Biology of Blood and Marrow Transplantation
Volume 10, Issue 11 , Pages 748-760, November 2004

Tracking ex vivo-expanded CD4+CD25+ and CD8+CD25+ regulatory T cells after infusion to prevent donor lymphocyte infusion-induced lethal acute graft-versus-host disease

Department of Pediatrics, Medical College of Wisconsin, and Children’s Research Institute, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin

Received 24 May 2004; accepted 22 July 2004.

Article Outline

Abstract 

Donor bone marrow (BM)-derived CD4+CD25+ regulatory T cells, maturing in the host thymus, are critical in inhibiting graft-versus-host disease (GVHD) after donor lymphocyte infusion (DLI) in murine BM chimeras. Data presented here demonstrate that fresh CD25+ cells isolated from donor-type mice can be expanded ex vivo by a variety of methods. Ex vivo-expanded CD4+CD25+ and CD8+CD25+ cells were potent suppressors of donor response to host alloantigens in mixed lymphocyte reaction assays. Both fresh and ex vivo-expanded CD4+CD25+ cells persisted long-term in vivo and effectively prevented DLI-induced GVHD in CD25−/− BM chimeras. Importantly, co-infused CD4+CD25+ cells with DLI cells migrated to peripheral lymphoid organs and survived long-term in DLI-treated CD25−/− chimeras, but not in DLI-treated CD25+/+ chimeras, indicating homeostatic control of CD25+ cells and an available niche required for their long-term persistence. Furthermore, maintenance of CD25 expression seemed necessary for suppressive function, because only the CD25+ cell fraction, but not the CD25− fraction isolated after adoptive transfer, was suppressive in vitro. Ex vivo-expanded CD8+CD25+ cells weakly prevented GVHD, apparently because of a rapid disappearance of these cells after adoptive transfer. Taken together, these data suggest that the therapeutic use of ex vivo-expanded CD4+CD25+ cells may be a feasible, nontoxic modality for controlling GVHD in the clinic. Because of strict homeostatic control, an available niche may be required for long-term persistence of infused regulatory T cells.

Keywords:  Donor lymphocyte infusion , Graft-versus-host disease , Bone marrow transplantation , Regulatory T cells , CD4+CD25+ , CD8+CD25+

 

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Introduction 

Generation of immune suppressor T (Ts) cells after bone marrow transplantation (BMT) has long been proposed to participate in the establishment and maintenance of donor-host tolerance [1]. Ts cells have been documented in various rodent tissue and organ transplantation models since the early 1970s [2, 3]. In experimental and clinical BMT settings, Ts cell activity has repeatedly been demonstrated in donor/recipient mixed lymphocyte reaction (MLR) assays. In the early 1980s, Tutschka and Santos [4] systematically characterized the kinetics and specificity of bone marrow (BM)-derived Ts cells after BMT in rats. Characteristics of those Ts cells included alloantigen (alloAg)-dependent generation and maintenance; both non-alloAg-specific and alloAg-specific development of suppressive activity; and passage of suppression from BMT recipients to naive rats by T-cell transfer. However, the absence of specific and reliable markers to discriminate Ts cells from other T cells prevented further characterization of these Ts cells.

Previous studies from our group have shown that donor BM-derived αβ T-cell receptor (TCR)+CD3+T cells, including CD4+CD8− and CD4−CD8− subsets, maturing in the host thymus are capable of inhibiting the development of graft-versus-host disease (GVHD) after delayed donor lymphocyte infusion (DLI) in allogeneic murine BM chimeras [5]. Further studies showed that donor BM-derived CD4+CD25+ cells are one of the principal Ts populations responsible for controlling GVHD initiated by DLI [6]. Interestingly, a population CD4+ cells coexpressing CD25 was shown in 1990 to be a primary mediator of cardiac graft tolerance in rats [7]. As the result of the seminal work by Sakaguchi et al. [8], CD4+CD25+ cells were shown to comprise a population of highly potent Ts cells. These Ts cells are now typically referred to as regulatory T (Treg) cells. Recently, several studies have shown that CD4+CD25+ cells can effectively suppress GVHD and graft rejection and facilitate the induction and maintenance of transplantation tolerance [9, 10, 11, 12, 13, 14, 15, 16, 17].

Strategies for inducing transplantation tolerance have recently focused on the generation or expansion of alloAg-specific CD4+CD25+ cells. Examples of such strategies include using monoclonal antibodies (mAb) specific for CD3, CD4, or CD154; a combination of 1α,25-dihydroxyvitamin D3 with mycophenolate mofetil; pretransplantation donor blood transfusion; and administration of tolerogenic or regulatory dendritic cells [16, 18, 19, 20, 21, 22, 23]. Also, a series of recent studies have clearly shown that it is possible to generate antigen-specific CD4+CD25+ clones in vitro and in vivo [24, 25, 26, 27, 28]. Furthermore, other recent studies have demonstrated that fresh or ex vivo-activated CD4+CD25+ T cells can prevent the development of GVHD and inhibit cardiac graft rejection in murine models [9, 10, 11, 12, 13, 14, 17, 29]. However, tracking and persistence of the adoptively transferred CD4+CD25+ cells was not investigated in these studies.

For ex vivo-generated CD4+CD25+ T cells to be used as clinical treatment for GVHD or autoimmune disease, the following cell properties would likely be necessary: sufficient numbers of cells, migration in vivo to sites of antigen reactivity, ability to suppress immune reactivity in an antigen-specific manner, and survival after infusion for some unknown period of time. To examine these issues, this study was aimed at, first, defining efficient methods for ex vivo expansion of CD4+CD25+ cells, because these cells are a rare cell population and are highly anergic to in vitro stimuli [29, 30, 31]. Second, we examined the suppressive function of fresh and ex vivo-expanded CD4+CD25+ and CD8+CD25+ cells towards alloAgs in vitro. Third, we studied in vivo dynamics of fresh and ex vivo-expanded CD25+ cells after adoptive transfer into syngeneic or allogeneic nude mice. Finally, immune-suppressive effects and in vivo persistence of fresh and ex vivo-expanded CD4+CD25+ and CD8+CD25+ cells were investigated after transfer into Treg-deficient CD25 knockout (KO; CD25−/−) BM chimeras undergoing lethal acute GVHD after DLI therapy.

