Department of Microbiology, Mount Sinai School of Medicine,1 Gustave L. Levy Place, New York, NY 10029, USA
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Abstract |
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Keywords: enzymatic engineering/MHC II-peptide multimerization/T-cell response
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Introduction |
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The major drawback of monovalent MHC IIpeptide ligands is that they are recognized by cognate TCRs with low avidity. The on-rates at 25°C vary from very slow (1000) to moderately fast (200 000), whereas the off-rates are in a relative narrow range (0.50.01) or to a t of 1230 s with an estimate of 23 times faster at physiological temperature (Matsui et al., 1991
, 1994
). In general, TCR exhibits a 23-times lower binding affinity for the monovalent MHC IIpeptide complex than for clonotypic antibodies to MHCpeptide complexes (Dadaglio et al., 1997
; Porgador et al., 1997
).
Recently, multivalent MHC IIpeptide ligands with increased avidity for the cognate TCRs have been generated. We engineered the first soluble bivalent MHC IIpeptide ligand on an immunoglobulin scaffold, which binds stably and specifically to cognate TCR on T-cells (Casares et al., 1997). Bivalent MHC IIpeptide ligands engineered on immunoglobulin scaffold exhibit ~2025-times lower off-rates than the monovalent forms (Appel et al., 2000
). Reich et al. (1997) expressed a BirA-dependent biotinylation site on the ß-chain of MHC class II molecules to engineer tetravalent MHC IIpeptide ligands through the streptavidin-mediated cross-linking (Reich et al., 1997
). Tetravalent MHC IIpeptide ligands were successfully used to identify low-frequency antigen-specific T-cells in the peripheral blood of patients with HIV infection (Crawford et al., 1998
). However, the tetravalent MHC IIpeptide ligands could not exceed the avidity of immunoglobulin-based, dimeric MHC IIpeptide ligands, presumably because of the rigidity of biotinstreptavidin bonds that may not provide optimal accommodation of the tetramers on the TCR motifs. In contrast, the immunoglobulin-based MHC IIpeptide ligands are endowed with high flexibility by virtue of their immunoglobulin hinge region.
Functionally, it has been shown that the valence of MHC IIpeptide ligands parallels the potency of T-cell activation in vitro (Boniface et al., 1998). We found that a soluble bivalent MHC IIpeptide ligand exhibited remarkable immunomodulatory effects in vitro and in vivo. At low TCR occupancy, the bivalent MHC IIpeptide ligand polarized the resting and activated, peptide-specific T-cells toward the T helper 2 (Th2) cell response by a mechanism of negative regulation of STAT4-dependent Th1 differentiation (Casares et al., 1999
). In contrast, at high TCR occupancy, the same ligand induced anergy of peptide-specific T-cells (Brumeanu et al., 2001
) by a mechanism of negative regulation of the cytokine gene transactivation (unpublished results).
Although the tetrameric MHC IIpeptide molecules generated through the biotinstreptavidin bonds are valuable tools for in vitro investigation, the non-covalent nature of this bond raises the concern of its stability in vivo. Here, we describe a new method to engineer covalent bonds between the N-glycan moieties of recombinant MHCpeptide dimers using a flexible diaminated polyethylene glycol polymer as cross-linker. The tetramers and octamers generated by this method bound stably and specifically to cognate T-cells, preserved their structural integrity in the lymphoid organs for 72 h and, depending on the TCR/CD4 occupancy and degree of TCR/CD4 co-clustering, exhibited distinct modulatory effects on antigen-specific CD4+ T-cells.
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Materials and methods |
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Transgenic (Tg) BALB/c mice expressing the 14.3d TCR that recognizes the HA110120 peptide of the hemagglutinin (HA) protein of PR8 influenza virus in association with I-Ed class II molecules were kindly provided by Dr H.von Boehmer (Dana Farber Institute, MS). The 78-week-old BALB/c mice were purchased from Jackson Laboratories, USA.
