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

Abstract

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.

Fig 1. Intrinsic MyD88 signaling regulates Tc17 cell responses.

Naïve purified OT-I cells (10⁶) were adoptively transferred into naïve congenic wild-type (WT) or MyD88^-/- mice (A & B). Naïve Thy1.1^+ve OT-I^wt or OT-I^myd88-/- T cells were transferred into naïve congenic Thy1.2^+ve WT mice (D). Mice were CD4⁺ T cell depleted and vaccinated with OVA-expressing (OT-I) #55 yeast (A, B, D) or #55 yeast (C). Draining LNs and spleens were harvested after 2–3 weeks to analyze percent and total number of cytokine producing CD8⁺ CD44^hi T cells by flow cytometry. CD4⁺ T cells were depleted throughout the experiment. Values are mean ± SD of 4–5 mice/group. *P≤0.05. Data is representative of 2–4 independent experiments.

Fig 2. Intrinsic MyD88 signaling in CD8 T cells is required for vaccine immunity.

(A & B) CD4⁺ T-cell depleted naïve mice were vaccinated with live (A) or heat-killed (B) #55 yeast (B). Vaccinated mice were challenged with virulent #26199 yeast, lungs were harvested for CFU analysis on indicated days, and data are shown in whisker plots (A & B; N = 7–14 mice/group). Lungs were harvested 4 days post-challenge to enumerate percentage (C) and total number of cytokine-producing CD8⁺ T cells (D) by flow cytometry. CD4⁺ T cells were depleted throughout experiment. Values are the mean ± SD of 5 mice/group. *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. Data is representative of 2 independent experiments.

Fig 3. Kinetics of Tc17 and Tc1 cell responses in vaccinated mice.

Naïve WT and MyD88ΔT mice were vaccinated and on indicated days. dLNs (A) and spleens (B) were harvested to analyze percent and total number of IFNγ⁺ and IL-17A⁺ CD8⁺ T cells. CD4⁺ T cells were depleted throughout the experiment. Data are mean ± SD of N = 4–5 mice/group. *P≤0.05.

Fig 4. The role of intrinsic MyD88 for effector CD8⁺ T cell survival.

Naïve WT and MyD88ΔT mice were CD4⁺ T cell depleted and vaccinated with strain #55. Two weeks later dLNs and spleen cells were collected, restimulated and stained for surface markers followed by intracellular staining using BD Perm/Fix and analyzed by flow cytometry. A. MFI of Bcl-2 and Bcl-xL expression in IFNγ⁺ (Tc1) and IL-17A⁺ (Tc17) cells. B. Active Caspase 3 expression. Numbers indicate percent active Caspase 3 expressing cells. C. Surface CD43 and CD27 expression in IL-17A⁺ and IFNγ⁺ cells. Values are the mean ± SD percentage; N = 4–5 mice/group. Data is representative of 2 independent experiments.

Fig 5. MyD88 signaling potentiates proliferation of Tc17 cells.

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.

Fig 6. Rapamycin treatment blunts Tc17 cell responses and proliferation.

Naïve WT and MyD88ΔT mice were CD4⁺ T cell depleted and vaccinated with strain #55. Rapamycin (~2 μg/mouse) was administered daily from day 4 by the i.p. route. On day 19 post-vaccination, dLNs and spleens were harvested to enumerate percent and total numbers of IL-17 and IFNγ producing CD8⁺ T cells (A). Cohorts of mice were pulsed with BrdU through drinking water from day 7–15. On day 16, dLNs and spleens were harvested to enumerate percent BrdU^+ve IL-17A and IFNγ producing CD8⁺ T cells (B). Values are the mean ± SD of 4–7 mice/group. *p≤0.05, ***p≤0.001 and ****p≤0.0001. Data is representative of 2 independent experiments.

Fig 7. MyD88 requires Akt1 signaling for mTOR activation.

A. Mice were CD4+ T cell depleted and vaccinated with strain #55. Akt1 inhibitor (A-443654) was administered s.c. from day 4 to 15 post-vaccination. On day 16, dLNs and spleen cells were collected, restimulated and stained for surface markers and intracellular cytokines. Numbers of IL-17A and IFNγ producing CD8⁺ T cells were analyzed by flow cytometry. A diamond represents an individual mouse and the bar is the mean of the group. *p≤0.05 and **p≤0.01. Data is representative of 2 independent experiments. B. Purified naive WT and MyD88ΔT OT-I cells were stimulated in vitro with anti-CD3 and yeast-stimulated BMDC supernatant. On day 4, supernatant was removed and replaced with culture medium. Cells were rested for 2.5 hours and medium was replaced with yeast stimulated BMDC supernatant either with or without Akt1 inhibitor (1μM) for 1 hour. Cells were surface stained prior to phospho-staining. Values in the histograms represent mean fluorescence intensity (MFI) of p-mTOR. Data in solid lines are from gating on activated cells (CD8⁺CD43⁺CD44^hiCD62L^lo), whereas data in dotted lines are from gating on naïve cells (CD8⁺CD43^negCD44^loCD62L^hi).

Fig 8. Role of IL-1R, IL-18R and TLR2 on Tc17 cell responses.

A. Purified naïve CD8⁺ T cells were incubated with WT DCs either with or without heat-killed yeasts for 5 days. Culture supernatants were harvested to quantify IL-17A production by ELISA. Error bars are mean ± SD of triplicate wells. Data are representative of two independent experiments. B. Overview of the experimental approach for generation of bone-marrow chimera mice and harvesting of organs from vaccinated mice. C. Number of cytokine producing cells in dLNs 20 days post-vaccination. D. Number of cytokine-producing cells in the dLNs in either control IgG or anti-IL-18R blocking antibody-treated, vaccinated-mice groups on day 14. CD4⁺ T cells were depleted throughout the experiment. Error bars are mean ± SD of N = 4–7 mice/group. *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.