Journal of Nutritional Health & Food Science

The human immune system possesses powerful surveillance functions to find and eliminate tumor cells. Several steps are involved, such as uptake and presentation of tumor antigens by dendritic cells (DCs) in order to activate T cells, trafficking and infiltration of the tumor with T cells and, finally, recognition and killing of tumor cells. Tumor cells release a number of soluble mediators that may disrupt these steps, thereby creating tolerance or immune escape. We previously published data on a rat animal model showing 1, 2-dimethylhydrazine (DMH) to inhibit DCs activation and enhance T regulatory cells (Treg) expression, ultimately inducing colon carcinogenesis. By feeding rats with mustard seeds (MS), the animals’ immune system shifted from a Th2 to a Th1 pattern, Tregs were inhibited and DCs activation enhanced. In the present study, we tried to isolate the functional fractions of MS and, to our surprise, we found that the oxidant and antioxidant fractions of MS showed synergistic effect on the activation of DCs and CD8⁺ effector T cells. Neither of the fractions alone, however, had any inhibitory effect on DMH-induced colon carcinogenesis. Differently from the antioxidant fraction, the oxidant fraction activated DCs (CD11c⁺CD80⁺, CD11c⁺CD86⁺), but not the CD8+ effector T cells. By adding Vitamin E, an acknowledged antioxidant, the oxidant fraction then became able to activate both spleenic DCs and effector T cells (CD8⁺CD28⁺). Taken together, these data suggest that the MS oxidant fraction might play an important role in activation of DCs, while also showing that oxidative signals may induce depletion of effector T cells. Addition of the antioxidant MS fraction, or Vitamin E, might restore the survival of effector T cells and eventually help eradicate tumor cells. Thus, MS oxidants, which normally are regarded as having negative effects, may actually function synergistically with antioxidants to prevent chemical-induced carcinogenesis.

Keywords: Oxidants; Antioxidants; Immuno-responses; Mustard seeds; Colorectal cancer; Chemoprevention; T cells; Dendritic cells

Abbreviations: AITC: Allyl Isothiocyanate; APCs: Antigen-Presenting Cells; CTLs: Cytotoxic T Lymphocytes; DCs: Dendritic Cells; DMH: 1, 2-Dimethylhydrazine; EGCG: Epigallocatechin Gallate; LPO: Lipid Peroxides ; MDA: Malondialdehyde; MHC: Major Histocompatibility Complex; MS: Mustard Seeds; MSE1: Ether Extract of Mustard Seeds; MSE2: Ethanolic Extract of Mustard Seeds; PBS: Phosphate-Buffered Saline; RBC: Red Blood Cell; ROS: Reactive Oxygen Species; TCRs: T Cell Receptors; Treg : T Regulatory Cells; VitE : Vitamin E

The immune response against tumors is provided by a multitude of cells, although T cells are considered the major mediators of anti-tumor immunity [1-6]. The exact type of T cell response required for optimal anti-tumor immunity is not entirely clear, but a potent CD8+ effector T cell response is certainly necessary. Additionally, a response directed by either CD4+ T helper 1 (Th1) cells or Th17 cells appears to promote the CD8+ effector T cell responses [7-9]. T cell response is initiated by specific recognition of cognate peptides presented by major histocompatibility complex (MHC) proteins on antigenpresenting cells (APCs) through T cell receptors (TCRs), which are referred to as the first signal for T cell activation. However, the ultimate magnitude and quality of T cell response is determined by a balance between co-stimulatory and co-inhibitory signals (collectively called co-signals) that are transduced into T cells and is referred to as the second signal [10,11]. Following TCR engagement by cognate peptide-MHC complexes, co-signaling receptors are often mobilized and colocalized with TCRs, forming immunological synapses between APCs and T cells. This synaptic interface is the place where cross-talks between co-signaling ligands and receptors synergize or antagonize with TCR signaling, rendering T cells activated or inhibited [12], it is also the place where oxidant radicals are considered transported from APCs to T cells, which may influence the activation and survival of effector T cells. Holmdahl and his colleagues reported that a decreased capacity to produce ROS, due to a natural Ncf1 polymorphism, was found to be a major factor behind severe arthritis and increased T cell-dependent autoimmunity [13]. Peroxides are immunologic transmitters secreted by antigen-presenting cells that down-regulate the responses by autoreactive T cells. Ncf1 polymorphism results in the release of less ROS from APCs with ensuing activation of autoreactive CD4+ T cells and resulting severe arthritis [14,15].

