ILC2s failed to function in <i>T</i>. <i>spiralis</i>–mice ablated CD4+ T cells.

<p>BALB/c mice were intraperitoneally given anti-CD4 or rat IgG antibody every other day after infection with <i>T</i>. <i>spiral</i>is. <b>(A)</b> Frequency and numbers of CD3⁺CD4⁺ cells at 7 and 14 days postinfection were evaluated in the mesenteric lymph node of infected mice treated with anti-CD4 or rat IgG antibody. <b>(B)</b> At 7 and 14 days postinfection, the small intestines were harvested and subjected for worm burden. <b>(C)</b> Detection and frequency of ILC2s (CD3^-CD4^-IL-17RB⁺CD127⁺T1/ST2⁺) in the mesenteric lymph nodes of infected mice treated with anti-CD4 or rat IgG antibody. <b>(D)</b> Mesenteric lymph node cells of <i>T</i>. <i>spiralis</i>-infected mice treated with anti-CD4 or rat IgG antibody were treated with IL-25 with or without <i>T</i>. <i>spiralis</i> antigen for 72 hours and the indicated cytokines in the supernatant were assessed using ELISA. <b>(E)</b> Lin^- cells from the mesenteric lymph node of <i>T</i>. <i>spiralis</i>-infected mice treated with anti-CD4 or rat IgG antibody were enriched using a MACS column. The enriched ILC2 cells were then restimulated with IL-25 or PMA/ionomycin for 72 hours, and the indicated cytokines in the supernatant were assessed using ELISA. Data represented one of three independent experiments (n = 4 mice each group). Error bars denote mean ± SD. Significance was determined by one-way ANOVA * p <0.05, ** p <0.01 and *** p <0.001.</p

Type 2 innate lymphoid cells (ILC2s) and CD4⁺Th2 cells are the major immune cells expressed IL-25 receptor (IL-17RB) in response to <i>T</i>. <i>spiralis</i> infection.

<p><b>(A)</b> BALB/c mice were infected with <i>T</i>. <i>spiralis</i> and their lamina propria cells were analyzed for the expression of IL-17RB at day 7 following infection. The gated IL-17RB⁺ population from infected mice was stained with lineage markers (CD11b, CD11c, B220, CD19, DX5, Gr.1, CD8), IL-7Rα, ST2, CD3 and CD4. The expression of IL-7Rα and ST2 on Lin^- cells from IL-17RB⁺ (open histogram) were compared with those from IL-17RB^- (shaded histogram) population. <b>(B and C)</b> Phenotypic analysis and frequencies of IL-17RB⁺GFP⁺ by CD3⁺CD4⁺ and Lin^- cells in the lamina propria of the small intestines <b>(B)</b> and mesenteric lymph nodes <b>(C)</b> of 4GET mice following <i>T</i>. <i>spiralis</i> infection at days 3, 7 and 14. <b>(D)</b> Cell surface marker expression (IL-7 receptor α, KLRG1, ST2, c-KIT and Thy1) in the IL-17RB⁺GFP⁺Lin^- cells in the lamina propria of the small intestines after <i>T</i>. <i>spiralis</i> infection for 7 days. Flow cytometric data shown are a representative histogram profile of one of three independent experiments. The solid gray line indicates the plot profile of isotype-matched control antibody and shaded histogram represents the profile of the indicated antibody. The data represent one of three independent experiments (n = 4 mice each group). Error bars denoted mean ± SD. Significance was determined using one-way ANOVA * p <0.05, ** p <0.01*** and p <0.001 (compared with data in naïve mice).</p

ILC2s activated by IL-25 promote antigen-specific Th2 and Th9 functions that contribute to the control of <i>Trichinella spiralis</i> infection

<div><p>IL-25, an IL-17 family cytokine, derived from epithelial cells was shown to regulate Th2- and Th9-type immune responses. We previously reported that IL-25 was important in promoting efficient protective immunity against <i>T</i>. <i>spiralis</i> infection; however, the cellular targets of IL-25 to elicit type-2 immunity during infection have not yet been addressed. Here, we investigated IL-25-responding cells and their involvement in mediating type-2 immune response during <i>T</i>. <i>spiralis</i> infection. ILC2 and CD4⁺ Th2 cells residing in the gastrointestinal tract of <i>T</i>. <i>spiralis</i> infected mice were found to express high levels of surface interleukin-17 receptor B (IL-17RB), a component of the IL-25 receptor. Following <i>T</i>. <i>spiralis</i> infection, activated ILC2s upregulated surface MHCII expression and enhanced capacity of effector T helper cell in producing antigen-specific Th2 and Th9 cytokines through MHCII-dependent interactions. Reciprocally, lack of CD4⁺ T helper cells impaired ILC2 function to produce type 2-associated cytokines in responding to IL-25 during <i>T</i>. <i>spiralis</i> infection. Furthermore, mice deficient in IL-17RB showed markedly reduced ILC2 numbers and antigen-specific Th2 and Th9 cytokine production during <i>T</i>. <i>spiralis</i> infection. The <i>Il17rb</i>^-/- mice failed to mount effective antigen specific Th2 and Th9 functions resulting in diminished goblet cell and mast cell responses, leading to delayed worm expulsion in the intestines and muscles. Thus, our data indicated that ILC2s and CD4⁺ Th2 cells are the predominant cellular targets of IL-25 following <i>T</i>. <i>spiralis</i> infection and their collaborative interactions may play a key role in mounting effective antigen-specific Th2 and Th9 cytokine responses against <i>T</i>. <i>spiralis</i> infection.</p></div

