Evaluation Of CXCL10-CXCR3 As Targets For Allograft Rejection Monitoring

ObjectiveOrgan transplantation is the most effective method for patients with end-stage diseases, but the occurrence of acute rejection (AR) is still very high, varies between 10 and 80% in the first year. AR is one of the most important negative factors that impact the graft and cause graft dysfunction. The specific noninvasive diagnosis of AR at the early stage is of great importance to protect the graft function and prolong graft survival time. Lots of studies have founded that the amount of CXCL10 in blood and urine before or after transplantation could prospect the prognosis of transplantation. CXCL10 along with its receptor CXCR3 plays an important role in CXCR3 positive immune cells (Thl cells) recruit, and their expression at the graft is closely related to the accordance of AR. Under the pro-inflammation microenvironment, CXCL10 could be secreted from a variety of cells, such as leukocytes, activated neutrophils, eosinophils, monocytes, endothelial cells, epithelial cells, stromal cells (fibroblasts) and keratinocytes in response to IFN-y. And the expression up-regulation of CXCL10 is earlier than CXCL9 (another ligand of CXCR3), so we suspect that the amount of CXCL10 can detect CXCR3 positive invasion earlier than CXCL9.Molecular imaging makes it possible to detect the distribution and relative amount of a target molecular noninvasively in vivo. After confirmed that CXCL10 is a target closely related to AR initiation, we successfully produced 131I-anti-CXCL10 mAb and used it to image the distribution of CXCL10; studied its use in early stage AR diagnosis. As a big-molecular,131I-anti-CXCL10 mAb distributes and eliminates slowly, its pharmacokinetic is not very well. So we studied the use of a small-molecular 125I-CXCL10 (8.5 Kd) targeted at CXCR3 to detect CXCR3 positive cells invasion of AR.Part ⅠNoninvasive Allograft Imaging of Acute Rejection:Evaluation of 131I-anti-CXCL10 mAbMethod1. Establishment of Isograft/allograft skin model:Isograft/allograft skin model was established with BALB/c/C57BL/6 skin as donor and BALB/c mice as receptor. Tacrolimus treated allografted model was treated with tacrolimus 1 mg/kg.d. And the rejection at the grafts was confirmed by HE staining.2. Expression of CXCL10:Expression of CXCL10 mRNA at day 10 was proved by RT-PCR. And the amount of CXCL10 protein in graft, spleen and blood was measured by ELISA and immunochemistry staining.3. Preparation of 131I-anti-CXCL10 mAb:131I-anti-CXCL10 mAb was produced by the Iodogen method; the radionuclide purity and stability of radiotracer was measured by a gamma counter after paper chromatography.4. Ex vivo biodistribution studies:Allograft/isograft mice were submitted to ex vivo biodistribution studies after tail vein injection of 131I-anti-CXCL10 mAb at day 8 after transplantation.5. Whole-body autoradioimaging studies:Allograft/isograft mice were submitted to whole-body autoradioimaging studies after tail vein injection of 131I-anti-CXCL10 mAb at day 8 after transplantation. For binding specificity studies, a separate group of mice was subcutaneously conducted with a blocking dose of anti-CXCL10 mAb.Results1. Isograft/allograft skin model was successfully established. The HE staining of allograft showed that there were obvious necrosis in epithelial cells and subcutaneous tissues, and there were a large number of lymphocyte and neutrophil infiltration in the epithelium and dermis. Tacrolimus treatment could decrease the inflammatory cells infiltration and protect the graft. And there was no obvious immune cells infiltration at isograft skin.2. The results of RT-PCR, ELISA, and immunohistochemical staining all suggested that the highest concentration/expression of CXCL10 was detected in allograft tissue compared with allograft treated with tacrolimus and isograft control. Tacrolimus treatment can obviously inhibit the expression of CXCL10 in graft, spleen, and serum.3.131I-anti-CXCL10 mAb was successfully produced. The radionuclide purity was 98% and above 95% till 72 h.4. The biodistribution results showed the highest uptake of radiotracer in allograft. T/NT (target/nontarget) ratio was 4.15±0.25 at 72h, apparently different from allograft treated with tacrolimus (2.29±0.10), P< 0.05.5. Allograft could be obviously imaged at all checking points from 6 h to 72 h after injection, much clearer than the other two groups. Blocking dose of anti-CXCL10 antibody (20 mg/kg) could significantly reduction the uptake of 131I-anti-CXCL10 mAb at graft.Conclusions1. CXCL10 is a promising target for early stage AR imaging.2.131I-anti-CXCL10 mAb can successfully image AR and monitor the effect of immunosuppressant.Part IIImaging CXCR3 Expression in Acute Rejection Graft:Evaluation of 125I-CXCL10Method1. Expression of CXCR3:Isograft/allograft skin model was established with BALB/c/C57BL/6 skin as donor and BALB/c mice as receptor. And the expression of CXCR3 was confirmed by RT-PCR and immunohistochemical staining at day 9.2. Preparation of 125I-CXCL10:125I-CXCL10 was produced by the Iodogen method; the radionuclide purity and stability of radiotracer was measured by a gamma counter after paper chromatography.3. Radioligand binding assay:The well specificity and affinity was confirmed by radioligand binding assay with spleen cells of allografted mice at day 8.4. The pharmacokinetics study:The pharmacokinetics study was performed with allografted mice after tail vein injection of 125I-CXCL10.5. Ex vivo biodistribution studies:Allograft/isograft mice were submitted to and ex vivo biodistribution studies after tail vein injection of 125I-CXCL10 at day 8 after transplantation.6. Whole-body autoradioimaging.Results1. The obvious higher expression of CXCR3 was detected in acute rejection allograft skin compared with isograft control.2. The 125I-CXCL10 was successfully produced. The radionuclide purity was 98% and above 95% till 72 h.3.125I-CXCL10 could bind to spleen cells specially, and the KD of 125I-CXCL10 was 3.48±0.31 nM, the immunoreactivity was about 90%.4. The pharmacokinetics result suggested 125I-CXCL10 distribution and excretion quickly in vivo (T1/2α was 0.34 h and T1/2β was 9.83 h).5. In accord with the image result, the biodistribution result showed the higher uptake of radiotracer in allograft. T/NT (Target/Non target) ratio of allograft group was 3.01±0.25 at 24 h, apparently higher than isograft group (1.14±0.10), p<0.05.6. Allograft could be imaged at all checking points, as early as 3 hour after injection, and it was much clear at 12 h and 24 h. On the contrast, there was no obvious radiotracer accumulation at the isograft.ConclusionThese data suggest that CXCR3 is a promising imaging target for early stage AR immune cells infiltration and 125I-CXCL10 can successfully image AR with good pharmacokinetics.