Cells
Primary T-cells specific for the immunodominant CD4 T-cell epitope HA110120 (TCR-HA T-cells) were obtained from the spleens of Tg mice. Approximately 32% of T-cells in the spleen of these mice express the TCR-HA transgene as determined by FACS using 6.5.2 anti-TCR-HA clonotypic monoclonal antibody (mAb). The clonotypic 6.5.2 mAb (rat IgG1/k) was a generous gift from Dr J.Caton (NIH). The 1431 T-cell hybridoma cells (TcH) expressing the 14.3d TCR-HA was a gift from Dr K.Karjalainen (Basel Institute for Immunology, Switzerland).
Soluble bivalent MHC IIpeptide ligand (DEF)
The genetically engineered soluble, bivalent MHC class IIpeptide chimera (DEF) consists of the I-Ed and I-Edß extracellular domains that were dimerized through a murine Fc
2a fragment at the C-termini of I-Edß chains. The HA110120 (SFERFEIFPKE) CD4 T-cell epitope of HA of influenza virus A/PR/8/34 (Haberman et al., 1990
) was covalently linked to the N-terminus of I-Edß chains (Casares et al., 1997
). The soluble, bivalent DEF protein was purified chromatographically on a goat anti-mouse
2a-Sepharose column from the cell culture supernatants of SF9 insect cells infected with baculovirus expressing both I-Ed
and I-Edß/HA110120/Fc
2a genes, as described previously (Casares et al., 1997
). The soluble dimeric DEF binds stably and specifically to cognate TCR on CD4+ T-cells (Casares et al., 1997
).
Enzymatically mediated synthesis of DEF multimers
Soluble, bivalent DEF protein (5 mg) was incubated overnight at 37°C with 500 mU of neuraminidases from Arthrobacter ureafaciens and Clostridium perfringens (Calbiochem-Novobiochem International, La Jolla, CA) in 5 ml of 0.1 M phosphate buffer, pH 5.5, containing 5 mM CaCl2. Free sialic acid released by neuraminidases was removed by dialysis against PBS, pH 7.4. Desialylated DEF was incubated for 48 h at 37°C with 100 U of galactose oxidase (GAO) (Sigma Chemical, St. Louis, MO) and 5 mg of diaminated polyethylene glycol bifunctional cross-linker with a molecular mass of 3400 Da [(NH2)2-PEG3400] (Shearewater Polymers, AL). The Schiff bases formed between the aldehyde groups generated by GAO at the sixth carbon of terminal galactose residues and the amino groups of PEG were stabilized on mild reduction with 80 mM pyridine borane (PB) (Aldrich) (Figure 1). The reaction mixture was dialyzed against PBS in SPECTRA/POR bags (100 000 MWCO) (Sigma) and DEF multimers were separated by size-exclusion chromatography.
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DEF multimers were separated by size-exclusion chromatography in a Superose 6 column (Amersham-Pharmacia Biotech) equilibrated in PBS. The reaction mixture (200 µl) was applied in the column at a flow rate of 1 ml/min and fractions were collected at 1 min intervals. The recovery yield for each protein peak was calculated on the chromatographic profile using UN-SCAN-IT analysis software version 5.1 (Silk Scientific, CA). To identify the PEG polymer, ~0.025 ml from each fraction was reacted with 0.025 ml of Nessler's reagent (Sigma). PEG polymer was detected as a whitish precipitate. The peak tubes were measured for the protein content using the biuret microassay, since the biuret reagent does not interfere with PEG polymers (Brumeanu et al., 1995).
