Tolerance Induction of Peripheral CD8 T Cell Transcriptional ...

The Journal of Immunology

Transcriptional Targeting of B Cells for Induction of Peripheral CD8 T Cell Tolerance1 Melanie Werner-Klein,* Christiane Dresch,* Peggy Marconi,† and Thomas Brocker2* Several mechanisms are in place to neutralize autoimmune CD8 T cells by tolerance induction. Developing self-specific CD8 T cells are eliminated in the thymus by Ag-presenting epithelial and dendritic cells (DCs). However, CD8 T cells escaping thymic central tolerance can also be inactivated by tolerance mechanisms in peripheral organs. In contrast to DCs, the role of B cells in generating CD8 T cell tolerance is not well-characterized. To investigate this question in more detail, we transcriptionally targeted Ag to B cells using B cell-specific retroviral vectors in vivo. Although Ag expression could be detected in B cells of thymus, lymph nodes, and spleen, B cells were unable to induce central tolerance of CD8 thymocytes. In contrast, in peripheral organs, we could identify clonal deletion and functional inhibition (anergy) of CD8 T cells as tolerance-inducing mechanisms. Although Ag expressed by B cells was acquired and cross-presented by DCs, B cells were also sufficient to tolerize CD8 T cells directly. These findings suggest exploitation of B cells for Ag-specific immunotherapy of CD8 T cell-mediated autoimmune diseases. The Journal of Immunology, 2007, 178: 7738 –7746.

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he CD8 T cell contributes to the pathology of autoimmune diseases such as diabetes (1, 2) and multiple sclerosis (3, 4) and CD8 T cell tolerance induction has been discussed as a therapeutic option in this context (1). Furthermore, CD8 T cell tolerance remains an appealing goal for prevention of allograft rejection (5). Inherent mechanisms for tolerance induction operate in the thymus and in peripheral organs. The recognition of self-Ag/MHC complexes by developing thymocytes with high-avidity TCR results in thymic-negative selection and central self-tolerance (reviewed in Ref. 6). However, not all self-Ag might be expressed in the thymus at sufficient levels and low-avidity T cells specific for “self” can escape central tolerance and be found in peripheral organs (7–9). Therefore additional peripheral tolerance mechanisms must be in place to maintain self-tolerance (reviewed in Ref. 10). In absence of inflammatory signals, dendritic cells (DC)3 are particularly potent inducers of T cell tolerance to self-Ag (reviewed in Ref. 11 and 12) and autoreactive CD8 T cells are tolerized either by physical deletion or rendered unresponsive or anergic. Though T cell removal by deletion is irreversible, ongoing thymic T cell output creates a continuous autoimmune “threat” by generating new potentially autoreactive T cells. In contrast to deletion, functional CD8 T cell anergy depends on persistence of Ag and CD8 *Institute for Immunology, Ludwig-Maximilians-University, Munich, Germany; and † Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy Received for publication November 21, 2006. Accepted for publication April 2, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Deutsche Forschungsgemeinschaft SFB 455 (to T.B.), the CAPES Foundation/Brazilian Ministry of Education (to C.D.), and the European Community FP6-2004-LIFESCIHEALTH-5, THOVLEN (to T.B. and P.M.). 2 Address correspondence and reprint requests to Dr. Thomas Brocker, Institute for Immunology, Ludwig-Maximilians-University, Goethestrasse 31, D-80336 Munich, Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: DC, dendritic cell; SIN, self interacting; RIP, rat insulin promoter; HSPC, hemopoietic stem and progenitor cell; sOVA, soluble OVA; BM-DC, bone marrow-derived DC; NTL, nontransgenic littermate.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org

T cells can regain their responsiveness if exposure to Ag vanishes (reviewed in Refs. 10 and 13). Taken together, effective induction of CD8 T cell tolerance needs to fulfill several requirements; first, the tolerizing Ag should be presented in the context of weak costimulatory signals and second, presentation should to be maintained as long as possible. Because B cells do not constitutively express costimulatory molecules, but have the capacity to present Ag, several studies used B cells to induce T cell tolerance. These approaches used adoptive transfer of Ag-loaded B cells or transfection and Ab targeting of B cells to test their tolerance-inducing capacities (14 –24). With the progress that has been made in visualizing and tracking transfer of Ag in vivo, it was shown that B cells can provide DC with Ag for presentation and tolerization via MHC class II (25, 26). However, in the light of the capacity of DC to cross-present exogenous Ag via MHC class I (27, 28), the possible “direct” tolerization of CD8 T cells by Ag-expressing B cells (29) remains to be revisited. To investigate the feasibility and mechanisms of CD8 T cell tolerance induction, we transcriptionally altered B cells in vivo using a retrovirus-based stem cell approach. As most B cell populations have a lifespan of weeks to months (30, 31) and adoptively transferred B cells might even survive only for shorter periods due to previous in vitro manipulation, a stem cell-based approach meets the requirements of enduring Ag persistence for efficient CD8 T cell tolerance induction. In the context of self-inactivating (SIN) retroor lentiviral control elements, B cell specificity using the CD19 promoter could be obtained in human cord blood-derived stem cell approaches (32) as well as in experimental bone marrow transfer systems in mice (33). Therefore, lethally irradiated mice were reconstituted with hemopoietic stem cells transduced with a retroviral vector encoding for a model Ag under the transcriptional control of the B cell-specific human CD19 promoter (33). Using soluble and membrane-bound forms of Ag, we demonstrate that targeted Ag expression in B cells cannot mediate central thymic CD8 T cell tolerance, but results in complete peripheral tolerance of CD8 T cells by clonal deletion and anergy. We demonstrate that retrovirally altered B cells not only provide Ag to DC, but also directly tolerize CD8 T cells in vivo and propose this B cell-specific gene-therapeutic approach as a potent means to functionally eliminate CD8 T cells with unwanted specificities.


