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GM-CSF as an adjuvant has been shown to promote antitumor immunity in mice and humans; however, the underlying mechanism of GM-CSF–induced antitumor immunity remains incompletely understood. In this study, we demonstrate that GM-CSF potentiates the efficacy of cancer vaccines through IL9-producing Th (Th9) cells. GM-CSF selectively enhanced Th9 cell differentiation by regulating the COX2–PGE2 pathway while inhibiting the differentiation of induced regulatory T (iTreg) cells in vitro and in vivo. GM-CSF–activated monocyte-derived dendritic cells converted tumor-specific naïve Th cells into Th9 cells, and delayed tumor growth by inducing antitumor CTLs in an IL9-dependent manner. Our findings reveal a mechanism for the adjuvanticity of GM-CSF and provide a rationale for the use of GM-CSF in cancer vaccines.

Supplementary Figs. 1-9

Increased expression of coinhibitory molecules such as PD-1 and Tim-3 on NK cells has been demonstrated in advanced cancer patients who harbor MHC class I–deficient tumors. However, even in preclinical models, the antitumor effects of checkpoint blockade on NK cells have not been clearly elucidated. Here, we show that anti–PD-1/anti–Tim-3 treatment suppressed tumor progression in mice bearing MHC class I–deficient tumors, and the suppression was further enhanced by recombinant IL21 (rIL21) treatments through an NK-cell–dependent mechanism. We also show that the intratumoral delivery of rIL21 attracted NK cells to the tumor site in a CXCR3-dependent fashion. A combination of IL21 and checkpoint blockade facilitated the effector function of exhausted NK cells in cancer patients. Given the effects of the checkpoint blockade and rIL21 combination on NK cells infiltrating into MHC class I–deficient tumors, we suggest that the efficacy of checkpoint blockade can be enhanced through the administration of IL21 for advanced cancer patients with MHC class I–low/deficient tumors. Cancer Immunol Res; 6(6); 685–95. ©2018 AACR.

S1. PD-L1 or galectin-9 knock-out MC38 MHC class I-deficient tumor cells. S2. Confirmation of CD8 T cell or NK cell depletion by depleting antibody treatment. S3. Cytokine treatment increases IFN-γ secretion of NK cells in MHC class I-deficient tumors. S4. Enhanced anti-tumor effects of the combination treatment of IL-21 and anti-PD-1/anti-Tim-3 therapy in TC-1 and CT26 tumor models. S5. The frequency and number of intratumoral NK cells in mice treated with combination or mono therapy. S6. IL-21 with checkpoint blockade additively foster anti-tumor effects in MC38 MHC class I & galectin-9 or PD-L1 knock out tumor models. S7. IL-21 treatment induces chemokine production by TC-1 tumor cells. S8. Analysis of IFN-γ production in Tim-3â"€PD-1â"€ intratumoral NK cells from normal tissues of cancer patients.

MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139–mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139−/− mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139−/− CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes. Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

Myeloid progenitor cells have generally been considered the predominant source of myeloid cells under steady-state conditions. Here we show that NK cells contributed to a myeloid cell lineage pool in naive and tumor-bearing mice. Using fate tracing of NKp46+ cells, we found that myeloid cells could be derived from NK cells. Notably, among mature CD11b+ CD27+ NK cells, c-Kit+ CD24+ NK cells were capable of differentiating into a range of myeloid lineages in vitro and produced neutrophils and monocytes in vivo. The differentiation was completely inhibited by NK-stimulating cytokines. In addition to the potential for differentiation into myeloid cells, c-Kit+ CD24+ NK cells retained NK cell phenotypes and effector functions. Mechanistically, GATA-2 was necessary for the differentiation of c-Kit+ CD24+ NK cells. Therefore, we discovered that GATA-2-dependent differentiation of c-Kit+ CD24+ NK cells contributes to myeloid cell development and identified a novel pathway for myeloid lineage commitment under physiological conditions.

GM-CSF as an adjuvant has been shown to promote antitumor immunity in mice and humans; however, the underlying mechanism of GM-CSF–induced antitumor immunity remains incompletely understood. In this study, we demonstrate that GM-CSF potentiates the efficacy of cancer vaccines through IL9-producing Th (Th9) cells. GM-CSF selectively enhanced Th9 cell differentiation by regulating the COX2–PGE2 pathway while inhibiting the differentiation of induced regulatory T (iTreg) cells in vitro and in vivo. GM-CSF–activated monocyte-derived dendritic cells converted tumor-specific naïve Th cells into Th9 cells, and delayed tumor growth by inducing antitumor CTLs in an IL9-dependent manner. Our findings reveal a mechanism for the adjuvanticity of GM-CSF and provide a rationale for the use of GM-CSF in cancer vaccines.

MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8(+) T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8(+) CTLs compared with naive CD8(+) T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139–mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139(2/2) mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8(+) T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8(+) T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139(2/2) CD8(+) T cells are outcompeted by wild-type CD8(+) T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes. Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

PD-1–based cancer immunotherapy is a successful example of immune checkpoint blockade that provides long-term durable therapeutic effects in patients with cancer across a wide spectrum of cancer types. Accumulating evidence suggests that anti-PD-1 therapy enhances antitumor immunity by reversing the function of exhausted T cells in the tumor environment. However, the responsiveness rate of patients with cancer to anti-PD-1 therapy remains low, providing an urgent need for optimization and improvement. In this study, we designed an anti-PD-1–resistant mouse tumor model and showed that unresponsiveness to anti-PD-1 is associated with a gradual increase in CD8 T-cell exhaustion. We also found that invariant natural killer T cell stimulation by the synthetic ligand α-galactosylceramide (αGC) can enhance the antitumor effect in anti-PD-1–resistant tumors by restoring the effector function of tumor antigen–specific exhausted CD8 T cells. IL2 and IL12 were among the cytokines produced by αGC stimulation critical for reinvigorating exhausted CD8 T cells in tumor-bearing mice and patients with cancer. Furthermore, we observed a synergistic increase in the antitumor effect between αGC-loaded antigen-presenting cells and PD-1 blockade in a therapeutic murine tumor model. Our study suggests NKT cell stimulation as a promising therapeutic strategy for the treatment of patients with anti-PD-1–resistant cancer. Significance: These findings provide mechanistic insights into the application of NKT cell stimulation as a potent adjuvant for immunotherapy against advanced cancer. Cancer Res; 78(18); 5315–26. ©2018 AACR.

Increased expression of coinhibitory molecules such as PD-1 and Tim-3 on NK cells has been demonstrated in advanced cancer patients who harbor MHC class I–deficient tumors. However, even in preclinical models, the antitumor effects of checkpoint blockade on NK cells have not been clearly elucidated. Here, we show that anti–PD-1/anti–Tim-3 treatment suppressed tumor progression in mice bearing MHC class I–deficient tumors, and the suppression was further enhanced by recombinant IL21 (rIL21) treatments through an NK-cell–dependent mechanism. We also show that the intratumoral delivery of rIL21 attracted NK cells to the tumor site in a CXCR3-dependent fashion. A combination of IL21 and checkpoint blockade facilitated the effector function of exhausted NK cells in cancer patients. Given the effects of the checkpoint blockade and rIL21 combination on NK cells infiltrating into MHC class I–deficient tumors, we suggest that the efficacy of checkpoint blockade can be enhanced through the administration of IL21 for advanced cancer patients with MHC class I–low/deficient tumors. Cancer Immunol Res; 6(6); 685–95. ©2018 AACR.

During cancer immunoediting, loss of major histocompatibility complex class I (MHC-I) in neoplasm contributes to the evasion of tumours from host immune system. Recent studies have demonstrated that most natural killer (NK) cells that are found in advanced cancers are defective, releasing the malignant MHC-I-deficient tumours from NK-cell-dependent immune control. Here, we show that a natural killer T (NKT)-cell-ligand-loaded tumour-antigen expressing antigen-presenting cell (APC)-based vaccine effectively eradicates these advanced tumours. During this process, we find that the co-expression of Tim-3 and PD-1 marks functionally exhausted NK cells in advanced tumours and that MHC-I downregulation in tumours is closely associated with the induction of NK-cell exhaustion in both tumour-bearing mice and cancer patients. Furthermore, the recovery of NK-cell function by IL-21 is critical for the anti-tumour effects of the vaccine against advanced tumours. These results reveal the process involved in the induction of NK-cell dysfunction in advanced cancers and provide a guidance for the development of strategies for cancer immunotherapy., Loss of major histocompatibility complex MHC-I expression contributes to cancer immune evasion. Here, the authors show that, in both mice and humans, MHC-I downregulation is associated with the induction of NK-cell exhaustion and that IL-21 restores NK-cell function and inhibits tumours progression.

Myeloid progenitor cells have generally been considered the predominant source of myeloid cells under steady-state conditions. Here we show that NK cells contributed to a myeloid cell lineage pool in naïve and tumor-bearing mice. Using fate tracing of NKp46

Although treatment with the glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) agonistic antibody (DTA-1) has shown antitumor activity in various tumor models, the underlying mechanism is not fully understood. Here, we demonstrate that interleukin (IL)-21–producing follicular helper T (Tfh) cells play a crucial role in DTA-1–induced tumor inhibition. The administration of DTA-1 increased IL21 expression by Tfh cells in an antigen-specific manner, and this activation led to enhanced antitumor cytotoxic T lymphocyte (CTL) activity. Mice treated with an antibody that neutralizes the IL21 receptor exhibited decreased antitumor activity when treated with DTA-1. Tumor growth inhibition by DTA-1 was abrogated in Bcl6fl/flCd4Cre mice, which are genetically deficient in Tfh cells. IL4 was required for optimal induction of IL21-expressing Tfh cells by GITR costimulation, and c-Maf mediated this pathway. Thus, our findings identify GITR costimulation as an inducer of IL21-expressing Tfh cells and provide a mechanism for the antitumor activity of GITR agonism.

Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in responses to chronic viral infection and melanoma tumors. Subsequently, our studies have focused on investigating the mechanisms by which PSGL-1 regulates T cell activation and exhaustion and evaluating PSGL-1 as a target for cancer immunotherapy. Using the CD4cre system in combination with PSGL-1-floxed mice, we find that T cell intrinsic deletion of PSGL-1 is sufficient to promote enhanced tumor growth control of mesothelioma as well as melanoma tumors. We have found that in vivo PSGL-1 blockade recapitulates the enhanced melanoma tumor growth control observed with PSGL-1-/- CD8+ T cells and limits the development of T cell exhaustion during chronic LCMV Clone 13 viral infection. Conversely, ligation of PSGL-1 in vitro or in vivo drives enhanced upregulation T cell exhaustion markers, most notably PD-1 expression. Phosphoproteomic analysis of early T cell activation revealed the rapid upregulation of more than 30 phosphorylated proteins with significantly enhanced expression following PSGL-1 ligation during T cell activation than T cell activation alone. GSEA and western blot analysis confirmed the increased expression of several regulators of T cell signaling including pAkt1 and pErk in both activated PSGL-1-/- T cells and following PSGL-1 ligation during T cell activation. Single-cell RNA-sequencing of melanoma tumor-specific PSGL-1-/- CD8+ T cells identified differential modulation of genes associated with T cell metabolism and effector responses, including increased Mtor and Hif1a in tumors and Tcf7 in tumor-draining lymph nodes. The Seahorse glycolysis stress test identified that PSGL-1-/- T cells show increased glycolysis after 72 hours of in vitro activation at both sub-optimal and optimal levels of αCD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1-/- CD8+ T cells within two hours of stimulation. Further, 2-NBDG uptake was increased by PSGL-1-/- CD8+ T cells ex vivo from melanoma tumors. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. We hypothesize that PSGL-1 serves as a key regulator of early T cell activation and repeated signaling through PSGL-1 promotes differentiation into exhausted CD8+ T cells by sustaining TCR signaling. Conversely, we hypothesize that the absence of PSGL-1 allows for faster initiation and termination of TCR signaling, inhibiting differentiation into exhausted CD8+ T cells. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile which may drive their enhanced responses to tumors. Citation Format: Jennifer L. Hope, Dennis C. Otero, Eun-ah Bae, Christopher J Stairiker, Ashley B. Palete, Hannah A. Faso, Petrus de Jong, Garth Powis, Linda M. Bradley. PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO014.

Endoplasmic reticulum (ER) stress is induced by the accumulation of misfolded proteins and other cellular stresses, which lead to the unfolded protein response to restore ER homeostasis. Previous studies have shown that the ER stress response is required for T cell activation and differentiation. However, recent studies demonstrated that inhibition or deletion of ER stress mediators in T cells actually promotes anti-tumor immunity. Therefore, this incongruity as to the role of ER stress response in effector T cell function warrants further investigation. Our lab previously identified that the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), acts as a T cell immune checkpoint regulator. PSGL-1-deficiency enhances T cell function, resulting in the clearance of chronic viral infection and dramatic tumor control. To better understand the contribution of ER stress to T cell function, we assessed T cell phenotype and function together with the ER stress mediators, XBP1s and ATF4, in highly-functional PSGL-1−/− T cells as well as exhausted wild-type T cells in both tumor and virus models. In melanoma models, ER stress was highly induced in activated tumor-infiltrating T cells (TILs). Effector CD8+ TILs producing IFNg displayed elevated XBP1s and ATF4 compared to IFNg− TILs. Upon chronic LCMV Cl13 viral infection in PSGL-1−/− mice, we found increased virus-specific T cells with enhanced multi-functional effector responses and increased memory/stem cell-like T cell populations characterized by the expression of the transcription factor TCF-1, as well as increased ER stress responses. Together, these data suggest that ER stress plays an essential role in effector T cell functions in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in response to chronic viral infection and tumors. Subsequent studies have focused on investigating the role of PSGL-1 signaling in T cell responses, with an emphasis on understanding the mechanisms by which PSGL-1 regulates T cell exhaustion. Single-cell RNA-sequencing of tumor infiltrating PSGL-1−/− CD8+ T cells identified the upregulation and differential modulation of several genes associated with T cell metabolism and enhanced intratumoral responses, including Mtor, Hif1a, and Mki67 in Gzmb/Ifng double-positive cells from tumors and Tcf7 in both tumor draining and non-draining inguinal lymph nodes. These data suggest an important role for PSGL-1 signaling in the development and maintenance of effective anti-tumor T cell responses to melanoma. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. In situ activation of PSGL-1−/− CD8+ T cells demonstrated that PSGL-1−/− CD8+ T cells have increased glycolysis and increased glycolytic capacity at both sub-optimal and optimal levels of α-CD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1−/− CD8+ T cells within two hours of stimulation. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile in response to melanoma tumors.

Cancer immunotherapy has emerged as an effective therapeutic strategy to treat cancer. Among diverse immune populations, invariant natural killer T (iNKT) cells have shown potent antitumor activity by linking innate and adaptive immune systems. Upon activation by lipid antigens on CD1d molecules, iNKT cells rapidly produce various cytokines and trigger antitumor immunity directly or indirectly by activating other antitumor immune cells. Administration of a representative iNKT cell ligand alpha-galactosylceramide (α-GalCer) or α-GalCer-pulsed APCs effectively stimulates iNKT cells and thereby induces antitumor effects. In this review, we will introduce the biology and importance of NKT cells in antitumor immunity. Previous studies have demonstrated that iNKT cells not only activate various immune cells but also reinvigorate exhausted immune cells in the tumor microenvironment. Furthermore, we will summarize the major clinical trials utilizing iNKT-based immunotherapies.

Aging is accompanied by altered T-cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD-1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T-cell exhaustion and aging-associated immune dysfunction. In this study, we demonstrate that T-cell immunoglobulin mucin domain-3 (Tim-3), another exhaustion marker, is up-regulated on aged T cells, especially CD8(+) T cells. Tim-3-expressing cells also produced PD-1, but Tim-3(+) PD-1(+) CD8(+) T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim-3(-) PD-1(+) cells. Tim-3(+) PD-1(+) CD8(+) T cells showed more evident properties associated with exhaustion than Tim-3(-) PD-1(+) CD8(+) T cells: an exhaustion-related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL-10 and induced normal CD8(+) T cells to produce IL-10, which might contribute to immune dysregulation in aged mice. The generation of Tim-3-expressing CD8(+) T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8(+) T-cell population with age-associated exhaustion was distinguishable by its expression of Tim-3. These results provide clues for understanding the alterations that occur in T-cell populations with age and for improving dysfunctions related to the aging of the immune system.

