Human regulatory T cells against minor histocompatibility antigens: ex vivo expansion for prevention of graft-versus-host disease

Graft-versus-host disease (GVHD) remains the main cause of treatment-related mortality after allogeneic bone marrow transplantation.^1,2  GVHD is mediated by donor CD4⁺ and CD8⁺ T cells, which inflict damage to the recipient target organs including the skin, intestines, liver, and lung.³  In the case of HLA-identical sibling transplants, the risk of acute GVHD is increased in male recipients of female grafts, denoting pathogenic alloreactivity against male-associated H-Y antigens. These and other minor histocompatibility antigens (mHAs) produce antigenic peptides presented by HLA molecules on recipient or donor antigen-presenting cells (APCs) that sensitize alloreactive donor T cells and cause GVHD.^4-7 

Regulatory T cells (Tregs) are naturally occurring or induced during a tolerogenic immune response.⁸  Tregs are distinguished by constitutive expression of the interleukin 2 (IL-2) receptor α chain (CD25)⁹  and the transcription factor Foxp3.^10,11  Their potent, antigen-driven immune suppression and their dominant role in transplantation tolerances have made Tregs an attractive candidate for adoptive immunotherapy.¹²  Studies in rodents with adoptive transfer of in vitro–expanded natural or induced Tregs have shown prevention of lethal GVHD^13,14  and, in most examples, preservation of graft-versus-tumor responses.^15,16  Tregs express a T-cell receptor repertoire that enables them to recognize self-antigens¹⁷  or alloantigens.^15,18  Given their low frequency in human blood, several groups have explored ex vivo Treg expansion for therapeutic application and cultured Treg-retained suppressive activity.^10,11,19-21  In contrast to polyspecific Tregs, antigen-specific Tregs produce selective suppression of alloresponses with no effect on third-party responses and facilitate alloantigen-specific tolerance after marrow transplantation and organ grafting in rodents.^14,15,22-24  Previously, we measured the frequency, growth requirements, and functional phenotype of ex vivo–expanded human Tregs against disparate HLA.¹⁸  While CD8 T cells specific for mHAs expressed on leukemic cells were isolated, expanded in vitro, and infused into allogeneic bone marrow transplant recipients to prevent or treat leukemia relapse,²⁵  there are no reports on the identification of mHA-specific Tregs in humans.

In the present study, we have detected mHA-specific, functional CD4 Tregs and cloned them. We measured the blood frequency of mHA-specific Tregs against HLA-identical siblings and used good manufacturing practice (GMP) for expanding mHA-specific Tregs in numbers sufficient for therapeutic application. The expanded Tregs maintained viability, antigen-specific suppression, transforming growth factor β (TGF-β) production, demethylation of the Treg-specific Foxp3 demethylation region (TSDR), and Foxp3 expression. The contaminating CD8 and CD4 conventional T cells in the final product were rare and anergic in response to specific antigen. With these data in hand, we have planned a first-in-humans phase 1 study for the prevention of acute GVHD in HLA-identical sibling transplants.

Eligible for the study were sibling pairs matched for HLA-A, B, C, DRB1, and DQB1. Typing for HLA-DPB1 was not performed because the probability of a DQB/DPB recombination is less than 1%. The study protocol was approved by the University of South Florida institutional review board. Subjects donated 100 mL of blood or cytapheresis after providing written informed consent in accordance with the Declaration of Helsinki. Tregs were isolated from blood samples using the CD4⁺CD25⁺CD127⁻ Treg isolation kit II (Miltenyi Biotech), involving negative selection of CD4⁺CD127⁻ T cells followed by positive selection of CD25. For some experiments, CD4⁺CD25⁺CD127⁻ Tregs were instead isolated on a BD FACSAria II high-speed cell sorter (BD Biosciences) under sterile conditions. Purified Treg populations obtained from magnetic or flow sort methods were 95% to 99% pure. For large-scale expansion, CD25⁺ Tregs were purified from leukapheresis products by single-step CD25-specific beads (catalog #274-01) using CliniMACS (Miltenyi Biotech) in the Cell Therapy Core Facility at the Moffitt Cancer Center. The CD25-specific beads were optimized by the manufacturer to select 6 × 10⁹ highly expressing CD25 cells from 40 × 10⁹ white blood cells from leukapheresis in a volume of 380 mL. Cells were washed with isotonic saline supplemented with EDTA and 0.5% human serum albumin, incubated with paramagnetic Fe₃O₄tagged CD25 monoclonal antibodies at 4°C to 8°C for 15 minutes, and loaded onto the CliniMACS for automated separation. The tagged cells were pooled in the collection bag, washed, and resuspended in RPMI medium supplemented with 10% human AB-positive serum.

