Single-Chain Bispecific Antibodies Retargeting of Human Regulatory T

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Retargeting of Human Regulatory T Cells by Single-Chain Bispecific Antibodies Stefanie Koristka, Marc Cartellieri, Anke Theil, Anja Feldmann, Claudia Arndt, Slava Stamova, Irene Michalk, This information is current as Katrin Töpfer, Achim Temme, Karsten Kretschmer, Martin of September 28, 2021. Bornhäuser, Gerhard Ehninger, Marc Schmitz and Michael Bachmann J Immunol 2012; 188:1551-1558; Prepublished online 19

December 2011; Downloaded from doi: 10.4049/jimmunol.1101760 http://www.jimmunol.org/content/188/3/1551

Supplementary http://www.jimmunol.org/content/suppl/2011/12/19/jimmunol.110176 http://www.jimmunol.org/ Material 0.DC1 References This article cites 43 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/188/3/1551.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Retargeting of Human Regulatory T Cells by Single-Chain Bispeciﬁc Antibodies

Stefanie Koristka,* Marc Cartellieri,* Anke Theil,† Anja Feldmann,* Claudia Arndt,* Slava Stamova,* Irene Michalk,* Katrin To¨pfer,‡ Achim Temme,‡ Karsten Kretschmer,† Martin Bornha¨user,x Gerhard Ehninger,x Marc Schmitz,*,† and Michael Bachmann*,†

Bispecific Abs hold great potential for immunotherapy of malignant diseases. Because the first components of this new drug class are now entering clinical trials, all aspects of their mode of action should be well understood. Several studies proved that CD8+ and CD4+ effector T cells can be successfully redirected and activated against tumor cells by bispecific Abs both in vitro and in vivo. To our knowledge, this study provides the first evidence that bispecific Abs can also redirect and activate regulatory T cells against a surface Ag, independently of their TCR specificity. After cross-linking, via a bispecific Ab, redirected regulatory T cells upregulate the activation markers CD69 and CD25, as well as regulatory T cell-associated markers, like CTLA-4 and FOXP3. The Downloaded from activated regulatory T cells secrete the immunosuppressive cytokine IL-10, but, in contrast to CD8+ and CD4+ effector T cells, almost no inflammatory cytokines. In addition, the redirected regulatory T cells are able to suppress effector functions of activated autologous CD4+ T cells both in vitro and in vivo. Therefore, the potential risk for activation of regulatory T cells should be taken into consideration when bispecific Abs are applied for the treatment of malignant diseases. In contrast, an Ag/tissue-specific redirection of regulatory T cells with bispecific Abs holds great potential for the treatment of autoimmune diseases and graft rejection. The Journal of Immunology, 2012, 188: 1551–1558. http://www.jimmunol.org/

