Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Introduction

T regulatory cells (Tregs) are dominant cellular compounds of the immune system protecting the body from autoimmune reactions. These cells are also involved in imposing tolerance to alloantigens such as transplanted allogeneic cells and tissues (1–5). For all these reasons, several Treg-based therapeutics are being tested in clinical trials as a prophylaxis or treatment of autoimmune diseases, graft-versus-host disease after hematopoietic stem cell transplants or rejections after solid organ transplants (6). The list of potential applications in the future is even wider. At the same time, manufacturing of Tregs for preclinical and clinical experiments varies considerably between different centers, which significantly diminishes possible comparisons between the trials. For this reason, future development of these therapies is hampered as it happens that the available results from different trials are contradictive. The specificity of cellular products makes it difficult to verify the results in huge multicentre trials and therefore better standardization of early-phase trials as well as cellular products themselves might facilitate the progress in this promising branch of medicine.

We propose here a tool for standardization of Tregs studies designed on the basis of so-called minimum information models (MIMs). These models have gained increasing popularity among scientists as they enable the interpretation of reported data, comparison between data from different studies and facilitate experimental reproducibility (7, 8). MIMs provide mechanisms that all laboratories report at least the key facts about their analysis in a clear and consistent manner, allowing a comparison across the whole field. Our consortium has already designed the MIM called minimum information about tolerogenic antigen-presenting cells (MITAP). This is a reporting framework that makes transparent differences and similarities of different tolerogenic antigen-presenting cells (tolAPC) (9). It provides minimum reporting guidelines for the production process of tolAPC used in preclinical and/or clinical studies. We have followed the MITAP experience and designed a MIM for the manufacture of Tregs. We call it minimum information about T regulatory cells (MITREG). MITREG will be a useful resource for investigators reporting their data on the use of in vitro expanded natural Tregs or induced Tregs in preclinical models or clinical trials.

Methods

Setting Up MITREG: Community Building and Initial Analysis

The community was mainly built on the experience of our completed MITAP initiative. For several years now, we have been working together in the field of tolerogenic cellular therapies under the umbrella of the consortium AFACTT (action to focus and accelerate cell-based tolerance-inducing therapies—http://www.afactt.eu/). It brings together European scientists and clinicians with the aim of jointly addressing issues related to the translation and clinical application of these new treatments. Having the experience of MITAP, we used this document as a template to describe Treg therapies. For MITREG, we also tried to extend the initiative beyond Europe and invited scientists working on tolerogenic cellular therapies from around the world. This way we ensured a broadly reflective discussion taking into account various opinions and current practices of many laboratories within the discipline.

The work on this MITREG document covered a series of “exercises” that provided some initial data. Like for MITAP, the exercises aimed at gathering “terms” in order to acquire basic vocabulary in use within the community. The first, so-called “sticky-note” exercise performed at several AFACTT meetings assumed that each participant wrote a term on a sticky-note; these were then collated and clustered on a wall by the whole group, identifying synonyms and related terms. Second, we used the MITAP template to incorporate the collected terms and created an initial version of MITREG. This document underwent several rounds of face-to-face and online consultations with AFACTT members to improve its clarity. Internally agreed version was circulated to external specialists in the field. This external feedback was collected and implemented in the final version of the MITREG document. Finally, we used the existing literature to obtain a picture of how well the required information has been described in published articles.

Results

Overview of the MITREG Document

The design of the MITREG document followed the concept of MITAP, which facilitated the whole process. It describes the manufacturing of Treg products in a chronological way. The document is divided into four sections highlighting critical points of the process and regulatory issues. The document describes the details that should be provided by investigators, which would allow other researchers to repeat the process. It also advises on the use of existing taxonomies and databases to provide the information in a uniform manner, and it suggests the use of other MIMs where appropriate. The full MITREG document can be found on archive.org (http://w3id.org/ontolink/mitreg) and it is also included in the Appendix A (MITREG document).

Section 1: Cells at the Start of the Procedure

This section describes characteristics of the biological material before it undergoes any manipulation. There are five subparts asking for (a) essential information about the donor, (b) source of the cells, (c) the methods used to separate Tregs, (d) the phenotype after separation, and (e) the number of Tregs after separation.

