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The Journal of Immunology, 2008, 180, 3601-3611
Copyright © 2008 by The American Association of Immunologists, Inc.
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Redirecting Therapeutic T Cells against Myelin-Specific T Lymphocytes Using a Humanized Myelin Basic Protein-HLA-DR2-{zeta} Chimeric Receptor1

Ioana Moisini*,{ddagger}, Phuong Nguyen*, Lars Fugger{dagger} and Terrence L. Geiger2,*,{ddagger}

* Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, 38105; {dagger} Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; and {ddagger} University of Tennessee Health Sciences Center, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84–102 epitope to HLA-DR2 and either incorporate or lack a TCR{zeta} signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The {zeta} signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic ({alpha}, β, green fluorescent protein) retroviral expression system. MBP-DR2-{zeta}-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84–102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-{zeta} chimeric receptors as a potential therapeutic in MS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Current therapies for multiple sclerosis and other autoimmune diseases are often limited by significant toxicities (1, 2). Regimens that selectively tolerize the autoreactive lymphocytes that orchestrate disease would be expected to have improved therapeutic indices and have been the subject of considerable efforts (3, 4). Various approaches to prevent or treat disease through the administration of tolerizing forms of Ag have been or are being tested in clinical trials, including the administration of oral Ag, altered peptide ligands, and Ag-coupled PBLs (reviewed in Ref. 5). Cellular immunotherapy with T lymphocytes may be a particularly promising alternative modality for Ag-specific immune therapy. T lymphocytes have a variety of immunomodulatory properties that may be therapeutically diverted. Trafficking of therapeutic T cells may mimic that of pathologic T lymphocytes. Furthermore, T lymphocytes have already been successfully used in the adoptive immunotherapy of infectious diseases and cancer (6, 7, 8).

Redirecting therapeutic T cells specifically against autoreactive T lymphocytes, however, represents a significant challenge. We and others have developed methods to redirect T cells using engineered TCR surrogates that link an extracellular ligand recognition domain with a cytoplasmic signaling tail (9, 10, 11, 12, 13). The extracellular domain, often a single chain Fv, binds a ligand on the targeted cell. The signaling domain, commonly a portion of the TCR complex such as the TCR-{zeta} cytoplasmic domain, activates the T lymphocyte after ligand engagement. These surrogate TCR may redirect receptor-modified T cells (RMTC)3 against tumors or other targets. Preclinical studies have confirmed that RMTC are functional. Phase I clinical trials have examined their toxicity in patients with HIV and cancer (14, 15, 16, 17). No significant toxicity has been described.

We have examined whether RMTC can similarly be redirected against autoantigen-specific T lymphocytes (10). To do this, we designed chimeric receptors that genetically link autoantigenic peptide, MHC, and TCR-{zeta} or other signaling domains. The MHC Ag serves as bait that specifically recognizes the TCR of autoantigen-specific T lymphocytes. Cognate TCR recognition cross-links the chimeric receptor, activating the RMTC through the receptor’s signaling domains.

In the initial studies, we used a T-lineage-specific promoter to express in transgenic (Tg) mice a chimeric receptor that included the immunodominant epitope of MBP in SJL mice linked to its restricting H-2As MHC and the signaling domain of TCR-{zeta} (18). MBP-specific T lymphocytes stimulated these Tg RMTC, inducing effector functions. CD8+, Th2, or CD4+CD25+ Tg RMTC showed therapeutic activity in experimental allergic encephalomyelitis (EAE), even after the dissemination of T cell responses through epitope spread (19, 20). These data supported the application of RMTC immunotherapy in autoimmune diseases.

Genetic studies have associated HLA-DR2 (DRA*0101/DRB1*1501) with susceptibility to MS in patients of European descent (21, 22, 23, 24). Furthermore, reactivity to a specific epitope, MBP84–102, restricted to the DR2 molecule has been well characterized in patients with MS (25). The relevance of this epitope to clinical disease is suggested by a clinical trial in which administration of an altered peptide ligand derived from it exacerbated MS in several patients (26). In this study, we took advantage of this established association to design two chimeric receptors specific for DR2-restricted, MBP84–102-specific T cells. Each heterodimeric receptor includes an extracellular and transmembrane (TM) domain comprised of HLA-DR2, with MBP84–102 peptide genetically linked to the β-chain (see Fig. 1). The MBP peptide folds into the HLA molecule’s peptide-binding groove, forming an antigenic complex recognizable by MBP-specific TCR. One receptor pair incorporates the cytoplasmic TCR-{zeta} signaling domain on both the {alpha}- and β-chains. The second receptor pair lacks these signaling chains, allowing us to define the role of receptor signaling in therapeutic cell function, which we were unable to do with the Tg cells. The receptors were placed in a murine stem cell virus (MSCV)-based retroviral vector to allow us to determine whether T lymphocytes transduced with tricistronic ({alpha}, β receptor, GFP) retrovirus are redirected against pathologic T lymphocytes and able to modulate autoimmune disease in a humanized model system.


Figure 1
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  		FIGURE 1. Structure and sequence of chimeric receptors. A, Structure and sequence of the MBP-DR2-{zeta} chimeric receptor. Full amino acid sequence is shown. Numbers in the left column indicate amino acid number. The construct was generated by linking segments from the HLA DRB1*1501 leader, MBP84–102, a linker peptide, HLA DRB1*1501 extracellular and TM domains, TCR-{zeta}, a T. asigna 2A self-cleaving peptidase sequence, HLA DRA*0101, and a synthetic TCR-{zeta}. These segments are indicated by arrows above the sequence. Cotranslational cleavage of the 2A sequence leads to the generation of two peptides, a β-chain comprised of MBP84–102, HLA-DRB1*1501, and TCR-{zeta}, and a separate {alpha}-chain comprised of HLA DRA*0101 and TCR-{zeta}. The location of restrictions sites inserted for cloning the segments together are shown, as is the location of the Kozak translation initiation site and stop codon (*). Nucleotide sequence information is provided only for DNA sequences altered during cloning. References to sequence information not shown can be found at the following GenBank accessions: HLA-DRB1*1501, NM_002124; hMBP, NM_002385; DRA*0101, NM_019111; and TCR-{zeta} NM_000734. The T. asigna 2A sequence has been reported previously (27 ). B, Structure and sequence of the MBP-DR2 chimeric receptor. The first and last four amino acids of each of the designated segments are shown. Sequence is identical with to of the MBP-DR2-{zeta} construct, except for the absence of the TCR-{zeta} segments.

