CD8+ T Cell-Mediated Therapeutic Antitumor Effect of an Herbal Mixture Containing Ganoderma lucidum

Although Kampo—a traditional Japanese herbal medicine—contributes in the control of tumor growth in vivo in experimental animals, most of the antitumor effects are prophylactic and not therapeutic. In this study, we determined whether oral administration of an herbal mixture containing Ganoderma lucidum (WTMCGEP; Wisteria floribunda, Trapae fructus, Myristica fragrans, Coicis semen, Ganoderma lucidum, Elfvingia applanata, and Punica granatum), anecdotally used in Japan for the palliative care of patients with cancer, exhibits a therapeutic effect on tumor growth in vivo in a hypodermic murine CT26 colorectal tumor model. An in vitro tumor assay revealed that WTMCGEP extract has some direct influence over suppression of tumor growth. In wild-type BALB/c mice, WTMCGEP did not show any antitumor effect in vivo. However, in BALB-CD1d−/− mice with partly mitigated immunosuppression by reason of them being devoid of both antitumoral type I and immunosuppressive type II natural killer T (NKT) cells, WTMCGEP therapeutically suppressed tumor growth. CD8+ T cell depletion significantly accelerated tumor growth in WTMCGEP mice; therefore, its antitumor activity was primarily in a CD8+ T cell-dependent manner. Regarding immunosuppressive cells in tumor-bearing CD1d−/− mice, WTMCGEP did not influence the abundance of tumor-infiltrating CD4+ and Forkhead box protein 3+ regulatory T cells. However, it reduced both intratumoral and splenic Ly6G+ Ly6Clo polymorphonuclear myeloid-derived suppressor cells, which were most likely involved in tumor growth inhibition related to higher frequency of intratumoral CD107a+ CD8+ T cells in these mice. Overall, these data illustrate that the deficiency of NKT cells urges WTMCGEP to exert a therapeutic antitumor effect mainly through CD8+ T cells. Our efforts are the first to scientifically demonstrate the WTMCGEP's contribution to tumor immunity.


Introduction
Kampo, traditional Japanese herbal medicine, is often used in patients with cancer based on the theory that it helps reduce their various complaints or adverse events associated with chemotherapies, surgical operation, and radiation therapies [1][2][3]. Moreover, some prescriptions show antitumor efect in several mouse tumor models [4][5][6][7][8][9][10][11], although they remain controversial. Recently, we demonstrated the antitumor efect of Juzentaihoto (JTT) (Shi-Quan-Da-Bu-Tang in Chinese and Sipjeondaebo-Tang in Korean) in CD1d −/− mice whose immune-suppressing efects were partly palliated because of the loss of both antitumoral type I and immunosuppressive type II natural remaining ingredients other than Trapae fructus have been reported to have some antitumoral activity [18][19][20][21][22]. Te origin formula of WTMCGEP included Wisteria foribunda, Terminalia chebulae, Trapa natans, and Coicis semen (WTTC), which has been used for treating cancers in Japan since the 1960s [23]. Hijikata then modifed WTTC to WTTCGE, a formula containing WTTC, Ganoderma lucidum, and Elfvingia applanata, and fnally, developed the present form to improve its antiviral efects [17,24,25]. In particular, it has been reported that a formula containing WTMCGEP plus Panax ginseng (WTMCGEPP) suppressed herpes zoster-related pain in fve Japanese patients with shingles [17]. Tese modifed WTTC formulas have also been empirically prescribed to mitigate various complaints associated with cancers, such as ascites, general fatigue, and pain. Although almost all reports on these formulas seem to be anecdotal, these formulas are believed to be relatively safe and could be used for a long time in patients with cancer. Moreover, clinicians and pharmacists can easily access WTMCGEP as formulated powders in Japan. Terefore, investigating on a more scientifc level whether WTMCGEP exerts anticancer efects in appropriate small animal models would be worthy.
In this study, in a hypodermic murine syngeneic CT26 colorectal tumor model, we evaluated the therapeutic antitumoral efect of oral administration of WTMCGEP.

