Induction Of Tolerance To Murine Tracheal Allograft By Intrasplenic Transplantation Of Donor Cells

Lung transplantation(LTx) is the final solution for end-stage pulmonary diseases. Unfortunately,LTx is faced with a dilemma between apparent inadequacy of immunosuppression and severe side-effect of immunotherapy.The long-term outcome of the LTx recipient is influenced severely by allograft rejection and immunotherapy- related diseases.It is no good escalate the immunosuppressive regimens.Induction of transplantation tolerance,characterized as donor-specific immune hyporeactivity,seems to be the most promising solution to this dilemma.Inspired by two tolerance phenomena associated with bone marrow transplantation(BMT) and donor-specific transfusion(DST),BMT-based protocols and DST-based protocols were developed into two classical types of induction regimens in the field of transplantation tolerance research.There was an appealing report in 2008 that tolerance has been induced successfully by a BMT-based protocol to 4 of 5 selected recipients in a clinical trial of kidney transplantation.Nonetheless,the adverse effect of radiation and/or the risk of graft versus host reaction(GVHR) remain giant obstacles for BMT-based or DST-based protocols to be applied to patients of organ transplantation,especially to those who suffered badly from end-stage pulmonary diseases.Over the past decade,non-specific immunosuppression has been reinforced further by the advent of co-stimulation blockade agents and new immunosuppressive drugs.However,the research on induction of donor-specific tolerance improved little.Imitating other tolerance phenomena may be another breakout for the research of donor-specific tolerance induction.Besides BMT-related and DST-related tolerance,there are another two known tolerance phenomena,namely spontaneous liver transplantation tolerance and immtmologic privilege provided by multi-organ transplantation.It is well known that allograft of liver,spleen or thymus can provide immunologic protection for simultaneous or secondary transplantation of other organ allograft from the same donor in rodent model.Considering the complicated operation and heavy traumatic burden on recipients,it is almost impossible to make combined multi-organ transplantation into a tolerance induction regimen just like BMT-based or DST-based protocols. One of new advances in transplantation field is that hepatocyte transplantation can take the place of solid liver transplantation in treating selected acute liver failure. Hepatocyte transplantation is conducted in murine model via intrasplenic injection of donor hepatocyte.Besides liver,both spleen and thymus can be made into single cell suspension.Therefore,it could be possible to replace solid organ transplantations of spleen and thymus with cell transplantation respectively.In this study,the solid organ transplantations of liver,spleen and thymus were replaced by respective cell transplantations to reproduce the immunologic protection on heterotopic tracheal allograft in a murine model.Partâ… :The feasibility of inducing transplantation tolerance to murine tracheal allograft by intrasplenic cell transplantation instead of solid organ transplantion.Objectives:To study the feasibility of inducing transplantation tolerance to murine tracheal allograft by intrasplenic cell transplantation instead of solid organ transplantion.Materials and methods:6～8-week-old male BALB/c mice(H-2d) and female C57BL/6 mice(H-2b) were used as donor and recipient.All recipients underwent total body irradiation(TBI) of 5.5Gy by ⁶⁰Co on Day -1,received heterotopic tracheal transplantation and cell transplantation on Day 0.A dose of 5×10⁶ bone marrow cells (200μl) were injected into tail vein of all recipients except control group(Con).The recipients of Group 5.5B,5.5BS,5.5BT and 5.5BH underwent intrasplenic injection of PBS(100μl),splenocytes(1×10⁷/100μl),thymocyts(1×10⁷/100μl),or heptocytes (2×10⁶/100μl),respectively.Mixed chimerism of peripheral blood monocytes were analyzed by flow cytometry on week 1,3,5,and 7.GVHR were monitored until the recipient was died or the 8 weeks of observation time was expired.Autopsy was performed on those who died of GVHR.Histological analysis was conducted on tracheal allograft, spleen,liver and lung.Results:The donor macrochimerism of peripheral blood was detected in the recipients of Group 5.5BS and 5.5BT,with a level of 54.3±13.1%and 47.6±17.1%at the end of 1 week.The level of mixed chimerism in Group 5.5BS and 5.5BT declined to 9.9±7.0%and 5.8±7.6%at the end of 7 weeks.The recipients of Group 5.5BS and 5.5BT represented severe GVHR after 7 weeks of transplantation,with a high mortality of 50% and 60%.Group 5.5B,5.5BH and Con had no chimerism detected,without GVHR and GVHR-related mortality.The pipe of tracheal allografts on 7 weeks was closed completely in