Functional role of memory Tc17 cells in resistance to fungal pneumonia.

<p>Recall: (<b>A</b>) Effector CD8⁺ T cells from vaccinated IL-17a^CreR26R^eYFP were purified from the dLNs and spleen on day 16 post vaccination and adoptively transferred into naïve WT recipients. After 5 months of rest, mice were challenged intratracheally with an isogenic virulent strain and after 4 days, lungs were harvested to analyze recall responses by flow cytometry. Values denote percent ± SD. Data is representative of 2 independent experiments. N = 4mice/group. Resistance: (<b>B</b>) Naïve WT mice were vaccinated with attenuated <i>B</i>. <i>dermatitidis</i> #55 strain and rested for ~5 months. Vaccinated mice and unvaccinated controls were challenged intratracheally with a virulent strain of <i>B</i>. <i>dermatitidis</i> (10⁴ CFU of #26199). Anti-IL-17A or control IgG antibody (300 μg) was administered intravenously every other day starting from day -1 of challenge. Lung homogenate was enumerated for CFU. Whisker plots represent data from two independent experiments harvested on day 6 and day 8 post-challenge. N = 17–23 mice/group. ***P≤0.001. CD4⁺ T cells were depleted throughout the entire 5-month experiment.</p

Impact of HIF-1α on homeostasis of memory Tc17 and Tc1 cells.

<p>Naïve IL17a^CreR26R^eYFP or chimeric mice were depleted of CD4⁺ T cells and vaccinated with strain #55. Mice were rested for at least 90 days. <b>A.</b> Echinomycin was administered for 10 days beginning 90 days after vaccination. On day 11, spleens were harvested to analyze cytokine producing CD8⁺ T cells. <b>B.</b> Mice were given Echinomycin or Mimosine every other day as above for 14 days during the memory phase, Percent cytokine-producing cells among polyclonal CD8⁺ T cells (<b>A & B</b>) <b>C.</b> Percent cytokine producing cells gated on CD8⁺CD44^hi T cells in single and mixed bone marrow chimera mice. Data represents percent cytokine-producing cells among CD8⁺ T cells in spleens. Values are mean ± SD. N≥5 mice/group. Data is representative of at least five (<b>A</b>) and two (<b>C</b>) independent experiments. *P≤0.05.</p

Long-term persistence, fidelity and plasticity of memory Tc17 cells.

<p>Naïve IL17a^CreR26R^eYFP mice were vaccinated with ~10⁵ CFU of #55 strain of B. <i>dermatitidis</i>. The draining lymph nodes (dLNs) and spleens were harvested on indicated days. Following staining, cells were analyzed by flow cytometry. Percent <b>(A)</b> and absolute numbers <b>(E)</b> of eYFP⁺ CD8⁺ T cells. Percent <b>(B)</b> cytokine producing cells among eYFP⁺ CD8⁺ T cells. N = 5 mice/group. <b>(C & F)</b> Percent and absolute numbers of eYFP⁺ cells in naïve WT or TCRα^-/- recipient mice in the spleens were analyzed by flow cytometry on indicated days following adoptive transfer of purified effector CD8⁺ T cells from IL17a^CreR26R^eYFP donor mice (day 23 post-vaccination, 12.7x10⁴ eYFP⁺ cells/mouse). Percent <b>(D)</b> cytokine producing cells among eYFP⁺ CD8⁺ T cells. N = 3–5 mice/group. Data is representative of at least two independent experiments. Mice were injected with GK1.5 throughout the experiment to deplete CD4⁺ T cells.</p

Role of Bcl-2 for memory Tc17 cells.

<p>IL17a^CreR26R^eYFP mice were vaccinated and rested for ~90 days. Mice were treated with either vehicle or Bcl-2 inhibitor ABT-199 (20 mg/kg body weight) for 10 days. Lymph nodes (<b>A</b>) and spleens (<b>B</b>) were harvested to analyze memory CD8⁺ T cells positive for eYFP, IL-17A and IFNγ. Data is representative of 4–5 mice/group. CD4⁺ T cells were depleted throughout the experiment. *P≤0.05 and **P≤0.01.</p

Impact of HIF-1α on proliferation of effector Tc17 and Tc1 cells.