Animals.
Inbred BALB/c and BALB/c CD1d −/− mice were procured from CLEA Japan (Tokyo, Japan) and Jackson Laboratory (Bar Harbor, ME, USA), respectively. Te mice were categorized in groups of fve and housed in flter cages, including females (>six weeks old), in all research and preserved in a temperature-restrained, specifcpathogen-free animal facility. Te mice were subjected to constant 12 h light/12 h dark cycle and allowed to have water without any obstacle and ad libitum reach to food as indicated. Te Animal Care and Use Committee at Nippon Medical School (Tokyo, Japan), which acquired the external validation from the Japanese Association for Laboratory Animal Science, approved the entire experiments (Permit No. 2020-020). All the animal experiments were appropriately conducted per the norms provided for the laboratory animals, Nippon Medical School, Act on Welfare and Management of Animals and Standards Related to the Care and Keeping and Reducing Pain of Laboratory Animals. Each experiment included 3-5 mice/group.

Te
Herbal Mixture WTMCGEP. Te herbal mixture containing Ganoderma lucidum, named WTMCGEP, comprises seven herbs (i.e., Wisteria foribunda, Trapae fructus, Myristica fragrans, Coicis semen, Ganoderma lucidum, Elfvingia applanata, and Punica granatum) ( Table 1). All herbs were extracted using 11-fold hot water (weight/weight) at 95°C for 2.5 h. After fltration, the fltrate was condensed through a spray drying. We purchased WTMCGEP from Tochimoto Tenkaido & Co. (Osaka, Japan) as preservative-free pure powder. Subsequently, we used a moderate-fat diet as a control (Oriental Yeast Co., Tokyo, Japan). Te mice were provided with a either the control diet or the mixture of control diet and WTMCGEP (2.0%; termed as WTMCGEP diet). To obtain the herbal extract for in vitro use, 100 mg of powdered WTMCGEP was dissolved in 1 ml of dimethyl sulfoxide (DMSO) (Nakarai tesque, Kyoto, Japan), and this mixture was nurtured for 24 h with shaking on a water bath at 37°C. Tereafter, it was centrifuged for 30 minutes at 20°C and 13,000 rpm. At last, its supernatant was collected as a stocked WTMCGEP extract.

In Vitro
Tumor Assay with WTMCGEP. CT26 cells were positioned along 96-well plates (5 × 10 3 cells/well) and cultured for 24 h at 37°C. Following discarding the culture supernatant, stocked WTMCGEP extract for in vitro use was diluted with CT26 culture medium, passed through 0.22 μm flters for sterilization, and then added to each well at various concentrations (2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml, and 0.03125 mg/ml). Since WTMCGEP extract was originally dissolved in DMSO, we also prepared a series of control wells with CT26 culture medium, including the corresponding concentration of DMSO (2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, and 0.03125%, respectively). Following a 24 h incubation, cell viability was assessed using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) in lines with the maker's guidelines. Because the prepared samples were divided into three types; those without WTMCGEP nor DMSO, those with DMSO, and those with WTMCGEP + DMSO, results were calculated as the percentage of viability � (OD at 450 nm of the sample with DMSO or WTMCGEP + DM-SO − OD at 450 nm of blank)/(OD at 450 nm of the sample without DMSO nor WTMCGEP − OD at 450 nm of blank) × 100.

In Vivo Tumor Assay and Antibody Treatment.
Te mice subcutaneously received 50,000 CT26 cells in 200 μL cold Dulbecco's phosphate-bufered saline (DPBS) (Termo-Fisher Scientifc) on day 0 and were fed with either the control or WTMCGEP diet for twenty-eight days soon after tumor challenge. In the subgroup of the experiments, some CD1d −/− mice were intraperitoneally injected with 0.2 mg of anti-CD8 mAb (clone, 2.43, cat. no.: BE0061; Bio X Cell, West Lebanon, NH, USA). Te following temporal targets were considered: frst and second day before tumor cell inoculation and 4 th , 7 th , 10 th , and 14 th days following tumor cell inoculation. A caliper gage was used to measure the tumor area twice a week per this calculation; tumor length × width measured in mm. When the tumor area was <100 mm 2 on 28 th day post tumor challenge, the mice were taken as "survived" [26].

Isolation of Tumor-Infltrating Lymphocytes.
For the isolation of tumor-infltrating lymphocytes (TILs), tumors were sectioned from the mice and assimilated using 1 mg/ mL collagenase (Roche Diagnostics GmbH, Mannheim, Germany) for forty-fve minutes at temperature of 37°C. Te resultant was subsequently softly creased to make the mass homogenous and sieved using a nylon flter mesh. For their purifcation, the collected leukocytes, via the latter process, were subsequently isolated from the polluting tumor cells using Lympholyte-M (CEDARLANE, Burlington, NC, USA) as per the maker's manual.

