Heparinized blood was used to obtain peripheral blood mononuclear cells (PBMC). PBMC were isolated by means of density gradient

centrifugation, and freshly isolated PBMCs were used for analysis of Tregs by flow cytometry. Routine blood samples included full leucocyte counts, alanine transaminase (ALT), HCV-RNA, anti-HCV antibodies, HCV genotype and IL-28B genotyping. Genotyping for the genetic polymorphism near the IL-28B gene (encoding IFN-λ), rs12979860 [34], was performed by allel discrimination with Taq-man 7900HT sequence Vismodegib mouse detection system (Source BioScience LifeSciences, Nottingham, UK). Flow cytometry.  For the determination of chronic-activated (CD38+ HLA-DR+) T cells and Th17 cells (CD3+ CD4+ CD161+), 100 μl of EDTA blood was incubated with 50 μl of fluorescent dye–conjugated monoclonal antibodies at room temperature for 15 min. Erythrocytes were lysed with 2 ml of Lysing Solution [Becton Dickinson

(BD), Franklin Lakes, NJ, USA] at room temperature for 20 min, and the samples were washed and resuspended in FACS flow (BD). Tregs (CD4+ CD25+ CD127lowFoxp3+ and CD8+ CD25+ Foxp3+) and CD4+ Treg subpopulations (resting Tregs CD45RA+Foxp3low, activated Tregs CD45RA−Foxp3high and non-suppressive Tregs CD45RA−Foxp3low) were determined by incubation with relevant PBMC surface marker antibodies for 20 min, followed by fixation and

permeabilization (Human Foxp3 Buffer Set; BD), and incubation with antibodies against intracellular Foxp3 (30 min). Gating strategy is shown in Fig. 1. Monoclonal antibodies used to determine high throughput screening assay lymphocyte subsets were isotype control IgG1/IgG2a Phycoerythrin (PE), IgG1 peridinin chlorophyll proteins – cyanine (PerCP-Cy5.5), IgG1/IgM fluorescein isothiocyanate (FITC), IgG1/IgG2b Allophycocyanin (APC), IgG1 PE-Cy7, Progesterone IgG1 APC-H7, CD161-PE, Foxp3-PE, CD8-PerCP-Cy5.5, CD25- PerCP-Cy5.5, CD3-FITC, CD127-FITC, HLA-DR-FITC, CD38-PE-Cy7 and CD4-APC-H7, all purchased from BD. Six-colour acquisition was performed using a FACS Canto, and data were processed using facs diva software (BD). For each sample, a minimum of 50,000 cells were acquired. Frequencies of activated T cells and Tregs are given as the frequency (%) of the cell population concerned (CD4+ cells or CD8+ cells), and frequencies of CD4+ Tregs subpopulation are given as the frequency (%) of CD4+ Tregs. Cytokines.  Samples were prepared by stimulating with phytohaemagglutinin (PHA). In brief, 0.4 ml full blood were cultured in 1.6 ml RPMI 1640 and 40 μl PHA (1 μg/μl) and incubated at 37 °C for 24 h after which the supernatant was isolated by centrifugation and stored at −80 °C until use. Interleukin-10 (IL-10), IL-17, TNF-α and TGF-β were measured by a bead-based multiplex sandwich immunoassay [35].

Comparison of WT and CD37−/− DC migration 18–20 h after oxazolone treatment revealed significant reductions in migratory function Cisplatin ic50 and random migration in CD37−/− DCs (see Oxa, Fig. 5A–C). This is further illustrated by comparison of the XY-displacement tracks of DC migration in WT and CD37−/− mice, which show extensive paths of migration in WT mice, in contrast to minimal responses in CD37−/− mice (Fig. 5D).