Materials and Methods Mice C57BL/6, OT-1 (34), CD11c-Rac1(N17) (35), and RIP-OVAlow (36) mice were maintained and bred in the animal facility of the Institute for Immunology (Munich, Germany). OT-1 mice are transgenic for a TCR recognizing OVA257–264 in the context of H-2Kb and can be identified by the expression of V␣2 and V␤5.1/5.2 TCR-␣ and ␤-chain. In CD11c-Rac1(N17) transgenic mice, the dominant-negative form of the Rho-GTPase Rac1 is expressed under control of the DC-specific CD11c promoter. In RIP-OVAlow mice, expression of low levels of OVA is controlled by the rat insulin promoter (RIP) and is restricted to the pancreas.

Cell lines Phoenix eco cells were cultured in high-glucose DMEM with glutamax, 10% FBS, and penicillin-streptomycin (Invitrogen Life Technologies). All cells were kept at 37°C in a humidified atmosphere in CO2 incubators (Heraeus) with the exception of virus generation, which was performed at 32°C.

Retroviral vectors SIN retroviral vectors (SIN vectors) lack the viral promoter elements located in the U3 region of the 3⬘ long terminal repeat sequence, which upon integration of the viral genome into the host DNA is transferred to the 5⬘ long terminal repeat resulting in the transcriptional inactivation of the provirus and allowing transcriptional regulation of a transgene from an internal (e.g., cell specific) promoter. The SIN vectors used here are based on SIN-CD19-W (33), which uses a 999-bp fragment (⫺1018 to ⫺19 bp) of the promoter region of the human CD19 gene as internal promoter to control transgene expression. In addition, the vector contains the WPRE sequence (W) to enhance the retroviral titer (33).To obtain SIN vectors encoding for the soluble secreted (37) or membrane-bound (38) form of OVA, the cDNA encoding GFP in SIN-CD19-W was replaced with a soluble OVA (sOVA) or transferrinreceptor (Tfr)OVA cDNA creating SINCD19-sOVA-W or SIN-CD19-TfrOVA-W. To generate retroviral supernatants, Phoenix eco cells were transfected using standard calcium phosphate transfection. Briefly, 7.5 ⫻ 106 cells were transfected with 60 ␮g of vector-DNA. Supernatants were routinely generated 24 – 48 h posttransfection by overnight incubation in Phoenix growth medium at 32°C. Vector stocks were filtered (0.45-␮m filter; Nalgene) and immediately used or snap-frozen and stored at ⫺80°C for later use.

Bone marrow chimeras Bone marrow cells from femurs and tibiae of female C57BL/6, OT-1, or CD11c-Rac1(N17) mice of 6 – 8 wk of age were harvested 4 days after i.v. injection of 5-fluorouracil (150 mg/kg body weight; Amersham Biosciences). The cells were prestimulated for 2 days in IMDM supplemented with 20% FBS, penicillin-streptomycin (Invitrogen Life Technologies), and a growth factor mixture containing human IL-6 (200 U/ml), murine IL-3 (10 ng/ml), and murine stem cell factor (50 ng/ml). Recombinant growth factors were purchased from Strathmann Biotech. After prestimulation, cells were transduced by spin infection (300 ⫻ g, 2 h, 30 –32°C) with cell-free supernatants of ecotropic retroviral particles in the presence of polybrene (4 ␮g/ml). The transduction procedure was repeated once 14 h after the first infection. One day after the final transduction, 1.5 ⫻ 106 cells/mouse were injected i.v. in lethally irradiated (split dose day ⫺2 and day 0: 550 rad) female C57BL/6 recipients (age 10 –15 wk). Chimeras routinely were analyzed or used for additional experiments 6 –10 wk after reconstitution with hemopoietic stem and progenitor cells (HSPC).

OVA-specific RT-PCR Total RNA was isolated and cDNA synthesized from CD19-positive and -negative cells using the PureLink Microto-Midi kit and RT-PCR was performed using the SuperScript One-Step RT-PCR Kit according to the manufacturer’s instructions (Invitrogen Life Technologies). CD19-positive and -negative cells were separated with CD19 microbeads (Miltenyi Biotec) from axial and inguinal lymph nodes, thymus, and spleen of SIN-CD19TfrOVA-W chimeras. The purity of the CD19-positive and -negative population was at least 90 and 99%, respectively, as measured by flow cytometry. The primers (MWG Biotech) OVA-forward 5⬘-cgtggattctc aaactgcaa-3⬘ and OVA-reverse 5⬘-gacttcatcaggcaacagca-3⬘ generated a 253-bp fragment from the TfrOVA transgene and the primers for the detection of ␤-actin (mouse/rat ␤-actin PCR primer pair; R&D Systems) amplified a 302-bp fragment. The PCR cycling conditions were as

7739 follows: 94°C/2 min, 40 ⫻ 94°C/30 s, 59°C/30 s, and 72°C/30 s. PCR products were separated by agarose-gel electrophoresis.