Flow Cytometric Analysis.
For the determination of the expression of the cell surface molecules, fow cytometry through a FACSCanto II fow cytometer (Becton Dickinson Immunochemical Systems, Mountain View, CA) was performed. Briefy, cold DPBS (2 mL) was used to wash the isolated TILs and spleen cells, then stained using dilute Zombie NIR ™ dye (BioLegend, San Diego, CA, USA) at 1:500 in DPBS at normal room temperature without light for a duration of ffteen minutes, and splashed again with DPBS with 2% heatinactivated FCS and 0.1% sodium azide (FACS bufer). For minimizing the union of nonspecifc antibody to the Fc receptors, the isolated cells were incubated with 0.5 μg anti-CD16/CD32 (clone 2.4G2, cat. no.: 14-0161; eBioscience, Inc., San Diego, CA, USA) at 4°C for a duration of ffteen minutes. Following that, the pertinent antibodies were utilized to stain for half an hour at 4°C. Te antibodies used were as follows: PE/ Cy7-conjugated anti-CD3 (clone 17A2, cat. no.: 100220), FITC- For the staining of intracellular Forkhead box protein 3 (Foxp3) to identify CD4 + Foxp3 + Tcells in TILs, after surface staining using anti-CD3, -CD4, and -CD45, 2 mL cold FACS bufer was used to wash the stained cells once. Following this, the cell units were secured with 1 mL fxation/permeabilization solution (eBioscience) at a temperature of 4°C for 30 min and then cleansed two times using 2 mL 1 × permeabilization bufer (eBioscience) and stained with 0.4 μg PE-conjugated anti-Foxp3 (clone FJK-16s, cat. no.: 12-5773-80; eBioscience) at 4°C for 30 min. Te cells were cleansed two times using 2 mL 1 × permeabilization bufer and suspended in cold FACS bufer.

Statistical Analysis.
Following tests were used for interpreting the study outcomes: for parametric data Student's t test was used, nonparametric data were analyzed using Mann-Whitney U-test, Log-rank test, and two-way repeated measures analysis of variance (ANOVA), followed by post hoc Sidak's multiple comparisons test using Prism 6 (version 6.0 d; GraphPad Software, Inc., La Jolla, CA, USA). Diferences with p values of <0.05 were considered statistically signifcant.

WTMCGEP Extract Has a Direct Impact on the Inhibition of CT26 Tumor Growth In Vitro.
To evaluate whether WTMCGEP afects tumor cell growth in vitro, CT26 cells were cultured in vitro with or without serially diluted WTMCGEP extract for 24 h. Ten, their viability was assessed by Cell Counting Kit-8 (Dojindo Laboratories), as described in the "Materials and Methods" section. As shown in Figure 1, the CT26 cell life was markedly reduced in the presence of various concentrations of WTMCGEP extract (1 mg/ml, 0.5 mg/ml, and 0.25 mg/ml). Tese data suggest that WTMCGEP has a direct impact on the suppression of tumor growth in vitro.

WTMCGEP Terapeutically Suppresses Tumor Growth in
We explored if the oral administration of WTMCGEP exerts antitumor efect in vivo. To attain this, WT BALB/c or BALB-CD1d −/− mice were hypodermically injected with 5 × 10 4 CT26 cells. Subsequently, the mice were nourished using the control diet or WTMCGEP diet for a duration of 4 weeks. As shown in Figures 2(a) and 2(b), WTMCGEP did not infuence tumor growth in the WT mice. In comparison, although we and other investigators have reported that tumors grew markedly slower in CD1d −/− mice than those in WT mice [26,27], WTMCGEP signifcantly accelerated tumor growth inhibition in such CD1d −/− mice (Figures 2(c) and 2(d)). To elucidate whether the antitumor efect of WTMCGEP depends on CD8 + T cells, CD1d −/− mice fed with or without the WTMCGEP diet were treated with anti-CD8 monoclonal antibodies in vivo before and after tumor challenge. As illustrated in Figures 2(c) and 2(d), the depletion of CD8 + T cells in vivo almost canceled the antitumor efect of WTMCGEP in the mice, although CD8-depleted mice fed the WTMCGEP diet still demonstrated a marginal but signifcant antitumor efect in vivo compared to those fed the control diet. Tese fndings indicate that CD8 + T cells play pivotal roles in maximizing the therapeutic antitumor efect of WTMCGEP in CD1d −/− mice.

