And Enhance Igg2a Production in Vivo HLA-DM-Sufficient Antigen

Chemical Chaperones Enhance Superantigen and Conventional Antigen Presentation by HLA-DM-Deficient as well as HLA-DM-Sufficient Antigen-Presenting Cells This information is current as and Enhance IgG2a Production In Vivo of September 24, 2021. Birinder Ghumman, Edward M. Bertram and Tania H. Watts J Immunol 1998; 161:3262-3270; ; http://www.jimmunol.org/content/161/7/3262 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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

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 Downloaded from 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 http://www.jimmunol.org/ 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. The Journal of Immunology, 1998, 161: 3262–3270.

olecular chaperones bind to non-native conformations heterodimers on an invariant trimer (3). In the absence of invariant of proteins and stabilize them against irreversible ag- chain, MHC II is poorly expressed and largely remains aggregated gregation. Through controlled cycles of binding and in the ER (4–6). Thus, invariant chain is considered to be a spe-

M by guest on September 24, 2021 release, molecular chaperones can facilitate the protein-folding cific molecular chaperone of the MHC II biosynthetic pathway. process. Once the native conformation of a protein is achieved, After assembly in the ER, the nonomeric ␣␤Ii complex travels molecular chaperones no longer bind. Thus, molecular chaperones via the Golgi to an endocytic compartment where invariant chain are important in the retention of misfolded proteins as well as in is removed by proteolysis, leaving an MHC II ␣/␤ complex bound the protein-folding process itself (1). to a fragment of invariant chain, class II associate invariant chain The assembly of MHC class II molecules with their peptide peptides, CLIP (7, 8). In the endosome, ␣␤CLIP or larger precur- ligands involves a complex biosynthetic pathway in which molec- sors are acted upon by another specialized molecular chaperone, ular chaperones are involved at several stages (2). Shortly after HLA-DM. HLA-DM binds to MHC II molecules and thereby fa- synthesis in the endoplasmic reticulum (ER),3 individual MHC ␣- cilitates release of CLIP and other unstably bound peptides from and ␤-chains associate with the molecular chaperone calnexin. MHC II molecules (9–19). In addition, HLA-DM can stabilize Heterodimers of MHC II ␣- and ␤-chains are then assembled upon empty MHC II dimers and therefore maintain them in a state suit- an invariant chain (Ii) trimer that also contains bound calnexin. able for peptide binding, hence its definition as a molecular Calnexin dissociates from the Ii-MHC ␣/␤ complex upon comple- chaperone (20, 21). tion of formation of a nonameric complex consisting of three ␣/␤ 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 Department of Immunology, University of Toronto, Toronto, Ontario, Canada from mature MHC II molecules as reflected in the instability of the Received for publication September 5, 1997. Accepted for publication May 27, 1998. CLIP-occupied MHC II dimer to SDS, at least for some MHC II The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance alleles (16, 22). HLA-DM-deficient APC present native protein with 18 U.S.C. Section 1734 solely to indicate this fact. Ags poorly (22, 25). However, HLA-DM deficiency does not im- 1 This work was supported by a grant from the National Cancer Institute of Canada pair surface expression of class II; therefore, DM-deficient cells (T.H.W.) with funds from the Terry Fox Foundation. T.H.W. is a senior research are capable of peptide presentation at the cell surface (22, 25). The scientist of the National Cancer Institute of Canada. affinity of CLIP for different MHC II alleles is quite variable, with 2 Address correspondence and reprint requests to Dr. Tania H. Watts, Department of k Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada. E-mail address: that for A being particularly low (26, 27). As a result, HLA-DM- k [email protected] deficient cells express A in the SDS-stable, CLIP-unoccupied 3 Abbreviations used in this paper: ER, endoplasmic reticulum; CLIP, class II asso- form, and the Ag presentation defect is not as severe as in DM- ciate invariant chain peptides; TMAO, trimethylamine oxide; CFTR, cystic fibrosis deficient cells expressing other MHC II alleles (28). However, in transmembrane conductance regulator protein; SEA, staphylococcal enterotoxin A; k PE, phycoerythrin; HB, homogenization buffer; HEL, hen egg lysozyme; KLH, key- the absence of HLA-DM, T cells specific for A and HEL46-61 hole limpet hemocyanin; TNP, trinitrophenyl. respond poorly to hen egg lysozyme (HEL) protein processed

