| | Isolation of CD4+CD25+ Regulatory T Cells for Clinical TrialsReceived 7 December 2005; accepted 9 January 2006. Abstract The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non–GMP-approved fluorescence-activated cell sorting.
Introduction  Murine CD4+CD25+ regulatory T (Treg) cells do not induce graft-versus-host disease (GVHD), even when transplanted in large numbers and across complete major histocompatibility complex barriers, but suppress GVHD induced by CD25− T cells when cotransplanted at high ratios (1:1 or 1:2) [1, 2, 3, 4, 5, 6]. T cells with similar phenotypic and functional characteristics in vitro have also been identified in human cord and adult peripheral blood, as well as in lymphoid organs [7, 8, 9, 10, 11, 12, 13]. Like their murine counterparts, they are CD4+ and constitutively express intracellular cytotoxic T-lymphocyte antigen (CTLA)–4 and the transcription factor forkhead box P3 (FOXP3), a key regulatory gene for the development and function of natural Treg cells in mice and humans [14, 15, 16]. In contrast to murine Treg cells, human Treg cells seem to reside predominantly in the subpopulation with high-level expression of CD25 and less so in the subpopulation with intermediate CD25 expression, which also contains memory and recently activated T cells [7, 17, 18]. In vitro, CD4+CD25+ Treg cells suppress the proliferation of CD4+ and CD8+ T cells after polyclonal or allogeneic stimulation in a cell contact–dependent and cytokine-independent manner [7, 9, 18]. On the basis of these similarities between human and mouse Treg cells, the adoptive transfer of donor CD4+CD25+ Treg cells after allogeneic stem cell transplantation (SCT) is anticipated to be beneficial for GVHD prophylaxis in humans. A prerequisite for the evaluation of this hypothesis in clinical trials is the development of cell-separation strategies for the efficient and reliable enrichment of this rare cell population from peripheral blood that comply with good manufacturing practice (GMP) guidelines. In this article, we demonstrate that CD4+CD25+ Treg cells can be isolated under GMP conditions from standard leukapheresis products by magnetic cell-separation technologies. A detailed phenotypic and functional comparison with fluorescence-activated cell sorting (FACS)–purified CD4+ T-cell subpopulations confirmed the preferential enrichment of natural Treg cells within the generated cell products. Materials and methods Antibodies and Flow Cytometry (FACS) FACS analysis was performed as published previously [18], with the following modifications. For analysis of CD25 expression after magnetic cell separation, anti-human CD25-biotin (M-T321; Miltenyi Biotec, Bergisch Gladbach, Germany) followed by phycoerythrin (PE)–labeled or allophycocyanin (APC)–labeled anti-biotin antibody (Bio3-18E7; Miltenyi Biotec) was used to enhance fluorescence intensity (directly PE- or APC-conjugated antibodies did not allow sufficient differentiation of CD25high and CD25int cells as a result of competition with attached beads; Figure 1A). FACS sorts were performed on a FACSAria (Becton Dickinson, Heidelberg, Germany). Sort gates for CD4+CD25high T cells were set as previously described [18] and included CD4+ T cells with higher CD25 expression levels than CD4−CD25+ cells within peripheral blood mononuclear cells (PBMCs). Sort gates for CD4+CD25int T cells included CD4+CD25+ T cells that did not reach the high CD25 expression level required for classification as CD4+CD25high T cells. All sorted subpopulations were >97% pure upon reanalysis. Expression of FOXP3 on a single cell level was analyzed by using the PE anti-human Foxp3 staining set (clone PCH101; eBioscience, San Diego, CA) according to the manufacturer’s instructions. PE-conjugated monoclonal rat immunoglobulin G2aκ (R35-95; BD Pharmingen, San Diego, CA) was used as an isotype control. Magnetic Cell Separation Leukapheresis products were obtained from 6 different volunteers after their informed consent and in accordance with approved protocols. For all magnetic cell separations, clinical-grade reagents were used, and 5 of 6 were performed in a certified GMP unit. Cells were washed and adjusted to 95 mL in phosphate-buffered saline (PBS), ethylenediaminetetraacetic