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The Journal of Immunology, 1998, 161: 3262-3270.
Copyright © 1998 by The American Association of Immunologists

Chemical Chaperones Enhance Superantigen and Conventional Antigen Presentation by HLA-DM-Deficient as well as HLA-DM-Sufficient Antigen-Presenting Cells and Enhance IgG2a Production In Vivo1

Birinder Ghumman, Edward M. Bertram and Tania H. Watts2

Department of Immunology, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide loading onto MHC II proteins and also stabilizes empty MHC II molecules prior to their acquisition of antigenic peptides. APC that lack HLA-DM exhibit quantitative defects in protein Ag as well as superantigen presentation. Here we show that both the superantigen and protein presentation defect in MHC II-transfected, HLA-DM-deficient T2 cells can be partially overcome by treating the APC with the chemical chaperones glycerol, DMSO, or trimethylamine oxide. These chemical chaperones also enhance superantigen and conventional Ag presentation by wild-type APC. In vivo, glycerol was found to act as an adjuvant and resulted in enhanced IgG2a production to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). In vitro, the enhancement of Ag presentation by chemical chaperones was found to take place at the level of the APC and took several hours to develop. Subcellular fractionation experiments show that HLA-DM enhances presentation of peptides by dense endosome fractions whereas chemical chaperones enhance presentation by light membrane fractions (early endosome or plasma membrane). The mechanism by which these chemical chaperones augment Ag presentation is not defined, but flow cytometric analysis suggests that the enhancement may be due to a subtle effect on the stability of several different proteins at the cell surface.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Molecular chaperones bind to non-native conformations of proteins and stabilize them against irreversible aggregation. Through controlled cycles of binding and release, molecular chaperones can facilitate the protein-folding process. Once the native conformation of a protein is achieved, molecular chaperones no longer bind. Thus, molecular chaperones are important in the retention of misfolded proteins as well as in the protein-folding process itself (1).

The assembly of MHC class II molecules with their peptide ligands involves a complex biosynthetic pathway in which molecular chaperones are involved at several stages (2). Shortly after synthesis in the endoplasmic reticulum (ER),3 individual MHC {alpha}- and ß-chains associate with the molecular chaperone calnexin. Heterodimers of MHC II {alpha}- and ß-chains are then assembled upon an invariant chain (Ii) trimer that also contains bound calnexin. Calnexin dissociates from the Ii-MHC {alpha}/ß complex upon completion of formation of a nonameric complex consisting of three {alpha}/ß heterodimers on an invariant trimer (3). In the absence of invariant chain, MHC II is poorly expressed and largely remains aggregated in the ER (4, 5, 6). Thus, invariant chain is considered to be a specific molecular chaperone of the MHC II biosynthetic pathway.

After assembly in the ER, the nonomeric {alpha}ßIi complex travels via the Golgi to an endocytic compartment where invariant chain is removed by proteolysis, leaving an MHC II {alpha}/ß complex bound to a fragment of invariant chain, class II associate invariant chain peptides, CLIP (7, 8). In the endosome, {alpha}ßCLIP or larger precursors are acted upon by another specialized molecular chaperone, HLA-DM. HLA-DM binds to MHC II molecules and thereby facilitates release of CLIP and other unstably bound peptides from MHC II molecules (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In addition, HLA-DM can stabilize empty MHC II dimers and therefore maintain them in a state suitable for peptide binding, hence its definition as a molecular chaperone (20, 21).

APC that lack HLA-DM have a defect in peptide loading within APC (22). MHC II molecules in these cells remain associated with CLIP (23, 24). MHC II-CLIP complexes differ in kinetic stability from mature MHC II molecules as reflected in the instability of the CLIP-occupied MHC II dimer to SDS, at least for some MHC II alleles (16, 22). HLA-DM-deficient APC present native protein Ags poorly (22, 25). However, HLA-DM deficiency does not impair surface expression of class II; therefore, DM-deficient cells are capable of peptide presentation at the cell surface (22, 25). The affinity of CLIP for different MHC II alleles is quite variable, with that for Ak being particularly low (26, 27). As a result, HLA-DM-deficient cells express Ak in the SDS-stable, CLIP-unoccupied form, and the Ag presentation defect is not as severe as in DM-deficient cells expressing other MHC II alleles (28). However, in the absence of HLA-DM, T cells specific for Ak and HEL46-61 respond poorly to hen egg lysozyme (HEL) protein processed within the APC (25) a defect that can be corrected, at least in part, by HLA-DM transfection (29).

