Stephan Schlickeiser

Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study

BackgroundImmune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry.MethodsSix leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons.ResultsOptimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies.ConclusionsLocal performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.

Monitoring tolerance and rejection in organ transplant recipients

To avoid toxic side effects caused by permanent immunosuppressive treatment, research in transplantation focuses on new treatment strategies inducing tolerance or allowing drug weaning. Implementing drug minimization into clinical routine can be only safely achieved when guided by biomarkers reflecting the individual immune reactivity. We review recently described biomarkers and assays allowing identification of patients suitable for drug weaning or at risk of rejection. However, the majority of described biomarkers and assays have not been validated in prospective clinical trials. Thus, collaborative efforts are needed to design and perform prospective multicenter trials to validate the identified biomarkers across different laboratories.

The taming of the shrew? The immunology of corneal transplantation

Corneal transplantation, first reported a century ago, is the oldest and most frequent form of solid tissue transplantation. Although keratoplasty is also considered as the most successful transplant procedure, several studies indicate that the long term survival of corneal grafts is even lower than that of transplanted parenchymatous organs. Despite the immune privilege enjoyed by the cornea and anterior segment of the eye, immunologic graft rejection is a major limitation to corneal transplantation. This review gives an update on corneal immunobiology and the mechanisms of corneal graft rejection, focusing on antigen presentation, as well as on the molecular and cellular mediators of this particular immune response.

Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-β, and RA treatment.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4+ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25+Foxp3+‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25+Foxp3+ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

Elevation of CD4+ differentiated memory T cells is associated with acute cellular and antibody-mediated rejection after liver transplantation.

Background It is now well known that the outcome after allogeneic transplantation, such as incidence of acute rejections, very much depends on the individual’s immune reactivity status. There is also increasing evidence that the presence of preexisting memory T cells can affect antigraft immune responses. Methods In a prospective study, we monitored peripheral CD4+ and CD8+ central memory, effector memory, and terminal differentiated effector memory (TEMRA) T cells in 55 patients who underwent deceased liver transplantation and received conventional immunosuppressive treatment with or without basiliximab induction. The primary endpoint of the study was acute allograft rejection during a 1-year follow-up period. Results We observed significantly increased proportions of CD4+ and CD8+ TEMRA cells in patients before transplantation compared with healthy controls (P=0.006 and 0.009, respectively). This characteristic was independent of the underlying disease. In patients with no signs of acute rejection, we observed an immediate reduction of CD4+ TEMRA cells. In contrast, patients who experienced acute cellular rejection, and especially antibody-mediated rejection, displayed persistent elevated TEMRA cells (P=0.017 and 0.027, respectively). Basiliximab induction therapy did not influence CD4+ and CD8+ TEMRA numbers. Conclusions Conventional immunosuppressive or basiliximab treatment cannot control the persistence of TEMRA T cells, which may contribute to acute cellular rejection and antibody-mediated rejection after liver transplantation. In the future, specific targeting of TEMRA cells in selected patients may prevent the occurrence of difficult to treat steroid-resistant rejections, thereby leading to improved patient outcome.

Pretransplant immune risk assessment.

Purpose of reviewDespite the introduction of advanced immunosuppressive drug therapies, clinical and subclinical rejections still occur in many graft recipients with a negative impact on the long-term transplant outcome. The immunological status of the patients awaiting the transplantation is a key factor for these processes. Here we summarize the recent efforts to identify and develop biomarkers and functional assays that allow an individual pretransplant risk assessment. Recent findingsNew sensitive techniques assessing T-cell memory and B-cell activation have been developed. Furthermore, the expression level of soluble and molecular markers reflecting the activation state of the immune system and donor graft intrinsic factors have been shown to influence graft outcome. SummaryA variety of parameters and assays that determine the pretransplant immune activation status has been developed. Some of these assays have already been used prospectively to define high-risk patients receiving advanced immunosuppressive induction therapy.However, the conflicting results obtained in different studies show that biomarker analysis and functional assays performance need to be further standardized and validated in large prospective trials before they can be routinely implemented into a pretransplant risk assessment. Subsequently, a combined effort to design pretransplant risk stratification algorithms should lead to personalized immunosuppressive treatment regimes and improved graft survival and long-term graft function.