 
We found that retrovirally transduced chimeric receptors are expressed at functional levels on RMTC and are able to mediate specific recognition of cognate MBP-DR2-specific T cells. Cognate T cell recognition mediates the activation of RMTC that express the signaling-capable MBP-DR2-{zeta} receptor, but not those expressing the {zeta}-deficient MBP-DR2 receptor. MBP-DR2-{zeta} RMTC are further able to eliminate Ag-specific T cells in vitro and in vivo and ameliorate EAE. These results provide support for the clinical application of RMTC in autoimmunity and specifically validate MBP-DR2-{zeta} RMTC as a potential cellular therapeutic for MS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Constructs

Constructs are shown in Fig. 1. mRNA was extracted from MGAR cells (HLA DRA*0101, DRB1*1501 homozygous, EBV-transformed B lymphocytes), cDNA prepared, and HLA cDNA sequence amplified by PCR. Fragments containing the DRB1*1501 leader, DRB1*1501 and DRA*0101 extracellular and TM domains, and human (h) TCR-{zeta} cytoplasmic domain (plasmid provided by D. Campana, St. Jude Children’s Research Hospital, Memphis, TN) were generated by amplifying cDNA with PCR using synthetic oligonucleotides specific for the desired sequence. Flanking restriction sites indicated in Fig. 1 were incorporated into the PCR oligonucleotides. The human MBP84–102 and linker sequence and the Thosea asigna 2A sequence (27) were generated using synthetic oligonucleotides. The sequence of the oligonucleotide containing the T. asigna 2A region overlapped that of the DRB1*1501 fragment and was linked to DRB1*1501 by PCR. Intermediate fragments were subcloned into the pBS-KS vector (Stratagene), sequenced, ligated together, and inserted into the MSCV-I-GFP vector (provided by E. Vanin, St. Jude Children’s Research Hospital, Memphis, TN).

Mice, cells, and Abs

DR2 (HLA DRA*0101, DRB1*1501) and Ob TCR (hTCR specific for MBP84–102/DR2) Tg mice have been described previously (28) and were backcrossed for >10 generations with C57BL/6 mice (The Jackson Laboratory) before in vivo analyses. Ob T hybridoma cells express the same MBP84–102-specific TCR as the Ob TCR Tg mice. 6F11 T hybridoma cells bear the same peptide specificity, although are restricted to H-2As (18). 4G4 T hybridoma cells are derived from BW5147 cells and have all invariant TCR chains although they are {alpha}β deficient (29) (gift from C. Janeway, New Haven, CT). EL4 thymoma cells were obtained from Dr. M. Blackman (Lake Saranac, NY). PE-conjugated anti-human TCR Vβ2 Abs were obtained from Immunotech. All other Abs were obtained from BD Pharmingen.

Retrovirus production and T cell culture

Ten micrograms of the MBP-DR2 or MBP-DR2-{zeta} chimeric receptor constructs and 10 µg of retrovirus helper DNA constructs were cotransfected into 293T cells by calcium phosphate precipitation. At 16 h, the cells were washed and cultured in DMEM/10% FCS for 48 h. Supernatant was collected twice daily and used to infect GP+E86 retroviral producer cells in the presence of 8 µg/ml polybrene (hexadimethrine bromide; Sigma-Aldrich). Transduced GP+E86 cells were flow cytometrically sorted for the presence of GFP and expanded, and supernatant was harvested. Logarithmically growing {alpha}β TCR-deficient 4G4 hybridoma or EL4 thymoma cells were transduced by the addition of viral supernatant and 8 µg/ml polybrene on 2 consecutive days. To transduce primary mouse T cells, isolated C57BL/6 lymph node cells or splenocytes were stimulated with anti-CD3{epsilon} and anti-CD28 Abs (BD Pharmingen) or 2 µg/ml Con A in the presence of 10 U/ml recombinant human IL-2 (rhIL-2; National Cancer Institute Biological Resources Branch, Bethesda, MD) for 24 h. Retroviral supernatant and 8 µg/ml polybrene was added, and the cells were centrifuged for 90 min at 660 x g in a Jouan CR422 tabletop centrifuge. The procedure was repeated at 48 h. Cells were sorted for expression of CD8 and GFP and expanded in 6-well plates by culturing in Eagle’s-Hanks’ amino acid medium (BioSource International) supplemented with 10% FCS, 100 U/ml penicillin G, 100 µg/ml streptomycin, 292 µg/ml L-glutamine (Invitrogen Life Technologies), and 50 µM 2-ME (Fisher Biotech) in the presence of 10 U/ml rhIL-2 for 5 days. The cells were restimulated every 7–10 days using 2 µg/ml Con A, 2 x 106/ml 3000-rad irradiated syngeneic splenocytes, and rhIL-2 for a maximum of two cycles. Transduced cells were washed and assayed 4–6 days after stimulation, unless otherwise indicated. Assays were performed in the absence of exogenously added IL-2.

Western blot analysis

Cells were thrice washed with ice-cold PBS and equal numbers of cells were lysed in 1% Brij 35 solution (Sigma-Aldrich) containing Protein Inhibitor Cocktail (Sigma-Aldrich) for 1 h at room temperature. Samples were centrifuged at 14,000 rpm in a microcentrifuge for 10 min and supernatant was collected. SDS-PAGE-loading buffer containing 2-ME was added to the cell lysate and protein derived from 5 x 106 cells/lane was separated by 12% SDS-PAGE, blotted, and probed with mouse anti-human TCR-{zeta} (clone 1D4; BD Pharmingen). Blots were washed twice with 0.1% Tween 20 in PBS, incubated with anti-mouse HRP-conjugated secondary Ab (catalog no. NA931V; Amersham Biosciences), washed again, and developed using chemiluminescence as per the manufacturer’s instructions (Amersham Biosciences).