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Materials and methods 

Mice 

The following strains of mice, 6 to 8 weeks of age, were purchased from Jackson Laboratories (Bar Harbor, ME): C57BL/6 (B6; H-2b; Thy1.2+), congenic B6.PL-Thy1a (H-2b; Thy1.1+), AKR/J (H-2k; Thy1.1+), AKR/Cum (H-2k; Thy1.2+), B6 nude (B6.Cg-Foxn1nu), BALB/c nude (CByJ.Cg-Foxn1nu/J; H-2d;Thy1.2+), and B6 CD25 KO (C57BL/6-IL2rarm/Dw; H-2b; Thy1.2+) heterozygote (CD25+/−) breeders. AKR/Cum mice and CD25 KO homozygotes (CD25−/−) were bred in-house, and all F(1) littermates derived from CD25 KO breeding pairs were screened for homozygosity by using polymerase chain reaction methods as suggested by the vendor. All mice were housed and cared for in the Medical College of Wisconsin’s Biomedical Resource Center (Milwaukee, WI).

Reagents and antibodies 

The following mAbs were purchased from BD Pharmingen (San Diego, CA): purified and fluorescein isothiocyanate (FITC)-conjugated hamster anti-mouse CD3ε (clone 145-2C11), allophycocyanin (APC)-conjugated rat anti-mouse CD4 (clone RM4-5), Cy-Chrome-conjugated rat anti-mouse CD8a (clone 53-6.7), phycoerythrin (PE)-conjugated rat anti-mouse CD25 (clone PC61), FITC-conjugated rat anti-mouse CD25 (clone 7D4), FITC-conjugated mouse anti-rat CD90/mouse CD90.1 (Thy1.1; clone HIS51), PE-conjugated rat anti-mouse CD90.2 (Thy1.2; clone 53-2.1), purified hamster anti-mouse CD28 (clone 37.51), and FITC-conjugated rat anti-mouse 4-1BB (clone 1AH2). In vivo-depleting anti-CD25 (clone PC61) and anti-Thy1.2 (clone 53-2.1) mAb-producing hybridoma cells were obtained from the American Type Culture Collection (Bethesda, MD). These mAbs were produced in our laboratory by using a Vivascience bioreactor system (Hanover, Germany). Rabbit anti-asialo GM1 serum was purchased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan). Collagenase D was obtained from Roche Applied Sciences (Indianapolis, IN). Recombinant human interleukin (IL)-2 was provided by the NCI Biological Resources Branch (Rockville, MD). Bovine serum albumin (BSA) and 2-mercaptoethanol were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), high-glucose Dulbecco modified Eagle medium (DMEM), HEPES buffer, sodium pyruvate, l-glutamine, l-arginine, l-asparagine, folic acid, nonessential amino acids, and penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA).

CD25+ cell isolation 

Single cells from B6 or B6.PL spleen and lymph nodes were enriched for T cells by using nylon wool fiber columns (Polysciences, Inc., Warrington, PA), and the nylon wool-enriched T cells were stained with PE/anti-CD25 mAb for 30 minutes on ice (0.1 μg/106 cells). After washing with 0.5% BSA/PBS, the cells were resuspended in 0.5% BSA/PBS and incubated with anti-PE-conjugated microbeads (1 μL/106 positive cells; Miltenyi Biotec Inc., Auburn, CA) for 15 minutes at 4°C. CD25+ cells were isolated with an automated magnetic cell sorter (AutoMACS; Miltenyi Biotec). With this strategy, CD25+ cell purity was >98%.

Ex vivo CD25+ cell expansion 

Two methods were used to expand CD25+ cells ex vivo.

Method I 

Ninety-six-well round-bottom plates were coated with anti-CD3ε mAb diluted in PBS (50 μL per well) at 4°C overnight. Freshly isolated CD25+ cells were suspended in complete DMEM (10% FBS, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES buffer, 0.05 mmol/L 2-mercaptoethanol, 2 mmol/L l-glutamine, 69 mmol/L l-arginine, and 100 U/mL penicillin/streptomycin) containing 100 U/mL IL-2 and 4 μg/mL anti-CD28 mAb. Aliquots of the cell suspension were cultured in anti-CD3ε-coated wells at final concentrations of 5 to 10 × 104 cells per well (200 μL per well). In selected experiments, irradiated (3000 rad) allogeneic strain AKR splenocytes were added as stimulators to the CD25+ cells. Specifically, irradiated CD11c-enriched AKR splenocytes (3 × 105 per well) were cocultured with 105 fresh CD25+ cells with or without IL-2 and/or anti-CD3/CD28 mAbs.

Method 2 

An established artificial antigen-presenting cell (aAPC) system—ie, a CD32 and 4-1BB ligand (4-1BBL) double-transfected K562 tumor cell line—has been described previously [32]. The rationale for using this system is that aAPCs loaded with anti-CD3ε and anti-CD28 mAb via CD32 molecules provide additional co-stimulatory signaling through 4-1BB ligation on CD25+ T cells [33]. The aAPCs were coated with anti-CD3ε (10 μg/mL) and anti-CD28 (4 μg/mL) mAb for 30 minutes in complete DMEM at room temperature. The cells were then irradiated (10000 rad) before coculture with fresh CD25+ cells in 100 U/mL IL-2 at a ratio of 1:2 (CD25+/aAPC) in 96-well plates.