In addition, a significant proportion of CD37−/− DCs were less motile displaying an increased frequency of cells with <5 μm displacement (Fig. 5E). Videos showing this impaired in vivo directional migration of CD37−/− DCs compared with that of WT controls are included in the Supporting Information (Supporting Information Fig. 3 and 4). Taken together, Figure 4 and 5 demonstrate that CD37 ablation induces a significant impairment in DC migration. Tetraspanins molecularly associate with integrins and regulate outside-in signaling and cytoskeletal rearrangement as evidenced by impaired adhesion strengthening under flow and cell spreading observed in tetraspanin-deficient cells [27-31]. To test if CD37 plays a similar role in DCs, we first measured DC adhesion to ECM substrates under low shear flow conditions. WT DCs adhered efficiently to fibronectin, but poorly

to laminin and collagen (Fig. 6A). However, despite normal expression of the fibronectin receptors CD49d and CD49e integrins (Fig. 6B), the EPZ-6438 purchase absence of CD37 resulted in significantly Celecoxib reduced BMDC fibronectin adhesion (Fig. 6A). Cell spreading upon adhesion and membrane protrusion formation are dependent on cytoskeletal rearrangement driven by actin polymerization. To assess the role of CD37 in these processes, activated BMDCs were allowed to adhere and spread on fibronectin. Actin-dependent cell spreading was visualized by Phalloidin staining (Fig. 6C and F), bright field imaging (Fig. 6F), and scanning electron microscopy (SEM) (Fig. 6G). The percentage of cells with membrane

protrusions and the area of adhered cells were quantitatively determined (Fig. 6D and E). While WT DC readily spread, formed membrane protrusions and showed a classical dendritic morphology, CD37−/− DCs had a smaller rounded morphology with a relative absence of protrusive membranes (Fig. 6C–G). We conclude that CD37 is essential for cytoskeletal-dependent processes such as adhesion under flow, cell spreading upon adhesion, and the formation of membrane protrusions. CD37−/− mice display poor adaptive cellular responses to live tumors, irradiated tumors, and soluble antigens (Fig. 1 and 2). These findings are difficult to reconcile with exaggerated T-cell proliferative [14] and DC antigen-presenting phenotypes [15] observed when examining CD37-deficient cells in vitro.

The differences in the complexity of the CD8+ T-cell response or the influence

of background genes (e.g. extent of IFN-γ production) may account for the results. Using LCMV infection of naïve C57BL-6-PKO mice Lykens et al. recently showed that heightened antigenic stimulation is responsible for exaggerated T-cell activation [[49]]. They suggested that perforin-dependent cytotoxicity, in addition to promoting viral clearance, regulates T-cell activation by modulating Ag presentation [[49]]. Despite the differences in susceptibility of naïve BALB/c and C57BL/6 PKO mice to LCMV infection, we also observed massive CD8+ T-cell expansion and accelerated LCMV-induced mortality in GP33-vaccinated compared with naïve C57BL/6-PKO Selleck Fludarabine mice (data not shown). Thus, the vaccine-induced sensitization to mortality associated with PKO memory CD8+ T cells after LCMV infection is not restricted to BALB/c background. In addition, functional exhaustion of antigen-specific CD8+ T cells is not always associated with chronic infection [[50, 51]]. Chronic infection may be pathogen or host specific and it does not necessarily lead to Ag-specific CD8+ T-cell

exhaustion in all the cases. Although we observed lesser degree of “exhaustion” as characterized by TNF and PD-1 expression in GP283-specific CD8+ T cells compared with NP118-specific CD8+ T cells, viral control was not achieved in the absence of perforin in both cases (Fig. 5). In the absence of perforin, the phenotype of GP283-specific CD8+ T cells appeared “less exhausted” at the time we analyzed them could reflect the Selumetinib extent that these cells can regulate cytokine production. In addition, it remained to be elucidated whether encounter with antigen is similar between the NP118- and GP283-specific memory CD8+ T

