Targeting major histocompatibility complex class II molecules to the cell surface by invariant chain...

Eur. J. Immunol. 1994.24: 873-883 MHC class I1 recycling 873

Targeting major histocompatibility complex class I1 molecules to the cell surface by invariant chain allows antigen presentation upon recycling”

Marga Nijenhuis, Jero Calafat , Karel C. Kuijpers., Hans Janssen, Marcel de Haas, Tommy W. Nordengo, Oddmund BakkeO and Jacques J. Neefjes

The Netherlands Cancer Institute, Amsterdam, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB)., Amsterdam, and MCB, Department of Biology, University of OSlOO, Oslo

We studied the functional consequences of targeting class I1 molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class I1 molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class I1 molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation.When class I1 molecules are targeted to the cell surface by Ii lacking either 15 (A15Ii) or 23 (A23Ii) amino acids from the cytoplasmic domain, a fraction of free class I1 molecules is also observed. Whereas A15Ii did not affect antigen presentation by class I1 molecules, A23Ii inhibited, but did not abrogate, the response. We show that class I1 molecules expressed in the presence of A23Ii can be internalized, followed by degradation of A23Ii and return of free class I1 aP heterodimers to the cell surface. A fraction of the resulting free class I1 molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class I1 molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class I1 molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class I1 molecules. The number of class I1 molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.

1 Introduction

MHC class I1 molecules associate with fragments of proteins in the endocytic route and present them to CD4+ T cells (for reviews see [1-31). Early during biosynthesis, the class I1 a and f~ chain associate with a third subunit, the invariant chain or Ii [4-61. This heterotrimeric complex is transported from the endoplasmic reticulum (ER) to the trans-Golgi reticulum, where it is sorted to endocytic compartments [7-91. Ii is responsible for the sorting of class I1 molecules to the endocytic route [lo, 111. Where

[I 124861

* This research was supported by grant 900-509-155 from the Netherlands Organisation for Scientific Research (JN) and grant 89-14 from the Dutch Cancer Foundation (MN).

Correspondence: Jacques J. Neefjes, The Netherlands Cancer Institute, Plesmanlaan 121, NL-1066 CX Amsterdam,The Nether- lands

Abbreviations: Ii: Invariant chain MIIC: MHC class I1 compartment Al5Ii, AZ3Ii: Ii lacking 23 (15) amino acids from the cytoplasmic domain B-LCL: B tymphoblastoid cell line CHX: Cycloheximide Tfr: Transfenin receptor

Key words: Major histocompatibility complex class I1 recycling I Invariant chain mutants /Antigen presentation / Major histocompatibility complex I1 compartment / Intracellular targeting

and how class I1 molecules associated with Ii enter the endocytic route is still unclear. Class II/Ii complexes have been reported to be sorted to different intracellular loca- tions: (1) to compartments named MIIC, which are multilaminar late endocytic compartments with lysosomal char- acteristics, found in B cells [12] and monocytes unpub- lished observations); (2) throughout the endosomal route in melanomas [13] or COS cells. In the COS cells the location in the endocytic route was dependent on the amount of Ii expressed [14]; (3) to early endosomes in B cells [15]; (4) to “sacs” in monocytes [16], and (5) to the cell surface [17, 181.

After arrival in endocytic compartments, Ii is degraded allowing class I1 molecules to bind peptides [19,20] that are generated in lysosomes [21]. Degradation of Ii is also required for egress of class I1 molecules from the endocytic pathway [22]. Class I1 molecules devoid of Ii and loaded with peptide are then transported to the cell surface.

Although the major route of intracellular transport of class I1 molecules seems to be clear, alternative pathways can not be excluded. For example, Ii-associated class I1 molecules have been found at the cell surface of B cells [17, 181 and are internalized [MI. Following degradation of Ii, peptide-loaded class I1 molecules can, in principle, reappear at the cell surface. Rapid recycling of class11 molecules through early endosomes has been observed [23]. However, no biochemical evidence has been obtained for peptide binding of class I1 molecules during recycling. Lanzavecchia et al. [24] have shown that the half-life of class I1 molecules in B cells is similar to that of associated

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1994 00 14-2980/94/0404-0873$10.00 + .25/0

874 N. Nijenhuis, J. Calafat, K. C. Kuijpers et al. Eur. J. Immunol. 1994. 24: 873-883

peptide, which suggests that recycling of class I1 molecules is insignificant for peptide binding. Other authors did observe peptide exchange [25], but it is unclear whether this is due to recycling or occurs at the cell surface. Another puzzling observation is that class 11-restricted presentation of antigen has been reported to be dependent on Ii in some cell types [26] but not in others [27-311 or seems to be dependent on the antigen [32-341. Recycling of class 11 molecules at low frequency in a cell type-dependent manner could be an explanation for these apparently contradictory data.

To investigate whether recycling of class I1 molecules affects antigen presentation, we have generated transfectants in which class I1 molecules are targeted directly to the cell surface by truncated forms of Ii. We show that class I1 complexes associated with Ii lacking 23 amino acids from the cytoplasmic domain (A23Ii) are internalized, followed by degradation of the truncated Ii and reappearance of free class I1 complexes at the cell surface. This cycle does not result in efficient antigen presentation. However, co- expression of Ii lacking 15 amino acids (Al5Ii) with class I1 molecules does result in efficient antigen presentation. In all transfectants (also those lacking Ii) class I1 molecules are found in characteristic endocytic multilaminar compartments that resemble the MIIC compartments observed in B cells [12]. The number of class I1 molecules present in the multilaminar MIIC-like compartments, as detected by immunoelectron microscopy, correlates with the efficiency of antigen presentation.

