Interferons (IFNs) perform a wide range of biological activities: anti-microbial, anti-proliferative, immunomodulatory etc. The IFN family includes three main classes: Type I, Type II and the recently identified Type III. The two main members of Type I class are IFNα and IFNβ, which are well known for their anti-viral roles. IFNλ, a member of the Type III class of IFNs, also exhibits antiviral activity. IFNγ, also known as immune IFN, is a Type II IFN which is secreted, primarily, by activated T cells, NK cells and macrophages. IFNγ is a potent immunomodulator which plays important roles in host defense. The diverse functions of this cytokine are demonstrated in Ifnγ-/- mice which display increased sensitivity to several pathogens, high incidences of tumors, reduced inflammatory response etc. IFNγ binds to its cognate receptors, which consist of two subunits, IFNγ receptor (IFNGR) 1 and IFNGR2. IFNγ mediates its multifarious biological actions by activating the Janus activated kinase (Jak)-Signal transducer and activator of transcription (Stat) 1 signaling pathway. Jaks belong to a family of non-receptor protein tyrosine kinases and phosphorylate the IFNγ receptor and the transcriptional co-activator, Stat. IFNGR1, the larger subunit, is required for ligand binding and its carboxyl terminus is involved in binding to Jak1, which in turn phosphorylates Stat1. The smaller subunit, IFNGR2, is required for signaling and contains the Jak2 binding site. After binding of IFNγ to its receptor, a series of phosphorylation events occur, resulting in Stat1 phosphorylation and homodimerization of Stat1 to form the gamma activating factor (GAF). These activated molecules translocate to the nucleus and bind to gamma activating sequence (GAS) present in the promoters of several IFNγ-modulated genes. Thus, the cellular responses mediated by IFNγ are, primarily, due to modulation of gene expression. Therefore, the identification and study of IFNγ stimulated genes, signaling mediators and their cross talk with other cellular pathways is an active area of research. The system of our study was a hepatoma cell line, H6, which is derived from a spontaneous tumor from B10.A mice and selected for in vitro cell culture. It is an IFNγ inducible system and has been used to study IFNγ-induced gene expression and functional responses. Treatment of H6 cells with IFNγ greatly enhanced MHC class I levels but also reduced cell growth. High amounts of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) play crucial roles in the growth suppressive effect of IFNγ. To better understand the signaling pathways involved in the generation of ROS and RNI, the involvement of Ras was investigated. Ras-GTP levels were determined by pull down assays using GST-Raf1-Ras binding domain fusion protein bound to glutathione agarose. Ras activation (conversion of Ras-GDP to Ras-GTP) was observed in H6 cells upon IFNγ treatment by ~12 hr. To assess the functional role of Ras activation, studies with Manumycin A, a farnesyl transferase inhibitor (FTI), were performed. The formation of functional Ras requires farnesylation, a post-translational modification, which is inhibited by FTIs. Treatment with Manumycin A blocked Ras activation but did not significantly modulate the IFNγ-induced MHC class I. However, the inhibitor reduced ROS amounts leading to increased cell growth in the presence of IFNγ. Together, these results delineated the role of Ras and ROS in modulating some functions of IFNγ. To further understand the mechanisms by which Ras mediates its functions during IFNγ mediated growth suppression, the activation and function of Ras effectors was evaluated. In particular, the role of Ras-like (Ral) guanyl nucleotide-binding proteins, RalA and RalB, was investigated. IFNγ induced transcripts of RalA but not RalB. Also, the induction of RalA and IFNγ induced growth suppression were Stat1-dependent. Studies involving chemical inhibitors and genetic studies revealed that Ras played a role in the induction of RalA during IFNγ treatment. The role of c-Jun N-terminal kinase (JNK), a stress induced kinase, was also elucidated in this system. Together, IFNγ induced activation of Ras and its effectors RalA and JNK, leading to high amounts of ROS that suppressed cell growth. To evaluate the physiological significance of Ras activation during inflammatory responses, the mouse model of acetaminophen (APAP) induced liver injury was established. Hepatotoxicity due to overdose of the analgesic and antipyretic, APAP, is a major cause of liver failure in adults. APAP is metabolized into a reactive metabolite which binds to glutathione. Consequently, the depletion of intracellular glutathione stores leads to oxidative stress and liver injury. Notably, Ifnγ-/- mice are resistant to APAP-induced liver damage demonstrating a crucial role for this cytokine. The role of Ras activation was evaluated after oral dosing of BALB/c mice with APAP. Ras-GTP was induced early and decreased amounts were observed upon treatment with L-methionine, which replenished glutathione amounts. Injection with L-methionine or Manumycin A rescued liver injury as assessed by lowered serum alanine aminotransferase amounts and histological analysis. Kinetic studies were also performed, under different treatment conditions, to estimate different biochemical parameters: glutathione amounts, JNK activation, protein carbonylation, ROS amounts, serum amounts of cytokines, TNFα and IFNγ etc. This study reveals a role of Ras activation in stimulating proinflammatory responses and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury. In addition the role of RalA during APAP-induced liver injury was also studied. In summary, this study, involving in vitro cell culture and in vivo liver injury model systems, sheds light on the significant contributions of Ras and its effector, RalA, during IFNγ mediated growth suppression and APAP-induced liver injury.

Classical midgut carcinoids belong to neuroendocrine tumors of the gastroenteropancreatic tract (GEP-NETs) and are associated with serotonin overproduction. The term midgut is derived from the tumors’ embryological site of origin: enterochromaffin cells in the lower jejunum, ileum, caecum and the ascending colon. Despite their rather benign nature, these tumors can metastasize to mesentery and liver, putting patients at risk for the so-called carcinoid syndrome. This syndrome is characterized by flushes, diarrhoea and valvular heart disease due to the excessive serotonin secretion by tumor cells. Treatment of metastatic disease is currently ineffective and T cell immunotherapy has been suggested as a novel approach. We propose a number of midgut carcinoid-associated proteins as potential antigens for immunotherapy. Chromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), islet autoantigen 2 (IA-2) and survivin represent interesting candidates based on their fairly restricted neuroendocrine tissue expression. In pursuit of potential antigens we identified a novel splicing variant of VMAT-1, lacking the second last exon. The variant, denoted VMAT1Δ15, encodes a differently translated C-terminal compared to the native form, is localized in the endoplasmic reticulum (ER) instead of large dense core vesicles and is unable to accumulate serotonin. We identify several immunogenic HLA-A*0201-binding peptide epitopes derived from our proposed antigens by analyzing CD8+ T cell responses in blood from midgut carcinoid patients. We demonstrate immune recognition of midgut carcinoid tumors in patients and in vitro generation of activated CD8+ T cells recognizing these peptide epitopes in blood from healthy controls. Patients also exhibit increased frequencies of circulating regulatory T cells (Tregs) with suppressive quality and patient lymphocytes display a decreased proliferative capacity compared to healthy controls. Midgut carcinoid tumors are frequently infiltrated by T cells, however always in the presence of Foxp3-expressing Tregs. Midgut carcinoid-associated antigens recognized by CD8+ T cells are of great interest for cellular therapies such as modified DC vaccines or adoptive T cell transfer. However, the systemic and local suppression of Th1 immunity must be considered and likely corrected in order to obtain clinically effective immunotherapies.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.

Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...