HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, J. Articles by Li, W. Search for Related Content PubMed PubMed Citation Articles by Chen, J. Articles by Li, W.

Recombinant Adenovirus Coexpressing Covalent Peptide/MHC Class II Complex and B7-1: In Vitro and In Vivo Activation of Myelin Basic Protein-Specific T Cells¹

Jiang Chen^*, Brigitte T. Huber, Richard J. Grand and Wei Li^2,*,

^* Division of Rheumatology and Immunology, Department of Pediatrics, and Center for Gastroenterology Research on Absorptive and Secretory Processes, Tufts University School of Medicine and New England Medical Center, and Immunology Program, Department of Pathology, Tufts University School of Medicine, Boston, MA 02111

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies have demonstrated that an MHC class II molecule with an antigenic peptide genetically fused to its -chain is capable of presenting this peptide to CD4⁺ T cells. We hypothesized that covalent peptide/class II complex may direct the accessory molecules to exert their function specifically onto T cells in a TCR-guided fashion. To test this hypothesis, we generated several recombinant adenoviruses expressing covalent myelin basic protein peptide/I-A^u complex (MBP_1–11/I-A^u) and the costimulatory molecule B7-1. Functional studies demonstrated that adenovirus-infected cells are capable of activating an MBP_1–11-specific T cell hybridoma. Coexpression of the B7-1 molecule and MBP_1–11/I-A^u by the same adenovirus leads to synergy in T cell activation elicited by virus-infected cells. Furthermore, studies in syngeneic mice infected with the various adenoviruses revealed that MBP_1–11-specific T cells are specifically activated by the coexpression of B7-1 and MBP_1–11/I-A^u in vivo. In conclusion, the coexpression of the covalent peptide/class II complex and accessory molecules by the same adenovirus provides a unique strategy to modulate the epitope-specific T cell response in a TCR-guided fashion. This approach may be applicable to investigate the roles of other accessory molecules in the engagement of the TCR class II molecule by substituting B7-1 with other accessory molecules in the recombinant adenovirus.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cells are activated by recognizing peptide fragments bound to the groove of MHC class I or II molecules. MHC molecules have the capacity to present a variety of peptide fragments to T cells (1). It was previously reported that the covalent peptide/MHC class II complex with the peptide genetically fused to its -chain activates specific CD4⁺ T cell populations (2, 3). These studies provide a new strategy for manipulating the CD4⁺ T cell response in an epitope-specific manner, different from conventional immunization.

The CD4⁺ T cell response to TCR ligation is subjected to regulation by several accessory molecules. In the absence of appropriate costimulation, Ag presentation to naive T cells in vivo results in a state of long-lasting Ag-specific unresponsiveness or anergy (4). One such costimulatory signal critical for T cell activation is provided by the members of the B7 family, B7-1 and B7-2 (5, 6). In contrast, the ligation of Fas/Fas ligand during Ag presentation may lead to TCR-mediated death of CD4⁺ T cells (7). Several other accessory molecules, such as CD30, CD27, CD40, TNF receptor type I and II, OX40, 4-1BB (CDw137), etc., were also reported to be expressed by CD4⁺ T cells and may play roles in the T cell response (7, 8, 9, 10, 11, 12).

CD4⁺ T cells have been implicated to play an important role in experimental autoimmune encephalomyelitis (EAE),³ an animal model for human multiple sclerosis with inflammation limited to the CNS white matter. EAE can be induced by myelin basic protein (MBP) (13, 14). The N-terminal aa 1–11 of MBP (MBP_1–11) have been demonstrated to reconstitute the pathogenic epitope in H-2^u mice (15). This epitope in the context of I-A^u is recognized by the T cell hybridoma (THy) 1934.4 (16). Transgenic mice with T cells bearing the MBP-TCR have been generated and are known to develop EAE upon challenge with MBP_1–11 (17, 18, 19). Purified, soluble covalent MBP_1–11/I-A^u complexes have been demonstrated to activate the 1934.4 THy (20).

We hypothesized that coexpression of the covalent peptide/class II complex and accessory molecules on the surface of the same cells may direct the accessory molecules to exert their function selectively on the T cells engaged through TCR ligation with the peptide/class II complex in a guided fashion. We chose MBP_1–11 and I-A^u as well as B7-1 as model system to investigate this hypothesis in vitro and in vivo. This approach represents a novel strategy, which may potentially be valuable not only to investigate the role of other accessory molecules in TCR ligation, but also to modulate the host immune response in a TCR-guided and Ag-specific fashion. In this study several replication-deficient recombinant adenoviruses have been constructed to express up to four different recombinant proteins, including I-A^u, the MBP_1–11/I-A^u, B7-1, and green fluorescence protein (GFP) reporter. The expression of recombinant proteins in adenovirus-infected cells was characterized biochemically. Functional engagement between the covalent MBP_1–11/I-A^u complex and the TCR was defined using the MBP_1–11-specific 1934.4 THy. The mechanism for MBP_1–11-specific T cell activation in a TCR-guided fashion was elucidated in syngeneic mice infected with the adenovirus coexpressing the MBP_1–11/I-A^u complex and B7-1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and culture conditions

The murine 1934.4 THy (16), which is specific for MBP_1–11 in the context of I-A^u, was a gift from Dr. D. Wraith (University of Bristol, Bristol, U.K.). The 3A9 THy, which specifically recognizes hen egg lysozyme peptide 46–61 (HEL_46–61) in the context of I-A^k, was obtained from Dr. E. Unanue (Washington University School of Medicine, St. Louis, MO) (21). TA-3 cells (22) and M5/114.15.2 cells (23) were obtained from Drs. B. Toole and M. Stadecker (Tufts University, Boston, MA), respectively. The 293 cell line (24) and Y-3P hybridoma (25) were purchased from American Type Culture Collection (Manassas, VA). All the cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS, penicillin/streptomycin (100 U/ml), 2 mM glutamine, and 5 x 10^-5 M 2-ME.

