Final Report Summary - ECP TOLERANCE (An Investigation into Tolerance Induction by Extracorporeal Phototherapy)
Final Summary Report ERG02-GA-2007-224841
Haematopoietic stem cell transplantation (HSCT) is one area of stem cell research where major advances in the cure of haematological disorders such as leukaemia and lymphoma, inherited immune disorders, and aplastic anaemia, has been made. Transplantation of stem cells from related or unrelated donors, where immature cells develop into white and red blood cells and platelets (i.e. haematopoiesis), leads to immune reconstitution and can be a life saving therapy. Currently over 12000 transplants are carried out each year in Europe. However, despite recent advances, the overall survival rate after HSCT is relatively poor. A 40 – 60% survival rate is the norm for such transplants, which involve the use of bone marrow, peripheral blood stem cells and umbilical cord blood as stem cell source. The cure of patients is hampered by clinical complications that arise post-transplant. These are largely due to a lack of understanding of tolerance mechanisms.
The current management in HSCT includes modifying pre-transplant therapies (conditioning regimens) to reduce toxic complications and a lethal form of host rejection; graft versus host disease (GvHD) where donor marrow cells attack foreign tissue in the patient (skin, liver and gut), causing destruction and increased susceptibility to infection. Leukaemia recurrence post-transplant (relapse) can be treated by the use of donor lymphocyte infusions (DLI), or specific T cell therapies against minor histocompatibility antigens or tumour antigens, giving rise to a graft versus leukaemia (GvL) response. Even with these relatively new methods of treatment, long term complications (chronic graft versus host disease) and poor survival rates are still observed.
Standard treatment of GvHD is administration of corticosteroids. However, up to one third of patients do not repond to steroid treatment. These steroid refractory patients have a very poor prognosis as second or third line conventional immunosuppression is associated with high mortality and morbidity due to infection and relapse rates.
In contrast, extracorporeal photopheresis (ECP) is an immunomodulatory treatment option that is not linked to generalised immunosuppression and has a very low side-effect profile. No increased replapse or infection rates have been reported to date.
The mechanisms of action of ECP in GvHD remains largely uncharacterised. Apoptosis, a specific form of cell death induced by ECP and other modalities, and cytokine modulation have been implicated as well as modulation of antigen-presenting cells (APC). Photochemotherapy/ECP has been reported to induce a shift towards anti-inflammatory cytokines. Data from a murine animal model and clinical data demonstrate the induction of regulatory T cells.
This project attempted to identify specific tolerogenic properties induced by ECP-treatment. An ex vivo human skin explant model mimicking ECP-treatment was established. In this model, donor allo-reactive lymphocytes are generated and incubated with patient skin. It could be demonstrated that donor-derived immature dendritic cells, after co-culture with ECP-treated allo-reactive cells, were able to significantly downregulate skin damage in the model accompanied by a decrease in the inflammatory cytokines IFN-gamma and IL-6.
It has long been speculated whether activated or allo-reactive lymphocytes were more susceptible to undergo apoptotic cell death upon ECP-treatment than resting cells. We tested this hypothesis, using single cell sorting and flow cytometry of allo-activated T-cells. The results obtained in this study reveal a significantly higher susceptibility of allo-activated T-cells to ECP-induced apoptosis. Moreover we demonstrate via cell sorting that this susceptibility is independent of bystander cell influence. The data suggest selective killing of activated cells, however, ECP also induces apoptosis in non-activated T-cells. Activation induced cell death could synergise with ECP efficacy but untreated activated T-cells had only slightly higher background apoptosis than non-activated T-cells (Holtick et al, Transplantation in press).
Concomitant to the skin explant model described before, isolated dendritic cells were analysed for tolerogeneic or regulatory markers which the cells may have acquired after co-culture with ECP-treated lymphocytes. Expression levels of ILT-3 and PD-L1 and IDO were analysed. These molecules have been described as molecules with a regulatory function and implicated in immunosuppressive processes in various conditions. ILT-3 and IDO expression remained unchanged. PD-L1 surface expression was non-significantly increased upon co-culture.