For small-scale experiments, monocytes were separated by CD14 immunoabsorption, whereas for large-scale experiments, monocytes were enriched by plastic adherence. Monocyte-derived DCs were produced by culture in X-VIVO 15 (Lonza) or CellGenix DC serum-free medium for 6 days in 1000 U/mL GM-CSF (R&D Systems) and 1000 U/mL IL-4 (R&D Systems). DCs were γ-irradiated (3000 cGy). Although the potential contaminating conventional CD4 T cells (Tconvs) are sensitive to rapamycin, Tregs express PTEN, a negative regulator of the PI3-K/AKT/mTOR pathway, and are relatively resistant to rapamycin.²⁶  The additive effect of IL-15 and IL-2 is critical for maximal Treg expansion, bcl-2 expression, and survival in the presence of rapamycin.¹⁸  Freshly isolated Tregs and HLA-matched sibling DCs or self-DCs were seeded at a 1:1 ratio in the presence of IL-2 (10 U/mL; R&D Systems), IL-15 (10 ng/mL; R&D Systems), and rapamycin (100 ng/mL; Millipore). The optimal DC:Treg ratio was determined in titration experiments (data not shown) and found to be between 2:1 and 1:2 at optimal cell density. Culture medium was RPMI 1640 plus glutamine supplemented with 0.02 mM pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated human pooled AB serum (SeraCare or Omega Scientific). Cells were cultured at 37°C, 95% humidity, and 5% CO₂ and in 96-well round- or flat-bottom plates (Nunc) or 225 cm² tissue culture flask, as indicated. For small-scale mHA-specific Treg expansion, approximately 1 × 10⁵ Tregs were cultured with 1 × 10⁵ irradiated HLA-matched recipient DCs in 96-well flat-bottom plates. Treg cultures were fed with fresh medium containing cytokines plus rapamycin on days 6, 8, and 10. To determine expansion kinetics of mHA-specific Tregs, the frequency of antigen-specific Tregs was calculated by limiting dilution analysis (LDA) at the start (day 0) and end (day 12) of the culture.

Proliferation was analyzed by ³H-thymidine incorporation using a gas scintillation counter (Matrix 96 β counter; Canberra Packard). Cells were pulsed with 1 μCi per well ³H-thymidine for the last 18 hours in culture and harvested on day 6 to measure proliferation. Results are expressed in counts per minute (CPM) of at least triplicate measurements. Where indicated, culture supernatants were collected and analyzed for TGF-β and tumor necrosis factor (TNF) levels by enzyme-linked immunosorbent assay (ELISA; eBioscience). To determine the fraction of replicating cells, cells were cultured in a glucose-free formulation of RPMI 1640 supplemented with the addition of [6, 6-²H₂] glucose (Cambridge Isotope Laboratories, Cambridge, MA). Deuterium-labeled glucose is taken up by replicating cells, metabolized through the pentose-phosphate pathway to phosphoribose-pyrophosphate, and incorporated into nucleic acids.^27,28  After 12 days, genomic DNA-associated deuterium was quantified by mass spectrometry as described elsewhere.²⁹  To assess for anergy, end-of-culture or sorted subpopulations were sorted with a fluorescence-activated cell sorter and cultured with 1 × 10³ irradiated allogeneic DCs or self-DCs in the presence or absence of IL-2 (10 U/mL) for 6 days. Culture supernatants were collected and tested for TGF-β or TNF, and remaining cells were pulsed with ³H-thymidine to measure proliferation. Suppression by Tregs was assayed in a mixed leukocyte culture assay. Expanded Tregs were titrated and mixed with 1 × 10⁵ effectors (CD4⁺CD25⁻ T cells) and mixed with 1 × 10⁴ irradiated DCs from an original or third-party stimulator. Proliferation was analyzed by ³H-thymidine incorporation.

