Should IFN-γ, IL-17 and IL-2 be considered predictive biomarkers of acute rejection in liver and kidney transplant? Results of a multicentric study.

Acute rejection (AR) remains a major challenge in organ transplantation, and there is a need for predictive biomarkers. In the present multicenter study, we prospectively examined a series of biomarkers in liver and kidney recipients. Intracellular expression of IFN-γ, IL-17 and IL-2 and IL-17 soluble production were evaluated both pre-transplantation and post-transplantation (1st and 2nd week, 1st, 2nd and 3rd month). 142 transplant patients (63 liver/79 kidney) were included in the study. Twenty-eight recipients (14 liver/14 kidney) developed AR. Pre- and post-transplantation intracellular expression of %IFN-γ(+) in CD4(+)CD69(+) and in CD8(+)CD69(+) and soluble IL17 identified liver and kidney transplant patients at high risk of AR. Pre-transplantation, %IL-2(+) in CD8(+)CD69(+) also identified kidney patients at high risk. We constructed pre- and post-transplantation risk prediction models, based on a composite panel of biomarkers, which could provide the basis for future studies and will be a useful tool for the selection and adjustment of immunosuppressive treatments.

Effect of immunosuppressant blood levels on serum concentration of interleukin-17 and -23 in stable liver transplant recipients.

INTRODUCTION: T(H)17 cells have been recently described to be involved in inflammatory and immune-mediated diseases, but there is no evidence of their role in human liver transplantation. Interleukin (IL)-23 is considered an inducer cytokine, whereas IL-17 is the main cytokine released by T(H)17 cells. The aim of our study was to measure the serum levels of IL-17 and IL-23 in stable liver transplant recipients and examine the influence of immunosuppressant concentrations. MATERIALS AND METHODS: Serum levels of IL-23 and IL-17 were determined in 38 healthy subjects and 35 stable hepatic transplant recipients who were free of rejection episodes for at least 8 years. The results were analyzed according to the simultaneous blood levels of cyclosporine (n = 20) or tacrolimus (n = 15). RESULTS: No significant differences were observed in the serum levels of IL-17 and IL-23 between healthy subjects and transplanted patients. In addition, patients with low blood levels of tacrolimus (<6 ng/mL), but not cyclosporine, showed significantly lower serum levels of the 2 cytokines. CONCLUSION: These preliminary results suggested a lack of activation of the T(H)17 pathway, which was more pronounced among the patient subgroup treated with tacrolimus.

T(H)17 versus Treg cells in renal transplant candidates: effect of a previous transplant.

INTRODUCTION: The T(H)1 and T(H)2 cells were described several years ago. However, this dichotomy has been disrupted by the description of other CD4(+) T cell subsets: the proinflammatory interleukin (IL)-17-producing T cells (T(H)17) and regulatory T cells (Tregs). The latter group inhibits the immune responses driven by T(H)1, T(H)2, and T(H)17 cells. IL-6 is involved in T(H)17 development, down-regulating Treg differentiation. Our hypothesis suggested that an imbalance between T(H)17 and Tregs enhances immune responses among renal transplant patients. MATERIALS AND METHODS: We studied 26 end-stage renal disease (ESRD) subjects and 10 patients awaiting a second renal transplant after previous graft dysfunction. We assessed the number of CD4(+)CD25(+)Foxp3(+) cells and serum levels of IL-17, the prototypic interleukin of T(H)17 cells. RESULTS: We observed a lower number of CD4(+)CD25(+)Foxp3(+) T cells among patients with previous graft dysfunction than those with ESRD (median 3.37 vs 8.63 cells/mm(3), P = .008). In contrast, IL-17 serum levels were augmented in graft dysfunction (median 4.45 pg/mL) compared with ESRD patients (1.39 pg/mL, P = .036), suggesting a proinflammatory state in patients awaiting a second renal transplant. CONCLUSION: The emerging alloresponse from a previous transplant favors the generation of T(H)17 instead of Treg cells. The enhanced activity of T(H)17 cells in retransplanted patients may down-regulate Treg cells, producing a proinflammatory environment that favors rejection of the next transplant.

Changes in the expression of the immunoglobulin-like transcript 3 (ILT3) and ILT4 receptors in renal allograft recipients: effect of donor and recipient aging.

