A single peptide-MHC complex positively selects a diverse and specific CD8 T cell repertoire

Abstract

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

Fig. 1

SCT 3KO primary CTLs are K^b-specific and non-MHC cross-reactive. (A) OVAp/K^b SCT 3KO splenocytes stimulated with B6 splenocytes were cultured with RMA (H2^b) target cells in the absence or presence of mAb. Data represent one experiment of 6. (B) OVAp/K^b SCT 3KO anti-B6 CTL clones were cultured with RMA (K^b+D^b+), RMA.S (TAP^−/−, H2^b) or R8.15 (K^b−D^b+) target cells. Data represent one experiment of 3. (C) K^b-reactive CTLs generated from control K^b−/−D^b+/+ splenocytes and SCT 3KO splenocytes, stimulated with K^b+/+D^b−/− splenocytes, were replated with indicated stimulator cells. Production of intracellular IFN-γ by CD8⁺ T cells was measured by flow cytometry. Data represent one experiment of 3. (D) CTL clones were generated from SCT 3KO mice and K^b−/−D^b+/+ mice stimulated with B6. The CTL clones were cultured with RMA (H2^b), P815 (H2^d), or R1.1 (H2^k) target cells. Data are one representative experiment. Clones were assayed 2 to 4 times.

Fig. 2

Recognition by SCT 3KO mice of K^b endogenous peptides complexed with H2-K^b. (A) The sequences of the 90 K^b-binding peptides are listed based on their ability to stimulate polyclonal T cells from the two strains of SCT 3KO mice. Peptides were assayed at least 3 times. (B) K^b binding was measured using an MHC class I surface induction assay as described (29). Binding is plotted as the concentration of peptide that resulted in a threefold increase in the expression level of K^b on RMA.S cells. The bar indicates the mean peptide concentration. Data represent one experiment of 3.

Fig. 3

SCT-selected cells are highly peptide-specific. Splenocytes from OVAp/K^b SCT 3KO mice were stimulated with 6 different peptides, indicated at the top of each panel, and tested with the stimulating peptide and 18 other peptides as indicated. Data represent one experiment of 3.

Fig. 4

T cell responses to differentially recognized peptides. (A) Primary SCT 3KO CTLs, stimulated with peptides recognized exclusively by OVAp/K^b (SAMVFSAM) or VSV/K^b (YQYSFPEL), were tested for reactivity over a range of peptide concentrations. Data represent one experiment of 3. (B) The same peptides in (A) were used to generate CTLs from OVAp/K^b SCT 3KO, VSVp/K^b SCT 3KO and (OVAp/K^b × VSVp/K^b) F1 SCT 3KO mice and were tested for lysis of peptide-treated RMA.S target cells. Data represent one experiment of 3.