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Stability of Surface H-2K^b, H-2D^b, and Peptide-Receptive H-2K^b on Splenocytes¹

Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

	Abstract

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We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of 1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, "peptide-receptive") belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-K^b, we found that the maximum rate for binding of exogenous peptide corresponds to a t_1/2 value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, 20 short-lived molecules must be exported to the cell surface for each long-lived molecule.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptide can bind directly to MHC class I (MHC-I)⁴ molecules on the surface of an intact viable cell. We found (1, 2) that MHC-I molecules capable of binding peptide can be rapidly generated. Cells were pulsed with a high concentration of a peptide that binds to MHC-I with high affinity and then incubated at 37°C in the absence of exogenous peptide for varying periods of time. On being repulsed with the same peptide, they not only retained the initially bound peptide but could bind an equivalent amount more after an incubation time as short as 2 h. However, if the export to the cell surface of newly synthesized MHC-I molecules was blocked, the ability to bind appreciable amounts more of the peptide was not regained. These observations prompted the current study in which we have examined, on a cell by cell basis, the cell surface dynamics of all MHC-I molecules, including those MHC-I molecules that can bind exogenous peptide. The objective was to obtain a better understanding of the interaction between exogenous peptide and cell surface MHC-I.

Stable MHC-I is a heterotrimer consisting of a polymorphic integral membrane glycoprotein (H chain, ) noncovalently associated with an invariant protein (₂-microglobulin, ₂m) and with a peptide of the correct length and motif to bind with high affinity. The -chain consists of three domains. The ₃ domain associates with ₂m, and the ₁ and ₂ domains constitute the Ag-binding groove (3, 4), which can accommodate peptides of 8–10 residues with the right binding motif (3, 5, 6, 7). Such peptide binds to -chain with high affinity and is here referred to as p_H. Peptides that are longer or contain only a partial binding motif may also bind, but do so with lower affinity (8, 9), such peptides being here referred to as p_L. MHC-I are constitutively synthesized in the endoplasmic reticulum (ER) and exported to the cell surface as heterotrimers containing peptides of varying affinity. When on the cell surface, both the bound ₂m and peptide can exchange freely and independently with ₂m and peptide from the surroundings (10, 11, 12).

Because the most well studied function of MHC-I is its ability to present peptide Ag to CD8-expressing T lymphocytes, many studies have focused on examining the kinetics of exogenous peptide binding to MHC-I. Studies using biochemically purified and refolded MHC-I subunits show that D^b binds to a D^b-specific p_H (Y-Flu-NP) with a t_1/2 of 13 h at 22°C (13). If ₂m is also present, the D^b binds Y-Flu-NP with a t_1/2 of 0.75 h. The study suggests that ₂m is critical in reconfiguring the to make it receptive to peptide. When preformed -₂m heterodimers were used in the binding, Y-Flu-NP bound much more efficiently, with a t_1/2 of <0.2 h at 22°C. Because most surface MHC-I contain bound peptide, Hörig et al. (12) examined the binding of exogenous peptides to preformed p--₂m heterotrimers. In a K^b-₂m complex loaded with PolyI peptide (p_H), the p_H was replaced by K6 peptide (a medium-affinity peptide, p_M) with a t_1/2 of 6 h, whereas in a K^b-₂m complex loaded with a K^b-restricted epitope of vesicular stomatitis virus nuclear protein (NP) (VSVp) (p_M), the p_M could be replaced by another p_M (E6 peptide) with a t_1/2 of 1.5 h. This study suggested that the rate of binding of exogenous peptide to MHC-I is determined by the off-rate of the prebound peptide.

Exogenous peptide binding to MHC-I on the cell surface has also been examined. The half-time required for the p_H, Y-Flu-NP, to bind to cell-surface D^b is 9.3 ± 1.1 min (peptide concentration, 1.68 µM; Ref. 8), very similar to results for Y-Flu-NP binding to purified -₂m heterodimers (13). MHC-I that are capable of binding exogenously added peptide are referred to as peptide-receptive MHC-I (PR-MHC-I) in this study (14). Luscher et al. (11) and Christinck et al. (8) showed that 10% of surface D^b on EL4 thymoma cells are available for binding by ¹²⁵I-Y-Flu-NP peptide during a 2-h incubation at 37°C. However, it is still unknown whether these PR-D^b molecules contain solvent in their peptide binding groove (i.e., -₂m) or a weakly bound peptide (i.e., p_L--₂m; p_L denoting a low affinity peptide).

