The FASEB Journal article fj.14-268094. Published online March 17, 2015.

The FASEB Journal • Research Communication

Dissociation of b2-microglobulin determines the surface quality control of major histocompatibility complex class I molecules Sebasti´an Montealegre, Vaishnavi Venugopalan, Susanne Fritzsche, Corinna Kulicke, Zeynep Hein,1 and Sebastian Springer1 Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany Major histocompatibility complex class I proteins, which present antigenic peptides to cytotoxic T lymphocytes at the surface of all nucleated cells, are endocytosed and destroyed rapidly once their peptide ligand has dissociated. The molecular mechanism of this cellular quality control process, which prevents rebinding of exogenous peptides and thus erroneous immune responses, is unknown. To identify the nature of the decisive step in endocytic sorting of class I molecules and its location, we have followed the removal of optimally and suboptimally peptide-loaded murine H-2Kb class I proteins from the cell surface. We find that the binding of their light chain, b2microglobulin (b2m), protects them from endocytic destruction. Thus, the extended survival of suboptimally loaded Kb molecules at 25°C is attributed to decreased dissociation of b2m. Because all forms of Kb are constantly internalized but little b2m-receptive heavy chain is present at the cell surface, it is likely that b2m dissociation and recognition of the heavy chain for lysosomal degradation take place in an endocytic compartment.—Montealegre, S., Venugopalan, V., Fritzsche, S., Kulicke, C., Hein, Z., Springer, S. Dissociation of b2-microglobulin determines the surface quality control of major histocompatibility complex class I molecules. FASEB J. 29, 000–000 (2015). www.fasebj.org

ABSTRACT

Key Words: endocytosis • receptor recycling • Brefeldin A peptide • dissociation

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IN ADAPTIVE CELLULAR IMMUNE responses of all vertebrates, cytotoxic T lymphocytes eliminate virally infected or malignantly transformed cells. Key to target recognition are major histocompatibility complex class I molecules, which present endogenous self- and nonself-peptides at the surface of target cells. The immunogenicity of presented peptides correlates with the stability of the class I/peptide complex (1). Binding of peptides to class I molecules in the endoplasmic reticulum (ER) is mediated by the chaperone tapasin and ensured by a cellular quality control mechanism (2). Complexes of low stability dissociate rapidly, and class I molecules are retained inside the cell (3). At the cell surface, Abbreviations: b2m, b2-microglobulin; bb2m, bovine b2microglobulin; BFA, brefeldin A; EEA1, early endosomal antigen 1; EndoF1, endoglycosidase F1; ER, endoplasmic reticulum; FCS, fetal calf serum; FHC, free heavy chain; hb2m, human b2microglobulin; HA, hemagglutinin; scKb, single-chain dimeric Kb; TAP, transporter associated with antigen processing