Section 2: Expansion/Differentiation

This section describes the protocol that has been used to expand or differentiate Tregs. The specificity of Tregs was a challenge here as different subsets can be obtained with a wide range of methods. Tregs can be either isolated and optionally expanded or can be induced from naive precursors. There are five subsections giving details on (a) preculture conditions, (b) culture conditions, (c) the protocol used to expand or differentiate cultured Tregs, (d) stimuli used during the process, and (e) the way Tregs are stored immediately after expansion/differentiation.

Section 3: Cells after Expansion/Differentiation

This section describes the characteristics of Tregs after the expansion or differentiation. It is mainly focused on the phenotype of the final Treg product as well as its suppressive activity verified in any form of functional assay. It also documents the cell yield from the entire process and, if the product is for clinical use or testing of adoptive transfer in animals, the details on administration of the cells to the recipient.

Section 4: About the Protocol

This final section describes remaining details of the experimental or clinical protocol such as primary or secondary goals as well as regulatory issues such as adherence to particular acts or directives including compliance with good practice requirements (GCP, GLP, or GMP guidelines). Finally, the name and contact details of the corresponding author(s) must be provided.

The MITREG document is accompanied by a handy checklist to assist investigators in ensuring that all the relevant detail is provided before submitting their manuscripts for publication. The checklist can be found at archive.org (http://w3id.org/ontolink/mitreg) and is also included in the Appendix B (MITREG checklist).

Prevalence of MITREG Data in Extant Published Articles

The purpose of the MITREG document is to ensure that authors provide sufficient basic information about their production protocol. An implicit assumption is that currently some or all of this information is not being routinely described. To test this assumption, we reviewed a number of articles about Treg products and for each we determined whether it included data described in the MITREG document.

In detail, 19 Treg articles were selected (predominantly from members of AFACTT or from researchers well known in the field) and read in detail. The articles are given chronologically in the references but the order in Figure 1 is different and anonymized (10–28). For each section of MITREG, we determined whether the information required was directly stated in the article (or referenced) (Figure 1: green squares), partly stated in the article (Figure 1: yellow triangles), not present at all (Figure 1: red circles), or whether information was not present due to lack of relevance for the publication (Figure 1: gray circles). For example, section 1-ai of MITREG describes the species used in the experimental setup. An article with the phrase “human” or “Homo sapiens” would fall into the first category (included in the publication). However, when mice are used and only the species is mentioned: “mouse” or “Mus musculus,” but not the strain, it would fall into the second category (included but details missing). Many articles do not describe their experimental methodology, but instead refer to another article (“as described previously”); in this case, we checked the article up to two references deep and if found, the information was considered as “present” (Figure 1: green squares), if not it was considered as “not present” (Figure 1: red circles). This work was performed by four independent scientists with experience in the field.

Figure 1

Agreement of published T regulatory cell (Treg) articles with the minimum information about T regulatory cell (MITREG) document. Graph showing the results of a total of 19 Treg articles (10–28). The order in the figure is anonymized and different from that in the references. MITREG data directly stated in the article ( green squares), partly stated in the article ( yellow triangles), not present at all ( red circles), or not present as it was not relevant for the publication ( gray circles).

Results are shown in Figure 1. This figure shows that in some sections like the species, characteristics, ethics, and cell dose transferred sections, reporting is good with almost all revised articles describing these. However, other sections are often very poorly reported. For example, storage of cells, anticoagulant used and the number/viability of cells after each separate step are not described in most articles. Moreover, important information (container type, concentration of cells) to repeat the performed experiments is missing in almost all articles.

Sustainability

We have taken particular care to consider the issues of digital sustainability for MITREG. A well-known problem with resources linked with URLs given in articles is that URLs are often lost over time: around a 25% loss 3 years after publication (29). We have, therefore, specifically addressed this issue by use of a stable identifier space; the MITREG document and checklist are hosted by archive.org, an organization committed to long-term digital preservation. In addition, we have used a permanent identifier (http://w3id.org/ontolink/mitreg) thereby providing a redirection-step.

Resources are available in a number of formats: both PDF and Word for manipulability, but also a simple HTML representation, ensuring vendor-neutrality and future-proofing, in so far as this is possible.

