Immunology 1993 80 566-573

Inhibition by chloroquine of the class II major histocompatibilitycomplex-restricted presentation of endogenous antigens varies according

to the cellular origin of the antigen-presenting cells, the nature of theT-cell epitope, and the responding T cell

S. LOMBARD-PLATLET,* P. BERTOLINO,* H. DENGt D. GERLIER* & C. RABOURDIN-COMBE**Immunobiologie Moleculaire, Lyons, France and tDepartment of Microbiology and Molecular Genetics, UCLA Los Angeles,

California, U.S.A.

Acceptedfor publication 11 August 1993

SUMMARY

Chloroquine treatment of antigen-presenting cells (APC) was explored as a tool to investigate theprocessing pathway for major histocompatibility complex (MHC) class II-restricted presentation ofthe endogenous secreted hen egg lysozyme (HEL) and transmembrane measles virus haemagglutinin(HA). A 72-hr pretreatment of the APC with 25 yM chloroquine blocked the presentation of theHEL(52-61) T-cell epitope generated from endogenous HEL to the I-Ak-restricted 3A9 T-cellhybridoma by MHC class II-transfected L cells expressing the invariant chain (Ii). The presentationofexogenously added HEL peptides was not affected. Under the same conditions, no inhibition ofthepresentation of HEL(106-116) to the I-Ed-restricted G28 high-avidity T-cell hybridoma, nor ofHAwhen synthesized by L cells, was observed. When B-lymphoid APC were used, inhibition was

observed in every case with a low number ofB APC pretreated for 48 hr with chloroquine prior to theT-cell stimulation test. Moreover, addition of chloroquine to untreated B APC during the T-cellstimulation assay was sufficient to inhibit completely the presentation of HEL(106-116) to theB10.D24.42 low avidity T-cell hybridoma. Altogether these studies suggest that an apparentresistance ofendogenous Ag presentation to chloroquine inhibition may not necessarily indicate theexistence ofa non-endosomal pathway but may be due to the nature of the T-cell epitope, to the use of'non-professional' APC such as L cells, to the use of T cells of high avidity, and to high amounts ofpre-existing MHC class II-peptide complexes expressed by the APC. We demonstrate here that, atleast in conventional APC such as B cells, class II-restricted presentation ofboth endogenous secretedHEL and transmembrane HA involves an endosomal pathway.

INTRODUCTION

It has been shown that endogenous antigens can be presentedeither by major histocompatibility complex (MHC) class I orclass II molecules (reviewed in ref. 1). These data are sustainedby the recent identifications of natural peptides which have beeneluted and sequenced from purified MHC class I or class IImolecules.2'3 Neosynthesized MHC class II molecules aretransitory associated with the invariant chain (Ii). It is thoughtthat the Ii chain shields the peptide-binding groove of class II

Abbreviations: Ag, antigen; APC, antigen-presenting cell; CTL,cytolytic T-cell; ER, endoplasmic reticulum; HA, haemagglutinin;HEL, hen egg lysozyme; Ii, invariant chain; MHC, major histocompati-bility complex; M6P-R, mannose-6-phosphate receptor; MTT, 3-4,5,dimethylthiazol-2yl-2,5-diphenyl tetrazolium bromide.

Correspondence: D. Gerlier, CNRS-ENS Lyon, UMR 49, 46 alled'Italie, 69364 Lyon Cedex 07, France.

molecules in the endoplasmic reticulum (ER) and prevents thebinding of immunogenic peptides in this compartment."4 Otherfunctions have also been proposed for the Ii chain. It is thoughtto act as a chaperone molecule to ensure the proper folding ofclass II molecules and their exit from the ER,5 and targets and/orretains class II molecules to the endosomes/lysosomes." 4 In thislatter compartment proteolytic enzymes responsible for thedegradation of Ii chain and antigens enable MHC class IImolecules to bind to antigenic peptides."16 Antigens which arepresent and thus degraded in the endosomal/lysosomal com-partment can derive from exogenous endocytosed antigens.'The pathways used by endogenous antigens for class II-restricted presentation are still undefined. Indeed, conflictingresults have been obtained using components such as theprotease inhibitor leupeptin or the weak base chloroquine whichblock endosomal proteolysis. Chloroquine acts by increasingthe endosomal/lysosomal pH, thereby inhibiting the activity ofacidic proteases present in this compartment (reviewed in ref. 7).