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 3263 within the APC (25) a defect that can be corrected, at least in part, tario, Canada) and 500 ng/ml Puromycin (Sigma, St. Louis, MO). SEA was by HLA-DM transfection (29). purchased from Toxin Technology (Sarasota, FL). Synthetic HEL46-61 In addition to an effect on protein Ag presentation, HLA-DM- was purchased from the Ontario Cancer Institute Biotechnology Facility (Toronto, Canada). The anti-Ak-producing hybridomas 10-2.16 and 11-5.2 deficient cells have a defect in presentation of the superantigen were obtained from the American Type Culture Collection. The anti-A␣k- staphylococcal enterotoxin A (SEA) by murine MHC II molecules producing hybridoma 39J (41) was kindly provided by W. Wade (Dart- (30, 31). The staphylococcal enterotoxins are 25-kDa proteins that mouth Medical School, Hanover, NH). Abs were purified from hybridoma bind as intact proteins to MHC II proteins outside the peptide- culture supernatants using protein A or G Sepharose (Pharmacia, Piscat- away, NJ). For biotinylation, Abs were dialyzed overnight against 0.1 M binding groove and activate T cells by simultaneously binding to sodium bicarbonate, pH 8.5, followed by incubation with a 10-fold molar the MHC II on the APC and the TCR on T cells expressing par- excess of N-hydroxysuccinimidyl-D-biotin for2hatroom temperature. ticular TCR V␤ segments (reviewed in Ref. 32). The binding of Free biotin was removed by dialysis against PBS. The superantigen SEA SEA to MHC II is peptide dependent, and the affinity of this toxin was biotinylated by the same method using a 10- or 20-fold molar excess for MHC II varies greatly with the peptide bound in the groove of N-hydroxysuccinimidyl-D-biotin over SEA. (33). Indeed, SEA does not appear to be presented efficiently by Treatment of APC with chemical chaperones and b CLIP-occupied A expressed on HLA-DM-deficient T2 cells (30). Ag/superantigen presentation assays Although not exclusively occupied with CLIP (28, 31), Ak molecules expressed on HLA-DM-deficient T2 cells also show a quan- The chemical chaperones DMSO, glycerol (BDH, Analar quality, distrib- uted by VWR Scientific, Mississauga, Canada), and TMAO (Sigma) were titative defect in their ability to bind and present SEA, a defect that diluted in serum-free culture medium and added to the cells at the con- is corrected by HLA-DM transfection (31). In this report we show centrations indicated in the figures. In preliminary experiments, the pres- that the protein and the SEA presentation defect in T2.Ak cells can ence of glycerol appeared to be toxic in the CTLL assay, so subsequent Downloaded from also be partially overcome by treatment of the cells with chemical experiments involved pretreating APC and washing out the chemical chaperones prior to setting up the coculture with T cells (data not shown). Most chaperones. experiments shown are for 20- to 24-h pretreatment. Chemical chaperones were recently defined in studies of dis- For Ag presentation assays, 105 T1.Ak, T2.Ak, T2.Ak/DM cells, or TA3 eases involving defects in protein folding (34). The first such ex- cells (with or without chemical chaperone pretreatment) were incubated ample was the chemical rescue of the ⌬F508 mutant of the cystic 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 fibrosis membrane conductance regulator (CFTR) protein (35, 36). http://www.jimmunol.org/ 18 to 24 h, supernatants were serially diluted and incubated with 104 IL- This mutant has a protein-folding defect that results in the protein 2-dependent CTLL cells. [3H]Thymidine (Amersham Canada, Oakville, being retained within the cell. Treatment of cells expressing this Ont, Canada) was added to the CTLL cultures