CD25+ cells expanded by the various methods were checked daily and split with fresh IL-2-containing media to maintain the cells at a concentration of 0.5 to 1 × 106/mL. After 5 days of culture in 96-well plates, the CD25+ cells were transferred to flasks for further culture. Ex vivo-expanded CD25+ cells were phenotyped by flow cytometry, assayed for suppressive activity in vitro, or used for adoptive transfer in vivo.

In vitro suppression assays 

Fresh and ex vivo-expanded CD25+ cells, including purified CD4+CD25+ and CD8+CD25+ cells (separated by magnetically activated cell sorting after culture) and CD4−CD8−CD25+ cells (isolated by flow cytometric sorting after culture), were assayed for suppressive activity in allogeneic MLR assays. To assay the in vitro suppressive activity of CD25+ and CD25− fractions of infused CD4+CD25+ cells (isolated from both adoptively transferred B6 nude and CD25−/− chimeric mice) and in vivo-expanded CD8+ cells (isolated from adoptively transferred B6 nude mice only), Thy1.1+CD4+CD25+, Thy1.1+CD4+CD25−, and Thy1.1+CD8+ cells were sorted by flow cytometry by using the following panel of mAbs: anti-Thy1.1 FITC, anti-CD25 PE, anti-CD8 Cy-Chrome, and anti-CD4 APC. Irradiated (3000 rad) AKR CD11c-enriched splenocytes, supplemented with irradiated CD11c-depleted splenocytes, were used as allogeneic stimulators (3 × 105 per well) in 96-well round-bottom plates. CD25-depleted (using an AutoMACS) naive B6 or B6.PL T cells were used as responder cells (105 per well). Variable numbers of CD25+ cells were added to a fixed number of responder cells and stimulator cells, and the CD25+ Treg/CD25− responder ratios ranged from 1:1 to 1:128. After 4 days in culture, 3H-thymidine was added to each well for an additional 18 hours. 3H-Thymidine incorporation was measured on a β-scintillation counter, and the results were expressed as counts per minute (CPM). Wells without CD25+ cells (responders and stimulators only) served as positive controls (ie, maximum CPM). Wells containing different dilutions of CD25+ cells and stimulator cells (no responders) served as baseline controls (ie, baseline CPM). Suppression was expressed as percentage of control MLR by using the following formula: [experimental CPM (wells containing CD25+ cells) − baseline CPM]/maximum CPM. The ratio of CD25+ Treg to CD25− responder T cells to achieve 50% suppression or inhibition (ID50) of control MLR was calculated with a linear regression method.

Adoptive transfer of CD25+ cells into nude mice 

Fresh or ex vivo-expanded CD25+ cells (5 × 106) were injected intravenously (IV) on day 0 into syngeneic B6 nude mice or natural killer (NK) cell-depleted allogeneic BALB/c nude mice. NK cell depletion was achieved by 5 consecutive injections of 50 μL of anti-asialo GM1 antibodies on days −2, 0, 2, 4, and 7.

Bone marrow transplantation 

Host AKR/cum mice were lethally irradiated with 1100 cGy 1 day before BMT. To help prevent graft rejection, a single intraperitoneal (IP) injection of 500 μg of anti-Thy1.2 mAb was given on the day of transplantation. A total of 107 fresh BM cells, harvested from CD25+/+, CD25+/−, or CD25−/− B6 donor mice, were injected IV via the tail veins. BM engraftment was confirmed by flow cytometry at 3 weeks after BMT.

GVHD induction 

GVHD was induced at 4 weeks after BMT in CD25+/+, CD25+/−, and CD25−/− BM chimeras by IV infusion of 5 × 106 CD25-depleted T cells (ie, DLI). In selected animals, fresh or ex vivo-expanded CD4+CD25+ or CD8+CD25+ cells, at CD25−/CD25+ ratios of 1:1 (5 × 106 CD25+) or 2:1 (2.5 × 106 CD25+) were co-infused with the DLI. In some CD25+/+ BM chimeras, mice were injected IP with anti-CD25 mAb at 7 days (500 μg per mouse) and 4 days (250 μg per mouse) before DLI. In these experiments, DLI cells consisted of 3 × 107 normal or in vivo-CD25-depleted (ie, treated with anti-CD25 mAb) B6 or B6.PL splenocytes. Approximately 70% of CD25+ cells were depleted by in vivo treatment with anti-CD25 mAb, and the CD25+ cells recovered to normal levels within 10 to 14 days after the last injection of anti-CD25 mAb.

Cell tracking and phenotypic analysis 

Infused fresh or ex vivo-expanded CD25+ cells from B6.PL mice (Thy1.1+) were monitored for in vivo persistence and CD25 expression for 6 months in syngeneic B6 (Thy1.2+) nude mice and allogeneic BALB/c (Thy1.2+) nude mice. Similarly, by using CD25+ cells from Thy1.1+ B6.PL mice and DLI cells from Thy1.2+ B6 mice, infused CD4+CD25+ or CD8+CD25+ cells were followed for persistence and CD25 expression in CD25−/− or CD25+/+ B6-into-AKR/Cum (Thy1.2+) BM chimeras for 15 weeks. Peripheral blood mononuclear cells and cells from spleen and lymph nodes were stained with the following panel of cell-surface markers: anti-Thy1.1 FITC, anti-CD25 PE, anti-CD8 Cy-Chrome, and anti-CD4 APC. The samples were analyzed on a Becton Dickinson FACSCalibur flow cytometer (San Jose, CA).

Statistics 

Survival curves were compared by log-rank analysis. Significance was defined as P <.05.