Antibodies

Biotin-labeled anti-mouse B7-1 mAb 16-10A1 (26) was purchased from BD PharMingen (San Diego, CA). Y-3P mAb recognizing conformational epitope of the -chain of I-A^u and M5/114.15.2 mAb against I-A^b were purified from the culture medium of Y-3P and M5/114.15.2 cells by a protein G column (obtained from Sigma, St. Louis, MO) according to the protocol described previously (27). The mAb was eluted using 100 mM glycine (pH 2.5) and immediately neutralized to pH 7.4 with 1 M sodium phosphate (pH 8.0). CTLA-4/Fc was purchased from Chimerigen (Allston, MA).

Synthetic peptides

Synthesis of MBP_1–11[4Y] (AcASQYRPSQRHG) peptide and HEL_46–61 (NTDGSTDYGILQINSR) peptide was performed by the Protein Core Facility, Department of Physiology, Tufts University. Both peptides were purified by reverse phase HPLC. Purified peptides were subsequently confirmed by mass spectrometry.

Construction of MBP_1–11/I-A^u -chain

Full-length I-A^u was amplified from a cDNA clone of I-A^u (from Dr. D. Wraith) by PCR using I-A^u-F1 primer 5'-CGA AGC TTC GCC ACC ATG GCT CTG CAG ATC CCC AGC-3' and primer 5'-TCG CGG CCG CGA GTC ACT GCA GGA GCC CTG-3', and subcloned directly into pGEM-T Easy plasmid (Promega, Madison, WI) to yield plasmid A (I-A^u/pGEM-T). The extracellular portion of I-A^u with MBP_1–11 fused to the N terminus through a flexible linker was generated by PCR from the plasmid of pAcUW51^u/^u-peptide (from Dr. E. S. Ward, University of Texas Southwestern Medical Center, Dallas, TX) (20) using I-A^u-F1 primer and primer 5'-AGT ACT CGG CGT CTG GCC-3', digested with HindIII and BstEII, and inserted into the plasmid A at HindIII/BstEII sites to yield plasmid B (MBP_1–11/I-A^u/pGEM-T).

Construction of expression plasmids for I-A^u and MBP_1–11/I-A^u

Rous sarcoma virus (RSV) long terminal repeat promoter (P_RSV) was generated from pREP4 vector (Invitrogen, Carlsbad, CA) by PCR using primers 5'-TTG TCG ACA AAG CGG GGC TTC GGT TG-3' and 5'-CCA AGC TTG GAG GTG CAC ACC AAT G-3', digested with SalI and HindIII, and inserted into pE1sp1B plasmid (Microbix Biosystems, Toronto, Canada) at SalI/HindIII sites to yield plasmid C. Bovine growth hormone (BGH) polyadenylation sequence (PAS) was amplified by PCR from pIRES1neo plasmid (Clontech Laboratories, Palo Alto, CA) using primers 5'-AGA AGC TTC TCG AGC TAG AGC TCG CTG ATC AGC C-3' and 5'-GAA GAT CTT CGA GCC CCA GCT GGT TCT TT-3', digested with HindIII and BglII, and inserted into plasmid C at HindIII/BglII sites to yield plasmid D. SV40 PAS was generated from pEGFP-N1 plasmid (obtained from Clontech) by PCR using primer 5'-CCA AGC TTG GCA TGG ACG AGC TGT AC-3' and SV40-R1 primer 5'-TAC TCG AGG GAT CCT AAG ATA CAT TGA T-3', digested with HindIII and XhoI, and inserted into plasmid D at HindIII/XhoI sites to yield plasmid E. CMV promoter (P_CMV) was amplified from pEGFP-N1 plasmid by PCR using primers 5'-CCG GAT CCT AGT TAT TAA TAG TAA TC-3' and SV40-R1 primer, digested with BamHI and XhoI, and inserted into plasmid E at BamHI/XhoI sites to yield plasmid F (P_RSV.PAS(SV40).P_CMV.PAS(BGH)/pE1sp1B).

Full-length I-A^u coding sequence was amplified by RT-PCR using RNA extracts from PL-8 cells (28) (from Dr. E. S. Ward) with primers 5'-GCG CTA GCG CCA CCA TGC CGT GCA GCA GAG CTC TG-3' and 5'-TCC TCG AGG ACT CAT AAA GGC CCT GGG TGT-3', digested with NheI and XhoI, and inserted into plasmid F at NheI/XhoI sites to yield plasmid G (P_RSV.PAS(SV40).P_CMV.I-A^u.PAS(BGH)/pE1sp1B).

A full-length I-A^u or MBP_1–11/I-A^u fragment was extracted from plasmid A or B by digestion of HindIII and NotI and was ligated into plasmid F at HindIII/NotI sites to yield plasmid H or I. I-A^u.PAS(BGH) fragment was digested out of plasmid G by NheI and BglII, and inserted into plasmid H or I at NheI/BglII sites to yield plasmid J (P_RSV.I-A^u.PAS (SV40).P_CMV.I-A^u.PAS(BGH)/pE1sp1B) or K (P_RSV.MBP_1–11/I-A ^u.PAS(SV40).P_CMV.I-A^u.PAS(BGH)/pE1sp1B).

Construction of adenoviral shuttle plasmids with inserts in the E1 region

Before expression cassettes were inserted into pAdTrack plasmid (gift from Dr. T.-C. He, The Johns Hopkins University, Baltimore, MD) (29), two XbaI sites in the plasmid were destroyed as followed. pAdTrack prepared from DH5 Escherichia coli strain was digested with XbaI, blunted, and religated. The resulting plasmid prepared from dam^- JM110 E. coli strain (Stratagene, La Jolla, CA) was digested with XbaI, blunted, and religated to yield a new pAdTrack plasmid (plasmid L), free of XbaI site. The expression cassettes for I-A^u and MBP_1–11/I-A^u were extracted from plasmids J and K by the digestion of ClaI and BglII, blunted, and inserted into plasmid L at EcoRV site to yield adenoviral shuttle plasmid M (P_RSV.I-A^u.PAS(SV40).P_CMV.I-A^u.PAS(BGH)/pAdTrack) and plasmid N (P_RSV.MBP_1–11/I-A^u.PAS(SV40).P_CMV.I-A^u.PAS(BGH)/pAdTrack; see Fig. 1).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Constructs of recombinant adenoviruses. PCMV, human CMV immediate early promoter; PAS, polyadenylation sequences from SV40 small T Ag (SV40), BGH, or TK.

