Large-scale in vitro expansion of polyclonal human CD4 CD25 high regulatory T cells

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1 TRANSPLANTATION Large-scale in vitro expansion of polyclonal human CD4 CD25 high regulatory T cells Petra Hoffmann, Ruediger Eder, Leoni A. Kunz-Schughart, Reinhard Andreesen, and Matthias Edinger Introduction CD4 CD25 regulatory T (T reg ) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4 CD25 T reg cells display phenotypic and functional characteristics similar to those of murine CD4 CD25 T reg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25 T cells. Thus far, the detailed characterization and potential clinical application of human CD4 CD25 T reg cells have been hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to fold expansion of highly purified human CD4 CD25 high T cells in vitro through the use of artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (IL-2). Expanded CD4 CD25 high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4 CD25 high T cells, and maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4 CD25 high T reg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell mediated diseases and transplantation medicine. (Blood. 2004;104: ) 2004 by The American Society of Hematology Immunosuppression is an intrinsic capacity of the immune system and is partially mediated by T cells. The best-defined T-cell population with immunosuppressive activity is CD4 and constitutively expresses the interleukin 2 receptor (IL-2R) -chain (CD25). 1 These thymus-derived suppressor cells 2,3 contribute to the maintenance of self-tolerance and thereby give protection from a variety of autoimmune diseases. 1,4 They control the size of the peripheral T-cell pool, 5 modulate immune responses after infection 6 and against tumors, 7,8 and contribute to tolerance induction after solid organ transplantation. 9,10 These findings confirm the central role of T reg cells in the modulation of immune responses under physiologic as well as pathologic conditions. We and others recently showed that donor-type CD4 CD25 T reg cells themselves do not induce graft-versus-host disease (GVHD) after major histocompatibility complex (MHC) mismatched bone marrow transplantation but suppress GVHD induced by nonregulatory donor T cells Importantly, cotransfer of CD4 CD25 T reg cells neither interferes with stem cell engraftment nor abrogates the beneficial antitumor activity of donor T-cell infusions. 15,18,19 These murine studies encouraged clinical transplanters to further investigate the role of donor CD4 CD25 T reg cells for the improvement of stem cell transplantation (SCT). CD4 T cells with high expression levels of CD25 have been isolated from human blood, peripheral lymphoid organs, umbilical cord blood, and thymus Phenotypically, they are comparable to their murine counterparts; that is, they constitutively express CD25, glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR) and intracellular cytotoxic T lymphocyte associated antigen-4 (CTLA-4). In addition, expression of the transcription factor forkhead box P3 (FoxP3), described recently as a key regulatory gene for the development of CD4 CD25 T cells in mice, 26 could now be demonstrated in human CD4 CD25 T reg cells. 27 Most importantly, however, functional characteristics of murine CD4 CD25 T reg cells could be confirmed for human CD4 CD25 /high T reg cells, such as hyporesponsiveness to T-cell receptor (TCR) mediated stimulation and cytokine-independent, cell contact dependent suppression of cocultured CD25 T cells. 20,21,23,25 Thus, immunoregulation by CD4 CD25 T reg cells seems to be a conserved mechanism, and their adoptive transfer or therapeutic manipulation in vivo might therefore be an attractive strategy for the prevention or treatment of T cell mediated diseases and, in particular, for the protection from GVHD after allogeneic SCT. The main obstacle for the clinical application of human CD4 CD25 T reg cells so far is their paucity in peripheral blood and, in consequence, the necessity to develop reliable expansion protocols. Although several studies demonstrated that exogenous IL-2 can abrogate the anergic state of human and murine CD4 CD25 T reg cells, 4,20,21,23 efficient long-term culture and polyclonal expansion of human CD4 CD25 T reg cells has not yet been reported. The few protocols published thus far for human CD4 CD25 T reg cells required stimulation either by allogeneic mononuclear cells (MNCs) combined with anti-cd3 antibodies, IL-2, and allogeneic lymphoblastic feeder cells 23 or by autologous in vitro differentiated dendritic cells (DCs), together with exogenous IL-2 and IL-7, 28 and resulted in only a limited expansion of From the Department of Hematology and Oncology and the Institute of Pathology, University Hospital Regensburg, Regensburg, Germany. Submitted January 8, 2004; accepted April 4, Prepublished online as Blood First Edition Paper, April 15, 2004; DOI /blood Supported by grants from the Dr Mildred Scheel Stiftung, the Wilhelm Sander Stiftung, and the Regensburg medical research program (ReForM). Reprints: Matthias Edinger, University Hospital Regensburg, Department of Hematology and Oncology, Franz-Josef-Strauss-Allee 11, Regensburg, Germany; matthias.edinger@klinik.uni-regensburg.de. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked advertisement in accordance with 18 U.S.C. section by The American Society of Hematology BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 895