he development of single-chain bispecific Abs for retar- not aware of any publication answering the question about whether geting of polyclonal T cells has opened the window for regulatory T cells (Tregs) can also be redirected and activated with T a new therapeutic strategy for the treatment of malignant these new therapeutic molecules. diseases (e.g., Ref. 1). In general, a single-chain bispecific Ab Tregs play a key role in immune homeostasis by establishing and molecule is composed of the variable L and H chains of two maintaining peripheral tolerance to self-Ags, as well as by con- distinct mAbs with different Ag specificities linked via flexible trolling the magnitude of immune responses to foreign Ags (7, 8). peptide chains. Thereby, these molecules can interact simulta- Nevertheless, in the context of malignant disorders, Tregs may have by guest on September 28, 2021 neously with two different Ags. Typically, they are directed, on the deleterious effects by suppressing antitumor responses and pro- one hand, to the CD3 complex of T cells and, on the other hand, moting tumor progression (9, 10). In fact, many studies revealed to a surface Ag on a target cell (e.g., a tumor-associated Ag). By that Treg-mediated immunosuppression is one of the main tumor cross-linking T cell and target cell, an activation signal is trans- immune-evasion mechanisms. Increased numbers of Tregs have mitted to the lymphocyte, which, in turn, triggers its effector been reported in tumor-bearing patients, and their accumulation functions and eventually leads to the specific elimination of the correlates with poor survival prognosis (11–13). Consequently, recognized target cell. Thus, polyclonal T cells are activated in- within the scope of tumor therapy with bispecific Abs, the activa- dependently of their TCR specificity. Bispecific Abs have been tion of Tregs in the tumor microenvironment is unfavorable and successfully applied for retargeting of CD4+ and CD8+ effector may have to be circumvented. T cells against tumors in vitro and in mice studies (e.g., 2–5). In contrast, given the fact that Tregs have many beneficial Moreover, the first clinical trials have proven the feasibility and effects (e.g., in preventing autoimmunity and transplant rejection efficacy of this strategy for tumor treatment (6). However, we are and limiting chronic inflammatory diseases), an Ag/tissue-specific retargeting of these cells via bispecific Abs may open novel therapeutic approaches concerning the aforementioned immuno- *Institute of Immunology, Medical Faculty, Carl Gustav Carus Technical University logic disorders. Ongoing efforts for the treatment of autoimmunity Dresden, 01307 Dresden, Germany; †Center for Regenerative Therapies Dresden, Carl Gustav Carus Technical University Dresden, 01307 Dresden, Germany; ‡Department of and graft-versus-host disease are based on the ex vivo expansion of Neurosurgery, University Hospital, Carl Gustav Carus Technical University Dresden, polyclonal Treg populations and their adoptive transfer back into x 01307 Dresden, Germany; and Medical Clinic and Policlinic I, University Hospital, the patient. This approach has proved successful in preventing or Carl Gustav Carus Technical University Dresden, 01307 Dresden, Germany attenuating inflammatory and autoimmune diseases in various Received for publication June 14, 2011. Accepted for publication November 17, 2011. animal models (14–16). However, with regard to the transfer of polyclonal Tregs into patients, one has to face the risk for unfa- Address correspondence and reprint requests to Prof. Michael P. Bachmann, Institute of Immunology, Medical Faculty, Carl Gustav Carus Technical University Dresden, vorable pan-immunosuppression. Moreover, experiments in mice Fetscherstrasse 74, 01307 Dresden, Germany. E-mail address: michael.bachmann@ showed that Ag-specific Tregs are far more efficient than poly- tu-dresden.de clonal populations (17–19). In light of these arguments, bispecific The online version of this article contains supplemental material. Abs could provide a promising tool for a target-specific redirec- Abbreviations used in this article: MFI, mean fluorescence intensity; PSCA, prostate tion of Tregs in settings like transplantation or autoimmune dis- stem cell Ag; scBsDb, single-chain bispecific diabody; Treg, regulatory T cell. eases. The application of polyclonal Tregs redirected in a target- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 specific manner might lower the risk for unwanted side effects, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101760 1552 RETARGETING OF TREGS WITH BISPECIFIC ANTIBODIES like increased risk for infections and cancers caused by a systemic scBsDbs (AbD Serotec, Du¨sseldorf, Germany), as described elsewhere (4, immunosuppression. 5). mAbs against CD3 (BW264/56; Miltenyi Biotec), human La-E7B6, and Over the past years, we successfully developed a series of re- human PSCA (7F5) (produced at the Institute of Immunology, Medical Faculty of the Technical University Dresden) served as positive controls. combinant bispecific Abs for redirecting immune effector cells against tumor cells (e.g., Refs. 4, 5). Performing binding studies to T cell isolation and expansion test the stability of these constructs at 37˚C, we noticed that the Human PBMCs were prepared by Ficoll-gradient centrifugation from buffy + bispecific Abs bound homogenously to all CD4 T cells in culture, coats (supplied by German Red Cross, Dresden, Germany) of healthy and no second peak could be observed (Supplemental Fig. 1). These volunteers after their informed consent. The study was approved by the local ethics committee of the university hospital of the medical faculty of Carl results prompted us to investigate whether singe-chain bispecific + + Gustav Carus Technical University Dresden (EK27022006). CD4 T cells Abs also bind to and, consequently, activate CD4 Tregs. were freshly isolated from PBMCs by negative selection and separated into To our knowledge, we provide the first experimental evidence CD25+ and CD252 cells by magnetic bead-activated cell sorting using the that both freshly isolated and in vitro-expanded human CD4+ CD4+/CD25+ Treg isolation kit from Miltenyi Biotec. To increase the CD25+ Tregs can be efficiently activated via bispecific Abs in a purity of isolated CD4+CD25+ cells, two consecutive column runs were target-specific, but TCR-independent, manner. We demonstrate that performed. The purity of the isolated Th cell and Treg populations was confirmed by FACS analysis and ranged between 85 and 94% (CD4+ incubation of freshly isolated Tregs with a bispecific construct in CD252) and 86 and 95% (CD4+CD25+), respectively. Before application, the presence of the respective target Ag leads to the upregulation of freshly isolated cells were maintained overnight in complete RPMI 1640 CD69, CD25, CTLA-4, and FOXP3, as well as the secretion of the medium in the presence of 300 U/ml rIL-2 (ImmunoTools, Friesoythe, immunosuppressive cytokine IL-10. Furthermore, bispecific Ab- Germany). For some experiments, highly pure expanded CD45RA+ Tregs were Downloaded from activated Tregs are hyporesponsive and proliferate only in the used. Prior to expansion, the isolated CD4+CD25+ cells were sorted on a presence of high doses of IL-2. Importantly, the redirected Tregs are FACSAria II cell sorter (BD Biosciences) to obtain the CD45RA+ Treg able to suppress the proliferation and cytokine production of au- subpopulation. After sorting, the resulting population showed .95% purity tologous CD4+ effector T cells both in vitro and in vivo. (CD4+CD25highCD45RA+). The expansion protocol was adapted and slightly modified from Putnam et al. (21). In brief, 2.5 3 105 cells were plated in X-Vivo 15 medium (Lonza, Cologne, Germany), supplemented Materials and Methods with 10% human AB serum (PAA Laboratories, Co¨lbe, Germany) and