A control plasmid for the expression of B7-1 was also constructed by inserting blunted B7-1 coding region into plasmid F at the blunted HindIII/XhoI sites. P_RSV.B7-1.PAS(BGH) fragment was extracted by digestion of ClaI and BglII, blunted, and inserted into pAdTrack at blunted XbaI/BglII sites to yield shuttle plasmid T (P_RSV.B7-1.PAS(BGH)/pAdTrack). All of the inserts in the constructed plasmids were confirmed by DNA sequencing.

Generation of recombinant adenoviral plasmids

Recombinant adenoviral plasmids were generated by homologous recombination in E. coli according to a previously published method (29) with the following modifications. Briefly, shuttle plasmids (plasmids M, N, R, S, and T) (1 µg each) were linearized with PmeI, purified using a Qiagen PCR purification kit, and transfected together with 0.5 µg pAdEasy-1 vector into 50 µl electrocompetent E. coli BJ5183 cells by electroporation. Cells were incubated in 1 ml L-broth (LB) medium at 37°C for 30 min, inoculated onto LB-agar plates containing 25 µg/ml kanamycin, and cultured at 37°C overnight. Several of the smallest colonies were picked from the plates and grown in 1 ml LB medium containing 25 µg/ml kanamycin at 37°C overnight. Miniprep DNA of the different clones were analyzed on 0.5% agarose gels and compared with the size of pAdEasy-1 or the shuttle plasmids. Only the clones similar in size to pAdEasy-1 were collected and further confirmed by PCR analysis for the presence of the inserts using the above-listed primer pairs or E2 region using the primer pair 5'-TAT TTA CCC CCA CCC TTG CC-3' and 5'-CAC GGT CAC CTT TTG ATG CC-3'. Once confirmed, the adenoviral plasmids were transformed into DH10B cells and purified by Qiagen Plasmid Maxi kit.

Recombinant adenoviruses

Generation of recombinant adenoviruses. Replication-deficient recombinant adenoviruses were generated in the 293 cells according to previously published methods (29) with the following modifications. Briefly, adenoviral plasmids (2 µg) were linearized with PacI, precipitated with ethanol, resuspended in dH₂O, and transfected into 50–70% confluent 293 cells in 60-mm dishes using Lipofectamine Plus reagent (Life Technologies) according to the manufacturer’s protocol. Transfected cells were monitored for GFP expression. At 7–10 days post-transfection, most dishes had the comet-like adenovirus-producing foci with green fluorescence. Cells along with the culture medium were collected from these dishes and pelleted. All but 1 ml of the supernatant was removed and saved. The viruses were released into the medium with three cycles of freezing in an ethanol/dry ice bath, with rapid thawing at 37°C and vortexing between each cycle. The lysates were combined with the saved supernatants, mixed, and centrifuged. To amplify adenoviruses and increase their titers, the supernatants were used to infect the 293 cells at 70–90% confluence in a 100-mm dish. The presence of adenoviruses and infection efficiency were monitored with the expression of GFP. At 3–5 days postinfection, cells were harvested, and lysates were prepared as described above. These virus amplification processes were conducted once more in two 150-mm dishes, and these third-round lysates were ready for large scale adenovirus preparation.

Large scale adenovirus purification and characterization. The third-round lysates were added to 10–30 dishes (150 mm) of 293 cells at 70–90% confluence (0.5–1.0 ml lysate/dish). At 48–96 h postinfection, cells were harvested and pelleted. The cell pellets were resuspended in 10 ml 10 mM Tris-HCl buffer (pH 8.1). The lysates were prepared as described above by freezing and thawing. Adenoviruses were purified from the supernatant by two sequential CsCl gradient centrifugations as described previously (31). The CsCl bands containing adenoviruses were collected and desalted through Sephadex G-25 M columns (PD-10 column from Amersham Pharmacia Biotech, Piscataway, NJ) using an elution buffer of 10 mM Tris-HCl (pH 7.4), 1 mM MgCl₂, and 10% glycerol. The fractions of purified viruses were verified for the presence of the inserts and E2 region by PCR using the primer pairs described above. The absence of wild-type adenovirus contamination was confirmed by the negative results of PCR (up to 35 cycles) for the E1 region using the primers 5'-TGA GTG CCA GCG AGT AGA GTT TTC-3' and 5'-ATA CAG TTC GTG AAG GGT AGG TGG-3'. Purified viruses were aliquoted and stored at -80°C. The viral titer (PFU per milliliter) for each adenovirus preparation was determined in 293 cells using the agarose overlay method described previously (31). Each plaque was also verified for GFP expression under an inverted fluorescence microscope.

Immunofluorescence and Northern blot

TA-3 cells infected with or lacking adenoviruses were detached from culture plates and stained by indirect immunofluorescence for cell surface expression of either I-A^u using purified mAb Y-3P followed by an F(ab')₂ goat anti-mouse IgG-PE, or B7-1 using biotin-labeled anti-B7-1 mAb followed by streptavidin-PE. Cells were analyzed by BD Biosciences (Mountain View, CA) FACSCalibur flow cytometer.

For Northern blot analysis, TA-3 cells were infected with adenoviruses at 200 multiplicity of infection (MOI) for 24 h. Total RNA extraction and Northern blot analysis were performed as previously described (32).

Adenovirus infection of cultured cells and THy activation

To express I-A^u or MBP_1–11/I-A^u, TA-3 cells (2 x 10⁶ cells in a 100-mm dish with 10 ml culture medium) were infected with various purified recombinant adenoviruses at the indicated dose or MOI (PFU per cell) at 37°C for 24 h. Infected cells were harvested by pipetting and washed twice with culture medium before the T cell activation study.

Ag presentation assays were performed in 96-well round-bottom plates in a total volume of 200 µl/well complete DMEM in quadruplicate. Virus-infected TA-3 cells (5 x 10⁴ cells/well or as otherwise indicated) were cocultured with the 1934.3 THy (1 x 10⁵ cells/well) at 37°C for 24 h.

Samples of the supernatants were either frozen or assayed immediately for IL-2. IL-2 was measured by ELISA using IL-2 Ab pairs from Endogen (Cambridge, MA), following the instructions of the supplier. The average and SD for each experimental condition were determined, and statistical analysis was performed using paired t test by GraphPad Instat (San Diego, CA).

