Pin1 Modulates the Type 1 Immune Response

BACKGROUND/ABSTRACT: Immune responses initiated by T cell receptor (TCR) and costimulatory molecule mediated signaling culminate in maximal cytokine mRNA production and stability. The transcriptional responses to co-stimulatory T cell signalling involve calcineurin and NF-AT, which can be antagonized by interference with the cis-trans peptidyl-prolyl isomerases (PPIase), cyclophilin A and FKBP. Signalling molecules downstream of CD28 which are essential for the stabilization of cytokine mRNAs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We now show that Pin1, a third member of the PPIase family mediates the post-transcriptional regulation of Th1 cytokines by activated T cells. Blockade of Pin1 by pharmacologic or genetic means greatly attenuated IFN-γ, IL-2 and CXCL-10 mRNA stability, accumulation and protein expression after cell activation. In vivo, Pin1 blockade prevented both the acute and chronic rejection of MHC mismatched, orthotopic rat lung transplants by reducing the expression of IFN-γ and CXCL-10. Combined transcriptional and post-transcriptional blockade with cyclosporine A and the Pin1 inhibitor, juglone, was synergistic. CONCLUSIONS/SIGNIFICANCE: These data suggest Pin1 inhibitors should be explored for use as immunosuppressants and employed with available calcineurin inhibitors to reduce toxicity and enhance effectiveness

Virally Activated CD8 T Cells Home to Mycobacterium bovis BCG-Induced Granulomas but Enhance Antimycobacterial Protection Only in Immunodeficient Mice

The effect of secondary infections on CD4 T-cell-regulated chronic granulomatous inflammation is not well understood. Here, we have investigated the effect of an acute viral infection on the cellular composition and bacterial protection in Mycobacterium bovis strain bacille Calmette-Guérin (BCG)-induced granulomas using an immunocompetent and a partially immunodeficient murine model. Acute lymphocytic choriomeningitis virus (LCMV) coinfection of C57BL/6 mice led to substantial accumulation of gamma interferon (IFN-γ)-producing LCMV-specific T cells in liver granulomas and increased local IFN-γ. Despite traffic of activated T cells that resulted in a CD8 T-cell-dominated granuloma, the BCG liver organ load was unaltered from control levels. In OT-1 T-cell-receptor (TCR) transgenic mice, ovalbumin (OVA) immunization or LCMV coinfection of BCG-infected mice induced CD8 T-cell-dominated granulomas containing large numbers of non-BCG-specific activated T cells. The higher baseline BCG organ load in this CD8 TCR transgenic animal allowed us to demonstrate that OVA immunization and LCMV coinfection increased anti-BCG protection. The bacterial load remained substantially higher than in mice with a more complete TCR repertoire. Overall, the present study suggests that peripherally activated CD8 T cells can be recruited to chronic inflammatory sites, but their contribution to protective immunity is limited to conditions of underlying immunodeficiency

Protein Kinase Cε, Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

To find clues about the mechanism by which kinase C epsilon (PKCε) may impart susceptibility to ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinomas (SCC), we compared PKCε transgenic (TG) mice and their wild-type (WT) littermates for (1) the effects of UVR exposures on percent of putative hair follicle stem cells (HSCs) and (2) HSCs proliferation. The percent of double HSCs (CD34+ and α6-integrin or CD34+/CD49f+) in the isolated keratinocytes were determined by flow cytometric analysis. Both single and chronic UVR treatments (1.8 kJ/m2) resulted in an increase in the frequency of double positive HSCs in PKCε TG mice as compared to their WT littermates. To determine the rate of proliferation of bulge region stem cells, a 5-bromo-2′-deoxyuridine labeling (BrdU) experiment was performed. In the WT mice, the percent of double positive HSCs retaining BrdU label was 28.4±0.6% compared to 4.0±0.06% for the TG mice, an approximately 7-fold decrease. A comparison of gene expression profiles of FACS sorted double positive HSCs showed increased expression of Pes1, Rad21, Tfdp1 and Cks1b genes in TG mice compared to WT mice. Also, PKCε over expression in mice increased the clonogenicity of isolated keratinocytes, a property commonly ascribed to stem cells

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

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

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

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