Basic And Clinical Research Of The Role Of TIM-3in Transplantation Immunity

Sequential monitoring the TIM-3gene expression inperipheral blood for diagnostic and prognostic evaluationof acute rejection in renal graft recipientsSubject: TIM-3has been demonstrated expression on primary T effectorscells which played essential roles in acute allograft rejection (AR), including Th1,CTL, and Th17. In this study we monitored sequential changes of TIM-3geneexpression in the peripheral blood leukocytes (PBL) from renal transplantrecipients to provide a deeper insight of the role of TIM-3in allograft rejection.Methods: The study consisted of AR-group (n=24), NO-AR group (n=20) andStable-group (n=18). Prospective, serial blood samples were collected in recentrecipients post allograft transplantation and during AR episode. The mRNAencoding for TIM-3was quantified using real time RT-PCR. Statistical analyseswere performed to investigate the gene transcripts measurements with the clinicalevents.Results: TIM-3mRNA in PBL was significantly higher expressed in AR-group comparing to NO-AR group and Stable-group (286.72±86.28vs126.10±28.31;286.72±86.28vs96.91±17.88, p=0.00). The ROC (receiver operatingcharacteristic) curve showed a specificity of90.91%and a sensitivity of90.00%for rejection diagnosis utility of TIM-3. Anti-rejection therapy decreased theTIM-3mRNA expression in all patients with AR at initial time. There was apositive correlation between TIM-3mRNA expression level and serum creatinine(r~2=0.716, p=0.00).Conclusions: TIM-3mRNA quantification by real time RT-PCR in PBL maybe a promising tool for a noninvasive diagnosis of AR. But the utility forpredicting the prognosis of AR after anti-rejection was limited due to the great variation of TIM-3mRNA expression during AR episode. Activated TIM-3-Galectin-9improves the survival of fullyallogeneic skin grafts and regulates the transplantationimmune balance between Teff and TregSubject: To explore the effect of Galectin-9on the survival of allogeneic skingrafts in mice and to explore the related mechanism for regulating the immunebalance.Methods: Construct the allogeneic skin transplantation model betweenBALB/c and C57BL/6mice. Median survival time of skin grafts in control-groupand study-group were observed. The lymphocytes infiltration in skins on7dayafter transplantation was investigated by hematoxylin-eosin staining. Analyzed theratio of CD4~+TIM-3~+T,CD4~+TIM-1~+T,Th17,CD4~+CD25~+Foxp3~+Treg in Tlymphocytes of PBL and spleen lymphocytes on7,14day after skintransplantation by FCM (flow cytometry). Evaluated the cytokine expression ofIL-2,IL-4,IL-17and IFN-γ in blood on7day after transplantation.Results:1. Galectin-9could prolong the survival time of allogeneic skin grafts in miceand the MST in control-group and study-group were12.5days and16daysseparately.2. Galectin-9could alleviate the lymphocytes infiltration in skin grafts on7day post-operation.3. Galectin-9could down-regulate the retio of Teff cells in CD4~+Tlymphocytes and have no effection on Treg cells. It was as followings:①CD4~+TIM-3~+T Galectin-9could down-regulate the CD4~+TIM-3~+T ratio inblood and spleen lymphocytes on7day after transplantation in study-group(spleen4.82±1.01%vs2.84±0.89%, blood7.13±1.82%vs4.94±1.56%, p<0.05);but have no effect on14day after transplantation (spleen3.34±1.10%vs 3.44±0.98%, blood2.79±0.87%vs2.67±0.84%, p>0.05).②Th17Galectin-9could down-regulate the Th17ratio in PBL on7dayafter transplantation in study-group (12.42±3.67%vs5.79±1.68%, p<0.05) andhave no difference in spleen lymphocytes (5.54±1.38%vs5.78±1.64%, p>0.05);on14day after transplantation, there was no significant differences in PBL(5.21±1.96%vs5.91±2.03%, p>0.05) and the Th17ratio was down-regulated inspleen lymphocytes in study-group (4.61±1.78%vs7.91±2.34%, p<0.05).③CD4~+TIM-1~+T Galectin-9had no effect on CD4~+TIM-1~+T ratio in spleenlymphocytes on7,14day after transplantation between two groups (7day3.79±1.35%vs4.27±1.52%,14day3.19±1.26%vs3.92±1.34%, p>0.05); no differencesin PBL was found (34.74±4.80%vs34.50±5.00%, p>0.05) on7day, but it wasup-regulated in study-group on14day after transplantation (52.80±8.79%vs34.86±5.67%, p<0.05).④CD4~+CD25~+Foxp3~+Treg Galectin-9had no effect on CD4~+CD25~+Foxp3~+Treg ratio in PBL and spleen lymphocytes on7,14day after transplantation(spleen7day10.57±2.56%vs10.02±3.04%,14day11.49±2.87%vs12.93±3.12%, p>0.05; blood7day8.00±1.78%vs8.20±2.01%,14day9.76±2.45%vs10.90±2.67%, p>0.05).4. Galectin-9could decreased the expression of IFN-γ (51.33±8.16pg/ml vs98.41±15.01pg/ml, p=0.00) and IL-17(48.50±8.48pg/ml vs15.17±5.46pg/ml,p=0.00) in blood on7day after transplantation; but have no effect on expressionof IL-2(23.17±4.22pg/ml vs26.50±3.78pg/ml, p=0.180) and IL-4(86.83±13.33pg/ml vs99.67±11.64pg/ml, p=0.107).Conclusions: Galectin-9could prolong the survival time of allogeneic skingrafts in mice. This was related to the down-regulation of CD4~+TIM-3~+T andTh17in PBL and spleen lymphocytes. Galectin-9had no effect of CD4~+CD25~+Foxp3Treg in vivo.