One further hypothesis on how ECP instigates a tolerogenic process is by induction of regulatory T cells (Treg), an important subset of immunosuppressive cells. Treg induction has been implicated in ECP treatment mainly by the use of animal models. Our human in vitro model tested ECP-treated or untreated lymphocytes, immature and mature dendritic cells as stimulators for allogeneic naïve T cells in the presence or absence of IL-10. Although no absolute increase in Tregs could be demonstrated, ECP treatment altered the balance between activated T cells and Tregs in favour of Tregs. The ratio of Tregs to activated T cells increased from 1/7 to about 1/4 when ECP-treated immature dendritic cells were used. When ECP-treated lymphocytes were used as stimulators, the ratio increased from 1/6 to 1/2.
In summary, the work of this project further substantiated the notion, that ECP-treatment strengthens the regulatory arm of the immune system. A novel finding is that allo-reactive lymphocytes are selectively killed in the process of ECP-treatment. Furthermore, it could be established that immature dendritic cells alone can transfer downregulatory properties after co-culture with ECP-treated lymphocytes and dampen severe skin inflammation ex vivo. The potential role of regulatory T cells is highlighted also in the human model. The exact cellular processes are still elusive. No significant results could be obtained by analysing regulatory molecules. The analysis of genetic polymorphisms in DNA of ECP-treated patients, which then can be linked to functional relevant molecules, response to treatment and outcome, is still ongoing.
The lack of generalised immunosuppression and the safety of the procedure make ECP an attractive treatment option. However, data from controlled randomised trials are scarce and ECP is mainly used as second-line option. A Cochrane meta-analysis is currently performed in our department to combine the clinical data available and clarify the effectiveness of ECP-treatment.
Further research into ECP mechanisms of action is warranted to identify patients and clinical settings holding the most benefit from ECP-treatment as cost and complexity of ECP are still relevant factors in times of limited health care budgets.
Haematopoietic stem cell transplantation (HSCT) is one area of stem cell research where major advances in the cure of haematological disorders such as leukaemia and lymphoma, inherited immune disorders, and aplastic anaemia, has been made. Transplantation of stem cells from related or unrelated donors, where immature cells develop into white and red blood cells and platelets (i.e. haematopoiesis), leads to immune reconstitution and can be a life saving therapy. Currently over 12000 transplants are carried out each year in Europe. However, despite recent advances, the overall survival rate after HSCT is relatively poor. A 40 – 60% survival rate is the norm for such transplants, which involve the use of bone marrow, peripheral blood stem cells and umbilical cord blood as stem cell source. The cure of patients is hampered by clinical complications that arise post-transplant. These are largely due to a lack of understanding of tolerance mechanisms.
The current management in HSCT includes modifying pre-transplant therapies (conditioning regimens) to reduce toxic complications and a lethal form of host rejection; graft versus host disease (GvHD) where donor marrow cells attack foreign tissue in the patient (skin, liver and gut), causing destruction and increased susceptibility to infection. Leukaemia recurrence post-transplant (relapse) can be treated by the use of donor lymphocyte infusions (DLI), or specific T cell therapies against minor histocompatibility antigens or tumour antigens, giving rise to a graft versus leukaemia (GvL) response. Even with these relatively new methods of treatment, long term complications (chronic graft versus host disease) and poor survival rates are still observed.
Standard treatment of GvHD is administration of corticosteroids. However, up to one third of patients do not repond to steroid treatment. These steroid refractory patients have a very poor prognosis as second or third line conventional immunosuppression is associated with high mortality and morbidity due to infection and relapse rates.
In contrast, extracorporeal photopheresis (ECP) is an immunomodulatory treatment option that is not linked to generalised immunosuppression and has a very low side-effect profile. No increased replapse or infection rates have been reported to date.