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Findings based on chloroquine sensitivity led some authors toclaim that transmembrane,8'9 secreted'0 or cytosolic antigens""2'2

are processed through an endosomal pathway whereas the classII-restricted presentation of other endogenous cytosolic'3 andtransmembrane'4'6 antigens are insensitive to chloroquine, thussuggesting the existence of a non-endosomal class II processingpathway.

The main difficulty which arises when trying to assess theeffects of chloroquine on class II-restricted presentation comes

from the pre-existence of MHC class II-peptide complexesexpressed at the surface of antigen-presenting cells (APC), sincethe cells are continuously synthesizing their endogenous anti-gens. Thus to test the blocking effects of chloroquine on theformation of neoformed complexes, the time of treatment withchloroquine should exceed the turn-over time of pre-existingclass II-peptide complexes. In addition, since chloroquineeffects are quickly reversible,'7 blocking effects should bemaintained during the T-cell stimulation assay. This last stepcan be achieved either by fixing the cells with an aldehyde or byfinding conditions for continuous exposure to chloroquine.Since fixation has been shown to alter the presentation capa-

bility of APC (ref. 18 and our unpublished data), we havedefined experimental conditions for long-term treatment ofAPC with chloroquine without toxic effects. When applied toMHC class II-transfected L cells which constitutively synthesizetransmembrane measles virus haemagglutinin (HA) or secretedhen egg lysozyme (HEL), the presentation of endogenous HAand HEL by MHC class II molecules to I-Ed-restricted T-cellhybridomas was not or hardly inhibited by chloroquine. On theother hand, the presentation to the same T-cell hybridomas ofendogenous HEL and HA synthesized by professional APCsuch as B cells was sensitive to chloroquine.

MATERIALS AND METHODS

Antigens and T-cell hybridomasGrade I HEL was purchased from Sigma Chemicals (St Louis,MO). A trypsin HEL digest was obtained as previouslydescribed.'9 HEL (52-61) synthetic peptide was kindly providedby Prof. J. Paris from the Faculty of Pharmacy in Lyons. Thelow avidity BO.D24.42, HEL(106-1 16)-specific, I-Ed-restricted,was kindly provided by Dr E. Sercarz (Los Angeles, CA), andthe high avidity G28 T-cell hybridoma, HEL(106-1 16)-specific,I-Ed-restricted, is described elsewhere (H. Deng, V. Calin-Laurens, B. Jamieson, R. Apple, C. Rabourdin-Combe, D.Gerlier and E. Sercaz, submitted). The 3A9 HEL(52-61)-specific, I-Ak-restricted T-cell hybridoma was a generous giftfrom Dr P. M. Allen (St Louis, Mo). HA plus I-Ed-specificT-hybridoma cells (TH5.124 and TH5.143) were obtained afterimmunization of mice with HA liposomes, as previouslydescribed. '9

Antigen-presenting cellsMouse CH27 B-lymphoblastoid cells were used as APC topresent exogenous HEL to 3A9 T cells. I-Ak-transfected L cellsand I-Ed-transfected L cells continuously secreting HEL,PC31.HEL and CA36.HEL, respectively, have been previouslydescribed,'9'20 as well as I-Ed-transfected L cells expressing lowamounts of endogenous HA, CA36.HA.2.6.20 Mouse M12.4.1B-lymphoblastoid cells synthesizing endogenous HEL or HA,

M12.HEL (clone 3) and M12.HA (clone 5.1), have beendescribed elsewhere.21

Chloroquine treatment and T-cell stimulation assayCulture medium was DMEM (Gibco BRL, Grand Island, NY)supplemented as previously described.'9 Chloroquine was pur-chased from Sigma Chemicals. A 10 mm stock solution wasprepared in culture medium, filter sterilized, and aliquots storedat - 20°. Fresh aliquots were used for each experiment. Forlong-term chloroquine treatment, APC were washed every dayand resuspended in culture medium containing newly addedchloroquine. Just before the T-cell stimulation assay, APC werewashed again and resuspended at a suitable concentration in theabsence or presence of chloroquine. 105 MHC class TI-restrictedT-hybridoma cells were cultured with 3 x 104 B APC or 105fibroblastic APC with or without the addition of exogenousantigen (Ag) and in the absence or presence of the indicateddoses of chloroquine, in a final volume of 200 MI in flat-bottomed microculture plates for 24 hr at 370 in a 7% CO2humidified incubator. The interleukin-2 (IL-2) content ofculture supernatants was measured using the CTL.L2 line.Proliferation of CTL.L2 cells was evaluated with a MTT

21colorimetric assay.