2 896 HOFFMANN et al BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 regulatory T cells (approximately 20-fold within 14 days and 3-fold within 7 days, respectively). Similarly, standard T-cell culture conditions or stimulation with autologous DCs resulted in only limited expansion rates in the murine system, 14,29,30 and only allogeneic stimulation combined with high-dose IL-2 or the use of a superagonistic anti-cd28 antibody allowed efficient expansion of murine and rat CD4 CD25 T reg cells, respectively. 19,31 We now demonstrate that repeated stimulation with cross-linked anti-cd3 and anti-cd28 antibodies together with high-dose IL-2 results in the profound polyclonal proliferation of highly purified human CD4 CD25 high T cells and their up to fold expansion within 3 to 4 weeks. Expanded CD4 CD25 high T cells retained their phenotype and showed even increased suppressive activity. In contrast to CD4 CD25 T cells, they maintained expression of the lymph node homing receptors L-selectin (CD62L) and CC chemokine receptor 7 (CCR7) during expansion. This expression profile might support the ability of expanded CD4 CD25 high T cells to enter lymphatic organs after reinfusion to mediate their suppressive activity in a site-specific manner in vivo. Materials and methods Cell lines and culture medium The L929-derived murine Ltk cell line stably transfected with human Fc RII (CD32) has been described previously 32 and was a kind gift from R. Levy, Stanford University (Stanford, CA). Cells were grown in RPMI 1640 (Bio Whittaker, Verviers, Belgium) with 10% fetal calf serum (FCS) (Gibco BRL, Karlsruhe, Germany), 2 mm glutamine, 10 mm HEPES (N-2- hydroxyethylpiperazine-n -2-ethanesulfonic acid), 1% nonessential amino acids (all PAN Biotech, Aidenbach, Germany), 50 U/mL penicillin, 50 g/ml streptomycin, and M 2-mercaptoethanol (2-ME) (all Gibco BRL) (crpmi). Hypoxanthine, aminopterin, thymidine (HAT) selection was performed in intervals to ensure CD32 expression. Cytokines and T-cell stimulatory antibodies Recombinant human IL-2 (rh IL-2) (aldesleukin) was from Chiron (Amsterdam, the Netherlands); anti-cd3 (OKT3) (muromonab-cd3) was a kind gift from Ortho Biotech (Neuss, Germany); and purified anti-cd28 (CD28.2) was purchased from BD Biosciences (Heidelberg, Germany). Clinical grade beads coated with anti-cd3 and anti-cd28 antibodies (T-cell expander) were purchased from Dynal Biotech (Hamburg, Germany). Antibodies and flow cytometry (FACS) For fluorescence-activated cell sorter (FACS) analysis, allophycocyanin (APC) anti-cd3 (UCHT1); APC, peridinin chlorophyll-alpha protein (PerCP), and fluorescein isothiocyanate (FITC) anti-cd4 (SK3); FITC anti-cd19 (4G7); APC and phycoerythrin (PE) anti-cd25 (2A3); PE anti- CD45RA (HI100); PE anti-cd45ro (UCHL1); APC anti-cd62l (DREG- 56); FITC anti-cd27 (L128); PE anti-cd152 (BNI3); and matched mouse isotype control antibodies were from BD Biosciences. FITC anti-ccr7 (150503) and PE anti-gitr (110416) antibodies were from R&D Systems (Abingdon, United Kingdom). For TCR V -repertoire analysis the IOTest Beta Mark kit (Beckman Coulter, Krefeld, Germany) was used. For intracellular staining, the Cytofix/Cytoperm kit (BD Biosciences) was used. All stainings were performed in phosphate-buffered saline (PBS)/2% FCS and 1% human immunoglobulin (Flebogamma) (Grifols, Langen, Germany) to block unspecific staining. Propidium iodide was added to exclude dead cells. Flow cytometry was performed on a FACSCalibur (BD Biosciences), and data were analyzed with CellQuest (BD Biosciences) or FlowJo (Treestar, Ashland, OR) software. Cell isolation and sorting Peripheral blood mononuclear cells (PBMCs) were isolated from leukapheresis products of healthy volunteers (after their informed consent and in accordance with protocols approved by the local authorities) by density gradient centrifugation over Ficoll/Hypaque (Pharmacia, Freiburg, Germany). Cells were stained with PE anti-cd25 and anti-pe magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), and CD25 cells were enriched with the use of the Midi-MACS system (Miltenyi Biotec). CD25-enriched and CD25-depleted cell populations were stained with FITC anti-cd4 and sorted into CD4 CD25 and CD4 CD25 high T cells on a FACStar Plus (BD Biosciences). Gates for the purification of CD4 CD25 high cells were set to exclude propidium iodide (PI) positive dead cells, to include CD4 T cells that exceed the CD25 expression level of CD4 CD25 cells contained in PBMCs (mostly CD19 B cells), and to include only cells with a slightly lower CD4 expression level within the CD4 CD25 high population (Figure 1). Cells were reanalyzed after sorting and routinely showed greater than 98% purity. T-cell expansion cultures FACS-purified CD4 CD25 high or CD4 CD25 T cells ( per well) were placed into 96-well flat-bottom plates with irradiated (70 Gy) CD32 L cells. 