Signaling through IL-17RB is essential to induce ILC2 and antigen specific Th2 and Th9 responses.

<p><i>Il17rb</i>^-/-/4GET or wild type 4GET mice were infected with <i>T</i>. <i>spiral</i>is. <b>(A)</b> Detection of IL-17RB in lineage negative (CD11b^-CD11c^-B220^-CD49b^-Gr.1^-CD335^-CD3^-CD4^-CD8^-) and CD3⁺CD4⁺ T cells. <b>(B)</b> Frequency and numbers of CD3⁺CD4⁺GFP⁺ cells at 7 and 14 days postinfection were evaluated in the mesenteric lymph node of infected <i>Il17rb</i>^-/-/4GET or wild type mice. <b>(C)</b> Detection and frequency of ILC2s (CD3^-CD4^-CD127⁺T1/ST2⁺KLRG1⁺GFP⁺) in the lamina propria of the small intestines of <i>Il17rb</i>^-/-/4GET or wild type 4GET mice infected with <i>T</i>. <i>spiral</i>is at 7 and 14 days postinfection. <b>(D)</b> Effector/memory (CD3⁺CD4⁺CD44⁺) T cells from mesenteric lymph nodes of <i>T</i>. <i>spiralis</i>-infected wild type and <i>Il17rb</i>^<i>-/-</i> mice were sorted and subjected to analyze the expression of indicated cytokine using real-time PCR. Data are expressed as fold induction over actin (<i>Actb</i>) expression, with the mRNA levels in the naïve group set as 1. <b>(E)</b> At day 7 and 14 postinfection, mesenteric lymph node cells were harvested and single cell suspensions were then cultured with or without <i>T</i>. <i>spiralis</i> extract antigen (10 μg/ml). After three days, the supernatant was collected and analyzed for <i>T</i>. <i>spiralis</i>-specific cytokine production by ELISA. Graphs depict mean±SD and are a representative of at least three independent experiments with three to four mice each group. Significance was determined by one-way ANOVA with Tukey’s post hoc analysis * p <0.05, ** p <0.01 and *** p <0.001.</p

Absence of IL-17RB resulted in reduced intestinal Th2 and Th9 effector responses and attenuated intestinal and muscle <i>T</i>. <i>spiral</i>is worm clearance.

<p><i>Il17rb</i>^-/- or BALB/c mice were infected with <i>T</i>. <i>spiral</i>is. <b>(A)</b> At days 7 and 14 postinfection, the small intestines (jejunum) were harvested and subjected to RNA extraction, followed by cDNA synthesis and cytokine gene expression using real-time PCR analysis. Data are expressed as fold induction over actin (<i>Actb</i>) expression, with the mRNA levels in the naïve group set as 1. <b>(B)</b> The small intestines (jejunum) were fixed with 10% formalin buffer and subjected to histological analysis of goblet cells by Periodic acid–Schiff (PAS) staining. Numbers of goblet cells were expressed per villus crypt unit (VCU). <b>(C)</b> At days 7 and 14 postinfection, the whole intestines of <i>T</i>. <i>spiral</i>is-infected <i>Il17rb</i>^-/- or BALB/c mice were harvested and analyzed for adult worms. <b>(D)</b> At day 30 postinfection, whole carcasses of infected mice from <i>Il17rb</i>^-/- or wild type groups were analyzed for muscle larvae burden. Graphs depict mean±SD and are a representative of at least three independent experiments with four mice each group. Significance was determined using one-way ANOVA with Tukey’s post hoc analysis * p <0.05, ** p <0.01 and *** p <0.001.</p

The effect of the JAK3 inhibitor on rhesus macaque NK cell function in vitro/ in vivo and on the frequencies of the major cell lineages in the blood.

