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

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections

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

Investigation of Genetic Susceptibility to Blastomycosis Reveals Interleukin-6 as a Potential Susceptibility Locus

Blastomycosis is a potentially life-threatening infection caused by the fungus Blastomyces dermatitidis. As with related fungal diseases, blastomycosis is noted to affect some populations more than others. These patterns of illness are often not related to predisposing conditions or exposure risks; thus, genetic differences are thought to underlie these health disparities. People of Hmong ancestry in Wisconsin are at elevated risk of blastomycosis compared to the general population. We studied the genetic codes of Hmong blastomycosis patients and identified candidate sites in their genomes that may explain their susceptibility to this infection. We further studied one particular region of the genome that is involved with the immune processes that fight B. dermatitidis. Our work revealed population differences in the response to fungi. A better understanding of the genetic underpinnings of susceptibility to infectious diseases has broader implications for community health, especially in the paradigm of personalized medicine.Genetic differences are hypothesized to underlie ethnic disparities in incidence rates of the endemic systemic mycoses, including blastomycosis. Individuals of Hmong ancestry display elevated risk for this serious fungal infection. Here, we interrogated the genomes of Wisconsin (WI) Hmong blastomycosis patients using homozygosity mapping to uncover regions of the genome that are likely shared among the greater Hmong population and filtered for variants with high potential to affect disease susceptibility. This approach uncovered 113 candidate susceptibility variants, and among the most promising are those in genes involved in the interleukin-17 (IL-17) response. In particular, we identified 25 linked variants near the gene encoding IL-6 (IL6). We validated differences in cytokine production between Hmong and European volunteers and formally demonstrated a critical role for IL-6 in the development of adaptive immunity to Blastomyces dermatitidis. Our findings suggest that the dysregulation of IL-17 responses underlies a recently reported and poorly understood ethnic health disparity

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

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

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