Limited Model Antigen Expression by Transgenic Fungi Induces Disparate Fates during Differentiation of Adoptively Transferred T Cell Receptor Transgenic CD4+ T Cells: Robust Activation and Proliferation with Weak Effector Function during Recall
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Mouse strains.
Fungi and growth conditions.
Engineering yeast expressing the model antigenic epitopes Eα and 2W1S.
Screening and characterization of Tg yeast.
Preparation of Eα-RFP from E. coli.
Generation and use of Y-Ae antibody.
Recombinant VSV-SED- and 2W1S-expressing Listeria monocytogenes.
Vaccinations with soluble Eα-RFP, 2W1S peptide plus LPS, Eα-mCh- and 2W1S-mCh-expressing yeast, and VSV-SED- and 2W1S-expressing L. monocytogenes.
Adoptive transfer of Tg CD4+ T cells and surface staining.
BrdU incorporation.
Intracellular cytokine staining.
Peptide–MHC-II tetramer-based enrichment to detect 2W1S-specific endogenous T cells.
Cytokine protein measurements.
Experimental infection.
Statistical analysis.
RESULTS
Generation and characterization of transgenic B. dermatitidis yeast.
Impact of precursor frequency and vaccine dose on T cell expansion and activation.
Migration of antifungal TEa cells to lung upon recall.
Expansion and activation of endogenous Ag-specific CD4+ T cells by recombinant yeast.
Migration of vaccine-induced 1807 cells into lung upon challenge.
Effect of Ag dose and duration on recall and function of primed TEa cells.
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
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