Xenogeneic Humoral Immune Responses to Human Mesenchymal Stem Cells in Mice.

Background and Objectives: Many preclinical studies have been conducted using animal disease models to determine the effectiveness of human mesenchymal stem cells (hMSCs) for treating immune and inflammatory diseases based on the belief that hMSCs are not immunogenic across species. However, several researchers have suggested xenogeneic immune responses to hMSCs in animals, still without detailed features. This study aimed to investigate a xenogeneic humoral immune response to hMSCs in mice in detail. Methods and Results: Balb/c mice were intraperitoneally injected with adipose tissue-derived or Wharton's jelly-derived hMSCs. Sera from these mice were titrated for each isotype. To confirm specificity of the antibodies, hMSCs were stained with the sera and subjected to a flow cytometic analysis. Spleens were immunostained for proliferating cell nuclear antigen to verify the germinal center formation. Additionally, splenocytes were subjected to a flow cytometric analysis for surface markers including GL-7, B220, CD4, CD8, CD44, and CD62L. Similar experiments were repeated in C57BL/6 mice. The results showed increased IgG1 and IgG2a titers in the sera from Balb/c mice injected with hMSCs, and the titers were much higher in the secondary sera than in the primary sera. These antibodies were specifically stained the hMSCs. Germinal centers were observed in the spleen, and flow cytometric analysis of the splenocytes showed higher frequencies of centroblasts (B220+ GL7+) and memory T cells (CD62L+ CD44+) both in CD4+ and CD8+ subsets. Similar results were obtained for C57BL/6 mice. Conclusions: hMSCs induced a humoral immune response in mice, with characters of T cell-dependent immunity.

Cytokine-Induced Killer Cell Immunotherapy Combined With Gemcitabine Reduces Systemic Metastasis in Pancreatic Cancer: An Analysis Using Preclinical Adjuvant Therapy-Mimicking Pancreatic Cancer Xenograft Model.

OBJECTIVES: To evaluate the efficacy and safety of cytokine-induced killer (CIK) cell therapy in pancreatic cancer. METHODS: An orthotopic murine model of pancreatic cancer and adjuvant therapy-mimicking xenograft murine model that underwent splenectomy was created. Eighty mice were randomized into four groups: the control, gemcitabine alone, CIK alone, and CIK with gemcitabine groups. The tumor growth was monitored using bioluminescence imaging once weekly. RESULTS: In the orthotopic murine model, the treatment groups showed a significantly longer survival than the control group (median: not reached vs 125.0 days; 95% confidence interval, 119.87-130.13; P = 0.04); however, the overall survival did not differ significantly among the treatment groups (P = 0.779). The metastatic recurrence rate and overall survival were also not significantly different among the groups in the adjuvant therapy-mimicking xenograft murine model (P = 0.497). However, the CIK and gemcitabine combination suppressed the metastatic recurrence effectively, with recurrence-free survival being significantly longer in the CIK with gemcitabine group than in the control group (median, 54 days; 95% confidence interval, 25.00-102.00; P = 0.013). CONCLUSIONS: The combination of CIK and gemcitabine suppressed systemic metastatic recurrence, with promising efficacy and good tolerability in an adjuvant setting of pancreatic cancer.

SB365, Pulsatilla Saponin D Induces Caspase-Independent Cell Death and Augments the Anticancer Effect of Temozolomide in Glioblastoma Multiforme Cells.

SB365, a saponin D extracted from the roots of Pulsatilla koreana, has been reported to show cytotoxicity in several cancer cell lines. We investigated the effects of SB365 on U87-MG and T98G glioblastoma multiforme (GBM) cells, and its efficacy in combination with temozolomide for treating GBM. SB365 exerted a cytotoxic effect on GBM cells not by inducing apoptosis, as in other cancer cell lines, but by triggering caspase-independent cell death. Inhibition of autophagic flux and neutralization of the lysosomal pH occurred rapidly after application of SB365, followed by deterioration of mitochondrial membrane potential. A cathepsin B inhibitor and N-acetyl cysteine, an antioxidant, partially recovered cell death induced by SB365. SB365 in combination with temozolomide exerted an additive cytotoxic effect in vitro and in vivo. In conclusion, SB365 inhibits autophagic flux and induces caspase-independent cell death in GBM cells in a manner involving cathepsin B and mainly reactive oxygen species, and its use in combination with temozolomide shows promise for the treatment of GBM.

Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro.

