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LBAL was developed as an adalimumab (Humira®) biosimilar using Chinese hamster ovary cell lines. Comparable quality, safety, and efficacy between a biosimilar and its reference product should be ensured for regulatory approval. Here, we present the results of a comprehensive physicochemical and biological characterization between LBAL and Humira®. As physicochemical attributes, primary and higher-order structure, N-glycan profile, and disulfide linkage were investigated. Biological attributes were evaluated by target/receptor binding analysis and in vitro/ex vivo cell-based assays, which are linked to mechanisms of action. As a result, LBAL had the identical amino acid sequence, similar post-translational modifications and N-/C-terminal variants, and comparable primary, secondary, and tertiary structures and disulfide linkage profile. However, some differences in N-glycan profiles were observed. Biological activities, including tumor necrosis factor (TNF) binding, TNF-neutralization, apoptosis, Fc receptor binding, and complement-dependent cytotoxicity, were largely consistent. Despite a slightly lower antibody-dependent cellular cytotoxicity activity in LBAL, this difference was not significant under physiological conditions. As indicated, this extensive analytical characterization and functional comparison assessment showed that LBAL was similar to Humira®, with minor differences of no clinical relevance. Taken together, our comparative assessment of physicochemical and biological attributes demonstrated that LBAL is structurally and functionally very similar to Humira®, supporting the biosimilarity of clinical efficacy and safety.

This study aims to test two hypotheses about the online processing of Gapping: whether the parser inserts an ellipsis site in an incremental fashion in certain coordinated structures (the Incremental Ellipsis Hypothesis), or whether ellipsis is a late and dispreferred option (the Ellipsis as a Last Resort Hypothesis). We employ two offline acceptability rating experiments and a sentence fragment completion experiment to investigate to what extent the distribution of Gapping is controlled by grammatical and extra-grammatical constraints. Furthermore, an eye-tracking while reading experiment demonstrated that the parser inserts an ellipsis site incrementally but only when grammatical and extra-grammatical constraints allow for the insertion of the ellipsis site. This study shows that incremental building of the Gapping structure follows from the parser’s general preference to keep the structure of the two conjuncts maximally parallel in a coordination structure as well as from grammatical restrictions on the distribution of Gapping such as the Coordination Constraint.

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/aims Adoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation. Methods To induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 105 T cell-depleted bone marrow cells and 5 × 105 CD4+CD25- splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 105 cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1). Results Systemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups. Conclusion Our findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.

Background: Cytomegalovirus(CMV)-related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). CMV-specific cytotoxic T lymphocytes (CMV-CTLs) have been reported as an alternative to antiviral drugs that provide long-term CMV-specific immunity without major side effects. However, their application has been limited by the prolonged manufacturing process required. Methods: In this study, we applied the IFN-γ cytokine capture system (CCS) using the fully automated CliniMACS Prodigy device for rapid production of CMV-CTLs, which may be applicable in clinically urgent CMV-related diseases. Five validation runs were performed using apheresis samples from randomly selected CMV-seropositive healthy blood donors. Successive processes, including antigen stimulation, anti-IFN-γ labeling, magnetic enrichment and elution, were then performed automatically using the CliniMACS Prodigy, which took approximately 13 h. Results: The original apheresis samples consisted mainly of CD45RA+ CD62L+ naïve T cells as well as 0.3% IFN-γ-secreting CD3+ T cells that showed a response to the CMV pp65 antigen (CD3+ IFN-γ+ cells). Following IFN-γ enrichment, the target fraction contained 51.3% CD3+ IFN-γ+ cells with a reduction in naïve T cells and selection of CD45RA- CD62L- and CD45RA+ CD62L- memory T cells. Furthermore, extended culture of these isolated cells revealed functional activity, including efficient proliferation, sustained antigen-specific IFN-γ secretion, and cytotoxicity against pp65-pulsed target cells. Conclusion: The findings reported here suggest that the IFN-γ CCS by the CliniMACS Prodigy is a simple and robust approach to produce CMV-CTLs, which may be applicable for the treatment of clinically urgent CMV-related diseases.

Supplemental material, Nayoun_Kim_et_al.-Processing_Gapping-Supplementary_Material for Processing gapping: Parallelism and grammatical constraints by Nayoun Kim, Katy Carlson, Mike Dickey and Masaya Yoshida in Quarterly Journal of Experimental Psychology

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.

