The Role And Mechanism Of Lymphocyte Clone Competetion In Composite Tissue Allotransplantation

Backgroud:Composite tissue allotransplantation (CTA) is a possible solution for major trauma tothe face and extremities which often leave massive defects in soft tissue and or bone thatare nearly impossible to correct with native tissue. Even if a cosmetic repair is achieved,often the functional result is far from satisfactory. There are often no good options availableand the only chance of restoring function, appearance, and dignity to these patients iscomposite tissue transplantation.Although the development of more efficacious immunotherapy moved the possibilityof successful CTA closer to reality, controversy remains as to whether the risks associatedwith the immunosuppressive drugs required to prevent rejection outweigh their benefits inCTA. Because CTA transplantations are not lifesaving procedures, much consideration isdevoted to the issue of minimizing or withdrawing immunosuppression. Hence, theunderstanding of the dynamic and mechanism of the immune response to CTA graft maygive a clue in the immunosuppressive treatment of CTA recipients.However, the specific mechanism of the immune response and tolerance to CTA graftis still not well demonstrated. CTA transplants differ from solid organ transplants in thatsolid organs present a relatively homogenous structure and tissue, whereas CTAs arehistologically heterogenous, that is, they are composed of different tissues types such asskin, muscle, bone, bone marrow, lymph nodes, nerve, and tendon. They also express adistinct immunogenicity of transplanted elements. Although CTA often consists of tissueswith strong immunogenicity such as skin and bone marrow, it is observed that less dose ofimmunosuppression agents is sufficient to maintain the survival of a CTA graft whencompared with single solid organ transplantation. These phenomena gave rise to thehypothesis that competetion and inhibition between different clones of lymphocyte mayplay an important role in the immune response to CTA graft. The idea that T cells competetion has its origin in transplantation study that one typeof minor histocompatibility antigen could inhibit the immune response to other minorhistocompatibility antigen. Recent study has confirmed that different clones of lymphocytecould compete against each other by intefering the access to APC, expressing immunesuppression co-stimulation moleculars or secreting immune suppression cytokines.However, the competetion and inhibition between lymphocytes was frequently observed inT cells for same antigen but rarely observed in T cells for different MHC or tissue antigen.As CTA consists of homogenous structure and tissue, it might be a appropriate modelfor studying the lymphocyte competetion in transplantation. And the low immunogenicityof CTA also give a clue that lymphocyte competetion may take an important role in CTAtolerance. However, the hypothesis is based on some indirect phenomena, lacking specificevidence to support it. In addition, most of these phenomena came from CTA models whichcontain bone marrow tissues. It is hard to probe the role of lymphocyte competetion in CTAtransplantation in these models, as the janus-faced immune regulation effect of allogeneichemopoietic stem cells (HSCs).To throw light onto this topic, we transplanted allogeneic grafts consisted of withdifferent combination of MHC or tissue antigen as an in vivo model. And we alsochallenged lymphocytes with multiple types of both MHC antigens in vitro. By this means,we may elucidate the lymphocyte competetion in CTA tranplantation.Methods:1. For testing the immune reaction to different combination of tissue antigen, CTAgrafts consisted with different tissue types from BN rats were transplanted to F344rats. Thesurvival situation was evaluated daily.2. Seven days after CTA transplantation, peripheral blood lymphocytes from the CTArecipients were stimulated by the lymphocytes from the donor rats in MLR model. And thecell proliferation was tested by WST-8colorimetric method.3. Isolating the spleen lymphocytes from BALB/c(H-2d), C3H(H-2k) and C3H(H-2k)mice, and stimulating the lymphocytes from C57BL/6mice with single or both twotypes of allogeneic lymphocytes in the mixed lymphocyte reaction(MLR) model. And thecell proliferation was tested by WST-8colorimetric method. 4. Collecting the liquid supernatant of nutrient medium at the72hour of MLR,examining the concentration of TGF-β1,TNF-α and IL-10by ELISA test.5. Collecting the lymphocytes at the72hour of MLR, extract the total RNA, andexamining the expression of IL-10and Foxp3mRNA by RT-qPCR.6. Skin grafts from BALB/c and C3H were transplanted to C57BL/6micesimutaniously or separately. The survival situation was evaluated daily.7. For evaluating the role of Foxp3+Tregs in lymphocyte competetion in vivo, thehistological structure and the infiltration of Foxp3+cell in the skin graft was checked byimmunohistochemical staining.Results:1. The MST of allogeneic skin graft was6.25±0.25days. While the MST of allogeneicgroin cutaneous flap and myocutaneous flap was9±0.707days and10.5±0.645days,significantly prolonged when compared with skin graft (p=0.006).2. Peripheral blood lymphocytes from allogeneic skin graft, cutaneous flap andmyocutaneous flap recipients show slight increased proliferation reaction to the stimulationof donor derived lymphocytes when compared to na ve F344rat. But no difference wasdetected among all recipients.3. Lymphocytes challenged by both two genotypes of MHC antigen showed slightlylower proliferation reaction when compared with single MHC stimulated lymphocytes inthe MLR model.4. The liquid supernatant of nutrient medium from lymphocytes challenged by bothtwo genotypes of MHC antigen showed significantly increased concentration of IL-2.While no obvious difference was found on the secretion of TGF-β1or TNF-α among allgroups.5. The expression of IL-10mRNA was significantly increased in the lymphocyteschallenged by both two types of allogeneic MHC antigen when compared with lymphocytesstimulated with only one genotype of MHC antigen (p<0.001). While the expression ofFoxp3gene was generally equivalent among all MLR reaction groups.6. The mean survival time (MST) of the single MHC genotype allogeneic skin graftswas7.60±0.51days and6.20±0.37days. While the MST of skin grafts in double MHC genotype simultaneous transplanted recipients was7.80±0.86days and6.80±0.80days. Nosignificant difference was observed in the rejection process or the infiltration of Foxp3+cellin the graft between two transplantation schemes.7. Inhibition of lymphocytes proliferation by two MHC antigens stimulation in MLRreaction was reversed by Qa-1knockout. But the rejection process of single MHC typeallogeneic skin graft was shortened in Qa-1KO recipients. The survival time of skin graftin double MHC transplanted group was similar in Qa-1KO and Qa-1WT recipients.Conclusion:1. The survival time of CTA graft is associated with the tissue complexity. However,simultaneous transplantation of skin grafts with two MHC genotypes could not significantlyprolong the survival time of the allogeneic skin graft as well as could not significantlyreduce the donor specific proliferation reaction of lymphocytes from the CTA recipients.2. Lymphocyte clones specific for different MHC antigen could compete and inhibitagainst each other in MLR model,in which IL-10produced by CD4+T cells but notinduction of Foxp3+Tregs was involved. And this inhibition could be reversed by theabsence of Qa-1.