CD8 T-cell ability to exert immunodomination correlates with T-cell receptor: Epitope association rate Guillaume Roy-Proulx, Chantal Baron, Claude Perreault

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CD8 T-cell ability to exert immunodomination correlates with T-cell receptor: Epitope association rate Guillaume Roy-Proulx, Chantal Baron, Claude Perreault Biology of Blood and Marrow Transplantation Volume 11, Issue 4, Pages 260-271 (April 2005) DOI: 10.1016/j.bbmt.2004.12.334 Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Anti-H7a and anti-HY CD8 T-cell precursors exhibit similar frequencies and CD5 expression levels in naive B10.H7b mice. A, Procedure for the analysis of lymphocytes labeled by H7a or HY tetramers. Autofluorescent cells and those that bind an irrelevant tetramer are gated out. B, One hundred eighty-three microcultures were set up with splenocyte suspension adjusted to 5 × 104 CD8 T cells per well. These cells were stimulated with HY or H7a as described in Materials and Methods. Plots show tetramer labeling of CD8 T cells in the positive anti-H7a (top row) and anti-HY (bottom row) wells. C, Gating procedure used to separate CD5lo, CD5int, and CD5hi CD8 T-cell subsets. Cells from these 3 subsets were plated in different wells. D, Frequency of positive microcultures obtained from CD5lo, CD5int, and CD5hi CD8 T cells. Culture conditions and cell gating procedures were similar to those used in the estimation of precursor frequency (A and B). Biology of Blood and Marrow Transplantation 2005 11, 260-271DOI: (10.1016/j.bbmt.2004.12.334) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 H7a (KAPDNRETL) and its allelic peptide H7b (KAPDNRDTL) are not cross-reactive at the TCR level. Splenocytes from B10.H7b mice primed against B10 cells (anti-H7a) and from B10 mice primed against B10.H7b (anti-H7b) were stained with H7a and H7b tetramers. Biology of Blood and Marrow Transplantation 2005 11, 260-271DOI: (10.1016/j.bbmt.2004.12.334) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 H7a-tetramer+ CD8 T cells use mainly TCR Vα8+ and Vβ8+ chains. Splenocytes from B10.H7b mice primed with B10 splenocytes were stained on day 14 with antibodies against various TCR Vα and Vβ chains. Nonimmune B10.H7b mice were used as controls. Results from nonimmune B10.H7b control mice (n = 5) are presented as the mean + SD. Results from mice primed against H7a are presented individually for the 8 mice tested. Biology of Blood and Marrow Transplantation 2005 11, 260-271DOI: (10.1016/j.bbmt.2004.12.334) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 TCR:pepMHC dissociation and association rates for (■) H7a- and (□) HY-specific CD8 T cells harvested at the time of primary response (day 14 after antigen priming). A, CD8 T cells labeled with H7a and HY tetramers were stained with antibody against CD8 and TCRαβ. B, One representative example of an H7a-tetramer decay plot series. C, Decay plot of the natural logarithm of the normalized total fluorescence versus time (after the addition of anti-Db monoclonal antibody) for primed splenocytes stained with H7a or HY tetramers. The rate of tetramer decay was similar for cells labeled with H7a and HY tetramers (P = .427). D, To evaluate the TCR:pepMHC association rate, day 14 splenocytes were incubated with graded concentrations of the relevant tetramer. C and D, Each point represents the mean of 5 to 10 mice tested. Biology of Blood and Marrow Transplantation 2005 11, 260-271DOI: (10.1016/j.bbmt.2004.12.334) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Kinetics of CD8 T-cell responses against H7a and HY. B10.H7b female mice were immunized with B10 or B10.H7b male cells to evaluate the kinetics of CD8 T-cell responses against H7a and HY, respectively. The number of tetramer+ CD8 T cells in the spleen was measured on day 5, 10, 15, 20, 25, and 30. The maximum response was defined as 100%, and other responses were plotted as a fraction of that maximum response. There were 4 to 7 mice per time point. *P < .05; Student t test. Biology of Blood and Marrow Transplantation 2005 11, 260-271DOI: (10.1016/j.bbmt.2004.12.334) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions