Volume 7, Issue 2, Pages 221-231 (August 1997) Specific Recognition of Thymic Self-Peptides Induces the Positive Selection of Cytotoxic T Lymphocytes  Qinghui Hu, Carthene R Bazemore Walker, Cristina Girao, Joseph T Opferman, Jiling Sun, Jeffery Shabanowitz, Donald F Hunt, Philip G Ashton-Rickardt  Immunity  Volume 7, Issue 2, Pages 221-231 (August 1997) DOI: 10.1016/S1074-7613(00)80525-7

Figure 1 Chromatography of Peptides Eluted from H-2Db Molecules of 427.1 Cells Peptides from anti-H-2Db affinity columns (H-2Db) or mock extracts (H-2Dd) were resolved by C18 narrow-bore RP-HPLC. Peptides in RP-HPLC fractions from peaks unique to H-2Db chromatographs (arrows; asterisk, pooled fractions 37 and 38) were further analyzed. Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)

Figure 2 Purification of H-2Db–Associated Self-Peptides from 427.1 Cells (A and B) Plots of total ion current obtained in each mass spectrum recorded on 1/25 of the material in the pooled RP-HPLC fractions 37 and 38 from anti-H-2Db (A) and anti-H-2Dd mock extracts (B). Mass spectra were recorded on a triple-quadrupole mass spectrometer equipped with an electrospray ion source and a C-18 microcapillary-HPLC column. The mass range, m/z 300–1400, was scanned repetitively every 1.5 sec, and the total ion signal in each spectrum was summed. Insets are plots of the ion current for the peptide (M+2H)2+ at m/z 541 (nominal mass). (C) Collision-activated dissociation mass spectrum of peptide (M+2H)2+ ions at m/z 541. Predicted masses for fragment ions of types b and y are shown above and below the deduced amino acid sequence (Hunt et al. 1986). Ions observed in the spectrum are underlined. Subtraction of m/z values for any two fragments that differ by a single amino acid generates a value that specifies the mass and thus the identity of the extra residue in the larger fragment. Since isoleucine and leucine are of identical mass, they cannot be differentiated on the triple-quadrupole instrument and are specified as Lxx. O indicates ions that represent single losses of water. Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)

Figure 2 Purification of H-2Db–Associated Self-Peptides from 427.1 Cells (A and B) Plots of total ion current obtained in each mass spectrum recorded on 1/25 of the material in the pooled RP-HPLC fractions 37 and 38 from anti-H-2Db (A) and anti-H-2Dd mock extracts (B). Mass spectra were recorded on a triple-quadrupole mass spectrometer equipped with an electrospray ion source and a C-18 microcapillary-HPLC column. The mass range, m/z 300–1400, was scanned repetitively every 1.5 sec, and the total ion signal in each spectrum was summed. Insets are plots of the ion current for the peptide (M+2H)2+ at m/z 541 (nominal mass). (C) Collision-activated dissociation mass spectrum of peptide (M+2H)2+ ions at m/z 541. Predicted masses for fragment ions of types b and y are shown above and below the deduced amino acid sequence (Hunt et al. 1986). Ions observed in the spectrum are underlined. Subtraction of m/z values for any two fragments that differ by a single amino acid generates a value that specifies the mass and thus the identity of the extra residue in the larger fragment. Since isoleucine and leucine are of identical mass, they cannot be differentiated on the triple-quadrupole instrument and are specified as Lxx. O indicates ions that represent single losses of water. Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)

Figure 3 Some but Not All Self-Peptides Induce the Positive Selection of CD8+ Cells The ability of thymic self-peptides (HH, BP, and RR) to restore the positive selection of CD8+ cells was tested in TAP1− FTOC. Thymocytes from TAP1+ and TAP1− FTOC were analyzed for the surface expression of CD8, CD4, and pan-TCRβ chain by MAb staining and FACS. Thymocytes that were CD4−CD8+ were gated and analyzed for the surface expression of pan-TCRβ, and then the overall percentage of CD4−CD8+ TCR+hi cells was determined (CD8+ cells). The levels indicated are based on the analysis of three to five separately cultured lobes (mean ± SEM). Addition of either HH or BP (300 μM) did not result in significant changes in the number of CD8+ cells (p > 5%) but the addition of RR (300 μM) did (1% > p > 0.5%). Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)