In addition to an effect on protein Ag presentation, HLA-DM-deficient cells have a defect in presentation of the superantigen staphylococcal enterotoxin A (SEA) by murine MHC II molecules (30, 31). The staphylococcal enterotoxins are 25-kDa proteins that bind as intact proteins to MHC II proteins outside the peptide-binding groove and activate T cells by simultaneously binding to the MHC II on the APC and the TCR on T cells expressing particular TCR Vß segments (reviewed in Ref. 32). The binding of SEA to MHC II is peptide dependent, and the affinity of this toxin for MHC II varies greatly with the peptide bound in the groove (33). Indeed, SEA does not appear to be presented efficiently by CLIP-occupied Ab expressed on HLA-DM-deficient T2 cells (30). Although not exclusively occupied with CLIP (28, 31), Ak molecules expressed on HLA-DM-deficient T2 cells also show a quantitative defect in their ability to bind and present SEA, a defect that is corrected by HLA-DM transfection (31). In this report we show that the protein and the SEA presentation defect in T2.Ak cells can also be partially overcome by treatment of the cells with chemical chaperones.

Chemical chaperones were recently defined in studies of diseases involving defects in protein folding (34). The first such example was the chemical rescue of the {Delta}F508 mutant of the cystic fibrosis membrane conductance regulator (CFTR) protein (35, 36). This mutant has a protein-folding defect that results in the protein being retained within the cell. Treatment of cells expressing this mutant with agents known to stabilize proteins in their native conformation, including DMSO (35) and glycerol (35, 36), were found to partially correct the protein-folding defect such that functional protein reached the plasma membrane. Similar results were obtained with the chemical osmolyte, trimethylamine oxide (TMAO) (35). Here, we show that treatment of APC with the same set of chemical chaperones (DMSO, glycerol, or TMAO) partially restores the ability of HLA-DM-deficient T2.Ak cells to present the superantigen SEA as well as HEL protein to T cells and has a general effect on enhancement of conventional Ag presentation. In vivo, we find that the chemical chaperone glycerol can act as weak adjuvant to promote an anti-TNP Ab response. In particular, we find that by the secondary response, glycerol results in enhanced IgG2a compared with immunizations in alum or PBS, consistent with a Th1-type response. The mechanism of action of chemical chaperones is not known, but treatment of APC with these chaperones appears to have a subtle effect on enhancing Ab binding to a number of proteins on APC. Thus, chemical chaperones may enhance Ag presentation by stabilizing protein structure at the cell surface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, Abs, and reagents

The BXT hybrid cell lines T1 and T2, transfected with Ak, T1.Ak, and T2.Ak, have been described (25). T1 is a wild-type cell line expressing human class II genes, whereas T2 has a large deletion in the MHC II region, deleting MHC II structural genes as well as HLA-DM. T1 and T2 cells transfected with Ak, as well as T2.Ak transfected with HLA-DM A and B genes (T2.Ak/DM, described in Ref. 31) were provided by Peter Cresswell and Lisa Denzin (Yale University School of Medicine, New Haven, CT). The HEL46-61-specific T cell hybrid A2.A2 (37) and the H-2d, H-2k B lymphoma TA3 (38) were obtained from L. Glimcher (Harvard University, Cambridge, MA). The Ad-restricted, OVA323–339-specific T hybrid DO-11.10 (39) was obtained from P. Marrack and J. Kappler (National Jewish Hospital, Denver, CO). The IL-2 indicator line, CTLL, was obtained from the American Type Culture Collection (Manassas, VA). All cell lines were grown in RPMI 1640 supplemented with 10% FCS, 2-ME, and antibiotics as previously described (40). T2.Ak/DM was grown in the presence of 250 µg/ml G418 (Life Technologies, Burlington, Ontario, Canada) and 500 ng/ml Puromycin (Sigma, St. Louis, MO). SEA was purchased from Toxin Technology (Sarasota, FL). Synthetic HEL46-61 was purchased from the Ontario Cancer Institute Biotechnology Facility (Toronto, Canada). The anti-Ak-producing hybridomas 10-2.16 and 11-5.2 were obtained from the American Type Culture Collection. The anti-A{alpha}k-producing hybridoma 39J (41) was kindly provided by W. Wade (Dartmouth Medical School, Hanover, NH). Abs were purified from hybridoma culture supernatants using protein A or G Sepharose (Pharmacia, Piscataway, NJ). For biotinylation, Abs were dialyzed overnight against 0.1 M sodium bicarbonate, pH 8.5, followed by incubation with a 10-fold molar excess of N-hydroxysuccinimidyl-D-biotin for 2 h at room temperature. Free biotin was removed by dialysis against PBS. The superantigen SEA was biotinylated by the same method using a 10- or 20-fold molar excess of N-hydroxysuccinimidyl-D-biotin over SEA.