32 Cultures were in 200 L crpmi with 100 ng/ml OKT3, 100 ng/ml anti-cd28 antibody (Ab), and 300 U/mL IL-2. After 5 to 6 days, cells were harvested, and T cells were cocultured with CD32 L cells in 500 L crpmi supplemented with Abs and IL-2 in 24-well plates. Cultures were supplemented with 200 L crpmi/il-2 after 4 days and split onto fresh CD32 L cells once per week. Helper cell free expansion cultures were performed in 96-well U- bottom plates by adding T-cell expander beads at a ratio of 4 beads per cell to CD4 CD25 high T cells for the first week, and at a 1:1 ratio with cells per well in 24-well plates thereafter. Cells were cultured in crpmi with 300 U/mL IL-2, fed with crpmi/il-2 after 4 days, and restimulated with fresh beads weekly. CD4 CD25 T cells were stimulated as CD4 CD25 high T cells for the first 2 weeks and later on with a reduced ratio of 1 bead per 10 cells. T-cell expansion was determined by counting trypan blue negative aliquots in approximately weekly intervals. Stimulation assay and MLR For polyclonal stimulation, purified CD4 responder T cells (T resp ) (selected from PBMCs with anti-cd4 MACS beads; Miltenyi Biotec) were cocultured in 96-well U-bottom plates with irradiated (30 Gy) autologous PBMCs in the presence of 100 ng/ml OKT3 in 200 L crpmi. Where indicated, titrated numbers of freshly purified or in vitro expanded autologous CD4 CD25 high T cells were added to obtain the ratios described. Expanded T cells were harvested and rested in IL-2 containing crpmi 48 hours prior to use. Cultures were incubated for 4 days and labeled with 0.5 Ci ( MBq) [ 3 H]thymidine ([ 3 H]TdR) (Hartmann Analytics, Braunschweig, Germany) per well for the last 18 hours. [ 3 H]thymidine incorporation was measured by liquid scintillation counting (Top Count; Perkin Elmer, Rodgau-Jügesheim, Germany). Mixed lymphocyte reactions (MLRs) were performed similarly, with CD4 T resp cells, graded numbers of autologous CD4 CD25 high T cells, and irradiated allogeneic PBMCs as stimulators, and were harvested after 5 days. All assays were performed in triplicate. For neutralization experiments, antibodies to human IL-4 (5 g/ml MP4-25D2; BD Biosciences), IL-10 (1 g/ml JES3-19F1; BD Biosciences), and transforming growth factor (TGF- ) (5 g/ml 1D11; R&D Systems) were added at the start of the cocultures. Antibody concentrations neutralize up to 200 ng/ml IL-4, 50 ng/ml IL-10, and 12.5 ng/ml TGF-. For select experiments, FACS-sorted CD4 CD25 high or CD4 CD25 T cells were seeded in 96-well U-bottom plates precoated with anti-cd3 (10 g/ml) and costimulated with soluble anti-cd28 (100 ng/ml), with or without IL-2 (300 U/mL), for 3 days. In parallel, FACS-sorted CD4 CD25 high or CD4 CD25 T cells were seeded in 96-well flat bottom plates with irradiated CD32 L cells, and stimulated with anti-cd3 and anti-cd28 (100 ng/ml each) and/or IL-2 (300 U/mL) for 3 days. Cultures were labeled with 0.5 Ci ( MBq) [ 3 H]thymidine for the last 18 hours.

3 BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 HUMAN T reg -CELL EXPANSION 897 Cytokine detection First, FACS-sorted or in vitro expanded (on either L cells or on beads) CD4 CD25 high or CD4 CD25 T cells were seeded in new wells and (further) incubated under the respective culture conditions (L cell based or bead based) for 3 days. For detection of endogenous IL-2, parallel cultures were set up without rh IL-2. Supernatants were collected and analyzed for IL-2, IL-4, IL-10, tumor necrosis factor- (TNF- ), and interferon- (IFN- ) using the human T-helper 1 (Th1)/Th2 competitive binding assay (CBA) kit from BD Biosciences. TGF- was determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) following the manufacturer s recommendations. Statistical analysis Differences in proliferation of T resp cells were analyzed by means of the 2-tailed Student t test. P less than.05 was considered significant. Results Highly purified human CD4 CD25 high T cells are anergic and suppressive Coexpression of CD4 and CD25 has been used to identify T reg cells in mice and humans. However, human PBMCs contain not only CD4 CD25 T cells with regulatory function, but also substantial numbers of CD4 CD25 int T cells, which predominantly represent recently activated cells 25 (Figure 1Ai). Since the goal of this study was the expansion of the T reg -cell population, we aimed at isolating them at highest purity. In consequence, only CD4 T cells with a CD25 expression level exceeding that of CD4 CD25 cells within PBMCs (predominantly activated B cells) were FACS sorted to greater than 98% purity (Figure 1Aiii). These CD4 CD25 high T cells represented, on average, 1.8% of PBMCs (range, 1.1% to 3.4%; n 20 analyses with cells from 13 different donors) and showed a slightly, but reproducibly, lower CD4 expression than the CD4 CD25 int cells (Figure 1Ai), probably owing to their smaller size, as revealed in the forward scatter channel (FSC) (data not shown). When compared, CD25 high and CD25 CD4 T cells differed not only in their CD25, but also in their intracellular CTLA-4 expression levels (Figure 1Bi), while both T-cell populations were predominantly double-positive for the lymph node (LN) homing receptors CCR7 and CD62L (Figure 1Bii-iii). We tested the suppressive activity of sorted CD4 CD25 high T cells through their ability to inhibit the proliferation of autologous CD4 T cells after allogeneic stimulation in MLRs. CD4 responder T cells, in contrast to FACS-purified CD4 CD25 high T cells, showed a vigorous proliferative response to irradiated PBMCs from an unrelated