Epstein-Barr virus (EBV)-positive extranodal NK/T-cell lymphoma is a rare and highly aggressive disease with a poor prognosis and strong resistance to anti-cancer drugs. Reactive oxygen species (ROS) are closely related to tumorigenesis and P-glycoprotein (P-gp) is highly expressed in various cancers. However, the exact relationship between ROS and P-gp in EBV-positive lymphoma remains unclear. In this study, we demonstrated that EBV latent infection induced intracellular ROS production and increased ROS levels triggered elevated P-gp expression, which resulted in strong resistance to existing anti-cancer drugs in EBV-positive lymphoma cell lines and in patients’ tissue samples. We also verified that regulation of intracellular ROS reduced P-gp expression and function via inhibition of STAT1 phosphorylation. These results indicate that treatment with a ROS scavenger is a potential therapeutic strategy to overcome resistance to anti-cancer drugs by downregulating the expression of P-gp in EBV-positive NK/T-cell lymphoma.

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 tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and macrophage inflammatory protein (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.

Interleukin (IL)-21, a proinflammatory cytokine, has been developed as an immunotherapeutic approach due to its effects on various lymphocytes, including natural killer (NK) cells and T cells; however, the clinical success in cancer patients has been limited. Recently, mesenchymal stem cells (MSCs) have emerged as vehicles for cancer gene therapy due to their inherent migratory abilities toward tumors. In the present study, we hypothesized that MSCs, genetically modified to express high levels of IL-21 (IL-21/MSCs), can enhance antitumor responses through localized delivery of IL-21. For tumor induction, BALB/c mice were injected intravenously with syngeneic A20 B-cell lymphoma cells to develop a disseminated B-cell lymphoma model. Then, 6 days following tumor induction, the tumor-bearing mice were treated with IL-21/MSCs weekly, four times. Systemic infusion of A20 cells led to hind-leg paralysis as well as severe liver metastasis in the control group. The IL-21/MSC-treated group showed delayed tumor incidence as well as improved survival, whereas the MSC- and recombinant adenovirus-expressing IL-21 (rAD/IL-21)-treated groups did not show significant differences from the untreated mice. These therapeutic effects were associated with high levels of IL-21 delivered to the liver, which prevented the formation of tumor nodules. Furthermore, the infusion of IL-21/MSCs led to induction of effector T and NK cells, while potently inhibiting immune suppressor cells. Our findings demonstrate that IL-21-expressing MSCs have the therapeutic potential to induce potent antitumor effects against disseminated B-cell lymphoma through localized IL-21 delivery and induction of systemic antitumor immunity.

Graft-versus-host disease (GVHD) is a major complication associated with allogeneic hematopoietic stem cell transplantation. Despite the prominent role of the adaptive immune system, the importance of controlling the innate immune system in the pathogenesis of GVHD has recently been rediscovered. High-mobility group box 1 (HMGB1) is a crucial damage-associated molecular pattern signal that functions as a potent innate immune mediator in GVHD. In the present study, we investigated treatment of experimental GVHD through HMGB1 blockade using the compound cyclopentylamino carboxymethylthiazolylindole (NecroX)-7. Treated animals significantly attenuated GVHD-related mortality and inhibited severe tissue damage. These protective effects correlated with the decrease in HMGB1 expression and lower levels of reactive oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF and IL-6, as well as the expression of TLR-4 and receptor for advanced glycation end products. We also observed increased regulatory T cell numbers, which may be associated with regulation of differentiation signals independent of HMGB1. Taken together, these data indicate that NecroX-7 protects mice against lethal GVHD by reciprocal regulation of regulatory T/Th1 cells, attenuating systemic HMGB1 accumulation and inhibiting HMGB1-mediated inflammatory response. Our results indicate the possibility of a new use for a clinical drug that is effective for the treatment of GVHD.

Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). 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 10 ENKTCL 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 (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.