Treatment of APC with chemical chaperones and Ag/superantigen presentation assays

The chemical chaperones DMSO, glycerol (BDH, Analar quality, distributed by VWR Scientific, Mississauga, Canada), and TMAO (Sigma) were diluted in serum-free culture medium and added to the cells at the concentrations indicated in the figures. In preliminary experiments, the presence of glycerol appeared to be toxic in the CTLL assay, so subsequent experiments involved pretreating APC and washing out the chemical chaperones prior to setting up the coculture with T cells (data not shown). Most experiments shown are for 20- to 24-h pretreatment.

For Ag presentation assays, 105 T1.Ak, T2.Ak, T2.Ak/DM cells, or TA3 cells (with or without chemical chaperone pretreatment) were incubated with SEA, native HEL or HEL46-61 at the concentrations indicated in the figure legends, together with 105 A2.A2 or DO.11.10 cells at 37°C. After 18 to 24 h, supernatants were serially diluted and incubated with 104 IL-2-dependent CTLL cells. [3H]Thymidine (Amersham Canada, Oakville, Ont, Canada) was added to the CTLL cultures after 16 h and thymidine incorporation was measured 8 h later.

Flow cytometry

Cell surface expression of Ak was determined by flow cytometry using a Becton Dickinson (San Jose, CA) FACSCalibur. Cells were incubated with primary Abs for 30 min at 4°C. In initial experiments, Abs were titrated to determine the saturating concentration. Biotinylated Abs were detected by phycoerythrin (PE)-streptavidin or by FITC-streptavidin staining (Molecular Probes, Eugene, OR). Following second-step incubation for 30 min at 4°C, cells were washed with PBS/1% FCS/2 mM NaN3 and fixed with paraformaldehyde. Sensitivity and alignment of the FACSCalibur were checked using CaliBRITE beads (Becton Dickinson) with the software program autoCOMP (Becton Dickinson) and Immuno-Check beads (Coulter, Hialeah, FL) with a standard protocol created with defined ranges for coefficient of variation and mean fluorescence. CellQuest software (Becton Dickinson) was used for acquisition and analysis of data. A threshold was set on forward scatter, and forward scatter and side scatter were used to gate on live cells. Data are reported as the geometric mean of the fluorescence intensity.

SEA binding to cells was measured using biotinylated SEA and PE-streptavidin. SEA was incubated with cells at 4°C for 30 min to 1 h. Ab blocking to assess specificity for Ak was carried out by preincubating cells with blocking Ab on ice for 15 to 30 min prior to addition of SEA. Samples were washed twice with PBS/1% FCS/2 mM NaN3 and incubated with PE-streptavidin for 20 min prior to washing, paraformaldehyde fixation, and analysis by flow cytometry.

Subcellular fractionation

T1.Ak, T2.Ak, and T2.Ak/DM (2–5 x 108) were washed with cold PBS followed by homogenization buffer (HB; 250 mM sucrose in 3 mM imidazole, pH 7.4), and resuspended at 108/ml in HB containing a cocktail of protease inhibitors (HB+) (10 µg/ml aprotinin, 1 µg/ml antipain, 1 µg/ml pepstatin, 1 mM PMSF, 17 µg/ml benzamidine, 10 µg/ml leupeptin, and 0.5 mM EDTA). Each cell suspension was then passed through a 22-gauge 1.5-inch needle using approximately 10 cycles of aspiration and expulsion against the wall of the test tube. The final number of cycles was based on an endpoint of no more than 1 or 2 intact cells per field in the final count. The homogenates were centrifuged at 2700 rpm for 10 min to pellet nuclei. The postnuclear supernatant was centrifuged for 20 min at 23,500 rpm (36,000 x g) in a Beckman L5-50 Ultracentrifuge, using a 50 Ti rotor. The membrane pellet in 1 ml of HB was loaded onto a 23-ml, 17% Percoll gradient (Pharmacia LKB Biotechnology, Piscataway, NJ) and centrifuged for 1 h at 20,000 rpm (36,000 x g) using a 50.2 Ti rotor. Samples (0.8 ml) were collected from the bottom of the tube using a tube piercer (ISCO model 184, Lincoln, NE), and aliquots of the gradient fractions were stored at -70°C immediately so that all assays could be done on fractions from the same preparation. All procedures were performed on ice or at 4°C.

For analysis of Ag presentation by gradient fractions, 50-µl aliquots of each gradient fraction per well of a 96-well plate were frozen (-70°C) and thawed to rupture membrane vesicles prior to setting up cocultures. HEL46-61 (100 µM) was added, followed by 105 A2.A2. After 10 to 24 h at 37°C, supernatants were serially diluted and incubated with 104 IL-2-dependent CTLL cells. [3H]Thymidine (Amersham Canada) was added to the CTLL cultures after 16 h and thymidine incorporation was measured 8 h later.