Carcinoembryonic antigen (CEA) is a well-known tumor antigen that is found in the serum of patients with various cancers and is correlated with an increased risk of cancer recurrence and metastasis. To understand the tumor environment and to develop antitumor therapies, CEA has been studied as an antigen to activate/tolerate specific T cells. In this study, we show that CEA can function as a coinhibitory molecule and can inhibit the activation of human peripheral blood mononucleated cell-derived T cells. The addition of CEA-overexpressing tumor cells or immobilized CEA dampened both cell proliferation and the expression of IL-2 and CD69 expression in T cells after TCR stimulation. The phosphorylation of ERK and translocation of NFAT were hampered in these cells, whereas the phosphorylation of proximal TCR signaling molecules such as ZAP70 and phospholipase Cγ was not affected by immobilized CEA. To determine the relevance of carcinoembryonic antigen-related cell adhesion molecule-1 and Src homology region 2 domain-containing phosphatase (SHP) molecules to CEA-mediated suppression, we tested the effect of the SHP inhibitor, NSC-87877, on CEA-mediated suppression of T cells; however, it did not reverse the effect of CEA. Collectively, these results indicate that CEA can function as a modulator of T-cell responses suggesting a novel mechanism of tumor evasion.

Various immune checkpoint blockades have shown great promise for cancer patients. Anti-PD-1 therapies are known to reinvigorate exhausted immune cells, which progressively have lost effector function after chronic exposure to antigen. Although anti-PD-1 blockades were tested in various clinical trials on a wide range of tumors, many have observed clinical efficacy in only a portion of the patients tested. As such, these anti-PD-1 resistance or non-responsiveness needs to be overcome by combination therapeutic approaches that can synergize with existing immune checkpoint blockades. In this study, we investigated the effects of cytokines induced by invariant Natural Killer T (iNKT) cell ligand stimulation, more specifically α-galactosylceramide (αGC), on exhausted CD8 T cells. We used a mouse tumor model expressing ovalbumin antigen (OVA) and tracked OVA-specific CD8 T cells to show the effects on exhausted CD8 T cells in the tumor. αGC-induced cytokines not only enhanced the effector functions, but also diversified the functionality of exhausted CD8 T cells in vitro. Also, administration of αGC-loaded antigen presenting cells (APCs) restored exhausted CD8 T cell functions and led to higher cytotoxic effects in vivo. Among αGC-induced cytokines, we found IL-2 and IL-12 to be crucial in enhancing exhausted CD8 T cell function in mouse tumors and patient tumors. Furthermore, we showed that αGC-loaded APCs bring synergistic effects with PD-1 blockade in a mouse therapeutic tumor model. Our studies suggest a new insight for applying αGC as an adjuvant in cancer immunotherapy.

To understand anti-allergic effect of Curcuma aromatica (family Zingerberaceae), which inhibited passive cutenous anaphylaxis (PCA) reaction in preliminary study, we isolated its main constituents, curcumin, demethoxycurcumin and bisdemethoxycurcumin, and investigated their inhibitory effects against PCA reaction and scratching behavior. These curcuminoids inhibited the PCA reaction induced by the IgE-antigen complex (IAC) and the scratching behavior induced by compound 48/80. These curcuminoids also inhibited degranulation, protein expression of TNF-alpha and IL-4, and transcription factor NF-kappaB activation in IAC-induced RBL-2H3 cells. Of these curcuminoids, curcumin exhibited the most potent inhibition, followed by demethoxycurcumin and bisdemethoxycurcumin. These findings suggest that curcuminoids, particularly curcumin, can improve the symptoms of allergic diseases, such as anaphylaxis and itching.

To understand the anti-allergic mechanism of compound K, which is a metabolite of ginsenoside Rb1, a main constituent of the root of Panax ginseng C.A. Meyer (family Araliaceae), its inhibitory effect against IgE-antigen complex (IAC)-induced passive cutaneous anaphylaxis (PCA) reaction in mice and mRNA and protein expressions of allergic cytokines in IAC-stimulated RBL-2H3 cells were investigated. Orally administered ginsenoside Rb1 more potently inhibited PCA reaction when administered at 5 h prior to the IAC treatment than when administered at 1 h before. However, compound K orally administered 1 h before IAC treatment showed a more potent anti-PCA reaction effect than when treated at 5 h before. Orally administered ginsenoside Rb1 more potently inhibited PCA reaction induced by IAC in mice than intraperitoneally treated one, apart from orally administered its metabolite, compound K, which was more potent than the orally administered one. The compound K, a metabolite of ginsenoside Rb1, inhibited mRNA and protein expressions of IL-4 and TNF-α and the activation of their transcription factor NF-κB and MAPK in IAC-stimulated RBL-2H3 cells. These findings suggest that orally administered ginsenoside Rb1 may be dependent on its metabolism by intestinal microflora in the intestine and the compound K may improve allergic diseases by the inhibition of IL-4 and TNF-α expresseion.