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Results 

Ex vivo expansion of CD25+ T cells (CD4+, CD8+, and CD4−CD8− subsets) by using anti-CD3 and anti-CD28 mAb 

Most (>90%) freshly isolated natural CD25+ T cells were CD4+; however, the CD25-enriched cells also contained small percentages of CD8+CD25+ (1%–3%) and CD3+CD4−CD8−CD25+ (2%–4%) T cells. As shown in Table 1, freshly isolated CD4+CD25+ cells were expanded only when activated through the TCR and CD28 pathways in the presence of IL-2; alloAgs alone or alloAgs plus IL-2 failed to expand the CD4+CD25+ population. In the presence of IL-2, the small numbers of CD8+CD25+ cells rapidly expanded in culture and became the dominant cell population. Overgrowth by CD8+CD25+ cells was not the main reason for poor expansion of CD4+CD25+ cells cultured with plate-bound anti-CD3, because removal of CD8+ cells from total CD25+ cells on days 3 to 5 in culture did not markedly enhance expansion of CD4+CD25+ cells (data not shown).

Table 1. Ex Vivo Expansion of Total CD25+ Cells
Stimulation*-Fold Expansion after 1 wk†
CD3+CD25+CD4+CD25+CD8+CD25+
AlloAgs0.35±0.120.27±0.080.29±0.12
IL-2 (100 U/mL)1.56±0.210.09±0.0650.4±6.21
IL-2/alloAgs2.43±0.510.42±0.2466.8±7.73
Anti-CD3/CD28Cells diedCells diedCells died
Anti-CD3/CD28/alloAgs0.70±0.180.61±0.201.89±0.44
Anti-CD3/CD28/IL-213.5±6.203.75±3.04301.2±14.2
Anti-CD3/CD28/IL-2/alloAgs23.4±14.265.98±3.42356.1±67.5

* The starting cell number for each culture was 2 × 106 freshly isolated CD25+ cells; see Materials and Methods for details.

† The averages±SD are the combined results of 6 experiments.

To achieve better ex vivo expansion of CD4+CD25+ cells, CD32/4-1BBL-transfected K562 cells were used as aAPC. Through loading of anti-CD3 and anti-CD28 mAb onto transfected CD32 molecules, a preferential (90%–95% CD4+) and marked expansion of CD4+CD25+ cells (10- to 15-fold between days +7 and +10 of culture) was obtained (Table 2). Ligation of 4-1BBL on aAPC to 4-1BB on CD4+CD25+ cells was important, because CD32-transfected K562 aAPC (no 4-1BBL transfection) loaded with anti-CD3/CD28 mAb failed to induce significant expansion of CD4+CD25+ cells (less than 3- to 5-fold by day +10; data not shown).

Table 2. Ex Vivo CD4+CD25+ Cell Expansion
Stimulation*-Fold Expansion†
Day +7Day +10Day +14
Plate-bound anti-CD3/soluble anti-CD28/IL-23.8±3.06.2±2.22.1±1.5
Plate-bound anti-CD3/soluble anti-CD28/IL-2/alloAgs5.9±3.410±3.54.9±2.9
Anti-CD3/CD28-coated K562 aAPC/IL-213.1±2.912.7±4.47.3±2.4

* The starting cell number for each culture was 2 × 106 freshly isolated CD25+ cells; see Materials and Methods for details. K562 aAPCs preferentially expanded CD4+CD25+ cells.

† The data (averages±SD) are the combined results of 6 experiments.

Ex vivo-expanded CD4+CD25+ and CD8+CD25+ cells, but not CD4−CD8−CD25+ T cells, were suppressive in MLR assays 

As shown in Figure 1A and B, activated/expanded CD25+ T cells exerted stronger suppression (ID50 was between 1:10 and 1:25) in MLR assays than fresh CD25+ T cells (ID50, 1:1), regardless of the activation signals, including alloAgs or anti-CD3/CD28 mAb. CD25+ T-cell subsets expanded in culture were sorted into CD4+CD25+, CD8+CD25+, and CD4−CD8−CD25+ subsets by flow cytometry by using the following panel of mAbs: anti-CD3 FITC, anti-CD25 PE, anti-CD4 APC, and anti-CD8 Cy-Chrome. CD4+CD25+ (ID50, 1:10) and CD8+CD25+ (ID50, 1:10) cells, but not CD4−CD8− cells, were potently suppressive in vitro (Figure 1C). The in vitro suppressive function of CD8+CD25+ cells was not overcome by the addition of 100 U/mL of exogenous IL-2 to the MLR assay (Figure 1D).

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  • Figure 1. 

    Fresh and ex vivo-expanded CD25+ T cells were suppressive in vitro. Fresh CD25+ cells, CD25+ cells activated by alloAgs, IL-2, anti-CD3/CD28 mAbs, or combinations of these stimuli (A and B), as well as ex vivo-expanded CD3+CD25+ or CD4+, CD8+, and CD4−CD8− subsets (C), were assayed for suppressive activity in MLR assays. In some experiments (D), exogenous IL-2 was added directly to the assays. All curves are expressed as percentage of control MLR and represent the combined average values of 10 to 15 individual experiments. The raw data values for the control MLRs ranged from 10 to 15 × 104 CPM.

Fresh or ex vivo-expanded CD4+CD25+ cells proliferated and trafficked through the peripheral lymphoid tissues in vivo after adoptive transfer into syngeneic or allogeneic nude mice 

Fresh CD4+CD25+ and CD8+CD25+ cells not only expanded ex vivo, but also seemed to proliferate in vivo after adoptive transfer into nude recipient mice. As shown in Figure 2A, freshly isolated Thy1.1+CD25+ cells (5 × 106 injected) could be detected in peripheral lymphoid organs, including the peripheral blood, spleen, and lymph nodes, after adoptive transfer into syngeneic B6 nude mice (Thy1.2+). Interestingly, only 20% to 30% of the injected CD4+CD25+ and less than 3% to 5% of the CD8+CD25+ cells maintained CD25 expression in syngeneic nude recipients (Figure 2B). Both fresh and ex vivo-expanded Thy1.1+CD4+CD25+ cells persisted and survived more than 6 months in peripheral blood after adoptive transfer into Thy1.2+ syngeneic B6 or NK cell-depleted allogeneic BALB/c nude recipients (Figure 2C). No homing capacity impairment was apparent: both fresh and ex vivo-expanded CD4+CD25+ cells persisted in the peripheral lymphoid organs equally well (Figure 2C). CD25 expression on fresh or expanded CD4+CD25+ cells was maintained on higher percentages of cells (60%–80%) in allogeneic nude recipients as compared with syngeneic nude recipients (20%–30%, Figure 2D).