To facilitate the study of peptide binding to PR-MHC-I, many investigators have used the murine mutant cell line RMA-S (15, 16). RMA-S cells are MHC-I-deficient somatic variants of the T lymphoma cell line, RMA resulting from a defect in the TAP, necessary for providing processed peptides for binding to newly synthesized MHC-I (17, 18). PR-MHC-I can be stabilized on RMA-S cells by culturing cells at 26°C but are unstable at 37°C (t_1/2 30–60 min) (14, 15, 16). However, the binding of exogenous peptide can stabilize the PR-K^b at 37°C and thus facilitate the study of peptide binding kinetics.

Stability of MHC-I complexes has generally been studied by monitoring changes in conformation that result in gain or loss of conformationally specific Ab-recognition epitopes on the ₁, ₂, and/or ₃ domains. Burshtyn et al. (13) examined the retention of ₁₂ antigenic epitopes on biochemically purified MHC-I complexes with an ₁₂-specific mAb. It was found that a p_H-containing heterotrimer, Y-Flu-NP-D^b-₂m, is relatively stable with a t_1/2 ranging from 2.5 to 5 h. At 37°C, Y-Flu-NP-D^b is slightly less stable (t_1/2 2 h) than the heterotrimer but is much more stable than D^b-₂m complexes (t_1/2 0.2 h). The stability of Y-Flu-NP-D^b-₂m heterotrimers on the EL4 cell surface was investigated and was found to be significantly more stable than the purified form with a t_1/2 greater than 10 h at 37°C (8, 11).

In the presence of peptide, -₂m complexes can be stabilized (i.e., can retain ₁₂ antigenic epitopes) by binding to peptide (15, 19). In the absence of peptide in the surroundings, the -₂m heterodimer quickly undergoes conformational changes leading to the loss of the ₁₂ antigenic epitopes (thereby denoted as ₁₂^- -₂m) (14, 15, 20). The half-time for the transition from the ₁₂⁺ state to the ₁₂^- state was estimated to be 30–60 min (14, 20). Abs that recognize specifically the ₃ antigenic epitopes are often used to detect the presence of ₁₂^- MHC-I on the cell surface because the ₃ domain is generally not affected by peptide or ₂m dissociation (20, 21). The ₂m exchange occurs independently of the peptide associated with the (i.e., it occurs for p_H--₂m at the same rate as for p_L--₂m or -₂m complexes) (11, 12). The dissociation of ₂m from the ₁₂^- -₂m complexes induces conformation changes in the leading to loss of the ₃ epitope (21). ₃^- K^b was detected on the cell surface by immunoprecipitation using a mAb recognizing an intracellular domain of H-2K^b ( p8) (22). These ₃^- have a t_1/2 of 1 h on the cell surface. It was found that one-third of surface K^b on EL4 or spleen cells are not associated with ₂m and are ₁₂^- (22). Surface expression of MHC-I is also lost through internalization (20). Internalization of MHC-I occurs constitutively on activated lymphoid cells (23, 24, 25, 26, 27). It appears that intact molecules are recycled to the cell surface and denatured molecules are shunted to a lysosomal pathway.

The above studies provide much detailed information on the different forms of MHC-I, their stability, and their ability to bind peptide. However, they do not give a clear overall picture of what happens to the whole population of MHC-I molecules on an intact cell and how this population is affected when it is exposed to exogenous peptide. In this study, we have used flow cytometry to examine the generation, stability, and peptide-binding capacity of MHC-I molecules on a per cell basis. For mouse MHC-I K^b molecules, we found that about half have a mean half-life of only 1 h with the remaining half having a half-life of 20 h. To maintain equilibrium, this implies that 20 short-lived molecules must be exported to the cell surface for each long-lived molecule. Nearly all PR K^b molecules are in the short-lived population and appear to have reached the cell surface containing a peptide bound with very low affinity.