SDSPAGE and Western blot analyses
About 5 µg of chromatographically purified DEF multimers were electrophoresed in 412% polyacrylamide gradient gels (PhastGels, Amersham-Pharmacia) under denaturing and non-reducing conditions and the gels were silver stained according to the manufacturer's instructions. In parallel experiments, 5 µl of blood serum and tissue extracts from mice injected intravenously (i.v.) with 125I-radiolabeled DEF multimers were analyzed at various intervals of time by SDSPAGE under denaturing and non-reducing conditions using 412% gradient PhastGels. Samples of tissue extracts containing 125I-labeled DEF multimers were prepared from spleen, thymus, lymph nodes and brain. The extracts were obtained by tissue homogenization and then cleared of debris by centrifugation. The supernatants were collected and precipitated for 2 h at room temperature with 50% saturated ammonium sulfate (SAS) and the SAS precipitates were dialyzed extensively at 4°C in SPECTRA/POR bags (100 000 MWCO) against PBS containing a cocktail of protein inhibitors (Complete kit, Boehringer Mannheim, Germany). The protein concentration in the dialyzed preparation was adjusted to 5 mg protein/ml with sample buffer containing 5% 2-mercaptoethanol (2ME) and separated by SDSPAGE in 412% gradient PhastGels. The gels were electrotransferred on to PVDF membranes (0.45 µm) and the radioactive bands were identified upon exposure of the membranes on Kodak X-OMAT films (Sigma).
The specificity of the glycosidic bonds generated by (NH2)2-PEG3400 linker between the N-glycan moieties of DEF dimers was determined by Western blot analysis. Samples of purified DEF multimers (5 µg) were digested for 2 h at 37°C with PGN-ase F (0.01 U/µg protein) (Sigma) in the presence of 5% 2ME and electrophoresed on 1015% gradient PhastGels. The gels were electrotransferred on to PVDF membranes (0.45 µm) and the membranes were blocked overnight at 4°C with 5% fat-free milk (Carnation, Nestlé Foods, Glendale, CA) in PBS, then washed with 0.05% Tween 20 in PBS and incubated for 2 h at room temperature with 125I-labeled goat anti-mouse 2a Ab (2x105 c.p.m. per 100 cm2 membrane) in PBS containing 1% BSA and 0.05% Tween 20. The membranes were washed with 0.05% Tween 20 in PBS and exposed on Kodak X-OMAT films.
Cytoflurometric analyses
The 1431 TcH (1x105) expressing 14.3d TCR-HA were incubated for 30 min on ice with 2 µg/ml of purified DEF multimers in PBS1% BSA containing or not 100 µg/ml 6.5.2 anti-TCR clonotypic mAb. The cells were washed in cold PBS1% BSA0.05% NaN3 and bound DEF molecules were stained for 30 min on ice with a goat anti-2aFITC conjugate (Boehringer Mannheim). The fluorescence intensity was measured among 10 000 cells in a FACSCalibur instrument (Becton Dickinson, CA) after subtraction of the background generated by the secondary AbFITC conjugate. To determine the extent of apoptosis induced by DEF multimers in TCR-HA T-cells, we used two-color FACS analysis. Cells were stained for 30 min on ice with 2 µg of 6.5.2 clonotypic mAbFITC conjugate and 2 µg of anti-Anexin V-PE conjugates (PharMingen, CA) and the 6.5.2+/Anexin V+ T-cells were scored among 10 000 events using the FACSCalibur instrument.
Thymidine incorporation assay
The proliferative capacity of TCR-HA T-cells on exposure to DEF multimers was determined by thymidine incorporation assay ([3H]TdR). Spleen cells (106) from TCR-HA Tg mice were incubated for 72 h with various concentrations of purified DEF multimers or medium alone. Tritiated thymidine (1 µCi/well) was added to the cultures for the last 24 h, cells were harvested on filter-paper (Squadron, Sterling, VA) and the radioactivity (c.p.m.) was measured in a -scintillation chamber (Amersham-Pharmacia Biotech).
Cytokine assays
The cytokine production was determined in the cell culture supernatants of spleen cells (106) from Tg mice incubated for 48 h with 10 µg/ml and 50 µg/ml of DEF multimers. The amount of IL-2, IL-4 and IFN- was measured by ELISA according to the manufacturer's instructions (Cytoscreen mIL-2 and Cytoscreen mIL-4 ELISA kits, Biosource International, CA).