The B7 protein family is a group of surface glycoproteins that share structural features with immunoglobulins (Ig). Their extracellular domains bear homology to the IgV and IgC domains of Igs. A hallmark of the B7 family molecules is their capability to co-stimulate or co-inhibit T cell responses in the presence of peptide/MHC complex-mediated TCR signaling [12]. B7 family members primarily bind to members of the CD28 protein family, including CD28, CTLA-4, PD-1, ICOS and BTLA that transmit cosignals to T cells [16]. The B7 co-stimulatory ligands are important for full activation of naïve T cells in the lymphoid organs, in which APCs, particularly dendritic cells, are the primary cellular source for providing ligands, including B7.1 (CD80) and B7.2 (CD86). In contrast, B7 co-inhibitory ligands are crucial for the termination of over activated T cell responses, maintenance of self-tolerance, and protection of tissues from damage induced by invading pathogens. In addition, co-inhibitory ligands expressed in pathological compartments, such as tumor microenvironments, actively inhibit the effector functions of cytotoxic T lymphocytes (CTLs) and the generation of regulatory T cells [10,11]; thus, playing the role of important immune checkpoint proteins that are involved in the immune resistance to cancer cells.

In this paper, in order to better understand the biochemistry behind the MS activity, we tried to check for crudely purified fractions of MS, using ether- and ethanol-soluble fractions of mustard seeds, on activation of DCs, CD80 and CD86 expression, and their positive activation ligand CD28 on CD8+ T cells.

MS has been used as a spice and herbal medicine since the Tang dynasty (618-907) [17]. Glucosinolates and their breakdown products, such as allyl isothiocyanate (AITC), sinigrin and sinapine are major components of MS and possibly responsible for inducing a reduced risk of cancer [18-21]. A previous study of ours indicated that MS reduced the incidence of DMH-induced colorectal cancer [22]. The proportion of spleenic B- and dendritic cells was amplified in the MS group. The expression of MHCI, as well as that of MHCII, was increased in dendritic cells and the percentages of CD8⁺CD28⁺ and CD4⁺CD28⁺ cells were increased in the MS group, while the CD4⁺CD25⁺Foxp3⁺ subset was depressed. Plasma analyses showed that DMH-exposure induced amplified amounts of IL-4, IL-5, IL-10 and TGF-beta, while MS feeding counteracted this effect but enhanced IL-2, IL12p70, IL21, TNFalpha, and IFN-gamma. On the SW480 colon adenocarcinoma cell-line, the cytotoxicity of spleenic T-cells from MS-fed animals was significantly increased. In DMH-exposed rats, the expression of perforin in the spleenic T-cells was dramatically decreased, while MS abolished this depression. These data indicated that dietary MS suppresses DMH-induced immuno-imbalance as well as colon carcinogenesis in rats. When we explored the activities of ether- and ethanol-extracted MS fractions with respect to how they activate DCs and effector T cells, and eventually execute tumor cells, we found that an ether-extracted MS fraction is an oxidant, rather than an antioxidant and activates DCs, although not T cells. The ethanol-extracted MS fraction, possessing antioxidant activity, did not activate DCs but might enhanced the survival of T cells that were activated by DCs when the latter present antigens to T cells, and also supply free radicals that might hurt T cells. Thus, both oxidant and antioxidant fractions of MS appear to play synergistic roles in activating the immune system against tumorgenesis.