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  • Figure 2. 

    Fresh and ex vivo-expanded CD25+ cells could be detected long-term after adoptive transfer into syngeneic or allogeneic nude mice. The peripheral blood of syngeneic B6 or allogeneic BALB/c nude recipient mice given fresh Thy1.1+ B6.PL CD25+ cells (A-D) or ex vivo-expanded CD4+CD25+ cells (C and D) was analyzed at various time points for the presence of infused cells (A and C) and for the maintenance of CD25 expression on infused cells (B and D). At 24 week after transfer, all mice were killed, and the peripheral blood (PB), spleen (SP), and lymph node (LN) were analyzed by flow cytometric analysis of gated lymphocytes. The results are the combined data of syngeneic B6 nude recipients given fresh (n = 5; A-D) or ex vivo-expanded CD4+CD25+ cells (n = 5; C and D) or NK cell-depleted allogeneic BALB/c nude recipients infused with fresh (n = 5; C and D) or ex vivo-expanded CD4+CD25+ cells (n = 5; C and D).

Permanent absence or temporary depletion of donor BM-derived CD25+ cells increased the severity of GVHD after DLI 

Wild-type (CD25+/+) and CD25 KO heterozygote (CD25+/−) BM chimeras were resistant to DLI-induced GVHD (Figure 3A). In contrast, the complete absence of CD25 molecules (CD25−/− bone marrow chimeras) on donor BM-derived T cells resulted in lethal acute GVHD after DLI with 5 × 106 CD3+CD25− T cells given 4 weeks after BMT (P <.000005; Figure 3A). The severity of GVHD was reflected by acute body weight loss (Figure 3B). Acute and lethal GVHD were also seen in CD25+/+ BM chimeras that had been temporarily depleted of CD25+ cells in vivo (by treatment with anti-CD25 mAb) before DLI at doses of 3 × 107 (Figure 3C) or 6 × 107 (Figure 3D) splenocytes. Depletion of CD25+ cells from the DLI inoculum did not increase the severity of GVHD in untreated or anti-CD25-treated hosts (Figure 3C and D). BMT controls (CD25+/+, CD25+/−, and CD25−/−) without DLI therapy (n = 5 for each group) all survived long-term, but the data are not shown in Figure 3.

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  • Figure 3. 

    CD25−/− BM chimeras or normal BM chimeras depleted of CD25+ cells developed lethal GVHD after DLI. The survival (A) and average body weights (B) for CD25+/+ (n = 10), CD25+/− (n = 10), or CD25−/− (n = 15) BM chimeras were monitored after DLI with 5 × 106 CD3+CD25− T cells. In (C) and (D), untreated or in vivo CD25-depleted CD25+/+ chimeras were given DLI with whole splenocytes (undepleted DLI) or CD25-depleted splenocytes (CD25-depleted DLI) at doses of 3 × 107 (C) or 6 × 107 (D) cells. In the CD25 depletion experiments, a total of 11 to 12 mice for each experimental DLI group were analyzed, representing the combined data from 3 independent experiments. The CD25-depleted, no-DLI control group in (C) and (D) consisted of 3 mice. BMT controls (CD25+/+, CD25+/−, and CD25−/−), composed of 3 to 5 animals for each group without DLI therapy, were followed up long-term but not shown on Figure 3.

Fresh or ex vivo-expanded CD4+CD25+ cells prevented lethal acute GVHD after cotransfer with DLI 

As previously shown in Figure 3, 5 × 106 CD25−CD3+ T cells given as DLI at 4 weeks after BMT induced lethal acute GVHD in CD25−/− BM chimeras. GVHD was significantly inhibited by co-infusion of CD25−/− BM chimeras with fresh (Figure 4A) or ex vivo-expanded (Figure 4B) CD4+CD25+ cells at doses of 5 × 106 (CD25−/CD25+ cell ratio of 1:1) or 2.5 × 106 (CD25−/CD25+ cell ratio of 2:1) cells. The BM chimeras co-infused with fresh or ex vivo-expanded CD4+CD25+ cells were healthy more than 15 weeks after DLI, with little or no body weight loss (Figure 4C and D) or any other signs of acute or chronic GVHD.

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  • Figure 4. 

    Adoptively transferred fresh or ex vivo-expanded donor CD25+ cells suppressed the development of DLI-induced GVHD in CD25−/− BM chimeras. Survival graphs (A and B) and body weight curves (C and D) are shown for CD25−/− chimeras given fresh (A and C) or ex vivo-expanded CD25+ cells (B and D) at the time of DLI (5 × 106 CD25− T cells given 28 days after transplantation). Fresh and ex vivo-expanded CD25+ cells were given at CD25−/CD25+ cell ratios of 1:1 (n = 9 mice per group) or 2:1 (n = 11 for fresh; n = 8 for expanded). Controls consisted of CD25−/− BM chimeras given DLI without co-infusion of CD25+ cells (n = 15).

