IL‐17 Expression by Tubular Epithelial Cells in Renal Transplant Recipients with Acute Antibody‐Mediated Rejection

Acute rejection is still a common complication of kidney transplantation. IL‐17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL‐17 graft expression in renal transplant recipients with acute antibody‐mediated rejection (ABMR), acute T‐cell‐mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin‐inhibitor toxicity (CNI). In acute ABMR, tubular IL‐17 protein expression was significantly increased compared to TCMR, where most of the IL‐17⁺cells were CD4⁺graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL‐17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL‐17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL‐17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a‐induced IL‐17 expression. The use of steroids and monoclonal antibodies reduced IL‐17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL‐17 in ABMR and this event might be mediated by the complement system activation featuring this condition.     

T helper 1, 2 and 17 cell subsets in renal transplant patients with delayed graft function

Ischemia‐reperfusion injury (IRI) in kidney transplantation is the major cause of delayed graft function (DGF), an event associated with an increased risk of acute rejection. The aim of this study was to evaluate T helper (Th) cell phenotype in renal transplants with DGF. T‐bet (Th1), GATA‐3 (Th2) and IL‐17 (Th17) protein expression was investigated in pretransplant biopsies, DGF and acute tubular damage (ATD) caused by calcineurin‐inhibitor toxicity. Intracytofluorimetric analysis of IFN‐γ, IL‐4 and IL‐17 was performed to analyze Th1, Th2 and Th17 responses in peripheral blood mononuclear cells of recipients with early graft function (EGF) and DGF, before (T0) and 24 h after transplantation (T24). In pretransplant biopsies, T‐bet⁺, GATA‐3⁺ and IL‐17⁺ cells were barely detectable. In DGF, T‐bet⁺ and IL‐17⁺ cells were significantly increased compared with pretransplant and ATD. More than 90% of T‐bet⁺ and less then 5% of IL‐17⁺ cells were CD4⁺. GATA‐3⁺ cells were increased to a lower extent. T‐bet⁺/GATA‐3⁺ cell ratio was significantly higher in DGF. Peripheral CD4⁺ IFN‐γ/IL‐4 ratio was significantly decreased in DGF, while CD4⁺/IL‐17⁺ cells did not differ between T0 and T24 in DGF. Our data suggest that DGF is characterized by a prevalent Th1 phenotype within the graft. This event might represent a link between DGF and acute rejection.     

The protective role of interleukin‐18 binding protein in a murine model of cardiac ischemia/reperfusion injury

IL‐18, a proinflammatory cytokine, is produced by macrophages, epithelial cells, T cells, neutrophils, NK‐T cells, and B cells, and has been implicated in the pathophysiology of a variety of inflammatory diseases including ischemia/reperfusion (IR) injury, transplant rejection, and autoimmune disease. Recent study indicated that neutralization of IL‐18 with anti‐IL‐18 antibody or IL‐18‐binding protein (IL‐18BP) ameliorates IR‐induced myocardial injury. However, the mechanism needs to be further investigated. In our current study, syngeneic heterotopic heart transplantation was performed by a modified non‐suture cuff technique. We found that IL‐18BP treatment ameliorated cardiomyocyte necrosis and infiltration of CD4⁺ T cells, neutrophils, and macrophages. IL‐18BP‐treated mice exhibited decreased expression of inflammatory cytokines including IL‐1β, IL‐23, IL‐18, and IL‐17. IL‐18BP treatment suppressed Th17 differentiation in vivo and in vitro. Adoptive transfer of T cells from IL‐18BP‐treated mice showed alleviated cardiac IR injury when compared with that transferred from control mice. Furthermore, the decreased infiltration of mononuclear cells and production of troponin T (TnT) induced by IL‐18BP treatment were both abrogated by additional administration of recombinant mouse IL‐17 (rmIL‐17). These data revealed a protective role of IL‐18BP in cardiac IR injury. Above all, IL‐18BP ameliorates cardiac IR injury in part through suppression of Th17 differentiation.     