INTRODUCTION: The aim of the present study was to investigate the number and phenotype of pre- and posttransplant peripheral blood dendritic cells (DCs) in kidney graft recipients to correlate with CD4(+)CD25(high) Treg and CD8(+)CD28(-) cells. Data were analyzed according to the age of the donor-recipient pairs. MATERIALS AND METHODS: A cohort of 49 cadaveric kidney transplant recipients was prospectively studied pretransplant and 6 months posttransplant by three-color flow cytometry with specific monoclonal antibodies. Patients were subgrouped according to age (elderly were considered above 60 years old and young below 55 years old) in the following donor-recipient pairs: aged/aged, young/aged, aged/young, young/young. RESULTS: At 6 months posttransplant, the proportion of cells tended to increase when the donor was young, regardless of the recipient. Importantly, there was a significant correlation between the numbers of immunoglobulin-like transcript 4(+) DCs and CD4(+)CD25(high) Treg cells before transplantation (r = .476, P = .004) and at 6 months (r = .408, P = .013). A significant association was also observed between ILT4(+) DCs and CD8(+)CD28(-) pretransplant (r = .540, P = .001) and at 12 months posttransplant (r = .609, P = .012). CONCLUSIONS: Renal grafts from young but not from aged donors seem to induce DC of a tolerogenic phenotype, both in aged and young recipients. These preliminary results suggested that donor age may have consequences in terms of tolerance induction.

Association between serum soluble CD30 and serum creatinine before and after renal transplantation.

OBJECTIVE: There is increasing evidence that circulating levels of soluble CD30 (sCD30) may represent a biomarker for outcome in kidney transplantation. The aim of this study was to measure the pre- and posttransplantation serum levels of sCD30 in cadaveric kidney transplant recipients and correlate them with serum creatinine. PATIENTS AND METHODS: Serum sCD30 was measured by a commercial enzyme-linked immunosorbent assay (ELISA) from prospective samples of 38 kidney allograft recipients serially transplanted at our center. Samples were collected at day 0 pretransplantation and at months 6, 12, 18, and 24 posttransplantation. We also studied sera from 29 patients with chronic kidney disease (CKD) at different stages of the K/DOQI guidelines, as a control group. RESULTS: Serum levels of sCD30 decreased significantly in samples posttransplantation compared with pretransplantation. The significant decrease after transplantation may be related to the improvement in renal function since we observed a significant correlation between serum levels of sCD30 and creatinine (sCr) at all times of the study. In addition, the patients with chronic renal failure showed a significant association between serum sCD30 and sCr (r = .454; P = .013). CONCLUSIONS: Our results did not suggest that the measurement of sCD30 may be used as a valuable biomarker in renal transplantation. Increased levels may be related to a decrease in its renal elimination.

Reduced numbers of blood natural regulatory T cells in stable liver transplant recipients with high levels of calcineurin inhibitors.

INTRODUCTION: In solid organ transplantation, most efforts are directed to achieve a state of tolerance and reduce the immunosuppressive load. Since the liver is thought to be more tolerogenic than other grafts, we examined the impact of various immunosuppressant regimens on induction of CD4+CD25(high)FOXP3+ regulatory T cells (Tregs). MATERIALS AND METHODS: We divided 35 liver transplant recipients with stable function and free of rejection episodes for at least 8 years into two main groups according to the blood levels of calcineurin inhibitors (CNIs) at the time of the study: 15 patients showing high concentrations of either cyclosporine or FK506 (high CNI: cyclosporine >80 ng/mL or FK506 >6 ng/mL) and another 20 patients with low levels (low CNI). Thirty-eight age-matched healthy subjects were used as normal controls. RESULTS: Circulating T cells of a regulatory phenotype (CD4+CD25(high)FoxP3+) were decreased in peripheral blood of liver transplant recipients compared with healthy donors. Importantly, those patients with high CNI demonstrated significantly lower levels of Tregs compared with those with low CNI. Furthermore, low CNI recipients showed no significant difference from healthy donors. CONCLUSIONS: A high load of immunosuppression may hamper the induction of tolerance in liver transplantation through interference with induction of Tregs in stable liver transplant recipients. Thus, quantification of blood Tregs may help to decide whether to lower immunosuppression.

Calcineurin inhibitors affect circulating regulatory T cells in stable renal transplant recipients.