Ex vivo-expanded CD8+CD25+ T cells weakly suppressed acute GVHD compared with ex vivo-expanded CD4+CD25+ cells 

Compared with ex vivo-expanded CD4+CD25+ cells, ex vivo-expanded CD8+CD25+ cells were relatively inefficient at suppressing lethal acute GVHD in CD25−/− BM chimeras given 5 × 106 CD25− T cells as DLI (Figure 5). Thirty percent of mice co-infused with 5 × 106 CD8+CD25+ cells (CD25−/CD25+ cell ratio of 1:1) survived 15 weeks after DLI, which was significantly better than survival of the noninfused DLI control mice (P = .005). The onset of GVHD was paralleled by body weight loss (Figure 5B). A lower dose of 2.5 × 106 CD8+CD25+ cells (CD25−/CD25+ cell ratio of 2:1) failed to protect any CD25−/− BM chimeras from DLI-induced GVHD (Figure 5A), and survival of these mice was not significantly different from that of DLI controls (P = .07).

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  • Figure 5. 

    CD8+CD25+ cells weakly suppressed the development of DLI-induced GVHD in CD25−/− BM chimeras. Survival (A) and body weights (B) were monitored for CD25−/− chimeras co-infused with DLI and 5 × 106 (n = 10) or 2.5 × 106 (n = 9) ex vivo-expanded CD8+CD25+ cells on day 28 after BMT. Controls consisted of CD25−/− chimeras given DLI only (n = 15).

Fresh and ex vivo-expanded CD4+CD25+ survived long-term and trafficked to lymphoid tissues during suppression of lethal acute GVHD, but ex vivo-expanded CD8+CD25+ disappeared within 2 to 3 weeks after infusion 

Fresh and ex vivo-expanded Thy1.1+CD4+CD25+ cells co-infused with DLI (Thy1.2+) into CD25−/− BM chimeras (Thy1.2+) persisted and survived equally well in peripheral blood and migrated through peripheral lymphoid organs during suppression of GVHD (Figure 6A). Stable percentages of CD4+CD25+ cells were observed in peripheral blood (0.5%–1% of peripheral blood mononuclear cells), and higher percentages were present in the spleen and lymph nodes (1.5%–3%) at 15 weeks after infusion. Both fresh and ex vivo-expanded CD4+CD25+ cells maintained CD25 expression on 50% to 70% of the adoptively transferred cells (Figure 6B). High percentages of adoptively transferred cells (60%–80%) found in the spleen and lymph node at 15 week after infusion also maintained CD25 expression (Figure 6B). Remarkably, ex vivo-expanded CD4+CD25+ cells could be readily detected in CD25−/− BM chimeras but were nearly undetectable in CD25+/+ BM chimeras during the entire experimental period (Figure 7), indicating that adoptively transferred CD4+CD25+ cells failed to persist in mice that had reconstituted their CD25+ cell compartment. In contrast to ex vivo-expanded CD4+CD25+ cells, ex vivo-expanded CD8+CD25+ cells typically disappeared within 2 to 3 weeks after co-infusion with DLI into CD25−/− BM chimeras (Figure 8).

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  • Figure 6. 

    Long-term persistence of fresh or ex vivo-expanded CD4+CD25+ cells given to suppress GVHD in vivo. Fresh Thy1.1+ B6.PL CD25+ cells or ex vivo-expanded CD4+CD25+ cells were tracked for in vivo persistence (A) and maintenance of CD25 expression (B) after co-infusion with DLI into CD25−/− Thy1.2+ BM chimeras. Flow cytometric analysis of gated lymphocytes in the peripheral blood (PB), spleens (SP), and lymph nodes (LN) was performed at the indicated time points.

  • View full-size image.
  • Figure 7. 

    Ex vivo-expanded CD4+CD25+ cells persisted and proliferated only in CD25−/−, but not CD25+/+, BM chimeras after co-infusion with DLI cells. Ex vivo-expanded CD4+CD25+ cells were tracked for in vivo persistence after co-infusion with DLI into CD25−/− or CD25+/+ Thy1.2+ BM chimeras.

  • View full-size image.
  • Figure 8. 

    Ex vivo-expanded CD8+CD25+ cells quickly disappeared after infusion to suppress GVHD in vivo. Ex vivo-expanded CD8+CD25+ cells were tracked for in vivo persistence after co-infusion with DLI into CD25−/− Thy1.2+ BM chimeras at 1:1 or 2:1 DLI/Treg ratios.

Only cells that maintained CD25 expression after adoptive transfer were suppressive in vitro 

As noted previously, 2 populations (CD4+ and CD8+) of CD25+ cells expanded after adoptive transfer into syngeneic nude mice, and more than half of the CD4+CD25+ cells lost CD25 expression. At 6 months after adoptive transfer, the CD4+ and CD8+ cells were reisolated from spleen and lymph node by flow cytometric sorting based on CD25 expression and assayed for suppressive activity in vitro by using naïve B6 CD25− T cells as responders and CD11c-enriched naïve AKR/J splenocytes as stimulators. Unexpectedly, only the CD4+CD25+ cells were still suppressive in MLR assays (Figure 9A); the CD4+CD25− and CD8+ cells either enhanced or failed to suppress MLR reactivity. CD25+ and CD25− fractions of infused CD4+CD25+ cells were also isolated from the spleen and lymph node by flow cytometric sorting from DLI-treated CD25−/− chimeric mice at 15 weeks after DLI and tested for suppressive activity in MLR assays. Again, only the CD25+ fraction of cells suppressed MLR reactivity (Figure 9B).

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  • Figure 9. 

    Only Treg cells that maintained CD25 expression in vivo were suppressive in vitro. Persisting Thy1.1+ Treg subsets (CD4+CD25+, CD4+25−, and CD8+) were isolated by flow cytometric sorting from the spleen (SP) and Lymph node (LN) of syngeneic nude mice 6 months after adoptive transfer (A) or from the SP and LN of DLI-treated CD25−/− chimeras 15 weeks after infusion (B). The reisolated cells were then assayed for suppressive activity in MLR assays. All curves are expressed as percentage of control MLR and represent the combined average values of 6 to 9 individual experiments. The raw data values for the control MLRs ranged from 8 to 10 × 104 CPM.

