Tc17 Cells Mediate Vaccine Immunity against Lethal Fungal Pneumonia in Immune Deficient Hosts Lacking CD4⁺ T Cells

<div><p>Vaccines may help reduce the growing incidence of fungal infections in immune-suppressed patients. We have found that, even in the absence of CD4⁺ T-cell help, vaccine-induced CD8⁺ T cells persist and confer resistance against <em>Blastomyces dermatitidis</em> and <em>Histoplasma capsulatum</em>. Type 1 cytokines contribute to that resistance, but they also are dispensable. Although the role of T helper 17 cells in immunity to fungi is debated, IL-17 producing CD8⁺ T cells (Tc17 cells) have not been investigated. Here, we show that Tc17 cells are indispensable in antifungal vaccine immunity in hosts lacking CD4⁺ T cells. Tc17 cells are induced upon vaccination, recruited to the lung on pulmonary infection, and act non-redundantly in mediating protection in a manner that requires neutrophils. Tc17 cells did not influence type I immunity, nor did the lack of IL-12 signaling augment Tc17 cells, indicating a distinct lineage and function. IL-6 was required for Tc17 differentiation and immunity, but IL-1R1 and Dectin-1 signaling was unexpectedly dispensable. Tc17 cells expressed surface CXCR3 and CCR6, but only the latter was essential in recruitment to the lung. Although IL-17 producing T cells are believed to be short-lived, effector Tc17 cells expressed low levels of KLRG1 and high levels of the transcription factor TCF-1, predicting their long-term survival and stem-cell like behavior. Our work has implications for designing vaccines against fungal infections in immune suppressed patients.</p> </div

Antifungal Tc17 cells are durable and stable, persisting as long-lasting vaccine memory without plasticity towards IFNγ cells.

Our understanding of persistence and plasticity of IL-17A+ memory T cells is clouded by conflicting results in models analyzing T helper 17 cells. We studied memory IL-17A+ CD8+ T-cell (Tc17) homeostasis, persistence and plasticity during fungal vaccine immunity. We report that vaccine-induced memory Tc17 cells persist with high fidelity to the type 17 phenotype. Tc17 cells persisted durably for a year as functional IL-17A+ memory cells without converting to IFNγ+ (Tc1) cells, although they produced multiple type I cytokines in the absence of residual vaccine antigen. Memory Tc17 cells were canonical CD8+ T cells with phenotypic features distinct from Tc1 cells, and were Ror(γ)thi, TCF-1hi, T-betlo and EOMESlo. In investigating the bases of Tc17 persistence, we observed that memory Tc17 cells had much higher levels of basal homeostatic proliferation than did Tc1 cells. Conversely, memory Tc17 cells displayed lower levels of anti-apoptotic molecules Bcl-2 and Bcl-xL than Tc1 cells, yet were resistant to apoptosis. Tc1 cells required Bcl-2 for their survival, but Bcl-2 was dispensable for the maintenance of Tc17 cells. Tc17 and Tc1 cells displayed different requirements for HIF-1α during effector differentiation and sustenance and memory persistence. Thus, antifungal vaccination induces durable and stable memory Tc17 cells with distinct requirements for long-term persistence that distinguish them from memory Tc1 cells

Role of IL-12 signaling on vaccine-induced Tc1 and Tc17 cells and resistance against fungal pneumonia.

<p>Mice were depleted of CD4⁺ T cells, vaccinated and challenged as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#ppat-1002771-g002" target="_blank">Fig. 2</a>. <b>A.</b> Three weeks after challenge, lung CFU were enumerated. CFUs are depicted in box and whisker plots for 10–17 mice/group. Data is representative of two independent experiments. <b>B.</b> On day 3–4 post-challenge, lungs were collected to enumerate cytokine producing CD8⁺ T cells by flow cytometry. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#s2" target="_blank">Results</a> are the mean ± SD of 4–8 mice/group from two independent experiments. *, p<0.05 vs. unvaccinated mice; and **, p<0.01.</p

CCR6 mediated recruitment of Tc17 cells into the lung after challenge.

<p>Mice were depleted of CD4⁺ T cells and vaccinated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#ppat-1002771-g002" target="_blank">Fig. 2</a>. <b>A.</b> Dot plots show the frequency of CCR6⁺ or CXCR3⁺ cells (middle and right panels) gated on the respective cytokine producing CD8⁺ T cells (left panel) in the dLNs after vaccination. <b>B.</b> Dot plots show the frequency of CCR6⁺ or CXCR3⁺ Tc17 cells (middle and right panels, respectively) in the lung 4 days after pulmonary challenge. <b>C.</b> The number of cytokine producing CD8⁺ T cells in the lung 5 days after pulmonary challenge as measured by flow cytometry. For neutralization of CCL20, vaccinated mice were given ∼90 µg of α-mouse CCL20 mAb or control antibody i.v. on days 0, 2 and 4 post-challenge. Values are mean ± SD of 5–6 mice/group. *, p<0.05.</p

Non-redundant role of Tc17 cells in anti-fungal vaccine immunity.