cells in the PKO mice, not just initially, but throughout the infection course. Previous studies using different models of infection showed that protective immunity mediated by pathogen-specific CD8+ T cells did not correlate with immunodominance hierarchies after infection [[36, 37]]. Based on the results with PKO mice vaccinated with dominant NP118 epitope, we expected that massive antigen-specific memory CD8+ T-cell expansion contributed to the LCMV-induced mortality independent of epitope specificity. Interestingly, Sodium butyrate PKO mice vaccinated with subdominant GP283 epitope survived the LCMV infection even though they contained similar starting memory CD8+ T-cell numbers and underwent similar expansion in numbers as NP118-specfic CD8+ T cells. These results suggested that epitope specificity dictates the LCMV-induced mortality in vaccinated PKO mice. Furthermore, we also observed less cytokine dysregulation, in particular IFN-γ, by GP283-specific CD8+ T cells following LCMV infection. It is unclear which specific parameter(s) influence the cytokine profile of these GP283-specific CD8+ T cells and subsequent vaccine-induced mortality in PKO mice.

Methods:  Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction. Results: 

Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became Midostaurin cost evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration. Conclusion:  HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation. “
“Kidney disease develops to renal failure over a period of days, months or years, hence, clinical markers that indicate

the real-time renal pathophysiological conditions is important. Liver type fatty acid binding protein (L-FABP) is much a 14 kDa molecule predominantly expressed selleck products in human proximal tubules. Clinical studies demonstrate that urinary excretion of L-FABP derived from the proximal tubules is an excellent biomarker for predicting and monitoring deterioration of renal function or for early detection of kidney

disease. However, in order to clarify the pathophysiological roles or dynamics of renal L-FABP in diseased settings, in vivo experimental studies of kidney diseases are indispensable. Since L-FABP is not endogenously expressed in murine kidneys, a transgenic (Tg) mouse model with expression of the human L-FABP gene was established. This review article summarizes the findings on the pathophysiological roles and dynamics of renal human L-FABP in the recent experimental studies performed using this Tg mouse model. The progression of kidney disease leads to renal failure, which requires renal replacement therapy with poor outcomes and at a high cost. Moreover, kidney disease is associated with the development and progression of cardiovascular1 or cerebrovascular disease.2 Therefore, clinical markers that accurately reflect the pathophysiological conditions of kidney disease are important in order to administer appropriate treatments and suppress the progression of kidney disease. Renal tubulointerstitial injury has been noted to have an important impact on the progression of kidney disease.

As vaccination of sheep with the H11 protein gives rise to protection and/or a reduction in worm burden after challenge infection (126), the authors investigated whether gene knock-down would have an effect on

worm development within the sheep host. Sheep infected with RNAi-treated larvae showed a 57% reduction in faecal egg count and 40% reduction in worm burden compared Neratinib to control dsRNA-treated larvae, providing evidence that the knock-down of a protective antigen mirrors the effect of vaccination (121). Thus, RNAi can potentially be utilized to elucidate gene function in vivo during actual infection, leading to the identification of crucial genes in parasite development and survival and parasite–host interaction. In conclusion, the susceptibility of parasitic nematodes to RNAi seems to be

dependent on how the trigger is delivered, the target gene, the life stage of the specific parasite and the presence of the RNAi machinery. Therefore, large-scale analysis and screening protocols might not be easily realized in nematodes but targeting genes at dsRNA accessible sites can be utilized to elucidate gene function in vivo. Therefore, RNAi has the potential to become a useful tool for the identification of vaccine candidates and drug targets. Transgenesis and RNAi have already made a tremendous impact on helminth research. Although the transgenesis techniques available today thus far have mainly been used to over-express reporter genes such as GFP and luciferase, they have also allowed analysis of the tissue-specific expression of parasite genes. In nematodes, we now see the emergence of functional

Selleckchem Vemurafenib studies using constructs to interfere with the corresponding endogenous genes to study signal transduction pathways. Heritable transgenesis is within our grasp, and once achieved, it will open the door to the study of the effect of transgenes on infections. It will, for example, enable the examination see more of the activation of host immune responses, and to understand where, when and how such responses are initiated. Likewise, the development of gene trap vectors will give us a better understanding of the sets of genes that are active in particular life cycle stages. Gene silencing using RNAi is now well established in many parasitic helminths and has for the first time allowed the direct study of parasite gene function. Nevertheless, there is still an urgent need for the development of more robust transgenic technologies for use in parasitic helminths in the post-genomic era. The wealth of information made available from genome sequencing projects will undoubtedly translate into functional genomic studies in these organisms. Such studies will not only provide a deep understanding of the molecular biology of the parasite, but could ultimately be used for the identification of proteins that could be targeted with drugs or vaccines.

