Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.     

Sub-mitogenic phorbol myristate acetate co-stimulation rescues the PHA-induced activation of both naïve and memory T cells cultured in the rotating-wall vessel bioreactor

T lymphocytes are unresponsive to T cell receptor (TCR) stimulation during culture in spaceflight or ground-based microgravity analogs such as the rotating-wall vessel (RWV) bioreactor. The TCR-induced activation of a subset of T cells can be rescued in the RWV by co-stimulation with sub-mitogenic doses of phorbol ester (PMA). We report that PMA co-stimulation of primary human T cells cultured in the RWV rescues the phytohemagglutinin (PHA)-induced activation of the CD8⁺ and CD4⁺ T cell subsets as well as naïve and memory CD4⁺ T cells. Importantly, T cells activated in the RWV by PHA + PMA contained these subsets in proportions strikingly similar to control cultures activated with PHA alone. The data indicate that rescuing T cell activation with PMA co-stimulation does not significantly perturb the heterogeneity of the responding cells, and represent an important proof of principle for the design of immune-boosting agents for use in spaceflight.     

Rhythmicity of engraftment and altered cell cycle kinetics of cytokine-cultured murine marrow in simulated microgravity compared with static cultures

Space flight with associated microgravity is complicated by "astronaut's anemia" and other hematologic abnormalities. Altered erythroid differentiation, red cell survival, plasma volume, and progenitor numbers have been reported. We studied the impact of microgravity on engraftable stem cells, culturing marrow cells in rotary wall vessel (RWV) culture chambers mimicking microgravity and in normal gravity nonadherent Teflon bottles. A quantitative competitive engraftment technique was assessed under both conditions in lethally irradiated hosts. We assessed 8-wk engraftable stem cells over a period spanning at least one cell cycle for cytokine (FLT-3 ligand, thrombopoietin [TPO], steel factor)-activated marrow stem cells. Engraftable stem cells were supported out to 56 h under microgravity conditions, and this support was superior to that seen in normal-gravity Teflon bottle cultures out to 40 h, with Teflon bottle culture support superior to RWV from 40 to 56 h. A nadir of stem cell number was seen at 40 h in Teflon and 48 h in RWV, suggesting altered marrow stem cell cycle kinetics under microgravity. This is the first study of engraftable stem cells under microgravity conditions, and the differences between microgravity and normal gravity cultures may present opportunities for unique future stem cell expansion strategies.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.     

Intact T cell receptor signaling by CD4+ T cells cultured in the rotating wall‐vessel bioreactor

T lymphocytes fail to proliferate or secrete cytokines in response to T cell receptor (TCR) agonists during culture in spaceflight or ground‐based microgravity analogs such as rotating wall‐vessel (RWV) bioreactors. In RWVs, these responses can be rescued by co‐stimulation with sub‐mitogenic doses of the diacyl glycerol (DAG) mimetic phorbol myristate acetate. Based on this result we hypothesized that TCR activation is abrogated in the RWV due to impaired DAG signaling downstream of the TCR. To test this hypothesis we compared TCR‐induced signal transduction by primary, human, CD4⁺ T cells in RWV, and static culture. Surprisingly, we found little evidence of impaired DAG signaling in the RWV. Upstream of DAG, the tyrosine phosphorylation of several key components of the TCR‐proximal signal was not affected by culture in the RWV. Similarly, the phosphorylation and compartmentalization of ERK and the degradation of IκB were unchanged by culture in the RWV indicating that RAS‐ and PKC‐mediated signaling downstream of DAG are also unaffected by simulated microgravity. We conclude from these data that TCR signaling through DAG remains intact during culture in the RWV, and that the loss of functional T cell activation in this venue derives from the affect of simulated microgravity on cellular processes that are independent of the canonical TCR pathway. J. Cell. Biochem. 109: 1201–1209, 2010. © 2010 Wiley‐Liss, Inc.     

Dynamic culture in a rotating-wall vessel bioreactor differentially inhibits murine T-lymphocyte activation by mitogenic stimuli upon return to static conditions in a time-dependent manner.

Depressed immune function is a well-documented effect of spaceflight. Both in-flight studies and ground-based studies using microgravity analogs, such as rotating wall vessel (RWV) bioreactors, have demonstrated that mitogen-stimulated T lymphocytes exhibit decreased proliferation, IL-2 secretion, and activation marker expression in true microgravity and the dynamic RWV-culture environment. This study investigates the kinetics of RWV-induced T lymphocyte inhibition by monitoring the ability of Balb/c mouse splenocytes to become activated under static culture conditions after concanavalin A (Con A) stimulation in an RWV. Splenocytes were stimulated with Con A and cultured for up to 24 h in the RWV before being allowed to "recover" under static culture conditions in the continued presence of Con A. The T-lymphocyte fraction of splenocytes was assayed during the recovery period for IL-2 secretion, expansion of the T-lymphocyte population, and expression of the activation marker CD25. Our results indicate that CD25 expression was not affected by any duration of RWV exposure. In contrast, proliferation and IL-2 secretion were inhibited by >8 and 12 h of exposure, respectively. Culture in the RWV for 24 h resulted in a near-complete loss of cellular viability during the recovery period, which was not seen in cells maintained in the RWV for 16 h or less. Taken together, these results indicate that for up to 8 h of RWV culture activation is not significantly impaired upon return to static conditions; longer duration RWV culture results in a gradual loss of activation during the recovery period most likely because of decreased T-cell viability and/or IL-2 production.     