Blood clearance and organ distribution
DEF multimers (100 µg in 100 µl of PBS) were radiolabeled with 125I using the conventional chloramine method. Two BALB/c mice per group were injected in the tail vein with 125I]DEF multimers (50x106 c.p.m.) in PBS (200 µl). The clearance rates and index of distribution in organs for the radiolabeled DEF multimers were calculated as described previously (Brumeanu et al., 1995). Briefly, 15 min after injection, the time required for uniform distribution of the radiolabeled material, the mice were bled from the tail vein and radioactivity (c.p.m.) in 20 µl of serum was measured in a
-counter (Pharmacia LKB). The total radioactivity injected per mouse (TRI) was estimated 15 min after injection, on the basis that 7.3% of body weight is blood and 55% of the blood volume is serum. The total residual radioactivity (TRR) was estimated in blood at the time of withdrawal. The index of distribution of DEF multimers in the organs was expressed as a percentage from TRR at the time of collection (% ID) according to the equation (Brumeanu et al., 1995
)
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Results and discussion |
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PEG polymers are not toxic, are immunologically inert and they were approved by the FDA as food ingredients. The enzymatically mediated cross-linking of DEF dimers via diamino-PEG3400 polymer led mainly to the generation of tetramers and octamers as found by size-exclusion chromatography (Figure 2). Their relative molecular masses estimated in the peak tubes were 375 and 720 kDa, respectively. Quantification of the corresponding chromatographic peaks showed a recovery of 55% for DEF tetramer and 32.5% for DEF octamer. About 2% of highly multimerized DEF (MW
800 kDa) and 9.5% of DEF dimer were also separated by chromatography. Accordingly, a small amount of DEF dimer (9.5%) did not react with PEG polymer either because of lower accessibility of the galactose acceptors or because of a smaller number of galactose acceptors per DEF molecule. Using a galactose oxidase (GAO)/tolidinehorseradish peroxidase coupled assay, we found that the number of galactose acceptors per molecule of DEF dimer was on average 10.5 (unpublished results). We previously separated by anion-exchange chromatography a small fraction of DEF dimers expressing smaller amounts of carbohydrates (Casares et al., 1997
), which contained on average 3.7 galactose acceptors per molecule. This may account for the lack of cross-linking by PEG polymer for 9.5% of DEF dimers.
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The SDSPAGE analysis confirmed the composition of the chromatographic peaks as consisting of DEF multimers with molecular masses of 170 kDa for DEF dimer, 365 kDa for DEF tetramer and 700 kDa for DEF octamer (Figure 2 inset, lanes 1, 2 and 3).
Digestion of DEF multimers with PGNase F under denaturing and reducing conditions followed by blotting with 125I-radiolabeled goat anti-2a Ab revealed a major component of ~80 kDa that corresponded to the monomeric unit of DEF dimer (Figure 2
inset, lanes 4, 5 and 6). This clearly demonstrated that PEG polymer was able to cross-link covalently the DEF dimer through the N-glycan moieties. Together, the results demonstrated that (1) the enzymatically mediated cross-linking of DEF dimers by diamino-PEG3400 polymer generated covalently linked DEF tetramers and DEF octamers through their carbohydrate moieties and (2) DEF multimers can be efficiently cleared of residual adducts by size-exclusion chromatography.
Binding specificity of DEF multimers to cognate T-cells
DEF multimers were tested by FACS for their ability to bind to TCR-HA T-cells. The fluorescence intensity of 1431 TcH expressing TCR-HA upon incubation with purified DEF multimers and secondary Ab (goat anti-2a-FITC) was between 33.2 and 38.7%. The fact that we did not detect differences in the fluorescence intensity of DEF multimers suggests that either FACS analysis cannot distinguish discrete alterations in their affinity binding constants to cognate TCR or the incorporated PEG cross-linker in DEF multimers can interfere with the binding of secondary Ab. However, none of DEF multimers bound to 1431 TcH when the cells were preincubated with 6.5.2 clonotypic mAb. This clonotypic mAb inhibits the binding of DEF dimer to 1431 TcH (Casares et al., 1999
). In aggregate, the results demonstrate that the enzymatically mediated multimerization of DEF did not affect its ability to bind specifically to cognate TCR.
