There is an urgent need for novel preventive and therapeutic strategies for graft versus host disease (GvHD) occurring after allogeneic hematopoietic cell transplantation (allo-HCT). T-cell-based immunotherapies have been developed, however there are still some hurdles for the use of currently availably regulatory T-cells in clinical practice (naturally occurring FOXP3 + nTregs and inducible regulatory T cells), mainly owing to the lack of specific cell surface markers. The hypomethylating agent azacytidine (5-aza-dC) has been shown to generate immunoregulatory T-cells ex vivo. Interestingly, it has been shown that genes other than FOXP3 are responsible for the suppressor function of 5-aza-dC induced T-regs. HLA-G is a surface molecule with potent immunoregulatory functions which is normally expressed during pregnancy protecting the “semi-allogeneic” fetus from maternal immune attack and then is epigenetically repressed. The aim of this study was the induction of HLA-G expression in T-lymphocytes with the use of the demethylating agent 5-Aza-dC and investigation of their possible immunoregulatory properties. Our results showed that short in vitro treatment of peripheral blood T-cells with 5-aza-dC induces HLA-G expression and, more importantly, these induced HLA-G + T-cells could suppress lymphoproliferation when added as third party cells in mixed lymphocyte cultures. This suppression seems to be reduced after HLA-G neutralization and cell-to-cell contact independent. Furthermore, these induced HLA-G + T-cells show a reduced proliferation to allogeneic stimuli. Taken together, our results indicate the ex vivo production of HLA-Gpos T-lymphocytes with immunoregulatory properties. Our long term goal is the use of this population as adoptive cellular therapy for GvHD and other T-cell mediated diseases. / -

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.

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.

Durch den Transfer der Gene des heterodimeren T-Zellrezeptors (TZR) mithilfe viraler Vektoren können T-Zellen programmiert werden, ein ausgewähltes Antigen spezifisch zu erkennen. In klinischen Studien wurden solche T-Zellen bereits mit Erfolg zur Immuntherapie von Krebs und viralen Infektionen eingesetzt. Genmodifizierte T-Zellen unterscheiden sich jedoch von normalen T-Zellen, weil sie neben den beiden zelleigenen auch die zwei übertragenen TZR-Gene exprimieren. Diese Situation erlaubt die Bildung vier verschiedener TZR-Heterodimere: der zelleigene TZR, der übertragene TZR und zwei gemischte TZR, bestehend aus je einer übertragenen und einer zelleigenen TZR-Kette. Gemischte TZR bergen das Risiko von Nebenwirkungen, weil sie durch Zufall gesundes Körpergewebe erkennen und so Autoimmunität auslösen könnten. In dieser Arbeit wurden deshalb virale Vektoren entwickelt, die gleichzeitig mit der Übertragung von neuen TZR-Genen den zelleigenen TZR durch RNA Interferenz (RNAi) unterdrücken. Mikro-RNA (miRNA), die in den Vektor MP71 eingefügt wurden, reduzierten den zelleigenen TZR in Maus-T-Zellen um mehr als 85%. Dies hatte zur Folge, dass beide Ketten des übertragenen P14-TZR in gleicher Menge auf der Zelloberfläche exprimiert wurden und die Bildung von gemischten TZR reduziert wurde. In einem Mausmodell der adoptiven T-Zelltherapie verhinderte die Unterdrückung des zelleigenen TZR die Entstehung von Autoimmunität, die andernfalls durch gemischte TZR verursacht wurde. Im Gegensatz dazu führte die Anwendung von gentechnisch optimierten P14-TZR-Genen weder zur angeglichenen Oberflächenexpression der P14-TZR Ketten noch zu weniger Autoimmunität im Mausmodell. Ein anderes Tierexperiment zeigte, dass die miRNA die Funktion der genmodifizierten T-Zellen nicht beeinträchtigte. Schließlich wurde ein viraler Vektor entwickelt und getestet, der die Expression des zelleigenen TZR in menschlichen T-Zellen effektiv unterdrückte und die Bildung von gemischten TZR reduzieren konnte. / T cells can be genetically modified using viral vectors. The transfer of genes encoding both chains of the heterodimeric T cell receptor (TCR) programs T cells to specifically react towards an antigen of choice. Such TCR gene-modified T cells were already successfully applied in clinical studies to treat cancer and viral infections. However, in contrast to nonmanipulated T cells these cells express the transferred TCR in addition to the endogenous TCR and this situation allows the assembly of four different TCR heterodimers: the endogenous TCR, the transferred TCR, and two mixed TCR dimers, composed of one endogenous and one transferred TCR chain. The formation of mixed TCR dimers represents a safety issue because they may by chance recognize self-antigens and thereby cause autoimmune side effects. To overcome this problem, an RNAi-TCR replacement vector was developed that simultaneously silences the endogenous TCR and expresses an RNAi-resistant therapeutic TCR. The expression of miRNA encoded by a retroviral MP71 vector in transduced mouse T cells reduced the surface levels of the endogenous TCR by more than 85%. The knockdown of the endogenous TCR in turn resulted in equal surface expression levels of both transferred P14 TCR chains and prevented the formation of mixed TCR dimers. Accordingly, the development of lethal mixed TCR dimer-dependent autoimmunity (TI-GVHD) in a mouse model of adoptive T cell therapy was dramatically reduced by the knockdown of the endogenous TCR. In contrast, the usage of genetically optimized TCR genes neither resulted in equal surface levels of both P14 TCR chains nor in reduced autoimmunity. A second mouse model demonstrated that the in vivo functionality of the transduced T cells was not negatively influenced by the expression of the miRNA. Finally, an RNAi-TCR replacement vector for human T cells was developed that effectively reduced the expression of the endogenous TCR and prevented the formation of mixed TCR dimers.

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