INTRODUCTION: Immunosuppression, although crucial for short-term management, has been described in renal transplantation to be a major hurdle for long-term graft survival. Efforts have been directed at achieving a true state of allotolerance, thereby reducing the load of immunosuppression. Recently, increased frequencies of CD4(+)CD25(high) regulatory T cells (Tregs) have been described as an additional mechanism to induce alloimmune tolerance. MATERIALS AND METHODS: We assessed 64 renal transplant recipients with stable renal function for at least 1 year, divided into two groups: one composed of patients receiving rapamycin but not calcineurin inhibitors (CNIs), and another, of those receiving CNIs but not rapamycin. RESULTS: We demonstrated that T cells with a regulatory phenotype were decreased in peripheral blood of renal transplant recipients under CNI therapy compared to those who were CNI-free. The Tregs in our patients showed a modest association with renal function as measured by the delta serum creatinine, which was not significant. CONCLUSIONS: CNIs, but not rapamycin, reduce the frequencies of circulating Tregs in renal transplant recipients. The use of rapamycin might be further exploited in strategies reducing immunosuppression in renal transplantation. Furthermore, quantification of blood Tregs may be a suitable tool to identify those recipients who are candidates for reducing immunosuppression.

[Autoimmune pancreatitis. An infrequent or underdiagnosed entity? Pathological, clinical and immunological characteristics]. / Pancreatitis autoinmune. 'Una entidad poco frecuente o infradiagnosticada? Características anatomopatológicas, clínicas e inmunológicas.

In the last few years, reports of pancreatic inflammatory diseases caused by immunological mechanisms and with good response to steroid treatment have increased. Although this entity has been known by a number of names, at present the most widely accepted is that of autoimmune chronic pancreatitis (ACP). The present report describes the clinical, immunological, morphological, functional and pathological characteristics of two patients recently studied at our unit and discusses currently used diagnostic tests. The two patients had a complete response to steroid therapy. In our opinion, ACP is probably underdiagnosed in Spain. The availability of morphological, pathological and serological diagnostic tools developed in recent years will help to precisely determine the epidemiology of this process. Thus, quantification of serum levels of anti-carbonic anhydrase II and IgG4 has greatly contributed to the diagnosis of ACP. These tests should be performed in patients with a possible diagnosis of ACP, those suffering from diabetes mellitus type I with impairment of exocrine function, and those with alcoholic pancreatitis and a poor response to alcohol elimination. Once we are able to diagnose and determine the real prevalence of ACP in our setting, the most appropriate therapy and prognosis of this disease can be established.

Antibody-Mediated Rejection in Kidney Transplantation Without Evidence of Anti-HLA Antibodies?

INTRODUCTION: The definition of antibody-mediated rejection (AMR) is based on serologic (presence and/or development of donor-specific anti-HLA antibodies [DSAs]) and histologic (C4d deposition and endothelial damage) criteria. However, several cases of AMR have been described without C4d deposition, and other cases of histologic AMR without DSAs, which could be driven by other non-HLA alloantibodies such as anti-MICA or anti-angiotensin II type I receptor (AT1R). Here we studied clinical and histologic humoral rejection in kidney transplant recipients without evidence of anti-HLA antibodies. MATERIALS AND METHODS: Fifteen kidney transplant recipients with AMR defined as C4d+ and/or histologic g+ptc without anti-HLA antibodies in screening test were studied. Sera at the moment of biopsy and 2 months earlier were studied for anti-HLA antibodies by Luminex, in neat, diluted 1/160, and sera after treatment with dithiothreitol (DTT) and confirmed by single-antigen test. The anti-AT1R was measured by enzyme-linked immunosorbent assay. RESULTS: A lack of anti-HLA and MICA antibodies was confirmed after anti-HLA screening test in all conditions (neat, diluted, and DTT-treated) and de novo development of AT1R antibodies was ruled out. Nevertheless, after single-antigen test, 3 patients were identified with a weak reaction against class I antigen and another 4 patients against class II antigen. Due to the lack of locus-C typing in the donors, the DSA assignment cannot be confirmed, whereas anti-HLA class II antigens were DSA. CONCLUSIONS: A low sensitivity in the screening of anti-HLA antibody testing was observed. Our results suggest performing single-antigen test in seronegative patients with clinical humoral rejection after screening to confirm the presence of DSA.

B-Cell-Activating Factor Levels Are Associated With Antibody-Mediated Histological Damage in Kidney Transplantation.