The frequency of mHA-specific Tregs or Tconvs in blood or apheresis samples was determined by LDA as described previously.²⁰  Briefly, T cells were seeded at various numbers increasing by 2-fold from 6 to 15 000 per well and cultured with a fixed concentration of DCs (2 × 10³) in a minimum of 10 replicates in U-bottomed 96-well plates. Treg cultures were supplemented with IL-2 (10 U/mL) and IL-15 (10 ng/mL) cytokines with rapamycin (100 ng/mL) and Tconv cultures with IL-2 alone. Wells were considered positive if CPM (or cytokine concentration) was greater than the mean plus 3 standard deviations (SDs) over the sum of negative control responder and stimulator cells, with each cultured alone. Online extreme LDA (http://bioinf.wehi.edu.au/software/elda/index.html) was used to calculate the frequency and 95% confidence interval (95% CI) of replicating T cells. We previously validated LDA by testing carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled Tregs sorted 8 days after alloantigen stimulation using DCs from HLA-incompatible donors.¹⁸  LDA showed enrichment of alloreactive Tregs among CFSE-negative (replicated) compared with CFSE-positive (resting) Tregs, but only 9% of CFSE-negative Tregs were alloreactive. Because CFSE estimates approach the frequency of antigen-specific T cells detected by HLA:peptide tetramers and γ-IFN release, the sensitivity of LDA may be as low as 9%.³⁰  T-cell cloning was performed as detailed in supplemental “Materials.” The H-Y–associated genes DBY, UTY, SMCY, or DFFRY-2, encoded by the Y chromosome, were cloned into a Moloney murine leukemia virus–based retroviral vector LZRS with green fluorescent protein tag and kindly provided by M. Griffioen and J. H. Falkerburg (Leiden University Medical Center, Netherlands).

Purity of Tregs and viability testing by Live/Dead Yellow (Life Technologies) were analyzed before and after Treg expansion using multicolor flow cytometry (LSR II; BD Biosciences). Cells were surface stained with CD3-PB, CD4-A700, CD127-A647, CD25-APC-Cy7, CD62L-PE-Cy7, CD45RA-PerCP-5.5, and CCR7-PE (BD Biosciences). As noted, cells were stained with CD3-PB, CD4-A700, CD127-A647, and CD25-PE-Cy7 and then fixed and permeabilized using the Fix and Fix/Perm buffer (eBioscience) according to the manufacturer’s instructions and stained with Foxp3-PE (BD Biosciences) or BCL-2-PE and Foxp3-A488. A separate panel included staining with CD3-APC-Cy7, CD8-PE, CD16-A700, CD56-PerCP5.5, CD19-PE-Cy7, CD14-PB, and CD11c-APC. DC purity was checked with CD3-APC-Cy7, CD8-PE, CD16-A700, CD56-PerCP5.5, CD19-PE-Cy7, CD14-PB, and CD11c-APC. Data were analyzed with FlowJo software version 9.3 (Tree Star). Isotype controls or fluorescence minus one was used for markers or gate settings.