Immunofluorescence analysis3 x I05 cells were incubated for 1 hr with MKD6 anti-I-AdmAb22 in 100 MI phosphate-buffered solution (PBS) containing1% bovine serum albumin (BSA) and 0-1% NaN3. Cells werethen washed three times in the PBS solution and incubated witha 1: 150 dilution of fluorescein isothiocyanate-conjugated goatanti-mouse immunoglobulin (Zymed, San Francisco, CA) in100 M1 PBS solution for 30 min at 4°. After three washes, cellswere fixed in 1% paraformaldehyde and flow cytometricanalyses were performed on a FACScan (Becton Dickinson,Mountain View, CA).

RESULTS

Establishment of experimental conditions for long-term chloro-quine treatment of APC

We first defined a dose range of chloroquine which could beperiodically added in the culture medium for several dayswithout cytotoxic effects. 20 JM or 30 pM of chloroquine for3 days was found to be the highest concentration not affectingcell viability and growth (data not shown) and these chloroquineconcentrations were chosen as working doses. MHC class I cell-surface expression was not affected by long-term chloroquinetreatment; indeed, immunofluorescence profiles obtained withtreated and untreated cells were strictly identical. Furthermore,target cells expressing endogenous transmembrane Ag andpretreated with chloroquine were lysed with the same efficiencyas untreated target cells by class I-restricted CTL (data notshown). Although it has been shown that growing APC inendosomal inhibitors leads to a depletion of MHC class IImolecules from the cell surface,23 under our conditions we didnot observe any significant variation in the level of cell-surfaceclass II molecules expressed by M 12.4.1 cells after 2 days ofchloroquine treatment (data not illustrated). However, the half-life of the Ii chain was increased in M 12.4.1 cells that had beentreated with 25 pM, chloroquine for 18 hr, since some intact Iichain was still immunoprecipitable after 36 hr of chase from

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Figure 1. Chloroquine inhibits presentation ofexogenous HEL, but notof tryptic HEL peptides. Exogenous HEL (a) or HEL tryptic peptides(b) were presented by untreated CH27 cells in the absence of chloro-quine (open squares) or in the presence of 25 pM chloroquine (closedsquares), or by CH27 cells pretreated with 25 pm chloroquine for 72 hrand kept in the presence of chloroquine during the assay (closedtriangles). IL-2 secretion by 3A9 T cells was measured as detailed in theMaterials and Methods.

103 104 105APC cell number/well

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Figure 2. Presentation ofHEL(106-1 16) by I-Ed-HEL-transfected L cellsis not blocked by chloroquine. Endogenous HEL was synthesized byCA36.HA.2.6, which were kept under high cellular density conditions.Presentation to the HEL(106-116) plus I-Ed-specific G28 T-cell hybri-doma was performed with untreated APC in the absence (open circles)or the presence (closed circles) of 25 uM chloroquine, or with APCpretreated with 25 pm chloroquine for 72 hr prior to the T-cellstimulation test (closed triangles). Pretreated APC were kept in thepresence of chloroquine during the assay. IL-2 secretion by G28 T cellswas measured as detailed in the Materials and Methods. Resultsrepresent the mean of triplicate cultures.

the inhibiting effects of chloroquine can be overcome by thepresence of a high concentration of HEL, indicating thatchloroquine doses compatible with cell viability do not totallyblock protein degradation in the endosomes. Similar results

021 (a)

chloroquine-treated metabolically labelled cells, and not fromcontrol cells (data not shown). MHC class II moleculesexpressed by chloroquine-treated cells are still functional in lightof their ability to efficiently present exogenous antigenic pep-tides, as discussed below. Expression of endogenous antigens(secreted HEL and cell-surface HA) by cell transfectants was

also found to be unaffected after prolonged treatment with30 gM chloroquine.

