Efficient and reproducible large-scale isolation of human CD4+ CD25+ regulatory T cells with potent suppressor activity

Journal of Immunological Methods 315 (2006) 27 – 36 www.elsevier.com/locate/jim

Research report

Efficient and reproducible large-scale isolation of human CD4 + CD25 + regulatory T cells with potent suppressor activity David G. Wichlan a , Philippa L. Roddam a , Paul Eldridge b , Rupert Handgretinger a,1 , Janice M. Riberdy a,⁎ a

Department of Hematology/Oncology, Division of Stem Cell Transplantation, Mail Stop 321, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA b Human Applications Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA Received 11 May 2006; received in revised form 16 June 2006; accepted 26 June 2006 Available online 21 July 2006

Abstract CD4+ CD25+ regulatory T cells have been the subject of intense investigation and have been shown to modulate immune responses in the settings of autoimmunity, cancer and transplantation. The assessment and optimization of purification schemes for specific cellular subtypes such as CD4+ CD25+ regulatory T cells is a critical consideration in developing cell-based therapies in the clinical setting. In the following studies, different strategies for magnetic isolation are compared and the parameters which affect the overall potency of purified human CD4+ CD25+ regulatory T cells are discussed. The data demonstrate that large-scale magnetic isolation can be used to efficiently and reproducibly purify human CD4+ CD25+ regulatory T cells capable of modulating alloreactive T cell responses. The ability to rapidly purify the desired cells from peripheral blood suggests that magnetic isolation may be a suitable alternative to cell sorting for clinical settings, where large numbers of CD4+ CD25+ regulatory T cells may be necessary. © 2006 Elsevier B.V. All rights reserved. Keywords: Human; CD4+CD25+ regulatory T cells; Transplantation; Autoimmunity; Purification; Suppression

1. Introduction CD4+ CD25+ regulatory T cells are critical mediators of peripheral tolerance and have recently been the topic of widespread investigation (Sakaguchi et al., 2001; Abbreviations: GvHD, graft versus host disease; TCR, T cell receptor; IRB, Institutional Review Board; PBS, phosphate buffered saline; PBMC, peripheral blood mononuclear cells; MLR, mixed lymphocyte reaction. ⁎ Corresponding author. Tel.: +1 901 495 5358; fax: +1 901 495 4023. E-mail address: [email protected] (J.M. Riberdy). 1 Current address: Children's University Hospital, University of Tuebingen, Hoppe-Seyler-Strasse 1, 72076 Tuebingen, Germany. 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.06.014

Wood and Sakaguchi, 2003; Maloy and Powrie, 2001). The pivotal role for CD4+ CD25+ regulatory T cells in modulating self-reactive responses was first observed in rodent models of neonatal thymectomy where mice developed a variety of autoimmune disorders in a strain dependent manner. In a seminal set of experiments it was observed that the transfer of CD4+ CD25+ regulatory T cells to such thymectomized animals prevented the onset of disease (Sakaguchi et al., 1995). These results reinvigorated an interest in suppressor T cells (currently referred to as CD4+ CD25+ regulatory T cells or Tregs) and bolstered support for clinical strategies of immune-based therapies. More recent studies have