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most class I molecules thus consist of the heavy chain, the light-chain b2-microglobulin (b2m), and a tightly bound peptide (trimers). Still, some suboptimally loaded class I molecules (i.e., without any or with a low-affinity peptide) do emerge from the cell both in mutant and wild-type cells (4–8). In addition, trimers at the cell surface eventually dissociate, and the resulting heavy chain/b2m dimers without peptide or free heavy chains (FHCs) are rapidly internalized (9) and degraded in acidic compartments (10). Dimers can be rescued from internalization by addition of a high-affinity peptide, which shows that the dissociation of the peptide from the trimer triggers the surface removal process (5, 8). Following dissociation of the peptide, dimers become unstable, and dissociation of b2m occurs (11, 12). The rapid removal of suboptimally loaded class I molecules, including dimers, at physiologic temperatures (8, 13) suggests an as yet undescribed cellular process for their specific recognition. At low temperatures (22–26°C), this mechanism appears not to work for the suboptimally loaded dimers of murine allotypes because they accumulate at the cell surface due to reduced endocytosis (9, 14). Internalization of class I molecules is clathrin independent (15) and does not seem to distinguish between different conformations because peptide-bound class I molecules are internalized spontaneously and continuously, despite their long life span at the plasma membrane, but are possibly recycled to the surface (16–18). In this study, we ask which form of the murine class I allotype H-2Kb is recognized for endocytic destruction and where in the cell this recognition occurs. Our results suggest that inside the cell, in an early endocytic compartment, b2m dissociates from suboptimally loaded dimers to yield FHCs that are then forwarded to lysosomes. MATERIALS AND METHODS Antibodies, peptides, and reagents Mouse monoclonal hybridoma supernatants Y3 (19), W6/32 (20), HC10 (21), BBM.1 (22), and hemagglutinin (HA) 12CA5 1 Correspondence: Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring, 28759 Bremen, Germany. E-mail: Z.H., [email protected]; S.S., [email protected] doi: 10.1096/fj.14-268094 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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(23) have been described previously. Rabbit monoclonal early endosomal antigen 1 (EEA1) was from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse conjugated with Alexa 488 was from Dianova (Hamburg, Germany). Anti-rabbit coupled to Cy3 was from Jackson ImmunoResearch Europe Limited (Suffolk, United Kingdom). The peptide SIINFEKL was from GeneCust (Dudelange, Luxembourg), purified by HPLC, and delivered at .90% purity.

without FCS and with BFA, and incubated at 25 or 37°C for the indicated time points. Finally, the cells were washed and processed for flow cytometry. Folding and addition of recombinant b2m hb2m was expressed in Escherichia coli as inclusion bodies, purified, solubilized, and stored at 220°C, as described previously (30).

Cells STF1 cells (24) (kindly provided by Henri de la Salle, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg, France), STF1/Kb, STF1/KbY84C, and STF1/single-chain dimeric Kb (scKb) cells were grown at 37°C and 5% CO2 in low-glucose (1 g/L) DMEM (GE Healthcare Europe, Freiburg, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. RMA-S cells (25) were grown in RPMI 1640 medium (GE Healthcare Europe) supplemented as above. For incubation at 25°C, CO2-independent medium (Gibco, Life Technologies, Darmstadt, Germany) supplemented as above was used.

Flow cytometry Cells were kept on ice throughout the staining process. Cells were fixed with 0.02% NaN3 in PBS for 5 minutes, washed, and incubated with primary antibody for 30 minutes. Anti-HA antibody 12CA5 hybridoma supernatant was used in a 1:5 dilution; W6/32, HC10, and Y3 hybridoma supernatants were used undiluted. After primary antibody incubation, cells were washed once with PBS, centrifuged, and stained with anti-mouse coupled to Alexa 488 for 30 minutes. Finally, the cells were washed with PBS as above and then resuspended in 1 ml PBS and counted with a CyFlow Space (Partec, G¨orlitz, Germany).

Generation of stable STF1 cell lines

Antibody-mediated internalization

A 2A ribosomal skipping sequence (GSGATNPSLLKQAGD VEENPGP) (26) was inserted between the coding regions of human b2m (hb2m) and H-2Kb in pKG5 background (27) using primers with BamHI overhangs. The hb2m-2A fusion was subcloned upstream HA-H-2Kb in pEGFP-N1 (28) via BamHI and SalI. The final construct was cloned into the lentiviral vector puc2CL6IPwo (29) via XhoI and AgeI. STF1 cells were transduced with lentiviruses and selected (28) generating the stable cell line STF1/Kb or STF1/KbY84C. The single-chain Kb construct was designed essentially as described (27). The coding region of the single-chain dimer was subcloned into pEGFP-N1 via XhoI-AgeI. An HA tag was inserted by site-directed mutagenesis (Agilent Technologies, Santa Clara, CA, USA) after the first GGGGS repeat in the linker region. HA-scKb was subcloned into puc2CL6IPwo via XhoI-AgeI, and STF1 cells were transduced as above.