Primary cell lines

CD4+ and CD8+ double Tg Ob TCR/DR2 T cells were flow cytometrically sorted and stimulated with 20,000-rad irradiated MBP-DR2-transduced EL4 cells and 3000-rad irradiated syngeneic splenocytes in medium with 20 U/ml rhIL-2. Stimulation was repeated every 10 days for a total of three cycles before experimentation. CD4 or CD8 purity in the expanded populations was confirmed by flow cytometry and, where needed, cells were resorted before analysis.

Cytokine analysis

Purified RMTC or control T cells were cocultured in a 96-well plate with irradiated (20,000 rad) Ob or 6F11 target cells. Alternatively, wells were precoated with goat anti-mouse IgG Ab for 3 h at 37°C and then washed; 10 µg/ml or no anti-HLA-DR2 Ab was subsequently added to the wells for 1 h at 37°C. The wells were again washed and transduced 4G4 cells were added in complete medium. Supernatant was harvested after 24 h and IL-2 and/or IFN-{gamma} were measured by Bio-Plex according to the manufacturer’s protocol (Bio-Rad).

Cytotoxicity assay

To assay cytotoxicity against hybridomas, target cells were labeled with 51Cr and washed three times with HBSS. RMTC or control effector cells were added at the indicated ratio to 105 target cells in round-bottom 96-well plates and cultured for 4–6 h in Eagle-Hanks’ amino acid/10% FCS. Maximal release wells had 0.1 M HCl added, whereas background wells had no effector cells added. Cell-free supernatant was harvested and released 51Cr was measured with a scintillation counter. Percent specific cytotoxicity was calculated as 100 x (cpm released – background cpm/maximal cpm – background cpm).

To assay cytotoxicity against primary T cell lines, 105 hMBP/DR2-reactive T cells were added to effector cells at the indicated ratio and cultured as above. After 6 h, the cells were stained with hVβ2, CD4, and CD8-specific Ab, washed once, 2000 Trucount beads (BD Biosciences) added, and viable cell counts determined by quantitative flow cytometry. Numbers of positive CD4+hVβ2+ or CD8+hVβ2+ cells were normalized to the Trucount bead event number to determine the total number of cells present in each well, thereby permitting quantitative comparison of event numbers between samples.

Proliferation assays

To assay the T cell response to MBP-DR2-transduced cells, 5 x 104 Ob TCR/DR2 responder cells were cocultured with different ratios of 20,000 rad irradiated, MBP-DR2-transduced target cells, pulsed with 1 µCi of [3H]thymidine at 72 h, and harvested onto filter mats (Wallac-PerkinElmer) after an additional 16 h. Proliferation was measured by liquid scintillation counting of 3H. Samples were analyzed in duplicates and means were plotted.

To assay the proliferation/expansion of primary Ob TCR/DR2 T cells to Ag in the presence of RMTC, freshly isolated splenocytes or lymph node (LN) cells from Ob TCR/DR2 mice were stimulated with 20,000 rad irradiated, Ag-expressing EL4-MBP-DR2 cells or control EL4 cells. Different quantities of RMTC or controls were added to the cultures to determine whether they could eliminate the MBP-specific responders. In some experiments, mice were treated in vivo with RMTC or control cells, and responder cells were then isolated and stimulated with Ag. After 5 days, the cells were stained with hVβ2-, CD4-, and CD8-specific Ab, 2000 Trucount beads (BD Biosciences) were added, and viable cell counts were determined by quantitative flow cytometry as above.

EAE induction and adoptive immunotherapy

To induce EAE, mice were injected s.c. in two sites at the tail base with a total of 300 µg of hMBP84–102 (St. Jude Hartwell Center, Memphis, TN) emulsified in 100 µl of CFA with 5 mg/ml Mycobacterium tuberculosis strain H37Ra (Difco). On the day of immunization and 48 h later, 400 ng of Bordetella pertussis toxin (List Biological Laboratories) was administered retro-orbitally. Mice were evaluated daily and disease was scored as: 1, flaccid tail; 2, hind limb weakness/paresis; 3, hind limb paralysis; 4, hind limb paralysis and body/forelimb paresis/paralysis; and 5, moribund or dead. For adoptive immunotherapy experiments, the indicated number of RMTC or control cells was adoptively transferred retro-orbitally 1–2 days before the induction of EAE. Transfer of cells through the retro-orbital venous plexus is an alternative to tail vein i.v. transfer. Although little data are available on differences between the techniques, in one study in which human T cells transduced with a luciferase-containing retrovirus were transferred into NOD severe-combined immunodeficient mice, retro-orbital injection resulted in improved engraftment compared with tail vein injection, with decreased cell trapping in the lungs and increased xenogeneic graft-versus-host disease development (30).

Statistics

Data are representative of at least three independent experiments except where otherwise indicated. SDs were calculated with Excel spreadsheet software (Microsoft). Mean values ± 1 SD are plotted where indicated. Area under the curve analysis was calculated with Excel spreadsheet software for EAE clinical disease from the time point disease was first detected in any mice until the end of the experiment. A trapezoidal estimation was used, with a calculation of {Sigma}(yn(tn + 1 – tn) + 1/2(yn + 1 – yn)(tn + 1– tn)) over all time points (tn) and disease scores (yn) measured. The Mann-Whitney U test was used to compare disease populations using a web-based Mann-Whitney calculator (http://elegans.swmed.edu/~leon/stats/ U test.html). Two-sided p values are reported with a p < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Design and expression of chimeric receptors