Blood clearance and organ distribution of DEF multimers
DEF multimers were tested in naive BALB/c mice for their span life and stability in blood circulation and lymphoid organs. All DEF multimers showed a longer half-life in blood circulation than a genetically engineered immunoglobulin (IgHA) expressing the HA110120 peptide in the CDR3 loop of VH domain (Brumeanu et al., 1993). The half-life of DEF multimers in blood was 50 h (Figure 3A
). Electrophoretic analysis showed similar patterns of degradation of DEF multimers in blood (Figure 3B
). During the first 24 h there was no detectable degradation, whereas after 24 h the degradation occurred progressively in all DEF multimers. However, intact molecules of DEF multimers were still detected 72 h after injection.
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The longer life of DEF multimers than the immunoglobulins in vivo may account for the large amount of carbohydrate moieties expressed by DEF multimers. The persistence of DEF multimers as intact molecules in blood and lymphoid organs also demonstrated that the PEGgalactose imidic bonds were resistant to endoglycosidases.
Immunoregulatory effects of DEF multimers on cognate T-cells
We compared the potency of DEF multimers in stimulating cognate T-cells at three different degrees of TCR/CD4 occupancy: 1, 10 and 50 µg/ml of DEF multimer per 106 splenic TCR-HA Tg cells. Since the frequency of TCR-HA T-cells in spleen of these mice is 3033%, the TCR/CD4 occupancy will correspond, respectively, to 0.7, 7.0 and 35 pmol DEF ligand per cell. At low occupancy (0.7 pmol/cell), DEF octamer was twice as potent as DEF tetramer and seven times more potent than DEF dimer in stimulating TCR-HA T-cells (Figure 4A). For as much as 10 times higher occupancy (7.0 pmol/cell), there was an inverse relation between the valence and immunopotency of DEF multimers. Thus, DEF dimer was 1.5 times more potent than DEF tetramer, whereas DEF octamer did not stimulate the cells. At the highest occupancy (35 pmol/cell), none of DEF ligands stimulated the cells.
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These results indicated that depending on the degree of TCR/CD4 occupancy, the increase in DEF valency correlated with distinct immunomodulatory effects on cognate T-cells. Thus, at low TCR occupancy, the increase in DEF valency paralleled its potency to induce T-cell proliferation and Th2-like cytokine secretion, whereas at high occupancy it was inversely proportional to the extent of T-cell unresponsiveness and cytokine secretion. This suggests that the order of TCR and CD4 co-clustering may represent an important quantitative parameter in dictating the strength of TCR signaling towards cell proliferation and differentiation. In addition, at the same TCR/CD4 occupancy, depending on the number of TCR and CD4 clusters per cell, qualitative changes may occur in TCR signaling machinery leading the T-cells toward anergy or apoptosis.
The fact that DEF multimers (1) exhibit a long life in blood circulation, (2) penetrate the lymphoid organs, (3) persist in these organs as intact molecules for as long as 72 h and (4) exhibit immunomodulatory effects on cognate T-cells in vitro, suggests that this new class of antigen-specific TCR/CD4 ligands can be used to modulate the effector functions of pathogenic T-cells in various infectious an autoimmune diseases. Also, antigen-specific CD4+ memory T-cells that can persist in vivo for long period of time at a low frequency could be identified and investigated functionally by using DEF-like multivalent chimeras.
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Notes |
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Acknowledgments |
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References |
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Received November 28, 2000; accepted December 23, 2000.