There were 2 3 104 STF1/Kb cells seeded in coverslips and incubated at 37°C. After 24 hours, cells were moved to 25°C or left at 37°C accordingly. The next day, cells were washed 3 times with PBS and incubated with antibodies for 5 minutes at room temperature. Unbound antibodies were removed by washing, and the cells were incubated in medium for the indicated time points at 25 or 37°C. Cells were then fixed with 4% paraformaldehyde in 200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, permeabilized with 0.1% saponin, and the secondary antibodies were added. Coverslips were mounted with Mowiol and observed under a Zeiss LSM 510 Meta confocal laser-scanning microscope, provided with Argon and Helium-Neon lasers (Carl Zeiss GmbH, Jena, Germany). Images were obtained at a pinhole setting of 1 Airy unit, 363 magnification, and a resolution of 1600 3 1600 pixels; they were further analyzed and processed using ImageJ 1.42h (National Institutes of Health, Bethesda, MD, USA).

Brefeldin A decay experiments There were 1.2 3 105 cells per well seeded into 6-well plates 2 days before the experiment and incubated at 37°C. The night before the experiment, cells were moved to 25°C for 16–20 hours. The next day, cells were washed in PBS, and the medium containing 5 mg/ml brefeldin A (BFA) (and 10 mM SIINFEKL peptide where indicated) was added. Afterward, cells were incubated at either 25 or 37°C for the indicated time points. To determine the baseline values at t = 0 in the presence of peptide, cells were incubated with peptide at 25°C for 30 minutes. At indicated time points, the cells were trypsinized, harvested, and processed for flow cytometry. Acid wash STF1/Kb cells were incubated overnight at 25°C as above. The next day, cells were trypsinized, washed, and placed on ice. Cells were resuspended in 500 ml acidic buffer [(pH 2.6) 22 mM Na2HPO4, 89 mM citric acid], and 0.5% bovine serum albumin or in neutral buffer [(pH 7.4) 187 mM Na2HPO4, 6.4 mM citric acid and bovine serum albumin] for 5 minutes on ice. Afterward, the pH was neutralized with 15 ml blocking buffer [(pH 7.4) 93 mM Na2HPO4 and 0.5% bovine serum albumin in medium without FCS]; the cells were then washed, resuspended in medium

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Pulse-chase experiments Experiments were performed as detailed in Fritzsche and Springer (31). b2m dissociation assay There were 1.6 3 107 STF1/Kb cells labeled with [35S]Met and Cys for 30 minutes at 37°C. Afterward, cells were lysed in lysis buffer [50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100] for 1 hour at 4°C. After lysis, the postnuclear supernatant was immunoprecipitated with BBM.1 antibodies for 30 minutes at 4°C. The beads were washed 3 times with wash buffer [50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100] and then distributed into different microcentrifuge tubes, spun down, and the supernatant was removed. Beads were resuspended in 50 ml wash buffer, and the tubes were transferred to 25 or 37°C accordingly. Finally, the beads were centrifuged, the supernatant was discarded completely, the beads were boiled at 95°C for 10 minutes, and the immunoisolates were separated by SDS-PAGE. Proteins were detected by autoradiography, and band intensities were determined by densitometry.

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Immunoprecipitation and immunoblotting Cells were lysed as above and immunoprecipitated with HA antibody. After immunoprecipitation, the samples were treated (or not) with endoglycosidase F1 (EndoF1), resolved by nonreducing SDS-PAGE, and immunoblotted against HA.