INTRODUCTION: Along with death engraftment, in recent years, antibody-mediated damage has been identified as the leading cause of loss of kidney transplants. Despite the recognition of the role of the B-lymphocyte subpopulation in the development of both tolerance and rejection, little is known about the trigger mechanisms and effectors of this humoral response. BACKGROUND: We analyzed the relationship between B lymphocyte subpopulations and levels of B-cell-activating factor (BAFF) with the histological findings in biopsies of renal transplantation. MATERIAL AND METHODS: We selected 35 patients whose kidney transplant biopsy was performed between January and November 2015. The biopsy specimens were classified according to Banff criteria. At the moment of the biopsy BAFF levels and B-lymphocyte subpopulations in blood were measured using enzyme-linked immunosorbent assay (ELISA) and using flow cytometry, respectively. RESULTS: Mean BAFF levels were 493 ± 245 pg/mL. The median performance of biopsy post-transplantation was 12.9 (11.7-23.9) months. BAFF levels correlated with pretransplantation antibodies (r = 0.523; P = .002) but not with kidney function. In biopsies performed more than 1 year after transplantation BAFF levels correlated with the severity of chronic glomerular (cg) involvement (r = 0.625; P = .003). Histological variables related to antibody-mediated injury selected by principal component analysis (glomerulitis, peritubular capillary, and chronic glomerulopathy) related to BAFF levels (B factor, 116; 95% confidence interval [CI], 12-220; P = .029). Biopsy specimens with transplant glomerulopathy (TG) showed lower levels of circulating naive CD19 + subpopulation, IgD+, and CD27- (32.7 ± 28.1 vs 87.9 ± 79.1; P = .017) compared with biopsy specimens without TG. CONCLUSIONS: Elevated levels of BAFF are associated with increased presence and severity of TG and a set of variables related to antibody-mediated histological damage. TG is associated with changes in circulating B-lymphocyte subpopulations that could contribute to its pathogenesis.

Serum Levels of Interleukin-34 During Acute Rejection in Liver Transplantation.

INTRODUCTION: Accumulating evidence indicates that interleukin (IL)-34 participates in T-cell homeostasis and tolerance due to the ability of IL-34 to trigger apoptosis of Th1, Th17, and Tc1 cells, but spare Th2 cells and Treg. In addition, IL-34 exerts anti-inflammatory effects by impairing leukocyte adhesion and transendothelial migration, and reducing the secretion of proinflammatory cytokines. The aim of our study was to investigate the time course of serum levels of IL-34 during hepatic allograft rejection. METHODS: Serum levels of IL-34 were determined in 20 healthy subjects and 45 hepatic transplant recipients. These patients were divided into 2 groups: group I was composed of 15 patients with acute rejection, and group II was composed of 30 patients without acute rejection. Samples were collected on days 1 and 7 after liver transplantation and on the day of liver biopsy. RESULTS: The concentrations of IL-34 were higher in the rejection group vs nonrejection group during the entire postoperative period. The whole transplant group, including those with stable graft function, showed higher IL-34 serum levels than the controls at all times after liver transplantation. CONCLUSIONS: Our preliminary results could be related to the recent finding that IL-34 may play an immune-suppressive role in liver transplantation. In our case, although we must be cautious with serum data, increased IL-34 would help to control alloresponse during rejection and protect from graft lost.

De Novo Donor-Specific Anti-Human Leukocyte Antigen Antibody Detection in Long-Term Adult Liver Transplantation.

INTRODUCTION: Information about the consequences of de novo donor-specific anti-human leukocyte antigen (DSA) antibody development in the long term after adult liver transplantation (LT) is scarce. We conducted a cross-sectional study in LT patients with a follow-up of at least 6 years. METHODS: A total of 28 adult LT patients were included, with a median follow-up of 77 months (range, 63 to 96) and without preformed anti- human leukocyte antigen (HLA) antibodies prior to LT. The anti-HLA identification was performed with LABScreen Single Antigen, whereas the ability to fix the complement was demonstrated with C1q test (One Lambda). In both assays, a value >3.500 mean fluorescence intensity (MFI) was considered positive. The anti-HLA antibody specificities were compared with donor HLA antigens to confirm them as DSA. Hepatic fibrosis was assessed by transient elastography. RESULTS: In 5 patients (17.8%), de novo DSA were detected, all them against DQ locus. In all of these cases (100%) the complement fixation was confirmed by C1q binding. The grade of hepatic fibrosis in de novo DSA patients was significantly higher compared with No-DSA patients (13.2 ± 9.2 KPa vs 7.3 ± 3.7 KPa; P = .02). It is noteworthy that in both groups of patients the levels of liver function tests (LFT) at the time of the study were normal or near the normal range with no difference between patients with or without de novo DSA. CONCLUSIONS: Our preliminary results are consistent with those previously demonstrated in pediatric LT, where de novo DSA production and humoral response could contribute to the liver fibrosis observed in the long term after LT in pediatric patients with normal or near-normal LFT.