after 16 h and thymidine mutant with agents known to stabilize proteins in their native con- incorporation was measured 8 h later. formation, including DMSO (35) and glycerol (35, 36), were found Flow cytometry to partially correct the protein-folding defect such that functional protein reached the plasma membrane. Similar results were ob- Cell surface expression of Ak was determined by flow cytometry using a tained with the chemical osmolyte, trimethylamine oxide (TMAO) 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 (35). Here, we show that treatment of APC with the same set of determine the saturating concentration. Biotinylated Abs were detected by by guest on September 24, 2021 chemical chaperones (DMSO, glycerol, or TMAO) partially re- phycoerythrin (PE)-streptavidin or by FITC-streptavidin staining (Molec- stores the ability of HLA-DM-deficient T2.Ak cells to present the ular Probes, Eugene, OR). Following second-step incubation for 30 min at superantigen SEA as well as HEL protein to T cells and has a 4°C, cells were washed with PBS/1% FCS/2 mM NaN3 and fixed with paraformaldehyde. Sensitivity and alignment of the FACSCalibur were general effect on enhancement of conventional Ag presentation. In checked using CaliBRITE beads (Becton Dickinson) with the software vivo, we find that the chemical chaperone glycerol can act as weak program autoCOMP (Becton Dickinson) and Immuno-Check beads adjuvant to promote an anti-TNP Ab response. In particular, we (Coulter, Hialeah, FL) with a standard protocol created with defined ranges find that by the secondary response, glycerol results in enhanced for coefficient of variation and mean fluorescence. CellQuest software IgG2a compared with immunizations in alum or PBS, consistent (Becton Dickinson) was used for acquisition and analysis of data. A thresh- old was set on forward scatter, and forward scatter and side scatter were with a Th1-type response. The mechanism of action of chemical used to gate on live cells. Data are reported as the geometric mean of the chaperones is not known, but treatment of APC with these chap- fluorescence intensity. erones appears to have a subtle effect on enhancing Ab binding to SEA binding to cells was measured using biotinylated SEA and PE- a number of proteins on APC. Thus, chemical chaperones may 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 enhance Ag presentation by stabilizing protein structure at the cell with blocking Ab on ice for 15 to 30 min prior to addition of SEA. Samples surface. were washed twice with PBS/1% FCS/2 mM NaN3 and incubated with PE-streptavidin for 20 min prior to washing, paraformaldehyde fixation, Materials and Methods and analysis by flow cytometry. Cell lines, Abs, and reagents Subcellular fractionation The BXT hybrid cell lines T1 and T2, transfected with Ak, T1.Ak, and T1.Ak, T2.Ak, and T2.Ak/DM (2–5 ϫ 108) were washed with cold PBS T2.Ak, have been described (25). T1 is a wild-type cell line expressing followed by homogenization buffer (HB; 250 mM sucrose in 3 mM imi- human class II genes, whereas T2 has a large deletion in the MHC II dazole, pH 7.4), and resuspended at 108/ml in HB containing a cocktail of region, deleting MHC II structural genes as well as HLA-DM. T1 and T2 protease inhibitors (HBϩ) (10 ␮g/ml aprotinin, 1 ␮g/ml antipain, 1 ␮g/ml cells transfected with Ak, as well as T2.Ak transfected with HLA-DM A pepstatin, 1 mM PMSF, 17 ␮g/ml benzamidine, 10 ␮g/ml leupeptin, and and B genes (T2.Ak/DM, described in Ref. 31) were provided by Peter 0.5 mM EDTA). Each cell suspension was then passed through a 22-gauge Cresswell and Lisa Denzin (Yale University School of Medicine, New 1.5-inch needle using approximately 10 cycles of aspiration