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shown that CD4+ CD25+ regulatory T cells can prevent the onset of autoimmune responses in several specific murine disease models including diabetes, colitis, gastritis, and experimental autoimmune encephalomyelitis (Salomon et al., 2000; Singh et al., 2001; Szanya et al., 2002; Kohm et al., 2002; Laurie et al., 2002). In addition, some experimental systems have demonstrated that CD4+ CD25+ regulatory T cells can ameliorate previously established pathology (Liu et al., 2003; Mottet et al., 2003). A further potential therapeutic function of CD4+ CD25+ regulatory T cells was described in murine bone marrow transplantation studies, where these cells were able to prevent graft versus host disease, GvHD (Taylor et al., 2002; Hoffmann et al., 2002). In sum, CD4+ CD25+ regulatory T cells are attractive candidates for immunotherapy because they can modulate the activity of multiple effector cell populations (T cells, B cells, NK cells and dendritic cells), and may therefore be therapeutic in a variety of disease settings (Wood and Sakaguchi, 2003). Despite the beneficial effects observed in animal models of disease, the precise mechanisms which control the functional activity of CD4+ CD25+ regulatory T cells remain unknown. However, three general features have been identified. First, these cells must be activated through their T cell receptor, TCR, to mediate regulatory function. Second, cell–cell contact is necessary for CD4+ CD25+ regulatory T cells to inhibit effector cell function in vitro (Thornton and Shevach, 1998). Third, once activated, CD4+ CD25+ regulatory T cells can suppress CD4+ and CD8+ T cells in vitro, regardless of the antigen specificity of the effector T cell (Thornton and Shevach, 2000; Piccirillo and Shevach, 2001). However, there may be some element of specificity in vivo, as recent studies suggest that activated CD4+ CD25+ regulatory T cells can inhibit alloreactive responses in an antigen-specific manner at low regulatory to effector cell ratios (Joffre et al., 2004). Finally, although our understanding of how human CD4+ CD25+ regulatory T cells mediate their function is limited, the therapeutic benefits demonstrated in animal models of disease illustrate the potential of these cells for future clinical therapies. One critical consideration for immunotherapy strategies employing CD4+ CD25+ regulatory T cells in human disease is the method of purification. Human CD4+ CD25+ regulatory T cells have been isolated from thymus, cord blood, peripheral blood, and lymph node by a variety of methods (Baecher-Allan et al., 2004). An important difference between murine and human studies is the source of cells. Most murine studies purify CD4+ CD25+ regulatory T cells from spleen or in some cases lymph

node, while the majority of studies using human cells isolate this population from peripheral blood. In human peripheral blood, regulatory activity is associated with the small fraction of CD4+ T cells expressing the CD4+ CD25bright phenotype (Baecher-Allan et al., 2001). Our studies and others have noted that CD4+ CD25intermediate T cells comprise a larger proportion of peripheral blood CD4+ T cells than found in lymphoid organs such as murine spleen or human thymus, thus complicating isolation strategies (Baecher-Allan et al., 2004; Kasow et al., 2004). We have further found that different methods co-purify variable numbers of CD4+ CD25intermediate T cells, which in turn alters the regulatory potency of the isolated CD4+ CD25+ T cell population. In contrast, murine studies have shown regulatory activity and/or Foxp3 expression in cells with CD4+ CD25intermediate or CD4+ CD25− phenotypes (Fontenot et al., 2005). Thus, several challenges will need to be overcome to isolate sufficient numbers of CD4+ CD25bright T cells for use in the clinical setting. Another approach to generate sufficient numbers of CD4+ CD25+ regulatory T cells has been to expand purified cells ex vivo and exploit the observation that activation through their TCR is necessary to acquire suppressor function. A number of studies have assessed a variety of large-scale culture conditions and demonstrated that human CD4+ CD25+ regulatory T cells can be expanded in vitro and maintain suppressor function with appropriate stimuli (Godfrey et al., 2004; Hoffmann et al., 2004; Earle et al., 2005). Thus, it is likely that combining optimized large-scale purification methods with ideal expansion conditions will be necessary to successfully use CD4+ CD25+ regulatory T cells in the clinic. We have focused on identifying a large-scale procedure that efficiently and reproducibly isolates CD4+ CD25+ regulatory T cells with potent suppressor activity. The parameters which affect the reproducible selection of highly active human CD4+ CD25+ regulatory T cells during magnetic isolation are discussed. 2. Materials and methods 2.1. Peripheral blood cells For analytical studies, peripheral blood cells from healthy platelet donors were obtained at St. Jude Children's Research Hospital (Memphis, TN), with permission from the Institutional Review Board (IRB). Mononuclear cells from the apheresis of healthy donors were purchased from Lifeblood Biological Services (Memphis, TN) for large-scale isolations.

