Epitope-specific enhancement of antigen presentation by invariant chain

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Epitope-specific Enhancement of Antigen Presentation by Invariant Chain By Frank Momburg, Serge Fuchs,* Johannes Drexler, Robert Busch, Markus Post, Gfinter J. H~immerling, and Luciano Adorinir From the Tumor Immunology Program, German Cancer Research Center, 19-69120

Heidelberg, Germany; *PredinicalResearch, Sandoz Pharma Ltd., CH-4002 Basel, Switzerland; and CRocheMilano Ricerche, 1-20132Milano, Italy

Summary The MHC dass II-associated invariant chain (Ii) is involved in the intracdlular sorting of class II molecules to the endocytic pathway where peptides from processed exogenous antigens are bound, and thereby Ii is thought to enhance antigen presentation. Here we demonstrate that presentation of only one out of five epitopes of a given antigen is augmented by Ii. We have compared the presentation of five different epitopes derived from hen egg white lysozyme (HEL) to Ak-restricted T hybridomas by rat-2 fibroblasts transfected with A~k and A~' (RKK) and RKK cells supertransfected with the mouse invariant chain (RKKI). Only the presentation of the HEL epitope 46-61 was enhanced whereas the presentation of the HEL epitopes 25-43, 3445, 112-124, and 116-129was unchanged or even slightly diminished in RKKI cells. The presentation of the epitopes 25-43 and 34-45 was virtually insensitive to the lysosomotropic reagent chloroquine. Brefeldin A (BFA), which inhibits protein egress from the endoplasmic reticulum, blocked the presentation of all epitopes tested in RKKI cells. In contrast, in Ii-negative RKK cells only the presentation of the epitope HEL(46-61) was inhibited by BFA and the presentation of the epitopes 25-43 and 34-45 was only slightly impaired. These findings suggest that Ii may target class II molecules to selected endosomal subcompartments involved in the processing of different peptides derived from an endocytosed antigen. As a result, the enhancement of the class II-restricted presentation in Ii expressing cells appears to be epitope specific rather than antigen specific.

eT~he MHC class II-associated invariant chain has been .L ascribed a number of different functions. One proposed role is to provide a sorting signal for the intracellular transport ofdass II molecules (1). In HeLa ceils, transport of class II molecules to the endocytic compartment was observed only when Ii was coexpressed (2, 3). Ii probably directs dass II molecules to a late endosomal compartment (4). A second role has been suggested by biochemical studies indicating that Ii can block the peptide binding to dass II o~ dimers (5, 6). Thus, Ii may prevent peptide loading onto class II molecules in preendosomal compartments. A third possible role of Ii is to act as a chaperone in the assembly and folding of dass II molecules, and in their egress from the endoplasmic reticulum (7, 8). These proposed functions suggest that Ii may facilitate the class II-restricted presentation of exogenous antigens to T cells. For Ltk- cells transfected with mouse class II genes in the presence or absence of Ii, Ii expression was reported to enhance the presentation of some antigens like the com1453

plement component C5 (9), hen egg lysozyme or measles virus hemagglutinin (10), but not of other antigens like ovalbumin or pork insulin (11). The crucial role of Ii in the presentation of certain antigens was further demonstrated with splenocytes from mice lacking the Ii gene (12). Recently, it was shown that the alternatively spliced Ii gene product p41 plays a role in the enhanced presentation of some antigens (13). Utilizing rat-2 fibroblasts expressing A k in the presence or absence of Ii, we previously reported that presentation of HEL but not of ribonuclease A (RNase) was enhanced in the presence ofli (14). Extending these findings we now show that the facilitating function of Ii is restricted to only one out of five epitopes derived from processing of HEL, suggesting that Ii may be involved in the fine tuning of the immune response to exogenous antigens. Materials and Methods Transfectants. Rat-2 fibroblasts transfected with cDNA con-