and expulsion Haven, CT). The HEL46-61-specific T cell hybrid A2.A2 (37) and the against the wall of the test tube. The final number of cycles was based on H-2d, H-2k B lymphoma TA3 (38) were obtained from L. Glimcher (Har- an endpoint of no more than 1 or 2 intact cells per field in the final count. vard University, Cambridge, MA). The Ad-restricted, OVA323–339-spe- The homogenates were centrifuged at 2700 rpm for 10 min to pellet nuclei. cific T hybrid DO-11.10 (39) was obtained from P. Marrack and J. Kappler The postnuclear supernatant was centrifuged for 20 min at 23,500 rpm (National Jewish Hospital, Denver, CO). The IL-2 indicator line, CTLL, (36,000 ϫ g) in a Beckman L5-50 Ultracentrifuge, using a 50 Ti rotor. The was obtained from the American Type Culture Collection (Manassas, VA). membrane pellet in 1 ml of HB was loaded onto a 23-ml, 17% Percoll All cell lines were grown in RPMI 1640 supplemented with 10% FCS, gradient (Pharmacia LKB Biotechnology, Piscataway, NJ) and centrifuged 2-ME, and antibiotics as previously described (40). T2.Ak/DM was grown for1hat20,000 rpm (36,000 ϫ g) using a 50.2 Ti rotor. Samples (0.8 ml) in the presence of 250 ␮g/ml G418 (Life Technologies, Burlington, On- were collected from the bottom of the tube using a tube piercer (ISCO 3264 EFFECT OF CHEMICAL CHAPERONES ON Ag PRESENTATION model 184, Lincoln, NE), and aliquots of the gradient fractions were stored for1hat37°C followed by incubation at 4°C overnight. Plates were at Ϫ70°C immediately so that all assays could be done on fractions from blocked with 5% skim milk in PBS/0.1% Tween-20 for2hat37°C. Eight the same preparation. All procedures were performed on ice or at 4°C. fivefold serial dilutions of serum in PBS/0.1% Tween-20/2% skim milk For analysis of Ag presentation by gradient fractions, 50-␮l aliquots of were added to wells for2hat37°C. After washing in PBS/0.1% Tween-20, each gradient fraction per well of a 96-well plate were frozen (Ϫ70°C) and horseradish peroxidase-conjugated anti-isotype Abs (Caltag Laboratories, thawed to rupture membrane vesicles prior to setting up cocultures. Burlingame, CA) were added for2hat37°C. Following washing, H2O2 HEL46-61 (100 ␮M) was added, followed by 105 A2.A2. After 10 to 24 h and 2,2Ј-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Sig- at 37°C, supernatants were serially diluted and incubated with 104 IL-2- ma) were added in citrate phosphate buffer, pH 5.0, and color development 3 dependent CTLL cells. [ H]Thymidine (Amersham Canada) was added to was measured after 20 min at OD405. The titer of each serum sample was the CTLL cultures after 16 h and thymidine incorporation was measured determined as the reciprocal of the last dilution that was 2.5 times the 8 h later. background value of normal serum at 1⁄5 dilution. Animals and immunizations Results Female C57BL/6 mice were obtained from Charles River Laboratory (St. k Constant, Quebec, Canada) and used at 8 to 10 wk of age. Mice were Effect of chemical chaperones on SEA presentation by T2.A immunized s.c. with 40 ␮g of TNP-KLH in either glycerol (0.75 M, 2.5 cells M), PBS, or bound to alum. Serum samples were prepared from blood taken by retro-orbital plexus puncture. As discussed above, HLA-DM has been described as a molecular chaperone for MHC II biosynthesis. Recent studies have shown Anti-TNP ELISA that the presence of HLA-DM is important for SEA presentation TNP-specific Abs were determined for IgM, IgG1, IgG2a isotypes. Each independent of its role in CLIP release (31). This led us to spec- well of 96-well plates was coated with 100 ␮lof5␮g/ml BSA-TNP in PBS ulate that it was a chaperone-like effect on protein folding that was Downloaded from http://www.jimmunol.org/