acid (EDTA), and 2% human albumin (HA), labeled with 7.5 mL of CliniMACS CD19 MicroBeads (Miltenyi Biotec) for 30 minutes at room temperature on an orbital shaker, washed again, and resuspended in 100 mL of PBS/EDTA/2% HA. B cells were depleted with the CliniMACSplus instrument by using a LS tubing set and the depletion program 2.1 (all Miltenyi Biotec). B cell–depleted cells were suspended in 190 mL of PBS/EDTA/2% HA, labeled with 7.5 mL of CliniMACS CD25 MicroBeads for 30 minutes at room temperature on an orbital shaker, washed, and resuspended in 100 mL. CD25+ cells were isolated by 3 automatic cycles of positive selection by using the enrichment program 3.1 of the CliniMACS device. Aliquots before and after each staining, depletion, or enrichment step were taken for FACS analysis. Proliferation and Suppression Assays Polyclonal proliferation was determined in cultures of 5 × 104 T cells (CD4+ responder T cells [Tresp] isolated from the CD19/CD25-depleted cell fraction with anti-CD4 MicroBeads [Miltenyi Biotec], CliniMACS-purified CD4+CD25+ T cells, or FACS-purified CD4+CD25high, CD4+CD25int, or CD4+CD25− T cells) that were stimulated with 100 ng/mL anti-CD3 (OKT3; kind gift from Ortho Biotech, Neuss, Germany) in the presence of 5 × 104 irradiated (30 Gy) autologous PBMCs in 96-well U-bottom plates in 200 μL of RPMI 1640 medium supplemented with 10% fetal calf serum (Seromed, Berlin, Germany), 2 mmol/L glutamine, 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 1% nonessential amino acids (all PAN Biotech, Aidenbach, Germany), 50 U/mL penicillin, 50 μg/mL streptomycin, and 5 × 10−5 mol/L 2-mercaptoethanol (all Gibco BRL, Karlsruhe, Germany). To determine their suppressive activity, titrated numbers of CliniMACS-purified CD4+CD25+ T cells or FACS-purified CD4+CD25high, CD4+CD25int, or CD4+CD25− T cells were added to cultures of 5 × 104 Tresp cells to obtain the indicated ratios and stimulated with 100 ng/mL anti-CD3 in the presence of autologous antigen-presenting cells. All cultures were incubated for 4 days and labeled with 18.5 MBq per well 3H-thymidine (Hartmann Analytics, Braunschweig, Germany) for the last 18 hours. The 3H-thymidine incorporation was measured by liquid scintillation counting (Top Count; Perkin Elmer, Rodgau-Jügesheim, Germany). All assays were performed in triplicate. Suppression was calculated by the following formula:
FOXP3 Quantitative Real-Time Polymerase Chain Reaction and Western Blotting Total RNA from 2 × 105 cells was extracted by using the RNeasy Micro kit (Qiagen, Hilden, Germany), and complementary DNA was synthesized by using the Reverse Transcription System (A3500; Promega, Mannheim, Germany) with oligo(dT)15 primers. Reverse transcriptase-polymerase chain reaction (PCR) was performed by using previously described primers for FOXP3 and glyceraldehyde-3-phosphate dehydrogenase [15], the QuantiTect SYBR Green PCR Kit (Qiagen), and Lightcycler 2.0 (Roche, Mannheim, Germany). For immunoblotting, lysates from 2 × 105 cells were separated in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Millipore, Schwalbach, Germany). Polyclonal goat anti-human FOXP3 (Abcam, Cambridge, UK) and horseradish peroxidase–labeled rabbit anti-goat immunoglobulin (Dako, Hamburg, Germany) were used together with the ECL detection system (Amersham, Freiburg, Germany). Blots were stripped and reprobed with polyclonal rabbit anti-actin (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) and horseradish peroxidase–labeled goat anti-rabbit immunoglobulin (Dako) for loading controls. Results Efficient Enrichment of CD4+CD25highFOXP3+ T Cells by Magnetic Cell Separation under GMP Conditions Human PBMCs comprise both CD4+ and CD4− cells that coexpress CD25, and activated B cells represent the main constituent of the CD4−CD25+ cell population. In laboratory-scale experiments using magnetic bead separation, we determined that a primary depletion of CD19+ B cells followed by 3 selection cycles for CD25 allows the preferential retention of CD4+CD25high T cells (as compared with CD4+ T cells that express low CD25 levels) in only 2 separation steps (data not shown). To test whether this isolation strategy is suited for potential clinical trials, we conducted a study