2 Materials and methods

2.1 Antibodies

Antibodies used were anti-class I1 mAb Tii36 [35, 361 and AB4 [37], anti-Ii mAb Bu45 [17], anti-transferrin receptor mAb 66Ig10 [38], anti-CD63 mAb 435 [39] and rabbit polyclonal anti-class I1 ci chain serum [S].

2.2 DNA constructs

cDNA encoding the DR1 a and f3 chain [40,41] were a kind gift from Dr. J.Trowsdale. cDNAencoding both the 35- and 33-kDa form of human Ii was isolated by Claesson et al. [42]. cDNA encoding A15Ii and A23Ii were generated as described [ 101. cDNA encoding full length human transferrin receptor (Tfr) was kindly povided by Dr. C. Schneider [43]. All cDNA were expressed in the vector pRc/CMV (Invitrogen) under the control of enhancer/promoter sequences derived from the immediate early gene of human cytomegalovirus [44].

The hybrid Tfr-Ii construct was made by PCR as follows: a DNA fragment encoding the cytoplasmic domain of Tfr [amino acids (aa) 1-61] and 18 nucleotides of the transmembrane domain of Ii was constructed by PCR amplification of theTfr cDNAwith the primers 5' CCT CAA AGC TTC GGG ATATCG GGT GGC GGC TC 3' (containing part of the leader sequence of the Tfr cDNA) and 5' GCC TGT GTA CAG GGC TCC CCT TTT TGG TTT TGT GAC 3' (with 18 nucleotides complementary to the Tfr cDNA and 18 nucleotides complementary to codon 31 to 36

of Ii [42]). A DNA fragment containing the lumenal part of Ii and 18 nucleotides of the transmembrane part of Tfr was constructed by amplification of Ii cDNA with the primers 5' CTT GGG AAG C'IT CAT GCG 3' (identical to codon 78 to 83 of Ii [42] and containing the internal HindIII site of the cDNA) and 5' GTC ACA AAA CCA AAA AGG GGA GCC CTG TAC ACA GGC 3' (complementary to the 36 nucleotide primer used for the amplification of Tfr cDNA). The final PCR fragment was constructed by annealing the fragments mentioned above and amplification with the two outer primers. The sequence was confirmed by M13 sequencing.The final fragment was digested with HindIII and cloned in the pRdCMV vector containing the part of the Ii cDNA downstream of the internal HindIII site.

2.3 Cell lines and transfections

The human embryonic kidney cell line 293 (ATCC CRL 1573) [45,46] was transfected with cDNA encoding DR1 ci and f3 chains using the calcium phosphate precipitation method [46] and selected with 1000 pg/ml G418. A clone expressing both class I1 a and f3 chains was selected by single-cell cloning and used as recipient for the Ii constructs, which were cotransfected with the plasmid pSV2a3.6 [47] and selected with 1 p~ ouabain. Positive clones were isolated and maintained in DMEM + 7.5 % FCS + 400 pg/ml G418 + 1 pM ouabain.

293 (Kb) cells transfected with the H2-Kb H-chain cDNA were obtained from Dr. H. G. Burgert (Hans Spemann Laboratory, Max-Planck-Institute for Immunobiology, Freiburg, FRG) and maintained in DMEM + 7.5% FCS + 400 pg/ml G418. The HLA-DR1 homozygous (3 lymphoblastoid cell line (B-LCL) used for flow cytometry and T cell proliferation assays has been established from a blood donor at the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service. The HLA-DR1 homozygous B-LCL C70 was obtained from Dr. E. Wiertz (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands).

2.4 Biochemical experiments

Transfectants were grown in different dishes and starved in CysMet-free RPMI medium for 0.5 h prior to labeling. Cells were labeled with 25 pCi of a mixture of [35S] Met and [35S] Cys (NEN) for the times indicated. Cells were lysed in NP40-containing lysis mix [8]. In pulse-chase experiments, 1 mM nonradioactive Cys and Met were added to stop incorporation of label and the cells were chased for the time periods indicated. Class I1 molecules and Ii were immunoprecipitated from equal amounts of trichloroacetic acid- precipitable radioactivity. When indicated, leupeptin (N- acetyl-Leu-Leu-Arg; Sigma) was added to the labeling medium during the pulse at a final concentration of 15 pglml.

To follow intracellular transport, cells were pulse labeled and the intracellular location of class I1 molecules was determined using treatment of intact cells with neuraminidase (Sigma type V), as described [8]. Cells were pulsed for 0.5 h and labeled at the time points indicated. Treatment

Eur. J. Immunol. 1994. 24: 873-883 MHC class I1 recycling 875

with neuraminidase in the medium was performed by adding neuraminidase to a final concentration of 5 U/ml during the last hour of chase. Cells were incubated with neuraminidase on ice after detachment from the dishes with PBYO.1 mM EDTA, and washing once with PBS. Cells were resuspended in PBS/10 mM CaClz containing 5 U/ml neuraminidase and incubated on ice for 0.5 h. After incubation with neuraminidase, cells were washed three times with PBS and lysed. The same procedure was followed for cells treated with neuraminidase in the medium and for the control cells but excluding neuraminidase during the 0.5-h incubation on ice.