Discussion

Minimum information models aid investigators by providing a specific guideline of what is required to interpret and compare experimental findings. Furthermore, reporting guidelines will facilitate independent validation of published results, a fundamental precept of scientific research. This is to our knowledge the first proposal of a minimum information standard on the description of experimental as well as clinical manufacturing and application of Tregs. The generation of MITREG was initiated by members of the European AFACTT consortium to fill a recognized gap in data reporting standards in the Treg community. MITREG was realized with the help of key international players in the Treg field.

Nine years after the first-in-man report, there are currently close to 30 recruiting or ongoing clinical trials administering Tregs in autoimmune settings, inflammatory diseases, transplantation and graft-versus-host disease (6). Clinical grade reagents for Treg isolation by magnetic activated cell sorting have become available to the growing community and off the shelf products and GMP-compatible fluorescence-based cell sorting is currently been available from multiple manufacturers of novel closed system devices, further increasing the diversity of isolation techniques (30). Given the low frequency of Tregs in the periphery, most clinical applications require an in vitro cell expansion culturing step classifying them as advanced therapy medicinal products. A growing number of culturing methods are being developed and published aiming at Treg induction, enhanced ex vivo expansion, alloreactivity and more recently, the implementation of specific T cell receptors or chimeric antigen receptors (17, 18, 25, 31–39). We are thus at a point where protocol diversity is growing exponentially, emphasizing the necessity to harmonize reporting regimens as a prerequisite of reproducibility and quality assurance. By analyzing extant articles according to the MITREG document (Figure 1), it also becomes clear that there is a big gap in what is currently being reported and what the community considers important and wants to receive in a Treg production/expansion protocol. For example, storage conditions, cell numbers and viability and anticoagulant used are almost never reported, but are most likely measured or known by the researcher. Moreover, essential information to allow experiments to be repeated is often missing.

Together with MITREG we provide a checklist that was designed with maximal flexibility to incorporate newly developed methodologies. While MITREG does not aim at uniform protocols or dictating quality checks, it is expected to enable a mere description of the growing diversity in production procedures. We expect it to mature as novel technologies arise and become a consensus guideline within the Treg community. Only by exact reporting we will be able to identify differences in Treg preparations that may help to understand results from clinical studies. We anticipate that MITREG will be a starting point for further joint efforts of the Treg community that will ultimately lead to optimized cellular therapy.

Author Contributions

As described in the Section “Methods,” these recommendations are the common effort of all the authors, who were involved in the design, acquisition, and interpretation of available data on Tregs as well as revised critically and approved final version of the MITREG document. In addition, AF, MG, NG, and RS were involved in collecting and analysis of the data sent by the contributors and SG, CH, GL, PL, EC, and PT supervised the work and edited the article.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LH declared a shared affiliation, with no collaboration, with several of the authors RS, JI, CH, and PL to the handling editor.