with clinical-grade reagents comprising 6 cell separations from leukapheresis products of different donors, 1 of which was performed outside a certified GMP laboratory and all others in strict compliance with GMP guidelines. Summarized results from all 6 separations are shown in Table 1. The initial leukapheresis products contained 4.4 to 15.8 × 109 nucleated cells. On average, 18.2% (range, 15.0%-21.5%) of the cells coexpressed CD4 and CD25, and on average 1.9% were CD4+CD25high T cells (range, 1.1%-2.9%). After magnetic cell separation, 63 to 262 × 106 cells were recovered in the target cell fraction (average, 1.72% of the starting population; range, 0.89%-2.47%), which contained less than 0.01% B cells, only 7.3% CD4− cells (range, 2.9%-14.3%), most of which were CD8+CD25+ T cells (data not shown), and 92% CD4+ T cells (range, 85.5%-96.0%). Only 0.5% of the isolated cells were CD4+CD25− (range, 0.2%-1.1%), whereas 91.5% (range, 85.2%-95.6%) of the cells expressed both CD4 and CD25. The target cell fractions were highly and preferentially enriched for CD4+CD25high T cells that constituted on average 49.5% (range, 30.3%-63.1%) of the isolated cells. The recovery of CD4+CD25high T cells as calculated from their frequencies in the initial leukapheresis products was 46.5% (range, 25%-66%). Representative FACS data illustrating the preferential retention of CD4+CD25high T cells after the repetitive magnetic enrichment for CD25-expressing cells are depicted in Figure 1A. Within the leukapheresis product (left panel), CD4+CD25high T cells are identified not only by their high CD25 expression, but also by their slightly lower CD4 levels as compared with CD4+CD25int or CD4+CD25− T cells. After B-cell depletion and incubation with anti-CD25 beads, the overall staining level for CD25 was decreased as a result of competition of the fluorescence-labeled antibody with the attached beads, but the CD25high population can still be clearly identified. In addition, the proportion of CD4−CD25+ cells was reduced because of the depletion of activated B cells coexpressing CD25 (second panel). After 3 repetitive enrichment cycles for CD25, an efficient retention of CD4+CD25high T cells in the target cell fraction was achieved (third panel), whereas residual CD4+CD25high T cells were almost undetectable within the CD25-depleted negative cell fraction (right panel). To verify the enrichment of Treg cells, leukapheresis products and target and CD25-depleted cell fractions were analyzed on a single cell level for the expression of the Treg cell–specific transcription factor FOXP3. As depicted in Figure 1B, FOXP3 was preferentially expressed by CD4+CD25high T cells within PBMCs, whereas CD4+ T cells with intermediate CD25 levels contained fewer FOXP3+ cells and the CD4+CD25− subpopulation contained almost none (left panel). As expected, the CD4+ T cells obtained after magnetic enrichment for CD25 were highly enriched in FOXP3+ cells (Figure 1B, middle panel), whereas CD25-depleted cells contained only very few FOXP3+ cells (Figure 1B, right panel). In summary, these data document a more than 25-fold preferential enrichment of CD4+CD25highFOXP3+ T cells from leukapheresis products by this 2-step, GMP-complying isolation strategy. Phenotypic and Functional Characteristics of Cells Isolated by Magnetic Cell Separation under GMP Conditions Apart from FOXP3, a constitutive, high expression of intracellular CTLA-4 represents another well-documented characteristic of natural CD4+CD25+ Treg cells that has also been suggested to contribute to their suppressive function [13]. Analysis of CD4+ T cells in the target cell fraction by flow cytometry revealed a biphasic expression pattern for intracellular CTLA-4, reflecting the uniformly high expression within the CD4+CD25high subpopulation versus the low expression in CD25int and CD25− T cells (Figure 2A). To analyze the potential effect of CD4+CD25intFOXP3−CTLA-4low T cells on the functional activity of the target cell product, we isolated CD25high, CD25int, and CD25− cells by high-speed FACS sorting from the CD4+ T-cell populations in the CD25-enriched target cell fraction and the CD25-depleted negative cell fraction (Figure 2B), and compared their FOXP3 expression