This highly conserved region within the human Foxp3 gene is demethylated exclusively on Tregs and not in any other blood-cell or non–blood-cell types. The percentage FoxP3 demethylation of naïve or expanded Tregs was measured using quantitative polymerase chain reaction (qPCR) as described by Wieczorek et al in 2009, with minor modification.³¹  Details of the assay are provided in supplemental “Materials.”

Differences in frequency or proliferation of alloreactive T cells were analyzed by the 2-tailed Student t test, and P < .05 was considered significant. A 2-way analysis of variance (ANOVA) model was used to calculate the specificity of the suppressive effect of each Treg clone.

We sought to characterize the responses of purified Tregs from normal individuals against mHAs expressed by their HLA-identical sibling APCs. CD4⁺/CD25⁺/CD127⁻ Tregs from volunteer donors were enriched by CD25 immunoabsorption, and cultured with the sibling DC, in the presence of IL-2, IL-15, and rapamycin. Under these conditions, we detected a 15- to 50-fold higher ³H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical, mHA-incompatible sibling DCs compared with self-DCs (Figure 1A).

To demonstrate that Tregs are specific for mHAs, we selected H-Y antigens as targets because they are biologically relevant and the frequency of H-Y–specific Tconvs is high,^4-7  and we surmised that H-Y-specific Tregs would be detectable. Blood Tregs were purified from a female donor, and Treg lines were expanded by stimulation with DCs from her HLA-identical brother. Responding T cells that secrete TGF-β were pooled and cloned by limiting dilution. We obtained 10 stable Treg clones that retained TGF-β secretion in response to the brother’s DCs, but not self-DCs, and produced only small amounts of TNF (Figure 1B). Conversely, Tconv clones secreted TNF, but not TGF-β, in response to the allogeneic DCs (Figure 1C). To assess the specificity, we tested cloned Tregs against self–Epstein-Barr virus–transformed B lymphoblastoid cell lines (self-LCLs) transduced with one of the H-Y-associated genes DBY, UTY, DFFRY-2, SMCY, or an empty vector. Of the 10 Treg clones tested, 2 were specific for DBY, 1 for UTY, and 1 for DFFRY-2, while the specificity of the other clones remains unknown (Figure 1D). Cloned Tregs expressed high levels of Foxp3 (Figure 1A inset) and demonstrated mHA-specific suppression (Figure 1E,F). For the first time, we demonstrated that it is possible to detect mHA-specific Treg alloresponses in humans.

We employed LDA to measure the precursor frequency of mHA-specific CD4 Tregs and CD4 Tconvs after stimulation of the purified subsets by HLA-identical sibling DCs. Both Tregs and Tconvs showed specific alloreactivity against sibling DCs when compared with self-DCs (Figure 2A,B). The median precursor frequency of mHA-specific Tregs is 1.6 (range, 0.4-8.4) cells per million total blood CD4 T cells, while the median frequency of mHA-specific Tconvs is 25.4 (range, 8.7-89) per million total blood CD4 T cells (Table 1). Among 16 HLA-identical sibling pairs tested in any gender combination, there was a 24-fold (range, 8-fold to 39-fold) excess of mHA-specific Tconvs over mHA-specific Tregs in the donor blood (Table 1), and the mHA-specific Tconv:Treg ratio had poor correlation (R² = 0.24) with the total Tconv:Treg ratio (not shown). The predominance of alloreactive Tconvs over Tregs likely drives the alloimmune responses causing GVHD and organ graft rejection, and the mHA-specific Tconv:Treg ratio may be a more precise predictor of alloreactivity than the total Tconv:Treg ratio.