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Discussion 

Among a variety of Ts or Treg cell populations, CD4+CD25+ Treg have been well characterized in the suppression of autoimmunity, prevention of GVHD and graft rejection, and induction of transplantation tolerance, as well as downregulation of immune responses against tumors and microbes [3, 29, 30, 34]. On the basis of their potent suppressive activity, therapeutic administration of CD4+CD25+ cells may be a viable approach to controlling GVHD and graft rejection or even inducing permanent transplantation tolerance without eliminating beneficial antitumor effects [12, 13]. These cells may also help to promote BM engraftment and thereby enhance immune reconstitution after BMT [13, 35]. However, it is essential to understand several unknown aspects of CD4+CD25+ cells before clinical use, including how these cells traffic after adoptive transfer and whether they persist, or how long they need to persist, to mediate their function. For example, can these cells be efficiently expanded ex vivo and still maintain their suppressive activity? Ex vivo expansion may be necessary because CD4+CD25+ cells are present in fresh lymphoid tissues at very low numbers [3, 29, 30]. Is systemic immune suppression after adoptive transfer a concern, or do these cells suppress antigen reactivity in a specific manner? In vitro data suggest that once CD25+ Treg become activated, their suppression is antigen nonspecific [31]. However, recent data have suggested that CD25+ Treg can suppress antigen reactivity in vivo in an antigen-specific manner [36].

Besides naturally occurring CD4+CD25+ cells, there are low numbers of CD8+ (1%–3% of total CD25+ cells) and CD4−CD8− (2%–4% of total CD25+ cells) cells in normal mice that coexpress CD25. After triggering the TCR and CD28 cosignaling pathways, all subsets of CD25+ cells are expandable, particularly CD4+CD25+ and CD8+CD25+ cells. Culture with plate-bound anti-CD3 and soluble anti-CD28 mAb was inefficient for expansion of the CD4+ subset from purified CD25+ cells. First, overgrowth of CD8+CD25+ cells inhibited expansion of CD4+CD25+ cells with this strategy. Second, no significant improvement in expansion was observed when the CD4+ fraction of CD25+ cells was purified (sorted by flow cytometry) before culture or purified after 3 days in culture. Activation with aAPC preferentially expanded CD4+CD25+ cells. Besides TCR signaling and CD28 co-stimulation, additional activation signals through 4-1BB/4-1BBL interactions may have driven activation and expansion, because aAPCs transfected with only CD32 and coated with anti-CD3 and CD28 mAbs were unable to efficiently drive the expansion of CD4+CD25+ cells. Compared with conventional T cells, signaling through CD28 seems to be particularly important for the activation and expansion of CD4+CD25+ cells. We were able to attain a relatively modest 13-fold expansion of CD4+CD25+ cells by using aAPC. A variety of other expansion strategies for mouse CD4+CD25+ cells have been used [9, 11, 36], but they also resulted in only modest cell expansions. In a recent report by Tang et al. [37], up to 200-fold expansion of mouse CD4+CD25+ cells could be achieved with a combination of anti-CD3, anti-CD28, and IL-2, indicating that high levels of expansion are feasible. Thus, if conditions are optimized, it seems that high levels of expansion can be achieved. We are currently modifying our protocols in an effort to achieve better expansion. CD28 co-stimulation has been shown to be critical for the generation and/or maintenance of CD4+CD25+ cells, and CD28 KO mice have few CD4+CD25+ cells [6, 24, 38, 39]. Moreover, binding of CD28 alone by a CD28 superagonist has been shown to efficiently expand rat CD4+CD25+ cells in vitro and in vivo [40]. Thus, continuous triggering through CD28 or other co-stimulatory molecules, including 4-1BB, OX40, CD30, and glucocorticoid-induced tumor necrosis factor receptor, may be critical for maintaining a stable CD4+CD25+ pool [17, 29, 41].

We observed approximately 50% suppression in MLR assays with freshly isolated CD25+ cells at a CD25+ Treg/CD25− responder ratio of 1:1 (Figure 1). This is different from the results of many studies in which suppression of MLR reactivity by CD25+ cells at a similar ratio has often been greater than 90%. It has been previously shown that strong T-cell signaling can overcome CD25+ cell-mediated suppression [42]. Perhaps the reason for the 50% suppression in our MLR assays with freshly isolated CD25+ cells was due to the degree of histocompatibility disparity between the stimulator and responder cells combined with our use of dendritic cell-enriched stimulators in the MLR assays. We did consistently see enhanced suppression when alloantigen-activated-expanded CD25+ cells were tested in the MLR assays (90%–98% suppression observed at a CD25+ Treg/CD25− responder ratio of 1:1). Suppressive activity of CD4+CD25+ cells expanded by CD32/4-1BBL-transfected aAPCs for 7 days was intact when they were tested for suppression in MLR assays, although recent reports have indicated that triggering the 4-1BBL/4-1BB pathway abrogates the suppressive activity of CD4+CD25+ cells [33, 43]. Perhaps 4-1BBL/4-1BB signaling was no longer present in our system at the time of testing (day 7 of culture or later), because irradiated aAPC cells disappear around day 5 of culture.

Unexpectedly, ex vivo-expanded CD8+CD25+ cells had suppressive activity in MLR assays equal to that of expanded CD4+CD25+ cells. This is consistent with a finding that rat CD8+CD25+ cells were suppressive in vitro, although the suppressive activity was weaker than that of CD4+CD25+ cells [14]. A suppressive population of fresh human CD8+CD25+ thymocytes has also been recently identified [44, 45]. It would be interesting to test freshly isolated murine CD8+CD25+ cells for suppressive activity; however, these cells are present at such low numbers (<5 × 104 per mouse) that it would be very difficult to isolate enough cells to assay their function in vitro. In contrast to CD8+CD25+ cells, CD4−CD8−CD25+ cells failed to mediate suppression in vitro. Thus, within the CD25+ cell population, expanded/activated CD8+CD25+ and CD4+CD25+ cells, but not CD4−CD8−CD25+ cells, exhibited suppressor function.