No. 219373) in a total volume of

100 μL of 10 mM sodium phosphate, 1% tryptic soy, at 37 °C, 5% CO2. The bactericidal reaction was terminated after 2 h by 1 : 10 dilution in 10 mM sodium phosphate. Viable counts of colony forming units were determined by plating serial dilutions of the pneumococcal culture on tryptic soy agar (TSA) plates supplemented with 250 U/mL Selleck BTK inhibitor bovine liver catalase (Sigma). All assays were performed in duplicate on at least three different days, at 37 °C, 5% CO2 without agitation. Following 2 h incubation with human neutrophil elastase wild-type encapsulated serotypes 2, 4 and 19F pneumococcal strains showed significantly less resistance to killing than the isogenic nonencapsulated derivatives (Fig. 1a).

Differences between encapsulated and nonencapsulated strains were analysed by Student’s t-test. A P value < 0.05 was considered statistically significant. Similarly following 2 h incubation with human neutrophil cathepsin G wild-type Rucaparib purchase encapsulated serotypes 2, 4 and 19F pneumococcal strains showed significantly less resistance to killing than the isogenic nonencapsulated derivatives (Fig. 1b). We observed an especially strong effect for the nonencapsulated serotype 2 strain (D39), for which we do not have a good explanation. The main finding of our study is that the absence of the pneumococcal polysaccharide capsule increases the

resistance of pneumococci to extracellular human neutrophil elastase- and cathepsin G-mediated killing. The pneumococcal targets of neutrophil protease have not yet been identified, Tideglusib but it is likely that essential pneumococcal surface proteins are degraded by neutrophil proteases. How the absence of capsule increases resistance to human neutrophil elastase- and cathepsin G-mediated killing is unclear. A potential explanation is that positive surface charges modifications, such as incorporation of positively charged d-alanine in lipoteichoic acids exposed on nonencapsulated pneumococci, repulses the positively charged proteases and thus increase resistance to degradation, whereas presence of pneumococcal polysaccharide capsule masks these positive charge modifications and increases susceptibility to the proteases. This mechanism is employed by different bacterial species including pneumococci to resist cationic antimicrobial peptides (Peschel, 2002; Beiter et al., 2008). An alternative explanation is the release of anionic bacterial decoys, specifically by nonencapsulated pneumococci, which may trap the positively charged (cationic) human neutrophil proteases. Before the role of neutrophil proteases in microbial killing was elucidated, it was shown that pneumococci release a highly charged polyanion that functions as a neutrophil elastase inhibitor during growth.

Thus, after LPS stimulation, miR-155 expression increases, SHIP1 levels fall, and AKT activity increases; as AKT downregulates miR-155, the initial high miR-155 levels are brought

back under control. miR-155 KO mice have been shown to have an impaired immune response to Salmonella typhimurium, and these mice cannot be successfully immunized against this pathogen 17. Further analysis revealed a defect in B- and T-cell activation, explaining the lack of immunization capacity in these mice. Furthermore, the failed T-cell response was, in part, due GPCR Compound Library datasheet to the failure of DCs to present antigen and due to an altered Th1 response in which the CD4+ T cells had impaired cytokine production 17. This was most likely due to the failure of DCs to functionally activate costimulatory signals and defective antigen presentation; miR-155 may be responsible for the impaired cytokine production. A second study showed that miR-155 KO mice exhibit reduced numbers of germinal centre (GC) B cells, whereas miR-155-overexpressing mice showed elevated levels 8. This study concluded that miR-155 achieves its response partly by regulating the expression of cytokines, e.g. TNF 8. A third study with