Animals and immunizations

Female C57BL/6 mice were obtained from Charles River Laboratory (St. Constant, Quebec, Canada) and used at 8 to 10 wk of age. Mice were immunized s.c. with 40 µg of TNP-KLH in either glycerol (0.75 M, 2.5 M), PBS, or bound to alum. Serum samples were prepared from blood taken by retro-orbital plexus puncture.

Anti-TNP ELISA

TNP-specific Abs were determined for IgM, IgG1, IgG2a isotypes. Each well of 96-well plates was coated with 100 µl of 5 µg/ml BSA-TNP in PBS for 1 h at 37°C followed by incubation at 4°C overnight. Plates were blocked with 5% skim milk in PBS/0.1% Tween-20 for 2 h at 37°C. Eight fivefold serial dilutions of serum in PBS/0.1% Tween-20/2% skim milk were added to wells for 2 h at 37°C. After washing in PBS/0.1% Tween-20, horseradish peroxidase-conjugated anti-isotype Abs (Caltag Laboratories, Burlingame, CA) were added for 2 h at 37°C. Following washing, H2O2 and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Sigma) were added in citrate phosphate buffer, pH 5.0, and color development was measured after 20 min at OD405. The titer of each serum sample was determined as the reciprocal of the last dilution that was 2.5 times the background value of normal serum at 1/5 dilution.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of chemical chaperones on SEA presentation by T2.Ak cells

As discussed above, HLA-DM has been described as a molecular chaperone for MHC II biosynthesis. Recent studies have shown that the presence of HLA-DM is important for SEA presentation independent of its role in CLIP release (31). This led us to speculate that it was a chaperone-like effect on protein folding that was responsible for the effect of HLA-DM on SEA presentation by T2 cells. To test this idea further, we treated DM-deficient T2 cells with several agents that had been previously defined as having chemical chaperone activity in studies of mutant CFTR proteins.

Figure 1Go shows the effects of overnight treatment of T2.Ak cells with either glycerol, DMSO, or TMAO on subsequent presentation of SEA. Coincubation of chemical chaperones with APC and T cells resulted in a lack of any response as measured in a CTLL assay, due to toxicity of these agents to the CTLL cells (data not shown). Similarly, overnight preincubation of the T cells with chemical chaperones followed by washing resulted in a slight inhibition in subsequent Ag presentation assays. Therefore, in all experiments reported here these agents were washed out of the APC cultures before adding T cells. It can be seen in Figure 1Go that both glycerol and DMSO pretreatment augment SEA presentation by T2.Ak cells in a dose-dependent manner. Addition of glycerol or DMSO in the absence of Ag had no effect on the T cell response (data not shown) (see Fig. 2Go). Glycerol-mediated enhancement of SEA presentation was optimal at between 0.75 and 1.0 M glycerol. Higher amounts were toxic to the APC. For DMSO, the optimal concentration was 1.5 to 2.0% v/v. The chemical osmolyte, TMAO, also showed enhancement of SEA presentation at 0.1 M, while higher levels were toxic to the APC and lower levels had little effect (data not shown).



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  		FIGURE 1. Effect of pretreatment of T2.Ak cells with the chemical chaperones glycerol, DMSO, or TMAO on superantigen presentation to A2.A2 T cells. T2.Ak cells (5 x 105/ml) were treated with glycerol, DMSO, or TMAO at the concentrations indicated in the figures. After overnight culture, T2.Ak cells were washed three times in PBS and then 105 cells were cocultured with 10 µg/ml SEA plus 105 A2.A2 T cells. After overnight incubation, supernatants were removed and serial dilutions of supernatant analyzed for induction of proliferation by CTLL. [3H]Thymidine was added for the last 6 h of culture. A, Results are shown as thymidine incorporation as a function of supernatant dilution for each chemical chaperone concentration. B, Thymidine incorporation by CTLL in response to undiluted culture supernatant is plotted as a function of chemical chaperone concentration.

 


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  		FIGURE 2. Kinetics of enhancement of Ag presentation by chemical chaperone pre-treatment. T2.Ak cells were treated with glycerol (0.5M), DMSO (1.5%) or TMAO (0.1M) for the times indicated in the figure. After preincubation at 37°C, APC were washed and assessed for presentation of SEA (10 µg/ml) to A2.A2 cells as described in Figure 1Go. As indicated in the figure, T2.Ak cells treated with chemical chaperones were also tested for T cell activation in the absence of SEA. Data shown are for thymidine incorporation by CTLL in response to undiluted culture supernatant.