In vivo study

Recombinant adenoviruses or PBS were injected into syngeneic mice of either PL/J or B10.PL through the tail vein at 2 x 10⁹ PFU/mouse in 0.1 ml PBS. Ten days after virus infection, spleen cells were isolated, washed, plated in 96-well plates at 1 x 10⁵ cells/well and restimulated in quadruplicate with PBS, HEL_46–61 (10 µg/ml), MBP_1–11 (10 µg/ml), Ad.GFP (50 MOI), or Con A (2 mg/ml) for 48 h. [³H]thymidine (1 µCi/well) was added to the medium. Cells were cultured for an additional 16 h and collected with a microplate cell harvester. T cell proliferation was determined by [³H]thymidine incorporation using a Packard (Downers Grove, IL) microplate scintillation counter.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation and characterization of adenoviruses

Recombinant adenoviruses generated for this study are illustrated in Fig. 1. All the viruses, purified by two sequential CsCl gradient centrifugations, were free of wild-type adenovirus contamination, as evidenced by the negative results of E1 region PCR (data not shown). The titers of the various recombinant adenoviruses were determined by the plaque overlay assay. Interestingly, the yield and titer for the MHC class II⁺ adenoviruses were 2- to 10-fold lower than those for the class II^- adenoviruses. Furthermore, the expression level of GFP by class II⁺ adenoviruses in the 293 cells during virus propagation was also relatively lower than that by class II^- adenoviruses, as judged by GFP intensity (data not shown).

The presence of all expression elements, including promoters, coding sequences and polyadenylation sequences, in purified adenoviruses was confirmed by PCR using various primer pairs (data not shown). Northern blot analysis (Fig. 2) using RNA extracts from TA-3 cells infected with purified viruses revealed high levels of I-A^u and I-A^u transcripts for Ad.MBP_1–11/I-A^u, Ad.MBP_1–11/I-A^u.B7-1, Ad.I-A^u, and Ad.I-A^u.B7-1, and a high level of B7-1 transcripts for Ad.B7-1, Ad.MBP_1–11/I-A^u.B7-1, and Ad.I-A^u.B7-1.

View larger version (67K): [in this window] [in a new window]   FIGURE 2. Northern blot analysis. TA-3 cells were infected with indicated adenoviruses at 200 MOI for 24 h. Total RNA extracts (5 µg/lane) from the infected TA-3 cells were analyzed by Northern blot using various cDNA probes. A, I-Au; B, I-Au; C, B7-1.

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Flow cytometric analysis. TA-3 cells were infected with indicated adenoviruses at 200 MOI for 24 h. Cells were stained with either anti-I-Au Y-3P mAb or biotin-labeled anti-B7-1 mAb as described in Materials and Methods. No Ab control represents secondary Ab-fluorescent conjugate staining without primary Ab.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Time course and dose response of THy activation. A, Time course of IL-2 production by THy activated by adenovirus-infected TA-3 cells. TA-3 cells were infected with Ad.MBP1–11/I-Au.B7-1 at 200 MOI for 24 h, plated in 96-well plate at 5 x 104 cells/well, and incubated with 1934.4 THy for the appropriate time as indicated. B, Dose-dependent activation of THy by adenovirus-infected TA-3 cells. The indicated amount of TA-3 cells, preinfected with Ad.MBP1–11/I-Au.B7-1 at 200 MOI for 24 h, was incubated with 1934.4 THy. C, THy activation by TA-3 cells infected with different MOI of the adenovirus. TA-3 cells (5 x 104 cells/well), preinfected with Ad.MBP1–11/I-Au.B7-1 at the indicated MOI for 24 h, were incubated with 1934.4 THy. The inset in C is the percentage of GFP+ TA-3 cells infected with Ad.MBP1–11/I-Au.B7-1 at different MOI for 24 h. The percentage of GFP+ cells was determined by flow cytometry. In all panels, 1934.4 THy were plated at 1 x 105 cells/well in quadruplicate and stimulated for 24 h before the IL-2 assay.

The sequence encoding peptide MBP_1–11[4Y], in which the lysine at position 4 was substituted by tyrosine, was fused to the 5' end of the I-A^u cDNA as previously described (20). This substitution was reported to increase the peptide affinity for I-A^u (34, 35), but does not appear to affect T cell recognition (36). The 1934.4 THy, specifically recognizing MBP_1–11 in the context of I-A^u, was used to evaluate the efficiency of gene expression in infected cells. If I-A^u and MBP_1–11/I-A^u were expressed in TA-3 cells, assembled and displayed appropriately on the cell surface, TA-3 cells would be able to present the antigenic peptide to T cells and activate them. Among all the recombinant viruses studied, Ad.MBP_1–11/I-A^u demonstrated some capability to stimulate the THy (Fig. 4), while Ad.MBP_1–11/I-A^u.B7-1-infected TA-3 cells showed strong stimulation. In contrast, cells infected with Ad.B7-1, Ad.I-A^u, or Ad.I-A^u.B7-1 did not have any stimulatory effect on this 1934.4 THy.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. THy stimulation assay with adenovirus-infected TA-3 cells. TA-3 cells were infected with the indicated adenoviruses at 200 MOI for 24 h. Infected TA-3 cells were detached from plate, washed twice with medium, replated in 96-well plate at 5 x 104 cells/well, and incubated with either 1934.4 or 3A9 THy (1 x 105 cells/well). Twenty-four hours later, culture supernatants were analyzed for IL-2 levels by ELISA. The results are the representative of four independent experiments, conducted in quadruplicate.

