How a Cytokine Is Chaperoned through the Secretory Pathway by Complexing with Its Own Receptor: Lessons from Interleukin-15 (IL-15)/IL-15 Receptor α▿

While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion

Novel Methods for Genetic Transformation of Natural Bacillus subtilis Isolates Used To Study the Regulation of the Mycosubtilin and Surfactin Synthetases▿ †

Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates competence development about 100-fold. The second method stimulates Campbell-type recombination of DNA ligation mixtures in B. subtilis by the addition of polyethylene glycol. We employed these novel methods to study the regulation of the synthetases for the lipopeptide antibiotics mycosubtilin (myc) and surfactin (srfA) in B. subtilis strain ATCC 6633. By means of lacZ reporter fusions, it was shown that the expression of srfA is >100 times lower in strain ATCC 6633 than in the laboratory strain B. subtilis 168. Expression of the myc operon was highest in rich medium, whereas srfA expression reached maximal levels in minimal medium. Further genetic analyses showed that the srfA operon is mainly regulated by the response regulator ComA, while the myc operon is primarily regulated by the transition-state regulator AbrB. Although there is in vitro evidence for a synergistic activity of mycosubtilin and surfactin, the expression of both lipopeptide antibiotics is clearly not coordinated

High dose CD11c-driven IL15 is sufficient to drive NK cell maturation and anti-tumor activity in a trans-presentation independent manner

The common gamma (γc)-chain cytokine interleukin 15 (IL15) is a multifunctional immune-modulator which impacts the generation, maturation and activity of many cell types of the innate, as well as the adaptive immune system, including natural killer (NK) and CD8(+) T cells. Using a new series of transgenic mice, we analyzed the in vivo potential of IL15 as an immune-regulator when available at different concentrations or delivery modes, i.e. soluble monomer or complexed to its specific receptor α (Rα)-chain. We have identified distinct effects on selected IL15-responsive populations. While CD8(+) T cells required complexed forms of IL15/IL15Rα for full functionality, mature NK populations were rescued in an IL15/IL15Rα-deficient environment by high levels of CD11c-restricted IL15. These IL15-conditions were sufficient to limit tumor formation in a lung metastasis model indicating that the NK cell populations were fully functional. These data underline the potential of "free" IL15 in the absence of Rα-complex as a powerful and specific immuno-modulator, which may be beneficial where selective immune-activation is desired.</p

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.</p

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

<div><p>Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα^−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.</p> </div

Figure 3. Gene transfer of APO-IL-15 and IL-15Rα sushi domain increase memory-phenotype CD8⁺ T cells and NK cells.

<p>(A) Absolute numbers of the indicated lymphocyte subsets in the spleen of mice treated with the indicated plasmids 5 days before splenectomy. Each point shows an individual mouse. This experiment was repeated twice with comparable results. (*** indicates p<0.0001 in a one-way ANOVA test followed by Bonferroni corrections). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052370#pone.0052370.s005" target="_blank">Figure S5</a> shows a time course analysis of these parameters (B) Experiments as in A measuring numbers of intrahepatic mononuclear leukocytes with the indicated surface phenotypes. Each point represents an individual mouse (*** indicates p<0.0001 in a one-way ANOVA test followed by Bonferroni corrections).</p

Partial rescue of the phenotype of IL-15Rα^−/− mice in NK cells and memory-phenotype CD8 cells.

<p>WT C57Bl/6 mice (WT) or IL-15Rα^−/− syngenic mice (IL-15Rα^−/−) were injected with the indicated plasmids by hydrodynamic injection. Four days later splenocytes were immunostained. (A) CD3⁺ CD8⁺ dot plots with the percentage of events in the gated rectangles. Results reflect the percentage referred to total spenocytes (B) Histograms showing CD44 specific immunofluorescence on the cells gated in A to assess the percentage of CD44^high CD8⁺ memory-phenotype T cells. (C) CD3 NK1.1 double staining showing the percentage of events in each dot plot quadrant. Histograms and dot plots were taken from a representative individual mouse. (D) Grouped sets of data from other individuals. Confirmatory results in a series of mice in B6129 background are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052370#pone.0052370.s008" target="_blank">figure S8</a>.</p

Immunotherapeutic activity of liver gene transfer with the APO-IL-15 constructions for established subcutaneous MC38 tumors.

<p>MC38 bearing mice were treated on day six after tumor cell inoculation with 10 µg of the indicated plasmids by hydrodynamic injection. (A) Individual follow-up of tumor sizes upon treatment with each plasmid. Graphics represent pooled data from 3 independent experiments. Fraction of surviving mice is indicated for each experimental group. Differences in tumor growth between the pApo-hIL15+ pSushi and the pApo group were found (p<0.001) in a nonlinear mixed effect model calculated with Monolix software. (B) Survival curves of the experimental groups from panel A (* indicates p<0.05 between pApo-hIL15+ pSushi and pApo in a Gehan-Breslow-Wilcoxon Survival Test).</p