Animals and Diets
Four weeks old male Kunming mice with an average body weight of 20 g were obtained from the Southern Medical University animal center. All animals were cared for according to the Institutional Animal Care Guidelines. The Committee for Animal Care and Use of the Southern Medical University, Guangzhou, China, approved the study. The mice were maintained under controlled conditions of temperature (24 ± 2 °C), humidity (50 ± 10%), 12 h light/12 h dark cycles and were acclimated to their new diet and environment for 2 weeks before experiments. Each cage housed three mice. Tap water was provided ad libitum. Animals were fed standard chow or standard chow containing 7.5% crushed fresh MS [21].

MS Extraction
MS fractions were prepared as previously reported [23]. Briefly, MS was first extracted in ether and then in ethanol. 300 g mashed MS were extracted in 1000 ml ether and the soluble fraction collected, filtrated, air dried by evaporation at room temperature (25 °C) and named MSE1, which was an oily material. The solid MS remains were then again extracted, but now in 1000 ml ethanol at 25 °C. The substances dissolved in ethanol were then precipitated at 4 °C over night; the sediment collected by centrifugation and named MSE2. The MSE2 was a solid substance that was dissolved in PBS to 1.5% at room temperature. Experiment I: Influence of mustard seeds and its fractions on DMH-induced colorectal cancer in mice

Experimental design: The mice were randomly divided into five groups:
Group I (n = 15): Mice were fed standard chow diet and received i.p. injections of normal saline (0.2 ml) once a week for 20 weeks. This group served as an untreated control group.
Group II (n = 15): Mice were fed standard chow diet and received i.p. injections of DMH (Sigma-Aldrich) at a dose of 20 mg/kg once weekly for 20 weeks.
Group III (n = 15): Mice were fed chow diet containing 7.5% MS. They received injections with the same dose of DMH as in group II once a week for 20 weeks.
Group IV (n = 15): Mice were fed standard chow diet and received i.p. injections of 50 μl MSE1 once a week, as well as the same DMH injections as the group II animals for altogether 20 weeks.
Group V (n = 15): Mice were fed standard chow diet and received i.p. injections of 100 μl MSE2, which was prepared at a concentration of 1.5% in PBS, once a week for 20 weeks as well as the same DMH injections as group II.

Mice were sacrificed by ether inhalation 12 weeks after their last injection of DMH or saline. Serum was collected for analysis of malondialdehyde (MDA) and Th1/Th2 cytokines. Spleens were removed and assayed for proportions of lymphocytes. Colons were removed and cut open longitudinally for further microscopical examination of selected areas.
Experiment II: Influence of MSE1 on dentritic cells in mice with DMH-induced colon tumors
Experimental design: The mice were randomly divided into groups as in Experiment I. The animals were sacrificed after 6 weeks of injections with DMH or saline. Spleens were removed, prepared and assayed for dendritic cells.
Experiment III: Influence of MSE1 with added VitE (vitamin E) on dentritic cells and CD8+ T cells in the spleens of DMH-exposed mice
Experimental design: The mice were randomly divided into the following groups:

Group I (n = 15): Mice were fed standard chow diet and received i.p. injections of normal saline (0.2 ml) once a week for 6 weeks. Group II (n = 15): Mice were fed standard chow diet and received i.p. injections of DMH at a dose of 20 mg/kg once a week for 6 weeks.
Group III (n = 15): Mice were fed standard chow diet and received i.p. injections of 50 μl MSE1 and 20 mg/kg DMH once a week for 6 weeks.
Group IV (n = 15): Mice were fed standard chow diet and received 50 μl MSE1, 100 mg/kg VitE and 20 mg/kg DMH once a week for 6 weeks.
Mice were sacrificed after 6 weeks and their spleens were removed for analyzes of dentritic and CD8+ T cells.
Experiment IV: Influence of different combination of mustard seeds extraction on DMH-induced colorectal cancer in mice Experimental design: The mice were randomly divided into the following groups:
Group I (n = 9): Mice were fed standard chow diet and received i.p. injections of phosphate buffer saline (0.2 ml) once a week for 20 weeks. This group served as an untreated control group. Group II (n = 9): Mice were fed standard chow diet and received i.p. injections of DMH (Sigma-Aldrich) at a dose of 20 mg/kg once weekly for 20 weeks.
Group III (n = 9): Mice were fed standard chow diet and received i.p. injections of 50 μl MSE1, 100 μl MSE2 once a week, as well as the same DMH injections as the group II animals for altogether 20 weeks.
Group IV (n = 9): Mice were fed standard chow diet and received i.p. injections of 50 μl MSE1, 100 mg/kg VitE once a week, as well as the same DMH injections as the group II animals for altogether 20 weeks.
Mice were sacrificed by continued given other medicine for 12 weeks after their last injection of DMH or saline. Colons were removed and then cut open longitudinally for further microscopical examination of selected areas. The area of tumor on the surface of colons were measured to assess the situation of colorectal cancer.