The humanized MBP-DR2-{zeta} and MBP-DR2 retroviral constructs are each tricistronic (Fig. 1). The β-chain is comprised of sequence encoding the HLA DRB1*1501 leader, MBP84–102 peptide, a linker peptide, and the DRB1*1501 extracellular and TM domains, either with (MBP-DR2-{zeta}) or without (MBP-DR2) the TCR-{zeta} cytoplasmic domain. The {alpha}-chain is comprised of the HLADRA*0101 leader, extracellular, and TM domains, similarly with or without a linked TCR-{zeta} cytoplasmic domain. The β- and {alpha}-chains are separated by the T. asigna virus 2A sequence (27). The 2A sequence is a 20-aa self-cleaving peptidase that cotranslationally releases the nascent chimeric receptor β-chain polypeptide from the ribosome while allowing read-through translation of the linked {alpha}-chain mRNA. This permits near stoichiometric production of the {alpha}- and β-chain chimeric receptor polypeptides. A linked internal ribosomal entry site-GFP in the constructs allows flow cytometric identification of transduced GFP+ cells. The chimeric receptors were each subcloned into the MSCV-based retroviral vector MSCV-I-GFP (31). To prevent viral recombination during retrovirus production due to the presence of two TCR-{zeta} sequences in the MBP-DR2-{zeta} construct, a synthetic human {zeta}-chain lacking nucleotide homology with the native sequence was incorporated into the {alpha} receptor chain.

Chimeric receptor or control vector containing retroviruses were transduced into the {alpha}β TCR-deficient T cell hybridoma cell line 4G4 or primary mouse CD8+ T lymphocytes. Transduction efficiencies of 5–50% were routinely observed for primary T cells and higher efficiencies for hybridoma cells. The chimeric construct’s internal ribosomal entry site-linked GFP permitted rapid fluorescent identification of transduced cells, which were flow cytometrically purified (primary T cells only). The cells were expanded, stained with an HLA-DR2-specific Ab, and analyzed by flow cytometry (Fig. 2, A and B). Receptors were well expressed on the cell surface, with expression levels proportional to GFP expression.


Figure 2
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  		FIGURE 2. Expression of chimeric receptor constructs. Control vector, MBP-DR2, or MBP-DR2-{zeta} constructs were transduced into 4G4 cells (A) or primary CD8 T cells (B). The cells were left unstained or stained with a chimeric receptor-specific Ab (anti-HLA-DR2) and analyzed by flow cytometry. GFP expression is displayed on the abscissa and chimeric receptor expression on the ordinate. C, Western blot analysis of MBP-DR2-{zeta} chimeric receptor. Primary mouse CD8+ T cells were transduced with MBP-DR2-{zeta}, or control MBP-DR2 or vector-containing retrovirus and expanded in culture. Protein from 5 x 106 cells was separated by SDS-PAGE per lane and blotted with Ab specific for human TCR-{zeta}.

 
The molecular masses of HLA class II {alpha}- and β-chains are ~33–35 and ~28–30 kDa, respectively (32). The additional MBP-linker, {zeta}, and 2A segments will add ~18 kDa to the construct β-chain and ~13 kDa to the {alpha}-chain, and we therefore anticipated that the {alpha}- and β-chain would each be ~46–48 kDa. Western blot analysis of MBP-DR2-{zeta}-transduced murine CTL with human {zeta}-specific Ab confirmed this. A doublet of anticipated size for the chimeric heterodimer was observed in cells transduced with the MBP-DR2-{zeta} construct (Fig. 2C). A single, less intense band of ~33 kDa also specifically stained with Ab, potentially representing a protein degradation product. As expected, protein from control MBP-DR2- or vector-transduced cells failed to stain with the anti-human TCR-{zeta} reagent. Cumulatively, these results demonstrate that the {alpha}- and β-chains of the chimeric receptor heterodimers are appropriately translated and expressed on the cell surface.

Ag presentation and RMTC stimulation by hMBP-DR2 chimeric receptors

To determine whether the extracellular MBP-DR2 domain of the chimeric receptors could present Ag, MBP-DR2-transduced EL4 (Fig. 3) or 4G4 cells (data not shown) were flow cytometrically purified, irradiated, and used to stimulate HLA-DR2-restricted, hMBP84–102-specific, TCR Tg T cells from Ob TCR/DR2 Tg mice. Prior analyses of the Ob TCR/DR2 double Tg mice demonstrated that most of their T cells expressed the Tg hVβ2 TCR-β chain; however, relatively few of the cells (~0.5%) were MBP specific in ELISPOT analyses (28). The poor allelic exclusion of these cells was also evident by the lack of detectable skewing of the T cells into the CD4 or CD8 lineage (data not shown). Therefore, we specifically analyzed the response of sorted populations of both CD4+ and CD8+ Ob TCR/DR2 T cells.


Figure 3
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  		FIGURE 3. Recognition and stimulation of hMBP/DR2-specific T cells by chimeric receptor. The MBP-DR2 chimeric receptor or retroviral vector was transduced into the B7+ EL4 thymoma cell line and GFP+ cells were purified by flow cytometry. The cells were irradiated and either cell type was used at a 1:1 ratio to stimulate flow-cytometrically purified CD4+ (A) or CD8+ (B) Ob TCR/DR2 Tg T lymphocytes. Alternatively, the responder T cells were stimulated with irradiated DR2 Tg splenocytes with or without 40 µg/ml MBP84–102 peptide. Proliferation was measured at 72 h by [3H]thymidine incorporation.

 
Interestingly, the MBP-DR2-transduced cells potently stimulated proliferation by both CD4 and CD8 populations of Tg T cells (Fig. 3, A and B). In contrast, vector-transduced control EL4 cells failed to stimulate the cells. Proliferation of Ob TCR/DR2 Tg T cells to the EL4 cells transduced with chimeric construct was vigorous, and the EL4-MBP-DR2 cells proved a superior stimulus to irradiated splenocyte APCs from DR2 Tg mice mixed with MBP84–102 peptide (Fig. 3). Thus, Ob TCR/DR2 Tg mice contain both CD4 and CD8 hMBP84–102/DR2-restricted T cells, each of which can engage the chimeric receptor. The coreceptor independence of the Ob TCR/DR2 T cells is somewhat surprising, although it may be explained by recent structural analyses of the Ob TCR bound to MBP/DR2, which revealed a unique binding register that partially blocks simultaneous coreceptor binding (33).