Cell lines stimulated using aCD3/aCD28-coated DynaBeads (Invitrogen) at a ratio of http://www.jimmunol.org/ 1:1 bead/cell at days 1 and 9. Every 2–3 d, 300 U/ml recombinant clinical The human embryonic kidney (HEK) cell line 293T was transduced with grade IL-2 (Proleukin S; Novartis Pharmaceuticals, Horsham, U.K.) was the reading frames of the single-chain bispecific diabodies (scBsDbs) added, assuming consumption of the cytokine. During the first week of CD3xPSCA and CD3xE7B6 and used for stable expression of both bi- expansion, 100 ng/ml rapamycin (Axxora Deutschland, Lo¨rrach, Germany) specific Abs (4). The prostate cancer cell line PC3 was genetically modified was added, together with IL-2, to further minimize the risk for CD4+ ef- for stable expression of the prostate stem cell Ag (PSCA), as also recently + fector T cell contaminations (22). After a 14-d expansion period, the described (4), and used as PSCA target cells for the redirection of Tregs. resulting cell population showed a purity .96% (CD4+CD25+CD127low Transductions were performed, as previously described, using a retrovi- FOXP3+). ral system based on Moloney murine leukemia virus (20). The modified cell line PC3-PSCA was maintained in complete RPMI 1640 medium Activation assays with bispecific Abs (Biochrom, Berlin, Germany), supplemented with 10% FCS, 100 mg/ml by guest on September 28, 2021 4 penicillin/streptomycin, 1% nonessential amino acids, 2 mM N-acetyl-L- A total of 5 3 10 Tregs or autologous Th cells was cocultured with the 4 alanyl-L-glutamine, and 1 mM sodium pyruvate (Biochrom). Bispecific prostate cancer cell line PC3-PSCA (1 3 10 cells) at a ratio of 5:1 in 96- Ab-secreting HEK293T cells were grown in DMEM (Invitrogen, Karls- well round-bottom plates in the presence or absence of 6 pmol the cross- ruhe, Germany), supplemented with 10% FCS and 100 mg/ml penicillin/ linking scBsDb CD3xPSCA in a total volume of 200 ml RPMI 1640 streptomycin. The original cell lines were purchased from the American medium, without additional cytokines. Bispecific Ab CD3xE7B6 was used Type Culture Collection. All cell lines were cultured at 37˚C in a humidi- as a control, which binds to an epitope of the intracellular autoantigen La fied atmosphere of 5% CO2. (23), which is not available on the target cells. Tregs or Th cells, activated with aCD3/aCD28-coated DynaBeads (Invitrogen) at a ratio of 1:1 bead/ Production and purification of soluble scBsDbs cell, served as a positive control. All samples were analyzed as triplets. The scBsDbs used in the current study were constructed, expressed, and CFSE-based suppression assays purified, as described elsewhere (4, 5). Briefly, culture supernatant of stably 2 transduced HEK293T cells was collected, and purification of the His-tagged Prior to their use in an in vitro suppression assay, CD4+CD25 Th cells Abs was achieved by affinity chromatography on Ni-NTA columns (Qiagen, were labeled with 1 mM CFSE (Sigma-Aldrich, Steinheim, Germany) for Hilden, Germany). Bound scBsDbs were eluted with ice-cold imidazol- 10 min at 37˚C and washed twice. For a suppression assay, 5 3 104 labeled containing PBS. Eluted fractions were dialyzed overnight against PBS. autologous responder T cells were incubated with 1 3 104 PC3-PSCA The purity of the elution fractions was analyzed after SDS-PAGE and tumor cells and the scBsDb CD3xPSCA in 96-well round-bottom plates subsequently stained with Coomassie brilliant blue solution. The concen- alone or in the presence of unlabeled autologous responder cells, at indi- tration of the scBsDbs in the fractions was determined by comparison to cated ratios, in a total volume of 200 ml RPMI 1640 medium, without a BSA standard curve on the same SDS gel (data not shown, see Ref. 4). adding any additional cytokines. For T cell suppression, in vitro expanded, autologous CD4+CD25+CD45RA+ Tregs were added at 1:1 and 4:1 (re- Flow cytometric analysis sponder to suppressor) ratios. All samples were analyzed as triplicates. Proliferation of CD4+ T cells was assessed by FACS on the basis of CFSE Flow cytometry was performed on a FACSCalibur (BD Biosciences) and dilution over time. Percent divided (precursor frequency) of responder a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells in the presence or absence of Tregs was calculated using FlowJo mAbs against human CD4 (RPA-T4), CD25 (2A3), CD45RA (HI100), 8.8.2 software (TreeStar, Ashland, OR). After 4 or 5 d, cocultures were CD69 (FN50), and CD152 (CTLA-4, BNI3) were obtained from BD finally harvested, stained for surface expression of CD127 and CD25, and Biosciences Pharmingen (Heidelberg, Germany). A mAb against human analyzed by flow cytometry. CD127 (eBioRDR5) was obtained from eBioscience (Frankfurt, Ger- many). mAbs against human FOXP3 (259D) and the respective isotype Determination of cytokine concentration control (IgG1k) were purchased from BioLegend (Uithoorn, The Neth- erlands). Staining for cell surface markers was carried out prior to intra- To determine amounts of secreted IFN-g, IL-10, TNF, and IL-2, super- cellular staining for FOXP3 and CTLA-4, according to the manufacturer’s natants were collected 24 h after the start of an activation or suppression instructions. assay and analyzed for cytokine secretion using OptEIA ELISA Sets (BD The binding of the scBsDbs was assessed by indirect immunofluores- Biosciences), according to the manufacturer’s protocol. The absorption of cence analysis using unfixed PC3-PSCA cells, La-decorated PC3 cells, the samples was measured after 30 min, and the obtained values were used PBMCs, or isolated CD4+ T cells. Binding was visualized by incuba- to calculate the concentration of the cytokines in the samples, according to tion with a FITC-conjugated mAb against the C-terminal myc-tag of the the values obtained for the standard series provided by the manufacturer. The Journal of Immunology 1553