Studies of chondrogenesis in rotating systems

A great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage. Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies. The bioreactor (rotating-wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation. Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 [Chesterman and Smith, J Bone Joint Surg 50B:184–197, 1965]. The ability to use large, tissue-like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders. In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations. Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground-based tool for studying how cell systems will react to microgravity. © 1993 Wiley-Liss, Inc.     

Epstein-barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

Summary Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein—Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4–7 d. The cells were then transferred to 50-cm³ tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated withn-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2–3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2–3 or 48 h with TPA. TPA reactivation of EBV following a 2–3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3–4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.     

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.     

Activation and expansion of cytotoxic T lymphocytes from tumor-draining lymph nodes

Summary Large numbers of cytotoxic T lymphocytes (CTL) could be generated from tumor-draining lymph nodes (DLN) from mice bearing PHS-5 tumor by culturing at low density with autologous tumor cell stimulators and 20 U/ml recombinant interleukin-2 (IL-2). Outgrowth of metastatic tumor cells in culture was prevented by use of this hypoxanthine/aminopterin/thymidine-sensitive mutant of P815, PHS-5. After 9 days in culture, lymphoid cells demonstrated specific cytotoxicity against autologous tumor target cells. Lymph node cells could be expanded continuously in culture with repeated tumor stimulation with up to 7500-fold increase in cell number by 6 weeks; although CTL could be activated from tumor-bearing host spleen cells in short-term culture, they showed no significant growth in long-term cultures. Phenotypically, DLN cells were a mixture of CD8⁺ and CD4⁺ cells immediately after harvest but after 2 weeks in culture they were predominantly CD8⁺ CD4^–. CTL could be generated from tumor-bearing mice 10–14 days after i.d. tumor inoculation into the abdominal wall, but the immune response declined both in spleen and DLN by 21 days. Much greater CTL activity could be generated from axillary DLN that contained metastases than from non-draining popliteal nodes that were free of metastatic tumor cells. Some CTL activity could be generated from DLN with the addition of IL-2 alone but was further increased by the addition of more tumor cells as stimulators. When adoptively transferred to a host with 3-day P815 liver metastases, lymphocytes from DLN activated in vitro were able to reduce or eliminate metastases with very little or no IL-2 administered concomitantly. As few as 10⁶ cells were therapeutically effective, and in vivo efficacy was tumor-specific, since L5178Y liver metastases were not affected.This work was supported in part by grants CA42443, CA48075 and T32-CA09210 from the National Cancer Institute, Department of Health and Human ServicesRecipient of the Canadian Cancer Society McEachern Fellowship.     

Identification of mechanosensitive genes in osteoblasts by comparative microarray studies using the rotating wall vessel and the random positioning machine

Weightlessness or microgravity of spaceflight induces bone loss due in part to decreased bone formation by unknown mechanisms. Due to difficulty in performing experiments in space, several ground-based simulators such as the Rotating Wall Vessel (RWV) and Random Positioning Machine (RPM) have become critical venues to continue studying space biology. However, these simulators have not been systematically compared to each other or to mechanical stimulating models. Here, we hypothesized that exposure to RWV inhibits differentiation and alters gene expression profiles of 2T3 cells, and a subset of these mechanosensitive genes behaves in a manner consistent to the RPM and opposite to the trends incurred by mechanical stimulation of mouse tibiae. Exposure of 2T3 preosteoblast cells to the RWV for 3 days inhibited alkaline phosphatase activity, a marker of differentiation, and downregulated 61 and upregulated 45 genes by more than twofold compared to static 1 g controls, as shown by microarray analysis. The microarray results were confirmed by real-time PCR and/or Western blots for seven separate genes and proteins including osteomodulin, runx2, and osteoglycin. Comparison of the RWV data to the RPM microarray study that we previously published showed 14 mechanosensitive genes that changed in the same direction. Further comparison of the RWV and RPM results to microarray data from mechanically loaded mouse tibiae reported by an independent group revealed that three genes including osteoglycin were upregulated by the loading and downregulated by our simulators. These mechanosensitive genes may provide novel insights into understanding the mechanisms regulating bone formation and potential targets for countermeasures against decreased bone formation during space flight and in pathologies associated with lack of bone formation.     