FIGURE 1. Effect of pretreatment of T2.Ak cells with the chemical chaperones glycerol, DMSO, or by guest on September 24, 2021 TMAO on superantigen presentation to A2.A2 T cells. T2.Ak cells (5 ϫ 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 ␮g/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. The Journal of Immunology 3265 responsible for the effect of HLA-DM on SEA presentation by T2 or DMSO in the absence of Ag had no effect on the T cell response cells. To test this idea further, we treated DM-deficient T2 cells (data not shown) (see Fig. 2). Glycerol-mediated enhancement of with several agents that had been previously defined as having SEA presentation was optimal at between 0.75 and 1.0 M glycerol. chemical chaperone activity in studies of mutant CFTR proteins. Higher amounts were toxic to the APC. For DMSO, the optimal Figure 1 shows the effects of overnight treatment of T2.Ak cells concentration was 1.5 to 2.0% v/v. The chemical osmolyte, with either glycerol, DMSO, or TMAO on subsequent presentation TMAO, also showed enhancement of SEA presentation at 0.1 M, of SEA. Coincubation of chemical chaperones with APC and T while higher levels were toxic to the APC and lower levels had cells resulted in a lack of any response as measured in a CTLL little effect (data not shown). assay, due to toxicity of these agents to the CTLL cells (data not Figure 2 shows the kinetics of induction of enhanced SEA pre- shown). Similarly, overnight preincubation of the T cells with sentation by chemical chaperone pretreatment of T2.Ak cells. It chemical chaperones followed by washing resulted in a slight in- should be noted, that the concentration of glycerol used for these hibition in subsequent Ag presentation assays. Therefore, in all experiments was suboptimal (0.5 M), since prolonged treatment of experiments reported here these agents were washed out of the APC with 0.75 M glycerol resulted in more than 50% cell death by APC cultures before adding T cells. It can be seen in Figure 1 that 72 h. It can be seen that the response of the T cells to SEA (10 both glycerol and DMSO pretreatment augment SEA presentation ␮g/ml) presented by T2.Ak was very weak in the absence of pre- by T2.Ak cells in a dose-dependent manner. Addition of glycerol treatment with chemical chaperones. For pretreatment of APC with Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Kinetics of enhancement of Ag presentation by chemical chaperone pre-treatment. T2.Ak cells were treated with glycerol (0.5M), FIGURE 3. Effect of glycerol pretreatment on presentation of SEA to DMSO (1.5%) or TMAO (0.1M) for the times indicated in the figure. After HLA-DM sufficient and HLA-DM-deficient cells. T1.Ak, T2.Ak and preincubation at 37°C, APC were washed and assessed for presentation of T2.Ak/DM cells were incubated with 0.75 M glycerol for 24 h at 37°C in SEA (10 ␮g/ml) to A2.A2 cells as described in Figure 1. As indicated in complete medium followed by washing and then assessed for presentation the figure, T2.Ak cells treated with chemical chaperones were also tested of the superantigen SEA as described in Figure 1. Results are reported as for T cell activation in the absence of SEA. Data shown are for thymidine thymidine incorporation by CTLL in response to undiluted culture incorporation by CTLL in response to undiluted culture supernatant. supernatant. 3266 EFFECT OF CHEMICAL CHAPERONES ON Ag PRESENTATION either DMSO or glycerol, there was little or no improvement in function for the three cell lines after overnight incubation (data not SEA presentation after 2 h, but after 24 h SEA presentation sig- shown). Figure 3 shows that pretreatment with glycerol enhances nificantly improved, with maximal effect by 48 h. The effect of SEA presentation by all three cell lines. Similar results were ob- pretreatment with TMAO on APC was slower to develop, but was tained using pretreatment with DMSO at 1.5% (data not shown). also maximal by 48 h. None of the chaperone-treated APC acti- Thus, the effects of chemical chaperones is not restricted to HLA- vated T cells in the absence of superantigen. DM-deficient cells, although the effect is greatest for the HLA- DM-deficient cell line, T2.Ak. The effect of chemical chaperones on superantigen presentation is not restricted to HLA-DM-deficient APC Effect of chemical chaperones on conventional Ag presentation Figures 1 and 2 show that chemical chaperone treatment can at To determine whether enhancement of Ag presentation by chem- least partially compensate for the SEA presentation defect ob- ical chaperones was unique to SEA presentation, we also tested served in T2.Ak cells. To determine whether this phenomenon was T1.Ak, T2.Ak, and T2.Ak/DM cells for presentation of HEL pro- restricted to HLA-DM-deficient cells, the experiments were re- tein, as well as HEL46-61 peptide, with or without glycerol pre- peated with HLA-DM-sufficient T1.Ak and DM-transfected T2.Ak treatment (Fig. 4). As has been observed by others (28, 31), T2.Ak (T2.Ak/DM) (Fig. 3). In contrast to T2.Ak cells, which have a large shows enhanced peptide presentation compared with T1.Ak, likely deletion in the MHC II region and thereby lack class II expression, due to a higher proportion of kinetically unstable MHC II-peptide T1.Ak cells expresses human class II molecules. Lower amounts of complexes on the surface of the DM-deficient cells. Separate ex- SEA could be used for activation of T cells by T2.Ak because periments showed that the optimum concentration of glycerol for human class II molecules have a much higher affinity for SEA than presentation of these Ags was similar to that seen for SEA (data Downloaded from do murine MHC II molecules (32). For these experiments, we not shown). It can be seen that HEL peptide as well as HEL protein chose a concentration of glycerol (0.75 M) that had been shown in presentation are enhanced by glycerol pretreatment of both HLA- a separate experiment to give the maximum augmentation of APC DM-deficient T2.Ak and HLA-DM-sufficient T1.Ak or T2.Ak/DM http://www.jimmunol.org/ by guest on September 24, 2021