Figure 4 Addition of Self-Peptides to TAP1− FTOC Restores the Positive Selection of Transgenic CD8+ Thymocytes (A) The positive selection of CD8+ F5 cells is impaired in fetal thymi from F5 TAP1− mice. Thymocytes from F5 TAP1+ and F5 TAP1− FTOC were analyzed for the surface expression of CD8, CD4 and TCR Vβ11 by MAb staining and FACS. Thymocytes that were CD4−CD8+ were gated and analyzed for the surface expression of TCR Vβ11, and then the overall percentage of CD4− CD8+ TCR Vβ11+hi (CD8+ F5 cells) was determined. The percentage of CD8+ F5 cells was reduced in F5 TAP1− (1%) compared to F5 TAP1+ (21%) FTOC. The percentage of CD8+ F5 cells could be partially restored by the addition of HH (3%) and BP (6%) peptides at 300 μM to F5 TAP1− FTOC. Similar results were obtained in four other separately performed experiments. (B) The positive selection of CD8+ P14 cells is impaired in fetal thymi from P14 TAP1− mice. Thymocytes from P14 TAP1+ and P14 TAP1− FTOC were analyzed for the surface expression of CD8, CD4, TCR Vα2, and TCR Vβ8.1 by MAb staining and FACS. Thymocytes that were CD4−CD8+ were gated and analyzed for the surface expression of TCR Vα2 and TCR Vβ8.1, and then the overall percentage of CD4−CD8+ TCR Vα2+hi TCR Vβ8.1+hi (CD8+ P14 cells) was determined. The percentage of CD8+ P14 cells was reduced in P14 TAP1− (1%) compared to P14 TAP1+(15%) FTOC and could be partially restored by the addition of the RR peptide (3%) at 300 μM. Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)

Figure 5 Differential Ability of Self-Peptides to Induce the Positive Selection of Transgenic CD8+ Thymocytes (A) The ability of thymic self-peptides (HH, BP, and RR) to restore the positive selection of CD8+ F5 cells was tested in F5 TAP1− FTOC, when added at 300 μM. The peptides BP (5% > p > 1%) and HH (5% > p > 1%) but not RR (p > 5%) were capable of generating CD8+ F5 cells above the level of F5 TAP1− FTOC incubated alone. There was no significant difference between the abilities of HH and BP to induce the positive selection of CD8+ F5 cells (p > 5%). (B) The ability of thymic self-peptides to restore the positive selection of CD8+ P14 cells was tested in P14 TAP1− FTOC when added at 300 μM. RR gave rise to numbers of CD8+ P14 cells above the level of P14 TAP1− FTOC incubated alone (1% > p > 0.5%), but HH (p > 5%) and BP (p > 5%) did not. The data represent the mean ± SEM of values obtained from four to six independently cultured thymi. Similar results were obtained in six other experiments. Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)

Figure 6 Summary of Self-Peptide Selection Experiments (A) The ability of thymic self-peptides (HH, BP, and RR) to restore the positive selection of CD8+ F5 cells was tested in F5 TAP1− FTOC, when added at 300 μM. Each point represents the mean number of CD8+ F5 cells recovered from thymic lobes from five separate experiments (four to six independent lobes per experiment, as in Figure 4 and Figure 5). The mean value for cell number is indicated with the SEM. The peptides BP (p < 0.1%) and HH (0.2% > p > 0.1%) but not RR (p > 5%) were capable of generating CD8+ F5 cells above the level of F5 TAP1− FTOC incubated alone. There was no significant difference between the abilities of HH and BP to induce the positive selection of CD8+ F5 cells (p > 5%). The number of CD8+ F5 cells recovered from F5 TAP1+ FTOC ranged from 16 to 59 × 103 cells per lobe (six experiments). (B) The ability of thymic self-peptides to restore the positive selection of CD8+ P14 cells was tested in P14 TAP1− FTOC, when added at 300 μM, over the course of seven separate experiments (as in A). RR gave rise to numbers of CD8+ P14 cells above the level of P14 TAP1− FTOC incubated alone (1% > p > 0.5%), but HH (p > 5%) and BP (p > 5%) did not. The number of CD8+ P14 cells recovered from P14 TAP1+ FTOC ranged from 17 to 60 × 103 cells per lobe (seven experiments). Immunity 1997 7, 221-231DOI: (10.1016/S1074-7613(00)80525-7)