Francisco Boix

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.

HLA-DRB1 and HLA–DQB1 genes on susceptibility to and protection from allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease that affects both patients with cystic fibrosis (CF) and those with asthma. HLA‐DRB1 alleles have previously been associated with ABPA–CF susceptibility; however, HLA‐DQB1 allele associations have not been clearly established. The aim of the present study was to investigate HLA class II associations in patients with ABPA–CF and determine their roles in susceptibility or protection. Patients with ABPA–CF, patients with CF without ABPA, patients with asthma without ABPA (AST), and healthy controls were included in this study. DNA was extracted by automatic extractor. HLA‐DRB1 and ‐DQB1 genotyping was performed by the Luminex PCR‐SSOP method (One Lambda, Canoga Park, CA, USA). Allele specific PCR‐SSP was also performed by high‐resolution analysis (One Lambda). Statistical analysis was performed with SSPS and Arlequin software. Both HLA‐DRB1*5:01 and ‐DRB1*11:04 alleles occurred with greater frequency in patients with ABPA–CF than in those with AST and CF and control subjects, corroborating previously published data. On the other hand, analysis of haplotypes revealed that almost all patients with ABPA–CF lacking DRB1*15:01 or DRB1*11:04 carry either DRB1*04, DRB1*11:01, or DRB1*07:01 alleles. In the HLA‐DQB1 region, the HLA‐DQB1*06:02 allele occurred more frequently in patients with ABPA–CF than in those with AST and CF and healthy controls, whereas HLA‐DQB1*02:01 occurred less frequently in patients with ABPA–CF. These data confirm that there is a correlation between HLA‐DRB1*15:01, –DRB1*11:04, DRB1*11:01, –DRB1*04 and –DRB1*07:01 alleles and ABPA–CF susceptibility and suggest that HLA‐DQB1*02:01 is an ABPA–CF resistance allele.

Influence of human leukocyte antigen mismatching on rejection development and allograft survival in liver transplantation: is the relevance of HLA-A locus matching being underestimated?

The influence of HLA matching on liver transplant is still controversial, as studies have failed to demonstrate an adverse effect of HLA mismatching on transplant outcome. We examined the effect of HLA mismatching on transplant outcome in a series of 342 consecutive liver transplants (224 finally analyzed). HLA typing was performed by serological and molecular methods. HLA-A matching was associated with an increased chronic rejection incidence (P=0.04). Indeed, HLA-A match also demonstrated a significant impact on allograft survival (P=0.03), confirming previous observation concerning to rejection, as complete HLA-A mismatching favored a better liver transplant outcome. Analysis of HLA-A+B+DR matching also demonstrated a significant impact on graft survival (P<0.05). Multivariate Cox regression analysis confirmed the effect of HLA-A and DPB1 matching as independent risk factors for graft loss. Another independent factor was a positive pre-transplant crossmatch. In conclusion, liver transplant outcome has not been found to be improved by HLA matching, however a poorer HLA compatibility favored a better graft survival and decreased rejection incidence, with a special relevance for HLA-A matching.

HLA-C antibodies are associated with irreversible rejection in kidney transplantation: Shared molecular eplets characterization

We report an interesting case concerning an irreversible antibody-mediated rejection (AMR), associated with anti-HLA-C DSA, which occurred after a second kidney transplantation despite having determined a low number of antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was then considered to be an indicator of low risk of AMR). A 63-year-old woman was re-transplanted with pre-transplant (PrT) sensitization. On day 7 post-transplantation, oligoanuria occurred and increased MFIs for the detected PrT antibodies and other antibodies (non-detected or detected with very low PrT MFI) were observed. SAB assay also showed antibodies against the second donor HLA-C mismatches and other HLA-C antigens. Nephrologists suspected AMR and the patient was therefore treated with methylprednisolone/plasmapheresis/IVIG/anti-CD20 without improvement, which led to transplantectomy. Histologic analysis confirmed acute AMR. Interestingly, it was possible to define exactly the potential immunizing epitopes whose recognition determines the specific antibody production. So, 1st donor DSAs (detected PrT with low MFI), 2nd donor DSAs (detected PTP), and non-DSA detected PTP have several shared eplets, being the 11AVR eplet the only one present on all alleles. Thus, the recognition of 11AVR eplet in the first transplant modeled the patients antibody response. Therefore, we propose that donor HLA-C typing should always be performed for recipients with anti-HLA-C antibodies, and specific shared-eplets should be investigated in order to determine previous transplant mismatches.

Activated Regulatory T Cells Expressing CD4+CD25highCD45RO+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.

CD28 biomarker quantification and expression level profiles in CD4+ T-lymphocytes in solid organ transplantation☆

The introduction of anti-calcineurin-based therapies has led to an increase in the one-year survival as well as graft function rates in patients undergoing solid organ transplantation (SOT). Nonetheless, early cellular acute rejection (EAR) incidence still remains a major challenge that irrevocably heads to poor outcomes. The mechanisms underlying CD4 T cell activation in SOT are still under research. In this sense, CD28 co-stimulatory molecule plays a pivotal role triggering CD4 T cell activation as well as survival maintenance. Previous own studies stated the role that CD4+CD28+ circulating T lymphocytes plays before and during EAR episodes. We assessed the percentage as well as the absolute number of CD28 molecules on CD4+ T cells as predictive surrogate biomarker of EAR in a prospective cohort of liver and kidney transplant recipients. Quantitative analysis of CD28 was carried out on whole peripheral blood samples by flow cytometry. Decreased pre-transplant expression of CD28 was associated with EAR in both study groups. Furthermore, the expression of CD28 within the rejected group, experimented an up-regulation upon transplantation. These preliminary results suggest that patients undergoing liver or kidney transplant can be stratified at high risk of EAR according to their CD28 molecule expression on peripheral CD4+ T lymphocytes.