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 1 and the methods. The Journal of Immunology 3267

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

Antibody Untreated ϩ Glycerol, 48 h ϩ TMAO, 48 h

TA3 Cells Control Ab 5 14 8 Anti-ICAM 1 (YN1) 77 97 92 Anti-LFA-1 (FD441.8) 97 138 127 Anti-B7-2 (GL1) 18.0 27.6 22.3 Anti-Ak (10-2.16) 508 636 595 Anti-Ak (11-5.2) 375 462 474 Anti-Ak (39J) 444 504 528 Anti-Ed/k (14-4-4S) 429 535 526 Anti-Ad (MKD6) 557 674 728 Anti-Ad (K24) 550 665 669 T2.Ak Control 6.4 8.1 7.8 Anti-Ak 10-2.16 2490 2369 2920 Anti-Ak 39J 2033 1995 2314

a Samples were incubated with biotinylated Ab as indicated in the table, followed

by FITC-streptavidin. Data are reported as the geometric mean of the ﬂuorescence Downloaded from intensity.

tially compensate for the lack of HLA-DM in allowing presentation of native HEL to A2.A2 T cells.

To further explore the timing of the effects of glycerol on protein http://www.jimmunol.org/ 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 continu- ing to be processed 24 h after addition of Ag, it appears that glyc- by guest on September 24, 2021 erol 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. 5) 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 FIGURE 5. Effect of chemical chaperones on Ag presentation by the enhance both native OVA and OVA323–339 presentation. SEA pre- murine APC, TA3. TA3 cells were either untreated (dashed lines) or presentation on TA3 can involve Ad and Ak as well as Ek/d molecules, treated overnight with 0.75M glycerol followed by washing (solid lines). with E molecules dominating at low SEA concentration (32, 42). A2.A2. T cells were stimulated with TA3 (Ϯglycerol pretreatment) plus Thus, glycerol pretreatment of TA3 seems to affect Ad-restricted and various concentrations of SEA or native HEL as indicated in each panel. perhaps E-restricted presentation to T cells, but has a lesser effect on Ϯ DO-11.10 T cells were stimulated with TA3 ( glycerol pretreatment) plus Ak-restricted presentation by these APC. 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 Does chemical chaperone treatment influence expression of or without chaperone treatment, and dashed lines (untreated) vs solid lines proteins on the APC? (glycerol-pretreated) should be compared for each symbol. One possible mechanism by which chemical chaperones might influence Ag presentation is by altering the expression of cells. For HEL46-61 presentation to the A2A2 T cells, glycerol proteins on the APC surface. We therefore analyzed MHC II pretreatment enhances T cell activation even when peptide is sat- expression as well as the expression of LFA-1, ICAM-1, and urating in the assay. Thus, for T2.Ak, glycerol treatment can par- B7-2, on TA3 cells with and without chaperone pretreatment 3268 EFFECT OF CHEMICAL CHAPERONES ON Ag PRESENTATION

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 6 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 ex- pected, 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. Downloaded from Thus, in contrast to HLA-DM, chemical chaperone treatment does not appear to act on the peptide-loading compartment within the APC.

Effect of glycerol on the immune response to TNP-KLH in vivo The effect of chemical chaperones on Ag presentation in vitro, led us http://www.jimmunol.org/ FIGURE 6. Effect of chemical chaperone pre-treatment on the ability of to hypothesize that these agents might be useful in augmenting im- low density but not high density membrane fractions to present HEL46-61 mune responses in vivo. To test this idea, we immunized mice with 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 TNP-KLH in PBS, alum, or glycerol and analyzed primary and sec- on Percoll as described in Materials and Methods. Each fraction was tested ondary Ab responses (Table II). We chose two concentrations of glyc- for presentation of HEL46-61 to A2.A2 T cells, results are plotted as thy- erol for these experients. We used a concentration that was optimal for midine incorporation by CTLL in response to undiluted culture supernatant the in vitro effects, 0.75 M. In addition, we used a higher concentra- (dilution of the supernatants indicated that the response of the CTLL were tion, 2.5 M, to take into account the dilution effect in vivo. In the not at saturation). Open symbols, no Ag; ﬁlled symbols, ϩ HEL46-61, 100