To determine whether the {zeta} signaling tail of the MBP-DR2-{zeta} chimeric receptor could signal into the RMTC, 105 purified transduced 4G4 cells were stimulated with plate-bound anti-HLA-DR2 Ab in 96-well plates and IL-2 production was measured 24 h later. MBP-DR2-{zeta}-transduced cells produced IL-2 in response to Ab stimulation (13,403 ± 546 pg/ml), but no detectable cytokine (<5 pg/ml) in the absence of Ab stimulation. Control vector or MBP-DR2-transduced cells failed to produce detectable levels of cytokine in the presence or absence of Ab stimulation. Therefore, the MBP-DR2-{zeta} chimeric receptor functionally signals into RMTC.

We next analyzed whether the engagement of the MBP-DR2 extracellular domain of the chimeric receptor by TCR on cognate Ag-specific T cells could also stimulate the RMTC. We cocultured CD8+ RMTC with irradiated MBP84–102/DR2-specific Ob T hybridoma cells or control cells. The Ag-specific Ob T cells induced strong proliferation of the MBP-DR2-{zeta} RMTC (Fig. 4, A and B). The RMTC failed to proliferate in response to control 6F11 hybridomas, which recognize the same peptide restricted by the mouse H-2As MHC molecule. In contrast to the MBP-DR2-{zeta} RMTC, MBP-DR2 or control vector-transduced cells did not respond to either stimuli. Similar results were obtained when IFN-{gamma} was analyzed as a measure of stimulation. Cytokine production required both the presence of cognate TCR on the T hybridomas as well as RMTC expressing chimeric receptor that included a functional signal transduction domain (Fig. 4, C and D). Therefore, the MBP-DR2-{zeta} chimeric receptor acts a surrogate TCR on the RMTC. The RMTC functionally engage cognate Ag-specific TCR and this engagement stimulates the RMTC to proliferate and secrete cytokines.


Figure 4
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  		FIGURE 4. Proliferation and cytokine production by RMTC in response to cognate MBP-specific T lymphocytes. Irradiated MBP/DR2-specific Ob hybridomas (A) or control MBP/IAs-specific 6F11 hybridomas (B) were cocultured with MBP-DR2-{zeta}, MBP-DR2, or vector-transduced CD8+ RMTC. Proliferation of the RMTC in response to the hybridomas was measured at 72 h by [3H]thymidine incorporation. Similar culture conditions were used to assess IFN-{gamma} production by the RMTC (C and D). Supernatants were harvested at 48 h for cytokine analysis.

 
Specific cytolysis of MBP84–102/DR2-reactive T lymphocytes by RMTC

To test the ability of the RMTC to specifically target and kill Ag-specific T cells, we cocultured purified CD8+ RMTC with 51Cr-labeled Ob or control 6F11 hybridomas. 51Cr release was measured 4 h later as an indicator of cell death. MBP-DR2-{zeta}-modified CTL potently killed the cognate Ag-specific T cells. Indeed, ~90% specific cytolysis was achieved at an E:T ratio of 5:1 and ~75% at a ratio of 1:1 (Fig. 5). In contrast, the MBP-DR2-{zeta} CTL were inactive against control 6F11 targets, and signaling-deficient MBP-DR2-modified cells showed no specific activity against either of the targets. Therefore, a retrovirally transduced, signaling-capable chimeric receptor can redirect cytolysis against Ag-specific T hybridomas.


Figure 5
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  		FIGURE 5. Cytolysis of MBP/DR2-specific T hybridomas by RMTC. MBP/DR2-specific Ob or control MBP/As-restricted 6F11 hybridoma cells were labeled with 51Cr and cocultured at the indicated E:T ratio for 4 h with MBP-DR2-{zeta} or MBP-DR2 RMTC. Supernatant was harvested and released 51Cr was measured by scintillation counting to determine specific cytotoxicity.

 
In vitro cytolysis of mouse DR2/TCR-specific T cell lines

To evaluate the response of the RMTC against primary MBP/DR2-specific T cells, we established MBP-specific T cell lines from the Tg mice. Purified CD4+ or CD8+ Ob TCR/DR2 cells were stimulated for three cycles with Ag, and the cell lines were repurified for CD4 or CD8 expression by flow cytometric sorting before analysis. To determine whether the RMTC could kill these lines, we cocultured them with RMTC for 6 h and measured the number of residual hVβ2+ Tg cells using quantitative flow cytometry. MBP-DR2-{zeta} RMTC showed potent activity against the CD4+ MBP-specific cell lines, whereas the MBP-DR2 and vector-transduced cells showed little or no activity (Fig. 6A). Thus, RMTC are able to kill CD4+ T cell lines, and, as for Ag-specific hybridomas, this activity requires chimeric receptor signaling.


Figure 6
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  		FIGURE 6. Cytolysis of MBP/DR2-specific T cell lines by RMTC. Ob TCR/DR2 Tg T cells were flow-cytometrically sorted into CD4+ and CD8+ populations, stimulated with Ag for three cycles, and resorted for CD4 or CD8 to produce hMBP/DR2-specific T cell lines. The CD4 (A) or CD8 (B) lines were cocultured for 6 h with MBP-DR2-{zeta}, MBP-DR2, or vector-transduced RMTC at the indicated E:T ratio. Residual CD4+hVβ2+ (A) or CD8+ hVβ2+ (B) target cell numbers were measured by quantitative flow cytometry. Postculture target cell counts are plotted.

 
In contrast to the CD4+ hMBP-specific T cell line, the MBP-DR2-{zeta} RMTC were unable to significantly kill the CD8+ cell line (Fig. 6B). This was not due to lysis of the RMTC by the CD8+ MBP-specific Ob TCR/DR2 cell line, since the number of RMTC present was also not significantly altered during the culture (data not shown). This shows that the MHC class II-restricted Ob TCR/DR2 CTL are resistant to the lytic activity of the RMTC.