Tumor Assay
The colon tumors that resulted in Experiment I were fixed in 10% buffered formalin, dehydrated and embedded in paraffin. Sections (4-μm-thick) were cut and stained with hematoxylin and eosin for histological examination. Sections of each tumor were examined under a light microscope (Olympus, Japan) by a pathologist blinded to the experimental design. If tumor cells invaded the submucosa, the tumor was diagnosed as an adenocarcinoma. When tumor cells did not invade the submucosa, the tumors were diagnosed as adenomas (low- or high-grade intraepithelial neoplasias).

Preparation of Mice Splenocytes for Flow Cytometry
Single cell suspensions of mice splenocytes were prepared by grinding one half of each spleen with the plunger of a disposable syringe. The homogenate was then passed through a 100-mesh screen. Erythrocytes were removed by suspending cells in red blood cell (RBC) lysis buffer (155 μM NH₄Cl, 10 μM KHCO3 and 100 μM EDTA) for 5 min followed by three washes in phosphatebuffered saline (PBS) and resuspending of the remaining splenocytes at 1×10⁷ cells/ml. Experiment I splenocytes were first surface-labeled with anti-mouse CD4-FITC, CD8-PE/Cy5, CD25-PE and CD28-PE/Cy7 (Miltenyi Biotec) for 15 min in a dark refrigerator (4°C). After fixation and permeabilization with a FoxP3 staining buffer set, cells were washed and intracellularly stained with APC-labeled anti-mouse Foxp3 for 30 min in the dark at 4°C according to the instruction offered by the manufacturer. Experiment II and III splenocytes were labeled with anti-mouse CD11c-APC, CD80-FITC, CD86-FITC, CD8-PerCP and CD28-PE/ Cy7 for 15 min in a dark refrigerator (4°C) before ensuing flow cytometry. Cells were run on a Becton Dickinson FACSVerse and data analyzed using the CellQuest software.

Assay for Malondialdehyde (MDA)
MDA is a degradation product of lipid peroxides (LPO). Blood samples from Experiment I were collected from the hearts and centrifuged for 10 min at 3000 × g The obtained serum was assayed for possible thiobarbituric acid-reactive substances using a colorimetric Diagnostic Reagent Kit (Nanjing Jiancheng Bioengineering, Nanjing city, P.R. China) according to the manufacturer’s instructions.

Assay for Cytokines
Plasma cytokines (IFN-γ, IL-12, IL-4, IL-5, IL-6, IL-10 and IL-17) from the different groups of mice of Experiment I were analyzed using the Milliplex map kit (Millipore Corporation, USA). The assay was performed according to the manufacturer’s protocol. Briefly, plasma was diluted 1:5 with PBS to maximize sensitivity at baseline levels. The diluted plasma (25 μl) was incubated with an antibody-immobilized mix of beads for 16 h at 4 °C. The beads were then washed twice and incubated with detection antibodies for 1 h at room temperature. Streptavidin–Phycoerythrin was then added and the mixture was incubated for an additional 30 min period at room temperature. The beads were again washed twice and finally analyzed on a Luminex-100 cytometer (Millipore Corporation, USA) using Start Station software (Version 2.0; Applied Cytometry Systems, Dinnington, UK). Cytokine concentrations were calculated from standard curves. The minimum significant value of the assay was 3.2 pg/ml.