Cell surface proteins were radioiodinated using lactoperox- idase 1481. Cells were lysed in NP40-containing lysis mix and class I1 molecules or Ii were isolated. To follow the fate of class I1 molecules associated with A23Ii, cells were cultured in the absence or presence of leupeptin (final concentration 15 pg/ml) for 48 h, followed by iodination.

2.6.2 Immunoelectron microscopy

Transfected 293 cells and human B-LCL C70 cells were fixed in 4% (v/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and embedded in 10 % (w/v) gelatin in PBS. Ultrathin frozen sections were incubated first with a mixture of rabbit anti-class I1 a-chain serum (11500) and mouse anti-CD63 mAb 435 ( W O O ) followed by incubation with a mixture of goat anti-rabbit IgG linked to 10-nm gold (1/40) and goat anti-mouse IgG linked to 5-nm gold (1/40) (both from Amersham Nederland, 's-Hertogenbosch, The Netherlands). Incubations were performed for 1 h at room temperature. After immunolabeling , the cryosections were embedded in a mixture of methylcellulose and uranyl acetate. All sections were examined with a Philips CM 10 electron microscope. Cycloheximide (CHX) treatment was performed by culturing the cells for 4 h in the presence of 100 PM CHX followed by fixation and staining as described above.

2.5 Immunological assays 3 Results

HLA-DR1-restricted influenza virus-specific T cell clones were generated as described before [49]. Briefly, responder cells obtained after two cycles of stimulation of peripheral blood mononuclear cells (PBMC) with influenza virus AMK/8/68 (at day 0 and day 7) were cloned by limiting dilution at day 14. These clones were stimulated with a mixture of autologous EBV-transformed B-LCL (105/ml, 50 Gy irradiated) infected with influenza virus and allo- geneic PBMC (106/ml, 30 Gy irradiated). In addition 20 % v/v of a supernatant of concanavalin A-stimulated PBMC was added as a source of T cell growth factors.

The T cell proliferation assays were performed as follows. The transfected cell lines were collected in PBS after firm agitation of the culture flasks, washed and subsequently irradiated (75 Gy). HLA-DR1-positive B-LCL (irradiated by 50 Gy) were used as controls. Cells (4.104/well) were plated in 96-wells flat-bottom plates (NUNC, Roskilde) to which influenza virus A/HK/8/68 were added as indicated in the figures. After 4 h of infection, 4 x lo4 clonal T cells were added per well and cultured for 72 h in a humidified incubator (5 % COz, 37 "C). Cells were pulsed with 0.2 pCi [3H]dThd/well during the last 4 h of the assay and were harvested with a Titertek multiwell harvester. Incorpora- tion of [3H] dThd was measured in a p-scintillation counter (LKB, Bromma). The results are given as the mean of duplicate cultures after subtraction of the background proliferation of the transfected cells.

2.6 Morphology

2.6.1 Immunofluorescence

Coverslips were incubated with 10 pg/ml poly-L-lysine for 30min at 4°C [50] and washed twice with PBS. The transfectants were cultured on these coverslips overnight up to 40-50 YO confluency, washed twice with PBS and fixed in methanol for 5 min at -20 "C. The cells were labeled with the mAb Bii45 (IgG) and AB4 (IgM) and stained with FITC-conjugated anti-mouse IgG antibody and Texas red-conjugated anti-mouse IgM antibody.

3.1 Expression of class I1 molecules and Ii constructs in different transfectants

To obtain cells expressing class I1 molecules that are transported to different intracellular compartments, we generated a transfectant expressing class I1 DR1 that was supertransfected with the following Ii molecules: full length Ii (to transport class I1 molecules to late endocytic vesicles [9]), Ii lacking 15 (A15Ii) or 23 N-terminal amino acids (A23Ii) (to transport class I1 molecules to the cell surface [51]) or a hybrid molecule containing the cytoplasmic tail of the Tfr and the transmembrane and extracellular portion of Ii (Tfr-Ii) (expected to impose recycling of class I1 molecules through early endosomes). Except for full length Ii, all Ii molecules were expressed in excess over class I1 molecules (not shown). Full length Ii was expressed in roughly equal amounts. The surface expression of class I1 molecules and Ii was determined by flow cytometry (Fig. 1A). A transfectant expressing H-2Kb heavy chains and a HLA-DRl homozygous B-LCL were included as controls. All transfectants expressed class I1 molecules at the cell surface in roughly similar amounts. Only A15Ii and A23Ii were expressed efficiently at the cell surface while Tfr-Ii showed a low level of cell surface expression.

To analyze whether the cell surface Ii molecules were free or associated with class I1 molecules, the transfectants were surface-iodinated. Class I1 molecules were immunoprecipitated first (Fig. lB, left panel), followed by Ii (Fig. lB, right panel). A15Ii, A23Ii (migrating just below the class I1 a chain) as well as some Tfr-Ii were recovered in association with surface class I1 molecules (Fig. lB, left panel). Only free iodinated A15Ii and A23Ii were recovered in a subsequent immunoprecipitation with the anti-Ii antibody Bu45 (Fig. lB, right panel).