WTMCGEP Augments Intratumoral CD8 + T Cell
Activity in CD1d −/− Mice. Because CD8 + TILs are known to back the enhancement of antitumoral immunity [28], we explored the abundance of intratumoral degranulation marker CD107a + CD8 + T cells in CD1d −/− mice nourished with a control or WTMCGEP diet 28 days after tumor challenge. As shown in Figures 3(a) and 3(b), the frequency of such TILs signifcantly increased in the WTMCGEPtreated mice compared to control mice. Te above results convey that the elevation of CD107a + CD8 + TILs is associated with the attenuation of tumor growth in WTMCGEPtreated mice.

WTMCGEP Has No
Efect on the Abundance of Intratumoral CD4 + Foxp3 + Regulatory T Cells. Various immunosuppressive cells are known to dampen antitumor immunity, among which CD4 + regulatory T (Treg) cells are one of the hurdles for immune surveillance against cancer [29]. Prophylactic depletion of such Treg cells augmented antitumoral immunity among both WT [26,30,31] and CD1d −/− [27] mice hypodermically injected with CT26 tumors. Moreover, a recent study has revealed that intratumoral Treg depletion by anti-CD25 immunotoxin contributes to the induction of CD8 + T cell-mediated systemic antitumor immunity [32]. Terefore, we evaluated the abundance of intratumoral CD4 + Treg cells in CD1d −/− mice nourished with the control or WTMCGEP diet on day 28 after the tumor challenge. As shown in Figures 4(a) and 4(b), there was no diference in the abundance of CD4 + Treg cells between the two groups. Tese fndings suggest that WTMCGEP did not afect the abundance of intratumoral Treg cells in CD1d −/− mice.  Subsequently, the mice were fed either the control or WTMCGEP diet for 4 weeks after the tumor challenge. In experiments with CD1d −/− mice, we added two more groups that included control-or WTMCGEP-diet mice treated with anti-CD8 monoclonal antibody (mAb) in vivo. Te tumor area was measured twice weekly with a caliper gage until 28 days after tumor inoculation, with the measurements being calculated as tumor length (mm) × width (mm). Mice were considered to have "survived" when the tumor area was less than 100 mm 2 after 28 days of tumor challenge. Each experiment had three-fve mice in each group. Te results were pooled from two (b) or three (d) independent experiments (n � 9 for the control group and n � 10 for the WTMCGEP group in (b); n � 14 for the control group, n � 13 for the WTMCGEP group, n � 6 for the control + anti-CD8 group, and n � 10 for the WTMCGEP + anti-CD8 group in (d)). In (a) and (c), lines represent the tumor growth of each individual mouse. In (d), the log-rank test between the control and WTMCGEP groups had a p value of <0.002, the log-rank test between the control and control + anti-CD8 groups had a p value of <0.0001, the log-rank test between the WTMCGEP and WTMCGEP + anti-CD8 groups had a p value of <0.0001, and the log-rank test between the control + anti-CD8 and the WTMCGEP + anti-CD8 groups had a p value of <0.02.  Figures 5(e), 5(f ) right, 5(g), and 5(h) right) in these mice. As our previous study has revealed that PMN-MDSCs derived from CT26-bearing mice could function as immunosuppressive cells [12], these observations imply that the reduction of both local and systemic PMN-MDSCs plays crucial roles in the enhancement of antitumor immunity in CD1d −/− mice fed with the WTMCGEP diet.