Since the effects of chloroquine have been reported to bequickly reversible,' we tested whether the effects of chloroquineon exogenous Ag presentation is also reversible. CH27 cellsgrown in the presence of 30 pm chloroquine for 72 hr and washedfree of chloroquine just prior to the Ag presentation testpresented exogenous HEL with the same efficiency as untreatedcontrol cells (data not shown). Moreover, APC pretreated withup to 500-pm chloroquine for I hr, then washed, remained viableand fully recovered their Ag presentation capability (data notshown). Thus, chloroquine needs to be constantly presentthroughout the T-cell stimulation test, even if cells have beenpretreated with chloroquine. We verified next that when 25 gMchloroquine was added to untreated CH27 cells or to CH27 cellswhich were pretreated with 25 gM chloroquine for 72 hr, thepresentation of exogenous HEL to 3A9 T cells was blocked tothe same extent (Fig. Ia), but the presentation of exogenouslyadded synthetic HEL peptides was not impaired (Fig. I b).Therefore, the presence of 25 pM or 30 gm chloroquine does notaffect the ability of the T-cell hybridoma to be activated and tosecrete IL-2 nor the CTLL-2 bioassay. Figure I a also shows that

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Figure 3. Presentation ofHEL(52-6 1) by I-Ak-HEL-transfected L cells issensitive to chloroquine. PC31.HEL cells were kept under high densitycellular conditions. (a) Presentation to the HEL(52-61) plus I-Ak_specific 3A9 T-cell hybridoma was performed with untreatedPC31.HEL cells in the absence (open circles) or the presence (closedcircles) of25 pm chloroquine, or with PC3 1 HEL cells pretreated with 25pM chloroquine for 72 hr prior to the T-cell stimulation test (closedtriangles). Pretreated cells were kept in the presence of chloroquineduring the assay. (b) Untreated PC3 1 HEL (closed circles) or pretreatedPC3 1 .HEL cells (closed triangles) were incubated in the presence of 25pM chloroquine with the indicated concentrations of HEL(52-61)synthetic peptide and used to stimulate 3A9 T cells.

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Chloroquine blocks class II presentation of endogenous Ag

were obtained using M 12.4.1 B cells to present exogenous HELto I-Ed- and I-Ad-restricted T cells (data not shown).

Therefore, the use of 25 or 30 yM chloroquine is compatiblewith cell viability and bioassays, and efficiently inhibits thepresentation of exogenous HEL without impairing the presen-tation of exogenously added synthetic peptides. Thus, 25 gM ofchloroquine was used to study the sensitivity of class II-restricted presentation of endogenously synthesized antigens.

MHC class II-restricted presentation of endogenously secretedHEL and transmembrane HA is slightly or not inhibited bychloroquine using transfected L fibroblasts as APC

To study the presentation ofendogenously secreted HEL, MHCclass JI-transfected L cells supertransfected with HEL cDNA,CA36.HEL and PC31.HEL, were used as APC. Since efficientpresentation ofendogenous HEL has been shown to require theexpression of the Ii chain, the experiments were performed incellular confluence conditions which induce high levels of theendogenous Ii chain in L cells.'9'20 I-Ed-transfected CA36.HELcould present HEL(106-116) to the high avidity G28 T-cellhybridoma. No inhibition of the presentation of HEL(106- 116)to G28 T cells was observed when 25 gm chloroquine was addedduring the T-cell stimulation test even if CA36.HEL cells were

pretreated for 72 hr with 25 gm chloroquine (Fig. 2). No longerchloroquine pretreatments were performed, due to toxic effectsseen after that time. On the other hand, when I-Ak-transfectedPC31.HEL cells were used as APC, the presentation of HEL(52-61) to 3A9 T cells was almost completely inhibited when theAPC were pretreated with 25 gM chloroquine for 72 hr prior to

the assay (Fig. 3a). A control experiment was performed to showthat PC31.HEL cells which underwent long-term chloroquinetreatment presented exogenous HEL(52-61) synthetic peptide to3A9 T cells with the same efficiency as untreated cells (Fig. 3b).The same results have been obtained with another fibroblasticcell line, KKI.HEL, which also secretes-endogenous HEL andhas been supertransfected with the Ii chain (data not shown). Noexperiments were performed in the absence of expression of theIi chain by the APC, since we have previously shown thatendogenous HEL is not or very poorly presented by MHC classI-transfected fibroblasts in that case.'9'20