Quantification of absolute cell numbers the binding of the bispecific Ab to the CD3 complex itself gen- In order to assess expansion rates of different T cell populations in activation erates an activation signal, a control scBsDb (CD3xE7B6) was or suppression assays, absolute T cell numbers were quantified by flow used, which binds to the same CD3 epitope on T cells as does the cytometry. At the indicated time points, cocultures in 96-well plates were CD3xPSCA scBsDb but is not able to mediate cross-linking to the carefully resuspended and an aliquot of 10 ml was removed for further tumor cells, because its second arm is directed to an intracellular analysis. Aliquots were diluted 1:10 with 1 mg/ml propidium iodide (Sigma- Ag (4). After 24 h of coculture, the T cell populations were har- Aldrich) immediately before measuring the cell number/ml of the different T cell subpopulations using the MACSQuant Analyzer and MACSQuantify vested, and the expression pattern of the activation markers CD69 software (Miltenyi Biotec). Living cells were distinguished from dying cells and CD25 was examined. Furthermore, intracellular stainings for and cellular debris by size exclusion and being propidium negative. the Treg-associated molecules FOXP3 and CTLA-4 were performed (Fig. 1). With regard to the expression of the early acti- In vivo-suppression assay of cytotoxic T cell activity in nude + + + 2 mice vation marker CD69, both CD4 CD25 Tregs and CD4 CD25 Th cells upregulated CD69 in the presence of the CD3xPSCA nu/nu Female 9-wk-old NMRI mice were provided by the animal facility of scBsDb (63.3 6 9.1% and 74.8 6 10.6%, n = 8, respectively). In the Technical University of Dresden. Animals were kept under specific contrast, without any bispecific Ab or with the unspecific control pathogen-free conditions. All experiments were carried out in accordance with the German animal protection law and were approved by the local scBsDb CD3xE7B6, no considerable upregulation of CD69 could authorities (Landesdirektion Dresden). A total of 1 3 106 mouse-adapted be observed. Similar results were obtained for the IL-2Ra–chain PC3-PSCA tumor cells was injected s.c. into the right flank of the mice. In (CD25). Although Tregs constitutively express high levels of 3 6 + 2 some of the animals, 1 10 CD4 CD25 Th cells and/or equal numbers this molecule on their cell surface, upon incubation with the of autologous, in vitro expanded CD4+CD25+CD45RA+ Tregs and/or 10 mg the scBsDb CD3xPSCA were coinjected with the PSCA+ tumor cross-linking bispecific Ab, the expression level is remarkably Downloaded from cells. Established tumors were measured weekly in both directions using enhanced (relative increase in mean fluorescence intensity [MFI]: a digital measuring slide. Mice were killed for ethical reasons when tumors 42.3 6 10.1%, n = 8). Th cells also exhibited increased CD25 exceeded 18 mm in one direction. expression following activation (61.8 6 17.2% positive cells, n = Statistical analysis 8). Similar to the enhanced expression of CD25 on the cell surface, redirected Tregs also exhibited increased intracellular amounts of Differences in the phenotype of Th cell and Treg populations or in the the transcription factor FOXP3 after cross-linking to target cells via http://www.jimmunol.org/ proliferation of responder T cells were analyzed using the two-tailed Student 6 t test for independent samples. Statistical significance of differences in tu- the CD3xPSCA bispecific Ab (relative increase in MFI: 58.6 + mor growth between the bispecific Ab-treated groups was also calculated 12.2%, n = 8). In comparison, CD4 Th cells showed only a mod- using the Student t test. In addition, data were analyzed by one-way ANOVA erate upregulation of FOXP3, from 5.8 6 2.5% to 13.2 6 7.0% and Bonferroni multiple comparison test using GraphPad Prism software. FOXP3+ cells (n = 8). A characteristic feature of Tregs is their The p values , 0.05 were considered significant. constitutive expression of high amounts of intra- and extracellular CTLA-4. In the presence of the CD3xPSCA scBsDb, Th cells Results (41.2 6 17.7%, n = 8), as well as Tregs, exhibited a markedly Tregs upregulate activation-associated markers after a increased expression level of intracellular CTLA-4 (relative in- cross-linkage with target cells via bispecific Abs crease in MFI: 31.9 6 13.0%, n = 8). Altogether, to our by guest on September 28, 2021 To elucidate whether the activation of Tregs can be induced by knowledge, these results provide the first evidence that Tregs can cross-linking bispecific Abs, CD4+CD25+ Tregs or autologous be activated via cross-linking of bispecific Abs. CD4+CD252 Th cells of eight donors were cocultured with the Additionally, we performed experiments in which we com- prostate cancer cell line PC3-PSCA (which was transduced for pared the activation status of Tregs cross-linked to target cells via permanent expression of PSCA; Materials and Methods) in the the bispecific Ab CD3xPSCA with Tregs stimulated with aCD3/ presence or absence of the scBsDb CD3xPSCA. To exclude that aCD28-coated beads. Analyzing the upregulation of the afore-