 
Figure 2Go shows the kinetics of induction of enhanced SEA presentation by chemical chaperone pretreatment of T2.Ak cells. It should be noted, that the concentration of glycerol used for these experiments was suboptimal (0.5 M), since prolonged treatment of APC with 0.75 M glycerol resulted in more than 50% cell death by 72 h. It can be seen that the response of the T cells to SEA (10 µg/ml) presented by T2.Ak was very weak in the absence of pretreatment with chemical chaperones. For pretreatment of APC with either DMSO or glycerol, there was little or no improvement in SEA presentation after 2 h, but after 24 h SEA presentation significantly improved, with maximal effect by 48 h. The effect of pretreatment with TMAO on APC was slower to develop, but was also maximal by 48 h. None of the chaperone-treated APC activated T cells in the absence of superantigen.

The effect of chemical chaperones on superantigen presentation is not restricted to HLA-DM-deficient APC

Figures 1Go and 2Go show that chemical chaperone treatment can at least partially compensate for the SEA presentation defect observed in T2.Ak cells. To determine whether this phenomenon was restricted to HLA-DM-deficient cells, the experiments were repeated with HLA-DM-sufficient T1.Ak and DM-transfected T2.Ak (T2.Ak/DM) (Fig. 3Go). In contrast to T2.Ak cells, which have a large deletion in the MHC II region and thereby lack class II expression, T1.Ak cells expresses human class II molecules. Lower amounts of SEA could be used for activation of T cells by T2.Ak because human class II molecules have a much higher affinity for SEA than do murine MHC II molecules (32). For these experiments, we chose a concentration of glycerol (0.75 M) that had been shown in a separate experiment to give the maximum augmentation of APC function for the three cell lines after overnight incubation (data not shown). Figure 3Go shows that pretreatment with glycerol enhances SEA presentation by all three cell lines. Similar results were obtained using pretreatment with DMSO at 1.5% (data not shown). Thus, the effects of chemical chaperones is not restricted to HLA-DM-deficient cells, although the effect is greatest for the HLA-DM-deficient cell line, T2.Ak.



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  		FIGURE 3. Effect of glycerol pretreatment on presentation of SEA to HLA-DM sufficient and HLA-DM-deficient cells. T1.Ak, T2.Ak and T2.Ak/DM cells were incubated with 0.75 M glycerol for 24 h at 37°C in complete medium followed by washing and then assessed for presentation of the superantigen SEA as described in Figure 1Go. Results are reported as thymidine incorporation by CTLL in response to undiluted culture supernatant.

 
Effect of chemical chaperones on conventional Ag presentation

To determine whether enhancement of Ag presentation by chemical chaperones was unique to SEA presentation, we also tested T1.Ak, T2.Ak, and T2.Ak/DM cells for presentation of HEL protein, as well as HEL46-61 peptide, with or without glycerol pretreatment (Fig. 4Go). As has been observed by others (28, 31), T2.Ak shows enhanced peptide presentation compared with T1.Ak, likely due to a higher proportion of kinetically unstable MHC II-peptide complexes on the surface of the DM-deficient cells. Separate experiments showed that the optimum concentration of glycerol for presentation of these Ags was similar to that seen for SEA (data not shown). It can be seen that HEL peptide as well as HEL protein presentation are enhanced by glycerol pretreatment of both HLA-DM-deficient T2.Ak and HLA-DM-sufficient T1.Ak or T2.Ak/DM cells. For HEL46-61 presentation to the A2A2 T cells, glycerol pretreatment enhances T cell activation even when peptide is saturating in the assay. Thus, for T2.Ak, glycerol treatment can partially compensate for the lack of HLA-DM in allowing presentation of native HEL to A2.A2 T cells.



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  		FIGURE 4. Effect of glycerol pretreatment on presentation of HEL protein or HEL46-61 to A2.A2 T cells. T1.Ak, T2.Ak, or T2.Ak/DM cells with or without pretreatment overnight with 0.75M glycerol were incubated with HEL46-61 or native HEL protein at the indicated concentrations and 24 h later culture supernatants were tested for IL-2 production using CTLL cells as described in Figure 1Go and the methods.

 
To further explore the timing of the effects of glycerol on protein Ag presentation, we compared the effects of adding glycerol before or after an Ag pulse with native HEL. HEL was added overnight and then the cells were washed and either left untreated or treated with glycerol for 24 h. We found that glycerol can exert its effect before or after addition of Ag (data not shown). Similarly, coincubation of HEL plus glycerol followed by washing and addition of T cells had similar effects on enhancement of Ag presentation (data not shown). Thus, assuming that residual Ag is not continuing to be processed 24 h after addition of Ag, it appears that glycerol does not need to exert its effects during Ag processing but can affect subsequent presentation by APC.