Impact of HLA antibodies on transplant glomerulopathy.

The influence of humoral rejection on the development of chronic allograft nephropathy (CAN) is controversial, especially in relation to transplant glomerulopathy. The aim of our study was to analyse the influence of anti-HLA antibodies on the development of transplant glomerulopathy (cg0, cg1, cg2, and cg3; Banff'97). We selected all renal transplants patients from 1975 to 2003 who had a functioning graft for at least 6 months and a clinically indicated graft biopsy with CAN and chronic glomerular changes (case group). We studied the presence of anti-HLA antibodies (Ab) in the last serum taken while the graft was functioning and divided them into three groups according to the severity of glomerular lesions. We also selected 52 contemporary and comparable cases without transplant glomerulopathy (control group). A total of 77 case had transplant glomerulopathy: 39 cg1, 29 cg2, and 9 cg3. Pretransplant Ab titers and number of previous blood transfusions were higher among the subgroup with the most severe glomerulopathy. Patients who developed posttransplant anti-HLA Ab more frequently showed transplant glomerulopathy. Serum creatinine and proteinuria were higher among cases with chronic glomerulopathy, and more grafts were lost in that group. Thus, the presence of HLA-Ab is a key factor in the development of transplant glomerulopathy and chronic allograft rejection.

Frequencies of circulating B-cell subpopulations before kidney transplantation identify patients at risk of acute rejection.

The response mediated by B lymphocytes has a crucial impact on kidney transplantation due to the role of anti-human leukocyte antigen antibodies in rejection and the contradictory observation of high B-lymphocyte numbers in tolerant kidney transplant recipients. The basis of the contradiction could lay in the different function of B-cell subsets depending on their degree of differentiation. We ought to measure circulating B-lymphocyte percentages in patients with end-stage renal disease before kidney transplantation to identify those with a high risk of acute rejection. Eighty patients on the waiting list for kidney transplantation followed up in our center were recruited from 2010, and samples were taken just before kidney transplantation. Eleven of 80 patients presented an episode of acute rejection (13.75%) and had an increased frequency of switched (SW) B cells compared with the rejection-free group (median [interquartile range] 24.5% [18.6% to 39.6%] vs 15.1 [8.45% to 23.4%]; P = .025). Subsequently, the frequency of SW B cells was assessed as a predicting factor of acute rejection. A value higher than 18.4% predicted patients at risk of suffering an acute rejection episode with a sensitivity of 81.8% and a specificity of 60.9% and an area under the curve of 71.2%. Moreover, a decrease in naïve B-cell subsets was related to patients at risk of acute rejection. The percentage of circulating B-cell subsets before kidney transplantation could be used as biomarker of risk to suffer acute rejection. These unicenter data must be validated in multicenter studies.

Galectin-1 in stable liver transplant recipients.

INTRODUCTION: The achievement of a state of tolerance and minimization of the immunosuppressive load form part of the "Holy Grail" in solid organ transplantation. Galectin-1 recently has been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of galectin-1 in stable liver transplant recipients. METHODS: Serum levels of galectin-1 were determined in 30 stable liver transplant recipients who had been free of rejection episodes for at least 8 years. Fifteen patients with an acute rejection episode and 34 healthy subjects were used as the control group. RESULTS: The concentrations of galectin-1 were significantly higher in stable liver transplant recipients compared with healthy subjects and with the acute rejection group. CONCLUSIONS: These preliminary results indicate that galectin-1 is upregulated in stable liver transplant recipients. Thus, our results extend the recent findings that galectin-1 may play an immune-suppressive role in liver transplantation. It remains to be established whether it might help to induce tolerance in liver transplantation.

Activated Regulatory T Cells Expressing CD4(+)CD25(high)CD45RO(+)CD62L(+) Biomarkers Could Be a Risk Factor in Liver Allograft Rejection.