Expression of I-A^u on cells infected with Ad.I-A^u was verified by the activation of 1934.4 THy in the presence of MBP_1–11 or HEL_46–61 peptide. The addition of MBP_1–11, but not HEL_46–61, to TA-3 cells infected with Ad.I-A^u or Ad.I-A^u.B7-1 fully restored the capacity of these infected TA-3 cells to stimulate 1934.4 THy, and similar levels of IL-2 were produced as with cells infected with Ad.MBP_1–11/I-A^u or Ad.MBP_1–11/I-A^u.B7-1, respectively (Fig. 5). These results proved that I-A^u, expressed by TA-3 cells infected with the recombinant adenoviruses (Ad.I-A^u and Ad.I-A^u.B7-1), was processed and displayed on the cell surface in appropriate conformation. However, the addition of MBP_1–11 did not further enhance the activation of 1934.4 THy by TA-3 cells infected with Ad.MBP_1–11/I-A^u or Ad.MBP_1–11/I-A^u.B7-1, suggesting that the Ag binding groove was fully occupied by the fused MBP_1–11 peptide. Furthermore, the expression of B7-1 on the surface of TA-3 cells infected with Ad.I-A^u.B7-1 or Ad.MBP_1–11/I-A^u.B7-1 was revealed by the enhancement of 1934.4 THy activation (Fig. 5).

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Functional expression of I-Au and MBP1–11/I-Au on the surface of TA-3 cells infected with various adenoviruses. TA-3 cells were infected with the indicated adenoviruses, washed and plated in 96-well plates at 5 x 104 cells/well, and incubated with 1934.4 THy (1 x 105 cells/well) as described in Fig. 5. MBP1–11 or HEL46–61 peptide at a final concentration of 100 µg/ml was added at the beginning of the incubation, as indicated. Twenty-four hours later, culture supernatants were analyzed for IL-2 levels by ELISA.

The activation of 1934.4 THy by TA-3 cells infected with Ad.MBP_1–11/I-A^u or Ad.MBP_1–11/I-A^u.B7-1 was dependent on the Ag-specific interaction between MHC class II and TCR. This was proven by the inability of virus-infected TA-3 cells to activate another THy, 3A9, which recognizes HEL_46–61 in the context of I-A^k (Fig. 4). These results showed that the T cell activation was highly specific, as there was no cross-recognition of the peptide-MHC class II complex (Fig. 4). Furthermore, the activation of 1934.4 THy by TA-3 cells infected with Ad.MBP_1–11/I-A^u.B7-1 was blocked by mAb Y-3P, which recognizes I-A^u, but not by mAb M5/114.15.2, which recognizes I-A^b (Fig. 6A), suggesting that Ag presentation to 1934.4 THy depends on the availability of the appropriate conformation of I-A^u. The functional role of B7-1 in the synergistic activation of T cells by adenovirus-infected cells was further revealed by blocking with CTLA-4/Fc (Fig. 6B). In the presence of CTLA-4/Fc, the activation of 1934.4 THy by TA-3 cells infected with Ad.MBP_1–11/I-A^u.B7-1 was reduced to the level seen with TA-3 cells infected with Ad.MBP_1–11/I-A^u, suggesting that B7-1 indeed contributes to the synergy in T cell activation in our system.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. A, THy activation by TA-3 cells infected with Ad.MBP1–11/I-Au.B7-1 was blocked by I-Au-specific mAb. TA-3 cells (5 x 104 cells) were infected with Ad.MBP1–11/I-Au.B7-1 at 200 MOI for 24 h, harvested, washed, preincubated with 10 µg of either purified Y-3P mAb or M5/114.15.2 mAb for 30 min at room temperature, pelleted, resuspended in culture medium, and plated in a 96-well plate. 1934.4 THy (1 x 105 cells/well) were incubated with the infected TA-3 cells for 24 h in the presence of 5 µg appropriate mAb. B, Enhancement of THy activation by B7-1 was blocked by the addition of CTLA-4. TA-3 cells (5 x 104 cells) were infected with indicated adenoviruses at 200 MOI for 24 h, harvested, washed, preincubated with either PBS or CTLA-4/Fc (2 µg/ml) for 30 min at room temperature, pelleted, and resuspended in culture medium in 96-well plates. 1934.4 THy (1 x 105 cells/well) were incubated with the infected TA-3 cells for 24 h in the presence of either PBS or 2 µg/ml CTLA-4/Fc. The results are representative of four independent experiments conducted in quadruplicate.

TA-3 cells infected with Ad.MBP1–11/I-Au.B7-1 stimulated 1934.4 THy in a dose-dependent manner (Fig. 7B). Infected TA-3 cells plated at 5 x 10⁴ or 1 x 10⁵ cells/well resulted in the most effective activation of 1934.4 THy. A higher number of infected TA-3 cells yielded less T cell activation, suggesting that the high density of TA-3 cells may have depleted nutrients in the culture medium necessary for IL-2 production by THy. It is possible, but less likely, that the high density of infected TA-3 cells induced T cell apoptosis through CTLA-4 or other mechanisms, resulting in the reduction of IL-2 production.

A similar dose-dependent response was observed for TA-3 cells infected with Ad.MBP_1–11/I-A^u.B7-1 at various MOIs (Fig. 7C). The most effective adenovirus dose to infect TA-3 cells was determined to be 200 MOI.

In vivo studies

To further define MBP_1–11-specific T cell activation in a TCR-guided fashion by our recombinant adenovirus, Ad.MBP_1–11/I-A^u.B7-1, along with other controls, was injected into H-2^u mice, either PL/J or B10.PL (male, 5–7 wk old, from The Jackson Laboratory). Ten days after virus infection, spleen cells were isolated from these mice, and T cell priming was analyzed, as described in Materials and Methods. T cells isolated from all the mice responded to the stimulation with Con A strongly and at a similar level, suggesting that T cells are functionally intact after the isolation procedure (data not shown). Our results indicate thatMBP_1–11-specific T cells were significantly activated in mice infected with Ad.MBP_1–11/I-A^u.B7-1 (p < 0.001; Fig. 8). However, Ad.MBP_1–11/I-A^u did not induce MBP_1–11-specific T cell activation in vivo, suggesting that naive T cell activation through TCR ligation requires costimulatory signals as previously described (7, 8, 9, 10, 11, 12). Furthermore, control viruses, Ad.I-A^u.B7-1 and Ad.B7-1, did not induce any MBP_1–11-specific T cell activation.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. In vivo T cell activation by recombinant adenovirus. H-2u mice, either PL/J or B10.PL, were infected with different adenoviruses, as described in Materials and Methods. Splenic T cells were isolated and restimulated by different reagents as indicated. T cell proliferation was determined by [3H]thymidine incorporation assay. The results are the typical representatives of three independent experiments for each strain of mice conducted in quadruplicate. A, PL/J mice; B, B10. PL mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we describe the expression and characterization of B7-1 and covalent MBP_1–11/I-A^u complexes by recombinant adenoviruses. We have successfully constructed replication-deficient recombinant adenoviruses, such as Ad.MBP_1–11/I-A^u.B7-1, that express up to four different proteins encoded in the adenoviral E1 region, including GFP. We have shown that MBP_1–11/I-A^u and I-A^u can be expressed, assembled, and displayed in appropriate conformation on the surface of nonprofessional APCs, as revealed by mAb Y-3P. Similarly, expression of B7-1 by recombinant adenoviruses was detected on the surface of infected TA-3 cells with anti-B7-1 mAb. Although the surface expression of MBP_1–11 fused to I-A^u cannot be confirmed by immunofluorescence due to the lack of specific mAb against the peptide, functional studies revealed that TA-3 cells infected with the adenoviruses expressing MBP_1–11/I-A^u are capable of activating 1934.4 THy. These data suggest that the MBP_1–11/I-A^u complex is expressed and displayed on the cell surface in appropriate conformation (Figs. 4 and 5). Similarly, cell surface expression of I-A^u was confirmed functionally by activation of 1934.4 THy in the presence of exogenous MBP_1–11 peptide (Fig. 5). Although previous studies had suggested that MHC class II is poorly expressed on the cell surface in transfected cell lines (33), our results reveal that recombinant adenovirus is capable of expressing and displaying MHC class II molecules on the surface of non-APC.