MSE1 and MSE2 do not inhibit DMH-induced formation of colon tumors, while MS does (Experiment I)
Table 1 depicts the effects of the two MS fractions and MS itself on colonic tumor formation in DMH-exposed mice. All DMH exposed mice developed tumors (100%) including adenocarcinomas (86.67%) and adenomas (13.33%). MS significantly inhibited tumor formation. By 40% it decreased the total tumor incidence and by nearly 50% the adenocarcinoma incidence, whereas neither MSE1 nor MSE2 had any significant effect on tumor incidences. Sample images of colon tumors are shown in figure 1. The macroscopic appearance of several solitary (upper illustration) and multiple (lower illustration) colonic tumors is shown in figure 1A, whereas figure 1B shows microscopic views of colon section stained with hematoxylin and eosin.

MS, but not MSE1 and MSE2, decreased CD4⁺CD25⁺Foxp3⁺ Treg cells. (Experiment I)
The immunologic action of MS and its two extracts in preventing colon carcinogenesis was investigated by analyzing the percentages of spleen CD4⁺CD25⁺Foxp3⁺ Treg cells (Figure 2). The DMH alone group showed increased numbers of Treg cells compared to the saline group (from 0.12% to 0.26%), indicating that tumor-infiltrating Treg cells are detrimental to the tumorbearing host. Treg cells of mice exposed to MS + DMH decreased to 0.16%, which is close to that of the saline group. Both MS fractions + DMH resulted in similar values for Treg cells as were found for the DMH alone group. Obviously, they had no effect.
MSE1 and MSE2 do not activate CD8⁺ T cells, while MS does (Experiment I)
CD8+ T cells functionally play a role as cytotoxic T lymphocytes (CTLs). CD28 molecules are highly expressed on the surface of naive T cells and initiate T cell activation. Mice in the DMH group had a 100% tumor incidence with a low ratio of CD8⁺ T cells (5.10%) and CD8⁺CD28⁺ cells (3.38%) as compared to the control (17.64% and 12.66%, respectively) and the MS group (11.23% and 7.31%, respectively). Mice exposed to DMH and simultaneously fed diets with MS had a lower total tumor incidence and an increased ratio of CD8⁺ T (11.23%) and CD8⁺CD28⁺ cells (7.31%), indicating that MS played a role in preventing carcinogenesis by increasing CTLs. Neither of the MS fractions caused any significant increase of CD8⁺ T or CD8⁺CD28⁺, indicating that they alone do not activate effector T cells (Figure 3).

MSE1 induces a Th1 reaction, while MSE2 induces type Th2 and Th17 reactions (Experiment I)
Our previous studies showed that DMH induced a Th2 pattern of immune responses while MS reversed it to a Th1 pattern. We next tried to explore the effects of the two MS fractions on formation of Th1, Th2 and Th17 type cytokines. As shown in figure 5, MSE1 significantly enhanced the Th1-type cytokines, including IFN-γ and IL-12, while MSE2 significantly induced Th2 and Th17 reactions, including formation of IL-4, IL-5, IL-6, IL-10 and IL-17, compared to DMH (Figure 4).

MSE1 is an oxidant, while MSE2 is an antioxidant (Experiment I)
The biochemical difference between MSE1 and MSE2 became better understood when we investigated their redox capability. By checking lipid peroxidation products (Figure 5), we found MSE1 to increase the level of MDA as did also DMH, while MSE2 had the opposite activity. MS behaved as MSE2, indicating that they both act mainly as antioxidants.

MSE1, but not MSE2, activate dendritic cells (Experiment II)
Dendritic cells (DCs) are sensors of immune responses; their main function is to process antigen and present it to T cells. CD11c is the most widely used specific marker of mouse DCs. Cell-surface receptors, such as CD80 and CD86 act as co-stimulating-ligands in T-cell activation and inducers of primary T cell responses. Figure 6 shows that MSE1 significantly enhances the DCs population and activates DCs (CD11c⁺CD80⁺ and CD11c⁺CD86⁺), while MSE2 does not.