Immunization with DNA, viral vectors, and cultured DC Naked DNA immunization of RIP-OVAlow mice with pCDNA3-OVA (39) was performed by gene gun administration (Bio-Rad). Cartridges of DNAcoated gold particles were prepared according to the manufacturer’s instructions. For each preparation, gold particles (25 mg; diameter, 1 ␮m) were coated with 200 ␮g of DNA. Mice were anesthetized with a mixture of ketaminhydrochloride and xylazinhydrochloride in PBS before vaccination. A total of 8 ␮g of plasmid DNA was delivered to the shaved abdominal skin of adult mice with a discharge pressure of 400 lb/inch (2). For immunizations with recombinant herpes simplex vectors encoding OVA or GFP, frozen virus stocks of TOH-OVA or TOZ-GFP (39) were thawed on ice, diluted in PBS, and 4 ⫻ 106 PFU/mouse were administered i.v. For immunizations with DC, total bone marrow was plated in 90-mm plates at 5 ⫻ 106 cells/ml in 10 ml of IMDM culture medium (Invitrogen Life Technologies) containing 5% FBS, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin, 50 ␮M 2-ME (Invitrogen Life Technologies) and 25 ng/ml GM-CSF for 9 days. Nonadherent cells were harvested and 3 ⫻ 106 cells/mouse were administered i.v. DC were loaded with OVA257–264 peptide for 1.5 h at 37°C and 5% CO2.

In vivo cytotoxic T cell assay This assay was performed as described before (40) and is based on the specific elimination of peptide-loaded spleen cells labeled CFSE (Molecular Probes) in vivo over control cells, which are not peptide loaded, but labeled with a lower CFSE intensity. C57BL/6 erythrocyte-depleted splenocytes were incubated in the presence or absence of 10 ␮M OVA257–264 for 1 h at 37°C and 5% CO2. To remove unbound peptide, the cells were washed three times with PBS. Peptide-loaded cells were labeled with a high (1.7 ␮M) concentration of CFSE, whereas unloaded cells were labeled with a low concentration (0.2 ␮M). Equal numbers of CFSEhigh and CFSElow cells were mixed and 30 ⫻ 106 cells/mouse were administered i.v. Fifteen hours later, mice were sacrificed and spleen cell suspensions were analyzed by flow cytometry. The percentage of percent-specific cytotoxic lysis was calculated as follows: percent-specific cytotoxic lysis ⫽ (1 ⫺ (r of nonimmunized mouse/r of immunized mouse) ⫻ 100); r ⫽ (% CFSElow/% CFSEhigh).

Adoptive T cell transfer Naive CD8-positive T cells were isolated from spleen and lymph nodes of OT-1 mice (Ly5.1⫹ or Ly5.2⫹) by negative selection using the MACS CD8-T cell isolation kit (Miltenyi Biotec). The purity of CD8 cells was ⱖ95%. For some applications CD8 T cells were labeled with 5 ␮M CFSE for 10 min at 37°C in PBS/0.2% BSA. Labeling was stopped with one volume of FBS and the cells were washed at least three times in PBS. 0.5–1 ⫻ 106 cells were injected into the lateral tail veins of the mice. In some experiments, 40 ␮g of LPS (Sigma-Aldrich) was coinjected.

Diabetes induction in RIP-OVAlow mice RIP-OVAlow mice received 0.75–1 ⫻ 106 OT-1 T cells and were immunized by naked DNA immunization with pCDNA3-OVA. The level of glucose in urine was measured with test sticks (Diabur; Roche Diagnostics) before and after immunization. Mice with glucose concentrations ⬎5.6 nM/L were considered diabetic.

In vitro T cell stimulation and Ag-presentation assays CD19⫹ B cells and CD11c⫹ DC were isolated from the spleen of sOVA or mock chimeras by positive selection using CD19 and CD11c microbeads (Miltenyi Biotec), respectively. A total of 4 ⫻ 105 B cells or DC were irradiated with 1000 rad and cultured with 1 ⫻ 106 CFSE-labeled OT-1 T cells in a 96-well flat-bottom plate at 37°C and 5% CO2. Proliferation was analyzed after 48 h by flow cytometry. OT-1 T cells were isolated from sOVA or control chimeras, labeled with CFSE, and restimulated in vitro with C57BL/6 splenocytes which had been pulsed with 100 pM SIINFEKL for 90 min at 37°C and 5% CO2. Forty-eight hours later, OT-1 T cells were analyzed by flow cytometry.

Flow cytometry The mAbs specific for CD4 (H129.19), CD8␣ (53-6.7 or CT-CD8␣ for tetramer-analysis), CD11c (HL-3), CD25 (7D4), CD44 (IM7), CD62L (Mel14), CD69 (H1.2F3), TCRV␣ 2 (B20.1), TCRV␤5.1/5.2 (MR9-4), IFN-␥, and TNF-␣ as well as streptavidin reagents were purchased from BD Biosciences/BD Pharmingen or Caltag Laboratories. H-2Kb/OVA257–264

