James A. Hutchinson

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.

Macrophages driven to a novel state of activation have anti-inflammatory properties in mice.

Recurrent episodes of inflammation underlie numerous pathologies, notably those of inflammatory bowel diseases. In this study, we describe a population of macrophages in a novel state of activation that mitigates colitis in mice. The cells responsible for this effect, called IFN-γ-stimulated monocyte-derived cells (IFNγ-MdC), derive from mouse spleen, blood, and bone marrow monocytes and are distinguished from known macrophage populations by mode of generation, cell surface phenotype, and function. IFNγ-MdC only arise when macrophages are cultivated in the presence of CD40L-expressing CD4+ T cells, M-CSF, and IFN-γ. IFNγ-MdC express markers including F4/80, CD11b/c, CD86, and CD274; they are negative for CD4, CD8, Gr1, CD19, CD80, and CD207. Functionally, IFNγ-MdC are defined by their capacity to enrich cocultured T cell populations for CD4+CD25+Foxp3+ regulatory cells; this enrichment, constituting up to 60% or more of residual lymphocytes, is attributed to an expansion, but also to a cell contact and caspase-dependent depletion of activated T cells. In mice, IFNγ-MdC delivered i.v. traffic to gut-associated peripheral lymphoid tissues, including the mesenteric lymph nodes, Peyer’s patches, and colonic mucosa, and promote the clinical and histological resolution of chronic colitis. We conclude that IFNγ-MdC represent macrophages in a novel state of activation, possessing multiple T cell-suppressive effects with therapeutic potential for the treatment of autoimmune inflammation.

A cell‐based approach to the minimization of immunosuppression in renal transplantation

Five renal transplant recipients were preoperatively treated with transplant acceptance‐inducing cells (TAICs) in a Phase‐I safety study of TAICs as an adjunct immune‐conditioning therapy in living‐donor kidney transplantation. Initially, patients received anti‐thymocyte globulin induction therapy in combination with tacrolimus and steroid immunosuppression. Over the course of 12 weeks, steroids were withdrawn and tacrolimus therapy was minimized. Three of the five patients were able to tolerate low‐dose tacrolimus monotherapy and one patient was withdrawn from all immunosuppression for over 8 months. No acute or delayed adverse events were associated with the infusion of TAICs. Monitoring of the recipient anti‐donor reactivity of TAIC‐treated patients in mixed lymphocyte cultures demonstrated that, during periods of clinically stable graft function, recipient T‐cell proliferation and cytokine secretion in response to stimulation with donor alloantigen was relatively suppressed. Therefore, although the TAIC‐II trial did not provide conclusive evidence of a beneficial effect of preoperative TAIC treatment, the results were encouraging because they suggest that TAICs promote a state of alloantigen‐specific unresponsiveness, which might allow safe minimization of pharmacological immunosuppression.

Hurdles in therapy with regulatory T cells.

Translating regulatory T cell therapy to the clinic in Europe is making progress but has remaining challenges. Improper activation of the immune system contributes to a variety of clinical conditions, including autoimmune and allergic diseases as well as solid organ and bone marrow transplantation. One approach to counteract this activation is through adoptive therapy with regulatory T cells (Tregs). Efforts to manufacture these cells have led to good maunfacturing practice–compliant protocols, and Treg products are entering early clinical trials. Here, we report the stance of the European Union Cooperation in Science and Technology Action BM1305, “Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies—A FACTT,” which identifies hurdles hindering Treg clinical applications in Europe and provides possible solutions.

Transplant acceptance-inducing cells as an immune-conditioning therapy in renal transplantation.

The transplant acceptance‐inducing cell (TAIC) is a type of immunoregulatory macrophage with the capacity to specifically dampen allogeneic rejection responses to a degree allowing safe minimization of conventional immunosuppressive therapy. In the first part of this report, the production and phenotype of the human TAIC is described. In the second part, an analysis is given of the TAIC‐I clinical trial, in which 12 recipients of renal transplants from deceased donors were treated with donor‐derived TAICs as an adjunct immune‐conditioning therapy. Conventional immunosuppression was gradually withdrawn from 10 of these 12 patients over a period of 8 weeks, starting in the fourth week after transplantation. All but two patients tolerated cessation of steroid therapy, while the remaining eight patients were first weaned from sirolimus and then, in six cases, were also weaned to low‐dose tacrolimus monotherapy. It is concluded that TAIC therapy is both safe and clinically practicable; however, the TAIC‐I trial was unable to provide evidence that postoperative TAIC administration has a beneficial effect.