MSE1 + VitE enhances the dendritic cell population and the activation of CD8+ T cells in spleens (Experiment III)
Since experiment I showed that MSE1 acts as an oxidant, while MSE2 performs as an antioxidant and since experiment II showed that MSE1 activates dendritic but not CD8⁺ T cells (Figures 3 and 6). We proposed that adding an antioxidant together with MSE1 might help dendritic cells to survive and activate CD8+ effector T cells. Figure 7 shows that MSE1 + VitE enhanced the numbers of dendritic and activated CD8⁺ T cells.

Both MSE1 + MSE2, or MSE1 + VitE decrease DMH-induced colon cancer area in Kunming mice model (Experiment IV).

Thus, components in MSE1 are good enough to activate Dc cells, and anti-oxidant components are good enough to survive CD8+ T cells. Whether they are good enough to suppress tumor-genesis? Although MS itself are good for inhibition of DMH-induced colon cancer incidents, we did not see similar effect from both MSE1 + MSE2, or MSE1 + VitE combinations (probably we need to do more optimal ratio of these combinations), but comparing the tumor size, we found both groups of MS extracts combination and MSE1 with VitE have good effect on inhibition of cancer growth (Figure 8).

Products from traditional medicinal herbs may act as potent anti-inflammatory, antioxidant and anticancer agents. Recent advances in genomics and metabolomics have enabled scientists to better understand the potential use of immuno-modulatory natural products for treatment or control of various malignancies. Plant products may block cancer initiation through several different mechanisms. These include prevention of attack of reactive oxygen species (ROS) on DNA, alteration of the metabolism of pre-carcinogens by phase-I drug metabolizing enzymes and excretion of reactive metabolites from the cell by a secondary line of defense that involves phase-II conjugating enzymes [such as glutathione-S-transferases (GST)] [24]. Paradoxically, some immunomodulatory plant products, such as epigallocatechin gallate (EGCG) of green tea and the polyphenol curcumin in turmeric and ascorbic acid of fruits act as both oxidants and antioxidants [25, 26]. Thus, oxidants and antioxidants derivatives obtained from natural products have been found to prevent tumorgenesis. Most agents were explored with respect to their effect on inflammation, cell cycle and apoptosis etc. Our data suggest that different fractions of natural products modulate different steps of the immune reactions. Oxidants, which commonly are regarded as inducers of inflammation and apoptosis, may actually activate dendritic cells in a positive way, while they probably damage T cells in close contact with the MHC-TCR complex. Therefore, antioxidants may be required for T cell survival.

Our previous publication indicated that MS reversed DMH-induced immune system Th2 profile to Th1 [22]. Next question is that which fraction of MS induces Th1 cytokines expression? Here we found that oxidant fraction did (inducing interferon-gamma expression), while anti-oxidant fraction actually induced IL-4, IL-5, IL-6, IL-10, etc., the Th2 profile of cytokines (Figure 4). This is another point of view to see oxidant in MS is necessary on preventing tumor-genesis. Although anti-oxidant fraction mainly induced Th2 profile of cytokines, but it also induced IL-17, so that anti-oxidant fracture may also have a tendency of a shift from Th2 to Th1. Thus, the difference in function of the two MS fractions on the expression of Th1 and Th2 cytokines suggest their synergistic role on immune regulation.

CD80 and CD86, which are expressed on DCs, bind to a cluster of members of the CD28 family, including CD28, CTLA-4, PD-1, ICOS and BTLA. Some of these ligands activate T cells (such as CD28), whilst others are negative regulators of T cells. We checked the effect of CD28 on CD8+ T cells, to see the activation of DC consequence is positive or negative activation of T cells. Activation signals may involve the expression of tumor-damaging enzymes, such as perforin in CD8+ T cells. We observed that mustard seeds reversed the DMH-induced down-regulation of perforin expression, while both the oxidant and antioxidant MS fractions were unable to replicate the effects of complete mustard seeds (data not shown), implicating that the expression of perforin by cytotoxic T cells may also be regulated by DCs activation.

