Characterizing Complex Polysera Produced by Antigen-Specific Immunization through the Use of Affinity-Selected Mimotopes

BACKGROUND: Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines

Elevated frequencies of self-reactive CD8+ T cells following immunization with a xenoantigen are due to the presence of a heteroclitic CD4+ T-cell helper epitope

Immunization of mice with human dopachrome tautomerase (hDCT) provides greater protection against melanoma than immunization with the murine homologue (mDCT). We mapped the CD8(+) and CD4(+) T-cell epitopes in both proteins to better understand the mechanisms of the enhanced protection. The dominant CD8(+) T-cell epitopes were fully conserved between both proteins, yet immunization with hDCT produced frequencies of CD8(+) T cells that were 5- to 10-fold higher than immunization with mDCT. This difference was not intrinsic to the two proteins because comparable frequencies of CD8(+) T cells were elicited by both antigens in DCT-deficient mice. Strikingly, only hDCT elicited a significant level of specific CD4(+) T cells in wild-type (WT) mice. The murine protein was not devoid of CD4(+) T-cell epitopes because immunization of DCT-deficient mice with mDCT resulted in robust CD4(+) T-cell immunity directed against two epitopes that were not identified in WT mice. These results suggested that the reduced immunogenicity of mDCT in WT mice may be a function of insufficient CD4(+) T-cell help. To address this possibility, the dominant CD4(+) T-cell epitope from hDCT was introduced into mDCT. Immunization with the mutated mDCT evoked CD8(+) T-cell frequencies and protective immunity comparable with hDCT. These results reveal a novel mechanism by which xenoantigens overcome tolerance. Our data also suggest that immunologic tolerance is more stringent for CD4(+) T cells than CD8(+) T cells, providing a mechanism of peripheral tolerance where autoreactive CD8(+) T cells fail to be activated due to a lack of autoreactive CD4(+) T cells specific for the same antigen

FOXN1GFP/w Reporter hESCs Enable Identification of Integrin-β4, HLA-DR, and EpCAM as Markers of Human PSC-Derived FOXN1+ Thymic Epithelial Progenitors

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1GFP/w line as a readout, we developed a reproducible protocol for generating FOXN1-GFP+ thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-β4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1+ TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1+ TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine