWYEQYF CASSLDWGQNTLYF CAGSLDGTGYEQYF CGARDGGYAEQFF CASSLTGQDTQYF CASSQDGYEQYF FGEG FGEG FGSG FGAG FGSG FGSG FGHG FGKG FGPG FGPG FGSG FGEG FGAG FGAG FGEG FGSG FGAG FGPG FGPG FGPG FGPG FGPG FGKG FGPG FGAG FGPG FGPG FGPG FGPGCD4+CD69+ (n = 50)CDR3 length12 11 12 12 14 12 12 12 12 12 14 13 12 11 12 13 12 11 13 10 11 12 12 11 12 12 11 1123 10 6 4 4 4.