Table 2. Classification of changes in predicted peptide HLA binding affinity due to amino acid changes.

Classification	High affinity binder status	Binding affinity before and after	Description of amino acid change impact
−2	New	>−1σ to ≤−1σ	Large increase in affinity (>1σ change) to surpass threshold and become a new high binder
−1	Retained	≤−1σ to ≤−1σ	Already a high binder, further affinity increase
0	No Change	≤−1σ to ≤−1σ	High affinity, no change
+1	Retained	≤−1σ to ≤−1σ	A high binder, affinity loss but remains above threshold as a high binder
+2	Lost	≤−1σ to > = 0	High affinity initially; large decrease to below high binder threshold

The impact on binding affinity associated with amino acid changes arising in cluster transition was determined. Based on impact of the mutations on predicted binding affinity a classification code was assigned to each peptide for each of the affected MHC molecules. The threshold of −1σ was selected based on the outcome of recursive partitioning (Figure S1). The peptides were then assigned to different one of five different classifications according to whether high affinity binding was gained, lost or retained above the threshold. Only high binders were considered, so some peptides that did not surpass the threshold were not included in the analysis of the particular transition.