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2.2. Small-scale magnetic purification of T cell subsets

2.4. Cell sorting and flow cytometry

Peripheral blood cells were centrifuged over a gradient of Ficoll–Paque Plus (Amersham Biosciences, Uppsala, Sweden). Peripheral blood mononuclear cells (PBMC) were collected, washed with Dulbecco's phosphate buffered saline without calcium or magnesium (PBS; Cambrex, Walkersville, MD), and resuspended in MACS buffer [PBS, 2 mM EDTA (Cambrex), and 0.5% bovine serum albumin (Sigma, St. Louis, MO)]. For the initial small-scale isolations, CD4+ CD25+ and CD4+ CD25− T cells were purified by magnetic bead separation using an AutoMACS cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) as follows: untouched CD4+ T cells were purified by magnetic depletion using a CD4+ T cell isolation kit (Miltenyi Biotec; #130-091-155), followed by fractionation into CD25+ and CD25− subsets by either using directly conjugated anti-CD25 microbeads (referred to as first generation anti-CD25 microbeads; Miltenyi Biotec; #130-090-445) or by a two-step purification where CD4+ T cells were incubated with the indicated PEconjugated monoclonal antibody [(mAb); ACT-1, Dako Cytomation, Denmark; 4E3, Miltenyi Biotec; B1.49.9, Beckman Coulter, Miami, FL; 3G10, Caltag Laboratories, Burlingame, CA], incubated 20 min on ice, washed once with MACS buffer, and isolated by anti-PE microbead selection. Second generation small-scale isolations used the human CD4+ CD25+ regulatory T cell isolation kit (#130-091-301, Miltenyi Biotec). All microbead isolations were performed at 4 °C and used an AutoMACS cell separator, following manufacturers' instructions (Miltenyi Biotec). Large-scale isolations using clinical grade beads were done by depletion of CD8+ T cells and CD19+ B cells, followed by positive selection using CD25 microbeads on the CliniMACS (Miltenyi Biotec; #308-01, #325-01, #193-01, respectively). All isolations followed manufacturers' instructions except where indicated in the text.

For CD4+ CD25+ T cells isolated by cell sorting, untouched CD4+ T cells were isolated as described above, incubated with the indicated anti-CD25-PE antibody for 20 min on ice, washed once, and sorted into CD4+ CD25+ and CD4+ CD25− fractions using a BD FACSAria cell sorter (BD Biosciences, San Jose, CA). To assess purity of the magnetically labeled CD4+ CD25+ cells, isolated populations were incubated with anti-CD4-APC (BD Biosciences) and the non-competing anti-CD25-PE clone 4E3 (Miltenyi Biotec) for 15 min at room temperature, washed once, and analyzed using a BD LSR or BD LSRII flow cytometer and Cell Quest Pro, BD FACSDiVa (BD Biosciences), or FlowJo software (Tree Star Inc, Ashland, OR).

2.3. Large-scale isolation of T cell subsets Clinical grade CD8 and CD19 microbeads were simultaneously incubated with white blood cells, WBC, from apheresis products following manufacturers' instructions (Miltenyi Biotec). CD8+ T cells and CD19+ B cells were depleted using the CliniMACS depletion 2.1 program. WBC were incubated with clinical grade anti-CD25 microbeads as indicated in the text and CD25+ cells were collected using CliniMACS enrichment program 3.1 except where flow rates were compared (program 2.1, 10 ml/min; program 3.1, 20 ml/min; and program 5.1, 40 ml/min).