3.2 Intracellular transport of class I1 molecules in the different transfectants

To follow intracellular transport of class I1 molecules in the absence or presence of the different Ii molecules, we

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Figure I. Biochemical characterization of the different transfectants. (A) Flow cytometry of the different transfectants. The transfectants described here and as controls a transfectant expressing H-2Kh and an HLA-DR1 homozygous B-LCL were stained with the anti-class I1 mAb Tu36 or the anti-Ii mAb Bii45. As a control no first antibody was added (white bars). The mean fluorescence intensity (MFI) is given. All class I1 transfectants and the B-LCL express similar amounts of class I1 molecules. Only the transfectants expressing A15Ii and A23Ii express considerable amounts of Ii at the cell surface. (B) Surface expression of free and MHC class 11-associated Ii molecules. The different transfectants as well as the B-LCL C70 were surface iodinated and class I1 molecules and free Ii were irnrnunoprecipitated sequentially with respectively mAb Tii36 and Bu45 (left and right panel, respectively).The isolated molecules were analyzed by 12 % SDS-PAGE.The marker positions are indicated as well as the positions of the class I1 a and F chain and the different Ii. Exposure times for ap A15Ii and ap A23Ii in the left panel were eight times shorter than for the other lanes. All transfectants express class I1 molecules at the cell surface. A15Ii, A231i and some Tfr-Ii are associated with class I1 molecules at the cell surface.

performed pulse-chase experiments (Fig. 2A). Intracellu- lar transport was monitored by a shift in apparent molecular weight on SDS-PAGE due t o carbohydrate modifications. Transport of class I1 molecules was inefficient in the absence of Ii [as confirmed by endoglycosidase H treatment

Eur. J. Immunol. 1994. 24: 873-883

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Figure 2. The effect of different Ii molecules on the intracellular transport of class I1 molecules. (A) Pulse-chase analysis. The different transfectants and the B-LCL C70 were labeled for 0.5 h and chased for the times indicated above the figure. Class11 molecules and associated Ii were isolated with, respectively, mAb Tii36 and mAb Bii45 and analyzed by 12% SDS-PAGE. The positions of the class I1 a and p chains and the different Ii molecules are indicated.The different Ii molecules induce transport of class I1 molecules. Note the differences in half-life of the different class 11-associated Ii molecules. (B) Intracellular localization of free and class 11-associated Tfr-Ii. The transfectant expressing class I1 molecules and Tfr-Ii was labeled for 0.5 h and chased for the times indicated above the figure. Cells were analyzed under three conditions: mock-treated (lanes '-'), incubated with neuraminidase on ice (lanes '1') or chased in the presence of neuraminidase in the medium (lanes 'M'). Tfr-Ii and associated class I1 molecules were immunoprecipitated with mAb Bii45 and analyzed by 1D-IEF. The anode is at the bottom. The positions of the a chain (boxed for every chase time), the p chain and Tfr-Ii are indicated. To control for the absence of remaining neuraminidase activity in the lysate, cells metabolically labeled and chased for 8 h, were mixed with unlabeled cells treated with neuraminidase in the medium. Class II/Tfr-Ii complexes were isolated from the mixed lysate (lane "mix 8 h ) . After 2 h of chaseTfr-Ii associated class I1 molecules are fully sialylated, partially susceptible to neuraminidase on ice (indicative for the cell surface population) and fully susceptible for neuraminidase in the medium (thus at the cell surface or in endocytic compartments).

(data not shown)]. All Ii constructs tested promoted intracellular transport of class I1 molecules and their products were rapidly converted (within 1 h) to a higher molecular weight (mature) form. Rapid transport of class I1 molecules and Ii was also observed in the B-LCL C70.


The intracellular vesicles observed in the different transfectants were further defined by immunoelectron microscopy (Fig. 3B) and compared to those found in the B-LCL C70. We had observed previously that class I1 molecules entered multilaminar endocytic compartments (named MIIC) in B cells that contained lgp 1 and CD63 molecules [12]. We, therefore, double labeled sections of the different transfectants with an anti-class I1 a-chain serum and an anti-CD63 antibody. Class I1 molecules were observed in multilaminar structures containing CD63 in the B-LCL C70 (Fig. 3B, panel D) as well as in the transfectants expressing class I1 molecules in the absence (panel A) or presence of A15Ii (not shown), A23Ii (panel C) and Tfr-Ii (panel B). These compartments were accessible to exogenously added horse- radish peroxidase (not shown), defining them as part of the endocytic pathway. Thus, class I1 molecules are found in endocytic compartments that resemble the MIIC found in B cell lines (like C70; panel D). Importantly, Ii is apparently not essential for targeting of class I1 molecules to these compartments.

Whether class I1 molecules enter these compartments during biosynthesis or whether they are recruited from a cell surface population was determined by culturing the different transfectants in the presence or absence of CHX for 4 h prior to fixation. CHX inhibits translation and thus should empty the biosynthetical pathway. We controlled for the inhibition of translation by CHX by metabolically labeling cellsprior to or after CHX addition.The amount of class I1 molecules in MIIC-like compartments should decrease considerably after CHX treatment in case they enter these compartments during biosynthesis [12]. The sections of the different transfectants were labeled with anti-class I1 a-chain serum. The amount of gold particles in MIIC from 15 random chosen cells were counted and compared to the amount of class I1 labeling in non-treated transfectants (Fig. 3C). CHX treatment did not affect the number of gold particles in MIIC compartments in any of the transfectants.This suggests that the class I1 molecules in MIIC are derived from a pool whose size is not influenced by a 4-h CHX treatment. Since transport of class I1 molecules from the E R is rapid for all but the aP transfectant (Fig. 2A), this pool is most likely the cell surface class I1 population. Note that most class I1 molecules are observed in the MIIC when they are expressed in the presence of A15Ii.