Tregs were enriched from blood samples of normal donors to >95% purity and expanded in culture for 12 days by stimulation with HLA-identical sibling DCs in the presence of IL-2, IL-15, and rapamycin. To determine the degree of expansion of mHA-specific Tregs, we measured their frequency by LDA in the start and end of culture populations. The frequency estimate of stimulator-specific Tregs was 1/14, 355 (95% CI, 1/24, 075-1/8,559) in the starting population and 1/37 (95% CI, 1/56-1/24) in the end population (Figure 2C). LDA results by ³H-TdR uptake and TGF-β release showed similar estimates of mHA-specific Treg frequency, and there was 98% concordance for culture positivity by ³H-TdR uptake and TGF-β. No TNF production was detected (Figure 2D). In 7 of 8 experiments, there was contraction of the total cell numbers by end of culture; however, the frequency of mHA-specific Tregs increased so the total numbers of mHA-specific Tregs increased a median of 330-fold (range, 155-fold to 405-fold; see Table 2). The ratio of mHA-specific Tconvs to Tregs in blood varies between 8 and 39 (median, 24; Table 1), so the magnitude of mHA-specific Treg expansion described here should allow exceeding the starting blood content of mHA-specific Tconvs.

In coculture suppression assay, expanded Tregs were titrated in various ratios with naïve autologous CD4⁺CD25⁻ Tconvs and stimulated by the original HLA-identical sibling DCs or HLA-incompatible third-party DCs. Tconv proliferation was reduced by 50% at a ratio of ∼1:5000 and by >90% at a Treg:Tconv ratio of ∼1:10. No suppressive effects were observed in the presence of a third-party stimulator (Figure 2E). Thus, Tregs maintained antigen-specific suppressive function after large-scale ex vivo expansion under GMP conditions as intended for in vivo use. To investigate whether expanded Tregs had maintained demethylation of the Foxp3 gene promoter TSDR that is required for stable Foxp3 expression, we sorted CD4⁺CD127⁻CD25⁺ Tregs and CD4⁺CD127⁺CD25⁻ Tconvs by flow cytometry before or after expansion and assessed the TSDR by qPCR. We found that Treg TSDR at the end culture was demethylated to the same degree as before culture (Figure 2F).

To assess whether the expanded Treg-enriched populations included immune-competent T cells, we flow-sorted Tregs, CD4 Tconvs, and CD8 T cells (termed “non-CD4 cells” in Figure 2G,H) and stimulated the entire end-of-culture population or each purified subset with allogeneic DCs or self-DCs in the presence or absence of IL-2. There was no significant response to antigen in any of the populations tested in the absence of IL-2. In the presence of IL-2, only the entire end-of-culture population and the purified Tregs responded to alloantigens by ³H-TDR uptake and TGF-β release (Figure 2G,H). We concluded from these experiments that contaminating CD4 T conv and CD8 T cells were tolerized to the specific mHAs while in culture with Tregs and rapamycin.

Expanded Tregs retained high expression of CD25 that is required for expansion in response to IL-2, Foxp3 that is required for suppressive function, BCL-2 that is required for survival, chemokine receptor CCR7, and lymphoid homing receptor CD62L, predicting that they can migrate and home to lymphoid tissue in vivo (Figure 3).

To assess the feasibility of Treg expansion to a clinical scale, we obtained mononuclear cells by apheresis from 3 donors. In each of the experiments, donor cells were incubated with GMP-grade anti-CD25 immunomagnetic beads in the cell therapy laboratory and enriched to a 76% to 82% purity using CliniMACS. The Tregs yield was a mean of 25% of the starting Tregs and only 3% of the starting mononuclear cells. Flow cytometry analyses of cells before and after CliniMACS purification and after ex vivo culture are shown in Figure 3A. We found contamination by CD4 Tconv (mean 7%), CD8 (mean 1.4%), and CD19 (mean 4%) cells after purification (Table 3). After freezing and thawing, the Treg recovery was a mean of 64% and the Treg phenotype and the frequencies of contaminants were unchanged. Before coculture, GMP-grade monocyte-derived DCs had an average purity of 59% (range, 30%-82%) based on a CD11c⁺/CD14⁻ phenotype and expressed HLA-DR, CD83, and CD86 (Figure 3B,C). A fraction of the purified Tregs were cultured with irradiated (30 Gy) DCs from an HLA-matched sibling at an approximate 1:1 ratio, with IL-2, IL-15, and rapamycin. After 12 days, the mean viability of the cells in culture was 89% and the mean Treg purity was 78% by flow cytometry. There was contamination by a mean of 6.9% CD4 T convs, 1.2% CD8 T cells, and 2.4% B cells from the original Treg-enriched population and 1.4% natural killer cells and 1.5% DCs from the irradiated APCs, and there were no detectable monocytes. The mHA-specific Tregs of the 3 pairs tested expanded by 281- to 405-fold, in each case exceeding the numbers of mHA-specific Tconvs in the starting apheresis (Table 4).

