Behcet’s disease (BD) is a chronic, multi-systemic disorder that has arthritic, intestinal, mucocutaneous, ocular, vascular, and central nervous system affects. BD takes a chronic course with periodic exacerbations and progressive deterioration (Shimizu, et al., 1978). The etiology of BD is unclear, but viral infection has long been postulated as one of the main factors. Since Hulusi Behcet first proposed a viral etiology, the viral hypothesis has been verified by detection of the virus in the saliva, intestinal ulcers, and genital ulcers of patients with BD (Behcet, 1937; Lee, et al., 1996; Bang, et al., 1997; Lee, et al., 1997). Subsequent to these findings, inoculation of the earlobes of ICR mice with herpes simplex virus (HSV) was found to result in the development of BD symptoms (Sohn, et al., 1998). Manifestations in mice after HSV inoculation include multiple symptoms such as oral ulcers, genital ulcers, skin ulcers, eye symptoms, intestinal ulcers, arthritis, and neural involvement, as well as skin crusting (Sohn, et al., 2001).
In BD patients, the frequencies of CD45RO+ cells were lower than disease control group has been reported (Takase, et al., 2006). By this time, there was no report related to the improvement accompanies regulation of memory T cells in BD patients except up-regulation of CD4+CD45RO+ cells after Thalidomide treatment (Direskeneli, et al., 2008).
Interleukin-15 (IL-15) is a pleiotropic cytokine that plays important roles in both innate and adaptive immunity. It is associated with a range of immunopathology, including rheumatoid arthritis and allograft rejection. IL-15 exerts its effects through binding to a
membrane receptor which consists of a high affinity binding α-chain (IL-15Rα), and the β-chain, common receptor subunit IL-2R also known as CD122, and the common γ subunit of different type I cytokine receptors (γC chain, CD132) (Tagaya, et al., 1996; Waldmann, et al., 1998; Wei, et al., 2001). Increased IL-15 production was associated with proliferation and survival of memory-phenotype CD8+ T cells (Judge, et al., 2002) and Interleukin-15 receptor alpha (IL-15Rα) supports memory T cells in vivo (Burkett, et al., 2003).
Polyinosinic:polycytidylic acid (Poly I:C) is a mismatched double-stranded RNA with one strand being a polymer of inosinic acid, and the other is a polymer of cytidylic acid. Poly I:C, immunostimulant, induces IL-15 and IL-15Rα (Lodolce, et al., 2001) which is essential for binding IL-15 (Lorenzen, et al., 2006) and boosts the generation of memory T cells (Wang, et al., 2010).
We previously observed the frequencies of CD8+CD44+ and CD8+CD62L- memory T cells in BD mice were significantly lower than BDN mice. In this study, we found that the frequencies of IL-15Rα were significantly lower in HSV-induced BD mouse model than control mice. Therefore, we investigated to determine whether Poly I:C supplementation could induce memory cells through up-regulation of IL-15Rα, and reduce inflammation in HSV-induced BD symptoms.
Ⅱ. MATERALS AND METHODS
protocol approved by the animal care committee of Ajou University School of Medicine.B. BD symptoms in a mouse model
Manifestations in mice after HSV inoculation involved multiple symptoms. Of the total number of HSV-infected mice, 15% developed BD symptoms. Symptoms in human patients including oral ulceration, genital ulceration, erythema, skin pustules, skin ulcerations, joint-arthritis, diarrhea, red eye (right, left), reduced vision (right, left), loss of balance, discoloration, and swelling of the face were selected and analyzed as BD symptoms.
Oral, genital, and other skin ulcers and eye symptoms were classified as major symptoms.
Arthritis, gastrointestinal ulcers, and neurological involvement were identified as minor symptoms. Mice with ≥ 1 major and 1 minor symptoms were classified as having BD. The score of each symptom was one and the sum of the symptoms were used to determine the severity of BD. The disappearance of symptoms or a decrease in the lesion size of more than 20% was classified as improvement. The severity of BD was followed by determination of
the modified Behcet’s disease activity index as outlined in the BD Activity Form (www.behcet.ws/pdf/BehcetsDiseaseActivity-Form.pdf). As a control group, HSV was inoculated, but asymptomatic healthy mice were used as BD Normal (BDN) as previously described.