	Materials and Methods

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Cells and culture

Normal C57BL/6 (B6, H-2^b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and kept in a specific pathogen-free environment. In most experiments, 6- to 10-wk-old female mice were used (although either sex gave similar results). Spleens harvested from B6 mice were pressed through a wire mesh screen with a disposable syringe plunger into -MEM medium (Life Technologies, BRL, Burlington, Ontario, Canada) supplemented with 10% FCS, 50 µM 2-ME, and 10 mM HEPES (10% complete medium, CM). Splenocytes were washed once with 1% CM (supplemented with 1% FCS instead of 10% FCS) and were resuspended in 5 ml of 10% CM, underlaid with 5 ml of Lympholyte M (CEDARLANE Laboratories, Hornby, Ontario, Canada), and centrifuged at 500 x g for 20 min to remove red cells and dead cells. Con A-activated lymphoblasts (Con A blasts) were generated by culturing 5 x 10⁶ splenocytes in 5 ml of 10% CM supplemented with Con A (2 µg/ml; ICN Pharmaceuticals Canada, Montreal, Quebec, Canada). For most experiments (unless stated otherwise), day 1.5 Con A blast cells were used.

Antibodies

The following Abs were used: PE-labeled mAb AF6-88.5 (1 µg/10⁶cells/100 µl) recognizing specifically the ₁₂ region of H-2K^b (28, 29) and FITC-labeled mAb KH95 (1 µg/10⁶ cells/100 µl) recognizing specifically H-2D^b (30) were purchased from BD PharMingen (San Diego, CA). FITC-labeled anti-biotin mAb was purchased from Sigma (St. Louis, MO). The mAb Y3 (IgG2a, 1.5 µg/10⁶ cells/100 µl) recognizing the ₁₂ domain of H-2K^b (31, 32), mAb 5F1 (IgG2b, 1 µg/10⁶ cells/100 µl) recognizing the ₂ domain of H-2K^b (32), and mAb 25D1.16 (IgG2a, 0.5 µg/10⁶ cells/100 µl) recognizing the SIINFEKL peptide associated with H-2K^b (33) were purified from hybridoma culture supernatants using Protein A (Sigma) chromatography. Purified Abs were labeled with FITC by adding 0.5–1 mg of FITC-CELITE (Calbiochem, La Jolla, CA) to the Ab solution (1–2 mg of purified mAb in PBS, adjusted to pH 9.0 with 5% sodium carbonate solution). The mixture was incubated in the dark for 30–45 min at room temperature. The FITC-labeled Ab was recovered by centrifugation at 500 x g for 5 min and fractionation on a P10 Bio-Gel exclusion column (Bio-Rad, Hercules, CA). The yellow mAb-containing fractions were collected, passed through a 0.2-µm filter (Gelman Sciences, Ann Arbor, MI), and stored at 4°C.

Flow cytometry and data analysis

Fluorescence and light scatter properties of individual cells were measured on a FACScan analyzer (BD Biosciences, Mountain View, CA) using logarithmic amplification of the fluorescence signals and linear amplification of the right angle/forward angle light scatter signals. Live splenocytes were gated (on the basis of right angle/forward scatter measurements) and analyzed for their fluorescence. Cells (5 x 10⁵) in 50 µl of 0.5% BSA/PBS were first incubated with 4 µl of reconstituted normal mouse serum (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 5 min. FITC- or PE-labeled mAbs were then added to the cells and incubated on ice in the dark for 30–45 min. The cells were then washed in 0.5% BSA/PBS, resuspended in 0.3 ml of 0.5% BSA/PBS, and analyzed in a FACScan analyzer. The number of Ab bound (Ab binding capacity, ABC) (for AF6-88.5 mAb, KH95 mAb, 5F1 mAb, Y3 mAb, and 25D1.16 mAb) per cell was calculated using a Quantum Simply Cellular kit (Sigma). Quantum Simply Cellular kit is a mixture of four populations of microbeads with different ABCs plus one nonbinding microbead population. The ABC is derived from covalently bound goat anti-mouse Ig on the microbeads. This goat anti-mouse Ig has equivalent reactivity to each mouse isotype (IgG1, IgG2a, and IgG2b). Quantum Simply Cellular microbeads were used in the experiments as an external calibrator. Microbeads (10⁵) in 50 µl of 0.5% BSA/PBS were stained with FITC- or PE-labeled mAbs in parallel with the cell samples. Staining of microbeads and cell samples was analyzed with the same instrumental setting except that light side scatter was set lower for microbeads. A regression calibration curve was constructed using the QuickCal program (provided by Sigma), which plots the mean fluorescence intensities of the Quantum Simply Cellular microbeads against the ABC values predetermined for each microbead population. The ABC values for cell samples were calculated using the regression calibration curve and the mean fluorescence intensity of mAb stainings. In studying the stability of surface K^b and D^b, the percentage of stable K^b (or D^b) molecules remaining on the cell surface was calculated as (ABC of AF6-88.5 (or KH95) per cell at time, t)/(ABC of AF6-88.5 (or KH95) per cell at t = 0). Because ABC is proportional to the number of Ags per cell, for other studies, the number of Ags per cell was used directly in plotting and statistical analysis.