We next analysed the effects of chloroquine on the presen-

tation ofanother endogenously synthesized antigen, the measlesvirus transmembrane haemagglutinin (HA). We have pre-

viously shown, using as APC class TI-transfected fibroblastswhich synthesize a low amount of HA, that the efficiency ofpresentation of this endogenous Ag is also dependent on thepresence of the Ii chain.20 The experiments were then performedin the same conditions as above, where endogenous Ii chain wasexpressed at a high level by the fibroblastic APC. We used twodifferent HA-specific T-cell hybridomas, which probably recog-

nize two different antigenic determinants.'9 No inhibition of thepresentation of endogenous HA to TH5.124 T cells andTH5.143 T cells was observed when CA36.HA.2.6 cells were

pretreated with 25 gM chloroquine for 72 hr prior to thestimulation test (Fig. 4a,b). Again, longer chloroquine treat-

ments of the APC could not be performed because of toxiceffects seen after this time. The absence of chloroquine sensiti-vity was also obtained in conditions where the presentation of

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102 103 104 65APC cell number/well

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Figure 4. Presentation of endogenous HA by I-Ed-transfected L cells isnot inhibited by chloroquine. CA36.HA2.6 cells were kept under highdensity cellular conditions. Presentation ofendogenous HA to TH5. 124(a) and TH5. 143 T-cell hybridomas (b) was performed with APC whichwere untreated (open circles) or pretreated with 25 juM chloroquine for72 hr (closed circles) prior to the T-cell stimulation test. Pretreated cellswere kept in the presence of chloroquine during the assay.

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Figure 5. The degree of inhibition ofchloroquine on the presentation ofHEL(106-116) from endogenous HEL by B cells varies according to

different T-cell hybridomas. HEL(106-116) was presented to G28 T cells(a) or BlO.D24.42 T cells (b) by untreated M12HEL cells in the absence(open circles) or in the presence (closed circles) of 25 pM chloroquine, or

by M 12HEL cells pretreated for 48 hr with 25 pM chloroquine prior to

the assay (closed squares). Pretreated APC were kept in the presence ofchloroquine during the assay.

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0-4-

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02-

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12 103 104 105APC cell number/well

Figure 6. Presentation of endogenous HA by B APC is sensitive tochloroquine. M 12.HA cells were left untreated (open circles), orpretreated with 25 gM chloroquine (closed circles) for 48 hr prior to theT-cell stimulation test. Pretreated cells were kept in the presence of freshchloroquine during the assay, performed with TH5.124 T cells (a) andTH5. 143 T cells (b).

endogenous HA does not require the expression of the Ii chain,20i.e. using MHC class II-transfected L cells as APC which expressa high amount of endogenous HA and no endogenous Ii chainwhen grown below confluence (data not shown).

These results demonstrate that, when using L cells as APC,chloroquine does not affect the MHC class II-restricted presen-tation of endogenous HEL(106-116) to I-Ed-restricted G28 Tcells nor the presentation of endogenous HA to TH5.124 andTH5.143 T cells, whereas inhibition of the presentation ofendogenous HEL(52-61) to I-Ak-restricted 3A9 T cells requiredlong-term pretreatment of the APC. We analysed then whetherthis relative lack of sensitivity to chloroquine was still observedwhen 'professional' APC such as B cells where used as APC.

Presentation of endogenously secreted HEL and transmembraneHA synthesized by B-lymphoid cells are both sensitive tochloroquine

The B-lymphoblastoid M 12.HEL cell line, which aftertransfection stably secretes HEL in the extracellular medium,was used as APC. When the APC cell number was equal orsuperior to 0-75 x 104 per well, no inhibition of the presentationof HEL(106-116) to G28 T cells was observed, whetherchloroquine was added to untreated M12.HEL cells or to cellsthat had been pretreated for 48 hr (Fig. Sa). However, below thisnumber of APC, a clear inhibition of the presentation was seen(Fig. Sa). The same degree of inhibition was obtained whenM12.HEL cells were pretreated for 72 hr with chloroquine (datanot shown). Surprisingly, results obtained with anotherHEL(106-1 16)-specific T-cell hybridoma, B10.D24.42, werestrikingly different, since in this case addition of 25 gmchloroquine solely during the T-cell stimulation test completelyinhibited the presentation of endogenous HEL, whatever thenumber of non-pretreated M 12.HEL cells used (Fig. Sb).