donor that was dose-dependently inhibited up to 60% at a 1:1 ratio upon coculture of the 2 cell populations (Figure 1Ci). In contrast, FACS-sorted CD4 CD25 T cells did not inhibit the proliferation of CD4 responder T cells in cocultures (Figure 1Ci). Similarly, freshly purified CD4 CD25 high T cells were hyporesponsive to polyclonal stimulation provided by anti-cd3 antibodies in the presence of autologous PBMCs, but suppressed the proliferation of CD4 responder T cells with even higher efficiency (89% at a 1:1 ratio) in this assay system (Figure 1Cii). Polyclonal expansion of highly suppressive CD4 CD25 high T reg cells after strong costimulation provided by artificial antigen-presenting cells As previously described for human CD4 CD25 T reg cells, the anergic state of pure CD4 CD25 high T cells was abrogated after simultaneous stimulation via TCRs and CD28 in the presence of IL-2, while neither IL-2 nor platebound anti-cd3 combined with soluble anti-cd28 could break their unresponsiveness (Figure 2A). Since these results suggested that CD4 CD25 high T cells were not per se incapable of proliferating, we next examined whether their long-term and large-scale expansion could be achieved under adequate costimulatory conditions. We applied a previously described culture system in which stimulatory antibodies are presented by Fc RII (CD32) bearing L cells. 32 Neither CD4 CD25 high nor CD4 CD25 T cells proliferated upon coculture with L cells alone or additional IL-2. However, the combined stimulation via CD3 and CD28 in the presence of CD32 L cells was sufficient to induce proliferation in both populations, which was further enhanced by IL-2 and reached maximum levels at 100 to 1000 U/mL (Figure 2A and data not shown). In consequence, saturating doses of 300 U/mL IL-2 were used in all subsequent experiments. CD4 CD25 high T cells cultured under these conditions expanded dramatically with an average fold (range, to fold) increase in cell numbers within 3 to 4 weeks (n 9 cultures from separate leukaphereses of 5 donors) (Figure 2B). Although CD4 CD25 high T-cell expansion usually slowed down after 4 weeks, individual cultures were propagated for more than 3 months Figure 1. Phenotype and function of CD4 CD25 high T reg cells from human peripheral blood. (A) Expression of CD4 and CD25 on human PBMCs and sort gates (i) used to isolate CD4 CD25 (ii) and CD4 CD25 high T cells (iii). Numbers indicate the precentage of cells in each quadrant. (B) Intracellular expression of CTLA-4 and surface expression of CD62L and CCR7 on sorted CD4 CD25 T cells (solid line histogram in i; also ii) and CD4 CD25 high T reg cells (gray histogram in i; also iii). Dotted line indicates isotype control. (C) CD4 T cells (T resp) were cultured either for 5 days with irradiated allogeneic stimulator cells (i) or for 4 days with irradiated autologous PBMCs and soluble anti-cd3 (ii) and variable numbers of CD4 CD25 high or CD4 CD25 T cells to obtain the indicated ratios. ***P.001 (Student t test). *P.05. Panels show 1 of at least 3 independent experiments using cells from different donors. Error bars represent standard deviation from the triplicate wells.

4 898 HOFFMANN et al BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 Figure 2. Large-scale expansion of polyclonal CD4 CD25 high T cells on CD32 L cells. (A) Proliferation of sorted CD4 CD25 high T cells and CD4 CD25 T cells in response to IL-2, anti-cd3/anti-cd28 (precoated and soluble, respectively), or both in the presence or absence of irradiated CD32 L cells. One of 3 independent experiments using cells from 3 different donors. (B) Expansion of sorted CD4 CD25 high T cells (n 9 individual cultures) cultured on irradiated CD32 L cells with anti-cd3 and anti-cd28 Abs plus IL-2. Symbols indicate different donors (n 5). (C) TCR V -repertoire of primary and 25-day expanded CD4 CD25 high and CD4 CD25 T cells. One of 5 individual cultures with cells from 5 different donors. with a maximum expansion rate of Expansion of CD4 CD25 T cells from the same donors cultured in parallel usually exceeded that of CD4 CD25 high T cells and resulted in an average fold expansion (range, to fold) within 3 to 4 weeks (n 9 cultures from separate leukaphereses of 5 donors; data not shown). To differentiate CD4 CD25 high T-cell expansion that was was polyclonal from that driven by individual T-cell clones, TCR V -analysis was performed. CD4 CD25 high T cells within unmanipulated PBMCs showed a polyclonal V -usage similar to that of CD4 CD25 T cells (Figure 2C). After isolation and in vitro expansion for 25 days, their TCR V -repertoire did not differ significantly either from that of freshly isolated CD4 CD25 high T cells or from that of freshly isolated or expanded CD4 CD25 T cells (Figure 2C). Thus, CD4 CD25 high T cells expanded polyclonally and without loss of clonotypes under these culture conditions. FACS analysis of CD4 CD25 high T-cell populations expanded beyond 2 and 3 weeks revealed a CD45RO high and CD45RA low phenotype, reflecting their activated state (data not shown). In addition, expression of CD25 and intracellular CTLA-4 was further up-regulated as compared with freshly isolated CD4 CD25 high T cells from the same donor (Figures 3A, i and iii, and 1A-B). In comparison, up-regulation of CD25 and CTLA-4 by expanded