Cytokine Expression Profile as Predictive Surrogate Biomarkers for Clinical Events in the Field of Solid Organ Transplantation

The morbidity and mortality after solid organ transplantation leads to poor outcomes in the long-term graft survival. There are many sources increasing bad outcomes within the post-transplant period reducing the quality of recipient´s life, such as rejection episodes, opportunistic infections as well as immunosuppression related morbidity. A complete understanding on the immune system responses against the allo-graft remains unknown. Recently, some pro-inflammatory cytokines, such as IFN-γ and IL-17, as well as IL-2, have been proposed as surrogate biomarkers able to predict the appearance of clinical event episodes. In this review we summarize the latest findings regarding the immune function monitoring in solid organ transplantation as well as the most common techniques (ELISPOT, ELISA and Flow Cytometry) that have been widely used across different clinical laboratories.

Killer immunoglobulin-like receptor (KIR) repertoire analysis in a Caucasian Spanish population with inflammatory bowel diseases

Immunological molecules are implicated in inflammatory disorders, including inflammatory bowel disease (IBD; Crohn disease [CD] and ulcerative colitis [UC]). Killer cell immunoglobulin‐like receptors (KIRs) are also genetically variable proteins involved in immune function. They are expressed by NK cells and certain T lymphocytes, regulate specificity and function by interaction with HLA Class I molecules, may be either inhibitory or activating and are polymorphic both in terms of alleles and haplotype gene content. Genetic associations between activating KIRs and certain autoimmune and inflammatory diseases have been reported; however, a possible association between KIR and IBD remains unclear. The aim of this study was to determine the relationship between KIR repertoire and IBD pathologies in a Spanish cohort. KIR variability was analyzed using PCR–sequence specific oligonucleotide probes (SSOP). Inhibitory KIR2DL5 was found more frequently in UC and IBD patient groups than in healthy controls (P = 0.028 and P = 0.01, respectively), as was activating KIR2DS1 (P = 0.02, Pc > 0.05, UC vs. Controls; P = 0.001, Pc = 0.01, IBD vs Controls; P = 0.01, Pc > 0.05, Controls vs CR), KIR2DS5 (P = 0.0028, Pc = 0.04, Controls vs UC; P = 0.0001, Pc = 0.0017, Controls vs IBD; P = 0.01, Pc > 0.05, Controls vs CD) and KIR3DS1 (P = 0.012, Pc > 0.05, Controls vs IBD). Our data suggest that imbalance between activating and inhibitory KIR may partially explain the different pathogeneses of these IBDs and that there is a hypothetical role for the telomeric B region (which contains both KIR2DS5 and KIR2DS1) in these diseases.

Killer immunoglobulin-like receptor repertoire analysis in a Caucasian Spanish cohort with inflammatory bowel disease: KIR repertoire analysis and IBD

Immunological molecules are implicated in inflammatory disorders, including inflammatory bowel disease (IBD; Crohn disease [CD] and ulcerative colitis [UC]). Killer cell immunoglobulin‐like receptors (KIRs) are also genetically variable proteins involved in immune function. They are expressed by NK cells and certain T lymphocytes, regulate specificity and function by interaction with HLA Class I molecules, may be either inhibitory or activating and are polymorphic both in terms of alleles and haplotype gene content. Genetic associations between activating KIRs and certain autoimmune and inflammatory diseases have been reported; however, a possible association between KIR and IBD remains unclear. The aim of this study was to determine the relationship between KIR repertoire and IBD pathologies in a Spanish cohort. KIR variability was analyzed using PCR–sequence specific oligonucleotide probes (SSOP). Inhibitory KIR2DL5 was found more frequently in UC and IBD patient groups than in healthy controls (P = 0.028 and P = 0.01, respectively), as was activating KIR2DS1 (P = 0.02, Pc > 0.05, UC vs. Controls; P = 0.001, Pc = 0.01, IBD vs Controls; P = 0.01, Pc > 0.05, Controls vs CR), KIR2DS5 (P = 0.0028, Pc = 0.04, Controls vs UC; P = 0.0001, Pc = 0.0017, Controls vs IBD; P = 0.01, Pc > 0.05, Controls vs CD) and KIR3DS1 (P = 0.012, Pc > 0.05, Controls vs IBD). Our data suggest that imbalance between activating and inhibitory KIR may partially explain the different pathogeneses of these IBDs and that there is a hypothetical role for the telomeric B region (which contains both KIR2DS5 and KIR2DS1) in these diseases.

High expression of CD38, CD69, CD95 and CD154 biomarkers in cultured peripheral T lymphocytes correlates with an increased risk of acute rejection in liver allograft recipients.

The mayor goal still outstanding into the solid organ transplantation field involves the search of surrogate biomarkers able to predict several clinical events, such as acute rejection (AR) or opportunistic infection. In the present multicenter study, a series of interesting surface antigens with important activator or inhibitory immune functions on cultured peripheral T cells were monitored in liver transplant recipients drawn at baseline and up to one year after transplantation. Sixty-four patients were included in the multicenter study during 3 years. Pre- and post-transplantation surface antigens levels displayed significant differences between AR and non acute rejection (NAR) groups, and also this differential expression was used to construct a risk predictive model based on a composite panel of outcome biomarkers (CD38, CD69, CD95 and CD154). The model was able to stratify these patients at high risk of AR. These preliminary results could provide basic information to improve the immunosuppressive treatment and it might better help to predict AR episodes.