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<table border="0" width="57%"><tbody><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f1.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f1.gif" alt="mcith_gzl008f1.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 1 </strong></span>Selective recovery of high affinity yeast after density centrifugation. (<strong>A</strong>) Percent of yeast recovered from the APC interface layer following centrifugation through Ficoll-Paque. Yeast cells expressing either the high affinity 2C-m80 scTCR or the non-binding C18-1 scTCR were incubated with T2-K<sup>b</sup> cells that had been either loaded with exogenous SIYR peptide or used without exogenous peptide loading. The number of yeast present before centrifugation, and after the first and second centrifugation, were determined by plating aliquots of cells at each stage. The percent recovery following the first centrifugation (white bars) and second centrifugation (black bars) was taken as: (# of yeast cells present at interface layer after centrifugation)/(# of yeast cells at interface layer before centrifugation) x 100. (<strong>B</strong>) Isolation of high affinity 2C-m80 yeast from excess non-binding yeast. A mixture of yeast comprising a 1 to 1000 ratio of 2C-m80 to C18-1 was incubated with SIYR-loaded T2-K<sup>b</sup> cells and subjected to two sequential rounds of density differential centrifugation. Aliquots of cells collected before centrifugation and after the second centrifugation were cultured and stained with antibodies specific for the V<sub>ß</sub> region of either C18-1 (1B3.3, white bars) or 2C-m80 (F23.2, black bars) and analyzed using flow cytometry. <em>Note:</em> The sums of the positive percentages do not equal 100 due to the negative population that is invariably observed in flow cytometric analysis in the yeast display system. See Figure 2.<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f2.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f2.gif" alt="mcith_gzl008f2.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 2 </strong></span>Enrichment of rare high affinity yeast. Yeast expressing the high affinity 2C-m6 were mixed with non-binding yeast at a ratio of (<strong>A</strong>) 1 to 10 000 or (<strong>B</strong>) 1 to 100 000. The mixtures were incubated with QL9-loaded T2-L<sup>d</sup> cells and subjected to two sequential rounds of density centrifugation. Aliquots of cells collected before centrifugation, after the first centrifugation and after the second centrifugation were cultured and stained with antibodies specific for the V<sub>ß</sub> region of either non-binding scTCR C18-1 (1B3.3, grey) or high affinity scTCR 2C-m6 (F23.2, black outline). <em>Note:</em> The negative population in each histogram is invariably observed in the yeast display system and serves as an internal control.<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f3.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f3.gif" alt="mcith_gzl008f3.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 3 </strong></span>Selection analyzed across a range of affinities. (<strong>A</strong>) T2-L<sup>d</sup> cells were loaded exogenously with the peptide variants R5, H5, and M5. Peptide-loaded APCs were then incubated with a yeast cell mixture comprising a 1 to 1000 ratio of 2C-m6 to C18-1. Cells were then subjected to two sequential rounds of density centrifugation. Aliquots of cells before centrifugation and after the second centrifugation were cultured and stained with antibodies specific for the V<sub>ß</sub> region of either C18-1 (KT-8C1) or 2C-m80 (F23.2) and analyzed using flow cytometry. The ratios of yeast cells staining positive for F23.2 (binder) to yeast cells staining positive for KT-8C1 (nonbinder) are shown before and after centrifugation (<em>K</em><sub>D</sub> value for each 2C-m6/pep/L<sup>d</sup> interaction is shown above bars). (<strong>B</strong>) R5-loaded APCs. Flow cytometry histogram overlays showing C18-1-positive yeast (grey) and 2C-m6-positive yeast (black outline) before and after centrifugation and following a second complete cycle of growth, induction, incubation with R5-loaded T2-L<sup>d</sup> and centrifugation.<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f4.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f4.gif" alt="mcith_gzl008f4.