CD4 CD25 T cells was more heterogeneous and did not show the uniformly high levels of CD4 CD25 high T cells, whereas both populations comparably up-regulated GITR expression after expansion (Figure 3A, i and iii, and data not shown). CD4 CD25 high T cells expanded for 2 weeks maintained high expression levels for both CD62L and CCR7, whereas CD4 CD25 T cells rapidly lost CCR7 expression upon in vitro culture and significantly downregulated CD62L within the first 2 weeks (Figure 3A, ii). Even after more than 3 weeks in culture, CD4 CD25 high T cells still remained CD25 high and CD62L, with close to 30% of them still expressing CCR7 (Figure 3A, iii and iv). Thus, in vitro expanded CD4 CD25 high T cells maintained phenotypic characteristics of T reg cells and clearly differed from expanded CD4 CD25 T cells. Next we examined whether expanded CD4 CD25 high T cells maintained their suppressive activity. To this end, expanded CD4 CD25 high T cells were rested in IL-2 containing medium without L cells and antibody stimulation for 48 hours. Rested CD4 CD25 high T cells reverted to their anergic state and did not proliferate in response to allogeneic stimulation (Figure 3Bi). In contrast, freshly isolated CD4 T cells proliferated well under these conditions, and their proliferation was dose-dependently suppressed in cocultures with in vitro expanded CD4 CD25 high T cells (Figure 3Bi). Interestingly, suppression by expanded CD4 CD25 high T cells on a per cell basis was stronger than that of freshly isolated CD4 CD25 high T cells from the same donor, still resulting in a highly significant reduction of [ 3 H]thymidine incorporation at a 1:8 ratio of CD4 CD25 high T cells to responder T cells (compare Figures 3Bi and 1Ci). In contrast to CD4 CD25 high T cells, in vitro expanded CD4 CD25 T cells did not suppress the proliferation of responder T cells. Similarly, expanded CD4 CD25 high T cells were also hyporesponsive to polyclonal stimulation but showed a significantly enhanced activity for the suppression of freshly isolated CD4 responder T cells. [ 3 H]thymidine incorporation was reduced by greater than 65%, even at a 1:16 ratio of CD4 CD25 high T cells to responder T cells (Figure 3Bii). Although never to the same extent as CD4 CD25 high T cells, CD4 CD25 T cells expanded beyond 3 weeks also diminished [ 3 H]thymidine incorporation in this assay system. This effect probably reflects different proliferation kinetics of long-term cultured T cells, resulting in earlier exhaustion of the cultures and/or higher cytokine consumption after their polyclonal restimulation, since it was only sporadically observed with CD4 CD25 T cells expanded up to 2 weeks (data not shown) and never occurred in MLR assays (Figures 3Bi and 4D). Helper cell free expansion of human CD4 CD25 high T reg cells For clinical applications, a helper cell free expansion protocol for CD4 CD25 high T cells would be preferable. We therefore applied clinical grade beads coated with anti-cd3 and anti-cd28 antibodies plus IL-2 to induce proliferation in CD4 CD25 high T cells. While the dose of beads recommended for non-t reg cells was insufficient for sustained CD4 CD25 high T-cell proliferation (data

5 BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 HUMAN T reg -CELL EXPANSION 899 Figure 3. T reg-cell characteristics in expanded CD4 CD25 high T cells. Expanded CD4 CD25 high T cells retain phenotypical and functional characteristics of T reg cells. (A) Surface expression of CD25, CD62L, and CCR7, and intracellular expression of CTLA-4 of CD4 CD25 high T cells (gray histograms and right dot plots) and CD4 CD25 T cells (solid line histograms and left dot plots) expanded for 2 weeks (i-ii) or 3.5 weeks (iii-iv) on CD32 L cells. Dotted line in i and iii indicates isotype control. Numbers indicate the percentage of cells within each quadrant. (B) CD4 T resp cells were stimulated with allogeneic PBMCs (i), or with anti-cd3 Ab and autologous PBMCs (ii) for 5 or 4 days, respectively. CD4 CD25 high or CD25 T cells, expanded on CD32 L cells for 23 days and then rested for 2 days, were added at variable numbers to obtain the indicated ratios. ***P.001 (Student t test). *P.05. One of at least 4 independent experiments using cells from different donors. Error bars represent standard deviations for triplicate wells. not shown), significant expansion was observed when a high bead-cell ratio was used for the initiation of cultures (4 beads per cell) as well as their long-term propagation (1 bead per cell). Although less efficiently than in L cell cultures, CD4 CD25 high T cells still expanded on average 2775-fold (range, 620- to fold) within 3 to 4 weeks (Figure 4A; n 7 cultures from individual leukapheresis products derived from 5 different donors). TCR V -screening performed on day 24 confirmed that beadcultured CD4 CD25 high T cells also expanded polyclonally, since the V -usage of expanded cells did not differ significantly either from that of freshly isolated CD4 CD25 high T cells or from that of freshly isolated or bead-expanded CD4 CD25 T cells (Figure 4B). Similarly to L cell expanded cells, both populations completely converted to a CD45RO state, and both showed upregulation of GITR expression after 2 and 3 weeks of expansion (data not shown). In addition, bead-expanded CD4 CD25 