The activation of 1934.4 THy by TA-3 cells infected with adenovirus expressing MBP_1–11/I-A^u is highly specific, because this activation was not observed for the 3A9 THy, specific for HEL_46–61 in the context of I-A^k, and can be blocked with mAb Y-3P, but not by the control mAb M5/114.15.2 directed against I-A^b. Although the quantity of mAb Y-3P used in our study was similar to that used in the previous study (20), blocking of 1934.4 THy activation by the mAb was not as complete as previously reported, as some residual T cell activation was observed (Fig. 6A). This may be due to the differences in the expression system used by us or by Radu et al. (20). In our study, TA-3 cells infected with recombinant adenovirus continuously express and display the MBP_1–11/I-A^u complex on the cell surface during Ag presentation, eventually exhausting the limited amount of Y-3P mAb in the culture medium. In contrast, in the previous study (20) soluble MBP_1–11/I-A^u complexes were purified and adsorbed to the culture plate to activate 1934.4 THy. Once the MBP_1–11/I-A^u complex was neutralized by Y-3P mAb, inhibition of T cell activation was complete.

Another difference between the two systems includes the different patterns in the time course of T cell activation. In the previous study IL-2 production nearly reached a plateau within 4 h, while in our study IL-2 secretion was not detectable at 4 h and only reached a plateau 24 h later. The reason for this difference is not clear. It is possible that purified soluble MBP_1–11/I-A^u complex, bound to the plate in the previous study, was present at higher density than adenovirus-infected TA-3 cells in our system, leading to differences in T cell activation. Such a difference is supported by the fact that apoptosis was only induced in 1934.4 THy after activation by the soluble MBP_1–11/I-A^u complex (20), but not by the I-A^u-expressing cells pulsed with antigenic peptide (28). Furthermore, the coexpression of B7-1 in our study significantly enhanced the production of IL-2 by 1934.4 THy (Fig. 4), suggesting that our system is similar to the natural process of Ag presentation (5, 6).

TA-3 cells were selected for this study because they are highly susceptible to adenovirus infection, even with virus titers as low as 0.5–2 MOI (Fig. 7C, inset). More importantly, cells of the H-2^k background are unable to present MBP_1–11, although the induction of endogenous MHC class II molecules by adenovirus infection has not been reported previously.

Among the five adenoviruses studied, only two, Ad.MBP_1–11/I-A^u and Ad.MBP_1–11/I-A^u.B7-1, were capable of activating 1934.4 THy without the addition of MBP_1–11 peptide. However, the former only possessed a weak capacity for activating T cells. When the costimulatory B7-1 molecule was coexpressed by the same adenovirus, Ad.MBP_1–11/I-A^u.B7-1, T cell activation by the virus-infected cells was significantly boosted, indicating that the two-signal pathway is required in our system for optimal T cell activation (4, 5, 6). The enhancement of 1934.4 THy activation by TA-3 cells infected with the B7-1-expressing adenovirus was verified by CTLA-4 blockade (Fig. 6B). Our approach of expressing B7-1 and MBP_1–11/I-A^u by the same adenovirus virtually guarantees that the cells infected by the adenovirus express both molecules on the cell surface. Thus, this strategy leads to significantly enhanced efficiency to activate epitope-specific T cells in our animal studies.

One of the reasons we chose an adenoviral expression system in this study is that the adenoviral vector can achieve high efficient gene transfer in vivo, as previously reported (39). However, the chance of viral infection for APCs in vivo is far less than that for non-APCs. The cells most susceptible to adenovirus infection are hepatocytes, endothelial cells, and muscle cells, but not naive lymphocytes (39, 40). Although previous studies suggested that APCs in in vitro culture are susceptible to adenovirus infection (41), we have failed to infect MHC class II⁺ cells in vivo with Ad.GFP at 2 x 10⁹ PFU/mouse (unpublished data), suggesting that APCs are not susceptible to adenovirus infection in vivo. Therefore, it can be assumed that most cells infected by adenovirus in vivo are non-APCs. Once infected, these non-APCs will gain the capacity of presenting the covalent MBP_1–11/I-A^u complex to T cells, as demonstrated in TA-3 cells. Our in vivo experiments revealed that Ad.MBP_1–11/I-A^u cannot elicit MBP_1–11-specific T cell activation in H-2^u mice (Fig. 8), despite moderate activation of 1943.4 THy by TA-3 cells infected with the virus (Fig. 4), suggesting that naive T cell activation through TCR ligation requires costimulatory signals, as previously reported (4, 5, 6). In vivo MBP_1–11-specific T cell activation by Ad.MBP_1–11/I-A^u.B7-1 was derived from the synergistic effect of both MBP_1–11/I-A^u and B7-1 in signaling to the T cell, as proposed in Fig. 9, because the expression of either B7-1 or MBP_1–11/I-A^u alone cannot evoke an MBP_1–11-specific T cell response (Fig. 8). Lack of significant enhancement of the T cell response against adenoviral Ags by B7-1-expressing adenoviruses suggests that viral Ags be mainly presented to T cells by professional APCs through endocytosis, rather than by the virus-infected cells directly. The possible induction of EAE by Ad.MBP_1–11/I-A^u.B7-1 in EAE-susceptible H-2^u mice or MBP-TCR transgenic mice (17) is currently under investigation.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. Proposed mechanism for the TCR-guided, epitope-specific T cell activation by recombinant adenovirus coexpressing the covalent MBP1–11/I-Au complex and B7-1.

