Mechanism Study Of Heterogeneous Hemopoietic Chimerism Preventing Acute GVHD

Introduction:The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution to the critical shortage of human organs for transplantation. However, graft-versus-host-disease (GVHD) is the major complication of xenogeneic bone marrow transplantation (BMT) mediated by more vigorous cellular and humoral response to hosts than allogeneic BMT.To explore whether the graft-versus-host disease could be alleviated by transplanting bone marrow from chimeras, we studied reverse BMT in a concordant rat-mouse model.Methods:1. First, SD rats received 800cGy total body irradiation (TBI),followed by injection intravenously of 2 X 10 BALB/c mouse bone marrow cells, then intraperitonially administered cyclophophamide(Cy, 50mg/kg). At day 30, donor engraftment in the peripheral blood of chimeric rats was 37. 39%~47. 26%, and the survival time of BALB/c mice skin graft in group B was longer than that in control group , showing that xenogeneic chimeric rats have been induced.2. Second, BALB/c mice received 900cGy TBI and divided into three groupsrandomly. Group A were injected intravenously with 4 ×10~7 bone marrow cells from naive rats, group B with 4 × 10~7 from chimeric rats, group C received injection of diluent only,. In addition, group D were B6 mice, which also received 900cGy TBI and were injected with 4 × 10~7 bone marrow cells from chimeric rats.3. Assessment of GVHD Transplanted mice weights were obtained and recorded on day +1 and weekly thereafter. A threshold of 10% weight loss was used to signify the presence of moderate GVHD. The degree of systemic GVHD was assessed by a scoring system which sums changes in five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity (maximum index =10). Individual mice were graded weekly from 0 to 2 for each criterion.4 Skin transplantation: BALB/c mice was transplanted skin graft (about 1 X Icm2)from SD and Whistar rats at day 30 after reverse BMT. Skin graft's colour and texture, scabbing and falling, fur growing were monitored everyday. Stiffening, scabbing or falling was assessed rejection, tenderness or fur growing was assessed tolerance and engraftment.5 Cytokine ELISAs Antibodies used in the TNF- α ,IFN- γ and IL-4 assays were purchased from PharMingen (San Diego, CA). All assays were performed according to the manufacturer's protocol. Briefly, TNF- α , IFN- γ and IL-4 were captured by the specific primary mAbs, and detected by horseradish peroxidase (TNF- α ) or biotin-labeled (IFN- γ and IL-4) secondary mAbs. The biotin-labeled assays were developed with strepavidin (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Plates were read at 450 nm using a microplate reader (Bio-Rad Labs, Hercules, CA). Recombinant murine TNF- a ,IFN- Y and IL-4 (PharMingen) were used as standards for ELISA assays. The sensitivity of the assays was 26 pg/ml for TNF- a , I5pg/ml for IFN- Y , and lOpg/ml for IL-4. Supernatants were collected after 4 h of culture for TNF- a , 48 h for IFN- y and IL-4 analysis.6 FACS analysis FITC-conjugated mouse anti rat RTla MHC class I mAb were purchased from Serotec Ltd (Kidlington,Oxford,UK), PE-conjugated anti-mouse H-2Dd mAb were purchased from PharMingen (San Diego, California, USA). Red cells in peripheral blood were removed with ammonium chloride red cell lysis buffer, then incubated with the relevant mAb for 30 minutes at 4°C, washed twice with PBS, and fixed with 1% paraformaldehyde in PBS. Finally, 2-color flow cytometry was performed on a FACScan cytometer.7 Mixed lymphocyte reacion Splenic T cells of group A or B animals were obtained by purification over a nylon wool column followed by red cell removal with ammonium chloride red cell lysis buffer, and were plated in flat-bottomed 96-well Falcon plates (Lincoln Park, NJ) at 4X105 cells per well with 2X105 irradiated (2,000 Rad) splenocytes lavaged from naive SD or Whistar rats. Cell culture was performed in 10% heat-inactivated fetal calf serum in RPMI-1640. Cultures were incubated for 5 days ( pH 7.75, at 37 °C humidified incubator supplemented with 5% CO2), and. were pulsed during the final 18 hours with 1 u Ci/well 3[H]-thymidine. Proliferation was determined in an auto-gamma counter (Beckman, USA)Results:1 -, Recipients of group A developed significant weight loss within the first week after BMT and continued to lose weight throughout the observation period. GVHD was severe, approximately 50% of group A survived the observation period of 60 days. By contrast, 87.5% of group B animals survived this period, and mild or moderate GVHD(clinical scores between 3 and 5 or <3) was induced . These results indicate that reverse BMT significantly prolongs survival by preventing acute GVHD.In addition, group D (B6 mice received BM cells from BALB/SD chimeric rats) also developed significant signs of GVHD, showing that this effect of reverse BMT on preventing GVHD was donor specific.2, Cytokine ELISAs result: Compared with group A , T cells from group B showed a significant increase in IL-4 production, with a simultaneous decrease in INF- y and TNF- a production (P<0.001), serum IFN-y and TNF-a levels in group B also reduced whereas serum IL-4 levels increased , consistent with the experiment in vitro.3n The survival of skin graft: Skin grafting was administered on 30 day after BMT. Compared with naive SD, the survival time of SD skin graft in group B and A was longer, furthermore, the survival time of skin graft in group B is longer than that in group A, but meanwhile, third party Whistar rat skin graft in group B is rejected during 2 weeks, showing that BALB/c recipients were specificly tolerant to donor SD rats.4^ The levels of chimerism: As time passed, the levels of chimerism gradually declined, but the rate of group A declined more rapidly than group B. To the day 90, levels of chimerism of group B animals were still (23.77+4.50)%, compared with group A (12.40 + 0.32)% , P<0.05.5> Histology: The liver of group A and D exhibit extensive hepatocellular necrosis and apoptosis, inflammatory cell infiltrate, the small bowel exhibits outright crypt destruction, loss of brush border, and marked depletion of surface epithelial mucous. In contrast, liver of group B exhibits dispersed hepatocellular steatosis , and a rare apoptotic cell, and the small bowel exhibits mild villous blunting and crypt regenerative changes.6> The result of MLR : Compared with naive BALB/c, proliferation of both group B and A splenic T cells to SD stimulators was significantly inhibited. Furthermore, the proliferation of group B T cells to naive SD stimulators in MLC was (4641.97+174.97) cpm, compared with group A (7160.56 ±797.89) cpm, P<0.001, indicating that further inhibition to donor xenoantigens was induced via reverse BMTin vitro.Conclusion:K The morbidity and mortality of acute GVHD was significantly reduced via reverse bone marrow transplantation, and survival were prolonged. These results indicate that Reverse bone marrow transplantation can prevented lethal GVHD in an xenogeneic bone marrow transplant model (rat-mouse)2^ reverse BMT is associated with decreases of TNF- a and IFN- Y , increase ofIL-4, further inhibition of proliferation of host T cells to donor xenoantigen in MLC.The mechanism may be that reverse BMT inhibited type-1 cytokine production (IFN-Y and TNF- a ), polarizing donor T cells toward the production of type-2 cytokines,which is associated with reduced severity of acute GVHD.3n Reverse bone marrow transplantation induces more stable chimerism over 90 days and promotes hematopoietic reconstitution more rapidly than conventional BMT after lethal conditioning. Considered together, we propose that reverse BMT may represent a novel and feasible approach to induce long-term xenogeneic chimerism without lethal GVHD, and has a potential application for clinical development.