During intracellular transport and entry of class II/Ii molecules in the endocytic route, Ii is degraded and removed [l-31. Full-length Ii was released from class I1 molecules between 2 and 4 h in the transfectants and the B-LCL C70, Tfr-Ii was released between 4 and 8 h and A15Ii and A23Ii after 8 h (Fig. 2A). Since degradation of Ii in endosomes can be inhibited by the protease inhibitor leupeptin [22], we followed the fate of the different Ii molecules in the presence of this inhibitor. It appeared that Ii and A23Ii associated with class I1 molecules are degraded both by leupeptin-sensitive proteases, but with different kinetics (corresponding to their respective half-lives; not shown).

Whereas A15Ii and A23Ii target class I1 molecules to the cell surface [51] (see also Fig. 1 and Fig. 5B), the effect of expression of Tfr-Ii on the intracellular distribution of class I1 molecules was less obvious. The transfectants express Tfr-Ii in excess over class I1 molecules and degradation of class II-associated Tfr-Ii is slow (Fig. 2A). How- ever, hardly any Tfr-Ii is found associated with class I1 molecules at the cell surface (Fig. 1A,B). In a pulse-chase experiment, we analyzed the accessibility of free and class II-associated Tfr-Ii in intact cells to neuraminidase added in the fluid phase,which desialylates molecules at the cell surface and in endosomes, or to neuraminidase added on ice, which only desialylates proteins at the cell surface [S] . Isolated molecules were analyzed by one-dimensional (1D) IEF (Fig. 2B). Desialylation of glycoproteins by neuraminidase results in a shift to a more basic position on 1D IEF. Tfr-Ii and associated class I1 a and p chains were fully sialylated after 2 h of chase. For each subunit multiple bands were observed due to heterogeneity in the extent of sialylation. At 2 h of chase, a fraction of the sialylated molecules was susceptible to neuraminidase at 0 “C, demon- strating their presence at the cell surface (lanes ‘I,). However, all molecules were susceptible to neuraminidase added to the medium, indicating their localization in endosomes and at the cell surface (lanes ‘M). Similar results were obtained for the Tfr isolated from the same lysates (not shown). We conclude that Tfr-Ii and class II/Tfr-Ii complexes are primarily routed to endosomes.

3.3 Intracellular distribution of class I1 molecules

The intracellular localization of class I1 molecules and the different forms of Ii were visualized by double immunofluorescence. Transfectants were labeled with the anti-Ii antibody Bu45 (labeled with FITC; Fig. 3A, left panel) and the anti-class I1 antibody AB4 (labeled with Texas red; Fig. 3A, right panel). Cells transfected only with class I1 a and fi chains did not stain for Ii (i) and class I1 labeling was observed at the cell surface and in intracellular vesicles 6). The class II/G transfectant stained for Ii (a) and class I1 molecules (b) in vesicles that show partial overlap (indicated by arrows).Tfr-Ii (c) and class I1 molecules (d) were found in the same vesicles, but vesicles staining for class I1 molecules only were also observed. Cotransfection of A15Ii (e,f) or A23Ii (g,h) resulted in surface staining for the respective Ii (e,g). Class I1 molecules were found in multiple vesicles when expressed in the presence of Al5Ii (f) but only few class II-containing vesicles were observed when a@ was expressed in the presence of A23Ii (h, see also below).

3.4 Presentation of influenza virus by class I1 molecules in the different transfectants

The effect of the different Ii molecules on presentation of influenza virus by HLA-DR1 was determined. As a negative control, the same cell line transfected with the heavy chain of H-2Kb was analyzed. Proliferation of three different HLA-DR1-restricted T helper clones, specific for either influenza virus neuraminidase or nucleoprotein [49], in the presence of different concentrations of influenza virus was determined (Fig. 4). In the kidney cells used as recipient for our transfections, expression of Ii is not required for, nor induces, a specific T cell response. Sur- prisingly, co-expression of Al5Ii did not negatively affect antigen presentation by class I1 molecules. Expression of Tfr-Ii resulted in a modest to no response, depending on the responding CD4+ T cell clone. Whereas no response was

878 N. Nijenhuis, J. Calafat, K. C. Kuijpers et al.

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Figure 3. Intracellular distribution of class I1 molecules in the absence or presence of different Ii molecules. (A) Stea- dy-state distribution of different Ii molecules and the ap chains in stably transfected 293 cells. The different transfectants were fixed and labeled with mouse IgG mAb (Bu45) against Ii and a mouse IgM mAb (AB4) against class I1 molecules. After labeling, the cells were stained with a FITC-conjugated anti- mouse IgG antibody and a Texas red- conjugated anti-mouse IgM antibody. The left panels (a,c,e,g and i) show the total distribution of Ii (FITC), and the right panels (b,d,f,h and j) show the total distribution of the a p chains (Texas red) in cells transfected with a0 and Ii (a and b), ap and Tfr-Ii (c and d), a0 and A15Ii (e and f), ap and A23Ii (g and h) and ap alone (i and j). Arrows indicate vesicles labeled for both class I1 molecules and Ii. (B) Immunoelectron microscopic characterization of class I1 containing vesicles. Ultrathin cryosections are shown of cells transfected with respectively class I1 ap (a), af3 Tfr-Ii (b) and ap A23Ii (c). As a control, cryosections of the B-LCL C70 are shown (d). All sections were double labeled with rabbit anti-class I1 a-chain serum (10-nm gold) and anti-CD63 mAb 435 (5-nm gold). In every transfectant, MIIC-like organelles are found containing abundant internal membrane sheets, arranged either in a concentric shape (a,b,d, closed arrows) or in a mixture of circular and small vesicles (c,d, open arrows). The organelles contain both class I1 and CD63 molecules. Other small vesicles are shown that label only for CD63 (b,c, arrowheads). Bar: 100 nm. (C) Effect of CHX treatment on MHC class I1 labeling in MIIC. Cell were cultured for 4 h in the presence or absence of CHX, fixed and labeled for class I1 molecules. For every quantitation, 15 cell pro- files were randomly selected for counting the gold particles in these organelles. No effect of CHX on class I1 labeling was observed. Note that most class I1 molecules are observed in MIIC when expressed in the presence of A15Ii.