MHC-I binding peptide

Peptides used were K^b-restricted epitopes of chicken OVA, SIINFEKL, (OVAp_258–265) (34) and VSV NP, RGYVYQGL, (VSVp_52–59) (35), and a D^b-restricted epitope of influenza nucleoprotein, ASNENMETM, (Flu-NP_366–374) (36). Chicken OVA, SIINFEKL (OVAp_258–265) was prepared by the Ontario Cancer Institute Biotechnology Laboratory, using an Applied Biosystems Peptide Synthesizer (Applied Biosystems, Foster City, CA). A derivative of SIINFEKL, biotinylated OVA peptide (SIINFEK(bio)L) was purchased from Alberta Peptide Institute (Edmonton, Alberta, Canada) and prepared by using an Applied Biosystems model 430A Peptide Synthesizer. Both VSVp_52–59 and Flu-NP peptides (>90% purity) were generous gifts from Dr. B. H. Barber (University of Toronto, Toronto, Canada). OVAp and VSVp peptides are natural ligands for K^b and bind to K^b with high affinities (34, 35). Flu-NP peptide is a natural ligand for D^b and binds with high affinity (36).

Measurement of the stability of MHC-I and PR-K^b

Con A blasts were washed twice with 1% CM, and the cell pellet was suspended in 10% CM (4 x 10⁶ cells/ml) supplemented with 5 µg/ml Brefeldin A (BFA; Sigma-Aldrich Canada, Oakville, Canada). BFA is a fungal metabolite that has been shown to block protein transport to the cell surface (37). Cells were then cultured at 37°C in a 4-ml polystyrene tube (10⁶ cells/250 µl/tube) for various lengths of time as indicated. For the 24-h time point, cells were spun down at t = 15 h and recultured in 10% CM supplemented with 2 µg/ml Con A to preserve cell viability and 0.5 µg/ml BFA, which is sufficient to maintain the BFA effect without provoking cytotoxicity. At the end of incubation, cells were washed twice with 0.5% BSA in PBS. The expression of K^b and D^b was examined by staining the cells with Abs (PE-AF6-88.5 mAb for K^b and FITC-KH95 for D^b). The expression of PR-K^b was studied, using a FACStain technique established in our previous study (2), by incubating the cells with OVAp (or OVAp_K-bio when cells were prepulsed with OVAp) (100 ng/ml/10⁶ cells) on ice for 45 min, washing twice, and then staining with FITC-25D1.16 mAb (or FITC-anti-biotin Ab when OVAp_K-bio was used). Note that mAb 25D1.16 is specific for K^b-OVAp (2, 33).

Prepulsing of cells with high affinity peptides (p_H)

Day 1 Con A blasts were washed once with 1% CM and then recultured in 10% CM supplemented with OVAp (or VSVp) Flu-NP peptides (1 µg/ml) and Con A (2 µg/ml) overnight (8–10 h). The cells were then washed three times and used to study the stability of surface K^b and D^b or the regeneration of surface PR-K^b. In experiments studying the stability of surface PR-K^b or OVAp binding to PR-K^b, the prepulsed cells were recultured in 10% CM supplemented with Con A (2 µg/ml) at 37°C for an additional 6 h in the absence of exogenous peptide.

Measurement of OVAp binding

Day 1.5 Con A blasts were washed twice and resuspended at 4 x 10⁶ cells/ml in 1% CM containing OVAp (1 µg/ml, 100 ng/ml, 10 ng/ml, or 1 ng/ml) with or without BFA (5 µg/ml). Cells were then cultured at room temperature in 4-ml polystyrene tubes (10⁶ cells/250 µl/tube) for various lengths of time as indicated. At the end of incubation, cells were washed twice with 0.5% BSA/PBS. OVAp binding was detected with FITC-25D1.16 mAb staining. Cells pulsed with VSVp, which should not be recognized by FITC-25D1.16 mAb, were used for background-staining control.