FIGURE 1. Expression of different activation-associated markers on redirected CD4+CD25+ Tregs and CD4+CD25– Th cells. Freshly isolated Tregs or Th cells cocultured with PC3-PSCA tumor cells were incubated without any scBsDb (dashed line), with a control CD3xE7B6 (gray line), or with the CD3xPSCA scBsDb (black line). Cell surface expression of CD69 and CD25, as well as intracellular FOXP3 and CTLA-4 expression, was assessed by flow cytometry analysis 24 h after starting the coculture. Numbers represent the MFI of total cells and percentage of positive cells under the marker for each condition. IgG isotype controls (filled gray area) were used to define gates. The results of one representative donor of eight are shown. 1554 RETARGETING OF TREGS WITH BISPECIFIC ANTIBODIES mentioned markers CD69, CD25, FOXP3, and CTLA-4, no con- the Th cells, very low amounts of inflammatory cytokines (IFN-g: siderable differences between the two activating agents could be 13.2 6 7.8%, TNF: 9.5 6 5.2%, IL-2: 15.5 6 6.1%). observed (Supplemental Fig. 2). Redirected Tregs proliferate only in the presence of high doses Tregs secrete high amounts of IL-10, but almost no IFN-g, of exogenous IL-2 TNF, or IL-2, upon TCR stimulation with a bispecific Ab For several years, it has been well known that Tregs are hypo- To analyze the cytokine-production profile of the different T cell proliferative in response to TCR stimulation. A prerequisite for populations, culture supernatants were collected 24 h after the their expansion is strong TCR signaling combined with high doses beginning of a coculture assay, and levels of secreted IL-10, IFN-g, of IL-2. To evaluate whether cross-linking of Tregs via a bispecific TNF, and IL-2 were measured by ELISA. Fig. 2A shows the Ab could induce an activation signal strong enough to initiate cytokine-production profile of Tregs and Th cells of one repre- proliferation of the Tregs, the doubling of Treg or Th cell pop- sentative donor. As expected, incubation of the cells without any ulations was assessed by coculturing them in the presence or ab- bispecific Ab or with the CD3xE7B6 control construct did not sence of the CD3xPSCA scBsDb, with or without the addition of result in cell activation; hence, no cytokines could be detected in exogenous IL-2 (Fig. 3). Cell numbers were determined every 24 h the supernatants. In contrast, culturing the cells in the presence using a MACSQuant Analyzer (Miltenyi Biotec), and the expan- of the bispecific CD3xPSCA Ab led to the induction of cytokine sion rate of the respective cell population was calculated. Incu- production. Although Th cells secreted substantial amounts of bation of Th cells or Tregs with target cells alone or with the the inflammatory cytokines IFN-g and TNF, as well as growth- control bispecific Ab CD3xE7B6 did not induce proliferation of promoting IL-2 and immunomodulatory IL-10, only the latter either cell population (Fig. 3A). However, culturing Th cells with Downloaded from cytokine is produced in considerable amounts by CD4+CD25+ target PC3-PSCA cells and the bispecific CD3xPSCA Ab resulted Tregs under the same culturing conditions. To compare the results in a significant expansion (4 6 1-fold) of the initial cell population of eight donors, the respective amount of cytokines secreted by (Fig. 3A,3B). In contrast, Tregs incubated under the same con- redirected Th cells was set to 100% and the corresponding amount ditions did not proliferate. Next, we investigated whether this lack of cytokines secreted by Tregs was calculated and given as a per- of proliferation was IL-2 dependent, although Tregs are activated

centage (Fig. 2B). The redirected Tregs of all donors produced more under these conditions, as already demonstrated. For this purpose, http://www.jimmunol.org/ IL-10 than did redirected Th cells (116 6 36%) but, compared with two concentrations of IL-2 (50 and 500 U/ml) were added to the by guest on September 28, 2021

FIGURE 2. Cytokine production proﬁle of redirected CD4+CD25+ Tregs or CD4+CD252 Th cells. A, Freshly isolated Th cells (black bars) or Tregs (white bars) were cocultured with PC3-PSCA cells and incubated without any scBsDb (-), with an unspeciﬁc CD3x7B6E (control), or with the cross-linking CD3xPSCA scBsDb (+). Cul- ture supernatants were collected 24 h later and analyzed by ELISA to determine the levels of secreted IL-10, IFN-g, TNF, and IL-2. Data shown represent means 6 SD of triplicate wells. The results of one representative donor are shown. B, Summary of the results of eight donors. The respective amount of cytokines secreted by Th cells was set to 100% and the corresponding relative amounts of cytokines secreted by Tregs were calculated. ***p , 0.001, Student t test. The Journal of Immunology 1555