Data analysis and processing Flow cytometry data were analyzed in FlowJo (Tree Star, Inc., Ashland, OR, USA). Gels were processed with ImageJ.

monitoring of the disappearance of existing surface class I molecules by flow cytometry (35). We incubated RMA-S cells overnight at 25°C, added BFA and incubated aliquots at either 25 or 37°C for different times, and measured surface Kb with Y3 staining and flow cytometry. At 37°C, Kb disappeared rapidly from the cell surface with a half-life of ;50 min, but at 25°C, it showed no significant decrease after 4 h (Fig. 1D). Like Day et al. (14), we conclude that suboptimally loaded Kb accumulates at the surface of RMAS cells at 25°C because of reduced endocytic destruction and not because of increased anterograde transport. At 25°C, FHCs are still destroyed by endocytosis

RESULTS Reduced endocytosis causes low-temperature cell surface accumulation of suboptimally loaded class I molecules In transporter associated with antigen processing (TAP)deficient cells, murine class I molecules are more abundant at the cell surface at 25°C than at 37°C (9, 14, 32). For H-2Kb (Kb) in TAP-deficient RMA-S cells, this 25°C accumulation is clearly visible (Fig. 1A). To compare the rates of anterograde transport of Kb, we performed pulse-chase analyses at 25 and 37°C and immunoprecipitated with the b2m-dependent antibody Y3 (31). The initial rates of EndoF1-resistance acquisition were very similar at both temperatures (Fig. 1B, C). At 25°C, the labeled Kb cell surface population continued to increase after 60 min of chase, but at 37°C, it leveled off and then decreased, presumably through endocytosis and lysosomal degradation. This suggests that at 25°C, suboptimally loaded Kb molecules are transported to the cell surface at a similar rate, but their endocytic destruction is decreased. We next tested this hypothesis in a BFA decay experiment. BFA disrupts vesicular transport between ER and Golgi, resulting in a rapid (2.5 min) block of anterograde vesicular transport without affecting endocytosis or recycling from endosomes (33, 34). BFA thus blocks the arrival of newly synthesized class I at the surface and allows simple

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We next tested whether this lack of endocytic destruction at 25°C is a general cellular feature. We thus compared the endocytosis of suboptimally loaded heavy chain/b2m dimers with that of FHCs of Kb. To enable the detection of FHCs, we expressed Kb with an N-terminal influenza HA tag in human TAP-deficient STF1 cells (24). Additional hb2m was expressed from the same mRNA with the help of a viral 2A ribosomal skipping sequence (26) (Supplemental Fig. 1A, B). We confirmed that in these cells, too, HA-Kb accumulated at the surface at 25°C (Supplemental Fig. 1C) and that HA-Kb was not significantly endocytosed at 25°C (Fig. 2A). To differentiate dimers and FHCs, we forced the dissociation of hb2m from cell surface Kb by incubating the cells at pH 2.6 for 5 minutes, such that signals for both Y3 and W6/32 (for STF1-endogenous human class I with bound hb2m) dropped to 40%, whereas the signal of the HA antibody (which recognizes all forms of HA-Kb) remained constant. As a control, the signal for HC10 (which recognizes human FHCs) increased to 3.5 times its preincubation level (Fig. 2B). Thus, our acid incubation generated significant amounts of FHC. We then followed the Y3, HA, and HC10 signals in a BFA decay experiment. After 1 hour, ;40% of HA-Kb molecules were endocytosed, as well as 30% of the human FHCs. In contrast, HA-Kb/b2m dimers remained entirely at the cell surface (Fig. 2C). As a control, both Y3-positive and HA-positive populations were rapidly endocytosed at 37°C (Fig. 2D).

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MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I SURFACE REMOVAL

Figure 1. Reduced endocytosis determines the presence of Kb molecules devoid of highaffinity peptides at the cell surface of TAPdeficient cells at 25°C. A) RMA-S cells were incubated overnight at 25 or 37°C, and surface levels of Kb/b2m were detected by flow cytometry with mAb Y3. B and C) RMA-S cells were pulse labeled with [35S]Met/Cys for 10 minutes, incubated at 25°C (upper panel) or at 37°C (lower panel), and chased for different times. After lysis, Kb was immunoprecipitated with mAb Y3 and treated with EndoF1. EndoF1r, EndoF1 resistant; EndoF1s, EndoF1 sensitive. D) BFA decay experiment. RMA-S cells were incubated overnight at 25°C, incubated with BFA, and after different times at 25 or 37°C, surface levels of Kb/b2m were detected by flow cytometry with mAb Y3. Error bars, SEM (n = 4 independent experiments).