miR-155-deficient mice revealed elevated levels of activation-induced cytidine diamine (AID) 18. AID is a strong mutation-causing component in the class switching Y 27632 process and therefore its Aspartate activity needs to be tightly regulated 19. AID initiates somatic hypermutation and is essential for class-switch recombination 19. The gene-encoding AID contains a miR-155 binding site in its 3′ UTR 8, 18. B cells undergoing class

switching express high, but controlled, levels of miR-155; genetically modified mice with a mutation in the 3′ UTR binding site for miR-155 in the AID gene that blocks miR-155 binding show increased AID levels, compared with WT cells, and increased numbers of Myx-Igh translocations and, as a result, have disrupted affinity maturation. miR-155 thus closely regulates AID expression in cells to prevent hypermutational activity. These in vivo experiments confirm that miR-155 is especially important for B-cell development and identify AID as a key target. miR-146 is one of the most prominent miRNAs induced by LPS in macrophages 3, 20. Resolvin D1, an anti-inflammatory lipid mediator, also induces miR-146 21. miR-146 expression is NF-κB dependent and, to date, IL-1R-associated kinase 1 (IRAK1), IRAK2, and TNFR-associated factor 6 (TRAF6) have been shown to be miR-146 targets 20. As shown in Fig. 1, these targets are components of the NF-κB pathway and control NF-κB expression. Irak1 has been validated as a target for miR-146 in in vivo studies 22.

Over the next 3 months, she maintained clinical and biochemical stability. Her Prednisolone dose was weaned down to X-396 chemical structure 10 mg by 6 months. A further biopsy at that time once again confirmed features of quiescent crescentic glomerulonephritis, without evidence of disease activity or allograft rejection. Her most recent serum creatinine, 9 months post-transplant, was 100 µmol/L. A MEDLine search was conducted using the keyword ‘ANCA’, and MESH terms ‘Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis’ and ‘kidney transplantation’. AAV is the most common cause of rapidly progressive glomerulonephritis. Since the introduction of Cyclophosphamide to the therapeutic armament, mortality

rates have improved significantly. Nevertheless, morbidity from this disease and its treatment remain significant.

Treatment may not necessarily prevent end-organ damage, especially if it is started late in the course of the illness. Indeed, in a large recent series by Lionaki et al. (n = 523), just over 25% of those who presented with AAV reached ESRD with peak serum creatinine at presentation predicting the likelihood of progressing Selleckchem BMS-936558 to ESRD.1 While kidney transplantation is a viable option for those who reach ESRD, there is debate concerning the timing of transplantation and the likelihood of recurrence of disease. Currently published data are limited to case series and opinion, with the general consensus being that the risk of relapse is lower in renal transplant recipients than patients

on maintenance dialysis, Cediranib (AZD2171) presumably because of the suppressive effect of their maintenance immunosuppression on vasculitis activity. Allen et al.’s retrospective analysis of 59 patients with AAV who were treated with chronic dialysis, transplantation or both, had rates of relapse of 0.02 and 0.09 per patient per year, respectively. Patient survival rates in this study at 1 and 5 years were 74%, 40% in the dialysis group, and 100%, 84% in the transplantation group.2 The first reported renal transplant in a patient with ESRD secondary to AAV was carried out in 1972. Since that time, despite hopes that standard transplantation immunosuppression might be sufficient to prevent relapses, numerous cases have been reported commencing with that of Steinman et al. in 1980, describing a patient on maintenance Prednisone and Azathioprine who developed recurrent vasculitis 4 years after transplantation.3 Reported rates of recurrence are quite variable since then perhaps because of increased transplant immunosuppressive regimens over time. The rate of recurrence with modern immunosuppression is unclear. A pooled analysis in 1999 by Nachman et al. described a recurrence rate of 17% among 127 patients, with an average time from transplant to relapse of 31 months (range 5 days to 13 years).4 Importantly, the target antigen (MPO or proteinase 3 (Pr3)) did not affect the rate of relapse, nor did ANCA positivity at the time of transplantation.