Mesenchymal stem cells (MSCs) have rapidly been applied in a broad field of immune-mediated disorders since the first successful clinical use of MSCs for treatment of graft-versus-host disease. Despite the lack of supporting data, expectations that MSCs could potentially treat most inflammatory conditions led to rushed application and development of commercialized products. Today, both pre-clinical and clinical studies present mixed results for MSC therapy and the discrepancy between expected and actual efficacy of MSCs in various diseases has evoked a sense of discouragement. Therefore, we believe that MSC therapy may now be at a critical milestone for re-evaluation and re-consideration. In this review, we summarize the current status of MSC-based clinical trials and focus on the discrepancy between expected and actual outcome of MSC therapy from bench to bedside. Importantly, we discuss the underlying limitations of MSCs and suggest a new guideline for MSC therapy in hopes of improving their therapeutic efficacy.

We investigate whether grammatical information is accessed in processing noun phrase ellipsis (NPE) and other anaphoric constructions. The first experiment used an agreement attraction paradigm to reveal that ungrammatical plural verbs following NPE with an antecedent containing a plural modifier (e.g. Derek’s key to the boxes … and Mary’s_ probably *are safe in the drawer) show similar facilitation to non-elided NPs. The second experiment used the same paradigm to examine a coordination construction without anaphoric elements, and the third examined anaphoric one. Agreement attraction was not observed in either experiment, suggesting that processing NPE is different from processing non-anaphoric coordination constructions or anaphoric one. Taken together, the results indicate that the parser is sensitive to grammatical distinctions at the ellipsis site where it prioritises and retrieves the head at the initial stage of processing and retrieves the local noun within the modifier phrase only when it is necessary in parsing NPE.

Conventional therapeutic approaches to post-transplant lymphoproliferative disorder (PTLD) occurring after solid-organ transplantation have shown only limited success in achieving durable response. Key factors driving the pathogenesis of PTLD include Epstein–Barr virus (EBV) reactivation and impaired immune surveillance due to prolonged immune suppression. Thus, EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach for the treatment of EBV-associated PTLD by enhancing EBV-specific immunity. We evaluated the safety and efficacy of EBV latent membrane proteins (LMP)-1- and 2-specific CTLs in two PTLD patients at high risk for relapse. Following diagnosis, patients were initially treated with a combination of chemotherapy and/or radiotherapy. Patients then received a total of eight doses of 2 × 107 EBV-CTLs/m2. Following initial therapy, both patients achieved complete remission confirmed by FDG-PET/CT imaging. Post-remission therapy using adoptive transfer of EBV-CTLs was safe without immediate or late toxicities. Infusion of EBV-CTLs led to an overall reduction in plasma EBV levels in the peripheral blood, which was associated with long-term remission of both patients during a follow-up of more than 65 months. Further prospective studies with larger number of patients will be needed to confirm the role of EBV-CTLs as post-remission therapy in high-risk PTLD.

Hypothalamic glial cells named tanycytes, which line the 3rd ventricle (3V), are components of the hypothalamic network that regulates a diverse array of metabolic functions for energy homeostasis. Herein, we report that TSPO (translocator protein), an outer mitochondrial protein, is highly enriched in tanycytes and regulates homeostatic responses to nutrient excess as a potential target for an effective intervention in obesity. Administration of a TSPO ligand, PK11195, into the 3V, and tanycyte-specific deletion of Tspo reduced food intake and elevated energy expenditure, leading to negative energy balance in a high-fat diet challenge. Ablation of tanycytic Tspo elicited AMPK-dependent lipophagy, breaking down lipid droplets into free fatty acids, thereby elevating ATP in a lipid stimulus. Our findings suggest that tanycytic TSPO affects systemic energy balance through macroautophagy/autophagy-regulated lipid metabolism, and highlight the physiological significance of TSPO in hypothalamic lipid sensing and bioenergetics in response to overnutrition. Abbreviations 3V: 3rd ventricle; ACAC: acetyl-Coenzyme A carboxylase; AGRP: agouti related neuropeptide; AIF1/IBA1: allograft inflammatory factor 1; AMPK: AMP-activated protein kinase; ARC: arcuate nucleus; Atg: autophagy related; Bafilo: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CNS: central nervous system; COX4I1: cytochrome c oxidase subunit 4I1; FFA: free fatty acid; GFAP: glial fibrillary acidic protein; HFD: high-fat diet; ICV: intracerebroventricular; LAMP2: lysosomal-associated membrane protein 2; LD: lipid droplet; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MBH: mediobasal hypothalamus; ME: median eminence; MEF: mouse embryonic fibroblast; NCD: normal chow diet; NEFM/NFM: neurofilament medium; NPY: neuropeptide Y; OL: oleic acid; POMC: pro-opiomelanocortin-alpha; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Rax: retina and anterior neural fold homeobox; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RER: respiratory exchange ratio; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TG: triglyceride; TSPO: translocator protein; ULK1: unc-51 like kinase 1; VCO2: carbon dioxide production; VMH: ventromedial hypothalamus; VO2: oxygen consumption