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Figure 2. Endocytosis at 25°C is blocked for suboptimally loaded HA-Kb dimers, but FHCs are slowly endocytosed. A) STF1/Kb cells were incubated overnight at 25°C, and a BFA decay experiment was performed at 25°C; cells were stained with Y3 and HA. B) STF1/Kb cells were incubated overnight at 25°C, acid treated, and stained with Y3 and HA; as a control, endogenous human class I was stained with W6/32 or HC10. After acid treatment, a BFA decay experiment was performed at 25°C (C) and in at 37°C (D). Cells were stained with Y3, HA, or HC10. Error bars, SEM (n $ 3).

The removal of FHC from the cell surface shows that endocytic destruction still operates at 25°C, in agreement with earlier reports (36); thus, at this temperature, a selective process—of unknown molecular mechanism— must be retaining suboptimally loaded HC/hb2m dimers at the surface or returning them to the surface from an early endocytic compartment. Because at 37°C, suboptimally loaded class I molecules are rapidly degraded (Fig. 1D), one might assume that at 37°C, this retention process does not function. Alternatively, at 37°C, suboptimally loaded class I molecules might rapidly lose their bound b2m and thus become substrates for the endocytic degradation of FHCs. At 37°C, b2m dissociation limits the rate of endocytic destruction of H-2Kb We next figured that if the latter hypothesis is correct, then suboptimally loaded class I molecules might be resistant to endocytic destruction at 37°C as long as they retained b2m. Thus, we decided to test in BFA decay experiments 2 variants of Kb with stronger b2m association, expecting that they would last longer on the cell surface, in suboptimally loaded form, than wild-type Kb. Those were initially Kb(Y84C/A139C) (KbY84C) (28), which has an especially high affinity to b2m, and then scKb in which hb2m is covalently attached to the N terminus of the heavy chain by means of a long glycine/serine linker (Supplemental Fig. 1A, B). Both KbY84C and scKb were resistant to endocytosis at 37°C, suggesting that b2m association is sufficient to prevent endocytic destruction of class I molecules (Fig. 3A, B and Supplemental Fig. 1D–H). If b2m association protects Kb from endocytic destruction, then the simplest hypothesis to explain the 25°C surface accumulation is that, at 25°C, b2m dissociation from the Kb heavy chain is slower than at 37°C. To test it, we radiolabeled STF1/Kb cells metabolically, lysed them with detergent, immunoprecipitated with the antibody BBM.1 (which recognizes both free and class I-bound hb2m), and incubated the beads at 25 or 37°C for up to 30 minutes to 4

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induce dissociation of the dimer. At different time points, we quantified the remaining bead-bound heavy chains by SDS-PAGE and autoradiography. It is noteworthy that after 30 minutes of incubation at 37°C, 75% of HA-Kb had dissociated from b2m, whereas at 25°C, there was almost no dissociation at all (Fig. 3C, D). Taken together, our data suggest that, at 37°C, suboptimally loaded Kb rapidly loses its b2m and thus becomes a target for endocytic destruction, whereas at 25°C, the slow dissociation of b2m causes its retention at the cell surface or its retrieval from an early endocytic compartment. Thus, b2m dissociation is the rate-limiting step for the endocytic destruction of Kb. Dissociation of b2m takes place in an intracellular compartment We next investigated where in the cell b2m dissociation occurs. We hypothesized that if it occurred at the cell surface, then the resulting FHCs should be detectable because they can bind exogenous b2m. First, we incubated the STF1/Kb cells overnight with 7 or 37 mM recombinant hb2m and found that the Kb levels (measured with the b2m-dependent Y3 antibody by flow cytometry) had increased considerably (Supplemental Fig. 1J). This confirms published data that FHCs exist at the cell surface that can bind to exogenous b2m (5, 6, 37–39). To estimate the amount of b2m-receptive FHCs at steady state, we next incubated STF1/Kb cells grown at 25 or 37°C with 7 or 37 mM hb2m (82 and 434 mg/ml, respectively) on ice (Fig. 4A). To our surprise, we did not find any increase at all in the Y3 signal after hb2m incubation; the same was true in RMA-S cells (data not shown). We thought that the lack of hb2m binding was perhaps attributed to rapid denaturation or endocytosis of the FHC, so instead of adding hb2m just before antibody staining, we cultured the cells in the presence of BFA and hb2m for up to 4 hours. We expected that the exogenous hb2m would bind to the freshly generated FHC and extend its surface lifetime. It is