primary response, the inclusion of glycerol during immunization re- by guest on September 24, 2021 ␮M; dashed lines, untreated APC; solid lines, APC that had been pretreated sulted in higher IgG1 and IgG2a relative to mice that were injected with glycerol. Fractions were collected from the bottom of the Percoll with TNP-KLH in PBS. The response was weaker than that obtained gradient, so that the lowest fraction numbers represent the densest using alum as an adjuvant. Upon secondary immunization with the membranes. same regimen, the difference between PBS-injected vs glycerol-injected mice was more substantial. Interestingly, the presence of glyc- (Table I). Forty-eight-hour pretreatment of TA3 cells with glycerol appeared to favor a higher ratio of IgG2a to IgG1 than in the erol or with TMAO led to a slight (10–20% enhancement) in alum-injected mice. Thus, although the adjuvant effect of glycerol in the geometric mean of the fluorescence intensity for Ab binding d k/d vivo is relatively weak, it may serve as a useful base from which to to A ,E , LFA-1, ICAM-1, and B7-2 molecules. However, build adjuvants favoring Th1 responses. glycerol or TMAO treatment also resulted in an increase in background staining with control Ab (line 1 of Table I). Thus the effect of chemical chaperones on Ag presentation is not Discussion explained by a large increase in expression of any one protein, In this report, we have shown that agents previously shown to have but might be due to the sum of a number of small effects on chemical chaperone activity can enhance the presentation of superan- many surface proteins. Similar experiments were also carried tigens, protein Ags, and peptide Ags to T cells. In contrast to molec- out to analyze MHC II expression on T2.Ak cells with and ular chaperones that bind to non-native conformations of proteins, without chaperone treatment. For glycerol-treated cells, there chemical chaperones are thought to act by stabilizing proteins in their was no significant change in binding of 10-2.16, 39J (Table I) native conformation. Glycerol is thought to stabilize proteins because or 11-5.2 or of the superantigen SEA (data not shown). For it is preferentially excluded from the immediate vicinity of proteins. TMAO-treated cells, the mean fluorescence intensity for bind- This results in an increase in relative hydration around the protein and ing to Ak was increased by 10 to 15%. For each of the Ak- thus enhances the hydrophobic effect that causes the polypeptide to specific Abs, analysis of Ab binding to T2.Ak as a function of maintain a minimum surface area and resist unfolding (discussed in concentration did not reveal significant differences between Ref. 34). Naturally occurring cellular osmolytes act similarly within glycerol-treated or untreated cells, thus it does not appear that cells to stabilize proteins (34). these agents change the affinity of the MHC II/Ab complex For the ⌬F508 mutation in the CFTR protein, chemical chap- (data not shown). erones are thought to subtly alter the conformation of the protein to allow release from molecular chaperones such as calnexin and Chemical chaperone treatment of intact cells does not enhance HSP73 in the ER, thereby allowing the molecules to reach the cell Ag presentation by late endosomes isolated from the cells surface (34). The mechanisms of action of chemical chaperones Previous work by Harding and Geuze (43) and from our laboratory leading to enhancement of Ag presentation in the present study (29) has shown that late endosomes can be used to present Ag to remain unknown. Examination of several proteins on TA3 cells for The Journal of Immunology 3269

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

Primary Response Secondary Response Mouse Adjuvant No. IgM IgG1 IgG2a IgM IgG1 IgG2a

PBS 80 625 Ͻ5 Ͻ5 25 15,625 625 81 125 5 125 5 15,625 625 82 625 5 5 25 78,125 3,125 Alum 76 3,125 125 125 Ͻ5 Ͼ390,625 625 77 3,125 3,125 625 25 Ͼ390,625 125 78 3,125 3,125 625 Ͻ5 Ͼ390,625 3,125 79 3,125 625 625 Ͻ5 Ͼ390,625 3,125 Glycerol 2.5 M 87 625 25 125 125 Ͼ390,625 15,625 88 625 625 125 125 Ͼ390,625 15,625 89 125 125 125 Ͻ5 Ͼ390,625 78,125 90 625 25 125 5 Ͼ390,625 15,625 0.75 M 83 125 25 125 Ͻ5 78,125 78,125 84 125 5 125 25 78,125 15,625

85 125 Ͻ5 125 125 78,125 15,625 Downloaded from 86 625 25 125 125 78,125 78,125

a Mice were immunized s.c. with 40 ␮g of KLH-TNP in adjuvant at days 1 and 21. Blood samples were taken at days 8 (primary) and 27 (secondary), and the sera were tested for the presence of TNP-speciﬁc Ab titers of IgM, IgG1, and IgG2a as described in Materials and Methods.