Generation of PR-K^b

Day 1.5 Con A blasts, after being prepulsed with OVAp or VSVp (1 µg/ml) overnight, were washed three times with 1% CM and recultured in 10% CM supplemented with Con A (2 µg/ml) at 37°C for various lengths of time as indicated. Prepulsed cells (250 µl, 4 x 10⁶ cells/ml) were cultured in a 4-ml polystyrene tube for each time point. At the end of incubation, the expression of surface PR-K^b was detected by incubating the cells with OVAp (or OVAp_K-bio when cells were prepulsed with OVAp) (100 ng/ml/10⁶ cells) on ice for 45 min, washing twice, and then staining with FITC-25D1.16 mAb (or FITC-anti-biotin Ab when OVAp_K-bio was used).

Data analysis

Curve fitting was performed using the GraphPad Prism data analysis program (version 2.0; Intuitive Software for Science, San Diego, CA). The following formulae were used: For two-phase exponential decay, (t) = A₁*exp(-k₁*t) + A₂*exp(-k₂*t). Here, (t) is the remaining fraction of surface MHC-I at time t; A₁ and A₂ represent the initial fractions (or numbers of ABCs) of the two MHC-I subpopulations; and k₁ and k₂ are the first-order decay rate constants. For loss of surface MHC-I, t_1/2 was calculated from the relation t_1/2 = ln(2)/k. For first-order decay, (t) = A*exp(-k*t). A represents the number of Ab binding sites per cell and k is the first-order rate constant. Data from the study of surface PR-K^b on VSVp-pulsed cells were fit to the equation (t) = A*exp(-k*t) + p, where p is the plateau value for the expression of the quasi-stable PR-K^b subpopulation. For peptide binding, the receptor-ligand association relation is (t) = P*(1 - exp(-k*t)). P represents the plateau value of OVAp binding. The rate of PR-K^b generation on the cell surface is the balance of the rate of PR-K^b export and the rate of PR-K^b decay on the cell surface. Data from the study of PR-K^b generation were fit to the relation (t) = /k*(1 - exp(-k*t)). is the rate of PR-K^b export, in molecules per hour and k is the rate of decay of cell surface PR-K^b. Note that the ratio, /k gives the plateau value.

	Results

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Stability of total MHC-I on the cell surface

Stability of total surface MHC-I on B6 Con A blasts was monitored as a function of time after blocking the export of newly synthesized MHC-I by addition of BFA. Abs and peptides used in this study are summarized in Table I. Optimal staining conditions were determined for each mAb used in staining 5 x 10⁵ cells in a 50-µl volume (data not shown). Examples of K^b and D^b staining with mAb AF6-88.5 and KH95, respectively, are shown in Fig. 1, A and B. Because the loss of Ab recognition epitopes is thought to be rapidly followed by the decay of the molecule (14, 20, 22), in this study the loss of Ab recognition epitopes is regarded as the loss of MHC-I.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Examples of FACscan data of splenocytes stained with mAb AF6-88.5, mAb KH95, mAb Y3, mAb 5F1, or mAb 25D1.16. A, B6 Con A blasts stained with PE-conjugated mAb AF6-88.5; B, FITC-labeled mAb KH95 (solid lines). Con A blasts stained with isotype control IgG were used as control (dashed lines). C, B6 Con A blasts stained with FITC-conjugated mAb Y-3, or (D) FITC-labeled mAb 5F1. E, To measure the number of PR-Kb, B6 Con A blasts pulsed with OVAp (100 ng/ml/106 cells) were stained with FITC-conjugated mAb 25D1.16. To control for the specificity of staining, B6 Con A blasts pulsed with VSVp (100 ng/ml/106 cells), which should not be recognized by mAb 25D1.16 (33 ) were used (dashed line). F, To measure re-expression of PR-Kb on cells prepulsed with pH (VSVp), cells were pulsed immediately (t = 0, solid line) or after 4 h at 37°C (dashed line) with OVAp and stained with mAb 25D1.16 as in E.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Stability of cell surface Db and Kb on spleen cells in the presence of BFA. The expression of Kb (•) and Db () on the cell surface was monitored using mAb AF6-88.5 and mAb KH95, respectively. Con A blasts were cultured in the presence of BFA (5 µg/ml), which blocks the export of newly synthesized MHC-I, for various lengths of time (as indicated). At the end of the incubation, the expression of Kb and and Db was assessed. Numbers of Ab-binding sites (ABC) were calculated for each experiment using a Quantum Simply Cellular Kit (Sigma). At t = 0, the number of ABC per cell ranged from 118,900 to 135,700 for Kb and from 68,000 to 75,200 for Db. The results are presented here as fractions of Kb and Db molecules remaining on the cell surface at each time point after the start of incubation. The data plotted are the mean values of three independent experiments and were fit to a two-phase exponential decay relation, the fits being indicated by the lines on the figures. The calculated fitting parameters are summarized in Table II (no. 1 and 2).