Acute Renal Endothelial Injury During Marrow Recovery in a Cohort of Combined Kidney and Bone Marrow Allografts

An idiopathic capillary leak syndrome (‘engraftment syndrome’) often occurs in recipients of hematopoietic cells, manifested clinically by transient azotemia and sometimes fever and fluid retention. Here, we report the renal pathology in 10 recipients of combined bone marrow and kidney allografts. Nine developed graft dysfunction on day 10–16 and renal biopsies showed marked acute tubular injury, with interstitial edema, hemorrhage and capillary congestion, with little or no interstitial infiltrate (≤10%) and marked glomerular and peritubular capillary (PTC) endothelial injury and loss by electron microscopy. Two had transient arterial endothelial inflammation; and 2 had C4d deposition. The cells in capillaries were primarily CD68⁺MPO⁺ mononuclear cells and CD3⁺CD8⁺ T cells, the latter with a high proliferative index (Ki67⁺). B cells (CD20⁺) and CD4⁺ T cells were not detectable, and NK cells were rare. XY FISH showed that CD45⁺ cells in PTCs were of recipient origin. Optimal treatment remains to be defined; two recovered without additional therapy, six were treated with anti‐rejection regimens. Except for one patient, who later developed thrombotic microangiopathy and one with acute humoral rejection, all fully recovered within 2–4 weeks. Graft endothelium is the primary target of this process, attributable to as yet obscure mechanisms, arising during leukocyte recovery.     

Plasma cell densities and glomerular filtration rates predict renal allograft outcomes following acute rejection

The contribution of T cells and graft‐reactive antibodies to acute allograft rejection is widely accepted, but the role of graft‐infiltrating B and plasma cells is controversial. We examined 56 consecutive human renal transplant biopsies classified by Banff schema into T‐cell‐mediated (N = 21), antibody‐mediated (N = 18), and mixed (N = 17) acute rejection, using standard immunohistochemistry for CD3, CD20, CD138, and CD45. In a predominantly African‐American population (75%), neither Banff classification nor C4d deposition predicted the return to dialysis. Immunohistochemical analysis revealed CD3⁺ T cells as the dominant cell type, followed by CD20⁺ B cells and CD138⁺ plasma cells in all acute rejection types. Using univariate Cox Proportional Hazard analysis, plasma cell density significantly predicted graft failure while B‐cell density trended toward significance. Surprisingly T‐cell density did not predict graft failure. The estimated glomerular filtration rate (eGFR) at diagnosis of acute rejection also predicted graft failure, while baseline eGFR ≥6 months prior to biopsy did not. Using multivariate analysis, a model including eGFR at biopsy and plasma cell density was most predictive of graft loss. These observations suggest that plasma cells may be a critical mediator and/or an independently sensitive marker of steroid‐resistant acute rejection.     

IL‐17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC‐Mismatched Renal Allograft Transplantation

IL‐17 is a pro‐inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL‐17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL‐17 in a fully MHC‐mismatched, life‐sustaining, murine model of kidney allograft rejection using IL‐17 deficient donors and recipients (IL‐17^?/? allografts). IL‐17^?/? allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN‐γ and histological attenuation of acute and chronic allograft rejection, as compared to wild‐type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL‐17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL‐17 showed a trend towards prolongation of survival only when recipients were IL‐17^?/?. Administration of a depleting anti‐CD25 antibody to IL‐17^?/? recipients abrogated the survival advantage conferred by IL‐17 deficiency, suggesting the involvement of a CD4⁺CD25⁺ T cell regulatory mechanism. Therefore, IL‐17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

     

Compartment‐specific expression of natural killer cell markers in renal transplantation: immune profile in acute rejection

Natural killer (NK) cells have been implicated in graft dysfunction. Here, we formulated hypothesis that distinct patterns of expression NK cells markers correlated with acute rejection in kidney transplantation. Therefore, we studied the pattern of NK cell markers CD56, CD57, and CD16 in different compartments of biopsies obtained from recipients diagnosed with acute graft rejection, with or without donor‐specific antibodies (DSA). DSA‐negative biopsies‐from patients with acute T‐cell mediated rejection (aTCMR) had an increased expression of CD56+ and CD57+ cells (P = 0.004 and P = 0.001) in the interstitial compartment in comparison with DSA‐positive biopsies from patients acute antibody‐mediated rejection (aABMR) with (aABMR C4d+) and without C4d deposition (aABMR C4d‐). CD16+ cells was increased (P = 0.03) in the glomerular compartment in DSA‐positive biopsies. We assume that CD16+ expression and antibody‐dependent cellular cytotoxicity (ADCC) in microvascular injury can be associated with aABMR. IFN‐γ release from cytoplasmic granules of NK cell could be associated with aTCMR. Our findings suggest that NK cells need to be carefully evaluated because variations in NK cell marker expression might imply the involvement of different immune system pathways in graft rejection.     