increased apparent expression showed that LFA-1, ICAM-1, B7 marginal effect on native HEL or HEL46-61 presentation by TA3 http://www.jimmunol.org/ family molecules, and MHC II molecules showed a 10 to 20% cells (Fig. 5) (data not shown). Thus for Ak-restricted responses, a increase in Ab binding after glycerol treatment. It is difficult to human APC may not be optimal for either MHC II folding or evaluate the significance of these effects as background staining A2.A2 T cell activation. However, treatment of murine TA3 cells also goes up after chemical chaperone treatment. While the en- with glycerol had a significant effect on presentation of SEA or hanced Ag presentation is not explained by an increase in surface OVA by TA3 cells. These Ags were presented by Ed/k or Ad, expression of any one molecule, it is possible that chemical chap- respectively. Thus, there may be allele-specific and species-spe- erones subtly stabilize the expression or folding of a number of cific situations in which Ag presentation is suboptimal and can be molecules on the APC surface involved in T cell activation and improved by chemical chaperones. Taken together, the data sug- thereby enhance Ag presentation. This would fit in with the idea gest a general role for chemical chaperones in augmenting presen- by guest on September 24, 2021 that these agents act to stabilize proteins in their native conforma- tation of conventional and superantigens by normal and abnormal tion. Alternatively, chemical chaperones might influence some APC, but the magnitude of the effect appears to depend on the other aspect of the cell surface such as the fluidity of the membrane Ag/MHC/APC combination. and thereby influence cell-cell contact. Glycerol and DMSO are sometimes used as protein stabilizers Treatment with chemical chaperones can partially overcome the and to facilitate solubilization of reagents used in Ag presentation quantitative defect in superantigen presentation by DM-deficient assays. The observation that as little as 1% DMSO or 0.75 M T2.Ak cells and also enhances the level of protein Ag presentation glycerol can have such a large effect on Ag presentation means that by T2.Ak cells (Figs. 1–3). HLA-DM is important in facilitating care should be taken to remove or control for these agents in Ag peptide loading within APC and in stabilizing empty class II mol- presentation experiments. ecules. However, the data presented here do not support a role for Finally, we have shown that immunization with a protein Ag in chemical chaperones in replacing DM function in peptide loading, glycerol leads to an enhanced Ab response in comparison with since glycerol pretreatment enhances Ag presentation whether protein immunized with PBS. In the secondary response, the in- given before or after Ag pulsing (data not shown). Furthermore, clusion of glycerol resulted in a higher ratio of IgG2a/IgG1 ob- the effect of chemical chaperones does not appear to be limited to served in glycerol-immunized vs alum- or PBS-immunized mice. abnormal APC lacking HLA-DM since chemical chaperones en- Enhanced IgG2a over IgG1 production is one of the hallmarks of hance superantigen and protein Ag by cells expressing HLA-DM a Th1 response (44). Alum is the only adjuvant currently licensed (Figs. 3–5). Using subcellular fractionation to separate low density for use in humans but has the drawback that it tends to favor Th2 membranes (early endosomes and plasma membrane) from high responses (45). In cases in which a Th1 response is preferable, density membranes (late endosomes and lysosomes), we find that alternate strategies are required. Although the effects of glycerol as HLA-DM enhances presentation by the dense endosome fraction, an adjuvant are modest at best, the observation that glycerol in- whereas chemical chaperones appear to augment peptide presen- clusion in the immunization protocol enhances the relative pro- tation only by the low density fraction. Thus, although chemical portion of IgG2a over IgG1 suggests that glycerol may provide a chaperones can partially compensate for the absence of HLA-DM, useful base from which to compose adjuvants capable of generat- they appear to act by a distinct mechanism. ing a Th1 response. It is conceivable that chemical chaperones enhance presentation In summary, we have shown that agents previously shown to by T1 and T2 cells because these cells express murine MHC II have chemical chaperone activity in studies of protein-folding mu- molecules in human APC, which might not provide the optimal tants augment presentation of conventional and superantigens by interaction for folding MHC II molecules. Indeed, glycerol pre- HLA-DM-deficient and HLA-DM-sufficient APC. The effect of treatment gave a substantial enhancement of native HEL or chemical chaperones on APC is slow to develop, with little effect HEL46-61 presentation by T1 or T2 cells (Fig. 4), but had only a by 2 h and optimal effect by 48-h chaperone treatment. Chemical 3270 EFFECT OF CHEMICAL CHAPERONES ON Ag PRESENTATION chaperones do not appear to exert their affect on Ag processing or 19. Katz, J. F., C. Stebbins, E. Appella, and A. J. Sant. 1996. 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