Effect of chemical chaperones on Ag presentation by murine APC

T2 cells are a human T-B hybrid and thus the ability of chemical chaperones to enhance presentation by T2 cells might be due to the folding of murine Ak being suboptimal in a human APC. Therefore, we tested the effect of glycerol pretreatment on subsequent Ag presentation by the murine B cell hybridoma TA3, which expresses Ad, Ak, Ed, and Ek molecules. It can be seen that presentation of SEA by TA3 cells to A2.A2 T cells is substantially augmented by glycerol pretreatment at low concentrations of SEA. However, the effect on HEL presentation to A2.A2 is much smaller (Fig. 5Go) and no enhancement of HEL46-61 presentation was observed (data not shown). For the Ad-restricted T cell DO-11.10, glycerol pretreatment was found to enhance both native OVA and OVA323–339 presentation. SEA presentation on TA3 can involve Ad and Ak as well as Ek/d molecules, with E molecules dominating at low SEA concentration (32, 42). Thus, glycerol pretreatment of TA3 seems to affect Ad-restricted and perhaps E-restricted presentation to T cells, but has a lesser effect on Ak-restricted presentation by these APC.



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  		FIGURE 5. Effect of chemical chaperones on Ag presentation by the murine APC, TA3. TA3 cells were either untreated (dashed lines) or pre-treated overnight with 0.75M glycerol followed by washing (solid lines). A2.A2. T cells were stimulated with TA3 (±glycerol pretreatment) plus various concentrations of SEA or native HEL as indicated in each panel. DO-11.10 T cells were stimulated with TA3 (±glycerol pretreatment) plus various concentrations of OVA323–339 or native OVA as indicated in the figures. In each panel, the same symbol is used for a given dose of Ag with or without chaperone treatment, and dashed lines (untreated) vs solid lines (glycerol-pretreated) should be compared for each symbol.

 
Does chemical chaperone treatment influence expression of proteins on the APC?

One possible mechanism by which chemical chaperones might influence Ag presentation is by altering the expression of proteins on the APC surface. We therefore analyzed MHC II expression as well as the expression of LFA-1, ICAM-1, and B7-2, on TA3 cells with and without chaperone pretreatment (Table IGo). Forty-eight-hour pretreatment of TA3 cells with glycerol or with TMAO led to a slight (10–20% enhancement) in the geometric mean of the fluorescence intensity for Ab binding to Ad, Ek/d, LFA-1, ICAM-1, and B7-2 molecules. However, glycerol or TMAO treatment also resulted in an increase in background staining with control Ab (line 1 of Table IGo). Thus the effect of chemical chaperones on Ag presentation is not explained by a large increase in expression of any one protein, but might be due to the sum of a number of small effects on many surface proteins. Similar experiments were also carried out to analyze MHC II expression on T2.Ak cells with and without chaperone treatment. For glycerol-treated cells, there was no significant change in binding of 10-2.16, 39J (Table IGo) or 11-5.2 or of the superantigen SEA (data not shown). For TMAO-treated cells, the mean fluorescence intensity for binding to Ak was increased by 10 to 15%. For each of the Ak-specific Abs, analysis of Ab binding to T2.Ak as a function of concentration did not reveal significant differences between glycerol-treated or untreated cells, thus it does not appear that these agents change the affinity of the MHC II/Ab complex (data not shown).


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  		Table I. Effect of glycerol or TMAO treatment on Ab binding to the surface of TA3 or T2.Ak cells1

 
Chemical chaperone treatment of intact cells does not enhance Ag presentation by late endosomes isolated from the cells

Previous work by Harding and Geuze (43) and from our laboratory (29) has shown that late endosomes can be used to present Ag to T cell hybridomas. To determine whether chemical chaperone pretreatment influenced the ability of MHC molecules in late endosomes to function in Ag presentation, we treated intact cells with glycerol prior to lysing cells and separating the membrane vesicles by density gradient sedimentation. Figure 6Go shows the ability of Percoll fractions isolated from glycerol-treated or untreated T2.Ak or T2.Ak/DM cells to present HEL46-61 peptide to A2.A2 T cells. As previously characterized (29), the peak of activity associated with high density fractions (fractions 2–5) represents late endosomes, whereas the peak of Ag presentation activity found in the low density fractions (fractions 19–22) represents early endosomes and plasma membrane. It can be seen that glycerol pretreatment enhances HEL46-61 presentation by low density fractions (plasma membrane/early endosomes) but did not enhance HEL46-61 presentation by dense endosome fractions. As expected, the presence of HLA-DM results in markedly enhanced Ag presentation by late endosomes (compare T2.Ak/DM vs T2.Ak presentation) but had little effect on peptide presentation by fractions containing early endosomes and plasma membrane. Thus, in contrast to HLA-DM, chemical chaperone treatment does not appear to act on the peptide-loading compartment within the APC.



