Unless otherwise specified, chemicals and laboratory equipment used in this study were obtained from Sigma Aldrich Chemical Co. (St. Louis, MO, USA) and SPL Life Sciences (Pocheon, Korea), respectively. BALB/c mice were purchased from Charles River Technology through Orient Bio, Inc. (Sungnam, Korea) and were maintained under general specific pathogen-free conditions with food and water ad libitum. Experimental procedures using mice were approved by the institutional Animal Care and Use Committee of Chonbuk National University (Approval No. CBU 2014-00060) and followed guidelines suggested by the committee.

A strain of Paecilomyces hepiali, CBG-CS-1, from the collection at the Chebigen, Inc. (Jeonju, Korea) was initially grown on potato-dextrose agar medium in Petri dishes. It was then transferred to a seed culture medium (4% dextrose, 1% yeast extract, and 1% peptone). The seed culture was again transferred to a 500-ml flask containing a nutritive medium with 100 mL 2% potato-dextrose broth and grown at 25℃ in a shaking incubator (150 rpm for 5 days). The culture was then transferred to a stirred tank fermenter containing a culture medium of 4% dextrose, 1% yeast extract, and 1% peptone with a 10% (v/v) working volume and then fermented for 5 days at 150 rpm. The fermented product was concentrated to 10% of the initial volume using a vacuum concentrator and an extractor at 80℃. The extract was freeze-dried and used as the extract (CBG-CS-2) of the cultured mycelium of Paecilomyces hepiali in this study. The main components of the CBG-CS-2 were analyzed to be cordyceps polysaccharide (30%), D-mannitol (7.3%), adenosine (1.3%), and cordycepin (0.01%).

Splenocyte proliferation was determined using a thymidine incorporation assay. Splenocytes were isolated from the mice (6~7 week-old) and distributed into each well of a 96-well culture plate (1×10⁵ cells) with 1 µg/ml concanavalin A (ConA) or 2 µg/ml LPS, together with various concentrations of CBG-CS-2. The plates were incubated for approximately 44 hours and then 0.5 µCi of [³H]-TdR (Amersham Life Science, Buckinghamshire, UK) was added to each well as a pulse to assess cell proliferation. After a 12-hour chase incubation, the cells were collected with a 96-well cell harvester (Inotech, Dottikon, Switzerland), and the incorporated tritium content was counted using a liquid scintillation counter (Perkin-Elmer, Waltham, MA, USA). Stimulation indices were calculated by dividing the tritium incorporation (cpm) in cells treated with the sample with the incorporation in control cells treated with PBS.

In order to analyze cytokine production in splenocytes, the cells (5×10⁶) were distributed into each well of a 24-well plate and stimulated with Con A (2 µg/ml), together with various concentrations of CBG-CS-2. The culture medium was collected 48 hours after stimulation and the expression levels of various cytokines were measured using a BD Cytometric Bead Array mouse cytokine kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's recommendation. The results were analyzed using FCAP Array software.

The non-specific cytotoxicity of CBG-CS-2 against an NK cell line, NK-92MI, kindly provided by Dr. D. Cho (Sookmyung Women's University, Seoul, Korea), was examined by determining the optimal concentration of CBG-CS-2 that activates the NK-92MI cells, without killing the cells. NK-92MI cells (1×10⁵ cells/ml) were incubated with various concentrations of CBG-CS-2 for 24-144 hrs at 37℃. Cells were then harvested and washed with PBS. Finally, the cells were stained with trypan blue and cell viability was calculated.

NK cell activity was measured by the ⁵¹Cr release assay, using K562 (American Type Culture Collection, Manassas, VA, USA) as target cells. Initially, K562 target cells were labeled with 100 µCi of chromium-51 radionuclide (Perkin-Elmer). In order to prepare effector cells, NK-92MI cells (1×10⁵ cells/ml) were incubated with various concentrations of CBG-CS-2 sample for 72-144 hrs and then harvested. The cells were washed and used for ⁵¹Cr release assay for measuring NK cell activity.

The ⁵¹Cr release assay was performed as described previously (22) with minor modifications. Various numbers of NK-92MI effector cells in 100-µl volumes were plated in the wells of a round-bottomed 96-well plate, and ⁵¹Cr-labeled K562 target cells (1×10⁴ cells/50 µl) were added and incubated for 4 hrs. After incubation, cell-free supernatants were collected and the level of ⁵¹Cr release was measured using a gamma counter (Packard Instrument, Meriden, CT, USA). Maximum release was induced by the addition of 100 µl of 5% Triton X-100 and spontaneous release was measured in the presence of 100 µl of medium only. NK cell-mediated cytotoxicity was defined as the percentage of specific lysis using the following equation: Specific lysis (%)=(Experimental release-Spontaneous release)/(Maximum release-Spontaneous release)×100.