2.5. Culture of isolated CD4+ CD25+ regulatory T cells 0.5–1 × 106 isolated CD4+ CD25+ regulatory T cells were cultured with 1 × 106 irradiated (25 Gy) allogeneic PBMC in complete medium [X-VIVO 15 (Cambrex Bio Science, Walkersville, MD), 15% human serum (Nabi, Miami, FL), and gentamicin (Cambrex Bio Science)] for 7–10 days. Recombinant human IL-2 (10 U/ml; R and D Systems Inc., Minneapolis, MN) and anti-CD3 (OKT3, 1 μg/ml, Ortho-McNeil Pharmaceuticals, Rariton, NJ) were initially added to the cultures. When necessary, cultures were expanded with complete medium supplemented with 10 U/ml recombinant human IL-2. 2.6. Mixed Lymphocyte Reaction (MLR) Maximum CPM response was determined by incubation of 105 CD4+ CD25− T cells with 105 irradiated (25 Gy) allogeneic PBMC in a standard primary MLR. Where indicated, 105 purified CD4+ CD25+ T cells were also added to the above responder and stimulator populations. All assays were performed in 96-well round bottom plates (Corning Inc., Corning, NY) in a final volume of 200 μl of complete medium. MLRs were pulsed with 1 μCi 3H thymidine (Amersham Biosciences, Piscataway, NJ) per well on day 5 and harvested on to glass fiber filters (Wallac, Turku, Finland) with a Tomtec cell harvester (Tomtec, Hamden, CT) on day 6. CPM were measured using a Wallac Trilux 1450 Microbeta Liquid Scintillation and Luminescence Counter (Perkin Elmer Life and Analytical Sciences, Shelton, CT). 2.7. FOXP3 Expression FOXP3 expression levels were determined by relative quantitative Real-time RT PCR using the ABI PRISM

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7700 Sequence Detection System (Applied Biosystems). Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA) and approximately 1 μg of total RNA was reverse transcribed using the Taqman Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA), according to kit instructions in a final volume of 50 μl. Real-time RT PCR reactions were carried out with 20 ng cDNA per 50 μl reaction, using pre-designed primer/probe assays for FOXP3 and the endogenous control gene, ABL1, and universal PCR master-mix (Applied Biosystems) in a 50 μl final volume. Reactions were run on an ABI Prism 7700 Sequence Detector (Applied Biosystems). Relative fold expression of FOXP3 was determined using the ΔΔCT method. 3. Results/discussion 3.1. Optimizing small-scale isolation on the AutoMACS The percentage of CD25+ T cells within the CD4+ T cell population varies among individuals. Although the CD4+ CD25bright subpopulation is typically 1–10% of the CD4+ T cells, the CD4+ CD25intermediate group is more variable (ranging from 14–46% of CD4+ T cells; n = 26; data not shown). Approximately 1–2 × 10 8 PBMC were enriched for CD4 + T cells by magnetically depleting the unwanted lineages. The degree of CD4 purity was highly homogeneous (N 94%; n = 8). In contrast, the percent of CD4+ T cells that were CD25+ after isolation with the first generation anti-CD25 microbeads (#130-090-445) had an unacceptably wide range in purity (22–74% CD25 + ; Fig 1A). These data are representative of many purifications. It should also be noted that the level of purity was highly variable whether the cells were isolated using an AutoMACS cell separator or manually with columns and a magnet (data not shown). In order to eliminate this high level of variability, a 2step labeling method was evaluated. Purified CD4+ T cells were incubated with anti-CD25-PE followed by magnetic isolation of CD4+ CD25+ T cells with anti-PE microbeads. Fig. 1B depicts CD4+ CD25+ T cells isolated by this 2-step methodology using three different anti-CD25 clones. The overall purity for CD4+ CD25+ T cells was consistently greater than 90% (total yields from 1 × 108 PBMC ranged from 2–6 × 106 cells; n = 12). Despite this high purity, CD4+ CD25+ T cells isolated via the 2-step labeling method lacked functional regulatory activity, regardless of the specific antibody clone (Fig. 1C). To address whether our cohort of anti-CD25 antibodies bind to different sites on the CD25 molecule, flow