Activated regulatory T cells (aTregs) are nowadays a hot topic in organ transplantation to establish their role during acute rejection (AR) episodes. The aim of this multi-center study was to monitor the frequency of aTregs within the first year after transplantation in a cohort of first-time liver transplant recipients enrolled from 2010 to 2012. aTregs frequency was analyzed by means of flow cytometry. Patients who had AR showed higher levels of aTregs during first year after transplantation in comparison with patients who did not have higher levels. High levels of aTregs in liver recipients might be used as a biomarker of AR; however, further studies must be done to address the potential role of aTregs as biomarkers of AR in liver transplantation.

[The effect of changes in nutritional status induced by age on oxidative liver metabolism]. / Influencia de los cambios en el estado nutritivo inducidos por la edad sobre el metabolismo oxidativo hepático.

INTRODUCTION: The hepatic oxidative metabolism is essential for the biotransformation of a large number substances, among which are found many drugs which are commonly used in clinical practice. The nutritional status of individuals has been shown to be of influence on this function. Aging produces a deterioration of the hepatic oxidative metabolism, without the cause for this situation having been clarified. Also, aging modifies the body composition of the individuals. The objective of this study is to evaluate whether the modifications which arise in the nutritive status due to age, can alter the hepatic oxidative capacity. MATERIAL AND METHODS: 165 elderly people of both sexes were studied, with an average age of 82 years, and 24 young people, with an average age of 29 years. All participants were subjected to a clinical questionnaire, along with an evaluation of anthropometric, biochemical, and immunological nutritive parameters. The study of the oxidative metabolism was conducted by evaluating the kinetics of antipyrine. RESULTS: The elderly people showed a decrease in the antipyrine clearance rate (Ap Cl) (P < 0.001), and a lengthening of their life-span (P < 0.05) with respect to the younger people. There was a significant correlation in the elderly people, between the Ap Cl and age, weight, size, the distribution volume, and the muscular area of the arm. A multiple regression analysis showed a predictive value which was independent for age, the AST, the lymphocytes, and size. CONCLUSION: Elderly people have a marked depression of the hepatic oxidative metabolism. The factors which participate in their nutritional situation, are of influence on this function. It is necessary to keep all the above in mind when it comes to prescribing drugs which require this type of biotransformation, in order to avoid adverse effects or drug interactions.

High regulatory T-cell levels at 1 year posttransplantation predict long-term graft survival among kidney transplant recipients.

INTRODUCTION: Regulatory T cells (Tregs) have gained an important role in mechanisms of tolerance and protection against the transplant rejection. However, only limited retrospective data have shown a relationship between peripheral blood Tregs and better long-term graft survival. The purpose of the present study was to investigate prospectively circulating Treg levels and their association with long-term graft survival. METHODS: Ninety kidney transplant recipients underwent measurement of Treg levels in peripheral blood before as well as at 6 months and 1 year posttransplantation. Receiver operating characteristic curves were applied to test the sensitivity and specificity of Treg levels to predict prognosis. RESULTS: Treg levels before transplantation correlated with those at 6 months and 12 months posttransplantation (P < .001 and P = .002, respectively). Patients who maintained high Treg levels (above 70th percentile) at both 6 and 12 months displayed better long-term graft survival at 4 and 5 years follow-up (P = .04 and P = .043 respectively). There was no effect on patient survival. CONCLUSION: Detection of high levels of peripheral blood Tregs was associated with better graft survival possibly using as a potential marker of prognosis.

Prospective study of biomarkers of immune response in lung transplant recipients.

INTRODUCTION: Studies on biomarkers of tolerance in organ transplantation have been widely performed during the last decade. AIM: To assess biomarkers in relation to evolution of the immune response among lung transplant recipients. METHODS: This multicenter study included 27 lung transplant recipients followed before as well as at 7, 14, 30, 60, 90, and 180 days posttransplantation. Biomarkers of the immune response based on flow cytometry technology were validated in each center. They included intracellular cytokine expression, regulatory T-cell level, as well as lymphocyte surface antigen and CD28 expressions. RESULTS: The 13 patients who developed acute rejection episodes showed increased numbers of regulatory T cells at 12 months posttransplant. Sixteen patients experiencing infections displayed decreased expression of CD69 on CD8 T cells within the first year of follow-up. CONCLUSION: High Treg levels in the peripheral blood of lung transplant recipients were associated with an increased risk of rejection but not infection. Inversely, we observed low levels of activated CD8 T cells in infected patients.