We subsequently tested the effects of chloroquine on the

presentation of endogenous transmembrane HA by M 12.HAcells. Again, when titrating the number ofAPC, the presentationof endogenous HA using APC which were pretreated for 48 hrwith 25 gM chloroquine was significantly inhibited (Fig. 6a andb). Inhibition ofTH5. 124 T-cell response was seen when I03 andbelow pretreated M12.HA cells were used per well (Fig. 6a),whereas inhibition of the TH5. 143 T-cell response was obtainedwith a higher number of pretreated Ml 2.HA cells per well(Fig. 6b). The same inhibition was obtained with a 72-hrpretreatment (data not shown). When 25 yM chloroquine wasadded to untreated M12.HA cells during the T-cell stimulationtest no inhibition of the presentation of endogenous HA wasobserved, whatever the number ofM 12.HA cells used (data notshown).

Taken together, these data indicate that the MHC class II-restricted presentation of endogenously secreted HEL andtransmembrane HA synthesized by B lymphoid cells is sensitiveto chloroquine.

DISCUSSION

The presentation of endogenously synthesized antigens byMHC class II molecules has been reported previously to involveeither an endosomal8-'2 or a non-endosomal (so-called 'endoge-nous')13 16 processing pathway. One of the tools which can beused to explore the pathway used by endogenous antigens is touse a drug able to block relatively specifically the endosomalpathway.

Chloroquine is a weak base that is rapidly taken up by cellsand accumulates reversibly in endocytic vesicles within I and 2',causing an increase in vesicle pH. Incubating fibroblasts with 25pM of chloroquine induces a pH elevation of 1-3 unit.'7 As aresult, the activity ofendosomal acidic proteases is inhibited, asshown by the inhibition of the degradation of endocytosedproteins; this inhibition can be observed with only 10 yMchloroquine (reviewed in ref. 7). In addition, neutralization ofacidic pH in endosomes by chloroquine can perturbate therecycling of receptors such as the asialoglycoprotein receptorand mannose-6-phosphate receptors (M6P-R).24 This inhibitionis ligand-dependant, and results in accumulation of ligand-receptor complexes in the endosomes. Prolonged treatment with10-30 pM chloroquine induces depletion of the pool of M6P-Rfree from ligands and as a consequence the neosynthesizedlysosomal proteolytic enzymes are secreted and are no longerdelivered to the lysosomes.24 However, chloroquine does notseem to affect the intracellular traffic between organelles.Indeed, weak base-mediated inhibition of M6P-R trafficking iscaused by the lack of ligand-receptor dissociation and not by ablockage of recycling per se, since blockage can be reversed byadding M6P as a competitive ligand.24 Furthermore, recyclingbetween cell-surface and early endosomes, as well as trafficbetween endosomes and lysosomes, is unaffected by chloro-quine.25 Finally, chloroquine does not inhibit constitutivesecretion and endocytosis (ref. 26, and C. Gimenez unpublisheddata). Side-effects of chloroquine, such as swelling of lysosomesand toxicity in several cell types, have been reported butoccurred only at high doses of chloroquine (reviewed in ref. 7).Collectively, these data show that any inhibiting effect on cellfunction observed with low doses of chloroquine is primarily, ifnot uniquely, due to the neutralization of acidic pH in

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endosomes and, as a consequence, to the inhibition of theproteolytic activity of endosomal/lysosomal enzymes.

