BTLA And ICOS As Early Indicators For Acute Rejection Following Kidney Transplantation

Kidney transplantation has become one of the methods for the therapy of patients with end-stage renal disease. Cadaveric renal allograft transplantation is common in clinical practice. Early acute rejection has been recognized as the most powerful predictor of graft loss, including those losses due to chronic rejection. The introduction of potent immunosuppressive drugs targeting T lymphocytes has led to a decrease in kidney transplant rejection and an increased quality of life. But the manifestations become more and more atypical. Kidney biopsy has been considered as the gold standard for pathological examination. It is significant to look for more convenient, highly sensitive and specific parameters for the occurrence of acute rejection following kidney transplantation.The mechanism of transplantation rejection is mainly correlated to the activation of cellular immunity of the recipient. The immune system recognizes non-self antigens and activates effecter T cell, which release cytokines to attack the allograft. Two signals are requested for T cell activation, proliferation and producing cytokines. One is the MHC-antigen complex between T cell receptor (TCR) and antigen presenting cells (APC). Another is costimulatory signal provided by soluble costimulatory molecules or ligands on the membrane of APC.The immunoglobulin super family (Ig SF) member B and T lymphocyte attenuator (BTLA/CD272) is a third inhibitory coreceptor expressed on T and B cells, together with cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1). Ligation of BTLA and its ligand inhibits activated T cells to prevent strong cellular immune response as a protective mechanism. BTLA negatively regulates humoral immune responses and also plays an important role in immune surveillance. Herpes Virus Entry Mediator (HVEM) is the specific ligand for BTLA. The combination of BTLA and HVEM produces coinhibitory signal, while ligation of LIGHT with HVEM produces a costimulatory signal. Inducible costimulator (ICOS) is a costimularoty molecule on the membrane of T cells. Its ligand is ICOSL, also named as GL-50,B7 related protein-1 (B7RP-1) or B7 homologous protein (B7h). ICOS/ICOSL pathway regulates T and B lymphocyte activation, proliferation and cytokine secretion. The blockade of ICOS/ICOSL leads to downregulation of several cytokines and chemokines.ObjectivesThe aim of this study was to analyze the expression patterns of BTLA/HVEM and ICOS/ICOSL on peripheral blood mononuclear cells (PBMC) and determine the concentrations of serum cytokines during immunosuppressive therapy. Evaluation would be made for BTLA/HVEM and ICOS/ICOSL as more convenient, highly sensitive and specific parameters in early predicting acute rejection following kidney transplantation. This study would benefit for the prevention and early treatment of acute rejection in patients receiving kidney transplantation and improve the survival of either transplanted kidney or the recipient to achieve a better quality of life.Methods1. Patients and Samples:In the current study,281 consecutive patients who received cadaveric renal allograft transplantation between February 2008 and February 2011 were enrolled. All the patients were Chinese and had transplant operations at the Department of Kidney Transplantation, Second Hospital, Shandong University. The warm ischemia time is less than 8 min and cold ischemia time was less than 10 h. MMF+CSA+Pred or MMF+FK506+Pred triple therapy were used in patients without any treatment or complications influencing the parameters. General conditions, urine volume, kidney functions, drug concentrations and T cell subsets were observed and acute rejection was diagnosed according to Banff2005 standard. Methylprednisolone (MP) was used for 3 days as the rescue therapy of acute rejection patients. PBMC and serum were separated and collected at the day of operation (before operation) and 3,7,14 and 28 days post operation. The study was approved by the Ethnics Committee of the second hospital of Shandong University.2. Reverse transcription (RT-PCR) and quantitative real-time PCR (qPCR) were performed to measure expression levels for BTLA/HVEM/LIGHT, ICOS/ICOSL, IL-2, IL-10, IL-4 and IFN-γmRNA in PBMCs. Total RNA were isolated using TRIzol Reagent. All cDNA was synthesized using the primers designed by Primer Primier 5 and PCR process was carried out as routinely. The products were analyzed on a 1% agarose gel that was stained with ethidium bromide. The bands were visualized in an Alpha Imager (Bio-Rad) and the optical density of each band was compared with the respectiveβ-action after background subtraction using the Alpha Imager software. The q-PCR reactions were performed using a preheated real-time PCR instrument (ABI7000).3. BTLA and ICOS protein expression levels were detected by flow cytometry.4. Enzyme-linked immunosorbent assay (ELISA) was used to determine IL-2, IFN-γ, IL-10, IL-4 concentration in the serum, which had been collected at day 0,3,7, 14 and 28. The experiment was carried out strictly according to the manufacturer's instructions. The value of O.D. was measured at 450 nm on the the microplate reader (Bio-Rad).5. The data were analyzed using SPSS 12, Microsoft Excel 2007 and GraphPad Prism v5.0. The data obtained were statistically evaluated by analysis of variance (ANOVA). For the analysis of two independent groups, Student's t-test was used with significance at p<0.05. The difference of costimulatory molecules and cytokine expression was analyzed among each time point and groups. The correlation was analyzed between the above expression difference and the occurrence of acute rejection following kidney transplantation.Results1. There was no significant difference between the groups including general conditions, sex, age, warm ischemia time, cold ischemia time and drug concentrations (P>0.05).2. BTLA/HVEM and ICOS/ICOSL expression on PBMC:RT-PCR, q-PCR and flow cytometry consistently showed that the expression of above molecules was obviously elevated at 3 days post operation, and decreased gradually at day 7,14 and 28 of the stable group. While in patients developing acute rejection, all molecules remained high level expressions compared with the renal stable group, especially the increased expression of BTLA and ICOS at 7 days post operation (P<0.01).3. IL-2, IFN-y, IL-10 and IL-4 mRNA expression on PBMC:RT-PCR results showed that the expression of above cytokines was obviously elevated at 3 days post operation, and decreased gradually at day 7,14 and 28 of the stable group. While in patients developing acute rejection, IL-2 and IFN-y remained high level expressions compared with the renal stable group, especially at 14 days post operation (P<0.01).4. IL-2, IFN-γ, IL-10 and IL-4 concentrations in the serum:ELISA showed that the concentrations of above cytokines were obviously elevated at 3 days post operation, and decreased gradually at day 7,14 and 28 of the stable group. While in patients developing acute rejection, IL-2 and IFN-y remained high concentrations compared with the renal stable group, especially at 14 days post operation (P<0.01).5. The correlation was analyzed between BTLA and ICOS expression and the occurrence of acute rejection following kidney transplantation:Acute rejection occurred at 14 days post operation, which was closely related to the expression change of BTLA and ICOS at 7 days.6. The correlation was analyzed between BTLA and ICOS expression and the concentration of cytokines:The sensitivity and specificity of each parameter was shown by Youden index. The combination of BTLA or ICOS expression levels has the best sensitivity and specificity to predict acute rejection.ConclusionsThis study analyzed the expression patterns of BTLA/HVEM, ICOS/ICOSL, IL-2, IL-4, IL-10 and IFN-y of patients presenting acute rejection and maintaining stable renal function following kidney transplantation. We showed that the expression of above molecules was obviously elevated at 3 days post operation, and decreased gradually at day 7,14 and 28 of the stable group. While in patients developing acute rejection at 12.6 days, all molecules remained high level expressions compared with the renal stable group. The increased expression of BTLA and ICOS occured 7 days before the appearance of clinical manifestations could be considered as early indicators for acute rejection following kidney transplantation.