7740

CD8 T CELL TOLERANCE BY B CELL-SPECIFIC GENE THERAPY

FIGURE 1. B cell specificity of retroviral SIN-CD19 vectors. a, Schematic representation of murine leukemia virus (MLV)-based SIN vectors, using ⬵1 kb of the human CD19 promoter to specifically direct the expression of soluble (sOVA) or membrane-bound (TfrOVA) chicken egg albumin (OVA) to B cells. (WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; A, AgeI; E, EcoRI; N, NotI; S, SacI). b, OVAand ␤-actin-specific RT-PCR from CD19-positive and -negative cells from axial and inguinal lymph nodes, thymus, and spleen of SIN-CD19TfrOVA-W chimeras. c, C57BL/6 mice (n ⫽ 5) received 2.4 ⫻ 106 OT-1 T cells and 1 day later 3 ⫻ 106 DC from in vitro bone marrow cultures from sOVA or mock chimeras. DC loaded with OVA257–264 served as positive control. Before and 4 days after the immunization with DC, the frequency of OT-1 T cells among all CD8 T cells in peripheral blood was determined by flow cytometry and is indicated in each dot plot. No expansion of OT-1 T cells in mice immunized with sOVA-DC or control DC was observed at later time points (data not shown). d, CFSE-labeled OT-1 T cells were stimulated with splenic CD11c⫹ DC or CD19⫹ B cells isolated from sOVA and mock chimeras (purity DC ⬎92%, purity B cells ⬎98%). DC and B cells isolated from C57BL/6 mice that had been injected with 100 ␮g of OVA protein 15 h before isolation served as positive control. Proliferation and activation of OT-1 T cells was analyzed 48 h later by flow cytometry. The percentage of cells that had divided more than once and did up-regulate CD44 is indicated in each dot plot. The data are representative for three independent experiments. To control for potential contamination of DC with OVA-expressing B cells and B cells with OVA-presenting DC, the respective percentage of contaminating cells was added deliberately to the cultures from mock chimeras (d, ⫹8% B cells, ⫹2%DC).

Statistical analysis

tetramers were provided by D. Busch (Institute for Microbiology, Immunology and Hygiene, Technical University, Munich, Germany). Flow cytometry was performed on a FACSCalibur instrument (BD Biosciences) and analyzed with CellQuest (BD Biosciences) or FlowJo software (Tree Star). For flow cytometry, organs were prepared as single-cell suspensions according to standard protocols.

Data were analyzed using the Student t test and the Mann-Whitney U test (GraphPad Prism 4.03; GraphPad Software). The Mann-Whitney U test was applied to data with non-Gaussian distribution. A value of p ⬍ 0.05 was considered to be significant.

Intracellular staining

Results

Intracellular staining for cytokines was performed using the Cytofix/Cytoperm kit (BD Biosciences/BD Pharmingen) after in vitro restimulation of 2–3 ⫻ 106 splenocytes with 1 ␮M SIINFEKL (OVA257–264) for 5 h in the presence of Golgi-Stop (BD Biosciences/BD Pharmingen, containing brefeldin A).

CD19-SIN vectors confer B cell-specific Ag expression in vivo To achieve persistent Ag expression and presentation by B cells, primary murine HSPC were transduced with B cell-specific retroviral vectors and bone marrow chimeric mice were generated.

The Journal of Immunology Transgene expression was controlled by the 1-kb fragment of the human CD19 promoter (Fig. 1a). Long-term transgene expression in 30 –50% of B cells in all CD19⫹ stages of B cell development can be achieved with these vectors (33). To study the effect of Ag expression in B cells on the fate of Ag-specific CD8 T cells, the secreted (sOVA) or membrane-bound (TfrOVA) forms of chicken OVA were chosen as model Ags (Fig. 1a). Bone marrow chimeras were generated with C57BL/6-HSPC transduced with retroviral vectors encoding for sOVA or TfrOVA. To confirm B cell-specific transgene expression from these vectors, RT-PCR for OVA and ␤-actin as control was performed on magnetically separated CD19-positive and -negative cells from lymph nodes, spleen, and thymus of bone marrow chimeras (Fig. 1b). Whereas ␤-actin transcripts were amplified in CD19⫹B cells and CD19⫺ non-B cells, OVA transcripts were detected selectively in CD19⫹ B cells in lymph nodes, spleen, and thymus, but not in CD19⫺cells of the same lymphoid organs. Therefore, these data confirm the B cellspecific transcriptional regulation by the CD19 promoter. B cells and DCs present virally encoded Ag in vivo As we could exclude expression of Ag in non-B cells from primary and secondary lymphoid organs by RT-PCR (Fig. 1b), we wanted to test to what extent in vivo DC would have access to OVA produced by B cells and be able to cross-present it to OVA-specific T cells. To further functionally test the possibility of OVA expression in DC, we generated bone marrow-derived DC (BM-DC) from sOVA or mock chimeras. During the culture period, generated DC had no access to OVA from B cells, as these cultures lacked CD19⫹ B cells. Subsequently, BM-DC were used for immunization of C57BL/6 mice that had received OT-1 T cells the day before. BM-DC derived from sOVA or mock chimera bone marrow could not stimulate the expansion and proliferation of OT-1 T cells, when used as DC vaccines (Fig. 1c). However, when DC from mock and sOVA chimeras had been loaded with exogenous OVA257–264 peptide before vaccination, OT-1 T cells expanded vigorously (Fig. 1c, p ⫽ 0.0042 and p ⬍ 0.001, respectively, Student’s t test). This experiment, together with the data from the RT-PCR (Fig. 1b) and the previously published data on the B cell specificity of this approach (33), argue against expression of OVA in DC from CD19-driven retroviral vectors. To analyze whether DC would have access to Ag produced by B cells in vivo, we purified CD11c⫹ DC and CD19⫹ B cells from spleens of OVA and mock chimeras and compared their capacity to stimulate OT-1 T cells in vitro. As a positive control for cross-presentation of Ag by DC, OVA protein was injected i.v. into control mice 15 h before isolation of DC and B cells (Fig. 1d). OT-1 T cells proliferated and up-regulated CD44 when DC from OVA chimeras or OVA-injected control mice were used, with the latter being more effective (Fig. 1d, p ⫽ 0,0008, Student’s t test). Resting B cells from the OVA-injected control induced only a marginal stimulation of OT-1 T cells, unless they were activated via CD40 ( p ⫽ 0.01, Student’s t test). In contrast, nonactivated B cells from sOVA chimeras could present OVA more efficiently ( p ⫽ 0.04, Student’s t test), probably reflecting the difference between endogenous Ag and the limited capacity of B cells to capture and cross-present exogenous Ag via MHC class I. In addition, we deliberately “contaminated” the B cells with DC and DC with B cells from CD19OVA mice to make sure that the observed proliferation of OT-1 T cells was not induced by the respective contaminating cell type (Fig. 1d). These results show that 1) B cells can directly present endogenously expressed OVA in context of MHC class I and that 2) OVA produced by B cells is efficiently cross-presented by DC. However, the presentation efficacy of B cells is much lower as compared with DC from the same mice.