Eur. J. Immunol. 1994.24: 873-883 MHC class I1 recycling 879

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observed for cells transfected with H-2Kb, a small, but significant response of clone 8D4 was observed for cells expressing class I1 molecules in the presence of A23Ii. Thus, the expression of class I1 molecules in the presence of A23Ii and Tfr-Ii considerably reduced the T cell response, but did not abrogate it. Note that the lower antigen- presenting capacity of the transfectants expressing class I1 aPA23Ii and apTfr-Ii (Fig. 4) correlates with the smaller amount of class I1 molecules observed in MIIC-like compartments in these transfectants (Fig. 3C).

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3.5 Do class I1 molecules recycle?

If A15Ii and A23Ii direct transport of class I1 molecules to the cell surface and inhibit peptide binding to class I1 molecules [51], how can we explain the observation that class I1 molecules in the presence of A15Ii and A23Ii do present exogenously added antigen? The transfectants used in this study express excess Al5Ii and A23Ii (Fig. lB), which suggests that class I1 molecules associate quantitatively with Ii in the endoplasmic reticulum [Sl] (see also Fig. 5C). To determine whether class I1 molecules were quantitatively associated with A15Ii or A23Ii at the cell surface of these transfectants, surface iodination was performed and class I1 molecules were recovered after depletion of Ii molecules (Fig. 5A). Despite the synthesis of excess A15Ii and A23Ii, free class I1 molecules were present at the cell surface. Moreover, a fraction of these class I1 molecules were SDS stable, which suggests proper peptide loading [52, 531 (Fig. 5A). Note the absence of SDS-stable complexes for the Ii-associated class I1 molecules (Fig. 5A, left two lanes). The band migrating at - 68 kDa in the left panel is the A23Ii disulfide-linked homodimer. A similar band can be observed for A15Ii after longer exposure of the autoradiogram (not shown).

The prese,nce of free class I1 molecules could be due to degradation of A15Ii and A23Ii after internalization of Ii-associated class I1 complexes (e.g. during the normal course of breakdown), followed by reappearance of free

0 0 1 035 1 3 3 10

i

class I1 molecules at the cell surface. The class I1 a6 A23Ii transfectant was selected for investigating this in detail. We first determined when class IIlA23Ii molecules appear at the cell surface by performing a pulse-chase experiment and incubating the cells at the end of every chase with (lanes ‘I7) or without (lanes ‘-’) neuraminidase on ice (Fig. 5B). When class I1 molecules appear at the cell surface, they become susceptible to neuraminidase and are desialylated, resulting in a shift to a more basic position on IEF. One hour after labeling, class IIlA23Ii molecules could be fully desialylated, indicating that these molecules are rapidly transported to the cell surface.

To follow the appearance of free class I1 molecules with time, we performed pulse-chase experiments and immunoprecipitated class I1 molecules prior to or after removal of Ii and Ii-associated class I1 molecules (Fig. 5C). Hardly any free class I1 molecules were isolated directly after labeling, indicating that the initial association of A23Ii with class I1 molecules was complete. Free class I1 complexes appeared only at later time points when class I1 molecules associated with A23Ii had already arrived at the cell surface. To assess whether the class 11-associated A23Ii was degraded in a similar fashion as wild-type Ii in B-LCL, we performed the same pulse-chase experiment in the presence of the endo- soma1 protease inhibitor leupeptin (Fig. 5C, right panel). Leupeptin inhibits degradation of class 11-associated A23Ii. The pattern of A23Ii degradation products was similar to that obtained after leupeptin inhibition of degradation of class 11-associated Ii in B-LCL [22]. We conclude that class I1 ap A23Ii complexes are internalized after transport to the cell surface, resulting in degradation of A231i in the endocytic route and generation of free class I1 complexes.

Degradation of Ii is essential for transport of class I1 molecules from the endocytic route to the cell surface [22,54]. If the free class I1 complexes generated in the endocytic route return to the cell surface, the amount of free surface class I1 molecules in cells cultured in the presence of leupeptin should decrease due to retention of

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x 0

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Figure 4. Presentation of influenza virus by the different transfectants. The different transfectants and an HLA-DR1-expressing B-LCL were infected with different titers of influenza virus (A/HK/8/68) and prolifera-

