Search

Interleukin (IL)-10-producing type 1 regulatory T (Tr1) cells, which are Foxp3-memory T lymphocytes, play important roles in peripheral immune tolerance. We investigated whether Tr1 cells exert immunoregulatory effects in a mouse model of acute graft-versus-host disease (GVHD). Mouse CD4+ T cells were induced to differentiate in vitro into Tr1 cells using vitamin D3 and dexamethasone, and these donor-derived Tr1 cells were infused on the day of bone marrow transplantation. The Tr1 cell-transferred group showed less weight-loss and a twofold higher survival rate than the GVHD group, together with markedly decreased histopathologic grades. It was associated with the expansion of CD4+IL-4+ type 2 T-helper (Th2) cells and CD4+CD25+Foxp3+ regulatory T (Treg) cells. Furthermore, Tr1 cells decreased the numbers of CD4+interferon-γ+ Th1 and CD4+IL-17+ Th17 cells. Recipient mice harbored some Foxp3+ Tregs due to adoptive transfer of Tr1 cells, together with the upregulated expression of costimulatory molecules, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and inducible T-cell costimulator (ICOS); however, the Treg cells did not show the plasticity. Therefore, adoptive Tr1 cell therapy may be effective against manifestations of GVHD, exert immunomodulatory effects in a manner dependent on CTLA-4 and ICOS, and induce differentiation of the transferred Tr1 cells into Foxp3+ Treg cells.

BACKGROUND Mesenchymal stem cells (MSCs) are an attractive tool to treat graft-versus-host disease because of their unique immunoregulatory properties. Although human bone marrow-derived MSCs (BM-MSCs) were the most widely used MSCs in cell therapy until recently, MSCs derived from human umbilical cords (UC-MSCs) have gained popularity as cell therapy material for their ethical and noninvasive collection. AIM To investigate the difference in mechanisms of the immunosuppressive effects of UC-MSCs and BM-MSCs. METHODS To analyze soluble factors expressed by MSCs, such as indolamine 2,3-dioxygenase, cyclooxygenase-2, prostaglandin E2 and interleukin (IL)-6, inflammatory environments in vitro were reconstituted with combinations of interferon-gamma (IFN-γ), tumor necrosis factor alpha and IL-1β or with IFN-γ alone. Activated T cells were cocultured with MSCs treated with indomethacin and/or anti-IL-10. To assess the ability of MSCs to inhibit T helper 17 cells and induce regulatory T cells, induced T helper 17 cells were cocultured with MSCs treated with indomethacin or anti-IL-10. Xenogeneic graft-versus-host disease was induced in NOG mice (NOD/Shi-scid/IL-2Rγnull) and UC-MSCs or BM-MSCs were treated as cell therapies. RESULTS Our data demonstrated that BM-MSCs and UC-MSCs shared similar phenotypic characteristics and immunomodulation abilities. BM-MSCs expressed more indolamine 2,3-dioxygenase after cytokine stimulation with different combinations of IFN-γ, tumor necrosis factor alpha-α and IL-1β or IFN-γ alone. UC-MSCs expressed more prostaglandin E2, IL-6, programmed death-ligand 1 and 2 in the in vitro inflammatory environment. Cyclooxygenase-2 and IL-10 were key factors in the immunomodulatory mechanisms of both MSCs. In addition, UC-MSCs inhibited more T helper 17 cells and induced more regulatory T cells than BM-MSCs. UC-MSCs and BM-MSCs exhibited similar effects on attenuating graft-versus-host disease. CONCLUSION UC-MSCs and BM-MSCs exert similar immunosuppressive effects with different mechanisms involved. These findings suggest that UC-MSCs have distinct immunoregulatory functions and may substitute BM-MBSCs in the field of cell therapy.