7741

FIGURE 2. Ag expression by B cells tolerizes polyclonal CD8 T cells. Chimeras based on SIN-CD19-sOVA-W, SIN-CD19-TfrOVA-W, or mock-transduced C57BL/6-HSPC were immunized with 4 ⫻ 106 PFU TOH-OVA or TOZ-GFP (n ⫽ 4). a, On day 7 after immunization, the frequencies of H-2Kb/OVA257–264-specific cells among all CD8 T cells were analyzed by flow cytometry and indicated in each dot plot. b, On day 8, a CFSE-based in vivo cytotoxic T cell assay was performed and the specific lysis of OVA257–264-loaded, CFSE-labeled target cells was determined by flow cytometry (n ⫽ 4). The data are representative for at least two independently performed experiments with similar outcome.

Ag-specific tolerization of polyclonal CD8 T cells To analyze whether a polyclonal CD8 T cell repertoire could be tolerized by B cells, chimeric mice were immunized with a recombinant herpes simplex virus encoding OVA (TOH-OVA), which was previously reported to induce strong OVA-specific CD8 T cell responses (39). The frequency of OVA257–264-specific polyclonal CD8 T cells was determined by H-2Kb/OVA257–264 tetramer staining at the response peak (Fig. 2a). Control mice immunized with a GFP-encoding control virus (TOZ-GFP) did not show expansion of OVA-specific T cells, indicating the OVA specificity of the H-2Kb-OVA MHC tetramer detection system (Fig. 2a). In contrast, immunization with TOH-OVA did elicit a significant expansion of tetramer-positive CD8 T cells in mock chimeras ( p ⫽ 0.010, Student’s t test), but not in sOVA- or TfrOVA-expressing mice. Subsequently, immunized mice were challenged with target cells for CTLs in vivo and the percentage of specific lysis was determined (Fig. 2b). In line with the results of the tetramer analysis (Fig. 2a), TOZ-GFP-immunized control mice and TOH-OVAimmunized TfrOVA or sOVA chimeras could not eliminate OVA257–264-loaded target cells, whereas mock chimeras immunized with TOH-OVA showed a high-specific killing (Fig. 2b, p ⫽ 0.0004 compared with sOVA and p ⫽ 0.0015 TfrOVA, Student’s t test). These data show that retrovirally mediated transcriptional targeting of B cells using transduced hemopoietic stem cells could induce Ag-specific tolerance in polyclonal CD8 T cells. However, from these experiments it was not clear which tolerance inducing mechanism was imposed by the treatment: thymic central tolerance, peripheral deletion, or functional anergy or combinations of these possibilities. No thymic central tolerance induction by B cells To study the underlying tolerance mechanism, we increased the number of Ag-specific CD8 T cells by using TCR-transgenic OT-1 mice as HSPC donors for the generation of sOVA- and TfrOVAexpressing bone marrow chimeras. Most CD8 T cells should express a transgenic TCR specific for the OVA257–264 epitope and can be easily monitored by flow cytometry. To determine whether

7742

CD8 T CELL TOLERANCE BY B CELL-SPECIFIC GENE THERAPY chimeras as compared with control mice ( p ⫽ 0.65, Student’s t test). These findings argue against a central deletion mechanism, although OVA transcripts were detected by RT-PCR in the CD19positive population of the thymus (Fig. 1b). In addition, these results indirectly confirm that other potent inducers of MHC class I-mediated negative selection such as thymic DC (41, 42) had neither access to circulating soluble OVA released by B cells nor to B cell-associated OVA in the thymus. Peripheral tolerance induction by B cells

FIGURE 3. B cell-specific Ag expression does not lead to central tolerance induction in TCR-transgenic CD8 T cells. HSPC from OT-1 mice were mock transduced (n ⫽ 3) or transduced with SIN-CD19-sOVA-W (n ⫽ 4) or SIN-CD19-TfrOVA-W (n ⫽ 3) and bone marrow chimeras were generated. Transgenic thymocytes were identified according to their expression of CD4, CD8, V␣2, and V␤5.1/5.2 by flow cytometry. a, The frequency (SEM ⱕ3%) and, b, the total numbers of single-positive OT-1 T cells were determined. Dot plots are gated on CD4⫺CD8⫹ thymocytes. The data are representative for three (sOVA) and two (TfrOVA) independently performed experiments.