high T cells showed a homogeneous up-regulation of CD25 and CTLA-4 and a sustained expression of CD62L, CCR7, and CD27 (Figure 4C), whereas bead-expanded CD4 CD25 T cells again differed in their CD25 and CTLA-4 expression levels, completely downregulated CCR7, and substantially decreased their CD62L and CD27 expression (Figure 4C). The suppressive activity of beadexpanded CD4 CD25 high T cells after allogeneic stimulation, as determined after 2 or 3.5 weeks of expansion, was comparable to that of L cell expanded CD4 CD25 high T cells and again significantly stronger than that of freshly isolated CD4 CD25 high T cells from the same donor (Figure 4D). Similar results were obtained after polyclonal stimulation (data not shown). Hence, CD4 CD25 high T cells expanded under helper cell free conditions retained their characteristic phenotype as well as their suppressive function. Next we examined, whether expanded CD4 CD25 high T cells suppress not only naive, but also preactivated, responder T cells. When polyclonally expanded CD4 CD25 T cells were rested for 2 days in IL-2 containing medium and then tested in the MLR, they responded well to allogeneic restimulation. Addition of in vitro expanded CD4 CD25 high T cells suppressed their alloresponse, while addition of in vitro expanded CD4 CD25 T cells further increased [ 3 H]thymidine uptake (Figure 5). Thus, in vitro expanded CD4 CD25 T cells are susceptible to restimulation and to suppression by CD4 CD25 high T cells, while expanded CD4 CD25 high T cells suppress both naive (Figures 3-4) and antigen-experienced T cells. Suppression by expanded CD4 CD25 high T cells is cytokine independent Cytokine secretion of CD4 CD25 high T cells expanded under both culture conditions was compared with that of freshly sorted CD4 CD25 high T cells stimulated for 72 hours in L cell cultures or with CD3/CD28 beads (Figure 6A). Neither fresh nor expanded CD4 CD25 high T cells produced significant amounts of IL-2 or TNF-, and both produced only marginal amounts of IFN-. TGF- production by fresh and expanded CD4 CD25 high T cells was slightly increased upon L cell based stimulation, while IL-4 production was increased in CD4 CD25 high T cells expanded under both conditions as compared with freshly sorted CD4 CD25 high T cells. Most strikingly, IL-10 secretion by expanded cells significantly exceeded that of freshly isolated CD4 CD25 high T cells. In comparison, freshly isolated CD4 CD25 T cells secreted at least 2 log higher amounts of IL-2, IFN-, and TNF-, but no detectable IL-4 or IL-10 and only marginal amounts of TGF-. After expansion, IL-2, IFN-, and TNF- stayed high while IL-4 and IL-10 were up-regulated to levels comparable to those of expanded CD4 CD25 high T cells (data not shown).

6 900 HOFFMANN et al BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 Figure 4. Polyclonal expansion of sorted CD4 CD25 high T cells with bead-coupled anti-cd3/ anti-cd28 Abs and high-dose IL-2. Sorted CD4 CD25 high T cells can be expanded polyclonally with bead-coupled anti-cd3/anti-cd28 Abs and high-dose IL-2 without down-regulation of LN homing receptors or loss of suppressive function. (A) Sorted CD4 CD25 high T cells (n 7 individual cultures) were stimulated with anti-cd3/anti-cd28 coated beads plus IL-2. Symbols indicate different donors (n 5; same as in Figure 2). (B) TCR V -repertoire of CD4 CD25 high and CD4 CD25 T cells before and after expansion for 24 days with Ab-coated beads and IL-2. One of 5 different cultures with cells from 5 different donors. (C) Surface expression of CD25, CD62L, CCR7, and CD27, and intracellular expression of CTLA-4 of CD4 CD25 high T cells (gray histograms in i and ii; and iv and vi) and CD4 CD25 T cells (solid-line histograms in i and ii; and iii and v) after expansion with Ab-coated beads and IL-2 for 3 weeks. Dotted line indicates isotype control. Numbers indicate the percentage of cells within each quadrant. (D) CD4 T resp cells were stimulated with allogeneic PBMCs for 5 days. CD4 CD25 high T cells, freshly isolated or expanded with Ab-coated beads for 13 or 22 days and then rested for 2 days, were added at variable numbers to obtain the indicated ratios. One of 3 independent experiments using cells from different donors. Error bars represent standard deviations from triplicate wells. Since it is well established that in vitro suppression of responder T cells by freshly isolated CD4 CD25 /high T cells is independent from secreted cytokines, we next examined whether the same was true for CD4 CD25 high T cells expanded either on L cells or with antibodycoated beads. To this end, neutralizing antibodies to IL-4, IL-10, and TGF- were added to MLR assays. None of the antibodies abrogated the suppressive activity of expanded CD4 CD25 high T cells, nor did the combined blockade of all 3 cytokines (Figure 6B). Thus, in vitro expanded CD4 CD25 high T cells seem to mediate suppression by the same, yet unidentified, cytokine-independent mechanism as freshly isolated CD4 CD25 high T cells. Discussion Thus far, the anergic state of human as well as murine CD4 CD25 T reg cells hindered their efficient long-term propagation and large-scale expansion in vitro. Yet, several animal studies have demonstrated that CD4 CD25 T reg cells can proliferate and expand in vivo: for example, in lymphopenic hosts, 33 after antigen (Ag) specific stimulation, 34,35 and even under steady-state conditions in unmanipulated animals. 