J. Exp. Med. 9 The Rockefeller University Press 9 0022-1007/93/10/1453/06 $2.00 Volume 178 October 1993 1453-1458

structs coding for the o~ and B chain of A k (RKK)have been deResults and Discussion scribed (14). Supertransfection with genomic DNA coding for muThe influence of Ii on the presentation of helper epitopes fine Ii was performed as described (14). RKKI done 4 which derived from HEL was studied using Ak-transfected Iiexpresses high amounts of Ii was used in this study. Ii expression negative rat-2 fibroblasts (RKK), or R K K ceils supertranswas determined by cytoplasmic immunoperoxidase staining and by fected with genomic D N A coding for mouse Ii (RKKI). In immunoprecipitation. The T cell hybridomas 2B6,31 (15), 1C5.1, RKKI cells a high level of Ii expression in excess of A k mol2D4.1 (16), 3A9 (17), 2C8.4 (18), and TS1.2 (19) have been deecuhs was shown by sequential immunoprecipitation with scribed. The T cell hybridomas 3Bl1.1, 4G4.1, 1G5.1, 2B5.1, and 1B9.1 were generated for the present study and characterized as monodonal antibodies against A k and Ii (Fig. 1 a). The exdescribed (18). pression of Ii in R K K I ceUs was homogenous as determined lraraunoprecipitations. Immunoprecipitations were performed as by intraceilular cytofluorometry (Fig. 1 b). The level of A k described (14). Briefly, 3 x 106 cells were labeled for 1 h with 100 cell surface expression was slightly lower in the R K K I done /~Ci of a mixture of 80% [35S]methionine and [35S]cysteine(ICNused in this study than in R K K parental cells (Fig. 1 b), posFlow, Cleveland, OH). The adherent ceils were harvested from the sibly due to intrinsic clonal variability of these ceils. Although dishes with TBS lysis buffer containing 1% NP-40. The lysates Ii is supposed to facilitate the egress of class II molecules from were precleared with Protein A-soluble (Sigma Chemical Co., St. Louis, MO) and a mixture of normal rat and mouse sera. For immunoprecipitations, Protein A-Sepharose beads (Pharmacia, Inc., Piscataway, NJ) preabsorbed with monoclonal antibodies were added to the lysates for 2 h. A k molecules were precipitated with mAb Hl16-32. To precipitate Ii, mAb In1 was used together with the protein A binding anti-rat kappa chain antibody MAR18.5 (for references see 13). The immunoprecipitates were analyzed on 12% SDS-PAGE. Cytofluorometry, Peptide Binding Assay. Surface immunofluorescence staining for A k was performed with mAb Hl16-32 and FITC-conjugated goat anti-mouse Ig (GIBCO-BRL Life Technols, Inc., Gaithersburg, MD). For intracelhlar staining of Ii, cells were permeabilized with 0.3% saponin (Sigma Chemical Co.) and stained with mAb In1 followed by FITC-conjugated goat anti-rat Ig (Southern Biotechnology, Inc., Birmingham, AL). Peptide binding to cell surface A k molecules was measured essentiaUy as described (20). RKK and RKKI ceils were mixed with varying concentrations of biotinylated HEL peptide 46-61 and incubated at 37~ for 3.5 h. Subsequently the cells were stained with successive layers of fluoresceinated avidin D, biotinylated goat anti-avidin D antibody, and again fluoresceinated avidin D (Vector Labs, Inc., Burlingame, CA). Staining was at 4~ for at least 30 min per layer followed by two washes. Class II expression was determined in the same experiment using saturating amounts of FITCconjugated mAb Hl16-32. 5000 cells per sample were analyzed for median fluorescence channel (MFC) intensity on a FACScan flow cytometer (Becton Dickinson and Co., Mountain View, CA). After background subtraction, the effects of variations in A k expression between cells was eliminated by calculating MFC ratios [(MFC with peptide - MFC without peptide)/(MFC with H116-32-FITC - MFC with control Ab)]. Cell Cultures. Antigen presentation assays were performed as Figure 1. (a) Immunoprecipitationof Ak and Ii moleculesfrom metadescribed (13, 14). Briefly, cultures containing 2.5 x 104 prebolically labded RKK and RKKI ceils. For control, untransfected rat-2 senting calls and 5 x 104 T hybridoma cells were set up in micells (R) were used. First Ak was precipitated with mAb Hl16-32 (anti-1crotiter plates with antigen in 200/~1 RPMI 1640 supplemented Ak), then Ii was precipitated from the same lysates with mAb In1 (antiwith 10% FCS. After 24 h of culture, 50/~1 of supernatants were I 0. The positions of the o~ and B chains of Ak and of the p31 form of assayed for the presence of IL-2 by culture with 104 CTLD2 cells li are indicated. Ii coprecipitateswith Ak in RKKI cells but is absent in for 24 h. During the final 4 h of culture, the CTLL-2 cells were RKK cells. As visible for the B chain of Ai, class II molecules are more rapidly processedinto higher glycosyhtedforms in the presenceof Ii. After pulsed with 1 /~Ci [3H]-methyl-thyraidine (Amersham Intl., cleating of class II molecules,high amounts of free Ii were recoveredfrom Buckinghamshire, England). Each point in the graphs of Figs. 2-4 RKKI cells. (b) Surfaceimmunofluorescencestaining of RKK and RKKI represent the mean from triplicate cultures. cells for Ak with mAb Hl16-32. Ii was intracelhlarly stained with mAb RKK and RKKI cells were preincubated with graded doses of Inl afterpermeabilizationof cellswith saponin. (c) Indistinguishablebinding chloroquine (Sigma Chemical Co.) for 30 min or with brefeldin of biotinylated peptide HEL(46-61)to RKK and RKKI cells. Cells were A (BFA) (Sandoz) for 15 min in complete medium followed by incubated with graded amounts of biotinyl-HEL(46-61)and stained with the addition of antigen for 4 h as described (19). Subsequently, the two layersof avidin-FITCsandwichedby biotinylated anti-avidinAb. The ceils were fixed in 0.05% glutaraldehyde for 30 s on ice, quenched MFC ratios shown representrehtive peptide binding, normalizedf~r differin 0.2 M glycine, and washed three times before T hybridoma cells ences in class II expressionthat were determined in the same experiment (see Materials and Methods). were added. 1454 Invariant Chain Enhances Presentation of SpecificEpitopes