<p>Mice were vaccinated with 10⁵ yeast of <i>Blastomyces</i> vaccine strain #55 subcutaneously (s.c.) and boosted once after 2 weeks. Two to three weeks later, mice were challenged intratracheally (i.t.) and lungs were harvested to enumerate CFUs. CD4⁺ T cells were depleted with GK1.5 mAb given i.v. weekly. <b>A.</b> Mice were given neutralizing anti-IL-17A mAb or rat IgG as control (100 µg each, i.v.) on days 0, 2 & 4 post-challenge. On day 6, lungs were analyzed for CFU. CFUs are from 8–14 mice/group. <b>B.</b> On day −3 and −1, mice were given 2–4×10⁹ pfu i.v. of recombinant adenovirus secreting mouse IL-17 receptor or control adenovirus expressing luciferase (AdLuc); a third dose was given on day 0 of challenge with virulent <i>Blastomyces</i> (i.t.). 10 days after challenge, lungs were harvested for CFU. CFUs are for 13–19 mice/group. <b>C.</b> IL-17RA^−/− and WT mice were infected i.t. with a lethal dose of WT <i>Blastomyces</i> yeast. 10 days later, lung CFU were enumerated. CFUs are from 10–14 mice/group from two independent experiments. <b>D.</b> IL-17A^−/− and WT mice were lethally challenged i.t. with WT <i>Blastomyce</i>s yeast. 13 days later, lung CFU were enumerated. CFUs are from 9–19 mice/group. In panels A–D, CFUs are shown in box and whisker plots. *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001.</p

Cross-regulation of Tc17 and Tc1 cells during anti-fungal vaccine immunity.

<p>Mice were depleted of CD4⁺ T cells, vaccinated and challenged as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#ppat-1002771-g002" target="_blank">Fig. 2</a>. Percentage (<b>A & B</b>) and number (<b>C &D</b>) of cytokine-producing CD8⁺ T cells in WT, IL-17A^−/− (A & C) and IL-17RA^−/− (B & D) mice as measured by flow cytometry. Values are the mean ± SD of 4 mice/group. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#s2" target="_blank">Results</a> are representative of two independent experiments. *, p<0.05 vs. respective control.</p

Tc17 cells are induced and recalled in the absence of CD4⁺ T cells.

<p>Mice were vaccinated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#s4" target="_blank">Methods</a>. CD4⁺ T cells were depleted using GK1.5 mAb i.v. weekly throughout the experiment. <b>A.</b> Percent CD8⁺ T cells producing IL-17 in draining LNs (dLNs) and spleen at 19 days post-vaccination in the presence (left panels) or absence (right) of CD4⁺ T cells as assessed by flow cytometry. <b>B.</b> In the left two panels, OT-I T cells (1×10⁶) were transferred to naïve congenic CD4<i>^−/−</i> mice and vaccinated with recombinant <i>Blastomyces</i> expressing the OT-I epitope SIINFEKL. At day 14, dLNs and spleens were harvested and stimulated <i>ex vivo</i> with OVA peptide. In the right three panels, dLNs were harvested and cultured with BMDCs in the presence or absence of heat-killed yeast. Yeast-specific OT-I and endogenous Tc17 cells were enumerated by flow cytometry. The percent (<b>C</b>) or number (<b>D</b>) of polyclonal and yeast-specific Tc17 cells during recall response in the lung 3–4 days after pulmonary infection with wild type virulent <i>Blastomyces</i>. Values are mean ± SD of 4 mice/group. * p<0.05 vs. unvaccinated mice; and ^† p<0.05 vs. vaccinated CD4⁺ T-cell sufficient mice.</p

Phenotypic attributes of anti-fungal effector Tc17 cells.

<p>Mice were depleted of CD4⁺ T cells and vaccinated once as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#ppat-1002771-g002" target="_blank">Fig. 2</a>. On day 19, dLNs cells were harvested and stimulated <i>ex vivo</i> with anti-CD3 and -CD28 in the presence of Golgi stop and TAPI-2 at 37°C for 5 hrs. Following incubation, cells were surface-stained before staining for intracellular cytokine or transcription factor using phospho-staining kit as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002771#s4" target="_blank">Methods</a>. <b>A.</b> Zebra plots showing percent CD27, CD43 & CD62L of IFN-γ⁺ and IL-17A⁺ CD8⁺ T cells. Values are mean ± SD of 4–5 mice/group. <b>B.</b> Zebra plots show frequency of IL-17⁺, KLRG-1⁺ and TCF-1⁺ among CD8⁺CD44⁺ cells. Values are mean ± SD of 4–5 mice/group. <b>C.</b> Left two panels show mean fluorescence (MFI) of RORγt and TCF-1 on IFN-γ⁺ and IL-17A⁺ CD8⁺ T cells. Right panel shows correlation of RORγt and TCF-1 expression on IL-17A⁺ CD8⁺ T cells. r = correlation coefficient derived from MFIs. Data are representative of two-independent experiments.</p

Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia

<div><p>Fungal infections have skyrocketed in immune-compromised patients lacking CD4⁺ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A⁺ CD8⁺ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4⁺ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ⁺ CD8⁺ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8⁺ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.</p></div

MyD88 signaling potentiates proliferation of Tc17 cells.

<p>Naïve WT or MyD88ΔT mice were CD4⁺ T cell depleted and vaccinated with strain #55. BrdU was pulsed through the drinking water on indicated interval days. Mice were sacrificed and dLNs were harvested at the end of each indicated pulse period. Cells were restimulated, stained for surface and intracellular cytokines before BrdU staining. Percent BrdU^+ve cells were analyzed by flow cytometry by gating on CD8⁺ IL-17- or IFNγ-producing cells. CD4⁺ T cells were depleted throughout the experiment. Values are percent mean ± SD of 4–5 mice/group. *P≤0.05. Data is representative of 3 independent experiments.</p