C. In vivo administration of Poly I:C
Polyinosinic-Polycytidylic acid (Poly I:C) (Sigma Chemicals, St. Louis, Mo, USA) was resuspended in physiological phosphate buffered saline (PBS) at a stock concentration of 10 mg/ml, aliquoted, and stored in freezer (-20°C) prior to use. 5 week aged male normal mice were randomly placed into the following treatment groups: PBS, Poly I:C 0.05, 0.2, 1 µg/g body weight. Normal mice were injected i.p. with Poly I:C or PBS for 2 times at 3-days interval. BD mice were injected i.p. with Poly I:C or PBS for 4 times at 3-days interval. Two days or seventeen days after the last administration of Poly I:C or PBS, peripheral blood mononuclear cells (PBMC), spleen cells, and lymph node cells were isolated for flow cytometric analysis and the sera were collected for ELISA.
D. Flow cytometric analysis of cell surface staining
Mouse peripheral blood mononuclear cells (PBMC), spleen cells, and lymph node cells were washed with phosphate buffered saline (PBS), after which 1x106 cells were incubated with 0.25 µg of APC-labeled anti-IL-15Rα (R&D system), FITC-labeled anti-CD4, CD122, PerCP-Cy5.5-labeled CD8, PE-labeled IL-7Rα, CD44, anti-CD62L (eBiosciences) for 30 min at 4°C. The stained cells were then washed with PBS and
analyzed by using a flow cytometer (FACSAria III; Becton Dickinson, San Jose, CA, USA) with X10,000 gated lymph node cells.
E. Enzyme-linked immunosorbent assay (ELISA)
Serum was analyzed by commercial ELISA kits for the detection of mouse IL-15 (eBioscience, San Diego, CA) and manual ELISA using monoclonal anti-mouse IL-17 antibody, biotinylated anti-mouse IL-17 antibody (R&D System, Minneapolis, MN), recombinant murine IL-17 (Peprotech, Rocky Hill, NJ), and was conducted according to the manufacturer’s recommendation. The means and standard deviations were calculated using ELISA values determined for each well. The ELISA values measured using a model 680 microplate reader (Bio-Rad, Hercules, CA,USA) and determined the absorbance at 450 nm.
F. Reverse transcription PCR (RT-PCR)
Total RNA was isolated with TRIzol (Life Technologies, Helgerman, CT), according to the manufacturer’s recommendations. An amount of 1 µg of total RNA was used as template for cDNA synthesis with AccuPowerTM RT PreMix for RT-PCR kit (Bioneer, Alameda, CA). The cDNA was amplified by PCR with the following primers: β-actin, sense:
5'-TGGAATCCTGTGGCATCCATGAAAC-3', antisense: 5'-TAAAACGCAGCTCAGTAA CAGTCCG-3'; mIL-23R (Kamiya, et al., 2007), forward primer: 5'-GCACTGCCGACCAA GGAATC-3' and reverse primer: 5'-GAGTTCTCCATGCCTAGGGA-3'. Amplified PCR products were visualized on 1.6-1.7% agarose gels.
H. Statistical analysis
All data shown represent the mean ± SE. Statistical differences between the experimental groups were determined using the Student’s t-test and Bonferroni correction.
Statistical analysis was conducted using MedCalc® version 9.3.0.0.
Ⅲ. RESULTS
A. The frequencies of IL-15Rα expressing cells in BD mice compared with BDN mice
The frequencies of IL-15Rα were analyzed in PBMC, splenocytes, and lymph node (LN) cells of BD and BDN mice (BD normal, HSV was inoculated but no symptomatic mice) by FACS analysis. The frequencies of IL-15Rα-expressing cells in PBMC from BD mice (n=11) were lower than BDN mice (n=10)(10.19 ± 3.22 vs. 13.58 ± 7.23%, p=0.03) (Fig.
1A). The expression pattern of frequencies was similar in splenocytes (n=3)(4.63 ± 0.12 vs.
5.7 ± 0.61%, p=0.04) (Fig. 1B). Whereas in lymph node cells, the frequencies were not significantly different between BD (n=8) and BDN mice (n=10)(11.55 ± 5.84 vs. 11.09 ± 5.66%, p=0.9) (Fig. 1C). The frequencies of IL-15Rα-expressing cells in PBMC and splenocytes from BD mice were significantly lower than BDN mice.
Fig. 1. The expression of IL-15R
and BDN mice. The data show the frequencies of IL cytometry in cells isolated from
BDN mice.