Mesenchymal stem cells (MSCs) have emerged as a therapeutic approach in a range of medical fields, including regenerative medicine, cancer, autoimmune diseases, and inflammatory diseases, because of their unique properties of tissue repair and major histocompatibility complex-unmatched immunosuppression. Because both in vitro and in vivo findings demonstrate that MSCs possess potent immunoregulatory functions, there has been increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation, especially in the prevention and treatment of graft-versus-host disease (GVHD). GVHD is a major cause of transplantation-related mortality, and conventional immunosuppressants frequently fail to treat patients suffering from GVHD. Following Ringden's pilot study that used third-party MSCs to treat a steroid-refractory GVHD patient, MSCs have created growing interest as a therapeutic agent for GVHD. There have been further studies which demonstrated the potentials of MSC treatment in steroid-refractory GVHD, de novo GVHD, and also GVHD prevention. However, MSCs still present limitations. The need for MSCs to be "licensed" in a pro-inflammatory environment, especially in the presence of interferon gamma, allows only a narrow window for their administration. Thus, their effects have been less clear as a preventive measure before the inflammatory environment of GVHD is established and also when administered during a chronic setting where MSCs may be alternatively licensed. In this review, we focus on the immunomodulatory properties of MSCs and their effects in relation to GVHD. Given the efficacy of MSCs in murine models of GVHD and their safety in clinical trials, it is crucial that larger clinical trials are conducted and further modifications are investigated.

Mesenchymal stem cells (MSCs) possess immunomodulatory properties and have potential, however, there have been conflicting reports regarding their effects in rheumatoid arthritis (RA), which causes inflammation and destruction of the joints. Through a comparative analysis of regulatory T (Treg) and IL-10-producing type 1 regulatory T (Tr1) cells, we hypothesized that Tr1 cells enhance the immunoregulatory functions of MSCs, and that a combinatorial approach to cell therapy may exert synergistic immunomodulatory effects in an experimental animal model of rheumatoid arthritis (RA). A combination of MSCs and Tr1 cells prevented the development of destructive arthritis compared to single cell therapy. These therapeutic effects were associated with an increase in type II collagen (CII)-specific CD4+CD25+Foxp3+ Treg cells and inhibition of CII-specific CD4+IL-17+ T cells. We observed that Tr1 cells produce high levels of IL-10-dependent interferon (IFN)-β, which induces toll-like receptor (TLR) 3 expression in MSCs. Moreover, induction of indoleamine 2,3-dioxygenase (IDO) by TLR3 involved an autocrine IFN-β that was dependent on STAT1 signaling. Furthermore, we observed that production of IFN-β and IL-10 in Tr1 cells synergistically induces IDO in MSCs through the STAT1 pathway. These findings suggest co-administration of MSCs and Tr1 cells to be a novel therapeutic modality for clinical autoimmune diseases.

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 stem cells (MSCs) are multipotent stromal cells with the potential to differentiate into different tissue lineages. In addition to their differentiation potential, MSCs possess immunomodulatory properties that have created growing interest in both pre-clinical and clinical research. Over the years, MSCs have been applied rapidly in the clinic in a wide variety of immune-mediated disorders; however, MSC therapy has shown contradictory results, often with poor clinical outcomes. Recently, studies on MSC-based immune modulation have provided possible explanations for the conflicting clinical reports. It is now generally recognized that the immunomodulatory properties of MSCs are not constitutive but are induced by various mediators present in the inflammatory microenvironment. Different inflammatory stimuli are able to polarize MSCs to elicit distinct immunomodulatory phenotypes. Thus, the concepts of plasticity and polarization of MSC-based immune modulation may have important therapeutic implications in the clinic. In this review, we focus on the underlying mechanisms of MSC-mediated immune regulation that contribute to their therapeutic potential. Importantly, we discuss novel strategic approaches that enhance the therapeutic potential of MSCs through a consideration of MSC plasticity in immune modulation.