FIGURE 3. Population doubling of redirected CD4+CD25+ Tregs or CD4+CD252 Th cells. A, Freshly isolated Th cells (left panel) or Tregs (right panel) were cocultured with PC3-PSCA target cells without any Ab (d), with an unspecific CD3xE7B6 scBsDb (:), or with the specific CD3xPSCA scBsDb (♦). The expansion rate of the respective cell populations over a period of 96 h was assessed by measuring cell numbers every 24 h after starting the coculture. Data shown represent means 6 SD of triplicate wells. The results of one representative donor are shown. B, Sum- mary of the results of five different donors 96 h after beginning of the coculture in the presence or absence of the cross-linking CD3xPSCA bispecific Ab. ***p , 0.001, Student t test. C, Population doubling of Tregs cultured with PC3-PSCA target cells in the presence or absence of the cross-linking CD3xPSCA scBsDb Downloaded from and different concentrations of rIL-2 (50 and 500 U/ml). cocultures (Fig. 3C). Only in the presence of the CD3xPSCA Bispecific Ab-redirected Tregs cause an extensive

bispecific Ab and high doses of IL-2 (500 U/ml) did Tregs expand downregulation of CD25 and CD127 on autologous Th cells http://www.jimmunol.org/ . 4-fold within 1 wk. To analyze the suppressive capacity of redirected Tregs in more Bispecific Ab-redirected Tregs are capable of suppressing detail, surface stainings for the a-chains of the receptors for the proliferation and cytokine production of autologous Th cells common g-chain (gc) cytokines IL-2 and IL-7 on suppressed Th cells were performed (Fig. 4D). With regard to the IL-2Ra–chain So far, these results supported the notion that activation of freshly CD25, upregulation on the surface of activated Th cells was isolated Tregs can be achieved in an Ag-specific manner by using markedly reduced in the presence of Tregs. A similar effect was bispecific Ab constructs. A prerequisite for the use of Tregs in observed for the a-chain of IL-7R. In response to CD3 stimulation clinical trials is their efficient in vitro expansion to sufficient caused by the bispecific CD3xPSCA Ab, Th cells downregulated numbers without losing phenotypic and functional characteristics. by guest on September 28, 2021 + CD127 expression, which is thought to be a homeostatic mecha- Previous studies showed that only highly pure human CD45RA nism to prevent excessive T cell expansion (26). Adding Tregs to Tregs maintain their suppressive phenotype over a long-lasting the culture strikingly enhanced the downregulation of the CD127 cultivation period (24, 25). Therefore, we next tested whether ex- molecule. In summary, bispecific Ab-redirected Tregs were capa- panded Tregs of three donors could also be activated via bispecific + ble of suppressing proliferation and cytokine production of autol- Abs and whether they exerted a suppressive capacity. CD45RA ogous Th cells and negatively influenced the expression of the Tregs were isolated by FACS sorting and expanded over 14 d using high-affinity receptor chains of the gc-cytokines IL-2 and IL-7. aCD3/aCD28-coated beads and high-dose IL-2. In an aCD3/ aCD28 bead-stimulated (1:75) suppression assay, these cells sup- Bispecific Ab-redirected Tregs suppress Th cell-induced pressed proliferation of autologous (data not shown), as well as antitumor response in nude mice 2 allogenic, CD4+CD25 effector T cells at suppressor/effector ra- The experiments conducted thus far showed that single-chain bi- tios of 1:1 to 1:32 (Supplemental Fig. 3). Using the bispecific specific Abs activated Tregs and triggered their suppressor func- CD3xPSCA Ab for the Ag-specific, but TCR-independent, acti- tion in vitro. To confirm the data in vivo, we tested whether redirected vation of Tregs also resulted in a significant reduction in autologous Tregs suppressed a Th cell-mediated antitumor response in athymic Th cell proliferation capacity (Fig. 4A,4B). nude mice. All animals were injected with PC3-PSCA tumor cells into In the experiment, 5 3 104 CFSE-labeled responder Th cells were their right flank, and developing s.c. tumors were measured weekly. cocultured in the presence of unlabeled responder Th cells and the Coinjection of PSCA+ prostate tumor cells with equal amounts of bispecific Ab, leading to a considerable proliferation of 34% (1:1 CD4+CD252 Th cells and the scBsDb CD3xPSCA resulted in re- ratio) and 40% (4:1 ratio) of the CSFE-labeled Th population after duced tumor outgrowth in this group of mice compared with those 120 h (Fig. 4A). When the unlabeled responder Th cells were groups in which tumor cells were injected alone or with Th cells in the replaced by Tregs at the same effector/suppressor ratios, prolifer- absence of the bispecific Ab. When autologous in vitro-expanded ation decreased significantly (Fig. 4A,4B). Furthermore, bispecific Tregs were added, tumor development was comparable to that of Ab-redirected Tregs influenced proliferation, as well as cytokine both aforementioned control groups. However, adding Tregs at an production, of autologous Th cells (Fig. 4C). Analysis of the effector/suppressor ratio of 1:1 in the presence of the activating bi- supernatants revealed that, in the presence of equal numbers of specific Ab strongly enhanced tumor growth (Fig. 5). Taken together, Tregs, Th cells secreted only 27% (IFN-g) and 2% (TNF) of the these results indicated that bispecific Ab-redirected Tregs can execute amount of inflammatory cytokines that they produced in their ab- their suppressive function in vitro, as well as in vivo posttransfer. sence. Moreover, under the same culturing conditions, almost no IL-2 could be detected in the culture supernatants, either because Discussion of IL-2 consumption by the Tregs or suppression of IL-2 produc- Bispecific Abs were originally designed for the recruitment of tion of Th cells. cytotoxic immune cells against target cells in an Ag-specific 1556 RETARGETING OF TREGS WITH BISPECIFIC ANTIBODIES Downloaded from http://www.jimmunol.org/