36 Decreased numbers or complete absence of CD4 CD25 T reg cells in various knockout (KO) mice revealed the relevance of IL-2 and CD28/B7 costimulation for their thymic generation 4,37-39 as well as for their peripheral function and expansion. 30,40-42 While stimulation with a superagonistic CD28- specific monoclonal antibody was shown to be sufficient for rat CD4 CD25 T-cell expansion in vitro and in vivo, 31 combined stimulation via TCR, CD28, and IL-2R was required to transiently brake the anergic state of human and murine CD4 CD25 /high T reg cells. 1,21,29,43 However, these strategies applying soluble or platebound antibody stimulation did not result in sustained T reg -cell proliferation, and even the most successful protocols reported so far involving allogeneic stimulation resulted only in short-term

7 BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 HUMAN T reg -CELL EXPANSION 901 Figure 5. Effect of expanded CD4 CD25 high T cells on the alloresponse of antigen-experienced responder T cells. Expanded CD4 CD25 high T cells suppress the alloresponse of antigen-experienced responder T cells. Sorted CD4 CD25 T cells were expanded with anti-cd3/anti-cd28 coated beads, rested for 2 days, and then stimulated with irradiated allogeneic PBMCs in the absence or presence of autologous expanded CD4 CD25 high or CD25 T cells at a 1:1 ratio for 5 days. ***P.001(Student t test). *P.05. One of 3 independent experiments using cells from different donors. proliferation and/or marginal expansion of human CD4 CD25 /high T reg cells. 23,28 In light of these results, we examined whether highly purified human CD4 CD25 high T reg cells could be propagated long-term and on a large scale in vitro through potent costimulation provided by 2 types of artificial antigen-presenting cells. To avoid contamination with recently activated CD4 CD25 int T cells, isolation of CD4 CD25 high T reg cells was carried out under stringent sorting conditions, with selection of only those CD4 cells that exceeded the CD25 expression level of CD4 CD25 cells (Figure 1) and that were slightly, but detectably, smaller than recently activated CD4 CD25 int T cells and discretely lower in CD4 expression. As described previously for murine as well as human CD4 CD25 T reg cells, 11,21,23,44 these CD4 CD25 high T cells constitutively expressed intracellular CTLA-4, were hyporesponsive to CD3-mediated or allogeneic stimulation, and dose-dependently suppressed cocultured CD4 responder T cells, thereby confirming earlier reports by Baecher-Allen et al. 25 Yet CD4 CD25 high T reg cells proliferated and expanded when cultured on L cells presenting anti-cd3 and anti-cd28 antibodies via human Fc RII for efficient TCR-synapse formation. 32,45 Addition of high-dose IL-2 further increased their proliferation and led to an average fold expansion within 3 to 4 weeks, which is several logs higher than any other T reg -cell expansion rate reported thus far. The use of anti-cd3/anti-cd28 coated beads and high-dose IL-2 also promoted profound and reproducible expansion, demonstrating that repeated stimulation via TCR and CD28 together with IL-2 is necessary and sufficient for the long-term propagation of human CD4 CD25 high T reg cells in vitro. Since these beads are approved for clinical use, the ex vivo expansion of human CD4 CD25 high T reg cells for clinical application now seems feasible. As reported earlier, 46 unmanipulated CD4 CD25 high T cells displayed a broad TCR V -usage similar to that of CD4 CD25 - T cells. This TCR V -repertoire remained virtually unchanged after in vitro culture, thereby documenting the polyclonal expansion of CD4 CD25 high T reg cells. In vitro expanded CD4 CD25 high T cells maintained phenotypic markers characteristic for primary CD4 CD25 high T reg cells, including uniformly high levels of CD25 and CTLA-4 as well as GITR expression. Most importantly, however, expanded CD4 CD25 high T cells could regain their anergic state, and their suppressive activity exceeded even that of primary CD4 CD25 high T reg cells from the same donor. These findings are consistent with previous reports demonstrating augmented suppressive capacity of short-term stimulated CD4 CD25 T reg cells 29,30 and support the observation that the proliferation and suppressive function of CD4 CD25 /high T cells are not mutually exclusive. 35 In model systems of autoimmunity, antigen-specific CD4 CD25 T reg cells are found predominantly in draining LNs of affected organs, 34 where their local proliferation is driven by Ag-presenting DCs expressing the costimulatory molecules CD80 and CD Suppression of effector T cells by CD4 CD25 T reg cells therefore seems to require their migration to secondary lymphoid organs where all 3 cell populations interact in close proximity. Expression of CD62L and CCR7 crucially determines the LN homing capacity of cells, including CD4 CD25 T reg cells. 47 Szanya et al 48 showed, that only CD62L CD4 CD25 T reg cells, which also expressed higher levels of CCR7 than the CD62L subpopulation, migrated to pancreatic LNs and delayed diabetes onset in an adoptive transfer setting. Similarly, we could show that only CD62L CD4 CD25 donor T reg cells provided protection from lethal GVHD after allogeneic bone marrow transplantation (BMT) although both CD62L and CD62L T reg cells were equally suppressive in vitro. 