Chronic graft-versus-host disease (cGVHD) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), which is characterized by autoimmune like inflammatory responses and reduced levels of regulatory T cells (Tregs). Recently, the use of low-dose IL-2 has been reported to selectively increase Tregs and therefore facilitate immune regulation and promote clinical improvements in cGVHD patients. In this report, we describe the case of a cGVHD patient who was treated with daily low-dose IL-2 therapy. Our observations demonstrate that low-dose IL-2 could induce significant expansion of Tregs in vivo leading to improved Treg/Th17 ratios. The patient showed moderate clinical benefits suggesting that multiple factors may be involved in the immunological responses. Therefore, while the therapeutic potential of low-dose IL-2 is promising, strategic approaches may be needed to induce a clinically significant and sustained Treg effect.

Graft-versus-host disease (GVHD) is a major complication associated with morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). GVHD can be fatal, as patients frequently fail to respond to conventional immunosuppressants and no established second-line therapy is available. Stem cells have emerged as a therapeutic approach in a range of medical fields, including regenerative medicine, cancer, autoimmune diseases, and inflammatory diseases, because of their unique properties. In particular, mesenchymal stem cells (MSCs) have created increasing interest as a therapeutic agent for various immune-mediated diseases including GVHD. MSCs demonstrate therapeutic potential by exerting potent immunosuppressive effects through interactions between lymphocytes associated with both the innate and adaptive immune system. Preclinical experiments using MSCs for the treatment of GVHD have provided critical insights into the mechanism of action of MSCs and evidence for clinical translation. In the clinical setting, Ringden’s pilot study using MSCs to treat a steroid-refractory GVHD pediatric patient was a milestone in the field of MSC therapy, which leads to numerous worldwide studies on steroid-refractory GVHD, de novo GVHD, chronic GVHD treatment, and GVHD prevention. In this chapter, we focus on the recent advances of MSCs for the treatment of GVHD in both preclinical and clinical settings. We discuss the current limitations and highlight the future considerations that should be made when using stem cell therapy for GVHD.

AIM: To investigate the effects of mesenchymal stem cells (MSCs) on dextran sulfate sodium-induced inflammatory bowel disease (IBD). METHODS: C57BL/6 mice were fed 3.5% (g/L) dextran sulfate sodium. On day seven, the mice received intraperitoneal injections of 1 × 106 MSCs. The survival rate, disease activity index values, and body weight, were monitored daily. On day ten, colon lengths and histopathologic changes were assessed. In addition, immunoregulatory changes following MSC administration were evaluated by determining the levels of effector T cell responses in the spleen and mesenteric lymph nodes, and the expression levels of inflammatory cytokines in homogenized colons. RESULTS: Intraperitoneal administration of MSCs did not prevent development of colitis and did not reduce the clinicopathologic severity of IBD. No significant difference was evident in either survival rate or disease activity index score between the control and MSC-treated group. Day ten-sacrificed mice exhibited no significant difference in either colon length or histopathologic findings. Indeed, the MSC-treated group exhibited elevated levels of interleukin (IL)-6 and transforming growth factor-β, and a reduced level of IL-10, in spleens, mesenteric lymph nodes, and homogenized colons. The IL-17 level was lower in the mesenteric lymph nodes of the MSC-treated group (P = 0.0126). In homogenized colons, the IL-17 and tumor necrosis factor-α (P = 0.0092) expression levels were also lower in the treated group. CONCLUSION: MSC infusion provided no significant histopathologic or clinical improvement, thus representing a limited therapeutic approach for IBD. Functional enhancement of MSCs is needed in further study.