Taken together, we found that the ether- and ethanol fractions of mustard seeds play different roles in activation of the immune system. When we found activation of DCs but not of CD8+ T cells, to be induced by the oxidant fraction MSE1, we hypothesized that the antioxidant effect needed to for T cells survival. We therefore exposed animals to the MSE1 + VitE, instead of the MSE2 fractions, and then successfully restored T cell activation. Therefore, we raise the theory that oxidants and antioxidants synergistically activate the immune system, possibly through immunological synapses between APCs and T cells. The activation of APCs needs oxygen radicals as second messengers, while the release of such radicals through MHC-TCR complexes may harm and damage T cells. Antioxidants seem to be required for secondary lymphocyte activation, which gives survival signals to activated T cells (Figure 9).

This work was supported by the National Natural Science Foundation of China (grants 81071549, 81272095, 81772074, 81974045), the Natural Science Foundation of Guangdong, China (grant 2012B050600002), the Guangdong Province Talent Recruitment Foundation and the Guangdong Innovative Research Team Program (No.201001Y0104675344), Guangzhou Science and technology project (201804020002).

Ethical Approval: The experiments were approved by the Animal Ethics Committee of Southern Medical University.

1. Jerome Galon, Anne Costes, Fatima Sanchez Cabo, Amos Kirilovsky, Bernhard Mlecnik, Christine Lagorce Pages, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006; 313(5795): 1960-1964. doi: 10.1126/science.1129139
2. Salman M Toor, Varun Sasidharan Nair, Reem Saleh, Rowaida Z Taha, Khaled Murshed, Mahmood Al Dhaheri, et al. Transcriptome of tumor-infiltrating T cells in colorectal cancer patients uncovered a unique gene signature in CD4 + T cells associated with poor disease-specific survival. Vaccines (Basel). 2021; 9(4): 334. doi: 10.3390/vaccines9040334
3. Chunling Ma, Qunyuan Zhang, Jian Ye, Fang Wang, Yanping Zhang, Eric Wevers, et al. Tumor-infiltrating γδ T lymphocytes predict clinical outcome in human breast cancer. J Immunol. 2012; 189: 5029-5036. doi: 10.4049/jimmunol.1201892
4. Y Naito, K Saito, K Shiiba, A Ohuchi, K Saigenji, H Nagura, et al. CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res. 1998; 58: 3491-3494.
5. Sytse J Piersma, Ekaterina S Jordanova, Mariette I E van Poelgeest, Kitty M C Kwappenberg, Jeanette M van der Hulst, Jan W Drijfhout, et al. High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res. 2007; 67: 354-361. doi: 10.1158/0008-5472
6. Zhang L, Conejo Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med. 2003; 348(3): 203-213. doi: 10.1056/NEJMoa020177
7. Natalia Martin Orozco, Pawel Muranski, Yeonseok Chung, Xuexian O Yang, Tomohide Yamazaki, Sijie Lu, et al. T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity. 2009; 31(5): 787-798. doi: 10.1016/j.immuni.2009.09.014
8. I Mellman, R M Steinman. Dendritic cells: specialized and regulated antigen processing machines. Cell. 2001; 106(3): 255-258. doi: 10.1016/s0092-8674(01)00449-4
9. Michael Valente, Camille Baey, Pauline Louche, Charles Antoine Dutertre, Lene Vimeux, Concepcion Maranon, et al. Apoptotic cell capture by DCs induces unexpectedly robust autologous CD4+ T-cell responses. Eur J Immunol. 2014; 44(8): 2274-2286. doi: 10.1002/eji.201344191