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  		FIGURE 6. Effect of chemical chaperone pre-treatment on the ability of low density but not high density membrane fractions to present HEL46-61 to T cells. T2.Ak or T2.Ak/DM cells were treated overnight with 0.75M glycerol followed by washing, lysis of cells, and separation of membranes on Percoll as described in Materials and Methods. Each fraction was tested for presentation of HEL46-61 to A2.A2 T cells, results are plotted as thymidine incorporation by CTLL in response to undiluted culture supernatant (dilution of the supernatants indicated that the response of the CTLL were not at saturation). Open symbols, no Ag; filled symbols, + HEL46-61, 100 µM; dashed lines, untreated APC; solid lines, APC that had been pretreated with glycerol. Fractions were collected from the bottom of the Percoll gradient, so that the lowest fraction numbers represent the densest membranes.

 
Effect of glycerol on the immune response to TNP-KLH in vivo

The effect of chemical chaperones on Ag presentation in vitro, led us to hypothesize that these agents might be useful in augmenting immune responses in vivo. To test this idea, we immunized mice with TNP-KLH in PBS, alum, or glycerol and analyzed primary and secondary Ab responses (Table IIGo). We chose two concentrations of glycerol for these experients. We used a concentration that was optimal for the in vitro effects, 0.75 M. In addition, we used a higher concentration, 2.5 M, to take into account the dilution effect in vivo. In the primary response, the inclusion of glycerol during immunization resulted in higher IgG1 and IgG2a relative to mice that were injected with TNP-KLH in PBS. The response was weaker than that obtained using alum as an adjuvant. Upon secondary immunization with the same regimen, the difference between PBS-injected vs glycerol-injected mice was more substantial. Interestingly, the presence of glycerol appeared to favor a higher ratio of IgG2a to IgG1 than in the alum-injected mice. Thus, although the adjuvant effect of glycerol in vivo is relatively weak, it may serve as a useful base from which to build adjuvants favoring Th1 responses.


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  		Table II. Anti-TNP Ab responses of mice immunized with KLH-TNP in glycerol1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have shown that agents previously shown to have chemical chaperone activity can enhance the presentation of superantigens, protein Ags, and peptide Ags to T cells. In contrast to molecular chaperones that bind to non-native conformations of proteins, chemical chaperones are thought to act by stabilizing proteins in their native conformation. Glycerol is thought to stabilize proteins because it is preferentially excluded from the immediate vicinity of proteins. This results in an increase in relative hydration around the protein and thus enhances the hydrophobic effect that causes the polypeptide to maintain a minimum surface area and resist unfolding (discussed in Ref. 34). Naturally occurring cellular osmolytes act similarly within cells to stabilize proteins (34).

For the {Delta}F508 mutation in the CFTR protein, chemical chaperones are thought to subtly alter the conformation of the protein to allow release from molecular chaperones such as calnexin and HSP73 in the ER, thereby allowing the molecules to reach the cell surface (34). The mechanisms of action of chemical chaperones leading to enhancement of Ag presentation in the present study remain unknown. Examination of several proteins on TA3 cells for increased apparent expression showed that LFA-1, ICAM-1, B7 family molecules, and MHC II molecules showed a 10 to 20% increase in Ab binding after glycerol treatment. It is difficult to evaluate the significance of these effects as background staining also goes up after chemical chaperone treatment. While the enhanced Ag presentation is not explained by an increase in surface expression of any one molecule, it is possible that chemical chaperones subtly stabilize the expression or folding of a number of molecules on the APC surface involved in T cell activation and thereby enhance Ag presentation. This would fit in with the idea that these agents act to stabilize proteins in their native conformation. Alternatively, chemical chaperones might influence some other aspect of the cell surface such as the fluidity of the membrane and thereby influence cell-cell contact.

Treatment with chemical chaperones can partially overcome the quantitative defect in superantigen presentation by DM-deficient T2.Ak cells and also enhances the level of protein Ag presentation by T2.Ak cells ( Figs. 1–3GoGoGo). HLA-DM is important in facilitating peptide loading within APC and in stabilizing empty class II molecules. However, the data presented here do not support a role for chemical chaperones in replacing DM function in peptide loading, since glycerol pretreatment enhances Ag presentation whether given before or after Ag pulsing (data not shown). Furthermore, the effect of chemical chaperones does not appear to be limited to abnormal APC lacking HLA-DM since chemical chaperones enhance superantigen and protein Ag by cells expressing HLA-DM ( Figs. 3–5GoGoGo). Using subcellular fractionation to separate low density membranes (early endosomes and plasma membrane) from high density membranes (late endosomes and lysosomes), we find that HLA-DM enhances presentation by the dense endosome fraction, whereas chemical chaperones appear to augment peptide presentation only by the low density fraction. Thus, although chemical chaperones can partially compensate for the absence of HLA-DM, they appear to act by a distinct mechanism.