Establishment of mixed chimerism is an ideal approach to induce donor-specific tolerance while expanding its potential in various clinical settings. Despite the developments in partial conditioning regimens, improvements are still needed in reducing toxicity and bone marrow transplantation-related complications. Recently, cell-based therapies, including mesenchymal stem cells (MSCs), have been incorporated in establishing noncytoreductive mixed chimerism protocols; however, its efficacy is only partial and shows reversed immunosuppressive properties. This study demonstrates a novel approach to induce mixed chimerism and tolerance through combinatory cell-based immune modulation (CCIM) of MSCs and regulatory T cells (Tregs). We hypothesize that the interaction between these cells may lead to greater inhibition of host immune responses. Compared with single cell therapy, CCIM induced a higher engraftment rate and robust donor-specific tolerance to skin allografts across full major histocompatibility complex barriers. These regulatory effects were associated with inhibition of natural killer cell cytotoxic activity, CD4(+)IL-17(+) cells, memory B cells, plasma cells, and immunoglobulin production levels along with increased frequencies of CD4(+)Foxp3(+) cells, IL-10-producing mature B cells, and myeloid-derived suppressor cells. Furthermore, CCIM was able to regulate mortality in a graft-versus-host disease model through reciprocal regulation of Treg/Th17. Taken together, we suggest CCIM as a clinically applicable strategy for facilitating the induction of mixed chimerism and permanent tolerance.

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.

Cytomegalovirus (CMV) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). As an alternative to antiviral drugs, CMV-specific cytotoxic lymphocytes (CMV-CTLs) can provide long-term CMV-specific immunity without major side effects. In this study, we apply the IFN-γ cytokine capture system (CCS) using the fully automated CliniMACS Prodigy device to rapidly produce CMV-CTLs. Five validation runs were performed using apheresis samples from randomly selected CMV-seropositive healthy blood donors. CliniMACS Prodigy automatically performed successive processes including antigen stimulation, anti-IFN-γ labelling, magnetic enrichment, and elution which took approximately 13 hours. The original apheresis samples consisted of 0.3% CD3+IFN-γ+ T cells which were mainly CD45RA+CD62L+ naïve T cells. Following IFN-γ enrichment, the target fraction contained 51.3% CD3+IFN-γ+ cells with reduction in naïve T cells and the selection of CD45RA-CD62L- and CD45RA+CD62L- memory T cells. Interestingly, the frequency of CMV pp65 specific CD3+ T cells was also increased by 1.7-fold. Furthermore, extended culture of isolated cells revealed efficient proliferation with sustained antigen-specific IFN-γ secretion and cytotoxicity against pp65 pulsed target cells. Therefore, we suggest IFN-γ CCS by CliniMACS Prodigy as a simple and robust approach to produce CMV-CTLs, which may be highly feasible and applicable for clinical use. Disclosures No relevant conflicts of interest to declare.

The dispreference for subject case ellipsis in OSV sentences has been analyzed as resulting from a violation of a structural requirement on the position of bare subject NPs (Ahn and Cho 2006a, 2006b, 2007). In this study, we present evidence from an acceptability rating experiment demonstrating that OSV sentences containing a case-ellipsed subject exhibit acceptability patterns different from ungrammatical sentences violating a core syntactic principle on case assignment and that these sentences are judged acceptable when the subject refers to expected, predictable information in context. This evidence supports the conclusion that the dispreference for subject case ellipsis in OSV sentences is due to violations of probabilistic constraints that favor case marking for rare types of subjects and such violations can be remedied by non-syntactic information.

In order to investigate the influence of neural blockade on tissue plasminogen activator (t-PA) release by venous occlusion, we compared the increase in t-PA and fibrinolytic activity in the euglobulin fraction initiated by cubital venous occlusion (100 mmHg for 10 min), in pre-neural block and post-neural block states, in 7 cases. In all patients, the supra-clavicular approach was used to obtain a brachial plexus block and venous occlusion was achieved at the cubital levels on the ipsilateral arm. The euglobulin fibrinolytic activity before venous occlusion was 100.9±27.5 BAU in the pre-nerve block state and 102.7±29.4 BAU in the post-nerve block state (p>0.5). The t-PA antigen level before venous occlusion was 3.5±1.2ng/ml in the pre-nerve block state and 4.0±1.0 ng/ml in the post-nerve block state (p>0.05). The increase in the euglobulin fibrinolytic activity after venous occlusion was 18.0±16.7 BAU in the pre-nerve block state, and 18.3±15.6 BAU in the post-nerve block state (p>0.5). The increase in the t-PA antigen levels after venous occlusion was 3.0±2.0 ng/ml in the pre-nerve block state and 3.0±2.1ng/ml in the post-nerve block state (p>0.5). These findings suggest that the peripheral nervous system does not exert any influence on t-PA release during venous occlusion.

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