Mesenchymal stem cells (MSCs) have been considered to be an ideal cellular source for graft-versus-host disease (GVHD) treatment due to their unique properties, including tissue repair and major histocompatibility complex (MHC)-unmatched immunosuppression. However, preclinical and clinical data have suggested that the immunomodulatory activity of MSCs is not as effective as previously expected. This study was performed to investigate whether the immunomodulatory capacity of MSCs could be enhanced by combination infusion of regulatory T (Treg) cells to prevent acute GVHD (aGVHD) following MHC-mismatched bone marrow transplantation (BMT). For GVHD induction, lethally irradiated BALB/c (H-2d) mice were transplanted with bone marrow cells (BMCs) and spleen cells of C57BL/6 (H-2b) mice. Recipients were injected with cultured recipient-derived MSCs, Treg cells, or MSCs plus Treg cells (BMT + day 0, 4). Systemic infusion of MSCs plus Treg cells improved clinicopathological manifestations and survival in the aGVHD model. Culture of MSCs plus Treg cells increased the population of Foxp3+ Treg cells and suppressed alloreactive T-cell proliferation in vitro. These therapeutic effects were associated with more rapid expansion of donor-type CD4+CD25+Foxp3+ Treg cells and CD4+IL-4+ type 2 T-helper (Th2) cells in the early posttransplant period. Furthermore, MSCs plus Treg cells regulated CD4+IL-17+ Th17 cells, as well as CD4+IFN-γ + Th1 cells. These data suggest that the combination therapy with MSCs plus Treg cells may have cooperative effects in enhancing the immunomodulatory activity of MSCs and Treg cells in aGVHD. This may lead to development of new therapeutic approaches to clinical allogeneic hematopoietic cell transplantation.

Background: Conventional therapeutic approaches, such as chemotherapy and radiotherapy, have been limited in successfully achieving durable response in Epstein Barr virus (EBV)-associated malignancies causing frequent relapse, low overall survival rate and poor prognosis. EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach to treat EBV associated lymphoma by enhancing EBV-specific immunity. Methods: To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated ten EBV+ ENKTCL patients and two EBV+ PTLD patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation were eligible to receive eight doses of 2 x107 LMP1/2a CTLs/m2. Results: Following infusion, there were no immediate or delayed toxicities. The seven-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71·4% to 100%) for ENKTCL patients respectively with a median follow-up of 94 months. Similarly, PTLD patients showed long-term remission with a follow-up of up to 70 months. Overall, circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Conclusion: Adoptive transfer of LMP1/2a CTLs in patients is a safe and effective post-remission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of EBV-associated lymphoma. Citation Format: Nayoun Kim, Hyun-Jung Sohn, Keon-Il Im, Young-Woo Jeon, Young-Sun Nam, Yunejin Song, Jun Seok Lee, Tai Gyu Kim, Seok-Goo Cho. Long term outcome of EBV-associated lymphoma patients treated with post-remission therapy using EBV LMP1 and LMP2a specific CTLs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT049.

Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here, we show that high-mobility group box 1 (HMGB1), a “danger signal” that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal-layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF-α, IL-1β and MIP-1β, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM. Citation Format: Keon-Il Im, Young-Sun Nam, Nayoun Kim, YuneJin Song, Young-Woo Jeon, Jung-Yeon Lim, Seok-Goo Cho. Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 465.