This result was not wholly unexpected. Pre-CTL are highly susceptible to fratricidal lysis. However, after stimulation, as the CD8+ T cells mature into CTL, they develop resistance to cytolysis (34). This results from the a number of mechanisms including poor perforin binding to the CTL membrane, expression of cell surface cathepsins that degrade perforin, altered expression of bcl-2 family members, and expression of granzyme-inhibitory serpins (35, 36, 37, 38, 39). The Ob TCR/DR2 CTL, like other CTL, would be anticipated to develop this resistance to lysis, protecting them from the lytic activity of the RMTC.

In vitro cytolysis of primary mouse DR2/TCR-specific T cells

We were next interested in determining whether the RMTC could also lyse freshly isolated MBP/DR2-specific T cells. Because of the low frequency of hVβ2+ Ag-specific T cells in Ob TCR/DR2 mice, limited to no cytolysis could be detected in cytolysis assays using freshly isolated T cells (data not shown). We therefore developed a functional assay to specifically assess for the presence of residual Ag-specific cells after RMTC treatment.

In preliminary studies, virtually all Ob TCR/DR2 Tg T cells cultured in the absence of Ag stimulation died by days 4 to 5 of culture. In contrast, in the presence of Ag, a subset of hVβ2+ cells survived and proliferated. To determine whether residual Ag-specific primary cells survived the RMTC, we therefore cocultured RMTC and Ob TCR/DR2 cells in the presence of Ag and 5 days later enumerated viable CD4+ or CD8+hVβ2+ target T cells by quantitative flow cytometry. If the Ag-reactive Tg cells were lysed by the RMTC, surviving hVβ2+ cells should not be detected after Ag stimulation. In contrast, if the RMTC failed to lyse Ag-specific cells, the Ag-specific cells should then proliferate and expand.

As expected, in control cultures of Ob TCR/DR2 and vector-transduced cells, expansion of the CD4+hVβ2+ cells was detected at day 5 in cultures containing Ag, but few cells were detected in the absence of Ag (Fig. 7A). This was true regardless of the E:T ratio tested. In contrast, few CD4+hVβ2+ cells were detected in cultures to which the MBP-DR2-{zeta} RMTC were added, and the number of cells present was not substantially different whether Ag was present or not. This implies that the RMTC killed the primary T cells, since T cells responsive to Ag were not detected. Cultures containing the MBP-DR2-modified RMTC demonstrated a distinct pattern. Expansion of the CD4+hVβ2+ population was observed regardless of the addition of exogenous Ag. Therefore, these RMTC, which express hMBP-DR2 Ag through their signaling-deficient chimeric receptor, provide an effective antigenic stimulus to the Ob TCR/DR2 T cells, causing the Tg cells to expand even in the absence of an alternative source of Ag.


Figure 7
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  		FIGURE 7. Cytolysis of freshly isolated MBP/DR2-specific T cells by RMTC. Ob TCR/DR2 Tg splenocytes were cocultured with MBP-DR2-{zeta}, MBP-DR2, or vector-transduced RMTC at the indicated E:T ratios in 96-well plates. Some wells were simultaneously stimulated with Ag (irradiated EL4-MBP-DR2 cells) to expand the Ag-specific Tg T cells. Others were left unstimulated (control irradiated EL4 cells added). After 5 days, numbers of CD4+ (A) or CD8+ (B) hVβ2+ target T cells were measured by quantitative flow cytometry and are plotted.

 
Similar results were observed with the CD8+hVβ2+ population of target cells. The loss of MBP-responsive CD8+ T cells was only observed with the addition of the MBP-DR2-{zeta} RMTC (Fig. 7B). This demonstrates that these RMTC are able to kill freshly isolated MHC class II-restricted CD8+ targets, but not, as shown above (Fig. 6B), CD8+ cell lines.

In vivo cytolysis by RMTC

Similar approaches were used to evaluate the ability of the RMTC to eliminate Ag-specific T cells in vivo. In brief, 10 x 106 freshly isolated TCR Tg splenocytes and LN cells (~50% T cells) from Ob TCR/DR2 mice and 30 x 106 MBP-DR2-{zeta}, MBP-DR2, or vector-transduced RMTC were adoptively transferred through contralateral retro-orbital sites into sublethally irradiated syngeneic recipients. After 2 days, spleens and LN were harvested and proliferation to Ag was measured either by [3H]thymidine incorporation (day3; Fig. 8, A and B) or by quantitative flow cytometry (day 5; Fig. 8, C and D). Results resembled our observations with the in vitro system. Cells from MBP-DR2-{zeta} RMTC-treated mice showed little response to Ag by 3H incorporation and had low numbers of residual CD4+ or CD8+hVβ2+ T cells that responded to Ag. As expected, both CD4 and CD8 T cells from the vector-control-treated animals responded well to Ag in both assays. Cells from the signaling-deficient MBP-DR2 RMTC also responded to Ag, indicating that these RMTC were unable to eliminate the primary Ag-specific T cells in vivo. These results demonstrate that the RMTC can eliminate Ag-specific T cells in vivo. They further show that signaling through the {zeta} cytoplasmic tail is essential for this activity.


Figure 8
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  		FIGURE 8. Targeting of MBP/DR2-specific T cells by RMTC in vivo. To assess the ability of RMTC to eliminate Ag-reactive T cells in vivo, 107 TCR Tg splenocytes were transferred retro-orbitally into sublethally irradiated DR2 Tg mice. Three x 107 RMTC or control cells were transferred through the contralateral retro-orbital site into each of two mice per treatment regimen. After 2 days, LN cells (A) and spleens (B) were harvested, 105 cells were cultured per well in a 96-well plate, and proliferation to Ag was measured at 72 h by [3H]thymidine incorporation. Additional MBP-stimulated or unstimulated splenocyte cultures were analyzed at day 5 by quantitative flow cytometry for absolute numbers of CD4+hVβ2+ (C) or CD8+hVβ2+ T cells (D).