Petasites japonicus (PJ, family Asteraceae) was fermented with lactic acid bacteria (LAB), Bifidobacterium breve (BB) or Lactobacillus acidophilus (LA), and its allergic effect was investigated. LAB-fermented PJs more potently inhibited IgE–antigen-stimulated degranulation of RBL-2H3 cells than non-fermented PJ, with BB-fermented PJ more potent than LA-fermented PJ. Fermented PJ more potently inhibited TNF-α and IL-4 cytokine expression and transcription factor NF-κB activation in IgE-induced RBL-2H3 cells. PJ and BB-fermented PJ also inhibited IgE–antigen-induced passive cutaneous anaphylaxis (PCA) reactions as well as compound 48/80-induced scratching behaviours in mice, with fermented PJ more potent than non-fermented PJ. These findings suggest that PJ may show anti-allergic effects by inhibiting degranulation, and TNF-α and IL-4 expression and its anti-allergic effects can be enhanced by LAB fermentation.

Lactic acid bacteria (LAB) can improve disturbances of indigenous microflora as well as inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease. We examined the anticolitic effect of Lactobacillus suntoryeus HY7801, which inhibited toll-like receptor (TLR)-4-linked NF-kappaB activation in human embryonic kidney (HEK) cells, in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitic mice.We measured the ability of commercial and intestinal LAB to inhibit lipopolysaccharide (LPS)-stimulated, TLR-4-linked NF-kappaB activation in HEK cells, as well as to inhibit colitis outcomes in TNBS-induced colitic mice. We also measured levels of the inflammatory markers, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6, and their transcription factor, NF-kappaB, in intestinal mucosa by enzyme-linked immunosorbent assay and immunoblotting.LAB inhibited TLR-4-linked NF-kappaB activation, and L. suntoryeus HY7801 was the most potent inhibitor. Intrarectal treatment of TNBS in mice caused colon shortening and also increased colonic expression of IL-1beta, IL-6, and TNF-alpha expression. However, oral administration of Lactobacillus HY7801 (100 mg/kg) inhibited colon shortening (p0.001) and myeloperoxidase activity in TNBS-induced colitic mice (p0.0002) and also decreased colonic expression of IL - 1beta (p0.003), IL-6 (p0.0001), and TNF-alpha (p0.0001). Lactobacillus HY7801 inhibited the NF-kappaB activation and TLR-4 expression induced by TNBS, as well as the expression of cyclooxygenase 2. Lactobacillus HY7801 also reduced the activity of intestinal bacterial glycosaminoglycan degradation and beta-glucuronidase induced by TNBS.L. suntoryeus HY7801 can improve colitis via the inhibition of TLR-4-linked NF-kappaB activation.

Here we fermented red ginseng (RG, the steamed root of Panax ginseng CA Meyer, Araliaceae) by Bifidobacterium longum H-1 and and investigated whether Bifidus fermentation could increase its anticolitic effect in 2,4,6-trinitrobenzenesulfonic acid- and dextran sulphate sodium (DSS)-induced colitic mice. The colitic mice had shortened colons, decreased body weight, and high myeloperoxidase activity, and administration of RG and fermented RG (FRG) significantly inhibited these changes. These agents also repressed expression of the pro-inflammatory cytokines, IL-1β, TNF-α, and cyclooxygenase (COX)-2, as well as the DSS-induced activation of NF-κB, with FRG more potently inhibiting the colitic markers than RG. The administration of RG and FRG also significantly reduced the activities of intestinal bacterial β-glucuronidase and chondroitin sulphate degradation activities, induced by a colitic inducer. These findings suggest that RG and FRG might improve colitis by regulating inflammatory cytokine expre...

To evaluate the effect of lactic acid bacteria (LAB) in inflammatory bowel diseases (IBD), inhibitory effect of several LAB isolated from intestinal microflora and commercial probiotics against NO production of lipopolysaccharide (LPS)-stimulated RAW264.7 cells was measured and anti-inflammatory effect of NO production-inhibitory LAB, Lactobacillus plantarum HY115 and L. brevis HY7401, in dextran sulfate sodium (DSS)-induced experimental colitic mice was investigated. The oral administration of the LAB to mice inhibited colon shortening and myeloperoxidase productivity in DSS-induced colitic mice. These LABs repressed the mRNA expressions of IL-1beta, TNF-alpha and IFN-gamma, as well as the protein expressions of IL-1beta and IL-6 proteins in the colon. The activation of the transcription factor, NF-kB, induced by DSS, was also inhibited by LAB. The administration of LAB reduced the degradation activities of chondroitin sulfate and hyaluronic acid of intestinal bacteria, induced by DSS, of which could induce the cytotoxic metabolites against intestinal cells. These findings suggest that NO-inhibitory LAB against LPS-stimulated RAW264.7 cells may improve colitis by the regulation of the inflammatory cytokine expression via the activation of transcription factor NF-kB as well as GAGs-degrading intestinal microflora.

The root of Paeonia lactiflora PALL (PL, Family Paeoniaceae) has been used frequently as an antipyretic and anti-inflammatory agent in traditional medicines of Korea, China and Japan. To evaluate antiallergic effect of PL, we isolated its main constituents, paeoniflorin and paeonol, and evaluated in vivo their inhibitory effects against passive cutaenous anaphylaxis (PCA) reaction induced by IgE-antigen complex and scratching behaviors induced by compound 48/80. PL, paeoniflorin and paeonol potently inhibited PCA reaction and scratching behaviors in mice. Paeoniflorin exhibited the most potent inhibition against scratching behaviors and the acetic acid-induced writhing syndrome in mice. Paeonol most potently inhibited PCA reaction and mast cells degranulation. These findings suggest that PL can improve IgE-induced anaphylaxis and scratching behaviors, and may be due to the effect of its constituents, paeoniflorin and paeonol.