We employed a second GMP-compliant assay to measure the fraction of Tregs replicating in response to antigen stimulation. Tregs were cultured in media that contained enriched [6, 6-²H₂] glucose, and the fraction of Treg-associated DNA that had incorporated deuterium was measured by mass spectrometry at end of culture.^27-29  We detected 4.4% and 10.2% deuterium enrichment in the DNA of the cultured Tregs in 2 large-scale expansion experiments, well over the 0.1% background signal in unlabeled culture and representing 7.1% and 16.5% newly divided cells, respectively. These data are in corroboration with the LDA results showing expansion, as evidenced by enrichment of mHA-specific Tregs up to 0.8% and 1.8% by the end of culture. Deuterium labeling did not affect Treg expansion as measured by LDA, suppressive activity, or Foxp3 demethylation. Deuterium-labeled Tregs secreted TGF-β and minimal TNF in the presence of allogeneic DCs (Figure 4). An advantage of this approach to measure DNA replication is that trace-labeled cells can be detected after in vivo transfer: T-cell subsets from blood or other tissues are sorted by flow cytometry, and DNA is extracted and tested by mass spectrometry for deuterium enrichment.

In this article, we identified, quantified, and cloned Tregs specific against human mHAs and developed a GMP-compliant protocol to expand sufficient numbers of mHA-specific Tregs for clinical trial testing. To our knowledge, this is the first report of mHA-specific human Tregs. Because mHAs drive transplantation responses, the technology reported here might be applied to prevent GVHD and organ graft rejection and adapted to control immune responses against pathogenic antigens in autoimmune disorders.

Among many human mHAs already identified,^4-7,32  we selected H-Y antigens to test the specificity of Treg clones because it has been unequivocally demonstrated that H-Y antigens are responsible for pathogenic transplant reactions of females against males and they elicit a broad immune response.^33,34  We isolated Treg clones specific for H-Y–associated DBY,⁶  UTY,^5,35  and DFFRY⁴  mHAs. The Treg clones against DBY or UTY showed significantly more suppression against Tconvs specific for the same mHA compared with Tconvs specific for another. Therefore, we envision that antigen-specific Tregs may be exploited to regulate GVHD-causing responses against ubiquitous mHAs, while selectively sparing antitumor responses against lineage-restricted mHAs or tumor-associated antigens. In contrast, polyspecific Tregs expanded with artificial APCs are expected to produce broader immune suppression and may be more likely to suppress antitumor immunity.^15,22 

Several cytokines have been implicated in Treg-mediated suppression, including TGF-β, IL-10, and IL-35³⁶ . IL-35 is expressed by a subset of ICOS-positive inducible Tregs,^37,38  whereas our investigation has focused on natural Tregs. Our initial experiments¹⁸  found that TGF-β production separates more clearly alloreactive Tregs from helper T cells than IL-10, and therefore in this study we adopted TGF-β production as a selection criterion for alloreactive Treg clones.