cytometry competition assays were done using antibodies conjugated to different fluorochromes. Although some anti-CD25 antibodies clearly inhibited the binding of others, there were several combinations that demonstrated no observable inhibition (data not shown). Thus, it is unlikely that lack of suppressor activity was simply due to a conformational change induced by binding the anti-CD25 antibody at a single epitope. Although some groups have successfully used the 2step labeling approach, the precise antibody and microbead concentrations vary considerably among the studies and may obscure potential threshold effects due to additional cross-linking mediated by the binding of antiPE beads (Baecher-Allan et al., 2004). To address this issue, purified CD4+ T cells were incubated with antiCD25-PE (mAb clone 3G10), split into two aliquots, and isolated by either cell sorting or magnetic isolation using anti-PE beads. Fig. 1D demonstrates that the sorted cells efficiently suppressed the alloreactive responses, while the CD4+ CD25+ T cells isolated with 2-step labeling did not exhibit detectable regulatory activity. The cells isolated by sorting versus those purified by 2-step magnetic isolation had increased CD25 expression (mean fluorescent intensity 5574 vs. 2955; Fig 1E), suggesting decreased co-purification of CD4+ CD25 intermediate T cells. Thus, it was unclear whether the lack of suppressor activity in the magnetically isolated population was potentially due to increased cross-linking via the antiPE beads or co-purification of CD4+ CD25intermediate T cells. To address this issue, CD4+ T cells were incubated with anti-CD25-PE and sorted for CD25bright cells. The sorted CD4+ CD25bright T cells were split into two aliquots and either mock treated or incubated with anti-PE beads. Fig. 1F shows that sorted CD4+ CD25bright T cells function to dampen alloreactive responses whether or not they have been previously incubated with anti-PE beads. Thus, enhanced cross-linking through a secondary reagent such as anti-PE microbeads does not alter the function of purified CD4+ CD25+ T cells. Rather, potent regulatory function appears to be associated with methods which isolate CD4+ CD25bright T cells without copurifying significant numbers of CD4+ CD25intermediate T cells. Our evaluation of the above small-scale magnetic isolation was not suitable to adapt for large-scale purification. Thus, an alternative strategy was evaluated for efficiency and reproducibility. A human CD4+ CD25+ regulatory T cell isolation kit was assayed for efficacy in isolating CD4+ CD25bright T cells with potent regulatory activity (Miltenyi Biotec; #130-091-301). This kit differed in the CD25 microbead component and uses the 3G10 anti-CD25 antibody directly conjugated to the

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Fig. 1. Parameters affecting the AutoMACS isolation of human CD4+ CD25+ regulatory T cells. 1A: CD25 expression on CD4+ CD25+ T cells isolated from 8 individuals by first enriching for CD4+ T cells followed by positive selection with anti-CD25 microbeads on the AutoMACS (#130090-445; Miltenyi Biotec). 1B: CD25 expression on CD4+ CD25+ T cells isolated by binding an anti-CD25 PE antibody to enriched CD4+T cells, followed by magnetic isolation with anti-PE microbeads (referred to as 2-step magnetic isolation). Three distinct anti-CD25 PE monoclonal antibodies are compared. 1C: mixed lymphocyte reaction with Tregs purified by 2-step magnetic isolation as described in 1B (representative of three experiments). 1D: mixed lymphocyte reaction with Tregs isolated by sorting (using 3G10 anti-CD25 PE) or 2-step magnetic isolation (also using 3G10 anti-CD25 PE). 1E: CD25 expression on Tregs isolated as described in 1D (representative of three experiements). 1F: mixed lymphocyte reaction with 3G10 sorted Tregs+/−incubation with anti-PE beads or Tregs isolated by 2-step magnetic isolation using 3G10 anti-CD25 PE (representative of two experiments).

microbeads. Fig. 2A demonstrates that CD4+ CD25bright T cells isolated per manufacturers' instructions were highly effective at modulating alloreactive T cell responses whether assayed immediately after isolation (mean % suppression = 59 ± 17; n = 4) or after expansion (mean % suppression = 78 ± 11; n = 5). It is important to note that isolation of CD4+ CD25bright T cells decreased the yield approximately 5-fold (overall recovery was 0.1–1 × 106 CD4+ CD25bright T cells from 1–2 × 108 PBMC).

