T Regulatory Cells Exhibit Surface Expression of FoxP3 Derived Peptides Presented within Class I MHC

Background:

FOXP3 is a transcription factor of central importance to the inhibitory function of CD4⁺, CD25⁺ FOXP3+ T regulatory cells (Tregs). Tregs function as critical inhibitory immunoregulatory cells and have been reported to be increased in tumor-bearing individuals, inhibiting optimal T effector mediated anti-tumor cytotoxicity. Treg depletion targeting CD25, a surface marker of Tregs as well as activated T cells, has been validated in pre-clinical models as a promising adjunctive therapeutic approach to improving T cell mediated therapy and is currently being explored in clinical trials. However, because CD25 expression is not limited to Tregs, CD25-directed therapies may also result in the undesired depletion of effector T cells. Although FOXP3 expression is more restricted to Tregs as compared to CD25, its intranuclear localization has made it challenging to target therapeutically.  Vaccination of mice against FOXP3 using FOXP3 mRNA-transfected dendritic cells has been explored and can elicit FOXP3 specific CTL mediated cytotoxicity. This work implies that FOXP3 mRNA-transfected dendritic cells process FOXP3 and present FOXP3 derived peptides on the cell surface. Direct expression of FOXP3 derived peptides on the surface of Tregs has however, not been previously demonstrated.

Methods:

We utilized mass spectrometry to investigate if FOXP3 derived peptides presented within MHC class I complex are expressed on the surface of Tregs. CD4⁺, CD25⁺ T cells were isolated from healthy donors using magnetic bead selection to enrich for Tregs. Surface class I MHC was immunoprecipitated using a pan-MHC class I anti- HLA-A, HLA-B and HLA-C antibody and associated peptides were eluted from their MHC complex using acid elution. Peptide and fragment masses were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the spectra of resulting epitopes were matched to known protein sequences by SEQUEST.

Results:

Four unique FOXP3 derived peptides were identified from the primary donor samples enriched for FOXP3⁺ cells as described. Predictive HLA binding algorithms demonstrate that the peptides identified are predicted binders to the specific HLA alleles of the original donors. No FOXP3 derived peptides were identified from the CD4⁺, CD25^- T cell fraction analyzed to date.

Conclusions:

This work directly demonstrates for the first time to our knowledge that, although FOXP3 is an intra-nuclear transcription factor, FOXP3 derived peptides are presented within class I MHC on the surface of Tregs, representing a targetable Treg associated surface protein complex.