“
“Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P<0.05) while enhanced soluble CD40 level in the culture supernatant (P<0.05) as compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced

the IL-12 and IFN-γ secretion by DCs (P<0.05) as compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Alvelestat cost Active Per a 10 stimulation caused low NF-κB activation in DCs as compared to heat-inactivated Per a 10. Inhibition of NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs further indicating that NF-κB is required

for CD40 up-regulation. CD40 expression activated the TRAF6, thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38MAPK activation that showed no significant effect check details on CD40 expression by DCs. However, inhibiting p38MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion

of IL-4, IL-6, IL-12 and TNF-α by CD4+ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels thereby modulating DC-mediated immune responses. “
“Natural killer (NK) cells play an important role in the innate immune system by eliminating infected and mutated cells. Their cytotoxic capacities vary markedly among individuals. The cytotoxic activity can be measured in peripheral blood mononuclear cells (PBMCs) using the NK cell–specific target cell line K562. In this chapter, Osimertinib solubility dmso we present a protocol for the standardization and normalization of cell preparation and NK cell cytotoxicity measurement in a 51Cr-release assay. By following these protocols, it is possible to compare the NK cell activity of numerous—if necessary selected—individuals in vitro. Curr. Protoc. Immunol. 100:14.32.1-14.32.11. © 2013 by John Wiley & Sons, Inc. “
“Integrins not only mediate cell–cell and cell–extracellular matrix adhesion, but also affect the multitude of signal transduction cascades in control of cell survival, proliferation, differentiation and organ development. Mutations in integrins or the major effectors of integrin signalling pathways cause defective organ development, immunodeficiency, cancer or autoimmune disease.

Based

on the aforementioned literature, finding a higher prevalence in patients with altered TCR Vβ repertoire could be expected. However, several lines of evidence suggest that viral infection and CMV infection in particular were not the main reason for the profound perturbation of the TCR Vβ repertoire observed. First, active inflammatory processes (including viral and bacterial infection) at the inclusion time and episodes of acute rejection were exclusion criteria for the recruitment of patients in the GenHomme cohort. The influence of CMV infectious episodes observed shortly after the transplantation in patients from the GenHomme cohort and thus at distance from the TcL analysis was studied. Similar prevalence of anti-CMV IgG was Adriamycin cell line found in operationally tolerant recipients and patients with chronic humoral Ivacaftor solubility dmso rejection despite exhibiting dramatically different repertoire usages. Furthermore, in these two groups, no correlation was found between TCR Vβ repertoire usage and CMV serology. Moreover, the analysis of the impact of the CMV pp65-specific T cells on the overall shape of the CD8+ repertoire showed that the TcL typology is not perturbed by CMV pp65-specific clones. Taken together, these data suggest that the TCR classification of the patients cannot be solely related to the CMV response. We then can

hypothesize that such peripheral expansions, and particularly in patients with chronic rejection, could be related to dominant indirect 3 or Carteolol HCl direct 30 alloimmune responses against the graft. The role of T cells and especially CD8+ T cells had been likely undermined in the process of chronic rejection, whereas several studies confirmed the presence of CD8+ T cells infiltrate in the graft 31–33. Moreover, we have shown that blood of animals (as reported here in patients) with

chronic rejection exhibited strong alteration of the CD8+ T-cell repertoire 34. The correlation between the Banff score and the shape of the TcL in this study reinforces the hypothesis that CD8+ T cells may be an instrumental player in chronic rejection. As the magnitude of the clonal selection in recipients with chronic rejection correlates with the severity of the rejection, TcL usage could be a useful tool for graft monitoring in these patients. Further studies on sorted Vβ families with strong alteration, on reactivity against donor cells and a long-term follow-up of the stable patient cohort are awaited for improving the interpretation of TCR alteration in long-term graft recipient. Combined with other biomarker data 9–11 and associated with the expression of inflammation or regulatory-related genes (GZMB, T-bet versus FOXP3) as shown, TCR repertoire categorization might be included in the calculation of a “composite score” for the follow-up of patients to prevent rejection or helping to decide upon immunosuppressant withdrawal.