Statistical analyses were performed using SigmaPlot™ (Systat Software, Chicago, IL, USA). Results are presented as the mean±standard error (SE). The unpaired Student's t-test was used to compare groups, and p-values less than 0.05 were considered statistically significant.

To assess the influence of CBG-CS-2 on in vitro immune cell stimulation, splenocyte proliferation was measured after in vitro incubation of the cells with various concentrations of CBG-CS-2 (Fig. 1). When we measured the in vitro CBG-CS-2-mediated splenocyte proliferation after adding T cell mitogen, Con A, approximately 40% increase in splenocyte proliferation was observed at 10 ng/ml of CBG-CS-2 compared to the control (Fig. 1A). Similarly, approximately 40% increase in splenocyte proliferation was observed compared to the control when 10 ng/ml of CBG-CS-2 was added together with a B cell mitogen, LPS, in vitro (Fig. 1B). Furthermore, the addition of CBG-CS-2 at 10-fold higher concentration (100 ng/ml) together with LPS, led to approximately 60% increase in splenocyte proliferation. However, addition of CBG-CS-2 at 1,000-fold higher concentration (1 µg/ml) slightly inhibited splenocyte proliferation in both Con A-treated and LPS-treated cells, although the levels were still higher than those obtained in control experiments without the addition of CBG-CS-2. Importantly, we could not detect any changes in splenocyte proliferation when either a placebo or a CBG-CS-2 alone was used to stimulate splenocytes in vitro (data not shown). These results suggest that CBG-CS-2 stimulates the non-specific proliferation of T and B lymphocytes in vitro.

We next determined the type of T cells stimulated in vitro by CBG-CS-2. Among the various cytokines measured from the cell culture supernatant after in vitro stimulation of splenocytes by CBG-CS-2, we detected increased expression of the Th1-type cytokine, IFN-γ (Fig. 2). As shown in Fig. 2, as low as 1 µg/ml CBG-CS-2 treatment significantly enhanced the expression (p<0.001, 27.78 pg/ml) of IFN-γ. However, we did not observe the same effects on IFN-γ expression when a placebo was added. Importantly, we could not detect the expression of the representative Th2-type cytokine, IL-4, at any CBG-CS-2 concentrations tested (data not shown). Consequently, we concluded that CBG-CS-2 stimulates Th1-type cells in vitro suggesting its capability to stimulate cell-mediated immunity.

We next determined whether in vitro treatment of cells with CBG-CS-2 could result in the stimulation of NK cell activity in NK-92MI NK cell line (Fig. 3). Incubation of NK-92MI cells with CBG-CS-2 over 24-48 hrs did not enhance the cytotoxicity; however, incubation of NK-92MI cells with CBG-CS-2 for 120 hrs resulted in the highest cytotoxicity against K562 target cells (data not shown). When we measured the NK cell activity using ⁵¹Cr-labeled K562 cells as target cells, incubation of NK-92MI cells with as little as 1 µg/ml CBG-CS-2 for 144 hrs significantly (p<0.001) enhanced the cytotoxic activity of the cells (Fig. 3A). In addition, we detected a strong correlation between E:T ratio and the level of CBG-CS-stimulated NK-92MI-mediated cytotoxicity against K562 target cells (Fig. 3B). Importantly, we could not detect NK-92MI-mediated cytotoxicity against K562 target cells when a placebo was used. Therefore, we concluded that in vitro treatment with CBG-CS-2 is capable of potentiating the cytotoxicity of NK-92MI NK cells against K562 target cells.

Collectively, to develop a standardized alternative to wild Ophiocordyceps, a well-known traditional Chinese medicine, we produced a refined product (CBG-CS-2) from a mycelial culture of Paecilomyces hepiali, which originated from wild O. sinensis. We tested its ability to stimulate T and B lymphocytes and NK cell activity. As was reported in in vivo oral administration experiments previously, we confirmed that CBG-CS-2 directly stimulates Th1-type immune cells. In addition, CBG-CS-2 enhances NK cells activity as was shown in in vivo oral administration experiments (21).