15Rα in PBMC, splenocytes and lymph node cells
The data show the frequencies of IL-15Rα were analyzed by Flow PBMC (A), spleens (B), and lymph nodes (C) from
lymph node cells from BD were analyzed by Flow (C) from BD and
B. Poly I:C up-regulates expression of IL-15Rα in normal mice
To determine whether the expression of IL-15Rα can be regulated by Poly I:C, Poly I:C or PBS (control) was injected into normal mice. At 2 days after the second injection of Poly I:C or PBS, the cells were isolated from blood of mice. Then, the frequencies of IL-15Rα were measured by flow cytometry (Fig. 2A). The frequencies of IL-IL-15Rα expression on PBMC from normal mice following Poly I:C 0.05 µg/g (n=2)(6.9 ±0.85), Poly I:C 0.2 µg/g (n=4)(8.3 ±2.45), Poly I:C 1 µg/g (n=2)(7.7 ±2.83), and PBS (n=3)(4.3 ±0.79) (Fig. 2B).
The frequencies of IL-15Rα on PBMC from 0.2 µg/g of Poly I:C treated mice were significantly increased than that found in PBMC from PBS administered control mice (p=0.04). 0.2 µg/g of Poly I:C most efficiently up-regulated the expression of IL-15Rα.
Therefore, we chose to apply 0.2 µg/g of Poly I:C for further experiments. In addition, the frequencies of IL-15Rα expression on splenocytes and lymph node cells from 0.2 µg/g of Poly I:C treated mice (n=5) were also up-regulated than that found from PBS administered control mice (n=3)(3.6 ±1.92 vs. 5.9 ±1.90%, p=0.1; 5.2 ±1.01 vs. 6.7 ±1.45%, p=0.2) but data was not significant.
Fig. 2. The expression of IL-15Rα were up-regulated by Poly I:C injection in a dose dependent manner in normal mice. 5 weeks aged male normal mice were randomly placed into the following treatment groups: PBS, Poly I:C 0.05, 0.2, 1 µg/g body weight for each group. (A) The mice were injected i.p. with PBS or Poly I:C for 2 times at 3-days interval.
Two days after the last injection, the mice were sacrificed and PBMC were isolated, and the expression of IL-15Rα was analyzed by Flow cytometry. (B) The data show the frequency of IL-15Rα in cells isolated from PBMC, spleens, and lymph nodes after Poly I:C or PBS injection.
C. Poly I:C ameliorates HSV-induced BD symptoms
To determine whether Poly I:C can improve the BD symptoms, we conducted intra-peritoneal injection of Poly I:C or PBS into BD mice for four times with three days interval (Fig. 3A). Before and 2 weeks after the last injection, the changes of BD symptoms in BD mice were photographed. The cutaneous symptoms were improved in Poly I:C treated BD mice compared to PBS injected BD mice (Fig. 3B). In Poly I:C injected BD mice, BD symptoms were ameliorated in 15 of 24 BD mice (63%) or not changed in 5 of 24 BD mice (21%) whereas deteriorated in 2 of 24 BD mice (8%) or died in 2 of 24 BD mice (8%).
However, in PBS injected BD mice, the BD symptoms were ameliorated in 3 of 20 BD mice (15%) or not changed in 7 of 20 BD mice (35%) whereas deteriorated in 6 of 20 BD mice (30%) or died in 4 of 20 BD mice (20%) (Table 1). Poly I:C administered BD mice with cutaneous symptoms showed diminished lesion size by 20% to 100% of the total area. The change of BD symptoms in BD mice was observed and scored according to the severity score of BD patients, which is outlined in the BD Current Activity Form. The scoring was followed by the changes of superficial symptoms because it is impossible to communicate with mice. In Poly I:C injected group, the severity score was decreased from 2.57 ±0.66 to 1.98 ±1.19 at 2 weeks after the first injection (n=22). Whereas, in the PBS injected group, the severity score was increased from 2.94 ±0.87 to 3.07 ±0.98 at 2 weeks after the first injection (n=14). At 2 weeks after the first injection, the severity score significantly lower in Poly I:C injected BD mice (n=22) than PBS injected BD mice (n=14)(1.98 ± 1.19 vs. 3.07
±0.98, p=0.006) (Fig. 3C).