MHC-I containing p_H have been shown to be very stable in vitro (12, 14, 21, 38), leading us to hypothesize that such p_H-K^b and p_H-D^b are the quasi-stable populations observed in Fig. 2 and that K^b and D^b populations not associated with p_H are unstable. Therefore, we examined whether prolonged exposure to OVAp, a peptide that binds K^b with high affinity, would influence the t_1/2 of surface K^b. Con A blasts were prepulsed overnight with OVAp to maximize the expression of p_H-bound K^b and minimize the expression of K^b not associated with p_H (12, 14). The cells were washed free of exogenous peptide, and BFA was immediately added to prevent export of newly synthesized MHC-I. The expressions of OVAp-K^b complexes and of total K^b were monitored for the next 24 h using FITC-labeled 25D1.16 mAb (33) and PE-labeled AF6.88.5 mAb, respectively (Fig. 3A). The short-lived population previously observed is no longer detectable. The data were successfully fitted to a first-order decay equation (see Materials and Methods), the results being summarized in Table II (no. 3 and 4). The t_1/2 value for OVAp-K^b complexes was calculated to be 45 ± 3 h (Table II, no. 3) and for total surface K^b was 29 ± 9 h. In comparison to quasi-stable K^b, OVAp-K^b seems to be more stable with a longer t_1/2, perhaps because OVAp binds with particularly high affinity.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Stability of cell surface OVAp-Kb complexes in the presence or absence of BFA. Day 1 B6 Con A blasts were prepulsed with OVAp (1 µg/ml) overnight and washed free of unbound peptide before being recultured in 10% CM with (A) or without (B) BFA for various lengths of time (as indicated). At the end of incubation, the expression of OVAp-bound Kb complexes (•) and total surface Kb molecules () were examined using 25D1.16 mAb and AF6-88.5 mAb, respectively. For each experiment, the number of ABC per cell was calculated. The data plotted are the number of ABC per cell against time and fit to an exponential decay relation, (t) = A*exp(-k*t). The results of the curve fitting are summarized in Table II (no. 3–6).