FIGURE 4. Suppressive capacity of bispecific Ab-redirected Tregs in vitro. A total of 5 3 104 CSFE-labeled CD4+ responder T cells were cultured with PC3-PSCA target cells and either unlabeled autologous CD4+ Th cells or unlabeled autologous expanded Tregs at effector/suppressor ratios of 1:1 or 4:1 in the presence or absence of the CD3xPSCA scBsDb. A, Proliferation of the labeled Th cells was monitored by levels of CFSE dilution. The graphs show the CFSE staining 120 h after starting the coculture in the presence (+) or absence (-) of the CD3xPSCA scBsDb. The percentage of proliferating cells is indicated in each graph. B, Relative proliferation of CFSE-labeled Th cells in the presence of unlabeled Th cells or Tregs after 96 h. The amount of proliferating cells in the presence of redirected Th cells was equalized to 100%, and the relative proliferation of labeled Th cells in the presence of redirected Tregs was calculated. Mean and SD from experiments with three donors are shown. *p , 0.05, ***p , 0.001, Student t test. C, Culture supernatants from the above-described by guest on September 28, 2021 experiments were collected after 24 h and analyzed by ELISA to determine the levels of secreted IFN-g, TNF, and IL-2. Results for one representative donor are shown; error bars represent SD from the triplicate wells. D, CD25 and CD127 surface expression of responder T cells 120 h after beginning of the coculturing period in the presence (solid line) or absence (dashed line) of the cross-linking Ab. Numbers represent the MFI of total cells and percentage of positive cells under the marker for each condition (filled gray area: isotype control). Data shown are representative for one of three donors. manner. Usually, T cells are redirected to target cells by cross- in vitro and in mouse models, that, in this way, cytotoxic T cells can linking the activating CD3 receptor of T cells and any chosen be very efficiently redirected and activated against tumor cells (e.g., Ag on the cell surface of the target cells. It was demonstrated, both Refs. 1–5), leading finally to the lysis of the target cells and production of inflammatory cytokines by the redirected T cells. Recently, the first clinical trials proved that this concept can be successfully applied for the treatment of malignant diseases. However, among various mechanisms of immune suppression by tumors, the recruitment of Tregs to tumor sites plays an important role. Accumulation of Tregs in tumors suppresses the activation and proliferation of immune effector cells, and increased numbers of Tregs correlate with poor survival prognosis for tumor patients. At this translational point toward a clinical application of bispecific Abs for tumor treatment, there is an urgent need to answer the question whether Tregs will be activated by bispecific Abs in a similar way as their effector counterparts. FIGURE 5. Suppressive capacity of bispecific Ab-redirected Tregs Using a recently described CD3xPSCA scBsDb, we demon- in vivo. Nude mice were inoculated s.c. with mouse-adapted PC3-PSCA strated that effector T cells, as well as CD4+CD25+ naturally 6 tumor cells (1 3 10 ). Animals received 10 mg of bispecific CD3xPSCA occurring Tregs, can be activated via bispecific Ab constructs. 3 6 1 n 5 3 6 1 Ab and either 1 10 CD4 Th cells ( , n 5) or 1 10 CD4 Th cells Experimental evidence was provided by monitoring the expression and in addition 1 3 106 in vitro-expanded Tregs (N, n 5 5). As controls, d of different activation markers upon the incubation of Tregs with tumor cells were injected alone ( , n = 5) or were coinjected with Th cells + or Th cells and Tregs without the cross-linking scBsDb (♦ and ), re- the scBsDb CD3xPSCA in the presence of PSCA target cells. spectively, n = 5). Tumor size was measured weekly. Statistical signifi- Only in the presence of the cross-linking bispecific Ab and target cance was determined for the bispecific Ab-treated groups with or without cells was an upregulation of the early activation marker CD69 Tregs at the final time point of the experiment using the Student t test and observed on both Tregs and Th cells. Moreover, the expression ANOVA with the Bonferroni multiple comparison test. *p , 0.05. levels of CD25 and CTLA-4 were markedly enhanced in both The Journal of Immunology 1557 populations under these conditions. In addition, an increased in- Finally, bispecific Ab-activated Tregs were able to abrogate the tracellular level of the transcription factor FOXP3 was observed in antitumor effector function of redirected Th cells and to promote the activated Tregs. In previous studies, it was shown that FOXP3 tumor growth in a mouse model. enhances the expression of CD25 and CTLA-4 (27, 28), which Although we clearly demonstrated in this study that bispecific might explain the increasing levels of both markers after acti- Abs are able to activate Tregs both in vitro and in vivo, the first vation in Tregs. Our observations of increased levels of these clinical trials showed their efficacy for cancer treatment (6). CD8+ markers in Tregs after activation are in accordance with experi- T cells respond much faster by cross-linking via bispecific Abs ments in which aCD3/CD28-coated beads were used for Treg than do conventional CD4+ T cells (M. Bachmann, unpublished stimulation, resulting in increased FOXP3, CD25, CTLA-4, and observations). Similar conclusions can also be drawn from data CD69 expression levels in activated Tregs (29). It was demon- published by other groups (37, 38). The time gap between the strated by a number of studies that the cross-linkage of conven- activation of cytotoxic CD8+ T cells and the activation of con- tional T cells to target cells via bispecific Abs triggers the ventional or regulatory CD4+ T cells might explain why, in the secretion of inflammatory cytokines (4, 5). In accordance with clinical trials performed thus far, the treatment with bispecific Abs these observations, our study showed that only in the presence of was rather successful, despite the most likely presence of Tregs. the specific bispecific Ab did the cross-linkage of conventional Nevertheless, strategies helping to circumvent or overcome the CD4+ T cells and Ag+ target cells result in the secretion of sig- potential activation of Tregs should improve the treatment of nificant amounts of IFN-g, IL-2, and TNF. In contrast, redirected malignant diseases with CD3-engaging bispecific Abs. One pos- Tregs secreted almost no proinflammatory cytokines or IL-2 upon sibility to avoid the unwanted stimulation of Tregs is the adoptive cross-linkage, although the flow cytometric analysis demonstrated transfer of ex vivo bispecific Ab-loaded effector T cells instead of Downloaded from the upregulation of activation markers under these conditions. a direct application of the bispecific Ab into a patient. This is in line with findings that constitutively expressed FOXP3 In view of their important role in establishing and maintaining in Tregs acts as a transcriptional repressor that blocks the pro- peripheral tolerance, an Ag-directed retargeting of Tregs using duction of a wide range of effector cytokines (30). Nevertheless, bispecific Abs may represent a promising therapeutic opportunity the activation of freshly isolated Tregs by bispecific Abs triggered for the treatment of autoimmunity and graft rejection. Based on