OVA-expressing B cells could induce central tolerance, thymocytes expressing the OT-1 TCR were analyzed (Fig. 3). In this study, a similar frequency (Fig. 3a) and total cell number (Fig. 3b) of transgenic OT-1 T cells was found in sOVA- and Tfr-OVA

FIGURE 4. Ag expression in B cells induces peripheral tolerance in TCR-transgenic CD8 T cells. HSPC from OT-1 mice were mock transduced (n ⫽ 3) or transduced with SIN-CD19-sOVA-W (n ⫽ 4) or SIN-CD19-TfrOVA-W (n ⫽ 3) and bone marrow chimeras were generated. OT-1 T cells in spleens were identified according to their expression of CD8, V␣2, and V␤5.1/5.2 by flow cytometry and (a) the frequency (SEM ⱕ2.2%) and (b) total number of transgenic T cells was determined. Dot plots are gated on CD8⫹ cells. c, The expression of activation markers by OT-1 T cells was analyzed with mAbs for CD62L, CD44, CD69, and CD25. The data are representative for four (sOVA) or two (TfrOVA) independently performed experiments with similar outcome.

In contrast to the findings in the thymus, peripheral lymphoid organs of the same animals showed a reduced frequency of OT-1 T cells in spleens (Fig. 4a) of sOVA ( p ⫽ 0.0001, Student’s t test, combined data from four independent experiments) and TfrOVA chimeras ( p ⫽ 0.033, Student’s t test, combined data from two independent experiments) compared with control chimeras (Fig. 4a). In contrast, the frequency of CD8⫹ non-OT-1 T cells with the TCR phenotypes V␣2⫹V␤5.1⫺, V␣2⫺V␤ 5.1⫹, or V␣2⫺V␤5.1⫺ was increased in OVA-expressing chimeras (Fig. 4a). More importantly, not only the percentage but also the total number of OT-1 T cells was significantly decreased in mice expressing OVA in B cells. In all experiments, the production of soluble Ag in CD19-sOVA chimeras induced a reduction in the total OT-1 numbers ranging from 28 to 75% (Fig. 4b, sOVA, p ⫽ 0.03, Student’s t test). In mice with B cells that expressed the membrane-bound Ag, OT-1 cells were reduced between 42 and 87% (Fig. 4b, TfrOVA, p ⫽ 0.03, Student’s t test). This reduction of total Agspecific CD8 T cell numbers indicates that Ag-expressing B cells induced tolerance by peripheral deletion. As not all OT-1 T cells had been deleted in OVA-expressing OT-1-chimeras (Fig. 4b), we analyzed the remaining cells in more detail. Recently activated T cells show up-regulation of CD25, CD69, and CD44 and down-regulation of CD62L. OT-1 cells from sOVA-expressing chimeras displayed no phenotype of recent activation as determined by the expression of CD44, CD62L, CD69,


7743 The role of cross-presenting DC in vivo

FIGURE 5. B cell-specific Ag expression induces anergy of Ag-specific TCR-transgenic CD8 T cells. RIP-OVAlow mice received (a) 1 ⫻ 106 OT-1 T cells from sOVA chimeras or 0.75 ⫻ 106 OT-1 T cells from TfrOVA chimeras (n ⫽ 4 – 6) or the same respective numbers of OT-1 T cells from control chimeras. One day later, mice were gene-gun immunized with pCDNA3-OVA encoding OVA. Diabetes induction was monitored and mice with ⬎5.6 nM/L glucose in urine were considered diabetic. The percentage of diabetic mice over time is shown. One representative experiment of two with similar outcome is shown. b, At day 43, splenocytes from mice that had received OT-1 cells from sOVA chimeras were restimulated with OVA peptide for 5 h and the frequency of IFN-␥- and TNF-␣-producing OT-1 T cells was determined by intracellular cytokine staining. Similar results were obtained for OT-1 T cells from TfrOVA chimeras (data not shown).

and CD25 (Fig. 4c; data for TfrOVA not shown, but similar). To determine whether these nondeleted OT-1 T cells from OVA-expressing chimeras could differentiate into effector T cells and exert autoimmune aggression in vivo, the RIP-Ovalow mouse model was used. In this strain, transgenic OVA-expression in the pancreas is controlled by the RIP and serves as a model self-Ag (43). When OT-1 T cells are transferred into RIP-OVAlow-mice, they are ignorant to OVA due to low expression levels of OVA (43). However, upon Ag-specific immunization, the transferred OT-1 T cells become activated, destroy the OVA⫹ pancreatic ␤-islet cells and mice develop diabetes (44). Eight to 9 days after transfer of OT-1 T cells from mock chimeras and immunization with an OVA-encoding vaccine, recipient mice developed diabetes as determined by urine glucose levels (Fig. 5a). In contrast, none of the mice became diabetic when the same numbers of OT-1 T cells from sOVA or TfrOVA chimeras were transferred (Fig. 5a). Then mice were sacrificed and lymphocytes were restimulated in vitro with OVA257–264 to measure cytokine production by the remaining OT-1 T cells. Although comparable numbers of OT-1 T cells were present in spleens of both groups, the frequency of IFN-␥- and TNF-␣-producing OT-1 cells ( p ⫽ 0.02 and p ⬍ 0.05, respectively; Mann-Whitney U) was reduced in spleens of mice receiving OT-1 T cells originating from sOVA chimeras (Fig. 5b). Similar results were obtained for OT-1 cells from TfrOVA-expressing mice (data not shown). These data show that B cells can induce peripheral tolerance by clonal deletion (Fig. 4) as well as by functional anergy of Agspecific CD8 T cells (Fig. 5).