3.2. Adapting large-scale isolation of CD4+ CD25+ T cells to the CliniMACS The reproducibility observed in small-scale preparations using the human CD4+ CD25+ regulatory T cell isolation kit with research grade clone 3G10 anti-CD25 microbeads prompted us to examine clinical grade clone 3G10 anti-CD25 microbeads to develop a large-scale purification method. The overall strategy was to deplete apheresis products using clinical grade anti-CD8 and

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Fig. 2. Comparison of Tregs isolated by AutoMACS (small-scale) or CliniMACS (large-scale). 2A: mixed lymphocyte reaction with Tregs isolated on AutoMACS using the human CD4+ CD25+ regulatory T cell isolation kit (#130-091-301; Miltenyi Biotec). 2B: CD25 expression on Tregs isolated as described in 2A. 2C: mixed lymphocyte reaction with Tregs isolated on CliniMACS by depleting CD8+ T cells and CD19+ B cells followed by CD25 positive selection. 2D: CD25 expression on Tregs isolated as described in 2C.

anti-CD19 microbeads, followed by positive selection with clinical grade clone 3G10 anti-CD25 microbeads. The initial experiment was to directly compare the potency of CD4+ CD25+ regulatory T cells isolated on the AutoMACS using the research grade isolation kit with those purified on the CliniMACS device using the clinical grade microbeads described above. Both isolation procedures were done following the recommended manufacturers' instructions. Surprisingly, the cells isolated from the large-scale procedure did not demonstrate any suppressor activity and co-purified a substantial amount of CD4+ CD25intermediate T cells (Fig. 2). Small scale AutoMACS studies demonstrated that increasing the flow rate over the magnetic column could decrease co-purification of CD4+ CD25intermendiate T cells (Fig. 3A). Therefore we examined whether increasing the flow rate during CliniMACS selections would similarly enhance selection of the CD4+ CD25bright T cells. A large-scale product was depleted of CD8+ T cells and CD19+ B cells and incubated with anti-CD25 clinical grade microbeads as delineated above, with the exception that the CD25 microbead incubation was done on ice instead of the manufacturer's recommended room temperature. The sample was split into three aliquots and separate CliniMACS selections were done with flow rates

of 10, 20, and 40 ml/min, respectively. Unlike what we observed with the AutoMACS, there was not a significant difference in the mean of CD25 expression with accelerated flow rates over the CliniMACS (Fig. 3B). Of note, there was some limited suppressor activity (less than 21%; Fig. 3C). Others have also shown suppressor activity in CD4+ CD25+ regulatory T cells isolated using a similar large-scale approach (Powell et al., 2005; Hoffmann et al., 2006). However, the potency of these purified cells varies greatly among the different studies and is likely to reflect the heterogeneity of CD4+ CD25+ regulatory T cell function among different individuals. Consistent with this notion, we have also observed great variability in the potency of CD4+ CD25+ regulatory T cells isolated under identical conditions, but deriving from different individuals (see below and data not shown). To further optimize the isolation of CD4+ CD25bright T cells with the aim of enhancing both reproducibility and potency of the isolated population, an alternative approach was assessed. CD8/CD19 depletion was done as per manufacturers' instructions, but the depleted sample was split and the bead:cell ratio for anti-CD25 microbeads was varied over the indicated range (see Fig. 4). AntiCD25 microbead incubations were done on ice here and for the remaining large-scale experiments. As the bead:

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Fig. 3. Increasing the flow rate over the magnetic column selects Tregs with higher CD25 expression on the AutoMACS but does not have an effect on CliniMACS isolations. 3A: CD25 expression on Tregs isolated with variable flow rates over the AutoMACS magnetic column. 3B: CD25 expression on Tregs isolated with variable flow rates over the CliniMACS magnetic column. 3C: mixed lymphocyte reaction with Tregs isolated as described in 3A and 3B.

