IL-15 Participates in the Respiratory Innate Immune Response to Influenza Virus Infection

Following influenza infection, natural killer (NK) cells function as interim effectors by suppressing viral replication until CD8 T cells are activated, proliferate, and are mobilized within the respiratory tract. Thus, NK cells are an important first line of defense against influenza virus. Here, in a murine model of influenza, we show that virally-induced IL-15 facilitates the trafficking of NK cells into the lung airways. Blocking IL-15 delays NK cell entry to the site of infection and results in a disregulated control of early viral replication. By the same principle, viral control by NK cells can be therapeutically enhanced via intranasal administration of exogenous IL-15 in the early days post influenza infection. In addition to controlling early viral replication, this IL-15-induced mobilization of NK cells to the lung airways has important downstream consequences on adaptive responses. Primarily, depletion of responding NK1.1+ NK cells is associated with reduced immigration of influenza-specific CD8 T cells to the site of infection. Together this work suggests that local deposits of IL-15 in the lung airways regulate the coordinated innate and adaptive immune responses to influenza infection and may represent an important point of immune intervention

Calcium- and polyphosphate-containing acidic granules of sea urchin eggs are similar to acidocalcisomes, but are not the targets for NAADP

Acidocalcisomes are acidic calcium-storage compartments described from bacteria to humans and characterized by their high content in poly P (polyphosphate), a linear polymer of many tens to hundreds of Pi residues linked by high-energy phosphoanhydride bonds. In the present paper we report that millimolar levels of short-chain poly P (in terms of Pi residues) and inorganic PPi are present in sea urchin extracts as detected using 31P-NMR, enzymatic determinations and agarose gel electrophoresis. Poly P was localized to granules randomly distributed in the sea urchin eggs, as shown by labelling with the poly-P-binding domain of Escherichia coli exopolyphosphatase. These granules were enriched using iodixanol centrifugation and shown to be acidic and to contain poly P, as determined by Acridine Orange and DAPI (4′,6′-diamidino-2-phenylindole) staining respectively. These granules also contained large amounts of calcium, sodium, magnesium, potassium and zinc, as detected by X-ray microanalysis, and bafilomycin A1-sensitive ATPase, pyrophosphatase and exopolyphosphatase activities, as well as Ca2+/H+ and Na+/H+ exchange activities, being therefore similar to acidocalcisomes described in other organisms. Calcium release from these granules induced by nigericin was associated with poly P hydrolysis. Although NAADP (nicotinic acid–adenine dinucleotide phosphate) released calcium from the granule fraction, this activity was not significantly enriched as compared with the NAADP-stimulated calcium release from homogenates and was not accompanied by poly P hydrolysis. GPN (glycyl-L-phenylalanine-naphthylamide) released calcium when added to sea urchin homogenates, but was unable to release calcium from acidocalcisome-enriched fractions, suggesting that these acidic stores are not the targets for NAADP

NK1.1+ NK cells are partially required for the subsequent accumulation of influenza-specific CD8 T cell accumulation at the site of infection.

<p>Beginning on the day of infection, mice received i.v. injections of either PBS vehicle control or αNK1.1 (PK136) every other day until day 6 p.i. (red arrows), and indicated tissues were collected on days 4, 6, and 8 p.i. (A). Mean number NK cells cells on day 4 p.i. in untreated (black bars) and PK136-treated (open bars) are shown ± SEM (n = 3 mice/group) (B). Mean number of NP-Tet⁺ CD8 T cells in the BAL, lung, and spleen were quantified and depicted ± SEM on day 6 (C) and day 8 (D) p.i. (n = 3 mice/group). Stars indicate statistical significance (*p = 0.009). Data are representative of three independent experiments.</p

Administration of IL-15c i.n. increases the number of NK cells recovered from the lung airways.

<p>On day 3 p.i. with 10³ pfu HKx31, mice were administered either PBS vehicle control (black bars), IL-15Rα alone (shaded bars) or IL-15c (open bars) intranasally. Twelve hours post treatment, CD3⁻ lymphocytes were analyzed for NK1.1 and NKp46 expression. These NK cells were quantified and analyzed for CD122 expression, BrdU incorporation, and Ki-67 staining. Representative flow plots are depicted in panel (A), and graphical representations of mean frequencies and numbers of NK cells ± SEM, as well as percentage of these cells positive for CD122 and BrdU are depicted in panel (B) (n = 3 mice/group; * = p<0.025). (C) Mean frequencies and numbers of NK cells in the spleen are plotted ± SEM (n = 3 mice/group). Data are representative of two independent experiments.</p

IL-15 is chemotactic for NK cells in vitro.

<p>Three days following infection with 10³ pfu HKx31 i.n., 1×10⁶ bulk lymphocytes from the pooled BAL, lung, and spleen of 8 mice were placed in the top chamber of a transwell with the bottom chamber containing 500 µL either media alone (black bars) or supplemented with 100 ng IL-15c (open bars). Mean percent migration of CD3⁻, NK1.1⁺ NK cells is depicted ± SEM (n = 3 replicates/group; *p = 0.046 and 0.003). Data are representative of three independent experiments. Significant differences in migration to media alone or media containing IL-15c are indicated by stars (*p<0.05).</p

Intranasal administration of IL-15c during the innate phase of the immune response against influenza improves early control of viral control.

<p>On days 1–4 post infection with 10³ pfu HKx31 i.n., animals received either PBS vehicle control (Black circles) or IL-15c (red squares) i.n. (A). Whole lungs were collected and analyzed via plaque assay for viral titer on days 2, 4, 6, and 8 p.i. (B). Significant differences between control and IL-15c-treated mice were observed on days 4 (C) and 6 (D) p.i. (*p = 0.022 and 0.013).</p

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells. We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vβ11+ iNKT cells. Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells. These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation. [Display omitted] •Human iNKT cells robustly expand for cytotherapy via an IL-2/IL-7-based protocol.•iNKT cells expanded through this protocol retain regulatory capacity.•Post-expansion, CD2/CD3/CD28 stimulation enhances iNKT cell Th2 cytokine secretion.•CD2/CD3/CD28 stimulation also enhances expanded iNKT cell anti-tumor cytotoxicity