To address the question of whether Ag presentation by B cells would be directly tolerogenic for CD8 T cells or if B cells rather provide Ag to cross-presenting DC for tolerance induction, we used a novel transgenic in vivo model (35, 45). The expression of a dominant negative form of the Rho-GTPase Rac1 under control of the DC-specific CD11c promoter severely impairs cross-presentation of exogenous Ag by DC in transgenic Rac-mice (35, 45). Bone marrow HSPC from Rac donors or nontransgenic littermates (NTL) were transduced with CD19-sOVA retrovirus and used to generate chimeras. We then adoptively transferred naive, CFSElabeled OT-1 cells into these recipients to analyze Ag-specific cognate interactions leading to OT-1 proliferation (29). We hypothesized that if B cells were not able to directly tolerize CD8 T cells, the level of T cell proliferation in OVA-expressing Rac1 chimeras should be reduced or even absent due to lack of functional DC. As published previously in the OT-1 T cell system (46), provision of an inflammatory signal such as IL-12 or LPS in vivo in addition to cognate Ag determines tolerance vs full activation of naive CD8 T cells. If no inflammatory signals were delivered, OT-1 T cells proliferated, but did neither increase in cell numbers, nor gain effector functions (46). Therefore, LPS was coadministered in one control group as an inflammatory stimulus (Fig. 6a). Two days posttransfer, OT-1 T cell proliferation could be observed in all OVA-expressing chimeras to a similar extent, while OT-1 T cells in mock chimeras did not proliferate (Fig. 6a). The analysis of total OT-1 T cell numbers in spleens 14 days after adoptive T cell transfer revealed productive T cell expansion in OVA-expressing mice that had received LPS as compared with mock chimeras (Fig. 6b, left panel, p ⫽ 0.0079, Mann-Whitney U test). In contrast, no significant T cell expansion was detected between OVA-negative mock chimeras and OVA-expressing NTL or Rac chimeras (Fig. 6b, p ⬎ 0.05, Student’s t test), despite T cell proliferation in the latter groups (Fig. 6a). These data indicate that T cell expansion in absence of inflammatory stimuli was comparable in NTL and Rac1 recipients (Fig. 6b, left panel). To test the functionality of the remaining OT-1 T cells from the different recipients, we restimulated them with antigenic peptide in vitro under conditions where only primed T cells were expected to produce significant amounts of IFN-␥ (Fig. 6b, right panel). As expected, OT-1 T cells from OVA-negative mock chimeras remained naive, did not proliferate in vivo (Fig. 6a), and could not respond to the brief ex vivo stimulation with antigenic peptide (Fig. 6b, right panel). In contrast, OT-1 T cells from the LPStreated mice had been primed in vivo and therefore readily produced IFN-␥ upon restimulation in vitro as compared with the mock control (Fig. 6b, right panel, p ⬍ 0.0001, Student’s t test). However, neither OT-1 T cells from NTL nor Rac chimeras had attained the ability to produce IFN-␥ during their encounter with OVA as no significant differences were observed between these groups and the mock control (Fig. 6b, right panel, p ⬎ 0.05, Student’s t test). Next, we monitored the surface CD44 expression as a marker for activation. In fact, the remaining OT-1 T cells in all OVA-expressing chimeras had similarly up-regulated their surface expression of CD44 (Fig. 6c) and down-modulated CD62L (data not shown) 14 days posttransfer. This was in contrast to OT-1 T cells in OVA-negative mock chimeras, which had retained their naive CD44lowCD62Lhigh phenotype due to absence of Ag (Fig. 6c). Taken together, these data indicate that proliferation in response to OVA had occurred similarly in Rac and NTL chimeras (Fig. 6a). In addition, this proliferation was nonproductive in both types of recipients, as OT-1 T cell numbers did not increase and

7744


FIGURE 6. B cells can directly tolerize CD8 T cells. HSPC from Rac and NTLs mice were used to create sOVA chimeras (n ⫽ 5). As negative control, C57BL/6 mock chimeras were generated (n ⫽ 4). CFSE-labeled CD44lowOT-1/Ly5.1⫹ T cells were transferred into these chimeras and (a) 48 h later proliferation in peripheral blood was analyzed by flow cytometry. b, After 14 days, splenocytes were isolated and the total number OT-1/Ly5.1⫹ T cells was determined by flow cytometry. To determine the percentage of IFN-␥-producing OT-1/Ly5.1⫹ T cells, splenocytes were briefly stimulated in vitro with OVA257–264 and analyzed by intracellular cytokine staining. c, Surface CD44 expression of OT-1 T cells isolated from spleens of the different recipients at day 14 posttransfer.

the remaining OT-1 T cells were activated (Fig. 6c), but functionally paralyzed (Fig. 6b). Therefore, we conclude from this experiment that transcriptionally targeted B lymphocytes are sufficient to induce CD8 T cell tolerance also when DC cannot cross-present Ag optimally.