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 4 </strong></span>Analysis of unique TCR mutants by density differential centrifugation. (<strong>A</strong>) QL9-loaded T2-L<sup>d</sup> cells were incubated with yeast expressing mutant scTCRs 2C-T7, 2C-m6 or 2C-mQ-1 through 2C-mQ-5 and (<strong>B</strong>) Y5-loaded T2-L<sup>d</sup> cells were incubated with yeast expressing mutant scTCRs 2C-mY-1 through 2C-mY-5. Cell mixtures were then subjected to one round of density differential centrifugation. The number of yeast present before and after centrifugation was determined by plating aliquots of cells at each stage. The percentages of yeast recovered from the interface, calculated as described for Figure 1, are shown as an average of three trials. The recovery percentages of 2C-m6 (high affinity) and 2C-T7 (wild-type affinity) when incubated with QL9-loaded T2-L<sup>d</sup> cells and subjected to density differential centrifugation from (A) are shown in (B) for comparison.<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f5.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f5.gif" alt="mcith_gzl008f5.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 5 </strong></span>Analysis of unique TCR mutants using flow cytometry. (<strong>A</strong>) Histograms of yeast cells expressing mutant scTCRs 2C-T7, 2C-m6, and 2C-mQ-1 through 2C-mQ-5 stained with either anti-V<sub>ß</sub>8.2 (F23.2) (left column) or QL9/L<sup>d</sup>-Ig pMHC dimer (right column) and PE-conjugated goat F(ab')2 anti-mouse Ig. (<strong>B</strong>) Histograms of yeast expressing 2C-T7, 2C-m6, and 2C-mY-1 through 2C-mY-5 stained with either anti-V<sub>ß</sub>8.2 (F23.2) (left column) or Y5/L<sup>d</sup>-Ig pMHC dimer (right column) and PE-conjugated goat F(ab')2 anti-mouse Ig. Mean fluorescence units for each histogram are indicated.<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f6.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f6.gif" alt="mcith_gzl008f6.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 6 </strong></span>Sequences of TCR mutants. The amino acid sequences of the CDR3<sub><img src="http://peds.oxfordjournals.org/math/alpha.gif" alt="{alpha}"></sub> of mutants 2C-mQ-1 through 2C-mQ-5 and 2C-mY-1 through 2C-mY-5 are shown. The CDR3<sub><img src="http://peds.oxfordjournals.org/math/alpha.gif" alt="{alpha}"></sub> amino acid sequences of previously isolated 2C mutants 2C-m6 and 2C-m12, as well as wild-type 2C CDR3<sub><img src="http://peds.oxfordjournals.org/math/alpha.gif" alt="{alpha}"></sub>, are shown for comparison. 2C-m12* was selected previously using QL9 peptide.<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr><tr style="border-top:1pt dotted #C0C0C0;border-bottom:1pt dotted #C0C0C0;padding-bottom:.1in;padding-top:.1in;"><td valign="top" style="padding-top:.1in;padding-bottom:.1in;"> <a href="http://www.biology-online.org/js/tiny_mce/plugins/imagemanager/files/boa002/gzl008f7.gif"><img src="/js/tiny_mce/plugins/imagemanager/files/boa002/mcith/mcith_gzl008f7.gif" alt="mcith_gzl008f7.gif"></a> </td> <td valign="top" style="padding-left:.1in;padding-top:.1in;padding-bottom:.1in;"><span style="font-family:Arial;"><strong> Figure 7 </strong></span>Enrichment of a high affinity MHC class II-restricted scTCR mutant. Yeast expressing the high affinity mutant scTCR 3.L2-M15 were mixed at a 1 to 1000 ratio of (<strong>A</strong> and <strong>B</strong>) non-binding mWT1-B7 yeast or (<strong>C</strong>) non-binding 2C-T7 yeast. The yeast mixtures were incubated with Hb peptide-loaded CH27 APCs. Cells were then subjected to one round of density differential centrifugation. Aliquots of cells that were collected before and after centrifugation were cultured and stained with antibodies specific for the V<sub>ß</sub> region of either the non-binding scTCRs mWT1-B7 (RR3-15-PE) or 2C-T7 (F23.2) or the high affinity scTCR 3.L2-M15 (1B3.3-PE in A and B, KT-8C1 in C). Stained yeast cells were analyzed using flow cytometry. (A) Histograms of yeast before and after centrifugation stained with antibody specific for V<sub>ß</sub> region of the high affinity 3.L2-M15 (1B3.3-PE, top row) or mWT1-B7 (RR3-15-PE, bottom row). Percent of cells staining positive in each histogram are shown in the upper right-hand corner. (B and C) Percent of yeast before and after centrifugation staining positive for non-binding scTCR (white bars) or high affinity scTCR (black bars) (average of three trials in B or two trials in C).<span style="font-family:Arial;"> <br></span><p> (Click image to enlarge) </p> </td> </tr></tbody></table><p> </p>
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