Table 1. Activation of A~-restricted T Cell Hybridoraas by Ii-negative RKK Cells and by Ii-positive RKKI Cells ECso for protein (gM) Hybridoma 2B6.31 3Bl1.1 1C5.1 4(34.1 1G5.1 2B5.1 3A9 2D4.1 2C8.4 1B9.1 TS1.2

ECs0 for peptide (gM)





ratio ECs0 RKK/RKKI



HEL 25-43 HEL 34-45 HEL 46-61 HEL 46-61 HEL 46-61 HEL 46-61 HEL 46-61 HEL 112-124 HEL 116-129 HEL 116-129 RNase 43-56

A~ Ak Ak Ak Ak Ak Ak Ak Ak Ak Ak

220 60 150 45 170 200 100 500 500 150 0.5

270 100 2 12 40 7 3 600 500 300 2

0.82 0.60 75.00 3.75 4.25 28.57 33.33 0.83 1.00 0.50 0.25

5.00 3.00 0.05 0.50 0.04 0.30 0.50 0.10 0.13 0.07 1.50

13.00 6.00 0.13 1.10 0.05 0.80 0.80 0.40 0.50 0.30 2.00

The responsiveness of T cell hybridomas to protein antigen (HEL or RNase) or to the indicated synthetic peptide presented by RKK or ILKKI cells was estimated by the antigen concentration (/~M) giving 50% of the maximal response (ECs0). A representative experiment out of three performed with similar results is shown.


cpm x 1000 250 200 150-





I 0.1



"P 1

"P T T 3 10 30 (Antigen) pM

T 100

T T 300 1000


cpm x 1000 120-

the endoplasmic reticulum we have observed similar levels of surface class II on a large variety of class II transfectants such as rat-2, HeLa, and RMA cells in the absence and presence of Ii. This is in contrast to splenocytes from Ii knockout mice, which express only low levels of class II on the cell surface (12). The reason for this discrepancy is not clear. In any case, when differences in I-A k expression were taken into account, the relative ability of surface class II molecules to bind peptides was similar in RKK and RKKI cells (Fig. 1 c). 10 T ceU hybridomas with specificity for five Ak-restricted HEL epitopes were used to study the effect of Ii on the presentation of individual epitopes. In Table 1, a quantitative evaluation of the response curves is given. The ratio of the antigen concentrations yidding 50% of the maximal responses (ECs0) for RICK vs. RKKI cells was greater than 3.0 for all five T cell hybridomas recognizing the HEL peptide 46-61, namely 1C5.1, 4G4.1, 1G5.1, 2B5.1, and 3A9, indicating a dear enhancement of the presentation of this epitope in the presence of Ii. A typical example is presented in Fig. 2 a. It can be seen that RKKI cells stimulated 3A9 T hybridoma



O.03 0.1


~ 1

'-,' ~ T 3 10 30 (Antigen) ~.M

3" 100




300 1000

Momburget al.

Figure 2. Efficiencyof the presentation of the Ak-restricted HEL epitopes 46-61 and 25-43 by Ii-negative and Ii-positive rat-2 cells. RKK cells (ape, symbols) or RKKI cells (closed symbols), 2.5 • 104/well, were incubated for 24 h with graded concentrations of HEL (squares) or the appropriate peptide (triangles) and 5 x 104/weUT hybridoma cells. For control fixed RKKI cells (circles) were incubated with HEL. In (a) hybridoma 3A9 (HEL 46-61) and in (b) hybridoma RB6.31 (HEL 25-43) was used. II~2 production was determined by [3H]thymidine incorporation into CTLL-2 cells. Data are presented as mean cpm from triplicate cultures. Brief Definitive Report

cells at an about 30-fold lower HEL concentration than RKK cells, but that at high HEL concentrations RKK stimulated almost as well as RKKI. For the hybridomas 1C5.1, 2B5.1, and 3A9, the ECs0 ratios were about 10 to 20 times greater than for the hybridomas 4G4.1 and 1G5.1. This difference did not correlate with the sensitivity of the hybridomas as determined by their responses to exogenously added peptide (Table 1). Exogenously added peptide was always presented somewhat more ei~ciently by RKK cells than by RKKI cells. This difference probably reflects the slightly higher level of A k expression in RKK because the degree of loading with peptide was similar for both cell lines (Fig. 1 c). The fine specificities of these hybridomas for shorter peptides within the sequence 46-61 have not been compared. Thus, it seems possible that the differences in the ECs0 ratios are due to

a %response

differentially enhanced presentation of subdeterminants of epitope 46-61. Using the hybridomas 2B6.31 (Fig. 2 b) and 3Bl1.1 recognizing the overlapping HEL epitopes 25-43 and 34-45, respectively, an ECs0 ratio dose to unity was found indicating that Ii did not facilitate the presentation of these epitopes. The hybridomas 2I)4.1, 2C8.4, and 1139.1 recognizing the overlapping COOH-terminal epitopes HEL 112-124 and 116-129, respectively, showed ECs0 ratios
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