Fig. 3. The change of symptoms in BD mice after injection of Poly I:C. (A) The used experimental schedule throughout this study. Poly I:C or PBS was injected i.p. into BD mice for 4 times at 3-days interval, and the symptoms were photographed and the severity score was analyzed. (B) The symptoms of BD mice were compared before and after injected with Poly I:C or PBS. (C) The severity score of Poly I:C or PBS injected BD mice. The disease score was estimated according to the Patients Index Score, Behcet’s disease current activity form 2006, ICBD.
Table 1. The change of symptoms in BD mice after injection of Poly I:C.
Table 1. The change of symptoms in BD mice after injection of Poly I:C.
D. Poly I:C up-regulates the frequencies of IL-15Rα but not IL-7Rα (CD127) in BD mice.
To confirm the frequencies of IL-15Rα in Poly I:C injected BD mice, the cells were isolated from PBMC, spleen, and lymph nodes at 2 and 17 days after injection. As has been reported Poly I:C elicited a brief phase of T cell proliferation peaking at day 2 post-injection and was followed for 7 to 32 days (Zhang, et al., 1998; West, et al., 2011). At 2 days after the last injection, the frequencies of IL-15Rα from Poly I:C injected group were significantly increased than PBS injected group in PBMC (16.8±2.6 vs. 9.2±3.5%, p<0.001), spleen (12.9±2.8 vs. 4.6±0.1%, p=0.001), and lymph nodes (21.8±2.3 vs. 9.6±6.0%, p<0.001). At 17 days after the last injection, the frequencies of IL-15Rα from Poly I:C injected group was significantly increased than PBS injected group in PBMC (27.4 ±9.8 vs. 9.2 ±3.5%, p<0.0001) but recovered in spleen (5.2 ±2.1 vs. 4.6 ±0.1%, p=0.7) and lymph node (11.0
±11.0 vs. 9.6 ±6.0%, p=0.7) (Fig. 4A). In PBMC of Poly I:C injected group, the up-regulated expression of IL-15Rα were sustained until 17 days (at 17 days vs. at 2 days, 27.4 ±9.8 vs.
16.8 ±2.6%, p=0.045). In spleen and lymph nodes of Poly I:C injected group, up-regulated IL-15Rα was decreased to control level at 17 days.
The frequencies of IL-7Rα expression in spleen cells were isolated from BD mice (n=5) were significantly lower than BDN mice (n=5)(28.5±3.8 vs. 20.0±1.4%, p=0.002) but not different in PBMC (42.6±12.5 vs. 37.8±7.8%, p=0.5) and lymph nodes cells (33.4±11.2 vs. 39.0±5.7%, p=0.3) between BD mice and BDN mice. Actually, IL-15 and IL-7 cytokines were involved with the generation and maintenance of memory CD8+ T cells (Kimberly, et al., 2003). These cytokines interact with IL-15Rα and IL-7Rα respectively, as has been
reported, the down-regulation of IL-15Rα and IL-7Rα expression is a contributing factor for the poor survival of memory CD8+ T cells in the airways (Shen, et al., 2008). To determine whether the injection of Poly I:C into BD mice can induce not only IL-15Rα but also IL-7Rα, we analyzed the frequencies of IL-7Rα in the same method. At 2 days after the last injection, the frequencies of IL-7Rα from Poly I:C injected group were not different compared with PBS injected group in PBMC (Poly I:C vs. PBS, 41.9 ±18.1 vs. 45.7 ±20.0%, p=0.7), spleen (26.1 ±6.4 vs. 28.0 ±12.9%, p=0.8), and lymph nodes (45.6 ±12.6 vs. 41.8 ±14.8%, p=0.6).
At 17 days after the last injection, the frequencies of IL-7Rα from Poly I:C injected group were also not different compared with PBS injected group in PBMC (Poly I:C vs. PBS, 51.0
±20.4 vs. 45.7 ±20.0%, p=0.6), spleen (23.4 ±14.0 vs. 28.0 ±12.9%, p=0.5), and lymph nodes (47.5 ±12.3 vs. 41.8 ±14.8%, p=0.4) (Fig. 4B). Poly I:C elevates the level of IL-15Rα but not IL-7Rα in BD mice. We next investigated the serum level of IL-7 and IL-15 by ELISA to determine whether the serum level of IL-7 and IL-15 was changed in BD mice after Poly I:C injection. Serum levels of IL-7 were 46.08±38.67 pg/ml in BDN (n=12) and 32.84±10.41 pg/ml in BD (n=13) by ELISA. The level of IL-7 was not different between BD mice and BDN mice (p=0.2). Serum IL-7 levels were elevated after injection of Poly I:C in BD mice at 2 days after the last injection (Poly I:C (n=6) vs. PBS (n=8))(48.8 ±18.22 vs.