the release of the immunosuppressive cytokine IL-10. It should be several animal experiments, it is increasingly evident that Ag- http://www.jimmunol.org/ noted that expanded CD45RA+ Tregs did not produce any IL-10 specific Tregs are more efficient in autoinflammatory prevention upon cross-linkage with the bispecific Ab (data not shown), which and treatment than are polyclonal Treg populations (39, 40). Ad- correlates with studies by Hoffmann et al. (25), who did not detect ditionally, adoptively transferred polyclonal Tregs might result in this cytokine in culture supernatants after in vitro expansion and an unfavorable systemic immunosuppression, with an increasing TCR stimulation of expanded CD45RA+ Tregs. risk for opportunistic infections and/or reactivation of persistent Activation of (memory) T cells via bispecific Abs results in virus infections. In contrast, Tregs with a certain Ag/tissue speci- profound proliferation as does normal TCR triggering, despite the ficity are able to suppress ongoing self-destructive immune re- fact that additional costimulatory signals are missing. As demon- sponses at the appropriate sites of inflammation. One of the main strated in this study, the cross-linkage of conventional CD4+ Th cells obstacles to the clinical application of Ag-specific Tregs is the by guest on September 28, 2021 to tumor cells via the CD3xPSCA bispecific Ab induced significant isolation of sufficient numbers. One possible approach is the gen- expansion of the Th cells, although the target cells used in this eration of genetically modified Tregs, which express an artificial study do not express the CD28 ligands CD80/CD86 (data not TCR against a desired Ag (e.g., Refs. 19, 41, 42). Still, the most shown) and, therefore, cross-linked T cells could not get any ad- efficient methods for the genetic manipulation of T cells make use ditional CD28 costimulatory signals from the target cells. In con- of virus-mediated gene transfer, which bears the risk of effecting trast, Ab-redirected Tregs were hyporesponsive and did not expand the expression of oncogenes or tumor-suppressor genes (43). In when cultured without the addition of exogenous cytokines. Most light of this, bispecific Abs may provide powerful tools to redirect likely, this hyporesponsiveness is due to their inability to produce adoptively transferred polyclonal Tregs in an Ag-specific and/or growth-promoting IL-2. As shown by other investigators, Tregs rely site-specific manner, without the need to genetically manipulate on high amounts of IL-2 and strong TCR ligation to proliferate the cells. A potential strategy could be the preloading of polyclonal (31, 32). Incubating Tregs with both CD3xPSCA and high doses Tregs ex vivo with a bispecific Ab directed against a desired Ag of exogenous IL-2 induced their expansion, further validating the (e.g., an autoantigen in the case of an autoimmune disease) and assertion that bispecific Abs can be used as activating molecules retransfer of the cells into a patient. The bispecific Ab would endow for Tregs. the decorated Tregs with a predefined Ag specificity and trigger One prerequisite for the treatment of graft rejection or autoim- their suppressive function locally at the site of the ongoing immune mune diseases with bispecific Ab-redirected Tregs is the suppres- reaction, thereby potentially enhancing the therapeutic effect and sive capacity of Tregs upon Ag-specific activation with bispecific minimizing the risk for unspecific immunosuppression. Abs. Therefore, we investigated the ability of bispecific Ab-redirected Tregs to inhibit the proliferation and cytokine production Disclosures of autologous Th cells, a well-described characteristic feature of The authors have no financial conflicts of interest. Tregs (33, 34). Indeed, the incubation of Tregs in the presence of a cross-linking bispecific Ab resulted in a significant suppression References of both proliferation and cytokine secretion of autologous Th 1. Baeuerle, P. A., P. Kufer, and R. Bargou. 2009. BiTE: Teaching antibodies to cells. The lack of IL-2, together with our observation that the engage T-cells for cancer therapy. Curr. Opin. Mol. Ther. 11: 22–30. Ab-activated Tregs strongly reduced the expression of the high- 2. Lutterbuese, R., T. Raum, R. Kischel, P. Lutterbuese, B. Schlereth, E. Schaller, affinity receptor a-chain for IL-2 (CD25), might account for the S. Mangold, D. Rau, P. Meier, P. A. Kiener, et al. 2009. 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