CD4⁺ T Cells Mediate Aspergillosis Vaccine Protection

Adaptive effector CD4⁺ T cells play essential roles in the defense against fungal infections, especially against invasive aspergillosis (IA). Such protective CD4⁺ T cells can be generated through immunization with specialized antifungal vaccines, as has been demonstrated for pulmonary Aspergillus fumigatus infections in mouse experiments. Adaptive transfer of fungal antigen-specific CD4⁺ T cells conferred protection onto non-immunized naive mice, an experimental approach that could potentially become a future treatment option for immunosuppressed IA patients, focusing on the ultimate goal to improve their otherwise dim chances for survival. Here, we describe the different techniques to analyze CD4⁺ T cell immune responses after immunization with a recombinant fungal protein. We present three major methods that are used to analyze the role of CD4⁺ T cells in protection against A. fumigatus challenge. They include (1) transplantation of CD4⁺ T cells from vaccinated mice into immunosuppressed naive mice, observing increasing protection of the cell recipients, (2) depletion of CD4⁺ T cells from vaccinated mice, which abolishes vaccine protection, and (3) T cell proliferation studies following stimulation with overlapping synthetic peptides or an intact protein vaccine. The latter can be used to validate immunization status and to identify protective T cell epitopes in vaccine antigens. In the methods detailed here, we used versions of the well-studied Asp f3 protein expressed in a bacterial host, either as the intact full length protein or its N-terminally truncated version, comprised of residues 15-168. However, these methods are generally applicable and can well be adapted to study other protein-based subunit vaccines.