<p>A) PBMC from 9 normal rhesus macaques was assayed for NK activity in the presence and absence of varying concentrations of the JAK-3 inhibitor. The mean lytic units/10⁶ effector cells were calculated. The S.D. was always <10%. B) Two normal rhesus macaques were orally administered 10 mg/kg of the JAK3 inhibitor and PBMC samples obtained prior to and at 1, 3, 24, 48, 72 and 168 hrs. post JAK3 administration and assayed for NK activity. Results reflect Mean lytic units of NK activity/10⁶ effector cells performed in triplicated with the S.D. <10%. C) Three normal rhesus macaques were orally administered the JAK3 inhibitor at 10 mg/kg daily for 14 days and an aliquot of their PBMC obtained prior to and at day 7 and 14 subjected to polychromatic flow cytometric analysis for the frequencies of CD4⁺ T cells, CD8⁺ T cells, CD20⁺ B cells and CD3⁻/CD8⁺/NKG2a⁺ cells. An aliquot was utilized for CBC and the absolute numbers of each cell lineage calculated and illustrated. Note the major depletion of the NK cells D). Another aliquot of the PBMC from C) were assayed for NK cell function and the data shows the Mean lytic units/10⁶ effector cells. The S.D. was always <10%.</p

<i>In Vivo</i> Administration of a JAK3 Inhibitor to Chronically SIV Infected Rhesus Macaques Leads to NK Cell Depletion Associated with Transient Modest Increase in Viral Loads

<div><p>Innate immune responses are reasoned to play an important role during both acute and chronic SIV infection and play a deterministic role during the acute stages on the rate of infection and disease progression. NK cells are an integral part of the innate immune system but their role in influencing the course of SIV infection has been a subject of debate. As a means to delineate the effect of NK cells on SIV infection, use was made of a Janus kinase 3 (JAK3) inhibitor that has previously been shown to be effective in the depletion of NK cells <i>in vivo</i> in nonhuman primates (NHP). Extensive safety and <i>in vitro/in vivo</i> PK studies were conducted and an optimal dose that depletes NK cells and NK cell function <i>in vivo</i> identified. Six chronically SIV infected rhesus macaques, 3 with undetectable/low plasma viral loads and 3 with high plasma viral loads were administered a daily oral dose of 10 mg/kg for 35 days. Data obtained showed that, at the dose tested, the major cell lineage affected both in the blood and the GI tissues were the NK cells. Such depletion appeared to be associated with a transient increase in plasma and GI tissue viral loads. Whereas the number of NK cells returned to baseline values in the blood, the GI tissues remained depleted of NK cells for a prolonged period of time. Recent findings show that the JAK3 inhibitor utilized in the studies reported herein has a broader activity than previously reported with dose dependent effects on both JAK2 and JAK1 suggests that it is likely that multiple pathways are affected with the administration of this drug that needs to be taken into account. The findings reported herein are the first studies on the use of a JAK3 inhibitor in lentivirus infected NHP.</p></div

Analysis of Th17⁺ cells in PBMC and corresponding colo-rectal biopsies from SIV infected nonhuman primates.

<p>Frequencies (%) and absolute numbers (#) of CD4⁺ Th17 cells in the PBMC and colorectal biopsies of groups of uninfected (Uninf), and SIV infected rhesus macaques classified as Elite Controllers (EC), those with plasma levels of Low Viral Loads (LVL), Intermediate Viral Loads (IVL), and High Viral Loads (HVL) in the left panels. The right panels reflect the same measurements in SIV seronegative (Negative) and SIV seropositive (Positive) sooty mangabeys assayed in parallel. A) Reflects the data on % CD4⁺ Th17 cells in PBMC B) the absolute number of CD4⁺ Th17 cells C) the % CD4⁺ Th17 cells in colorectal biopsy tissues and D) the % CD4⁺ T cells in the same aliquot of biopsies. Statistically significant data are reflected by the number of asterisks with p<0.02 (*). p<0.05 (**), p<0.01 (***), p<0.001 (****) and p<0.0001 (*****) for all figures.</p

Analysis of CD4⁺ Tregs cells in PBMC and corresponding colo-rectal biopsies from SIV infected nonhuman primates.

<p>Nomenclature identical to Fig. 1 except the data reflects values for CD4⁺ Tregs. Please note D) reflects the frequencies of total CD4⁺ T cells in the same sample.</p

Analysis of IFN-α⁺ pDC cells in PBMC and corresponding colo-rectal biopsies from SIV infected nonhuman primates.

<p>The frequencies of IFN-α synthesizing pDCs in the PBMC (A) and colo-rectal biopsy tissues (B) of the same group of animals as depicted in Fig. 1. The blank bars reflect the frequencies of pDCs that constitutively express IFN-α and the dark bars reflect the frequencies of pDCs that are induced to express IFN-α.</p