levels, anergy, and suppressive capacity with those of the corresponding unfractionated target cell product. As anticipated from the phenotypic analyses shown in Figure 1B, the target cell product was considerably enriched in FOXP3-expressing cells, as determined on the messenger RNA level by quantitative real-time PCR and on the protein level by Western blotting (Figure 2C and D, respectively). Further subdivision of the target cell product by FACS into CD4+CD25high and CD4+CD25int cells resulted in a coseparation of FOXP3-high–expressing and FOXP3-low–expressing cell populations, respectively. In contrast, neither the CD25-depleted cell fraction obtained by magnetic separation nor the CD4+CD25− T cells isolated from this population showed any substantial FOXP3 expression. The functional characteristics of the target cell products obtained by magnetic separation under GMP conditions and of CD4+CD25high T cells FACS-purified from these products were surprisingly similar. Both populations showed a comparable hypoproliferative response (anergy) after polyclonal stimulation, whereas purified CD4+CD25− T cells proliferated vigorously and CD4+CD25int T cells proliferated at intermediate levels (Figure 3A). In addition, the suppressive capacity of the target cell product, as measured in standard suppression assays, was comparable to that of FACS-purified CD4+CD25high T cells, whereas CD4+CD25int cells showed only moderate activity and CD4+CD25− T cells showed no suppressive activity (Figure 3B). In summary, cells isolated from leukapheresis products by this GMP-complying magnetic cell separation protocol were highly enriched in CD4+ T cells with phenotypic and functional characteristics of natural CD4+CD25+ Treg cells. Discussion CD4+CD25+ Treg cells are pivotal for the induction and maintenance of self-tolerance during ontogeny and thereby protect from autoimmunity [19]. It is therefore conceivable that their regeneration also contributes to tolerance induction after allogeneic SCT [20, 21]. In mouse models of allogeneic SCT, we and others have shown that their suppressive activity could be exploited for protection from GVHD. Several of these studies confirmed that donor CD4+CD25+ Treg cells themselves do not induce GVHD but protect recipients from GVHD induced by CD25− T cells when cotransplanted in high numbers [1, 2, 3]. Depending on the strain combinations and experimental setups, protection ranged from reduced GVHD severity to complete inhibition of clinical manifestations and GVHD-associated lethality. However, cotransplanted Treg cells did not induce an overall paralysis of the immune system, because both the graft-versus-hematopoiesis effect and the graft-versus-tumor effect of transplanted CD25− T cells were maintained [4, 22]. Even their delayed transfusion ameliorated GVHD, thus suggesting that Treg cells could also be administered for the treatment of GVHD provided that the disease course is not too aggressive [6]. To test these findings in clinical trials, we established an isolation strategy for human Treg cells under GMP conditions based on magnetic cell separation. Because of the lack of exclusive surface makers for CD4+FOXP3+ Treg cells, their constitutive expression of CD25 was used for magnetic enrichment. Although this approach has already been applied in many laboratories for experimental purposes, it has been unclear whether it would be suited for the large-scale purification of Treg cells under GMP conditions for clinical applications. To this end, we sought to develop a strategy that ensures the efficient and reliable enrichment of human CD4+CD25high T cells from leukapheresis products with a minimal number of reagents and selection steps. The final protocol presented here comprises 1 cycle of B-cell depletion with anti-human CD19 beads followed by 3 repetitive enrichment cycles for CD25+ cells. B-cell depletion was included to eliminate activated CD25+ B cells that would contaminate Treg cell products to various degrees, depending on the blood composition of the donor. More importantly, we reasoned that depletion of B cells from such products might reduce the risk of Epstein-Barr virus–associated lymphoproliferative disease in patients receiving Treg cells early after