Discussion
Recently, we reported that JTT has antitumor efects on BALB-CD1d −/− mice in a prophylactic setting, in which mice had been fed with a JTT-containing diet from 14 days before  Figure 3: WTMCGEP augments intratumoral CD8 + T cell activity in CD1d −/− mice. CD1d −/− mice fed the control or WTMCGEP diet were subcutaneously challenged with 5 × 10 4 CT26 cells. Twenty-eight days after tumor inoculation, the recovered TILs were stained with anti-CD45, -CD3, -CD8β, and -CD107a. Te CD107a + CD8 + cell proportion was determined using fow cytometry. Te representative fow plots are demonstrated as CD107a + CD8β + cell percentage in TILs (a). Te cumulative data of two independent experiments (n � 9 for the control group and n � 7 for the WTMCGEP group) were shown in (b). Te data are expressed as means ± standard deviations. * p < 0.04 (Student's t test). 6 Evidence-Based Complementary and Alternative Medicine  Figure 4: WTMCGEP has no efect on the CD4 + Foxp3 + Treg cell frequency in TILs. CD1d −/− mice fed the control or WTMCGEP diet were subcutaneously challenged with 5 × 10 4 CT26 cells. Twenty-eight days after tumor inoculation, the recovered TILs were stained with anti-CD45, -CD3, -CD4, and -Foxp3. Te CD4 + Foxp3 + cell proportion was determined using fow cytometry. Te representative fow plots are demonstrated as CD4 + Foxp3 + cell percentage in TILs (a). We used three-fve mice for each group to perform three independent experiments and pooled the results (b). Each symbol indicates one data point. Te data are expressed as means ± standard deviations. Evidence-Based Complementary and Alternative Medicine the CT26 tumor challenge for 42 days [12]. Based on these fndings, we have sought an herbal mixture, other than JTT, to show the therapeutic antitumor efect in murine tumor models and conducted this study. In vitro, WTMCGEP extract had some direct antitumor efects (Figure 1). In vivo, we exemplifed that oral administration of this herbal mixture therapeutically inhibited tumor growth in CD1d −/− mice but not in immune-competent BALB/c mice (Figures 2(b) and 2(d)). Furthermore, CD8 + T cells were necessary for the maximization of WTMCGEP's antitumor efect (Figure 2(d)), and the frequency of CD107a + CD8 + TILs was signifcantly elevated in WTMCGEP-treated mice compared to that in control mice (Figure 3). Tese fndings emphasize the pivotal role of WTMCGEP in reinforcing antitumor immunity through CD8 + T cells in a tumorbearing host on the condition that immunosuppression is partly mitigated because of defciency of NKT cells. Although six of the seven ingredients of WTMCGEP have already been known to possess some antitumor properties [13][14][15][16][18][19][20][21][22], only anecdotal evidence on the antitumor efect of this mixture itself exists. Terefore, to our best knowledge, this is the frst time when WTMCGEP's contribution to enhancing the immunological antitumor efect in vivo is shown.
In a hypodermic CT26 tumor model, CD4 + Foxp3 + Treg cells are the predominant immunosuppressive cells. In particular, a recent study revealed that the regulation of intratumoral Treg cells enhances systemic antitumor immunity [32]. Terefore, we planned to explain whether WTMCGEP afects intratumoral CD4 + Treg cells in CD1d −/− mice. However, this herbal mixture had no infuence over the Treg cells numbers in CT26-bearing CD1d −/− mice (Figure 4).
MDSCs have been regarded as a barrier to many cancer immunotherapies [36]. In our previous research, we reported that the abundance of intratumoral, but not splenic, PMN-MDSCs was signifcantly reduced in CT26-bearing CD1d −/− mice fed the JTTdiet related to the control diet [12]. Tus, we next measured the frequency of MDSCs in these mice nourished with the control or WTMCGEP diet. Distinct from JTT, the proportion of PMN-MDSCs was substantially lesser in TILs and spleens resulting from   [37]. Although a precise mechanism is yet to be elucidated, the reduction in PMN-MDSCs in tumor-bearing WTMCGEP mice may be partially mediated by Ganoderma lucidum, which is one of the major components of this mixture. Among the seven ingredients of WTMCGEP, Ganoderma lucidum may be the most studied as an anticancer agent and is known to have anticancer efects in vitro and in vivo, controversies remain [13][14][15][16]. However, the fve remaining ingredients, other than Trapae fructus, have also been reported to have anticancer properties [18][19][20][21][22]. Terefore, WTMCGEP's antitumor efect may not depend on or may be more potent than Ganoderma lucidum alone.