Ex vivo-expanded CD8+CD25+ cells only weakly suppressed DLI-induced GVHD, although these cells were as suppressive as ex vivo-expanded CD4+CD25+ cells in MLR assays. Infused CD8+CD25+ cells typically disappeared within 2 weeks after infusion. We speculate that this may be due to IL-2 withdrawal. Occasionally, CD8+CD25+ cells could be detected at low levels 2 weeks after infusion, and this was always seen in mice that developed less severe GVHD. Overall, our results suggest that therapeutic application of CD8+CD25+ cells may be limited.

Ex vivo-expanded CD4+CD25+ cells persisted in vivo as efficiently as fresh CD4+CD25+ cells after adoptive transfer into syngeneic or allogeneic hosts, thus suggesting that their maintenance can be driven by self-antigens or alloAgs [46, 47]. Interestingly, the in vivo persistence of both fresh and ex vivo-expanded CD4+CD25+ seems to be under strict homeostatic regulation, because infused CD4+CD25+ cells were nearly undetectable in CD25+/+ BM chimeras, whereas they persisted in CD25−/− BM chimeras after coadministration with DLI cells. Thus, exogenous CD4+CD25+ cells may compete with endogenous CD4+CD25+ cells for niches to colonize and survive. This is consistent with the finding that exogenous CD4+CD25+ cells survived in IL-2Rβ−/− but not in wild-type recipients [48]. Importantly, both fresh and ex vivo-expanded CD4+CD25+ cells effectively prevented lethal acute GVHD. Thus far, the exact mechanism of GVHD protection/suppression in our model is unclear, although multiple mechanisms have been implicated in different model systems in vitro and in vivo [17, 29, 31, 39, 49].

It is unclear from our experiments whether the infused CD25+ cells expanded in vivo after adoptive transfer, but the increasing percentages of infused cells in nude recipients over time (Figure 1) suggested that they were expanding in these mice. However, in DLI-treated CD25−/− BM chimeras, the infused CD4+CD25+ cells had already achieved maximum levels in the peripheral blood by 2 weeks after infusion (Figure 7). We did not look at earlier time points in these mice, so if expansion of the infused cells did take place, it occurred during the first 2 weeks after infusion. The percentages of infused CD25+ cells in the peripheral blood and other lymphoid tissues (spleen and lymph nodes) of CD25−/− BM chimeras were not remarkably different from the percentages of CD25+ cells in normal mice, suggesting that the infused cells filled the vacant CD25 cell compartment and expanded no further. We speculate that the relatively large numbers of infused CD25+ cells (2.5 − 5 × 106 cells) rapidly filled the vacant CD25 compartment and that, therefore, vigorous homeostatic expansion was not required. The situation in the nude mice is different because the total T-cell compartment is severely deficient. Perhaps homeostatic expansion would have been more evident in the CD25−/− BM chimeras had we given smaller numbers of CD4+CD25+ cells.

Investigation is ongoing to define whether the suppression mediated by CD4+CD25+ cells in our DLI model is alloAg specific. On the basis of recent findings that antigen-specific suppression can be mediated by CD4+CD25+ cells in vivo, we hypothesize that the suppression observed in our system is alloAg specific [27, 36, 50, 51]. It does not seem that administration of expanded/activated CD4+CD25+ cells results in systemic immune suppression, because treated mice in our experiments remained in good health and had no apparent increase in infections. Future studies will address whether fresh or ex vivo-expanded CD4+CD25+ cells can be used to treat ongoing DLI-induced GVHD and prevent its occurrence. On the basis of recent data published by Jones et al. [11], it seems promising that CD4+CD25+ cells, particularly after ex vivo expansion, could be used alone or in combination with other forms of immunosuppression to target ongoing GVHD.

Interestingly, infused CD4+CD25+ cells are dynamic in vivo [46, 52]. Maintenance of CD25 expression seems useful for defining the suppressive subset of infused CD4+CD25+ cells toward alloAgs, because in our studies the cells that lost CD25 expression after adoptive transfer were no longer suppressive in MLR assays. This is consistent with a previous study showing that transfer of the CD25+ fraction, but not the CD25− fraction, of previously infused CD4+CD25+ cells (parked in lymphopenic mice for 7 weeks) could control CD4+ cell expansion in secondary lymphopenic mice [52]. In our model, it is uncertain whether the cells that lost CD25 expression were suppressive against antigens other than alloAgs, because the proliferation of infused CD4+CD25+ cells was likely driven by both alloAgs and self-antigens. Other published data have shown that both CD25+ and CD25− cell fractions from CD4+CD25+ cells previously transferred to lymphopenic mice were still capable of suppressing concanavalin A-stimulated T-cell proliferation in vitro [46]. We will need to perform similar studies to determine whether the same is true in our model.

In conclusion, the anergic status of fresh CD4+CD25+ cells can be overcome by strong activation signals induced through the TCR and CD28. Ex vivo-expanded CD4+CD25+ cells migrate to peripheral lymphoid tissues, undergo dynamic changes, and can potently suppress the development of DLI-induced GVHD. Thus, the therapeutic use of ex vivo-expanded CD4+CD25+ cells may be a feasible, nontoxic modality for controlling GVHD in the clinic.

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Acknowledgments 

This work was supported by US Public Health Services grant no. CA90286 and the Midwest Athletes against Childhood Cancer Fund (Milwaukee, WI).

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PII: S1083-8791(04)00375-1

doi:10.1016/j.bbmt.2004.07.004

Biology of Blood and Marrow Transplantation
Volume 10, Issue 11 , Pages 748-760, November 2004

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