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remarkable that excess hb2m addition slowed down the decrease of Kb only slightly (Fig. 4B). Taken together, the data suggest that overall surface levels of Kb can indeed be stabilized by incubation of cells with hb2m over a long time, but that at any given time point, the steady-state level of b2m-receptive FHCs is very low. Because a given cohort of surface Kb is not efficiently rescued by exogenous b2m, we conclude that dissociation of b2m from the heavy chain occurs mainly inside the cell, perhaps in an early endosomal structure. Peptide-bound and suboptimally loaded Kb accesses an early endocytic compartment We next reasoned that if dissociation of b2m from the Kb heavy chain occurs inside the cell, then peptide-empty or suboptimally loaded Kb dimers should be able to access these internal compartments, perhaps cycling between the cell surface and an endocytic compartment. Such internalization was shown previously for H-2Ld (40). To see whether it occurs in our system, we incubated STF1/Kb cells at 25°C to accumulate dimers at the cell surface, added the b2m-dependent Y3 antibody for 5 minutes, and then washed the cells and shifted to 37°C. At different time points, we then fixed the cells, stained with secondary antibody, and observed the cells by immunofluorescence microscopy. Within minutes of the temperature shift, Kb moved to punctate structures that colocalized with the early endosomal marker EEA1 (Fig. 5A). The same movement and colocalization were observed when we left the cells at 25°C after antibody incubation, albeit at longer times. These results suggest that, even at 25°C, when the cell surface levels of class I molecules appear constant over time, Kb dimers are internalized, presumably returning to the cell surface. To investigate the internalization of peptide-bound Kb, we then repeated the experiment but added SIINFEKL peptide to the cells prior to antibody binding. At 37°C, we found colocalization with EEA1 after 30 minutes (Fig. 5B). Thus, the simplest model to explain our observations is that suboptimally loaded dimers and

MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I SURFACE REMOVAL

Figure 3. b2m is the rate-limiting factor for the endocytic destruction of Kb molecules. A) BFA decay experiments in STF1/Kb, STF1/KbY84C, and STF1/scKb cells. Staining was performed with Y3. B) As in (A), but in the presence of 10 mM SIINFEKL. Error bars, SEM (n $ 3). C) STF1/Kb cells were radiolabeled; after coimmunoprecipitation of b2m molecules with BBM.1, the beads were heated to 25 or 37°C, and the immunoisolates were separated by SDS-PAGE. D) Quantification of (C). Error bars, SEM (n = 3).