10. Lieping Chen, Dallas B Flies. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013; 13: 227-242. doi: 10.1038/nri3405
11. Drew M Pardoll. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012; 12 (4): 252-264. doi: 10.1038/nrc3239
12. Takashi Saito, Tadashi Yokosuka, Akiko Hashimoto Tane. Dynamic regulation of T cell activation and co-stimulation through TCR-microclusters. FEBS Lett. 2010; 584 (24): 4865-4871. doi: 10.1016/j.febslet.2010.11.036
13. Rikard Holmdahl, Outi Sareila, Angela Pizzolla, Susann Winter, Cecilia Hagert, Noora Jaakkola, et al. Hydrogen peroxide as an immunological transmitter regulating autoreactive T cells. Antioxid Redox Signal. 2013; 18: 1463-1474. doi: 10.1089/ars.2012.4734
14. Peter Olofsson, Jens Holmberg, Jesper Tordsson, Shemin Lu, Bo Akerstrom, Rikard Holmdahl. Positional identification of Ncf1 as a gene that regulates arthritis severity in rats. Nat Genet. 2003; 33(1): 25-32. doi: 10.1038/ng1058
15. Malin Hultqvist, Peter Olofsson, Jens Holmberg, B Thomas Backstrom, Jesper Tordsson, Rikard Holmdahl. Enhanced autoimmunity, arthritis, and encephalomyeslitis in mice with a reduced oxidative burst due to a mutation in the Ncf1 gene. Proc Natl Acad Sci U S A .2004; 101: 12646-12651. doi: 10.1073/pnas.0403831101
16. Keunok Jung, Inhak Choi. Emerging co-signaling networks in T cell immune regulation. Immune Netw. 2013; 13: 184-193. doi: 10.4110/in.2013.13.5.184
17. W E Court. A history of mustard in pharmacy and medicine. Pharm Hist (Lond). 1986; 16 (2): 4-7.
18. Rong Tsao, Qing Yu, John Potter, Mikio Chiba. Direct and simultaneous analysis of sinigrin and allyl isothiocyanate in mustard samples by high-performance liquid chromatography. J Agric Food Chem. 2002; 50(17): 4749-4753. doi: 10.1021/jf0200523
19. C Manesh, G Kuttan. Effect of naturally occurring allyl and phenyl isothiocyanates in the inhibition of experimental pulmonary metastasis induced by B16F-10 melanoma cells. Fitoterapia. 2003; 74 (4): 355-363. doi: 10.1016/s0367-326x(03)00055-8
20. Maria Uhl, Brenda Laky, Evelyn Lhoste, Fekadu Kassie, Michael Kundi, Siegfried Knasmüller. Effects of mustard sprouts and allylisothiocyanate on benzo(a)pyrene-induced DNA damage in human-derived cells: a model study with the single cell gel electrophoresis/Hep G2 assay. Teratog Carcinog Mutagen. 2003; Suppl 1: 273-1282. doi: 10.1002/tcm.10051  
21. Endalkachew Nibret, Sonja Krstin, Michael Wink. In vitro anti-proliferative activity of selected nutraceutical compounds in human cancer cell lines. BMC Res Notes. 2021; 7; 14(1): 18. doi: 10.1186/s13104-020-05435-1
22. Minggu Zhu, Haifeng Yuan, Wen Guo, Xinyan Li, Lin Jin, Ulf T Brunk, et al. Dietary mustard seeds (Sinapis alba Linn) suppress 1,2-dimethylhydrazine-induced immuno-imbalance and colonic carcinogenesis in rats. Nutr Cancer. 2012; 64: 464-472. doi: 10.1080/01635581.2012.658948
23. Kandan Aravindaram, Ning-Sun Yang. Anti-inflammatory plant natural products for cancer therapy. Planta Med. 2010; 76(11): 1103-1117. doi: 10.1055/s-0030-1249859
24. Francesca Tosetti, Douglas M Noonan, Adriana Albini. Metabolic regulation and redox activity as mechanisms for angioprevention by dietary phytochemicals. Int J Cancer. 2009; 125(9): 1997-2003. doi: 10.1002/ijc.24677
25. Mary E Waltner Law, Xiaohui L Wang, Brian K Law, Robert K Hall, Masao Nawano, Daryl K Granner. Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J Biol Chem. 2002; 277(38): 34933-34940. doi: 10.1074/jbc.M204672200