Background: Immune 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. Methods: Six 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. Results: Optimized 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. Conclusions: Local 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.

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. © 2016 International Society for Advancement of Cytometry

All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4⁺ memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1⁺KLRG1⁺GPR56⁺ CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRβ repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4⁺ memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development.