Mesenchymal stromal cells (MSCs) are seen as an ideal source of cells to induce graft acceptance; however, some reports have shown that MSCs can be immunogenic rather than immunosuppressive. We speculate that the immunomodulatory effects of regulatory T cells (Tregs) can aid the maintenance of immunoregulatory functions of MSCs, and that a combinatorial approach to cell therapy can have synergistic immunomodulatory effects on allograft rejection. After preconditioning with Fludarabine, followed by total body irradiation and anti-asialo-GM-1(ASGM-1), tail skin grafts from C57BL/6 (H-2k(b)) mice were grafted onto the lateral thoracic wall of BALB/c (H-2k(d)) mice. Group A mice (control group, n = 9) did not receive any further treatment after preconditioning, whereas groups B and C (n = 9) received cell therapy with MSCs or Tregs, respectively, on days -1, +6 and +13 relative to the skin transplantation. Group D (n = 10) received cell therapy with MSCs and Tregs on days -1, +6 and +13. Cell suspensions were obtained from the spleens of five randomly chosen mice from each group on day +7, and the immunomodulatory effects of the cell therapy were evaluated by flow cytometry and real-time PCR. Our results show that allograft survival was significantly longer in group D compared to the control group (group A). Flow cytometric analysis and real-time PCR for splenocytes revealed that the Th2 subpopulation in group D increased significantly compared to the group B. Also, the expression of Foxp3 and STAT 5 increased significantly in group D compared to the conventional cell therapy groups (B and C). Taken together, these data suggest that a combined cell therapy approach with MSCs and Tregs has a synergistic effect on immunoregulatory function in vivo, and might provide a novel strategy for improving survival in allograft transplantation.

Interleukin (IL) 21 plays a key role in the development of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. Therapeutic manipulation of IL-21 activity may improve acute GVHD during the early-posttransplant period. We investigated the mechanisms regulating T- and B-cells during IL-21 blockade in acute GVHD. Interleukin 21 blockade enhanced regulatory T and T helper (Th) 2 cell differentiation and inhibited Th1- and Th17-derived transcription factors and cytokines as a modulator of activated T-cells. Interleukin 21(-/-) cell recipients showed increased mature B- and marginal-zone B-cells, but decreased memory B-cells, germinal center formation, and plasma cells that did not lead to immunoglobulin production. B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are involved in the induction and maintenance of T- and B-cell responses. We observed decreased levels of only BAFF during acute GVHD and confirmed that mammalian target of rapamycin complex 1 was reduced by the BAFF/BAFF-receptor pathway. Therefore, this study suggests that IL-21 blockade modulates activated T- and B-cell homeostasis via BAFF-pathway-mediated inhibition in acute GVHD following murine allogeneic bone marrow transplantation.

NecroX-7 is a novel small molecule of the NecroX series, its chemical compound is C25H32N4O4S2 with molecular weight 516.67 based on the indole-moiety. It prevents from oxidative stress-induced cell death. NecroX-7 prevented the release of HMGB1 or ameliorated hepatic IR injury. It is shown that NecroX-7 can inhibit NAPDH oxidase activity and osteoclast differentiation. Furthermore, NecroX-7 protected against APAP-induced hepatic injury by directly binding to NAPQI. However, the function and the underlying mechanisms of NecroX-7 in cancer cell growth have not been well established. This study was aimed at evaluating the efficacy as anticancer agent in human lymphoma cells. Human Burkitt lymphoma cell lines were treated NecroX-7 in vitro. The effects of NecroX-7 treatment were evaluated using MTS assay for cell viability and annexin-V/propidium iodide labeling for apoptosis. Cell cycle was analyzed by Flow cytometry. Western blotting was used to look for NecroX-7 induced protein changes. As results, NecroX-7 treatment efficiently inhibited cell growth at dose- and time- dependent and induced apoptosis in cell lines. Apoptosis was showed by accumulation of annexin-V and PI-positive cells, caspase-3 activation and induction of PARP cleavage. The IC50 values of NecoX-7 in cell lines were around 50 uM. NecroX-7 induced G2/M phase cell cycle arrest in Ramos and Daudi cells.These results suggest that NecroX-7 is a potential anticancer agent for treatment of human Burkitt lymphoma cells and provide a rational for preclinical and clinical evaluation of NecoX-7 for lymphoma therapy. Citation Format: Eun- Jung Kim, Eun Sol Lee, Keon- Il Im, Soon Ha Kim, Jung- Yeon Lim, Nayoun Kim, Seok- Goo Cho. In vitro evaluation of a novel chemotherapeutic agent, NecroX-7, in human Burkitt lymphoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2720. doi:10.1158/1538-7445.AM2014-2720