 
Prevention of EAE with RMTC

Considering that the MBP-DR2-{zeta} RMTC could eliminate both CD4 and CD8 naive MBP-specific T cells, pretreatment of mice with these RMTC should be able to ameliorate clinical EAE. To determine the impact of the RMTC on autoimmune disease, 3 x 107 flow cytometrically purified RMTC or control cells were directly transferred into Ob TCR/DR2 double Tg mice and EAE was induced 1–2 days later. Clinical disease was subsequently followed. In three of three experiments, the MBP-DR2-{zeta} RMTC substantially reduced or prevented EAE disease (Fig. 9A and Table I). Mean time to disease was longer in mice receiving the MBP-DR2-{zeta} RMTC compared with control cells (15.1 ± 1.5 vs 8.8 ± 1.5 d). Peak disease score and average daily disease was also diminished. Further mortality in the RMTC-treated animals was reduced from 75 to 0%. In contrast to the MBP-DR2-{zeta} RMTC, the MBP-DR2 RMTC had more mild, nevertheless significant, effects when compared with vector-control cell-treated animals.


Figure 9
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  		FIGURE 9. EAE blockade mediated by RMTC. A, Three x 107 RMTC or control cells were transferred into Ob TCR/DR2 Tg mice (experiment 1, Table I). After 2 days, the mice were immunized with hMBP84–102 peptide to induce EAE. Clinical disease was monitored. B, Experiment performed as in A, except MBP-As RMTC were compared with vector-transduced control cells. C, CD4+ T cells were flow-cytometrically purified from Ob TCR/DR2 Tg mice. In brief, 1.3 x 107 cells were injected per mouse retro-orbitally and 3 x 107 RMTC or vector-transduced control cells were injected through the contralateral retro-orbital plexus. EAE was induced 2 days later and clinical disease was monitored.

 

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  		Table I. EAE blockade mediated by RMTCa

 
We have previously developed a receptor analogous to the MBP-DR2 receptor in which a similar core fragment of MBP is linked to H-2As (MBP-As) (18). As a further control, we assessed whether MBP-As RMTC could prevent EAE development in this model system. In contrast to the MBP-DR2-transduced RMTC, these MBP-As RMTC did not show a beneficial effect (Fig. 9B and Table I; p > 0.05). Cumulatively, these data show that transferred RMTC are able to alleviate clinical disease and that disease modulation is dependent on cognate recognition of the chimeric receptor, though not wholly dependent on chimeric receptor signaling.

Because the Ob TCR/DR2 mice have a substantial CD8+, MBP-specific/DR2-restricted T cell response, a response not anticipated to be substantial in patients with MS, we also tested in a single pilot experiment whether the RMTC would be active against a pure population of CD4 T cells. CD4+ T cells were flow cytometrically sorted from Ob TCR/DR2 double Tg mice and 1.3 x 107 transferred into sublethally (450 rad) irradiated DR2 Tg recipients. Approximately 3 x 107 MBP-DR2-{zeta} or vector-transduced RMTC were then transferred and EAE was induced. Severe disease was observed in mice receiving control vector-transduced RMTC. In contrast, mice treated with the MBP-DR2-{zeta} RMTC were fully protected from disease development (Fig. 9C and Table I). Thus, the RMTC are specifically active against CD4+ effector cells, blocking their ability to induce autoimmune disease.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report several new findings supporting the development of an Ag-specific cellular therapy for autoimmune disease. Specifically, retrovirally driven chimeric Ag-MHC-{zeta} receptor heterodimers are expressed at functional levels on T lymphocytes and can be readily detected through direct staining or cotranscribed GFP. When transduced into CTL, receptors incorporating a functional signaling tail specifically respond to cognate Ag-specific T lymphocytes, promoting RMTC proliferation, cytokine production, and target cell cytolysis. Chimeric receptors lacking a {zeta} signaling tail are ineffective in this regard, though do engage cognate Ag-specific TCR, thereby stimulating target T cells.

Using a humanized mouse model of MS, we detected both CD4+ and CD8+ MHC class II-restricted, MBP-specific T cells. We demonstrate that our RMTC can target either of these cell lineages when the target cells are naïve; however, CD8+ T cells that have been stimulated and matured into CTL are resistant to the lytic activities of the RMTC. Therefore, in this specific model system, the RMTC target cognate T cells in a coreceptor-independent manner, although effective cytolysis is dependent on target cell susceptibility to lysis. In adoptive transfer models, the signaling-competent RMTC were able to eliminate Ag-specific T lymphocytes and were able to block EAE disease. Interestingly, RMTC expressing a chimeric receptor lacking the {zeta} signaling tail also ameliorated disease, but were less effective than cells expressing a receptor with the signaling chain.

Previous studies of RMTC redirected against T cells have not analyzed signaling-deficient receptors (18, 19, 20, 34, 40) and activity of these cells was unexpected. One possibility was that the MBP peptide Ag was released from the chimeric receptors and induced tolerance independently of its context on RMTC, as free peptide has been shown to induce tolerance in other systems (41). This would seem unlikely. The quantity of free peptide released from MBP-DR2-bearing RMTC would be expected to be low. Furthermore, in contrast to the MBP-DR2 RMTC, MBP-As RMTC were unable to inhibit EAE development. More extensive studies will be needed to determine how the MBP-DR2 RMTC, which lack the {zeta} signaling tail, modulated disease. Although these cells showed no activity against naive Ag-specific CD4+ T cells in vivo (Fig. 8), in preliminary studies they were capable of eliminating adoptively transferred CD4+ (but not CD8+) T cell lines in vivo (I. Moisini and T. Geiger, unpublished data), suggesting that stimulation of cell lines through the chimeric receptor of the CD8+ MBP-DR2 RMTC promotes a veto-like effect, inducing the death of target cells. Overall, our data support the feasibility of producing RMTC in adequate quantity and purity for immunotherapy, demonstrate that these cells can identify and tolerize cognate Ag-specific T cells in vivo, and suggest an important although nonobligate role for the chimeric receptor signaling domain in the immunotherapeutic effect.

