Reverse Genetics Modification of Cytomegalovirus Antigenicity and Immunogenicity by CD8 T-Cell Epitope Deletion and Insertion
Figure 3
Verification of the loss-of-presentation phenotype. (a1) Loss of MHC-I H-2-Ld binding by replacement of the C-terminal anchor residue Leu with Ala in the IE1 peptide. The response of a polyclonal IE1-specific CTLL, representing a broad TCR affinity repertoire, was measured in an IFN-γ-based ELISpot assay performed with Ld-expressing stimulator cells loaded exogenously with the indicated synthetic peptides at graded molar concentrations. Recognition of pMHC complexes by CTL serves as an indirect measure for pMHC formation by peptide binding to the presenting MHC-I molecule ∅, no peptide added. Note that presented IE1-Ala analog is recognized only by high-avidity CTL after high-dose peptide loading. (a2) Loss of endogenous peptide presentation by the mutation L176A. The response of an IE1-CTLL (see above) was measured with Ld-expressing stimulator cells that were infected with the indicated WT, mutant, or revertant viruses (see also Table 1). n.i., not infected as a negative control. Note the selective lack of recognition of stimulator cells infected with mutant virus mCMV-IE1-L176A. (b) Loss of in vivo immunogenicity by the mutation L176A. BALB/c (H-2d haplotype) mice were infected with “wobble” revertant virus or with mutant virus (see Figure 2), and ex vivo isolated memory CD8 T cells were used as effector cells in an IFN-γ-based ELISpot assay performed with Ld-expressing stimulator cells that were loaded exogenously with saturating doses of the indicated synthetic peptides ∅, no peptide added. Note the selective loss of IE1-specific CD8 T-cell priming after infection with mutant virus mCMV-IE1-L176A. See Simon et al. [8] for further explanation and experimental details. Reproduced in modified arrangement from reference [8] with permission by the Journal of Virology (American Society for Microbiology).