In summary, we have demonstrated that coexpression of the covalent MBP_1–11/I-A^u complex and B7-1 by adenovirus will direct B7-1 to exert its costimulation to MBP_1–11-specific T cells in a guided fashion. This novel approach may not only be applicable to investigate the roles of other accessory molecules in TCR-mediated T cell signaling, but may also be valuable to modulate host immune responses in an epitope-specific manner.

	Acknowledgments

 
We thank Drs. E. S. Ward, D. Wraith, T.-C. He, G. Freeman, B. Toole, M. Stadecker, and E. Unanue for cDNA clones and cell lines; Dr. A. Rosenzweig for adenovirus technique; and Drs. H. Wortis, N. Sutkowski, and A. Meyer for scientific discussion.

	Footnotes

 
¹ This work was supported by a New Investigator Award from New England Medical Center, a Charlton Award from Tufts University, Pediatric Gastroenterology Research Training Grant T32DK07471, a pilot research grant from Center for Gastroenterology Research on Absorptive and Secretory Processes (National Institutes of Health Digestive Disease Center P30DK34928), and the Lincoln Foundation.

² Address correspondence and reprint requests to Dr. Wei Li, Division of Rheumatology and Immunology, Box 406, New England Medical Center, 750 Washington Street, Boston, MA 02111. E-mail address: wli{at}lifespan.org

³ Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; BGH, bovine growth hormone; GFP, green fluorescence protein; HEL, hen egg lysozyme; LB, L-broth; MBP, myelin basic protein; MOI, multiplicity of infection; PAS, polyadenylation sequence; RSV, Rous sarcoma virus; P_RSV, RSV long terminal repeat promoter; THy, T cell hybridoma; TK, thymidine kinase.