peptide-bound trimers both travel from the cell surface to an endocytic compartment and from there either return to the cell surface or—perhaps concomitant with the loss of peptide and b2m—move on toward lysosomal degradation. DISCUSSION We have asked how suboptimally peptide-loaded dimers (called dimers in the following) of H-2Kb heavy chain and b2m are selected for endocytic destruction from the plasma membrane. Our results are most simply explained by the model in Fig. 6. Dimers, along with trimers (Kb/b2m complexes with high-affinity peptide) and FHCs, are internalized from the plasma membrane (arrows 1, 2, and 3 in Fig. 6) and reach an endocytic compartment. There, dimers lose b2m (arrow 4 in Fig. 6), and the resulting FHCs travel on toward lysosomal destruction (arrow 5), whereas trimers with stably bound peptide are returned to the cell surface (arrow 6). At 25°C, dissociation of b2m is much slower, and dimers return to the surface (arrow 7 in Fig. 6). Internalization (15–17, 41–44) and cell surface return (18, 45–48) of b2m-bound class I molecules have been shown by several groups who used b2m-dependent conformation-specific antibodies for class I such as W6/ 32 (for HLA) and Y3 (for Kb), which also bind to suboptimally loaded dimers (19, 20). In contrast, explicit demonstrations of internalization of peptide-bound class I (49, 50) and its return to the surface (50, 51) are rare. In agreement with previous data for human class I (18), our microscopy suggests that, at steady state, relatively little Kb/SIINFEKL trimer is inside the cell (Fig. 5), but this does not demonstrate differential internalization of dimers versus trimers from the cell surface; rather, the return of the trimers from endosomes to the cell surface may be very efficient (arrow 6 in Fig. 6). If dimers and trimers indeed spend 5–10% of their time cycling through endocytic compartments, as assumed previously (18), it is very likely that the dissociation of b2m from the heavy chain takes place in acidic endosomes 5

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Figure 4. Dissociation of b2m occurs in an intracellular compartment. A) STF1/Kb cells were incubated overnight at 25 or 37°C, trypsinized at room temperature, washed, incubated with 7 or 37 mM recombinant hb2m on ice for 15 minutes, washed, stained with Y3, and fluorescence was recorded by flow cytometry. A.U., arbitrary units. B) BFA decay experiments with STF1/Kb cells in the presence of 7 or 37 mM recombinant hb2m. Error bars, SEM (n $ 3).

because it is dramatically accelerated below pH 5.5 (41, 49). Most FHCs thus generated might never reappear at the surface but instead become routed to lysosomal destruction directly (arrow 5 in Fig. 6). This matches our observation that extended incubation with exogenous b2m cannot rescue suboptimally loaded dimers from destruction (Fig. 4B). Likewise, in the BFA decay experiments with HA-Kb in STF1 cells, the HA and Y3 signals decayed at almost the same rate, which suggests that no significant amount of an intermediate HA+Y32 species of Kb (i.e., FHCs) is present at either temperature (Fig. 2D and Supplemental Fig. 1F). In our hands, incubation with exogenous b2m at 4°C does not lead to an increase in the surface Y3 signal on STF1/Kb cells (Fig. 4A). 6