The mechanisms of action of ECP in GvHD remains largely uncharacterised. Apoptosis, a specific form of cell death induced by ECP and other modalities, and cytokine modulation have been implicated as well as modulation of antigen-presenting cells (APC). Photochemotherapy/ECP has been reported to induce a shift towards anti-inflammatory cytokines. Data from a murine animal model and clinical data demonstrate the induction of regulatory T cells.
This project attempted to identify specific tolerogenic properties induced by ECP-treatment. An ex vivo human skin explant model mimicking ECP-treatment was established. In this model, donor allo-reactive lymphocytes are generated and incubated with patient skin. It could be demonstrated that donor-derived immature dendritic cells, after co-culture with ECP-treated allo-reactive cells, were able to significantly downregulate skin damage in the model accompanied by a decrease in the inflammatory cytokines IFN-gamma and IL-6.
It has long been speculated whether activated or allo-reactive lymphocytes were more susceptible to undergo apoptotic cell death upon ECP-treatment than resting cells. We tested this hypothesis, using single cell sorting and flow cytometry of allo-activated T-cells. The results obtained in this study reveal a significantly higher susceptibility of allo-activated T-cells to ECP-induced apoptosis. Moreover we demonstrate via cell sorting that this susceptibility is independent of bystander cell influence. The data suggest selective killing of activated cells, however, ECP also induces apoptosis in non-activated T-cells. Activation induced cell death could synergise with ECP efficacy but untreated activated T-cells had only slightly higher background apoptosis than non-activated T-cells (Holtick et al, Transplantation in press).
Concomitant to the skin explant model described before, isolated dendritic cells were analysed for tolerogeneic or regulatory markers which the cells may have acquired after co-culture with ECP-treated lymphocytes. Expression levels of ILT-3 and PD-L1 and IDO were analysed. These molecules have been described as molecules with a regulatory function and implicated in immunosuppressive processes in various conditions. ILT-3 and IDO expression remained unchanged. PD-L1 surface expression was non-significantly increased upon co-culture.
One further hypothesis on how ECP instigates a tolerogenic process is by induction of regulatory T cells (Treg), an important subset of immunosuppressive cells. Treg induction has been implicated in ECP treatment mainly by the use of animal models. Our human in vitro model tested ECP-treated or untreated lymphocytes, immature and mature dendritic cells as stimulators for allogeneic naïve T cells in the presence or absence of IL-10. Although no absolute increase in Tregs could be demonstrated, ECP treatment altered the balance between activated T cells and Tregs in favour of Tregs. The ratio of Tregs to activated T cells increased from 1/7 to about 1/4 when ECP-treated immature dendritic cells were used. When ECP-treated lymphocytes were used as stimulators, the ratio increased from 1/6 to 1/2.
In summary, the work of this project further substantiated the notion, that ECP-treatment strengthens the regulatory arm of the immune system. A novel finding is that allo-reactive lymphocytes are selectively killed in the process of ECP-treatment. Furthermore, it could be established that immature dendritic cells alone can transfer downregulatory properties after co-culture with ECP-treated lymphocytes and dampen severe skin inflammation ex vivo. The potential role of regulatory T cells is highlighted also in the human model. The exact cellular processes are still elusive. No significant results could be obtained by analysing regulatory molecules. The analysis of genetic polymorphisms in DNA of ECP-treated patients, which then can be linked to functional relevant molecules, response to treatment and outcome, is still ongoing.
The lack of generalised immunosuppression and the safety of the procedure make ECP an attractive treatment option. However, data from controlled randomised trials are scarce and ECP is mainly used as second-line option. A Cochrane meta-analysis is currently performed in our department to combine the clinical data available and clarify the effectiveness of ECP-treatment.
Further research into ECP mechanisms of action is warranted to identify patients and clinical settings holding the most benefit from ECP-treatment as cost and complexity of ECP are still relevant factors in times of limited health care budgets.