(MITREG) Checklist
Must	Should	May
			(1) Cells at the start of procedure
			(a) Essential information about the donor
			(i) Species and strain
			Species
			Strain (if applicable)
			(ii) Characteristics of the organism
			Health
			Age
			Treatment/Environment
			Individual identifier number
			Source of purchase (if applicable)
			(b) Source of cell material
			Organ, tissue, fluid, or blood product
			Source (if applicable)
			Quantity (volume, size, or weight)
			Anti-coagulant (if applicable)
			If using cryopreserved sample
			Method and duration of storage
			Initial cell counts
			Ethical committee approval/written informed consent
			(c) Cell separation process
			(i) Cell handling and labeling
			Cell extraction method
			Tissue conditions between tissue retrieval and cell separation
			Duration
			Temperature
			Container
			Fluid
			Cell labeling
			Buffers and reagents (incl. source)
			Cell suspension volume and concentration
			Incubation temperature and duration
			Washing steps
			(ii) Cell separation equipment and process
			Methodology
			Equipment
			Presence of target cells in starting material described
			(d) Phenotype
			For any of the below, indicate the percentage of cells displaying the characteristic (if known)
			(i) Cell surface and intracellular markers
			Molecules measured [using cluster of differentiation (CD) names]
			Details of reagents used and source (incl. mAb clone, fluorochrome)
			Methodology
			Stimulus and time of stimulation (if applicable)
			Gating strategy to determine positive cells
			(ii) Secreted molecules
			Molecules measured
			Details of reagents used (incl. mAb clone, conjugate) and source
			Methodology
			Cell density/ml of medium and type of tissue culture plate
			Time point of supernatant collection
			Stimulus and time of stimulation (if applicable)
			(iii) Epigenetic modifications
			Epigenetic modification relevant to the characteristics
			(iv) Specificity
			Specificity of the cells (polyclonal or antigen-specific)
			Methodology used to obtain specificity
			Methodology used to confirm specificity
			(e) Cell numbers
			(i) Absolute cell number
			Total number of cells at the end of the isolation process
			Methodology
			(ii) Viability
			Percentage of viable cells
			Methodology
			(2) Expansion/differentiation
			(a) Pre-culture conditions
			Storage conditions
			Fluid
			Type of container
			Temperature
			Fresh or thawed
			Storage time
			(b) Culture conditions
			(i) Cell number
			The total number of cells put into culture
			(ii) Cell concentration
			The number of cells per ml of medium at start of culture
			(iii) Culture medium
			Type(s) of medium
			Source(s)
			Additives (excluding agents to maintain/induce T regulatory cells)
			Refreshment of the medium
			(iv) Culture container
			Type of container
			Size
			Manufacturer
			Cell culture volume per container or well
			Total number of containers or wells
			(v) Culture environment
			Temperature and CO2 concentration
			Use of pre-warmed medium
			Equipment
			(c) Differentiation/tolerization protocol
			Name of cytokine(s) or other agent(s) used
			Concentrations
			Time point(s) added to cell culture
			Total length of the culture period
			Rounds of stimulation
			Number of cell splitting
			(d) Stimulus
			Polyclonal/antigen-specific/alloantigen
			Stimulus (agent and/or accessory cell)
			Source
			Concentration
			Time point(s) added to culture
			Restimulation conditions (if applicable)
			(e) Storage
			Storage time
			Storage conditions
			If fresh
			Fluid
			Container
			Temperature
			If cryopreserved
			Freezing/thawing process
			Freezing medium
			Cell recovery and viability after thawing
			Time point at which cells are stored if different to the end of the culture process
			(3) Cells after expansion/differentiation
			(a) Phenotype
			For any of the below, indicate the percentage of cells displaying the characteristic (if known)
			Stability of the phenotype (if tested)
			Phenotype tested on fresh or thawed cells
			(i) Cell surface and intracellular markers
			Molecules measured (using CD names)
			Details of reagents used and source
			Methodology
			Stimulus and time of stimulation (if applicable)
			Gating strategy to determine positive cells
			(ii) Secreted molecules
			Molecules measured
			Details of reagents used and source
			Methodology
			Cell density/milliliter of medium and type of tissue culture plate
			Time point of supernatant collection
			Stimulus and time of stimulation (if applicable)
			(iii) Epigenetic modifications
			Epigenetic modification relevant to the characteristics
			(b) Functional assay
			Response of the cells to a defined stimulus
			Behaviour of other biological entities after exposure to the cells
			If using accessory cells, describe phenotype and source
			(c) Cell numbers
			(i) Absolute cell number
			Total number of cells at the end of the expansion process
			Methodology
			(ii) Viability
			Percentage of viable cells
			Methodology
			(d) Dosing
			Dose of cells transferred into organism (if applicable)
			Vehicle (solvent/medium) and intermediate components (for clinical trials only)
			(e) Quality control (for clinical trial only)
			Specificity
			Purity
			Sterility
			Potency
			(4) About the protocol
			(a) Regulatory authority
			External authority that approved the protocol
			Does protocol follow Good Manufacturing Practice?
			(b) Purpose
			The disorder for which the cell treatment has been manufactured
			(c) Relationship between the source organism for the cells and the target organism
			Allogeneic/autologous/xenogeneic/syngeneic
			(d) Contact details
			Name and contact information of the corresponding author(s)
			(e) Citation
			Acknowledge the MITREG reporting guidelines