Characteristics of human dendritic cells generated in a microgravity analog culture system

Summary Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected, to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR⁺ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytoseAspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of Dc expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

Recycled addition of CD4+ T cell-rich population for induction of human autologous cytotoxic T lymphocytes: A practically efficient method

When CD4⁺ T cell-rich population appears in theinitial trial in induction cultures of humanautologous cytotoxic T lymphocytes (CTL), the cultureresults frequently in no or weak killing activity andtherefore usually be discarded as an `unsuccessful'CTL induction culture. However, addition of theinitial CD4⁺ T cell-rich population enabledefficient induction of the autologous CTL in theensuing trials. The CTL thus generated exhibitedstronger killing activities against autologous braintumor cells and ovarian tumor cells than previouslyobserved. This simple recycling of the primed butinert CD4⁺ T cell-rich population for CTLinduction will promote clinical practice of adoptiveimmunotherapy of human tumors with autologous CTL.     

Simulated microgravity suppresses osteoblast phenotype,Runx2 levels and AP-1 transactivation

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood but involve the suppression of osteoblast function. To elucidate the influence of mechanical unloading on osteoblasts, a rotating wall vessel (RWV) was employed as a ground based model of simulated microgravity. Mouse MC3T3-E1 osteoblasts were grown on microcarrier beads for 14 days and then placed in the RWV for 24 h. Consistent with decreased bone formation during actual spaceflight conditions, alkaline phosphatase and osteocalcin expression were decreased by 80 and 50%, respectively. In addition, runx2 expression and AP-1 transactivation, key regulators of osteoblast differentiation and bone formation, were reduced by more than 60%. This finding suggests that simulated microgravity could promote dedifferentiation and/or transdifferentiation to alternative cell types; however, markers of adipocyte, chondrocyte, and myocyte lineages were not induced by RWV exposure. Taken together, our results indicate that simulated microgravity may suppress osteoblast differentiation through decreased runx2 and AP-1 activities.     

Effects of lamivudine on the hepatitis B virus specific CD8+ cytotoxic T lymphocyte responses via peptide-MHC tetrameric complexes assay

Summary Lamivudine, an oral nucleoside analogue, inhibits hepatitis B virus (HBV) replication. It has been shown to be able to restore T cell responsiveness and to induce a type 1 T helper cell (Th 1) immunity in chronic HBV patients. To further examine the effects of lamivudine on cytotoxic T Imphocyte (CTL), responses, two HBV antigenic peptide-HLA-A2 tetrameric complexes containing peptides derived from HBV core protein (residues 18–27; FLPSDFFPSV) and polymerase (residue 551–559; YMDDVVLGA) were constructed. These two tetramers were used to serially determine the frequency of HBV antigen-specific CD8⁺ T cells before and during the treatment of lamivudine. The specificity of these tetramers was confirmed by (a) nonstaining of CD8⁺ T cells from HLA-A2-negative HBV patients, (b) having variable frequency data in the different teteramer measurement, and (c) showing peptide-specific CTL activity in the sorted tetramer-stanining CD8⁺T cells. Low frequency of HBV-specific CTLs was measured for both tetramers before lamivudine treatment. However, the number of CD8⁺ T cells specific for HBV core 18–27 increased significantly during lamivudine treatment. In contrast, relatively lower frequency of HBV pol 551–559 specific CD8⁺T cells was persistently measured after lamivudine treatment. These results indicated that the lamivudine treatment could enhance HBV specific CTL responses.     

Increased PRAME-Specific CTL Killing of Acute Myeloid Leukemia Cells by Either a Novel Histone Deacetylase Inhibitor Chidamide Alone or Combined Treatment with Decitabine

As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8⁺ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201⁺ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME^100–108 or PRAME^300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.     

Cloning of Murine T Cells

T-cell clones have been extremely useful in studying the cellular arm of the mammalian immune response. A method for generating homogeneous long-term, antigen-specific cultures of murine T cells is discussed, with emphasis on obtaining CD4⁺T-cell clones. Some procedures and assays that will be helpful in characterizing the T-cell clones are also included.     

A surfactin cyclopeptide of WH1fungin used as a novel adjuvant for intramuscular and subcutaneous immunization in mice

WH1fungin, a surfactin cyclopeptide from Bacillus amyloliquefaciens WH1, is firstly reported as a novel immunoadjuvant, which can markedly enhance the immune response when given in mixture with antigens. After intramuscular or subcutaneous immunization, WH1fungin can help to induce both of durable humoral and cellular immune response, even as strong as Freund's adjuvant. Both IgG1 and IgG2a antigen-specific antibodies were elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice intramuscularly immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8⁺TNF-α⁺ and CD8⁺IFN-γ⁺ T cell populations, and also an increase in CD4⁺TNF-α⁺ T cells and CD4⁺IFN-γ⁺ T cell populations was found from mice subcutaneously immunized with OVA plus WH1fungin when responded to OVA Th peptide stimulation. These results further suggest that WH1fungin helps to elicit humoral and cellular responses to OVA. The potential mechanism of WH1fungin as an immunoadjuvant was investigated. In vitro assays showed that WH1fungin could enter into RAW 264.7 cells, induce ROS accumulation, and increase the expression of cell surface markers and cytokines in cells. Further investigation suggested that WH1fungin might exert its adjuvant activity by ligating with TLR-2 in antigen present cells such as RAW 264.7. Taken together, WH1fungin is very potent as a novel adjuvant for development of vaccines in the future.