Single Cell Tracking of Gadolinium Labeled CD4+ T Cells by Laser Ablation Inductively Coupled Plasma Mass Spectrometry

Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of hematopoietic stem cell (HSC) transplantation, autoimmune disease, and solid organ transplantation. Determining the persistence of cell-based therapies in vivo is crucial to understanding their regulatory function and requires the combination of an extremely sensitive detection technique and a stable, long-lifetime cell labeling agent. This paper reports the first application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform single cell detection of T cell populations relevant to cellular immunotherapy. Purified human CD4(+) T cells were labeled with commercially available Gd-based magnetic resonance imaging (MRI) contrast agents, Omniscan and Dotarem, which enabled passive loading of up to 10(8) Gd atoms per cell. In mixed preparations of labeled and unlabeled cells, LA-ICP-MS was capable of enumerating labeled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained a sufficient label to remain detectable for up to 10 days post-labeling both in vitro and in vivo in an immunodeficient mouse model.

Human Regulatory Macrophages

Regulatory macrophages (M regs) are a novel type of suppressor macrophage which may be a particularly suitable cell for inducing tolerance of solid organ transplants. In this article, we provide a detailed description of the generation of human M regs from peripheral blood monocytes and methods for the assessment of their phenotype. The uniqueness of the human M reg is best appreciated when the M reg is compared to macrophages in other states of activation; therefore, protocols are provided for generating five comparator macrophage types which have been used as cell type-specificity controls in our work.

Alternative approaches to myeloid suppressor cell therapy in transplantation: comparing regulatory macrophages to tolerogenic DCs and MDSCs

Several types of myeloid suppressor cell are currently being developed as cell-based immunosuppressive agents. Despite detailed knowledge about the molecular and cellular functions of these cell types, expert opinions differ on how to best implement such therapies in solid organ transplantation. Efforts in our laboratory to develop a cell-based medicinal product for promoting tolerance in renal transplant patients have focused on a type of suppressor macrophage, which we call the regulatory macrophage (M reg). Our favoured clinical strategy is to administer donor-derived M regs to recipients one week prior to transplantation. In contrast, many groups working with tolerogenic dendritic cells (DCs) advocate post-transplant administration of recipient-derived cells. A third alternative, using myeloid-derived suppressor cells, presumably demands that cells are given around the time of transplantation, so that they can infiltrate the graft to create a suppressive environment. On present evidence, it is not possible to say which cell type and treatment strategy might be clinically superior. This review seeks to position our basic scientific and early-stage clinical studies of human regulatory macrophages within the broader context of myeloid suppressor cell therapy in transplantation.

Now or never? The case for cell-based immunosuppression in kidney transplantation.

By exploiting mechanisms of immunological regulation against donor alloantigen, it may be possible to reduce the dependence of kidney transplant recipients upon calcineurin inhibitor-based maintenance immunosuppression. One means to strengthen regulatory responses is treating recipients with preparations of regulatory cells obtained by ex vivo manipulation. This strategy, which is a well-established experimental method, has been developed to the point that early-phase clinical trials in kidney transplantation are now feasible. Cell-based therapies represent a radical departure from conventional treatment, so what grounds are there for this new approach? This article offers a three-part justification for trialing cell-based therapies in kidney transplantation: first, a clinical need for alternatives to standard immunosuppression is identified, based on the inadequacies of calcineurin inhibitor-based regimens in preventing late allograft loss; second, a mechanistic explanation of how cell-based therapies might address this clinical need is given; and third, the possible benefit to patients is weighed against the potential risks of cell-based immunosuppressive therapy. It is concluded that the safety of cell-based immunosuppressive therapy will not be greatly improved by further basic scientific and preclinical development. Only trials in humans can now tell us whether cell-based therapy is likely to benefit kidney transplant recipients, but these should be conservative in design to minimize any potential harm to patients.

Cell therapy as a strategy to minimize maintenance immunosuppression in solid organ transplant recipients.

Purpose of review This review presents a clinically focussed introduction to cell-based immunotherapy in solid organ transplantation. The potential benefits and risks of cell-based immunotherapeutics are critically discussed. Recent findings The use of immunoregulatory cells as medicinal agents is very much in its infancy, but the field is expanding rapidly. In principle, this approach permits manipulation of specific immunological functions, opening new possibilities in the field of tolerance-promoting therapies. Several immunoregulatory cell types have reached the point of preclinical and clinical development that should allow them to be tested in early-phase clinical trials. Solid organ transplantation represents an important potential indication for the use of cell-based immunosuppressive agents because promoting immunological regulation towards allografts remains a promising strategy for preventing chronic rejection. Summary Remarkable progress is being made in the implementation of novel cell-based immunotherapeutics in solid organ transplantation studies. It is hoped that these new immunoregulatory therapies will afford better long-term transplant outcomes by mitigating chronic graft injury.