It is conceivable that chemical chaperones enhance presentation by T1 and T2 cells because these cells express murine MHC II molecules in human APC, which might not provide the optimal interaction for folding MHC II molecules. Indeed, glycerol pretreatment gave a substantial enhancement of native HEL or HEL46-61 presentation by T1 or T2 cells (Fig. 4Go), but had only a marginal effect on native HEL or HEL46-61 presentation by TA3 cells (Fig. 5Go) (data not shown). Thus for Ak-restricted responses, a human APC may not be optimal for either MHC II folding or A2.A2 T cell activation. However, treatment of murine TA3 cells with glycerol had a significant effect on presentation of SEA or OVA by TA3 cells. These Ags were presented by Ed/k or Ad, respectively. Thus, there may be allele-specific and species-specific situations in which Ag presentation is suboptimal and can be improved by chemical chaperones. Taken together, the data suggest a general role for chemical chaperones in augmenting presentation of conventional and superantigens by normal and abnormal APC, but the magnitude of the effect appears to depend on the Ag/MHC/APC combination.

Glycerol and DMSO are sometimes used as protein stabilizers and to facilitate solubilization of reagents used in Ag presentation assays. The observation that as little as 1% DMSO or 0.75 M glycerol can have such a large effect on Ag presentation means that care should be taken to remove or control for these agents in Ag presentation experiments.

Finally, we have shown that immunization with a protein Ag in glycerol leads to an enhanced Ab response in comparison with protein immunized with PBS. In the secondary response, the inclusion of glycerol resulted in a higher ratio of IgG2a/IgG1 observed in glycerol-immunized vs alum- or PBS-immunized mice. Enhanced IgG2a over IgG1 production is one of the hallmarks of a Th1 response (44). Alum is the only adjuvant currently licensed for use in humans but has the drawback that it tends to favor Th2 responses (45). In cases in which a Th1 response is preferable, alternate strategies are required. Although the effects of glycerol as an adjuvant are modest at best, the observation that glycerol inclusion in the immunization protocol enhances the relative proportion of IgG2a over IgG1 suggests that glycerol may provide a useful base from which to compose adjuvants capable of generating a Th1 response.

In summary, we have shown that agents previously shown to have chemical chaperone activity in studies of protein-folding mutants augment presentation of conventional and superantigens by HLA-DM-deficient and HLA-DM-sufficient APC. The effect of chemical chaperones on APC is slow to develop, with little effect by 2 h and optimal effect by 48-h chaperone treatment. Chemical chaperones do not appear to exert their affect on Ag processing or peptide loading, since superantigen presentation at the cell surface is also sensitive to enhancement by chemical chaperone pretreatment. Chemical chaperones were found to induce a subtle enhancement of Ab binding to several surface proteins and thus they might act as general stabilizers of proteins on the APC surface. Regardless of mechanism, the observation that Ag presentation can be enhanced by chemical chaperone treatment suggests that for some Ag/MHC combinations some aspect of the APC surface is suboptimal with respect to T cell activation. These data suggest that Ag presentation can be manipulated with chemical chaperones to facilitate improvement of immune responses, and we have provided evidence that this approach may form the basis of an immunization strategy leading to IgG2a/Th1 responses.


   Acknowledgments
 
We thank David Williams for helpful discussion and for critical reading of the manuscript and William Welch for advice on starting conditions for the chemical chaperone experiments.


   Footnotes
 
1 This work was supported by a grant from the National Cancer Institute of Canada (T.H.W.) with funds from the Terry Fox Foundation. T.H.W. is a senior research scientist of the National Cancer Institute of Canada. Back

2 Address correspondence and reprint requests to Dr. Tania H. Watts, Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada. E-mail address: Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; CLIP, class II associate invariant chain peptides; TMAO, trimethylamine oxide; CFTR, cystic fibrosis transmembrane conductance regulator protein; SEA, staphylococcal enterotoxin A; PE, phycoerythrin; HB, homogenization buffer; HEL, hen egg lysozyme; KLH, keyhole limpet hemocyanin; TNP, trinitrophenyl. Back

Received for publication September 5, 1997. Accepted for publication May 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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