Retinoic acid attenuates acute heart rejection by increasing regulatory T cell and repressing differentiation of Th17 cell in the presence of TGF‐β

Retinoic acid (RA), in a transforming growth factor beta (TGF‐β)‐dependent manner, promotes differentiation of regulatory T cells (Tregs) but inhibits the differentiation of Th17 cells in vitro from naive CD4⁺T cells. In addition, transfer of induced Tregs (iTregs) reduces rejection. We therefore examined whether RA could attenuate acute cardiac transplant rejection in vivo in a mouse model by regulating the reciprocal differentiation of Tregs and Th17 cells. The iTregs and naive T cells were respectively transferred into congenic mice. Two weeks later, the percentages of transferred cells and Forkhead box P3 (FoxP3)+ Tregs were measured in spleen. Mice with cardiac transplants were treated with TGF‐β alone, RA alone, both or none. The percentage of Tregs or Th17 cells in CD4⁺T cells, the level of FoxP3 protein or serous interleukin (IL)‐17A, or suppressive function of Tregs from recipient mice were assessed. The percentage of Th17 cells and level of serum IL‐17A both increased significantly during acute rejection. RA favored differentiation to Tregs over Th17 cells. Unlike naive T cells, only a few transferred iTregs remained after transfer. Treatment with RA plus TGF‐β prolonged graft survival, increased the percentage of Tregs, and decreased the percentage of Th17 cells in peripheral T cells. Tregs from all recipients had normal suppressive function. In conclusion, treatment with RA plus TGF‐β attenuates acute rejection by promoting the differentiation of Tregs and inhibiting the differentiation of Th17 cells.     

The Effect of Costimulatory and Interleukin 2 Receptor Blockade on Regulatory T Cells in Renal Transplantation

Regulatory T cells (Treg) are critical regulators of immune tolerance. Both IL‐2 and CD28‐CD80/CD86 signaling are critical for CD4⁺CD25⁺FOXP3⁺ Treg survival in mice. Yet, both belatacept (a second‐generation CTLA‐4Ig) and basiliximab (an anti‐CD25 monoclonal antibody) are among the arsenal of current immunotherapies being used in kidney transplant patients. In this study, we explored the direct effect of basiliximab and belatacept on the Tregs in peripheral blood both in the short term and long term and in kidney biopsies of patients with acute rejection. We report that the combined belatacept/basiliximab therapy has no long‐term effect on circulating Tregs when compared to a calcineurin inhibitor (CNI)‐treated group. Moreover, belatacept‐treated patients had a significantly greater number of FOXP3⁺ T cells in graft biopsies during acute rejection as compared to CNI‐treated patients. Finally, it appears that the basiliximab caused a transient loss of both FOXP3⁺ and FOXP3^? CD25⁺ T cells in the circulation in both treatment groups raising important questions about the use of this therapy in tolerance promoting therapeutic protocols.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

Rabbit anti‐rat thymocyte immunoglobulin preserves renal function during ischemia/reperfusion injury in rat kidney transplantation

Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction in humans. Increases in cold and warm ischemia times lead to a higher risk of early post‐transplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long‐term kidney graft loss. The protective effect of rabbit anti‐rat thymocyte immunoglobulin (rATG) was evaluated in a rat model of I/R injury following syngeneic kidney transplantation. Serum creatinine concentration was evaluated at 16 h and 24 h post‐transplant. Animals were sacrificed 24 h post‐transplant for evaluation of histology, infiltrating leukocytes, nitrotyrosine staining, and apoptosis. rATG was effective in preventing renal function impairment, tissue damage and tubular apoptosis associated with I/R only when was given 2 h before transplantation but not at the time of reperfusion. Pretransplant rATG treatment of recipient animals effectively reduced the amount of macrophages, CD4⁺, CD8⁺ T cells and LFA‐1⁺ cells infiltrating renal graft subjected to cold ischemia as well as granzyme‐B expression within ischemic kidney. On the other hand, granulocyte infiltration and oxidative stress were not modified by rATG. If these results will be translated into the clinical setting, pretransplant administration of Thymoglobuline^® could offer the additional advantage over peri‐transplant administration of limiting I/R‐mediated kidney graft damage.     