880 N. Nijenhuis, J. Calafat, K. C. Kuijpers et al. Eur. J. Immunol. 1994. 24: 873-883

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removal Ii (hr) T - i ’ C - i ’ T T T a f t e r

removal li P

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Figure 5. Cell surface class I1 molecules can recycle. (A) Surface expression of free SDS-stable class I1 molecules and Ii. Left panel: To establish whether class I1 molecules are quantitatively associated with A15Ii or A23Ii, the respective transfectants were surface iodinated. Class I1 molecules associated with Ii were isolated by the anti-Ii mAb Bii45 (left two lanes), remaining AW23Ii and associated class I1 molecules were removed by two additional rounds of immunoprecipitation with Bii45 (isolated molecules from the third round shown in the middle two lanes) followed by isolation of (free) class I1 molecules with mAb Tu36 (right two lanes). Immunoprecipitates were incubated in SDS sample buffer at room temperature and analyzed by 12 % SDS-PAGE.The position of the markers, the class I1 a and chains, (AlY23)Ii and the class I1 nfi dimer are indicated. Free class I1 molecules not associated with (A15/23)Ii are isolated.Whereas the class I1 molecules associated with AWA23Ii do not express SDS-stable class I1 molecules, a fraction of the free class I1 molecules is SDS stable. The band migrating at approximately 68 kDa in the left panel is the A23Ii disulfide-linked homodimer. A similar band can be observed for Al5Ii after longer exposure of the autoradiogram. (B) Surface appearance of class I1 molecules associated with A23Ii.The appearance of class II/A23Ii complexes at the cell surface was monitored by labeling the cells for 0.5 h and chasing them for the times indicated above the figure. Cells were incubated on ice with (lanes ‘1’) or without (lanes ‘-’) neuraminidase. Class I1 molecules were immunoprecipitated with mAbTii36 and analyzed by 1D-IEF. Anode is at the bottom.The position of the respective subunits is indicated. Class IUA23Ii complexes are desialylated by neuraminidase after 1 h of chase, indicative for surface appearance. (C) Conversion of class 11 molecules associated with A23Ii to free class I1 molecules. Cells expressing class I1 molecules and A23Ii were labeled for 1.5 h and chased in the absence (left panel) or presence (right panel) of leupeptin for the times indicated above the figure. Class I1 molecules were immunoprecipitated with mAbTii36 either directly from one fourth of the lysate or after removal of A23Ii-associated molecules from the remaining lysate, as indicated. The middle two lanes show that A23Ii was quantitatively removed. Whereas class I1 molecules associated with A23Ii could be isolated at the first chase points, free class I1 molecules are recovered from 6 h of chase. Inclusion of leupeptin in the medium inhibits the degradation of A23Ii, resulting in partial degradation products (A23Ii*). A23Ii is slowly further degraded in the presence of leupeptin and small class 11-associated products accumulate (see arrow). Note that the half-life of class I1 molecules in longer than that of class I1 molecules associated with A23Ii. (D) Inhibition of surface appearance of free class I1 molecules by leupeptin. Cells were cultured for 48 h in the presence (lanes ‘ +’) or absence (lanes ‘-’) of leupeptin followed by cell surface iodination. Class I1 molecules were either immunoprecipitated directly from one fourth of the lysate (left two lanes) or after removal of A23Ii-bound molecules from the remaining lysate (right two lanes). The middle two lanes show that A23Ii was quantitatively removed. The amount of free class I1 molecules at the cell surface is decreased by culturing cells in the presence of leupeptin.


internalized class IIlA23Ii molecules in endocytic vesicles [22]. Cells expressing class I1 molecules in association with A23Ii were cultured in the absence or presence of leupeptin for 48 h followed by cell surface iodination. Class I1 molecules were recovered prior to or after removal of Ii-associated class I1 molecules (Fig. 5D). Whereas similar amounts of class IIlA23Ii complexes could be recovered (left panel), the amount of iodinated free class I1 molecules was clearly reduced in cells cultured in the presence of leupeptin. This indicates that removal of A23Ii by leupeptin-sensitive proteases is essential for appearance of free class I1 molecules at the cell surface. We conclude that cell surface class I1 ufi/A23Ii complexes can be internalized, followed by degradation of A23Ii and return of free class I1 ap heterodimers to the cell surface.

4 Discussion

During biosynthesis of class I1 molecules Ii is degraded in endocytic compartments, allowing class I1 molecules to associate with peptide [lY, 201. The consequence of this model is that each class I1 molecule is allowed to bind one peptide during its life [55], which is confirmed by biochemical experiments [24]. However, a number of observations do not fit this model: peptide exchange on class I1 molecules has been observed [25] and certain antigens can be presented by class I1 molecules in the absence of Ii [27-30, 32, 34, 561. These phenomena can be explained if, under certain conditions, class I1 molecules do recycle.

Here, we have supertransfected a HLA-DR1-expressing human kidney cell line with different Ii molecules that were expected to result in targeting of class I1 molecules either to late endocytic compartments (full length Ii), to endosomes (Tfr-Ii) or to the cell surface (A15Ii or A24Ii). Targeting class I1 molecules to the cell surface by A231i significantly decreased the capacity of the transfectants to present antigen by HLA-DR1 . Interestingly, co-expression of A15Ii did not inhibit the capacity of class I1 molecules to present antigen, although a large portion of class I1 molecules at the cell surface were associated with AlSIi, which should abolish proper peptide binding (Fig. 5A) [51]. Also a fraction of free class I1 molecules is observed in these transfectants being the result of internalization of A15l23Ii- associated class I1 molecules. A15l23Ii is degraded in endosomes and free class I1 molecules appear at the cell surface. Because Roche et al. [51] did not deplete the Ii-bound fraction before analysis of the free class I1 pool, they did not detect free surface class I1 afi dimers in presentation competent cells expressing class I1 a@ A15Ii or up A20Ii. The presence of a fraction of free class I1 molecules at the cell surface, as observed in our study, could have explained the residual presentation of exogenously added peptide.

Tfr-Ii targets class I1 molecules to endocytic compartments, which does not result in efficient antigen presentation. Apparently, not every part of the endocytic route is involved in inducing a proper antigen-presentation response. The absence of efficient proteolytic degradation of antigens in early endosomes has been demonstrated [21,57]. Early endosomes seem to lack the proteolytic enzymes necessary to degrade (Tfr-)Ii, explaining the long half-life of Tfr-Ii associated with class I1 molecules (Fig. 2).