Notably, WTMCGEP had no efect on tumor development in immunocompetent WT mice (Figure 2(b)). Moreover, compared with CD1d −/− mice, tumor-bearing WT mice presented with no diference in the PMN-MDSC population in the presence or absence of WTMCGEP (Figures 5(f) left and 5(h) left). Te antitumor type I NKT and immunosuppressive type II NKT cells found in WT mice functionally regulated each other. However, it is known that type II NKT cells lead type I NKT cells in tumor immunity [38]. Consequently, in several tumor models, CD1d −/− mice falling short on both kinds of NKT cells showed an antitumor or antimetastatic efect at one level or another [26,[39][40][41][42][43]. Furthermore, IL-13 manufacture by type II NKT cells has been reported to contribute to the maintenance of CD11b + Gr-1 + MDSCs via the IL-13R-STAT6 pathway in tumor-bearing mice [41,44]. Terefore, even if WTMCGEP had some negative efects on the survival of PMN-MDSCs in tumor-bearing hosts, type II NKT cells might more strongly support the maintenance of such MDSCs. In WT mice, WTMCGEP treatment might be unable to reduce the population of PMN-MDSCs, probably due to the presence of type II NKT cells, thereby leading to failure of tumor suppression. In contrast, IL-13 inhibitor has been observed to be a pivot in the enhancement of antitumor immunity in some tumor models [39,40,45]. Given that IL-13 is expected to support the maintenance of MDCSs, a trial combining IL-13 blockade with oral administration of WTMCGEP for promoting effective tumor eradication in immunocompetent hosts should be considered. Te efectiveness of such a combination needs to be confrmed in further studies.
Furthermore, the outstanding antitumor efect of WTMCGEP observed in CD1d −/− mice suggests that combination therapy using this herbal mixture and other current cancer therapies prevents immunosuppression. Consistent with our concept, it has been reported that JTT augments the antimetastatic efect of anti-PD1 antibody against B16 mouse melanoma cells [46]. Moreover, JTT and Ninjin'yoeito (NYT; Ren-Shen-Yang-Rong-Tang in Chinese) have been described to synergize the antitumor efect in conjunction with tumor vaccines [9,11]. Particularly, our previous study has reported that NYTmay contribute to the reduction of CD4 + Treg cells in the spleens and tumor-draining lymph nodes derived from CT26-bearing WT BALB/c mice inoculated with irradiated tumor vaccine prophylactically [11]. As the components of these two Kampo medicines and WTMCGEP do not overlap, WTMCGEP may be an attractive option to synergize the antitumor efects in conjunction with these Kampo medicines and tumor vaccines.
However, there may be limitations in this study. First, our study about WTMCGEP was based on the results of one tumor cell line (CT26). Moreover, we were unable to check whether WTMCGEP shows antitumor efects in mouse strains such as C57/BL6 and C3H/HeN, other than BALB/c. To generalize our fndings, studies are needed in the future to elucidate the in vivo antitumor efect of WTMCGEP against tumor cells derived from various mouse strains. Second, given that the duration of our in vivo tumor assay was 28 days, the long term antitumor efect of WTMCGEP could not be clarifed. Tird, it should be noted that WTMCGEP still possessed a marginal but signifcant antitumor efect in vivo in the absence of CD8 + T cells (Figures 2(c) and 2(d)). Terefore, the precise mechanism of the CD8-independent antitumor efect by WTMCGEP should be elucidated in the future. Finally, the safety of WTMCGEP should be also considered. WTMCGEP is believed to be almost free from toxicity because this mixture or its derivatives have been empirically used for more than 60 years in Japan [23] for various complaints of patients with cancer as anecdotally described as well as for palliating symptoms in various viral infections, like herpes simplex virus, varicella zoster virus, Epstein-Barr virus, and human cytomegalovirus [17,24,25]. Moreover, WTMCGEPtreated mice seemed to be healthy throughout our study. Nonetheless, WTMCGEP should be still consumed carefully because, for example, in Ganoderma lucidum alone, there are few cases of adverse efects and drug interactions reported in the literature [15]. Terefore, large-scale controlled studies will be necessary to elucidate the safety of this mixture.

Conclusions
To the best of our knowledge, it may be concluded that our study is the frst to scientifcally demonstrate WTMCGEP's contribution to the augmentation of CD8 + T cell-mediated antitumor immunity in CD1d −/− mice with partly attenuated immunosuppression by reason of the defciency of NKT cells. Although the characteristics of WTMCGEP as an anticancer agent has yet to be fully elucidated, current study sheds light on this herbal mixture as a promising and practical antitumor adjuvant.