Age and gender leucocytes variances and references values generated using the standardized ONE-Study protocol

Flow cytometry is now accepted as an ideal technology to reveal changes in immune cell composition and function. However, it is also an error‐prone and variable technology, which makes it difficult to reproduce findings across laboratories. We have recently developed a strategy to standardize whole blood flow cytometry. The performance of our protocols was challenged here by profiling samples from healthy volunteers to reveal age‐ and gender‐dependent differences and to establish a standardized reference cohort for use in clinical trials. Whole blood samples from two different cohorts were analyzed (first cohort: n = 52, second cohort: n = 46, both 20–84 years with equal gender distribution). The second cohort was run as a validation cohort by a different operator. The “ONE Study” panels were applied to analyze expression of >30 different surface markers to enumerate proportional and absolute numbers of >50 leucocyte subsets. Indeed, analysis of the first cohort revealed significant age‐dependent changes in subsets e.g. increased activated and differentiated CD4+ and CD8+ T cell subsets, acquisition of a memory phenotype for Tregs as well as decreased MDC2 and Marginal Zone B cells. Males and females showed different dynamics in age‐dependent T cell activation and differentiation, indicating faster immunosenescence in males. Importantly, although both cohorts consisted of a small sample size, our standardized approach enabled validation of age‐dependent changes with the second cohort. Thus, we have proven the utility of our strategy and generated reproducible reference ranges accounting for age‐ and gender‐dependent differences, which are crucial for a better patient monitoring and individualized therapy.

The use of novel diagnostics to individualize immunosuppression following transplantation.

Despite major improvements in short‐term survival of organ allografts, long‐term graft survival has not changed significantly. It is also known that toxic side effects of current immunosuppressive drugs (IS) especially calcineurin inhibitors (CNI) contribute to the unsatisfactory graft and patient survival following transplantation. Thus, clinicians strive to reduce or wean IS in potentially eligible patients. Research in the last 10 years has focussed on identification of biomarkers suitable for patient stratification in minimization or weaning trials. Most of the described biomarkers have been run retrospectively on samples collected within single‐centre trials. Thus, often their performance has not been validated in other potentially multicentre clinical trials. Ultimately, the utility of biomarkers to identify potential weaning candidates should be investigated in large randomized prospective trials. In particular, for testing in such trials, we need more information about the accuracy, reproducibility, stability and limitations of the described biomarkers. Also, data repositories summarizing crucial information on biomarker performance in age‐ and gender‐matched healthy individuals of different ethnicity are missing. This together with improved bioinformatics tools might help in developing better scores for patient stratification. Here, we will summarize the current results, knowledge and limitations on biomarkers for drug minimization or weaning trials.

Wild immunology assessed by multidimensional mass cytometry.

A great part of our knowledge on mammalian immunology has been established in laboratory settings. The use of inbred mouse strains enabled controlled studies of immune cell and molecule functions in defined settings. These studies were usually performed in specific‐pathogen free (SPF) environments providing standardized conditions. In contrast, mammalians including humans living in their natural habitat are continuously facing pathogen encounters throughout their life. The influences of environmental conditions on the signatures of the immune system and on experimental outcomes are yet not well defined. Thus, the transferability of results obtained in current experimental systems to the physiological human situation has always been a matter of debate. Studies elucidating the diversity of “wild immunology” imprintings in detail and comparing it with those of “clean” lab mice are sparse. Here, we applied multidimensional mass cytometry to dissect phenotypic and functional differences between distinct groups of laboratory and pet shop mice as a source for “wild mice”. For this purpose, we developed a 31‐antibody panel for murine leukocyte subsets identification and a 35‐antibody panel assessing various cytokines. Established murine leukocyte populations were easily identified and diverse immune signatures indicative of numerous pathogen encounters were classified particularly in pet shop mice and to a lesser extent in quarantine and non‐SPF mice as compared to SPF mice. In addition, unsupervised analysis identified distinct clusters that associated strongly with the degree of pathogenic priming, including increased frequencies of activated NK cells and antigen‐experienced B‐ and T‐cell subsets. Our study unravels the complexity of immune signatures altered under physiological pathogen challenges and highlights the importance of carefully adapting laboratory settings for immunological studies in mice, including drug and therapy testing.

Demethylation of the TSDR Is a Marker of Squamous Cell Carcinoma in Transplant Recipients

Malignancy is an important cause of death in transplant recipients. Cutaneous squamous cell carcinoma (cSCC) causes significant morbidity and mortality as 30% of transplant recipients will develop cSCC within 10 years of transplantation. Previously we have shown that high numbers of regulatory T cells (Tregs) are associated with the development of cSCC in kidney transplant recipients (KTRs). Demethylation analysis of the Treg‐specific demethylated region (TSDR) provides a more accurate association with cSCC risk after transplantation. Age, gender and duration of immunosuppression matched KTRs with (n = 32) and without (n = 27) cSCC, were re‐analyzed for putative clinical and immunological markers of cancer risk. The proportion of FOXP3+ CD4+ cells was higher in the population with a previous SCC. Major T cell subsets remained stable over time; although B cell, CD8 and CD4 subpopulations demonstrated age‐related changes. TSDR methylation analysis allowed clarification of Treg numbers, enhancing the association of high Treg levels in KTRs with cSCC compared to the cSCC‐free cohort. These data validate and expand on previous findings in long‐term KTRs, and show that immune markers remain stable over time. TSDR demethylation analysis provides a more accurate biomarker of cancer posttransplantation.