Ginseng (the root of Panax ginseng CA Meyer, family Araliacease), which is used in Korea, China and Japan as a herbal medicine, was fermented with Ganoderma lucidum (GL) and their antiallergic effects were investigated. Of GLs used for fermentation, KCTC 6283 potently produced ginsenoside Rh2, followed by KFRI M101. KCTC 6532, and ginsenoside Rd, followed by KFRI M101. Oral administration of these fermented ginseng extracts inhibited allergic reactions, passive cutaneous anaphylaxis reaction induced by IgE and scratching behaviors induced by compound 48/80. Of them, the ginseng extract fermented by KCTC 6532 and KFRI M101 potently inhibited allergic reactions compared to that fermented by KCTC 6283. These findings suggest that the fermentation of ginseng with GL can increase its antiallergic activity and the increment of its antiallergic effect may be due to the biotransformation of ginseng saponins to ginsenosides Rd and Rh2.

To evaluate the antiatopic effect of Korea red ginseng (RG, steamed root of Panax ginseng CA Meyer, Family Araliaceae) fermented by Bifidobacterium longum H-1 (FRG), its inhibitory effect on passive cutaneous anaphylaxis (PCA) reaction and itching in mice was measured. FRG and its ingredient saponin fraction (FSF) potently inhibited PCA reaction and scratching behaviors. FRG at a dose of 200 ㎎/㎏ and FSF at a dose of 50 ㎎/㎏ significantly inhibited the scratching frequency by 45% and 47%, respectively. FRG and FSF also inhibited the degranulation and protein expression of tumor-necrosis factor-α and interleukin-4 of RBL-2H3 cells induced by IgE-complex. However, polysaccharide fraction of FRG (FPF) weakly inhibited it, compared with FSF. The inhibitory effect of FRG against PCA reaction and scratching behaviors more potently inhibited than that of RG. Based on these findings, FRG can improve allergic skin disorders atopic dermatitis by the regulation of TNF-α, and IL-4 produced by mast cells and basophils and its degranulation.

Licorice (Glycyrrhiza glabra L., Leguminosae) is frequently used in traditional medicine to treat inflammatory and allergic diseases. In this study, the main components (glycyrrhizin, 18beta-glycyrrhetinic acid, isoliquiritin, and liquiritigenin) were isolated from licorice, and their anti-allergic effects, such as antiscratching behavior and IgE production-inhibitory activity, were evaluated both in vitro and in vivo. Liquiritigenin and 18beta-glycyrrhetinic acid most potently inhibited the degranulation of RBL-2H3 cells induced by IgE with the antigen (DNP-HSA) and rat peritoneal mast cells induced by compound 48/80. Liquiritigenin and 18beta-glycyrrhetinic acid potently inhibited the passive cutaneous anaphylactic reaction as well as the scratching behavior in mice induced by compound 48/80. These components inhibited the production of IgE in ovalbumin-induced asthma mice but liquiritigenin had little effect. This suggests that the antiallergic effects of licorice are mainly due to glycyrrhizin, 18beta-glycyrrhetinic acid, and liquiritigenin, which can relieve IgE-induced allergic diseases such as dermatitis and asthma.

When the inhibitory effect of ginsenoside (G) Re isolated from ginseng and its metabolites G-Rg1, G-F1, GRh1 and protopanaxatriol in mouse ear skin psoriasis stimulated by oxazolone was investigated, G-Re and its metabolites suppressed mouse ear swelling stimulated by oxazolone. Among these agents tested, G-Rh1 most potently suppressed ear swelling as well as mRNA expression of COX-2 and proinflammatory cytokines IL-1β, TNF-α and interferon-γ. These findings suggest that G-Rh1 may improve chronic dermatitis and psoriasis.

Artemisia princeps Pampanini, which is called Ssajuarissuk in Korean (SS-1), was fermented with lactic acid bacteria (LAB) and their passive cutaneous anaphylaxis reaction-inhibitory activity was investigated. Of these fermented agents, SS-1 extract fermented withBifidobacterium infantis K-525 (F-SS-1) most effectively inhibited the release of β-hexosamindase from RBL-2H3 cells induced IgE. In IgE-induced RBL-2H3 cells, F-SS-1 inhibited proinflammatory cytokines IL-6 and TNF-α mRNA expression. Oral administration of SS-1 and F-SS-1 to mice inhibited passive cutaneous anaphylaxis (PCA) reaction induced by IgE and scratching behaviors induced by compound 48/80. The inhibitory activity of F-SS-1 against scratching behaviors induced by compound 48/80. The inhibitory activity of F-SS-1 against scratching behaviors was more effective than that of SS-1. These findings suggest that the fermentation of SS-1 with LAB can increase its antiallergic activity.

The effect of a main constituent ginsenoside Rg5 isolated from red ginseng and its metabolite ginsenoside Rh3 in a chronic dermatitis model was investigated. Ginsenosides Rg5 and Rh3 suppressed swelling of oxazolone-induced mouse ear contact dermatitis. These ginsenosides also reduced mRNA expressions of cyclooxygenase-2, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. The inhibition of ginsenoside Rh3 was more potent than that of ginsenoside Rg5. These findings suggest that ginsenoside Rh3 metabolized from ginsenoside Rg5 may improve chronic dermatitis or psoriasis by the regulation of IL-1beta and TNF-alpha produced by macrophage cells and of IFN-gamma produced by Th cells.