We report here that different parameters must be taken intoconsideration when evaluating the inhibiting properties ofchloroquine on the presentation of endogenous Ag; first, thecellular origin of the APC; second, the nature of the T-cellepitope; third, the global amount of MHC class IT-peptidecomplexes; and fourth, the avidity of the interactions betweenthe APC and the T cells. When we used class II-transfectedL cells as APC, the chloroquine sensitivity varied according tothe T-cell epitope and/or to the avidity of the responding T cell.No inhibition of the presentation ofendogenous HEL(106-116)and HA to I-Ed-restricted T cells was obtained, even when theAPC were pretreated for 72 hr before the T-cell stimulation test,although under the same conditions presentation ofendogenousHEL(52-61) to I-Ak-restricted 3A9 T cells was blocked. There-fore the lack of sensitivity to chloroquine is not a property ofL cells per se, but seems to be related to the T-cell epitope.Several hypotheses can be made to explain this difference; eitherthis is evidence of a non-endosomal processing of de novosynthesized HEL and HA in L cells, giving rise to the HEL(106-116) T-cell epitope and two HA T-cell epitopes; or endogenousHEL and HA can be partly degraded even at the more basic pHinduced by the presence of chloroquine; or, finally, in fibro-blastic APC MHC class I-peptide complexes are exceptionallylong-lived due to high-affinity peptides and absence of class IIrecycling.27 We do not favour the first hypothesis since it seemsunlikely that the processing ofendogenous HEL in L cells wouldresult both from a non-endosomal and an endosomal/lysosomalmechanism, resulting in the generation of HEL(106-116) andHEL(52-61) T cell epitopes, respectively. There is more evidencefor the second and third hypotheses, which are not mutuallyexclusive, together with the existence of an endosomal process-ing mechanism. We believe that the lack of chloroquinesensitivity observed in the presentation of endogenous HA orHEL(106-116) using L cells as APC, reflects a steady degrada-tion ofHA and HEL in the endosomes/lysosomes still occurringat the pH buffered by chloroquine, together with a long half-lifeof MHC class II-peptide complexes pre-existing at the surfaceof L cells. Experiments performed after the disappearance ofthese supposedly long-lived complexes would require very long-term chloroquine treatments incompatible with cell viability.On the other hand, the generation of the HEL(52-61) determi-nant has been shown to require the integrity of lysosomalfunctions,28 and is therefore more sensitive to elevation of pH.Furthermore we have previously shown that the presentation ofendogenous HEL(52-61), HEL(106-116) and HA, when synthe-sized in low amounts, is dependent on the expression of the Iichain by the APC (ref. 20 and data not shown). Among differentfunctions attributed to the Ii chain, one is to prevent the bindingof peptides onto neosynthesized MHC class II molecules in theER;',4 the other is to target/or retain class II molecules in theendosomes/lysosomes where it is subsequently degraded, there-by allowing the binding of peptides to class II molecules beforetheir cell-surface expression"4. Therefore, altogether our dataindicate that in L cells, the generation of endogenous HEL(52-61), HEL(106-116) and HA T-cell epitopes, which are depen-dent on Ii expression for class II-restricted presentation (whensynthesized in low amounts), should occur through an endoso-mal processing mechanism. The only other report published sofar which studies chloroquine effects on the MHC class II-

restricted presentation of an endogenous Ag synthesized byfibroblastic L cells shows that processing ofmeasles virus matrixproteins, in the absence of the expression of the Ii chain, istotally unaffected by a 12-hr treatment with chloroquine.'3 Weextend these data with the presentation of high amounts ofendogenous measles virus HA, which does not require theexpression of the Ii chain (data not shown). The possibility of anon-endosomal processing in these last two examples remainsopen, but cannot be solely inferred from the insensitivity tochloroquine inhibition.

Since fibroblastic L cells do not express MHC class IImolecules in physiological conditions, and are unable to recyclethese molecules when transfected,27 they are not considered as'professional' APC. The same experiments were thereforeperformed using conventional APC such as B cells. In this casethe presentation ofboth endogenous HEL andHA was sensitiveto chloroquine. However, the inhibition of presentation to theHEL-specific G28 T cells and HA-specific TH5.124 andTH5.143 T cells was only observed in the presence of a lownumber of titrated B APC, i.e. with a low number ofMHC classTI-peptide complexes. This is consistent with the very highefficiency of MHC class II-restricted presentation of endoge-nous HEL and HA,20 and is reminiscent of the observationshown in Fig. la, where chloroquine inhibition of the presen-tation of exogenous HEL was only seen for low amounts ofHEL, i.e. when the number ofMHC class II-peptide complexesis limited. Another report has claimed that MHC class II-restricted presentation of the transmembrane fusion glycopro-tein of measles virus synthesized by B lymphoid cells isinsensitive to chloroquine, therefore reflecting a non-endosomalendogenous processing pathway.'6 This observation was madewith one single number of APC probably synthesizing highquantities ofendogenous Ag. In light ofour data, we believe onemust be very cautious before interpreting experiments per-formed with chloroquine in the presence of high doses of Agand/or a high number of APC. Furthermore, there was astriking difference between the responses obtained in thepresence of chloroquine with the HEL(106-116)-specific G28and B IO.D24.42 T-cell hybridomas. Addition ofchloroquine tountreated M 12.HEL cells during the T-cell stimulation test onlywas sufficient to completely block the response of BlO.D24.42 Tcells whereas, as said above, a 48-hr pretreatment with chloro-quine together with titration of M 12.HEL was necessary toinhibit the response of G28 T cells. Since HEL(106-116) is adominant antigenic determinant, the different results obtainedwith G28 and BIO.D24.42 T cells are probably due to differentavidities resulting from the molecular interactions involvedbetween M12.HEL cells and the T-cell hybridomas. Therefore,the effects of chloroquine on the formation of MHC class II-peptide complexes are seen more drastically when interactionsof low avidities are involved between APC and T cells. Finally,since the presentation of the HEL(106-116) and HA T-cellepitopes was not impaired by chloroquine when the Ag weresynthesized by L cells, whereas both were sensitive to chloro-quine when the Ag were synthesized by B cells, it can bequestioned whether recycling of MHC class II moleculesoccurring in murine B cells,27'29 which has been reported to beunaffected by chloroquine,29 may lead to class II-peptidecomplexes which possess a shorter half-life than the complexesexpressed in fibroblastic cells.