The observation that Treg clones totally inhibit responses by Tconv clones at the ratio of 1:3 (Figure 1E,F) is consistent with murine data demonstrating that adoptive transfer of polyspecific Tregs at a Treg:Tconv ratio of approximately 1:1 is necessary for preventing GVHD.^13,14,19  These data predict that the minimum number of mHA-specific Tregs effective for the prevention of GVHD has to approximate the number of mHA-specific Tconvs in the stem cell graft. A few studies estimated the frequencies of alloreactive Tconvs in the blood of hematopoietic cell transplant donors against the graft recipient.^39,40  In HLA-identical siblings, Theobald et al⁴¹  determined that the precursor frequency of mHA-specific, IL-2–secreting donor T cells in the blood is 1 per 100 000. Using a more sensitive and rapid (6 days vs 14 days) limiting dilution assay that employs DCs as APCs, we found that the frequency of mHA-specific, TNF-producing proliferative Tconvs is 2.5 per 100 000 blood CD4 T cells (Table 1). We report here that the ratio of mHA-specific Tregs:Tconvs in normal donor blood is on average 1:24. These findings are largely applicable to mobilized blood stem cell grafts, because granulocyte-colony stimulating factor, which is used to mobilize stem cells, barely favors Treg over Tconv mobilization.⁴²  Our data are consistent with the hypothesis that the predominance of alloreactive Tconv over Treg in the graft drives the immune response to cause GVHD and that the expansion of mHA-specific Tregs by an average of 24-fold in a blood cell graft may be adequate to prevent GVHD. Others computed that much greater expansion of polyspecific Tregs is required for effective immunotherapy.^21,43 

A high degree of Treg purity can be attained by multistep immunoabsorption on magnetic columns using multiple antibodies or fluorescence-activated cell sorting.^18,44-46  Although these procedures are highly effective in the research laboratory, the cost of multiple GMP-compatible immunobeads as used by Di Ianni et al,⁴⁶  or the lack of broadly available cell sorters for clinical use as proposed by Sagoo et al,⁴⁴  would make the routine clinical application unfeasible. Therefore, we elected to employ a single-step purification using clinical-grade CD25-specific paramagnetic beads that attained an average 80% Treg purity (range: 75%-82%; Table 3) and relied upon the subsequent T cell culture with alloantigen, excess Tregs, and rapamycin to tolerize contaminating Tconvs and CD8 (Figure 2 G,H).⁴⁷  By the end of culture, the Treg purity met previously accepted specifications⁴⁸  and the Tconv and CD8 cells appeared totally anergic in response to alloantigen and IL-2. A carefully titrated dose-escalation phase 1 study of GVHD prophylaxis is planned to test the in vivo safety of alloreactive Tregs grown under the conditions described here (NCT01795573).

We employed 2 GMP-compliant techniques to measure Treg expansion against mHAs: LDA and DNA labeling with deuterium-containing glucose. We¹⁸  and others^49,50  found that LDA underestimates the frequencies of allospecific Tregs and Tconvs. However, by measuring frequencies before and after culture, the LDA has the ability to providing an accurate measure of the expansion. Although in most cases the total number of cells contracted slightly by the end of culture, the frequency of mHA-specific Tregs increased by 155- to 405-fold (Table 2). Large-scale expansion experiments in the GMP-compliant Cell Therapy Core Laboratory at Moffitt showed that numbers of mHA-specific Tregs in the final product consistently exceeded the numbers of mHA-specific Tconvs in the start apheresis (Table 4) and the numbers of Treg that we have planned to administer for GVHD prophylaxis. DNA labeling with deuterium has major advantages over CFSE or 5-bromo-2'-deoxyuridine to be GMP compliant, because it is a stable, naturally occurring isotope and it incorporates into the DNA, providing a safe method to measure cell replication in vitro down to 0.1% sensitivity. Also, deuterium allows subsequent in vivo cell tracking for months.²⁹  The fraction of deuterium-labeled Tregs correlated with the mHA frequency measured by LDA, and labeled cells had preserved suppressive function (Figure 4). We plan to use deuterium labeling for assessing cell replication in culture and Treg longevity after transfer.