Only when class I1 afi Tfr-Ii molecules enter late endocytic compartments, isTfr-Ii removed and free class I1 molecules can associate with antigen. This process is apparently inefficient, since the class I1 molecules in the ap Tfr-Ii transfectant are not abundantly present in MIIC-like compartments that resemble those observed in B cells (as visualized by immunoelectron microscopy) [ 121.

The characteristic multilaminar, class II-positive compartments observed in all transfectants by immunoelectron microscopy, resemble MIIC as described in B cells: they label for CD63 and are part of the endocytic route [12]. However, Ii is not essential for targeting of class I1 molecules ot the endocytic pathway, since class I1 molecules are observed in MIIC-like compartments in the absence of Ii. This does not necessarily imply that class I1 molecules contain a specific sorting signal, since they might arrive in these compartments after normal internalization preceding degradation. The observation that prolonged treatment with CHX did not decrease the amount of class I1 molecules in MIIC-like compartments, suggest that class I1 molecules in MIIC in these transfectants are derived from a steady-state pool, which is most probably the cell surface. Also, class I1 molecules expressed in the presence of Tfr-Ii and A231i were observed in MIIC-like compartments. Still, this did not result in efficient presentation of influenza antigen. Apparently, not the mere presence but the amount of class I1 molecules in MIIC-like compartments correlates with the efficiency of antigen presentation. Co-expression of A151i resulted in three times more class I1 molecules in MIIC than co-expression of Tfr-Ii or A23Ii. The difference in amount of antigen resulting in equal presentation by a good (up A15Ii) or a bad APC (a@ A23Ii of TFr-Ii) is between three and ten (Fig. 4; clone 8D4, for up TFr-Ii also clone 492). Although there is not necessarily a linear relationship between class II-peptide loading and T cell stimulation, this is in line with threefold difference in amount of class I1 molecules in MIIC.

For the up A23Ii complexes we have determined in detail how free class I1 molecules are generated from cell surface Ii-containing complexes. These complexes are rapidly transported to the cell surface, followed by slow internalization. A23Ii is degraded involving leupeptin-sensitive endosomal proteases and the resulting free class I1 molecules can recycle to the cell surface. Indeed, class I1 molecules expressed in the presence of A23Ii present antigen to T cells, albeit rather inefficiently compared to class I1 molecules expressed in the absence of Ii. If A23Ii reduces the rate of internalization of class I1 molecules, then at any time less class I1 molecules would be available for association with antigen. This is suggested by the decreased amount of class I1 molecules in MIIC-like intracellular compartments in the transfectant co-expressing A23Ii. On the other hand, since class I1 molecules expressed with Al5Ii present antigen efficiently, AlSIi should not affect the rate of internalization of class I1 molecules and the amount of class I1 molecules in MIIC- like compartments. In Al5Ii-transfected HeLa cells, Al5Ii has been detected in peripheral vesicles [l l].These vesicles were different from the perinuclear vesicles where full- length Ii accumulated [ l l ] . This suggests that A15Ii may contain (part of) an internalization signal that would be completely removed in A23Ii. We were, however, not able to show a significant difference in internalization rate of

882 N. Nijenhuis, J. Calafat, K. C. Kuijpers et al.

class I1 molecules associated with either A15Ii or A23Ii in our transfectants. Alternatively, a function of Ii may be to control the rate of degradation of class I1 molecules while they transit via the endocytic route.This has been observed in normal class 11-expressing cells, where a fraction of class I1 molecules was degraded en route to the cell surface [22,52]. This fraction could be rescued by inhibition of Ii breakdown by leupeptin [22]. Thus, recycling of class I1 molecules may be the result of internalization as a first step to degradation. In the 293 kidney cells used in this study, a fraction of class I1 molecules escapes degradation, asso- ciates with peptide and is transported back to the cell surface. The efficiency of degradation of internalized class I1 molecules may be cell type specific, which would explain why class I1 molecules, expressed in the absence of Ii, present antigen efficiently in certain cell types [27, 331, but hardly at all in other cell types [26] including normal murine B cells [31, 341. Interestingly, recycling of class I1 molecules has been claimed to occur in murine B cells [58], although these molecules are not loaded with peptide in B cells from Ii-deficient mice [31, 341, excluding recycling of class I1 molecules as a major source of peptide uptake in these cells. Another interesting pool of class I1 molecules, whose fate is usually ignored, is the small amount of class I1 molecules associated with Ii that is found at the cell surface of certain B cell lines [17, 181 and may be part of the normal transport route to endosomes of class I1 molecules associated with Ii. These heterotrimers internalize with a half-life of approximately 1 h [MI. The resulting free class I1 molecules may recycle back to the cell surface after removal of Ii and basically follow a pathways as described here for class I1 molecules associated with A23Ii.

Eur. J. Immunol. 1994.24: 873-883

We thank Drs. G. Moldenhauer, B Momburg and J. Drexler (DKFZ, Heidelberg) for samples of the rnAb Bii45, S. Funderud for the m A b AB4, S. H. Ong for preparation of the micrographs, Dr. J. Borst and Prof. H. L. Ploegh for critically reading the manuscript and J. M. Overwater for typing the manuscript. The 293(Kb) transfectant was a kind gift of Dr. H. G. Burgert.

Received November 12,1993; in revised form December 16,1993; accepted December 17, 1993.

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