Vitamin C is an essential micronutrient that affects immune responses. T cells are one of the main players in acquired immunity and have been reported to be influenced by in vivo vitamin C supplementation. Yet, the way by which T cells uptake vitamin C and what direct effects vitamin C exerts on the cells are not known. To elucidate, we isolated human peripheral blood T cells and analyzed the expression of sodium-dependent vitamin C transporters (SVCT). T cells were activated in vitro in the absence or presence of vitamin C, before or after activation. As results, human T cells expressed SVCT2, but not SVCT1, and the expression level increased following activation. Vitamin C added in the culture media generally did not affect T-cell behaviors following activation, such as proliferation, apoptosis, expression of CD25 and CD69, and interleukin 2 secretion, regardless whether it was added before or after activation. However, exceptionally, high concentration vitamin C, when it was added before activation, but not after activation, did exert toxic effects on cell activation with respect to the above-mentioned parameters. In conclusion, we showed the expression of SVCT2 in human T cells for the first time. Vitamin C exerted toxic effects, at least in vitro, when the concentration was high and when it was given before activation. These toxic effects are not thought to be via anti-oxidant effects of vitamin C.

Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-ß Production and Th17 Differentiation.

It has recently been reported that the CD40-CD40 ligand (CD40L) interaction is important in Th17 development. In addition, transforming growth factor-beta (TGF-ß) promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-ß. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-ß production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-ß production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells.

Vitamin C treatment of mouse bone marrow-derived dendritic cells enhanced CD8(+) memory T cell production capacity of these cells in vivo.

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4(+) T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8(+) T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ(+) CD8(+) T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44(high) CD62L(low) CD8(+) effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors.

Lack of transglutaminase 2 diminished T-cell responses in mice.

Transglutaminase 2 (TG2) has been reported to play a role in dendritic cell activation and B-cell differentiation after immunization. Its presence and role in T cells, however, has not been explored. In the present study, we determined the expression of TG2 on mouse T cells, and evaluated its role by comparing the behaviours of wild-type and TG2(-/-) T cells after activation. In our results, naive T cells minimally expressed TG2, expression of which was increased after activation. T-cell proliferation, expression of activation markers such as CD69 and CD25, and secretions of interleukin-2 and interferon-γ were suppressed in the absence of TG2, presumably due, in part, to diminished nuclear factor-κB activation. These effects on T cells seemed to be reflected in the in vivo immune response, the contact hypersensitivity reaction elicited by 2,4-dinitro-1-fluorobenzene, with lowered peak responses in the TG2(-/-) mice. When splenic T cells from mice immunized with tumour lysate-loaded wild-type dendritic cells were re-challenged ex vivo with the same antigen, the profile of surface markers including CD44, CD62L, and CD127 strongly indicated lesser generation of memory CD8(+) T cells in TG2(-/-) mice. In the TG2(-/-)  CD8(+) T cells, moreover, Eomes expression was markedly decreased. These results indicate possible roles of TG2 in CD8(+) T-cell activation and CD8(+) memory T-cell generation.

Chronic vitamin C insufficiency aggravated thioacetamide-induced liver fibrosis in gulo-knockout mice.

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo(-/-) mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells.

Suppression of in vitro murine T cell proliferation by human adipose tissue-derived mesenchymal stem cells is dependent mainly on cyclooxygenase-2 expression.

Mesenchymal stem cells (MSCs) of human origin have been frequently applied to experimental animal models to evaluate their immunomodulatory functions. MSCs are known to be activated by cytokines from T cells, predominantly by interferon-γ (IFN-γ), in conjunction with other cytokines such as tumor necrosis factor-α (TNF-α) and interlukin-1ß. Because IFN-γ is not cross-reactive between human and mouse species, the manner in which human MSCs administered in experimental animals are activated and stimulated to function has been questioned. In the present study, we established MSCs from human adipose tissue. They successfully suppressed the proliferation of not only human peripheral blood mononuclear cells but also mouse splenic T cells. When these human MSCs were stimulated with a culture supernatant of mouse T cells or recombinant murine TNF-α, they expressed cyclooxygenase-2 (COX-2), but not indoleamine 2,3-dioxygenase. The dominant role of COX-2 in suppressing mouse T cell proliferation was validated by the addition of COX-2 inhibitor in the co-culture, wherein the suppressed proliferation was almost completely recovered. In conclusion, human MSCs in a murine environment were activated, at least in part, by TNF-α and mainly used COX-2 as a tool for the suppression of in vitro T cell proliferation. These results should be considered when interpreting results for human MSCs in experimental animals.