After expansion, Tregs retained potent antigen-specific suppression, with 50% inhibition of T-effector responses at ratios approaching 1:2500 and 90% inhibition at 1:100. The suppression was selective for the specific alloantigens, because inhibition of third-party responses was 1000 times less effective. Expanded Tregs produced TGF-β and minimal amounts of TNF when restimulated with allogeneic DCs and exhibited high expression of CD25, which is required for the exquisite sensitivity to IL-2, and Foxp3,²¹  which is required for the suppressive function. Under stimulation with phorbol ester and ionomycin, cultures released higher amounts of TNF, presumably secreted by monocytes and monocyte-derived DCs. However, the ratio TGF-β/TNF was significantly higher in the supernatant of cultures containing Tregs compared with Tconvs (Figure 4 D,E; P = .01). As previously shown with allogeneic-HLA–specific Tregs, a sizeable fraction of expanded Tregs expressed CD45RA, because Treg conversion from CD45RA to CD45RO after T-cell receptor activation is inhibited by rapamycin.^51,52  This is important because CD45RA-positive Tregs retain Foxp3 TSDR demethylation and more potent suppressive function.^45,53  In addition, we found cultured Treg expression of lymphoid homing receptor CD62L and chemokine receptor CCR7 that predicted the ability of Tregs to migrate to lymphoid tissues in vivo.⁵⁴  A highly conserved region within the human Foxp3 gene is demethylated exclusively on Tregs and not in any other blood-cell or non–blood-cell type, and Tregs require persistent demethylation of this region to express Foxp3 and maintain suppression function.⁵⁵  The levels of TSDR demethylation were similar in the Tregs before and after expansion, predicting lineage stability after transfer.

In the present study, we used allogeneic recipient-type DCs to present mHAs to establish proof of principle for their ability to stimulate specific donor Tregs. However, DCs from patients would also present mHAs associated with hematopoietic malignancies, and therefore methods for loading patient mHAs from GVHD target organ to donor DCs will have to be developed for clinical translation so as to minimize the risk of stimulating donor Tregs that suppress graft-versus-leukemia responses.

This report demonstrates that mHA-specific Tregs can be expanded ex vivo to large scale under GMP conditions and retain sufficient purity, potency, and immune attributes that are sufficient to proceed to testing in clinical trials for GVHD prevention.

The authors thank all of the research subjects who volunteered for this study; Ashur-Dee Brown for identifying sibling pairs; Darlene Rhan for the leukapheresis collections; Renee Smilee, Albert J. Ribickas, Emily Hopewell, and Andrea Carney for CD25 separation using CliniMACS; Anupama Shastry and Sabine S. Ellwanger for large-scale Treg expansion under GMP in the cellular therapy laboratory at Moffitt Cancer Center; and Mark Fitch (Department of Nutritional Science and Toxicology, University of California, Berkeley) for measurement of deuterium enrichments by mass spectrometry.

This work was supported in part by grants from the National Institutes of Health: National Cancer Institute; National Heart, Lung, and Blood Institute; and National Institute of Allergy and Infectious Diseases (CA132197 and HL114994) (C.A.), (3P30CA076292) (W.J.), (AI43866) (M.H.), (CA118116) (X.-Z.Y.); the American Cancer Society (MRSG-11-149-01-LIB) (J.P.); and was facilitated by the flow cytometry, molecular biology, and cell therapy core facilities at the Moffitt Cancer Center.

Contribution: A.V. participated in the design and performed most of the experiments; F.B. performed the flow cytometry studies; W.J. directed the large-scale Treg expansion experiments; M.H directed the experiments of measuring deuterium enrichment; A.V., F.B., B.B., J.T., J.K, and C.A. analyzed the results and produced figures; A.V., J.P., W.J., X.-Z.Y, and C.A. designed the research; A.V. and C.A. wrote the manuscript; and all authors read and edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Claudio Anasetti, Moffitt Cancer Center, FOB3-BMT 12902 Magnolia Dr, Tampa, FL 33612; e-mail: claudio.anasetti@moffitt.org