cell ratio was decreased the number of co-purified CD4+ CD25intermediate T cells was also decreased (Fig. 4A) and a modest increase in suppressor activity was observed, peaking at 38% suppression (Fig. 4B). It is important to note that as the bead:cell ratio decreased a correlative reduction in yield was also observed (data not shown). We have also noted that the spread of CD25 expression within the CD4+ CD25bright T cell population appears to contribute to the overall yield, in that isolations from individuals with a broad range of CD25 expression tend to have a greater yield than those with a compressed CD4+ CD25bright population (data not shown). A striking increase in suppressor activity was observed in purified CD4+ CD25bright T cells which had been ex vivo expanded for 7 days prior to assessing functional activity in the MLR (Fig. 4C). Since there is a direct correlation between relative FOXP3 RNA levels and CD25 expression (Table 1), we investigated whether the enhanced suppressor activity after ex vivo culture was simply due to preferential expansion of CD4+ CD25bright FOXP3bright T cells. Relative FOXP3 RNA levels remained similar before and after ex vivo expansion (Table 2), suggesting that the increased suppressor activity after expansion was not simply due to increased

FOXP3 RNA levels. The increase in suppressor capability observed after ex vivo expansion is consistent with the observation that CD4+ CD25+ regulatory T cells require activation through the TCR to function and indicates that pre-activation during culture may allow the additional time necessary to mature into cells with maximal suppressor function. In the above experiment a CD8/CD19-depleted product from a single large-scale donor was split among five CD25 microbead isolation conditions (5 × 108 WBC cells/condition). We next verified that an entire CD8/ CD19-depleted preparation (7.3 × 109 WBC) could be scaled up to isolate the CD4+ CD25bright T cells with similar efficacy. Indeed, using the lowest bead:cell ratio yielded CD4+ CD25bright T cells (Fig. 5A) that had potent suppressor activity both before and after expansion (Fig. 5B). However, the yield (1.2 × 107 CD4+ CD25bright T cells) was small and we sought to address how variable the yield and efficacy would be with a different individual. From a second individual 1.7 × 107 CD4+ CD25bright T cells were obtained from 5 × 109 CD8/CD19 depleted WBC (indicating an increase in yield from 0.16% of the depleted product to 0.34%). Fig. 5 highlights the variability in potency when CD4+ CD25bright T cells are

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Fig. 4. Decreasing the bead:cell ratio when selecting with anti-CD25 microbeads on the CliniMACS isolates Tregs with increased CD25 expression and enhanced suppressor function. 4A: CD25 expression on Tregs isolated on the CliniMACS by depleting CD8+ T cells and CD19+ B cells followed by positive selection with variable amounts of clinical grade anti-CD25 microbeads per 5 × 108 cells. 4B: mixed lymphocyte reaction with freshly isolated Tregs as described in 4A. 4C: mixed lymphocyte reaction with ex vivo expanded Tregs (representative of two experiments).

freshly isolated from different individuals using identical bead:cell ratios (compare Fig. 5B and D; 80% versus 55% suppression). We also investigated whether there was a threshold whereby increasing the bead:cell ratio could increase the yield without significantly affecting the potency of the purified CD4+ CD25bright T cells. As expected, decreasing the bead:cell ratio decreased the selection of CD4+CD25intermediate T cells (Fig. 5C), but decreased the yield from 3 × 107 to 1.7 × 107, respectively. In contrast, there was not a substantial difference in suppressor activity (55% versus 48%; Fig. 5D), suggesting that there may be a threshold where yield can be increased without substantially impairing the potency of the isolated population. Importantly, the ex vivo expanded cells again demonstrated enhanced suppressor activity (94% and 89%; Fig. 5D), further suggesting that pairing optimal large-scale isolation with specific ex vivo expansion conditions may be a potential strategy to obtain sufficient numbers of potent CD4+ CD25bright regulatory T cells for therapeutic use. 3.3. Summary A key consideration for developing therapeutic strategies utilizing human CD4+ CD25+ regulatory T cells is

the optimization of isolation methods. Two characteristics pose unique challenges for harnessing maximal activity of CD4+ CD25+ regulatory T cells in the setting of human disease. First, the percentage of CD4+ CD25+ regulatory T cells possessing potent suppressor function is relatively small and represents only 1–10% of the CD4+ T cell population in peripheral blood. Second, human CD4+ T cells in peripheral blood have a spectrum of CD25 expression ranging from dim to bright, and cells with potent regulatory activity partition into the CD25bright fraction (Baecher-Allan et al., 2001). Thus, a rapid method to selectively isolate CD4+ CD25bright T cells from a large pool of peripheral blood cells will be Table 1 Relative FOXP3 expression in CD4+ CD25bright and CD4+ CD25intermediate populations compared to CD4+ CD25dim populations