Group 5.5B,5.5BH and Con,whereas closed only 25%in Group 5.5BS and 5.5BT.Conclusion:It was concluded that intrasplenic cell transplantation could induce tolerance to murine heterotopic tracheal allografts instead of organ transplantation of spleen or thymus.Without the protection of immunosuppression,this hemostasis of immune tolerance was so fragile that it resolved soon.Hepatocyte transplantation can take the place of liver transplantation technically,but failed to facilitate chemerism and tolerance.Partâ…¡:Comparision of the intrasplenic route of cell transplantation with others.Objectives:To determine which route is the best one among intrasplenic,intrathymic and intravenous cell transplantation.Materials and methods:6～8-week-old male BALB/c mice(H-2d) and female C57BL/6 mice(H-2b) were used as donor and recipient.All recipients underwent TBI of 5.5Gy by ⁶⁰Co on Day -1,received heterotopic tracheal transplantation and cell transplantation on Day 0.A dose of 5×10⁶ bone marrow cells(200μl) were injected into tail vein of all recipients except control group(Con).The recipients of Group 5.5BT and 5.5BBs underwent intrasplenic injection of thymocyts(1×10⁷/100μl) and unseparated bone marrow cells(1×10⁷/100μl),respectively.The recipients of Group 5.5BTt underwent intrathymic injection of thymocyts(half in one lobe,1×10⁷/100μl totally).The recipients of Group 5.5BB received additional dose of unseparated bone marrow cells(1×10⁷/100μl) by injection into tail Vein.Mixed chimerism of peripheral blood monocytes were analyzed by flow cytometry on week 1,3,5,and 7.GVHR were monitored until the recipient was died or the 8 weeks of observation time was expired.Autopsy was performed on those who died of GVHR.Histological analysis was conducted on tracheal allograft,spleen,liver and lung.Results:The donor macrochimerism of peripheral blood was detected in the recipients of Group 5.5BT,5.5BTt and 5.5BBs,with a level of 47.6±17.1%,40.1±8.3% and 33.2±11.5%at the end of 1 week.The level of mixed chimerism in Group 5.5BT, 5.5BTt and 5.5BBs declined to 5.8±7.6%,0.06±0.03,and 0.08±0.02%at the end of 7 weeks.The recipients of Group 5.5BT represented severe GVHR after 7 weeks of transplantation,with a high mortality of 60%.Group 5.5BB and Con had no macrochimerism detected,without GVHR and GVHR-related mortality.The pipe of tracheal allografts by week 7 was closed almost in Group 5.5BTt and 5.5BBs,and was closed completely in Group 5.5BB and Con,whereas closed only 25%in Group 5.5BT.Conclusion:In conclusion,intrasplenic cell transplantation could provide more effective immunologic privilege in comparison with intrathymic or intravenous route. Moreover,the more immunoreactive cells were transplanted,the more significant immunomodulation was obtained.The intrasplenic cell transplantation seemed to be the optimal means in using donor's immunoreactive cells to induce transplantation tolerance.Partâ…¢:The magnitude of immunologic privilege provided by intrasplenic cell transplantation.Objectives:To determine the magnitude of immunologic privilege provided by intrasplenic cell transplantation.Materials and methods:6～8-week-old male BALB/c mice(H-2d) and female C57BL/6 mice(H-2b) were used as donor and recipient.All recipients underwent TBI of 3.0 Gy by ⁶⁰Co on Day -1,received heterotopic tracheal transplantation and cell transplantation on Day 0.A dose of 5×10⁶ bone marrow cells(200μl) were injected into tail vein of all recipients except control group(Con).The recipients of Group 3.0B,3.0BS, 3.0BT,3.0BST,3.0BSTB and 3.0BSTBL underwent intrasplenic injection of PBS(100μl), splenocytes(1×10⁷/100μl),thymocyts(1×10⁷/100μl),splenocytes+thymocyts (1×10⁷/100μl totally),splenocytes+thymocyts+bone marrow cells(1×10⁷/100μl totally), or splenocytes+thymocyts+bone marrow cells+peripheral blood monocytes (1×10⁷/100μl totally),respectively.Mixed chimerism of peripheral blood monocytes were analyzed by flow cytometry on week 1,3,5,and 7.GVHR were monitored until the recipient was died or the 8 weeks of observation time was expired.Autopsy was performed on those who died of GVHR.Histological analysis was conducted on tracheal allograft, spleen,liver and lung.Results:The donor macrochimerism of peripheral blood was not detected in all groups at the end of weekl,3,5,and 7.There was no GVHR or GVHR-related death occurred.The pipe of tracheal allografts by week 7 was closed completely in all groups. Conclusion:When the dose of TBI decreased from 5.5Gy to 3.0 Gy,the protocol of BMT(5×10⁶/mouse) and intrasplenic cell transplantation(1×10⁷/mouse) could not induce macrochimerism of peripheral blood and transplantation tolerance.In combination with partâ… andâ…¡,it was concluded that intrasplenic cell transplantation could provide immunomodulation equivalent to a dose of 3～5.5Gy TBI,probably inferior to solid organ transplantation,but superior to DST.