34.33 ±13.12 pg/ml, p=0.1) and at 17 days after the last injection (Poly I:C (n=6) vs. PBS (n=8))(54.75 ±15.89 vs. 34.33 ±13.12 pg/ml, p=0.02) (Fig. 4C). Although the frequency of IL-7Rα were not affected, the serum level of IL-7 significantly up-regulated by Poly I:C injection in BD mice. The serum level of 15 was not analyzed because reliable mouse
IL-Fig. 4. The expression of IL-15Rα, IL-7Rα in PBMC, splenocytes and lymph node cells isolated at 2 and 17 days after the last injection of Poly I:C in BD mice. The cells were isolated from PBMC, spleen and lymph nodes at 2 and 17 days after the last injection of Poly I:C or PBS. The expression of IL-15Rα and IL-7Rα were measured by Flow cytometry.
The data show the frequencies of IL-15Rα (A) and IL-7Rα (B) in PBMC, splenocytes, and lymph node cells from PBS or Poly I:C injected BD mice. (C) The serum levels of IL-7 were measured by ELISA in BDN, Poly I:C or PBS injected BD mice.
E. Poly I:C induces CD8+CD44+ memory T cells in BD mice
There has been reported, IL-15Rα on bone marrow derived cells trans-present IL-15 and it mediates the basal proliferation of memory CD8+ T cells (Schluns, et al., 2004). We previously observed the frequencies of CD8+CD44+ and CD8+CD62L- memory T cells frequencies of memory cell types were measured by flow cytometry. At 2 days after the last injection, the frequency of CD4+CD44+ memory T cells in Poly I:C injected BD mice significantly increased than PBS injected BD mice in spleen (Poly I:C vs. PBS, 24.1 ±5.9 vs.
15.9 ±8.4%, p=0.03) and lymph node (44.7 ±6.8 vs. 33.7 ±10.9%, p=0.02) but was not significant at 17 days after the last injection in PBMC (Poly I:C vs. PBS, 34.2 ±16.3 vs. 22.8
±16.6%, p=0.1), spleen (26.6 ±14.1 vs. 15.9 ±8.4%, p=0.07), and lymph nodes (41.5 ±12.6 vs. 33.7 ±10.9%, p=0.5) (Fig. 5A). Whereas, the frequencies of CD8+CD44+ memory T cells were not different between Poly I:C and PBS injected group at 2 days after the last injection in PBMC (Poly I:C vs. PBS, 9.4 ±2.4 vs. 7.2 ±4.0%), spleen (3.8 ±1.2 vs. 3.4
±2.0%) and lymph node (7.8 ±2.6 vs. 10.5 ±5.5%). At 17 days after the last injection, the frequencies of CD8+CD44+ memory T cells from Poly I:C injected group were significantly increased than PBS injected group in PBMC (Poly I:C vs. PBS, 18.6 ±9.6 vs. 7.2 ±4.0%, p=0.003) and spleen (7.2 ±1.5 vs. 3.4 ±2.0%, p=0.0008) though there was no difference in
lymph node (14.0 ±9.2 vs. 10.5 ±5.5%, p=0.3) (Fig. 5B). The injection of Poly I:C significantly elevated CD8+CD44+ memory T cells in BD mice. We conducted to determine whether injection of Poly I:C in BD mice can induce not only CD8+CD44+ memory T cells but also CD4+CD62L- or CD8+CD62L- memory T cells. CD4+CD62L- and CD8+CD62L- memory T cells were measured in the same method. The frequencies of CD4+CD62L- memory T cells were not different between Poly I:C and PBS injected group in PBMC, spleen and lymph nodes (Fig. 5C). Whereas, the frequencies of CD8+CD62L- memory T cells significantly increased in Poly I:C injected group than PBS injected group at 2 days (Poly I:C vs. PBS, 8.8 ±2.4 vs. 2.4 ±1.6%, p=0.0004) and 17 days (10.9 ±3.9 vs. 2.4 ±1.6%, p=0.0006) after the last injection in PBMC but there was no difference in spleen and lymph nodes (Fig. 5D).
