An understanding of the factors that influence Ag presentation, priming of Ag-specific T cells, and memory T cell development is essential for the rational design of vaccines. In work described here, we sought to establish a heterologous adoptive transfer system to quantitatively and qualitatively monitor the development of fungus-specific CD4+
T cell memory. To establish a system to study antifungal CD4+
T cell development in vivo
, we performed extensive testing of the variables that influence T cell phenotypes and function. It is now recognized that the input frequency of naïve TCR Tg precursors and the availability of Ag are the most important factors that dictate the fate of T cell development. A high level of initial TCR Tg precursor has frequently led to an intrinsic defect in the T cells and resulted in reduced division, altered kinetics of expansion, reduced cytokine production, and the lack of a detectable memory population (4
). In those studies, T cell competition could be overcome by adding more Ag (7
) or Ag-bearing APCs (36
). Below, we discuss our results obtained using a heterologous, adoptive transfer system to study antifungal CD4+
T cell immunity. We analyzed selected stages of the antifungal T cell response, including activation, proliferation, differentiation, memory, and migration to the lung upon a recall challenge.
We found that Eα-mCh expressed on vaccine yeasts did not impair their ability to confer resistance and that transgene expression on wild-type yeast did not impair their virulence during rechallenge (data not shown). Eα-mCh on yeast was processed and presented on APCs, and the peptide triggered the activation and proliferation of corresponding TEa cells in an Ag-specific manner. TEa cells expanded maximally using an input precursor frequency of 5 × 105 Tg cells and a vaccine dose of 106 to 107 yeast cells. Transfer of ≤5 × 103 TEa precursors did not yield a detectable number of primed TEa cells in the skin-draining lymph nodes at the burst of expansion (at day 7 postvaccination). Fungus-specific TEa cells expanded a maximum of 1,000- to 3,000-fold using a precursor frequency of 5 × 105 naïve cells and less than 1,000-fold using higher and lower precursor frequencies (A). It is conceivable that the expansion of 5 × 104 TEa precursors might have been higher than we calculated since the number of TEa cells in the unvaccinated group was near the detection limit, and therefore, the calculations of the n-fold expansion factor might not have been as precise as in the higher-precursor-frequency groups. Thus, 5 × 104 to 5 × 105 TEa precursors had to be transferred to yield a measurable pool of primed TEa cells at the peak T cell response in the skin-draining lymph nodes.
Antifungal TEa cells primed with Eα-mCh yeast alone failed to efficiently acquire memory and migrate to the lung upon challenge. In contrast, adoptive transfer of comparable numbers (1 × 105
to 5 × 105
) of 1807 cells that recognize an endogenous fungal antigen led to the induction of memory and recruitment of effector cytokine-producing 1807 cells into the lung (C). We hypothesize that the amount of Eα peptide expressed by vaccine yeast and/or the duration of Ag presentation was sufficient to induce proliferation, but not differentiation and memory development. This idea is compatible with earlier reports indicating that the signaling threshold for proliferation of naïve T cells is lower than the threshold necessary to drive the differentiation of memory cells (4
). Ag limitation characterized by the amount of Ag available per T cell precursor can be the result of transferring too many initial precursor T cells into recipient mice or limited availability of the cognate Ag. However, we found that reducing the precursor frequency to 104
naïve TEa cells and enriching the cells with magnetic beads (17
) at the time of harvest did not yield sufficient numbers of trackable TEa cells (data not shown). Rather, we found that delivery of recombinant VSV-SED vaccine restored TEa functions, indicating that the vehicle (virus versus fungus) and/or the delivery route (i.v. versus s.c.) critically impacts the levels of Ag available per naïve T cell precursor. Interestingly, VSV-SED-primed TEa cells were efficiently recruited to the lung in response to rechallenge with Eα-mCh yeast. These results suggest that the antigen threshold for recruiting already primed TEa cells is lower than it is for TEa cells initially primed naïve during vaccination.
To verify that poor TEa recall resulted from limited Ag expression by recombinant Eα-mCh vaccine yeast, we added exogenous Eα-RFP to the vaccine yeast. The addition of Eα-RFP alone or with IFA significantly boosted the number of cytokine-producing TEa cells that migrated to the lung and expanded upon challenge, whereas administration of just Eα-RFP plus IFA (without yeast) yielded poor recall responses and secondary expansion in the lung. These results are consistent with our hypothesis that the amount of Ag expressed by Eα-mCh yeast alone during vaccination limited the quality and quantity of functional memory TEa cells. However, because vaccine yeast added to Eα-RFP enhanced lung recall, our results also highlight the fact that the yeast is a potent adjuvant for inducing functional effector T cells.
We used an additional strategy to exclude the possibilities that the high frequency of transferred cells or the identity of the model Ag may have skewed our results. Here, we controlled for the means of Ag expression (yeast) while changing the antigen and using tetramer staining to track endogenous T cells. We engineered yeasts that express the 2W1S peptide, an epitope that is recognized by the most frequent population of endogenous Ag-specific CD4+
T cells known to date (27
). The expansion and activation of 2W1S tetramer-positive T cells in response to vaccination with recombinant yeast were modest compared to those in response to vaccination with recombinant L. monocytogenes
infection, and the population of primed (CD44+
) 2W1S-specific T cells was too small to evaluate memory and recall functions. This result supports our interpretation that the amount of yeast-displayed model Ag was limiting and did not exceed the necessary threshold.
We conclude that the heterologous adoptive transfer system described here is a powerful method to study the earliest events of Ag presentation to antifungal CD4+
T cells. We showed that Y-Ae MAb nicely detected yeast-derived Eα–pMHC-II complexes on DCs. These levels of Ag display corresponded with robust T cell activation, proliferation, and expansion of antifungal CD4+
TEa cells during the first week after vaccination. However, the downstream effector, memory, and recall functions of TEa cells were blunted by insufficient Ag display on the vaccine yeast. Thus, this TEa Tg system is ideally suited to studying fungal Ag presentation and early T cell priming, whereas T cell effector and memory development may instead be studied with the autologous TCR Tg 1807 cells described recently (37