The anti-inflammatory effect of ginsenosides Rg3 and Rh2, which improves ischemic brain injury induced by middle cerebral artery occlusion, was investigated in lipopolysaccharide (LPS) and IFN-gamma-induced murine BV-2 microglial cells. Ginsenoside Rh2 inhibited the production of NO, with an IC50 value of 17 microM. The inhibitory effect of Rh2 on NO correlates with the decreased protein and mRNA expression of an inducible NO synthase (iNOS) gene. Additionally, ginsenoside Rh2 inhibited the expression of COX-2, pro-inflammatory TNF-alpha and IL-1beta in BV-2 cells induced by LPS/IFN-gamma, while it increased the expression of the anti-inflammatory cytokine IL-10. Electrophoretic mobility shift assays revealed that ginsenoside Rh2 significantly inhibited the LPS/IFN-gamma-induced AP-1 DNA binding activity, while it enhanced the protein binding to CRE sequences. However, it did not affect NF-kappaB binding activity. Thus, the anti-inflammatory effect of Rh2 appears to depend on the AP-1 and protein kinase A (PKA) pathway. The anti-inflammatory effect of ginsenoside Rg3 against LPS/IFN-gamma-activated BV-2 cells was less potent than that of ginsenoside Rh2. These findings suggest that the in vivo anti-ischemic effect of ginsenoside Rg3 may originate from ginsenoside Rh2, which is a main metabolite of ginsenoside Rg3 by intestinal microflora, and that of ginsenoside Rh2 may be due to its anti-inflammatory effect in brain microglia.

Background/Aim: The main component of Panax ginseng, which have been reported by many researchers, are ginsenoside Rb1, Rb2 and Rc. Orally administered ginsenosides are metabolized to 20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol (compound K) by intestinal bacteria and absorbed to blood. To understand its hepatoprotective effect and its mechanism, the effects of ginsenoside Rb1 and its metabolite compound K on chemically injured HepG2 cells and mice were investigated. Methods: Ginsenoside Rb1 and compound K were isolated from ginseng. Hepatotoxicity of HepG2 cells and mice was induced by tert-butyl hydroperoxide (t-BHP). Cytotoxicity for HepG2 cells and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for mice as markers of hepatoprotective activity were measured. Results: Compound K protected HepG2 cell cytotoxicity induced by t-BHP. However, ginsenoside Rb1 did not inhibit cytotoxicity. Nevertheless, both ginsenoside Rb1 and compound K significantly inhibited the increment of ALT and AST induced by t-BHP in mice, when it was orally administered. However, intraperitoneally administered ginsenoside Rb1 did not inhibit the increment of plasma ALT and AST induced by t-BHP in mice. These compounds did not exhibit antioxidant activity. However, compound K showed the potent membrane stabilizing activity more than ginsenoside Rb1. Conclusion: Compound K, which was produced from ginsenosides of Panax ginseng in intestine, could protect liver injury.

During the screening program to discover antipsoriatic agents from natural products, ginseng was found to show inhibitory activity in oxazolone-induced mouse ear dermatitis. Therefore, the effects of a main constituent ginsenoside Rb1 isolated from ginseng and its metabolite compound K on oxazolone-induced mouse ear dermatitis were investigated. Compound K at concentrations of 0.02% and 0.05% also potently suppressed mouse ear swelling by 54% and 76% at 16 days, respectively, although ginsenoside Rb1 did not significantly show the inhibitory activity. The compound K also significantly reduced the levels of mRNA of cyclooxygenase (COX)-2, IL-1beta, TNF-alpha, IFN-gamma and IL-4 increased in oxazolone-applied mouse ears. Based on these findings, the compound K may improve contact dermatitis or psoriasis by the regulation of COX-2 produced by macrophage cells and interferon-gamma and IL-4 induced by Th cells.

Antiallergic and antipsoriatic effects of Korean Red Ginseng (KRG, steamed root of Panax ginseng CA Meyer, Family Araliaceae) were measured. Orally administered KRG water extract potently inhibited passive cutaneous anaphylaxis (PCA). KRG water extract also showed the potent inhibition in oxazolone-induced mouse dermatitis, and suppressed mouse ear swelling by 39% at 16 days at a dose of 0.1 %. KRG water extract reduced the levels of mRNA of cyclooxygenase (COX)-2, IL-1β, TNF-α and IFN-γ increased in oxazolone-applied mouse ears, however, did not inhibit that of IL-4. KRG water extract also inhibited iNOS and COX-2 mRNA expression level of RAW264.7 cell induced by lipopolysaccharide. Based on these findings, we suggest that KRG can improve atopic and contact dermatitis by the regulation of IL-1β and TNF-α produced by macrophage cells and interferon-γ produced by Th1 cells.