If, as shown by the sensitivity to chloroquine, degradation of

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572 S. Lombard-Platlet et al.

endogenous antigens in B cells occurs in the endosomal/lysosomal compartment, how can they reach this compartment?We have previously reported a very efficient MHC class II-restricted presentation by B-lymphoid cells of endogenoustransmembrane HA and secreted HEL.2' Transmembrane HAmay be diverted from the secretory pathway to the endosomesand/or reach the endosomes after internalization from the cellsurface. Although we have shown that presentation of endoge-nous HEL is not due to recapture of the secreted molecule fromthe extracellular medium, it is possible that processing ofendogenous HEL results from two different mechanisms. Assuggested by us and others,'0'2' some degree of intersectionbetween the secretory and endocytic pathways may occur. Aportion of neosynthesized HEL may escape the secretorypathway and enter the endosomal compartment; concomi-tantly, some secreted HEL molecules may be locally trapped inthe cell coat and rapidly endocytosed. Alternatively, peptidesderived from endogenous antigens may be produced outside theendosomal/lysosomal compartment but then have to be routedthere for efficient binding to MHC class II molecules. Thuschloroquine may act not by inhibiting endogenous antigendegradation in the endosomal compartment but simply bymodifying the physico-chemical conditions present duringpeptide binding to MHC class II molecules in this samecompartment. Indeed, most peptides bind more efficiently toMHC class II molecules at an acidic pH.30

Altogether these studies show that an apparent resistance ofendogenous Ag presentation to chloroquine inhibition may notnecessarily indicate the existence of a non-endosomal pathwaybut may be due to the nature of the T-cell epitope, to the use of'non-professional' APC such as L cells, to the use of T cells ofhigh avidity, and to high amounts of pre-existing MHC class II-peptide complexes expressed by the APC. After careful controlof these parameters, we have provided evidence that twodifferent endogenous Ag, one which is transmembrane the othersecreted, when synthesized by B lymphoid cells, are processedfor MHC class II-restricted presentation through a chloro-quine-sensitive pathway. This was observed with every T-cellhybridoma tested, but under the condition that the number of BAPC was low, i.e. that the number of MHC class II-peptidecomplexes was low. We have previously reported that everyMHC class II-restricted T cell recognizing exogenous HA orHEL does also recognize their endogenous counterparts whensynthesized and translocated into the ER, indicating that the setof peptides derived from the degradation of exogenous andendogenous forms are similar if not identical.2' Taken together,these data favour the interpretation that, like the processing ofexogenous antigens, the formation ofcomplexes between MHCclass II and peptides deriving from endogenous antigens in Bcells occurs in the endosomal/lysosomal compartment irrespec-tive of the delivery mechanism to this cell compartment.Whether binding of peptides derived from endogenous antigenswith MHC class II molecules can occur physiologically in'professional' APC in a non-endosomal compartment remainsto be established.

ACKNOWLEDGMENTSThis work was supported in part by grants from INSERM (CRE no.92061) and ARC (CRC no. 92-6108). The authors are grateful to J.Mariansky for providing cytotoxic T cells and targets used as controlsfor this study, E. Sercarz for the BlO.D24.42 and 6D3.9 T-cell

hybridomas, J. Paris for the generous gift of HEL(52-61) syntheticpeptide, A. Thomas for the flow cytometry analyses, and C. Gimenez forperforming a control experiment. The authors wish to thank K.L. Rockfor his advice and K. Belorisky, I. Fugier, D. Naniche, M.-C. Trescol-Biemont and G. Varior-Krishnan for helpful discussions and criticalreading of the manuscript.

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