Fig. 5. The frequencies of memory T cells in PBMC, splenocytes and lymph node cells isolated at 2 and 17 days after the last injection of Poly I:C in BD mice. The cells were isolated from PBMC, spleen and lymph nodes at 2 and 17 days after the last injection of Poly I:C. The cells were stained for memory T cell markers and analyzed by Flow cytometry.
The data show the frequencies of CD4+CD44+ (A), CD8+CD44+ (B), CD4+CD62L- (C), CD8+CD62L- (D) in PBMC, splenocytes, and lymph node cells after Poly I:C injection.
F. Poly I:C up-regulate CD122(IL-15/2Rβ) and CD8+CD122+ T cells in BD mice
IL-15 binding to IL-15Rα likely leads to recruitment of CD122(IL-15/2Rβ) and common γ chain, and initiation of signaling cascades from these shared chains (Bamford, et al., 1994). We next investigated the frequency of CD122-expressing cells in Poly I:C injected BD mice to determine whether the expression of CD122 that demanded to signaling cascades of IL-15-15Rα complexes could be induced by Poly I:C.
The frequencies of CD122-expressing cells in PBMC (8.4±7.6 vs. 15.3±9.7%, p=0.2) and lymph nodes cells (4.6±2.0 vs. 5.7±1.1%, p=0.3) did little different between BD mice and BDN mice. At 2 days after the last injection, the frequencies of CD122-expressing cells were not different between Poly I:C and PBS injected BD mice in PBMC (Poly I:C vs. PBS, 12.1±4.8 vs. 8.4±7.6%) and lymph nodes (7.3±4.4 vs. 4.6±2.0%). At 17 days after the last injection, the frequencies of CD122-expressing cells from Poly I:C injected group were significantly elevated than PBS injected group in PBMC (Poly I:C vs. PBS, 25.9±12.0 vs.
8.4±7.6%, p=0.01) and lymph nodes (7.8±3.3 vs. 4.6±2.0%, p=0.05) (Fig. 6A). In result, the frequencies of CD122-expressing cells were significantly up-regulated in lymph nodes cells isolated form Poly I:C injected BD mice at 17 days after the last injection.
CD8+CD122+ T cells are newly identified regulatory T cells (Saitoh, et al., 2007) and reported the effect which involved anti-inflammatory responses (Rifa'i, et al., 2008) in the recovery phase of EAE mouse model (Lee, et al., 2008). The frequencies of CD8+CD122+ T cells in lymph nodes cells of BD mice (n=6) were significantly lower than BDN mice (n=5)(1.5±0.7 vs. 3.8±1.6%, p=0.009) but not different in PBMC between BD mice and
BDN mice (4.0±2.4 vs. 5.7±2.6%, p=0.4). At 2 days after the last injection, the frequencies of CD8+CD122+ cells were not different between Poly I:C and PBS injected BD mice in PBMC (Poly I:C vs. PBS, 3.9±3.1 vs. 6.1±1.3%) and lymph nodes (4.0±2.9 vs. 3.3±0.1%, p=0.07). At 17 days after the last injection, the frequencies of CD8+CD122+ cells from Poly I:C injected group (n=4) were significantly increased than PBS injected group (n=6) in lymph node (3.6±1.2 vs. 1.5±0.7%, p=0.008) though there was no difference in PBMC (5.8±1.0 vs. 4.0±2.4%, p=0.2) (Fig. 6B). In the result, the frequencies of CD8+CD122+
regulatory T cells were significantly higher in lymph nodes cells isolated from BDN mice compared with BD mice. Administration of Poly I:C into BD mice up-regulated the frequencies of CD8+CD122+ regulatory T cells in lymph nodes cells.
Fig. 6. The frequencies of CD122(IL-15/2Rβ) and CD8+CD122+ T cells in lymph node cells isolated at 2 and 17 days after the last injection of Poly I:C into BD mice. The cells were isolated from lymph nodes at 2 or 17 days after the last injection of Poly I:C. The frequencies of CD122- and CD8+CD122+-expressing cells were analyzed by Flow cytometry. The data shows the frequencies of CD122+ (A), CD8+122+ cells in PBMC and lymph node cells (B) and the dot plot of CD8+CD122+ T cells in lymph node cells from BDN, PBS or Poly I:C injected BD mice group (C).