The optimal format for applying RMTC will require careful consideration. Paramount is the type of effector cell the chimeric receptor is expressed on. In the current study, therapeutic CTL were used. CD8+ T cells are present in large quantities, easy to transduce, and grow more readily in culture than other T cell types, thus remaining pure with progressive stimulation cycles. However, although CTL grow rapidly, they also die rapidly when depleted of sustaining promitiotic and antiapoptotic cytokines. Indeed, studies of adoptively transferred CTL suggest that the majority of effector cells die within several days after transfer (42). This may lead to concerns as to whether Ag presented by chimeric receptors on dead or dying cells may be stimulatory or transferred to stimulatory APCs, thereby exacerbating rather than ameliorating autoimmunity. We have never observed exacerbation of EAE with RMTC treatment, suggesting that the tolerogenic effect of the transduced cells exceeds any immunogenic effects that may result from Ag introduced through the chimeric receptor. Nevertheless, potential immunogenicity of chimeric receptors is a concern that will need to be addressed as this and other Ag-specific approaches to induce tolerance are tested.

Alternative effector cell types may also be used to counteract pathologic T cells. Adoptive immunotherapy with forkhead box p3-positive (Foxp3+) regulatory T lymphocytes (Treg) has generated a significant amount of interest because these cells appear to uniformly be immunomodulatory and would therefore be unlikely to have untoward effects due to the production of proinflammatory or mixed function biological response modifiers (43). Indeed, we have demonstrated that Tg CD4+CD25+ MBP-H-2 As-{zeta} RMTC from chimeric receptor Tg mice are robust suppressors of EAE disease in the SJL model system, and that the chimeric receptor enhances the potency of Treg by at least 10-fold (20, 40). Other studies have shown similarly increased potency when the suppressive activity of Treg-expressing Ag-specific TCR are compared with nonspecific Treg (44). However, acquiring quantities of Treg sufficient for therapeutic purposes has posed a significant challenge and, in our hands as those of others, even a small contaminating population of Foxp3– nonregulatory cells will rapidly overgrow these cells in culture.

As an alternative, we and others have examined whether therapeutic Treg may be generated ex vivo. Stimulation of naive T cells in the presence of IL-2 and TGF-β up-regulates Foxp3 and promotes immunoregulatory function (45, 46, 47). This should permit the generation of large numbers of regulatory cells. In preliminary work, we have demonstrated the feasibility of retrovirally transducing these "induced" regulatory T cells. Induced Treg have also been able to ameliorate immunopathologic responses in several models (48, 49, 50). However, these cells remain incompletely characterized and their functional similarity with natural regulatory T cells is unknown.

A second feature of RMTC that must be better defined is the structure of the chimeric receptor. We demonstrate that signaling is important to RMTC-mediated inhibition of EAE, although not an absolute requirement. The signaling domain is required for redirected cytolysis and RMTC activation. Various permutations of the signaling domain have been tested. Our current studies assayed a domain that only included regions of TCR-{zeta}. We demonstrated that it is possible to link signaling regions from multiple proteins in tandem to generate receptors with enhanced signaling properties (51). Indeed, in T hybridomas, signaling regions incorporating TCR, coreceptor, and costimulatory domains had the highest sensitivity and strongest response to ligand. Similarly, CD28-{zeta} tandem signaling domains were shown by several groups to enhance RMTC function and survival in vivo when compared with {zeta}-only signaling domains (52, 53, 54). We have produced a heterodimeric chimeric receptor including hMBP-DRB1*1501-CD28-{zeta} and DRA*0101. In preliminary in vitro studies, however, we have not detected enhanced activity compared with the {zeta}-{zeta} signaling domain described here (I. Moisini and T. Geiger, unpublished observations).

The generation of RMTC for clinical trials would require specific ex vivo modification of autologous cells in good tissue practice conditions and the facilities and mechanisms to do this are in place in a number of centers, including our own. Indeed, many stem cell transplant regimens require the selection and, in some cases, genetic modification and/or outgrowth of specific cell populations. Because the HLA types of patients with MS and several other autoimmune diseases are skewed and because in previous studies we have shown that RMTC directed against a single Ag may be used to treat disease in the context of a complex autoreactive repertoire, small numbers of receptor constructs may be useful to treat a large fraction of diseased patients. RMTC immunotherapy is already being or has been tested for activity in patients with other disease conditions, particularly cancer (14, 16). Although efficacy has yet to be demonstrated, clinical trials remain at early stages with only phase I trial results published. Therefore, RMTC immunotherapy represents a viable approach with potential as a treatment of autoimmune conditions. Our results provide support for the further clinical translation of this therapeutic modality.


   Acknowledgments
 
We thank Richard Cross, Jennifer Smith, and Yuxia He for assistance with flow cytometric cell sorting and Divya Mekala, Rajshekhar Alli, Christine Duthoit, Ramesh Selvaraj, and Meredith Steeves for technical support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 National Institutes of Health Grant R01 AI056153 (to T.L.G.) and by American Lebanese Syrian Associated Charities/St. Jude Children’s Research Hospital (to I.M., P.N., and T.L.G.). Back

2 Address correspondence and reprint requests to Dr. Terrence L. Geiger, Department of Pathology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, D-4047, Memphis, TN 38105. E-mail address: terrence.geiger{at}stjude.org Back

3 Abbreviations used in this paper: RMTC, receptor-modified T cell; MBP, myelin basic protein; MS, multiple sclerosis; Tg, transgenic; h, human; rh, recombinant human; EAE, experimental allergic encephalomyelitis; TM, transmembrane; Treg, regulatory T lymphocyte; Foxp3, forkhead box protein P3; AUC, area under the curve; NA, not applicable; MSCV, murine stem cell virus. Back

Received for publication September 18, 2007. Accepted for publication December 28, 2007.


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 Discussion
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