49 Reisolation of donor T cells from transplant recipients confirmed the impaired ability of CD62L CD4 CD25 T reg cells to enter mesenteric lymph nodes. These findings suggest that lymphatic organs are the crucial sites for protection from GVHD by donor CD4 CD25 T reg cells and that host antigen-presenting cells not only are responsible for the priming of alloaggressive T cells, 50,51 but also provide the strong costimulation required for the activation and in vivo expansion of alloantigen-specific donor T reg cells. 30,34 Since the suppressive activity of CD4 CD25 high T reg cells depends crucially on their own activation, we examined whether in vitro expanded CD4 CD25 high Figure 6. Immunosuppressive cytokines of CD4 CD25 high T cells. CD4 CD25 high T cells produce immunosuppressive cytokines, but their mode of suppression is cytokine independent. (A) First, CD4 CD25 high T cells, freshly sorted or expanded either on CD32 L cells or with anti-cd3/anti-cd28 coated beads (TCE) were stimulated (or restimulated) under the indicated culture conditions for 3 days. Supernatants were harvested and analyzed by ELISA (TGF- ) or with a CBA kit (all others). One of 2 independent experiments with cells from 2 different donors is shown. (B) CD4 T resp cells were stimulated with allogeneic PBMCs for 5 days in the absence or presence of bead-expanded (left) or L cell expanded CD4 CD25 high T cells (right) at a 1:1 ratio and neutralizing antibodies, as indicated. P.001 for all cocultures versus T resp cells alone.

8 902 HOFFMANN et al BLOOD, 1 AUGUST 2004 VOLUME 104, NUMBER 3 T cells maintained lymph node homing receptors. Our data clearly demonstrate sustained expression of CD62L and CCR7 on in vitro expanded human CD4 CD25 high T reg cells, suggesting that they maintain the ability to enter lymphatic tissues to exert their suppressive function in a site-specific manner. The combined expression of CD62L, CCR7, and CD27 on Ag-experienced CD45RO T cells has recently been described as being characteristic for central memory T cells that continuously screen lymphoid organs for recall antigens. 52,53 Their comparable phenotype suggests a similar capacity of activated CD4 CD25 high T reg cells. Trenado et al 19 recently demonstrated improved protection from lethal GVHD by in vitro expanded CD62L CD4 CD25 T reg cells primed to host alloantigens as compared with T reg cells primed by third-party stimulators in murine models. Those experiments confirmed that in vitro expanded T reg cells survive after reinfusion and that they still depend on TCR stimulation to mediate suppression. Taking these observations into account, we purposely aimed at expanding human CD4 CD25 high T cells polyclonally rather than using allogeneic stimulator cells for future clinical trials in BMT patients. This decision was based on our previous finding that in vitro priming is not required for GVHD inhibition, since activation of alloantigen-specific T reg cells occurs efficiently in vivo. 11,15 The potential advantage of this in vivo selection is the maintenance of TCR specificities reactive to nonhematopoietic minor alloantigens that are not necessarily presented by recipient stimulator cells isolated from peripheral blood. In contrast to polyclonally expanded T reg cells, in vitro expanded T reg cells primed by host PBMCs are expected to recognize predominantly References hematopoietic alloantigens. However, suppression of the graftversus-hematopoiesis effect of donor T cells is not intended in allogeneic BMT and might even result in graft rejection or higher relapse rates in patients receiving transplants for hematologic malignancies. Owing to the lack of protocols for the efficient polyclonal expansion of CD4 CD25 T reg cells, a systematic comparison of differentially expanded CD4 CD25 T reg cells in GVHD models could not be performed thus far, but is now under investigation. In summary, we have shown that highly purified CD4 CD25 high T cells are a polyclonal T reg -cell population that rapidly expands in vitro upon strong costimulation. Expanded CD4 CD25 high T reg cells retain their phenotypic characteristics, maintain expression of LN homing receptors, and show even increased suppressive activity when compared with freshly isolated cells. These findings will facilitate the elucidation of their mode of action and allow the detailed examination of their Ag specificity. 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10 : doi: /blood originally published online April 15, 2004 Large-scale in vitro expansion of polyclonal human CD4 + CD25 high regulatory T cells Petra Hoffmann, Ruediger Eder, Leoni A. Kunz-Schughart, Reinhard Andreesen and Matthias Edinger Updated information and services can be found at: Articles on similar topics can be found in the following Blood collections Immunobiology and Immunotherapy (5619 articles) (577 articles) Transplantation Immunotherapy (2313 articles) Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Blood (print ISSN , online ISSN ), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Copyright 2011 by The American Society of Hematology; all rights reserved.