To clarify the hepatoprotective effects of tectoridin and tectorigenin from Puerariae Flos, their effects on tert-butyl hyperoxide (t-BHP)-injured HepG2 cells and mice were investigated. When tectorigenin at a dose of 50 mg/kg was intraperitoneally administered to mice injured by t-BHP, it significantly inhibited the increase the activities of plasma ALT and AST by 39% and 41%, respectively, in the t-BHP-treated group. The inhibitory effect of tectorigenin is much more potent than that of a commercially available dimethyl diphenyl bicarboxylate. Orally administered tectoridin showed hepatoprotective activity. However, when tectoridin was intraperitoneally administrated to mice, no hepatoprotective activity was observed. Tectorigenin also protected against the cytotoxicity of HepG2 cells induced by t-BHP. This protection may have originated from the inhibition of apoptosis. Tectorigenin may be hepatoprotective and tectoridin should be a prodrug that is transformed to tectorigenin. Keywords:: tectorigenin, HepG2, hepatic injury

To evaluate the antipsoriatic effect of Chunghyuldan (CHD, Daio-Orengedokuto in Japanese), which exhibited anti-inflammatory and antiischemic actions, the inhibitory activity of CHD metabolized with and without human intestinal microflora was investigated in oxazolone-induced mouse ear dermatitis. The CHD and metabolized CHD (MCHD) at concentrations of 0.1% also potently suppressed mouse ear swelling by 52.7% and 63.2% at 16 d, respectively. The antipsoriatic effect between CHD and MCHD was not significantly different, although that of CHD weakly increased by the metabolism of human intestinal microflora. Both CHD and MCHD also potently reduced the mRNA levels of cyclooxygenase (COX)-2, interferon (IFN)-gamma and IL-4 increased in oxazolone-applied mouse ears, but weakly inhibited that of IL-1beta and TNF-alpha. Based on these findings, CHD may improve contact dermatitis or psoriasis by the regulation of COX-2 produced by macrophage cells and IFN-gamma and IL-4 produced by Th cells.

Red ginseng and fermented red ginseng were prepared, and their composition of ginsenosides and antiischemic effect were investigated. When ginseng was steamed at 98-100 degrees C for 4 h and dried for 5 h at 60 degrees C, and extracted with alcohol, its main components were ginsenoside Rg3> ginsenoside Rb1 > ginsenoside Rb2. When the ginseng was suspended in water and fermented for 5 days by previously cultured Bifidobacterium H-1 and freeze-dried (fermented red ginseng), its main components were compound K > ginsenoside Rg3 > or = ginsenoside Rh2. Orally administered red ginseng extract did not protect ischemia-reperfusion brain injury. However, fermented red ginseng significantly protected ischemica-reperfusion brain injury. These results suggest that ginsenoside Rh2 and compound K, which was found to be at a higher content in fermented red ginseng than red ginseng, may improve ischemic brain injury.

Background/Aim: To understand the relationship between the fluctuation in serum β-glucuronidase and hepatotoxicity, an inhibitor of β-glucuronidase was isolated from the flowers of Pueraria thunbergiana and its hepatoprotective activity was measured. Method: Tectorigenin was isolated from the flowers of pueria thunbergiana as an inhibitor of β-glucuronidase, and serum ALT, AST and biological parameters as markers for its hepatoprotective activity were measured on CCl4-induced liver injury in mice. The relationship between serum β-glucuronidase and hepatoprotective activities in mice was measured. Results: When tectorigenin at a dose of 100 mg/kg was intraperitoneally administered on CCl4-induced liver injury in mice, it significantly inhibited the increase of plasma ALT, AST and LDH activities. The inhibitory effect of tectorigenin is much more potent than that of dimethyl diphenyl bicarboxylate (DDB), which has been used as a commercial hepatoprotective agent. When tectoridin transformed to tectorigenin by intestinal bacteria was orally administered to mice, it showed hepatoprotective activity. However, when tectoridin was intraperitoneally administrated to mice, it did not exhibit hepatoprotective activity. Moreover, orally administered tectoridin not only inhibited β-glucuronidase but also increased GSH content and GST activity on CCl4-induced hepatotoxicity of mice. Conclusion: We insist that an inhibitor of β-glucuronidase tectorigenin may be hepatoprotective and tectoridin should be a prodrug transformed to tectorigenin.

When ginsenoside Rg3 was anaerobically incubated with human fecal microflora, all specimens metabolized ginsenoside Rg3 to ginsenoside Rh2 and protopanaxadiol. The main metabolite was ginsenoside Rh2. 20(S)-ginsenoside Rg3 was quickly transformed to 20(S)-ginsenoside Rh2 or 20(S)-protopanaxadiol in an amount 19-fold that compared with the transformation of 20(R)-ginsenoside Rg3 to 20(R)-ginsenoside Rh2 or 20(R)-protopanaxadiol. Among the bacteria isolated from human fecal microflora, Bacteroides sp., Eubacterium sp., and Bifidobacterium sp. metabolized ginsenoside Rg3 to protopanaxadiol via ginsenoside Rh2. However, Fusobacterium sp. metabolized ginsenoside Rg3 to ginsenoside Rh2 alone. Among ginsenoside Rg3 and its metabolites, 20(S)-protopanaxadiol and 20(S)-ginsenoside Rh2 exhibited the most potent cytotoxicity against tumor cell lines, 20(S)- and 20(R)-protopanaxadiols potently inhibited the growth of Helicobacter pylori, and 20(S)-ginsenoside Rh2 inhibited H+/K+ ATPase of rat stomach.

When kalopanaxsaponin K (KPK) from Kalopanax pictus was incubated for 24 h at 37 degrees C with human intestinal microflora, KPK was mainly metabolized to kalopanaxsaponin I (KPI) via kalopanaxsaponin H (KPH) rather than via kalopanaxsaponin J (KPJ), and then transformed to kalopanaxsaponin A (KPA) and hederagenin. Bacteroides sp., and Bifidobacterium sp. and Fusobacterium sp. transformed KPK to KPI and KPA and hederagenin via KPH or KPJ. However, Lactobacillus sp. and Streptococcus sp. transformed KPK to KPI, KPA, and hederagenin only via KPJ. The metabolite KPA of KPK showed potent antirheumatoid arthritis activity.