<p>Naïve IL17a^CreR26R^eYFP or chimera mice were depleted of CD4⁺ T cells and vaccinated with strain #55. Splenocytes were re-stimulated, surface-stained, and stained for intracellular cytokines. (A) Mice were given Echinomycin intraperitoneally every other day (starting day 4 post-vaccination) for 10 days to inhibit HIF-1α. Percent cytokine-producing cells among polyclonal CD8⁺ T cells (day 15 post-vac). <b>B.</b> Percent cytokine producing cells gated on CD8⁺CD44^hi T cells in single and mixed bone marrow chimera mice (day 17 post-vac). <b>C</b>. Mice were given Echinomycin every other day starting at -7 days (pre-vac) or at +4 days (post-vac) of vaccination. Percent cytokine producing cells gated on CD8⁺CD44^hi T cells (day 17 post-vac). N≥5 mice/group. Data is representative of at least five (<b>A</b>) and two (<b>C</b>) independent experiments. Values are mean ± sd. *P≤0.05, ***P = 0.001, ****P≤0.0001.</p

Multicytokine producing CD8⁺ eYFP⁺ T cells.

<p>Cells from mice portrayed in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006356#ppat.1006356.g001" target="_blank">Fig 1</a></b> were analyzed for expression of IL-17A, IFNγ, TNFα and GM-CSF cytokines. <b>(A)</b>. Naïve IL17a^CreR26R^eYFP mice were vaccinated with #55 strain. The draining lymph nodes (dLNs) and spleens were harvested on indicated days enumerate multi-cytokine producing CD8⁺ cells by flow cytometry. <b>(B)</b>. Naïve IL17a^CreR26R^eYFP mice were vaccinated, effector CD8⁺ T cells were purified (as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006356#ppat.1006356.g001" target="_blank">Fig 1</a>) and transferred into naïve WT or TCRα^-/- mice. On indicated days, cells were analyzed for multicytokine producing CD8⁺ T cells by flow cytometry. Values in the pie diagrams represent percent cytokine-expressing cells among all cytokine-producing cells.</p

Proliferation and apoptosis profile of anti-fungal memory CD8⁺ T cells.

<p>Naïve IL17a^CreR26R^eYFP mice were depleted of CD4⁺ T cells and vaccinated as noted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006356#ppat.1006356.g001" target="_blank">Fig 1</a>. <b>A.</b> Proliferation of anti-fungal effector cells, and early and late memory CD8⁺ T cells. Vaccinated mice were pulsed with BrdU in drinking water (DW) for 12 days. On the following day, spleens were harvested, processed and re-stimulated. Cells were surface-stained and stained intracellularly for cytokines before analysis of BrdU staining. Numbers represent the percent BrdU⁺ of eYFP⁺/IFNγ⁺ CD8⁺ T cells. N = 5 mice/group. <b>B.</b> On day 76 post-vaccination, surface-stained splenocytes were stained intracellularly for active Caspase3/8. Dot plots show the staining of pro-apoptotic caspases gated on CD8⁺CD44^hi T cells. N = 4–5 mice. <b>C.</b> On day 76 post-vaccination, splenocytes were stained for surface markers before staining for intracellular anti-apoptotic factors. Values indicate MFIs gated on CD8⁺CD44^hi eYFP^+/- T cells. Dotted lines indicate staining controls. Data are representative of two independent experiments. *P≤0.05, ***P≤0.001, and ****P≤0.0001.</p

Phenotype and transcription factor profile of CD8⁺eYFP⁺ T cells.

<p>Splenocytes from vaccinated IL17a^CreR26R^eYFP mice (day 46 post-vaccination) were surface stained <i>ex vivo</i> for <b>(A)</b> CD8β, TCRβ, CD3ε, and NK1.1 expression on CD8α⁺ eYFP⁺ cells and <b>(B)</b> cytokine, chemokine, adhesion, co-stimulatory and terminal differentiation markers/ receptors. <b>C.</b> Naïve IL17a^CreR26R^eYFP mice were depleted of CD4⁺ T cells and vaccinated with strain #55. On day 346 after vaccination, splenocytes were re-stimulated and stained for surface markers, Bcl-2, intracellular cytokines and transcription factors. CD8⁺ T cell populations were analyzed by flow cytometry. Values represent mean florescence intensity (MFI) ±SD.</p