Interleukin‐33 prolongs allograft survival during chronic cardiac rejection

Interleukin‐33 (IL‐33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL‐33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C‐H2bm12/KhEg hearts were transplanted into MHC class II‐mismatched C57Bl/6J mice; IL‐33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL‐33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft‐infiltrating cells. Proinflammatory IL‐17A production was decreased with IL‐33 treatment, while increased levels of IL‐5, IL‐10, and IL‐13 were observed. After IL‐33 therapy, flow cytometry showed a direct induction of CD4⁺ Foxp3⁺ Treg, whereas the number of B220⁺ CD19⁺ B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL‐33 treatment, a significant decrease in graft‐infiltrating CD11b^highGr1^high granulocytes coincided with a significant increase in CD11b^highGr1^intermediate myeloid‐derived suppressor cells (MDSC). In conclusion, IL‐33 treatment in the setting of chronic rejection promotes the development of a Th2‐type immune response that favors MDSC and Treg expansion, reduces antibody‐mediated rejection (AMR), and ultimately, prolongs allograft survival.     

The extent of HLA‐DR expression on HLA‐DR+ Tregs allows the identification of patients with clinically relevant borderline rejection

Regulatory T cells (Tregs) were shown to be involved into the pathogenesis of acute rejection after transplantation. The suppressive activity of the total regulatory T cell pool depends on its percentage of highly suppressive HLA‐DR⁺‐Treg cells. Therefore, both the suppressive activity of the total Treg pool and the extent of HLA‐DR expression of HLA‐DR⁺‐Tregs (MFI HLA‐DR) were estimated in non transplanted volunteers, patients with end‐stage renal failure (ESRF), healthy renal transplant patients with suspicion on rejection, due to sole histological Bord‐R or sole acute renal failure (ARF), and patients with clinically relevant borderline rejection (Bord‐R and ARF). Compared to patients with only Bord‐R or only ARF, the suppressive activity of the total Treg cell pool was exclusively reduced in patients with clinically relevant Bord‐R. In parallel, the HLA‐DR MFI of the DR⁺‐Treg subset was significantly decreased in these patients, due to a significantly lower proportion of DR^high+‐Tregs, which were shown to have the highest suppressive capacity within the total Treg pool. Our findings clearly demonstrate that the determination of the HLA‐DR MFI of the HLA‐DR⁺‐Treg subset allows a highly sensitive, specific and non‐invasive discrimination between patients with clinically relevant Bord‐R (Bord and ARF) and patients with subclinical rejection or other causes of transplant failure.     

Paneth and intestinal stem cells preserve their functional integrity during worsening of acute cellular rejection in small bowel transplantation

Graft survival after small bowel transplantation remains impaired due to acute cellular rejection (ACR), the leading cause of graft loss. Although it was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, no results of Paneth and intestinal stem cells localized at the crypt bottom have been shown so far. Therefore, we wanted to elucidate integrity and functionality of the Paneth and stem cells during different degrees of ACR, and to assess whether these cells are the primary targets of the rejection process. We compared biopsies from ITx patients with no, mild, or moderate ACR by immunohistochemistry and quantitative PCR. Our results show that numbers of Paneth and stem cells remain constant in all study groups, whereas the transit‐amplifying zone is the most impaired zone during ACR. We detected an unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki‐67⁺ IL‐22R⁺ stem cells revealing cell functionality in moderate ACR samples. We conclude that Paneth and stem cells are not primary target cells during ACR. IL‐22R⁺ Ki‐67⁺ stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients.     

Expression of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4⁺Foxp3⁺ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4⁺CD25⁺CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4⁺CD39⁺ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4⁺ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4⁺CD25⁺CD39⁺ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.     

Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation

Bone marrow‐derived mesenchymal stromal cells (MSC) have emerged as useful cell population for immunomodulation therapy in transplantation. Moving this concept towards clinical application, however, should be critically assessed by a tailor‐made step‐wise approach. Here, we report results of the second step of the multistep MSC‐based clinical protocol in kidney transplantation. We examined in two living‐related kidney transplant recipients whether: (i) pre‐transplant (DAY‐1) infusion of autologous MSC protected from the development of acute graft dysfunction previously reported in patients given MSC post‐transplant, (ii) avoiding basiliximab in the induction regimen improved the MSC‐induced Treg expansion previously reported with therapy including this anti‐CD25‐antibody. In patient 3, MSC treatment was uneventful and graft function remained normal during 1 year follow‐up. In patient 4, acute cellular rejection occurred 2 weeks post‐transplant. Both patients had excellent graft function at the last observation. Circulating memory CD8⁺ T cells and donor‐specific CD8⁺ T‐cell cytolytic response were reduced in MSC‐treated patients, not in transplant controls not given MSC. CD4⁺FoxP3⁺Treg expansion was comparable in MSC‐treated patients with or without basiliximab induction. Thus, pre‐transplant MSC no longer negatively affect kidney graft at least to the point of impairing graft function, and maintained MSC‐immunomodulatory properties. Induction therapy without basiliximab does not offer any advantage on CD4⁺FoxP3⁺Treg expansion ( ClinicalTrials.gov number: NCT 00752479).     

Infection with the Intracellular Bacterium,Listeria monocytogenes,Overrides Established Tolerance in a Mouse Cardiac Allograft Model

Infections and TLR signals at the time of transplantation have been shown to prevent the induction of tolerance, but their effect on allografts after tolerance has been established is unclear. We here report that infection with Listeria monocytogenes precipitated the loss of tolerance and the MyD88‐ and T cell‐dependent rejection of accepted cardiac allografts in mice. This loss of tolerance was associated with increases in the numbers of graft‐infiltrating macrophages and dendritic cells, as well as CD4⁺FoxP3^? and CD8⁺ T cells. Rejection was also associated with increased numbers of graft‐infiltrating alloreactive as well as Listeria‐reactive IFNγ‐producing T cells. Rejection of the established grafts required both IL‐6 and IFNß, cytokines produced during acute Listeria infection. However, IL‐6 and IFNß alone, even when present at higher concentrations than during Listeria infection, were insufficient to break tolerance, while the combination of IL‐6 and IFNß was sufficient to break tolerance. These and in vitro observations that IL‐6 but not IFNß enhanced T cell proliferation while IFNß but not IL‐6 enhanced IFNγ production support a hypothesis that these cytokines play nonredundant roles. In conclusion, these studies demonstrate that the proinflammatory effects of infections can induce the loss of tolerance and acute rejection of accepted allografts.     

HLA‐G on peripheral blood CD4+ T lymphocytes: a potential predictor for acute renal rejection

HLA‐G Expression in grafts and serum has been shown to improve graft acceptance. However, its expression on peripheral blood lymphocytes (PBLs) during acute rejection (AR) remains unknown. In this study, we serially monitored HLA‐G expression on CD4⁺ and CD8⁺ PBLs of 66 recipients undergoing renal transplantation using flow cytometry at different time points before and after transplantation, as well as during AR episode. In stable recipients, HLA‐G expression on CD4⁺ PBLs declined during the first week after transplantation and increased continuously with immunosuppressive therapy. Then, expression declined gradually after 1 month and remained at a higher level compared with pretransplantation. When AR occurred, HLA‐G expression decreased significantly compared with the stable level. In three recipients suffering from recurrent rejection, it remained at a low level despite impact immunosuppressive treatment. With mix lymphocyte assay, HLA‐G⁺ CD4⁺ T cells showed inhibitory role on proliferation of peripheral blood mononuclear cell. HLA‐G expression on CD8⁺ PBLs was almost undetectable at different time points in the recipients and healthy controls. Our results suggest that HLA‐G on CD4⁺ PBLs might provide a potential marker for the early diagnosis of renal AR and for the immunosuppressive status of recipients.