Discussion We have shown that transcriptional targeting of Ag to B cells leads to Ag-specific tolerance induction of CD8 T cells in vivo. Using an OT-1-based TCR-transgenic model, we revealed that tolerance is induced in peripheral lymphoid organs, but not in the thymus. We could show that peripheral tolerance was established by peripheral deletion and induction of anergy. Our findings are in contrast to a previous report, where transfer of peptide-loaded B cells failed to anergize OT-1 T cells, while partially deleting them (29). Bennett et al. (29) described activation-induced deletion of OT-1 T cells by B cells as a CD95-dependent mechanism and we assume by analogy, although not having investigated this point in detail, that in the system presented here this mechanism is also important. The discrepancies between the previous study (29) and our report are probably due to the timely limited availability of loaded Ag in a B cell transfer approach, as maintenance of peripheral CD8 T cell tolerance depends on continuous presence of the tolerizing Ag (24, 47, 48). We have previously demonstrated that the CD19-driven B cell-specific retroviral approach leads to long-term Ag expression in reconstituted mice (33). Therefore, using a stem cell-based approach rather than transfer of ex vivo peptide-loaded or transduced B cells, continuous Ag expression can be achieved, imposing and maintaining tolerance, which is the result of combined peripheral deletion and induction of T cell paralysis. Why are B cells good targets for tolerizing approaches? A large proportion of developing B cells undergo apoptosis during development in bone marrow and spleen. Only 5–10% of all B cells are selected into the long-lived pool with a half-life of weeks to

months (31). In turn, DC efficiently acquire apoptotic material (e.g., from B cells) for cross-presentation (25, 26), which is important for tolerance induction in the steady state (49, 50). Hence, it seems to be very likely that DC cross-present B cell-derived Ag in bone marrow and spleen. Indeed, we could demonstrate efficient cross-presentation of B cell-derived OVA by purified DC (Fig. 1). It is therefore possible that Ag uptake and presentation could occur already in the bone marrow, as this primary hemopoietic organ was shown to possess the micromilieu necessary for T cell priming and proliferation (51–53). We excluded that unspecific Ag expression from the retroviral vector and consecutive direct presentation in DC could account for the observed tolerance induction in CD8 T cells (Fig. 1). Although Ag was undetectable in B cells and serum of chimeric mice by highly sensitive biochemical methods (Western blot, ELISA, data not shown) as well as flow cytometry, B cells efficiently presented retrovirally encoded Ag (Fig. 1). Although soluble as well as membrane-bound Ag has access to the endoplasmic reticulum lumen in DC (54), cross-presentation of soluble Ag has been reported to be less efficient than direct presentation (55). In line with this, deletion of OT-1 T cells was more efficient in TfrOVA chimeras as compared with sOVA chimeras, indicating that indirect tolerization by DC might be involved in this system. Thymic-negative selection is a central mechanism for tolerance induction in developing thymocytes. Using targeted expression of MHC class II molecules to DC, we could previously show that transgene-expressing thymic DC are sufficient to delete self-specific CD4 T cells (56). In addition, thymic DC have been shown to take up ectopically expressed self-Ag from medullary thymic epithelial cells to induce thymic tolerance in CD4 and CD8 thymocytes (41). In contrast, targeting transgenic MHC class II to thymic B cells was only partially sufficient to delete I-E-reactive CD4 thymocytes, but not CD8 T cells (57). Also, other studies described B cells as inefficient inducers of thymic-negative selection of CD4 T cells (58). Although it was demonstrated that thymic DC are not

The Journal of Immunology necessary to induce central tolerance in CD8 thymocytes if epithelial cells could present Ag (41), it is unclear at the moment, why OVA-producing thymic B cells could not provide thymic DC with Ag in a similar way as epithelial cells to delete CD8 T cells (Fig. 3). Peripheral deletional tolerance is characterized by T cell proliferation without increase in T cell numbers (46). Proliferation of OT-1 T cells without clonal expansion was observed in both OVAexpressing Rac1 and NTL chimeras (Fig. 6, a and b). In addition, the remaining OT-1 T cells were similarly unable to produce IFN-␥ in types of recipient (Fig. 6b). These data argue for the capacity of B cells to directly tolerize CD8⫹ T cells. In conclusion, we think that transcriptional targeting of Ag to developing B cells takes advantage of apoptosis induction during B cell development, leading to efficient uptake and DC-mediated cross-presentation of apoptotic material from dying B cells. Therefore, targeting Ag to B cells will also exploit the professional APC capacities of DC for tolerance induction. Although cross-presentation by DC might be sufficient to induce tolerance to certain Ag, additional direct tolerization by B cells might play an important role when particular protein epitopes cannot be crosspresented efficiently, as reported for epitopes within protein signal sequences (59, 60). Several gene therapeutic approaches for induction of tolerance have been studied previously (61– 66). However, the desired tolerizing effects were obtained by using conventional retroviral vectors. This leads to broad and nonspecific transgene expression and could therefore be disadvantageous and dangerous due to viral enhancer activities (67). For example, retrovirus vector integration in the proximity of a proto-oncogene-promoter induced uncontrolled exponential clonal proliferation of T cells in a high percentage of gene-therapy patients (68). The observed premalignant T cell proliferative disorder was most likely driven by retroviral enhancer activity on endogenous cellular promoters, eventually leading to leukemia (69). Such danger could be minimized with retroviral vectors devoid of viral enhancers and focusing gene expression to a restricted, but functionally relevant, cell type by using an appropriate eukaryotic promoter. We now provide strong evidence that such approaches are a potent means to induce and maintain Ag-specific CD8 T cell tolerance and may be of clinical relevance for therapeutic application in transplantation or autoimmunity where CD8 T cell tolerance is required to limit tissue pathology.

Acknowledgments We thank Dirk Busch for providing H-2Kb/OVA257–264 tetramers, Reinhard Obst, and Susan King for critically reading the manuscript, and A. Bol and W. Mertl for excellent care of mice.

Disclosures The authors have no financial conflict of interest.

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