A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The Systemic Immune State of Super-shedder Mice Is Characterized by a Unique Neutrophil-dependent Blunting of T_H1 Responses

<div><p>Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism <i>Salmonella enterica</i> serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened T_H1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (T_regs), fewer T-bet⁺ (T_H1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened T_H1 response with fewer T-bet⁺ T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated T_H1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet⁺ T_H1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the T_H1 immune response in the spleens of mice that are colonized by high levels of <i>S.</i> Typhimurium in the gastrointestinal tract.</p></div

Distinct systemic immune profiles associated with fecal shedding states.

<p>Summary of the results in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003408#ppat-1003408-g001" target="_blank">Figures 1</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003408#ppat-1003408-g002" target="_blank">2</a> presenting a super-shedder immune state that is distinct from the moderate-shedder immune state in the parameters mentioned. It should be noted that the plus signs indicate greater frequency of the cell type or better cytokine responsiveness with respect to uninfected mice. Thus while moderate-shedders have high levels of splenic neutrophils (+) compared to uninfected mice, they have fewer neutrophils compared to super-shedder mice (+++). Similarly, the frequency of T_regs decreases during infection compared to uninfected. Thus, moderate shedders have fewer T_regs (−−−) compared to super-shedders (−).</p

G-CSF treatment mimics the super-shedder phenotype by inhibiting IL-2 mediated T_H1 expansion.

<p>A,B: Mice were identified as moderate (<10⁶ cfu/gm) or super-shedder (>10⁸ cfu/gm) between day 15 and day 30 post infection. After 30 days of infection, mice were injected with 15 µg IL-2 antibody complexed with 4.5 µg IL-2 cytokine in a 15 minute incubation period at room temperature. Data shown is from a single experiment with a total of 12 mice, 4 untreated and 8 treated. A: Splenocytes were fixed and permeabilized and pSTAT5 MFI quantified in all CD4 T cells. B: T_H1 and T_reg levels were expressed as a percentage of total splenic CD4 T cells. Untreated mice (in square) showed no expansion and IL-2 antibody complex- treated mice included both super-shedders (open circles) and moderate-shedders (filled circles). IL-2 mediated T_H1 expansion was significantly lower in super-shedder mice (p = 0.02), calculated using a one-tailed Mann Whitney U test. C, D, E: Moderate shedders were treated with either G-CSF or PBS for 3 days and then injected with IL-2 antibody complex on the 4^th day. Mice were sacrificed on the 6^th day, 36 days post-infection. Data shown is representative of 2 independent experiments with a total of 8 mice in each condition. Significance was determined using a two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing p<0.01. C: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 for 15 minutes and fixed and permeabilized and the frequency of pSTAT5⁺ CD4 T cells was quantified. D: Splenocytes were fixed and Ki-67⁺ CD4 T cells were quantified as a percentage of total CD4 T cells. E: T_H1 and T_reg levels were expressed as a percentage of total splenic CD4 T cells in G-CSF treated mice (open squares) and control mice (closed squares). T_H1 cells were significantly lower in the G-CSF pretreated mice (p = 0.02) calculated using a two-tailed Mann Whitney U test.</p

G-CSF treatment mimics the splenic super-shedder immune phenotype.

<p>Mice were identified as moderate (<10⁶ cfu/gm) or super-shedder (>10⁸ cfu/gm) between day 15 and day 30 post infection. Moderate shedders were injected with 1 µg G-CSF per day for three days and sacrificed on the 4^th day. Data shown is representative of 2 independent experiments with a total of 8–10 mice in each condition. Significance was determined using a two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing a p<0.01. A: Splenic Gr1⁺ cells are represented as a percentage of total cells in G-CSF- and PBS-treated control mice. B: Tbet⁺ T_H1 cells from splenocytes were quantified as a percentage of CD4 T cells. C, D: Splenocytes were stimulated ex vivo for 15 minutes with 40 ng/ml of IL-6 and IL-2 and total CD4 T cell pSTAT1 and pSTAT5 MFI measured. E: Fecal bacterial burden was quantified.</p

Neutrophils control the dampened systemic T_H1 response of super-shedders.

<p>In super-shedders, high levels of <i>Salmonella</i> in the gastrointestinal tract induce systemic neutrophilia with extensive granulopoiesis occurring in the bone marrow, resulting in elevated neutrophil counts in the blood, GI tract and spleen. This is accompanied by dampened IL-2 and IL-6 cytokine responsiveness in splenic CD4 T cells, fewer T_H1 cells and more T_regs. In moderate shedders, low levels of gastrointestinal <i>Salmonella</i> induce very few neutrophils systemically, leading to an active IL-2 and IL-6 response with increased T_H1 cells and fewer T_regs. Induction of granulopoiesis in moderate-shedders results in the super-shedder immune phenotype with the exception of T_regs, which remain unchanged.</p

Neutrophil depletion results in increased CD4 pSTAT5 and Tbet expression.

<p>After 30 days of infection, mice were injected with 1 µg RB6 depletion antibody every day for 3 days and sacrificed on the 4^th day. Data shown is representative of 2 independent experiments with a total of 8–10 mice in each condition. Significance was determined using a Two-tailed Mann-Whitney U test with one asterisk representing p<0.05 and two representing a p<0.01. A: Tbet⁺ CD4 T (T_H1) cells from splenocytes were quantified as a percentage of CD4 T cells. B: Splenocytes were stimulated ex vivo with 40 ng/ml IL-2 and fixed and permeabilized and total CD4 T cell pSTAT5 MFI measured. C: FoxP3⁺ CD4 T cells (T_regs) were quantified as a percentage of CD4 T cells. D: Fecal pellets were collected and bacterial burden quantified.</p