Donor 1 Donor 2 Donor 3

Bright

Intermediate

Dim

258 135 58.5

9 7.7 3

1 1 1

Magnetically enriched CD4+ T cells were sorted into CD25bright, CD25intermediate, and CD25dim populations. Relative FOXP3 levels were determined by using the ΔΔCT. CD25bright and CD25intermediate populations are compared relative to the CD25dim population.

D.G. Wichlan et al. / Journal of Immunological Methods 315 (2006) 27–36 Table 2 Fold difference in FOXP3 expression in fresh vs. cultured populations of CD4+ CD25+ Cells isolated with variable bead:cell ratios

4 ml 1 ml 0.5 ml 0.25 ml 0.1 ml

Fresh

Cultured

1 1 1 1 1

0.87 0.5 0.81 0.81 0.81

Large-scale depletion of CD8+ T cells and CD19+ B cells was followed by CD25 positive selection using the indicated concentration of antiCD25 microbeads per 5 × 108 cells. All magnetic isolations were done on the CliniMACS. Cultured cells were expanded ex vivo for 7 days. Relative FOXP3 levels were determined by using the ΔΔCT method.

necessary to maintain optimal function of the these cells. Although cell sorting has been a traditional approach to isolating cells with high levels of purity, separation of rare cells can require extended periods of time if the total number of cells is large (N 1010 PBMC). The speed of magnetic separation is advantageous in the ability to rapidly isolate desired cells from large samples (4 × 1010) and has precedent for being used in the clinical setting of hematopoietic stem cell transplantation (Handgretinger et al., 2001; Lang et al., 2003; Lang et al., 2004). However, there are disadvantages associated with magnetic isolation

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of CD4+ CD25+ regulatory T cells. Whereas cell sorters can be easily programmed to select cells expressing antigen at a defined density, magnets will collect all cells sufficiently labeled. Thus, the ability to selectively isolate CD4+ CD25bright T cells without substantial co-purification of the CD4 + CD25 intermediate subset is more problematic. The data presented herein clearly demonstrate that magnetic separation can be manipulated to reproducibly isolate human CD4+ CD25bright regulatory T cells with potent functional activity if parameters such as the density of CD25 labeling are carefully considered. Finally, the observation that specific expansion conditions can greatly increase the regulatory potency, if copurification of CD4+ CD25intermediate T cells is minimized, suggest that combining optimal large-scale isolation with ex vivo expansion may provide a therapeutic strategy for using CD4+ CD25bright regulatory T cells in the clinical setting. Acknowledgments The authors thank Marti Holladay and Jim Houston for their expert assistance with the flow cytometry, members of the Human Applications lab (SJCRH) for their technical assistance, and Drs. Shayna Street, and Kimberly Kasow for their critical comments on the

Fig. 5. Decreasing the anti-CD25 bead:cell ratio during large-scale magnetic isolation of Tregs enhances the selection of CD4+ CD25bright regulatory T cells with potent suppressor activity. 5A: CD25 expression on Tregs after large-scale magnetic isolation using 0.1 ml clinical grade antiCD25 microbeads per 5 × 108 CD8/CD19-depleted WBC. 5B: mixed lymphocyte reaction with fresh and ex vivo expanded Tregs isolated as described in 5A. 5C: CD25 expression on Tregs after large-scale magnetic isolation using 0.1 ml or 0.25 ml clinical grade anti-CD25 microbeads per 5 × 108 CD8/CD19-depleted WBC. 5D: mixed lymphocyte reaction with fresh and ex vivo expanded Tregs isolated as described in 5C.

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