Phosphorylated peptides are naturally processed and presented by major histocompatibility complex class I molecules in vivo

Abstract

Posttranslational modification of peptide antigens has been shown to alter the ability of T cells to recognize major histocompatibility complex (MHC) class I-restricted peptides. However, the existence and origin of naturally processed phosphorylated peptides presented by MHC class I molecules have not been explored. By using mass spectrometry, significant numbers of naturally processed phosphorylated peptides were detected in association with several human MHC class I molecules. In addition, CD8(+) T cells could be generated that specifically recognized a phosphorylated epitope. Thus, phosphorylated peptides are part of the repertoire of antigens available for recognition by T cells in vivo.

Figure 1

MS/MS spectrum of the HLA-A*0201–associated peptide RLDpSYVRSL. Predicted masses for the ions of type b and y are shown above and below the sequence, respectively. Those observed are underlined. ▵, loss of phosphoric acid from the corresponding ion of type b or y.

Figure 2

Discrimination of phosphorylated peptides by CD8⁺ T lymphocytes. (A) IFN-γ production by phosphopeptide-specific CD8⁺ T cells. CD8⁺ T cells (generated by long-term culture with the indicated peptides as described in Materials and Methods) were stimulated for 5 h with CIR-AAD that had been pulsed with 25 μM of one of the indicated phosphorylated peptides and then assayed for the intracellular accumulation of IFN-γ. Nonspecific IFN-γ accumulation was determined by the stimulation of CD8⁺ T cells with unpulsed CIR-AAD and was <100/10⁵ CD8⁺ T cells. (B) Specificity of GLDpSYVRSL-specific CTLs. CD8⁺ T cells elicited by stimulation with 1 μM GLDpSYVRSL were analyzed for cytolytic activity using ⁵¹Cr-labeled CIR-AAD targets (E/T ratio of 3:1) that had been pulsed with the indicated concentrations of phosphorylated GLDpSYVRSL, RLDpSYVRSL, GLDpYYVRSL, and GLDpTYVRSL, or nonphosphorylated GLDSYVRSL. Specific lysis of unpulsed CIR-AAD targets was <2%.