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Although we find very little FHC that is able to bind b2m (called in the following b2m-receptive FHC) at the plasma membrane at steady state, this does not necessarily contradict older observations of b2m binding to the surface of RMA-S and other (including human) cells (5, 6, 37–39, 52) for the following reasons. First, in the cited experiments, incubations with exogenous b2m for several hours might cause much b2m binding even if the steady-state amounts of b2m-receptive FHC are small (Supplemental Fig. 1J). Second, those short-term incubations with b2m that are documented in the literature confirm our findings that only small amounts of b2mreceptive FHC are present at steady state (53). Third, wherever different cell types were compared, the surface amounts of b2m-receptive FHCs were found to depend on the surface amount of class I in the particular cell type, and on the growth phase (54, 55). Fourth, because we used human cells to express Kb, the high affinity of hb2m to Kb might cause slower dissociation of b2m; therefore, on our STF1/Kb cells, fewer peptidereceptive FHCs might be present than in RMA-S cells (52, 56). Finally, in our experiments, we cultured the cells in FCS-containing medium that contains bovine b2m (bb2m), which binds to murine class I (6, 57). Thus, any small amounts of FHC generated on our cells might rapidly bind bb2m from the medium and thus not be available to bind exogenous hb2m. In our experiments, the FHCs at the cell surface were not removed by trypsin treatment because addition of b2m before trypsinization did not change the outcome (Supplemental Fig. 1K). For our conclusion, in summary, it is most important that incubation of STF1/Kb over several hours with additional exogenous hb2m does not rescue a cohort of suboptimally loaded dimers at the cell surface in a BFA decay experiment (Fig. 4B), which demonstrates that steady-state levels of b2m-receptive Kb FHCs at the surface of STF1 cells are low. Such low steady-state levels may have 2 of the following reasons: 1) FHCs are generated at the cell surface, but once generated, they rapidly denature to lose their ability to bind b2m or 2) alternatively, FHCs are mostly generated inside the cell where exogenous hb2m does not reach them. Because our Kb molecules are tagged with the conformation-independent HA epitope, we were able to show that during a BFA decay, Y3 and HA epitopes decay at almost the same rate, which suggests that in STF1/Kb cells, no significant accumulation of Y32HA+ FHCs exists. Thus, in these cells, dissociation of b2m from the heavy chain most likely occurs mainly in an internal compartment (arrow 4 in Fig. 6). The question of how FHCs are selected for routing to lysosomes and eventual destruction will require substantial further work. Our data suggest a mechanism that acts in the early endocytic compartment (arrow 5 in Fig. 6). That mechanism may require acidification because in the presence of the acidification inhibitor concanamycin B, the surface levels of FHC increase (10). The low-temperature surface level increase of murine class I (9) is explained in our model by the significantly slower dissociation of b2m at lower temperatures (Figs. 2C and 3C) and the resulting return of dimers to the surface (arrow 7 in Fig. 6). This agrees with our previous in vitro experiments, which show that the peptide-empty dimer of the Kb lumenal domain and b2m is conformationally stable

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Figure 5. Peptide-bound and suboptimally loaded Kb accesses an early endocytic compartment. STF1/Kb cells were incubated overnight at 25°C. A) Cells were then incubated with Y3 for 5 minutes at 25°C. The antibody was washed off, and internalization of the Y3-Kb complexes was followed for 0, 10, and 60 minutes (top 3 panels, respectively) at 25°C or 10 and 30 minutes (lower 2 panels, respectively) at 37°C. After each time point, cells were fixed, permeabilized, and stained with secondary antibody antimouse IgG conjugated with Alexa 488 (green) against Y3 and anti-EEA1 in combination with Cy3-conjugated anti-rabbit IgG (red). B) Cells were preincubated with SIINFEKL (10 mM) at 25°C for 15 minutes, and internalization was followed as in (A). Boxes with white outlines are enlarged and displayed on adjacent columns on their right. Scale bars, 20mm.

below 32°C (30), and with the decrease in the amounts of sialylated Kb FHC that is visible in RMA cells at 26°C (10). Finally, it nicely concurs with our model that Luˇcin and collaborators (50) showed the separation of 64-3-7+ (suboptimally loaded) and 30-5-7+ (peptide-bound) H-2Ld molecules in endosomes, with the former proceeding to lysosomes and the latter recycling to the surface. Identification of the precise subcompartments in which these

processes occur is currently ongoing (58). It remains to be seen what differences in class I surface quality control exist between cell types and to what extent cell surface quality control is mechanistically connected to the loading of class I molecules in the endocytic tract that occurs in crosspresenting cells. The authors thank Ursula Wellbrock for excellent technical assistance; Henri de la Salle for cells; and Linda Janßen, Venkat Raman Ramnarayan, and Malgorzata Garstka for discussions. The work was supported by the Deutsche Forschungsgemeinschaft (SP583/2-3 and 7-1 to S.S.), the German Academic Exchange Service (to S.M.), and the Thyssen Foundation (to Z.H.).

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Figure 6. Model of endocytic trafficking of the 3 forms of Kb. On the left, the Kb heavy chain associates with b2m and peptide in the ER. On the right, loss of b2m and peptide and internalization followed by recycling or endocytic destruction of the 3 forms of Kb are shown. Dashed arrows represent routes of low significance; this may vary with the cell type.

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