Received for publication November 2, 2000. Accepted for publication May 25, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Hunt, D. F., H. Michel, T. A. Dickinson, J. Shabanowitz, A. L. Cox, K. Sakaguchi, E. Appella, H. M. Grey, A. Sette. 1992. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-A^d. Science 256:1817.[Abstract/Free Full Text]
2. Kozono, H., J. White, J. Clements, P. Marrack, J. Kappler. 1994. Production of soluble MHC class II proteins with covalently bound single peptides. Nature 369:151.[Medline]
3. Ignatowicz, L., G. Winslow, J. Bill, J. Kappler, P. Marrack. 1995. Cell surface expression of class II MHC proteins bound by a single peptide. J. Immunol. 154:3852.[Abstract]
4. Linsley, P. S., J. A. Ledbetter. 1993. The role of the CD28 receptor during T cell responses to antigen. Annu. Rev. Immunol. 11:191.[Medline]
5. Boussiotis, V. A., G. J. Freeman, J. G. Gribben, L. M. Nadler. 1996. The role of B7-1/B7-2:CD28/CLTA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune response. Immunol. Rev. 153:5.[Medline]
6. Sperling, A. I., J. A. Bluestone. 1996. The complexities of T-cell co-stimulation: CD28 and beyond. Immunol. Rev. 153:155.[Medline]
7. Krammer, P.. 1999. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv. Immunol. 71:163.[Medline]
8. Tarkowski, M.. 1999. Expression and function of CD30 on T lymphocytes. Arch. Immunol. Ther. Exp. 47:217.
9. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
10. Hintzen, R. Q., R. de Jong, S. M. Lens, R. A. van Lier. 1994. CD27: marker and mediator of T-cell activation?. Immunol. Today 15:307.[Medline]
11. Walker, L. S., A. Gulbranson-Judge, S. Flynn, T. Brocker, P. J. Lane. 2000. Co-stimulation and selection for T-cell help for germinal centres: the role of CD28 and OX40. Immunol. Today 21:333.[Medline]
12. Vinay, D. S., B. S. Kwon. 1998. Role of 4-1BB in immune responses. Semin. Immunol. 10:481.[Medline]
13. Zamvil, S. S., L. Steinman. 1990. The T lymphocyte in experimental allergic encephalomyelitis. Annu. Rev. Immunol. 8:579.[Medline]
14. Martin, R., H. F. McFarland, D. E. McFarlin. 1992. Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10:153.[Medline]
15. Zamvil, S. S., D. J. Mitchell, A. C. Moore, K. Kitamura, L. Steinman, J. B. Rothbard. 1986. T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis. Nature 324:258.[Medline]
16. Wraith, D. C., D. E. Smilek, D. J. Mitchell, L. Steinman, H. O. McDevitt. 1989. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunology. Cell 59:247.[Medline]
17. Goverman, J., A. Woods, L. Larson, L. P. Weiner, L. Hood, D. M. Zaller. 1993. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell 72:551.[Medline]
18. Pearson, C. I., W. van Ewijk, H. O. McDevitt. 1997. Induction of apoptosis and T helper 2 (Th2) responses correlates with peptide affinity for the major histocompatibility complex in self-reactive T cell receptor transgenic mice. J. Exp. Med. 185:583.[Abstract/Free Full Text]
19. Dittel, B. N., R. M. Merchant, Jr C. A. Janeway. 1999. Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis. J. Immunol. 162:6392.[Abstract/Free Full Text]
20. Radu, C. G., B. T. Ober, L. Colantonio, A. Qadri, E. S. Ward. 1998. Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases. J. Immunol. 160:5915.[Abstract/Free Full Text]
21. Allen, P. M., E. R. Unanue. 1984. Differential requirements for antigen processing by macrophages for lysozyme-specific T cell hybridomas. J. Immunol. 132:1077.[Medline]
22. Nagy, J. A., E. S. Morgan, K. T. Herzberg, E. J. Manseau, A. M. Dvorak, H. F. Dvorak. 1995. Pathogenesis of ascites tumor growth: angiogenesis, vascular remodeling, and stroma formation in the peritoneal lining. Cancer Res. 55:376.[Abstract/Free Full Text]
23. Bhattacharya, A., M. E. Dorf, T. A. Springer. 1981. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. 127:2488.[Abstract]
24. Graham, F. L., J. Smiley, W. C. Russell, R. Nairn. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36:59.[Abstract/Free Full Text]
25. Jr Janeway, C. A., P. J. Conrad, E. A. Lerner, J. Babich, P. Wettstein, D. B. Murphy. 1984. Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells. J. Immunol. 132:662.[Abstract]
26. Razi-Wolf, Z., G. J. Freeman, F. Galvin, B. Benacerraf, L. Nadler, H. Reiser. 1992. Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells. Proc. Natl. Acad. Sci. USA 89:4210.[Abstract/Free Full Text]
27. E. Harlow, and D. Lane, eds. Antibodies: A Laboratory Manual 1988511. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
28. Wraith, D. C., D. E. Smilek, S. Webb. 1992. MHC-binding peptides for immunotherapy of experimental autoimmune disease. J. Autoimmun. 5:(Suppl. A):103.
29. He, T. C., S. Zhou, L. T. da Costa, J. Yu, K. W. Kinzler, B. Vogelstein. 1998. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95:2509.[Abstract/Free Full Text]
30. Freeman, G. J., G. S. Gray, C. D. Gimmi, D. B. Lombard, L. J. Zhou, M. White, J. D. Fingeroth, J. G. Gribben, L. M. Nadler. 1991. Structure, expression, and T cell costimulatory activity of the murine homologue of the human B lymphocyte activation antigen B7. J. Exp. Med. 174:625.[Abstract/Free Full Text]
31. Graham, F. L., E. J. Prevec. 1991. Manipulation of adenovirus vectors. E. J. Murray, ed. Gene Transfer and Expression Protocols 109. Humana Press, Clifton, NJ.
32. Li, W., S. D. Krasinski, M. Verhave, R. K. Montgomery, R. J. Grand. 1998. Three distinct mRNA distribution patterns in human jejunal enterocytes. Gastroenterology 115:86.[Medline]
33. Cresswell, P.. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259.[Medline]
34. Fairchild, P. J., H. Pope, D. C. Wraith. 1996. The nature of cryptic epitopes within the self-antigen myelin basic protein. Int. Immunol. 8:1035.[Abstract/Free Full Text]
35. Fugger, L., J. Liang, A. Gautam, J. B. Rothbard, H. O. McDevitt. 1996. Quantitative analysis of peptides from myelin basic protein binding to the MHC class II protein, I-Au, which confers susceptibility to experimental allergic encephalomyelitis. Mol. Med. 2:181.[Medline]
36. Wraith, D. C., B. Bruun, P. J. Fairchild. 1992. Cross-reactive antigen recognition by an encephalitogenic T cell receptor: implications for T cell biology and autoimmunity. J. Immunol. 149:3765.[Abstract]
37. Yang, Y., S. E. Haecker, Q. Su, J. M. Wilson. 1996. Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle. Hum. Mol. Genet. 5:1703.[Abstract/Free Full Text]
38. Yang, Y., J. M. Wilson. 1996. CD40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD40. Science 273:1862.[Abstract/Free Full Text]
39. Hitt, M. M., C. L. Addison, H. L. Graham. 1997. Human adenovirus vectors for gene transfer into mammalian cells. Adv. Pharmacol. 40:137.
40. Huang, S., R. I. Endo, G. R. Nemerow. 1995. Upregulation of integrins _v3 and _v5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. J. Virol. 69:2257.[Abstract]
41. Zhang, H.-G., X. Su, D. Liu, W. Liu, P. Yang, Z. Wang, C. K. Edwards, H. Bluethmann, J. D. Mountz, T. Zhou. 1999. Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells. J. Immunol. 162:1423.[Abstract/Free Full Text]

This article has been cited by other articles:

				G. T. Gurda, L. Guo, S.-H. Lee, J. D. Molkentin, and J. A. Williams Cholecystokinin Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo Mol. Biol. Cell, January 1, 2008; 19(1): 198 - 206. [Abstract] [Full Text] [PDF]

				E. C. Carlson, M. Lin, C.-Y. Liu, W. W-Y. Kao, V. L. Perez, and E. Pearlman Keratocan and Lumican Regulate Neutrophil Infiltration and Corneal Clarity in Lipopolysaccharide-induced Keratitis by Direct Interaction with CXCL1 J. Biol. Chem., December 7, 2007; 282(49): 35502 - 35509. [Abstract] [Full Text] [PDF]

				J. Ouyang, Y.-C. Shen, L.-K. Yeh, W. Li, B. M. Coyle, C.-Y. Liu, and M. E. Fini Pax6 overexpression suppresses cell proliferation and retards the cell cycle in corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2397 - 2407. [Abstract] [Full Text] [PDF]

				T. Kawakita, E. M. Espana, H. He, R. Smiddy, J.-M. Parel, C.-Y. Liu, and S. C. G. Tseng Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-{beta} Signaling in a Low-Calcium, Serum-Free Medium Invest. Ophthalmol. Vis. Sci., May 1, 2006; 47(5): 1918 - 1927. [Abstract] [Full Text] [PDF]

				Q. Liu, F. Shang, E. Whitcomb, W. Guo, W. Li, and A. Taylor Ubiquitin-conjugating enzyme 3 delays human lens epithelial cells in metaphase. Invest. Ophthalmol. Vis. Sci., April 1, 2006; 47(4): 1302 - 1309. [Abstract] [Full Text] [PDF]

				F. C. Kurschus, T. Oelert, B. Liliensiek, P. Buchmann, D. C. Wraith, G. J. Hammerling, and B. Arnold Experimental autoimmune encephalomyelitis in mice expressing the autoantigen MBP1-10 covalently bound to the MHC class II molecule I-Au Int. Immunol., January 1, 2006; 18(1): 151 - 162. [Abstract] [Full Text] [PDF]

T. Kawakita, E. M. Espana, H. He, A.