1. Eric S. Huseby et al, Cell (2005); Nature Immunol. (2007), compared the T cells of normal mice, with mice genetically engineered to present only one type of peptide in their thymus. T cells selected in the thymus are challenged with an antigenic peptide, and reactive T cells identified. Does a reactive T cell remain reactive upon mutating the peptide’s amino acids? If mutations to an amino acid destroy reactivity with at least half the T cells, the amino-acid is labeled a “hot spot”. Main results: – Single peptide selection: few hot spots – cross-reactive T cells – Many peptide selection: many hot spots – specific T cells Specificity

2. Specificity to antigen peptide: – Single peptide: mutations don’t matter – cross-reactive T-cells – Many peptides: mutations destroy reactivity – specific T-cells

3. † Hot-spots are defined as locations along the sequence, where mutations of a peptide amino acid destroy reactivity with more than half the reactive T cells frequency Numerical results for hot-spots † mirror the experimental situation

4. Frustration during negative selection constrains TCR sequences TCR One peptide E p < E < E N selected

5. Frustration during negative selection constrains TCR sequences TCR One peptide selected

6. Frustration during negative selection constrains TCR sequences TCR One peptide selected E > E N negatively selected Many peptides Optimizing interactions with one peptide can lead to “bad” interactions with another – FRUSTRATION. Positive selection does not involve frustration. TCR