SCT. In fact, B cells were almost undetectable in the cell products generated under GMP conditions, and the low percentage of CD4−CD25+ cells still present in the target cell fraction mainly consisted of activated CD8+ T cells and a few natural killer cells (data not shown). It was recently shown that a single magnetic cell-separation step efficiently depletes human Treg cells from leukapheresis products but is insufficient for their enrichment [23]. In our protocol, the repetitive enrichment for CD25 ensured the preferential retention of cells with high CD25 expression as compared with those with intermediate CD25 levels and resulted in an almost-complete elimination of CD25− T cells (Figure 1A). Of note, the 3 positive selection cycles are performed in an automated fashion such that retained cells are re-exposed to the magnet without additional washing or bead incubation steps that would prolong the procedure and risk increased cell loss. Recovery of CD4+CD25high T cells from leukapheresis products was on average 46.5%, and the target cell products contained 63 to 262 × 106 cells, thus clinically relevant numbers for adoptive T-cell transfer studies. Because these cellular products contained not only CD4+CD25high T cells, but also CD4+ T cells with intermediate CD25 expression levels, we FACS-purified these subpopulations and examined them in detail. As expected, CD4+CD25high T cells were uniformly positive for FOXP3 and CTLA-4 and potently suppressed non-Treg cells in functional assays. Although the molecular mechanisms responsible for the potent suppressive activity of CD4+CD25+ Treg cells are still not identified, it is now well established that FOXP3 expression is essential for their thymic generation and peripheral function. A lack of function mutation in the FOXP3 gene results in the loss of CD4+CD25+ Treg cells and lethal autoimmunity in mice and humans, as observed in the scurfy mouse strain [14, 16] and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients [24]. The CD4+CD25int cell population was more heterogeneous than the CD4+CD25high T cell population and contained FOXP3+CTLA-4+ Treg cells as well as FOXP3−CTLA-4low memory and recently activated T cells (compare Figure 1B and 2A). In functional assays, these CD4+CD25int T cells showed a less pronounced but still substantial suppressive effect, thus suggesting that the FOXP3+ cells within this subpopulation indeed represent functional Treg cells. Hence, the frequency of Treg cells within the target cell products is even higher than that deduced from the quantification of CD4+CD25high T cells (Table 1; Figure 1). This might explain why magnetically enriched Treg cells and FACS-purified CD4+CD25high T cells show almost the same suppressive activity in a standard in vitro suppression assay (Figure 3). Because protection from GVHD in murine studies was observed when donor Treg cells were cotransplanted at 1:1 or 1:2 ratios with non-Treg cells, we aimed at a purity of at least 50% for FOXP3+Treg cells when developing our isolation strategy. As this goal was achieved, we have now initiated a phase I clinical trial examining the adoptive transfer of donor Treg cells in recipients of allogeneic stem cell grafts. Such studies aim to reduce the risk for GVHD after allogeneic SCT without completely abrogating the beneficial donor T-cell effects, such as immunity to infection, facilitation of engraftment, and graft-versus-tumor activity. Acknowledgments We thank Kathrin Dummer, Andrea Havasi, Nancy Hahn, Angelika Lukas, and Doris Haas for excellent technical assistance. 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1 Department of Hematology and Oncology, University Hospital Regensburg, Regensburg, Germany 2 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Regensburg, Germany 3 Miltenyi Biotec, Bergisch Gladbach, Germany 4 Institute of Immunology, University Regensburg, Regensburg, Germany  Correspondence and reprint requests: Matthias Edinger, MD, Department of Hematology and Oncology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
PII: S1083-8791(06)00043-7 doi:10.1016/j.bbmt.2006.01.005 © 2006 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved. | |
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