Stability of PR-K^b on the cell surface

Besides p_H-MHC-I, a population of MHC-I called "peptide-receptive" has been documented, these being MHC-I on the cell surface that can bind appropriate exogenous peptides (8, 12, 14). We next studied the stability of these PR-K^b on the cell surface. In the presence of BFA, the surface expression of PR-K^b was monitored for 6 h using OVAp and FITC-labeled 25D1.16 mAb as described previously (2, 33). An example of PR-K^b staining at t = 0 is shown on Fig. 1E. The mean values of measurements from three independent experiments were plotted as a function of time. As seen in Fig. 4 (open circles), the surface expression of PR-K^b decreased drastically during the first 2 h (24 ± 1% PR-K^b remained) followed by a much slower rate of decline. The data suggest the existence of two subpopulations of PR-K^b with very different half-lives. They were fitted successfully to a two-phase exponential decay equation (Table II, no. 7). The less stable subpopulation (74% of total) has a t_1/2 of 0.6 ± 0.2 h, and the more stable subpopulation (26% of total) has a t_1/2 of 4.0 ± 1.4 h. We hypothesized that the longer-lived subpopulation of PR-K^b represented K^b molecules stabilized with intermediate affinity peptides that could become PR on losing the peptide. To test this, cells were pulsed overnight with a p_H (VSVp). Con A blasts prepulsed with VSVp (p_H) were washed three times and then recultured at 37°C for 6 h to allow the constitutive export and accumulation of newly synthesized PR-K^b on the cell surface. At the end of the incubation, BFA was added and surface PR-K^b expression was monitored for 6 h. The mean values of measurements from two independent experiments were plotted as a function of time (Fig. 4, closed circles). There was a rapid decay of surface PR-K^b in the first 2 h (5.3 ± 0.1% PR-K^b remained) followed by a pseudo plateau with a much slower decline rate. The data were successfully fitted to a first-order decay equation that included a plateau. The t_1/2 of the unstable PR-K^b on VSVp-prepulsed cells was calculated to be 0.5 ± 0.1 h (Table II, no. 8), not different from that found for unpulsed cells. By 6 h, there were only 4.1 ± 0.1% PR-K^b remaining on the cell surface, suggesting only a small population of quasi-stable PR-K^b on VSVp-prepulsed cells in comparison with non-prepulsed cells (24 ± 1%). This implies that quasi-stable PR-K^b are K^b associated with p_M or p_L, the expression of which was greatly reduced on VSVp-prepulsed cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Stability of PR-Kb on spleen cells. The expression of PR-Kb on day 1.5 B6 Con A blasts after being cultured in the presence of BFA () was examined as previously described. Briefly, at each time point, cells were pulsed with OVAp (1 µg/ml) for 45 min, and then stained with 25D1.16 mAb. The expression of PR-Kb on Con A blasts prepulsed with VSVp overnight was also examined (•). Day 1 Con A blasts were prepulsed overnight with VSVp (1 µg/ml), washed three times with 1% CM, recultured in 10% CM for 6 h, and then recultured again in 10% CM supplemented with BFA (5 µg/ml) for various lengths of time as indicated. The expression of PR-Kb was then examined. The data for no prepulse were fit to a two-phase exponential decay model, (t) = A1*exp(-k1*t) + A2*exp(-k2*t) () and for VSVp prepulse to a one-phase exponential model, (t) = A*exp(-k*t) + p where p is a plateau value (•). The results of the curve fittings are summarized in Table II (no. 7 and 8).

We next studied the kinetics of p_H binding to surface PR-K^b. Con A blasts were pulsed with OVAp peptide for various lengths of time, and the formation of OVAp-K^b complex was detected using FITC-labeled 25D1.16 mAb. Using an OVAp concentration of 100 ng/ml, it was found that binding to H-2K^b followed biphasic kinetics with a pseudo plateau being reached by 1 h (Fig. 5A). The initial binding appeared to follow first-order kinetics (t_1/2 of 15–16 min), whereas the latter binding occurred at a slower steady rate (t_1/2 of 45–53 min). The half-times for OVAp binding were derived by fitting the data to a receptor-ligand association equation (see Materials and Methods) and are summarized in Table III (line 1).

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Kinetics of OVAp binding to surface PR-Kb on spleen cells. A, Binding of OVAp to PR-Kb on day 1.5 Con A blasts was examined by pulsing the cells with OVAp (100 ng/ml) for various lengths of time as indicated. The OVAp-Kb complexes were detected using 25D1.16 mAb. This is representative of two independent experiments. B, Binding of OVAp to PR-Kb on Con A blasts was repeated in the presence of BFA, which blocks the export of newly synthesized proteins. Four different concentrations of OVAp were used in the assay: 1 µg/ml (not shown in the graph, see Table III), 100 ng/ml (), 10 ng/ml (), and 1 ng/ml (). ABCs were calculated, plotted against time, and fitted to a binding equation, (t) = p*(1 - exp(-kt)) where p is the plateau value and k is the rate constant. The results of curve fitting are summarized in Table III.

We also examined OVAp binding to surface PR-K^b using concentrations of OVAp ranging from 1 to 1000 ng/ml (Fig. 5B). The plateau values for OVAp-K^b formation were the same (15,000 molecules) for all peptide concentrations. For 1 ng/ml, 29 min was required to reach one-half the plateau value. This time decreased to 17 min for 10 ng/ml and 11 min for 100 ng/ml but remained the same for 1000 ng/ml. Thus, the rate of the initial phase of peptide binding to PR-K^b was dependent on the peptide concentration at lower peptide concentrations but becomes independent of peptide concentration at higher concentrations. The OVAp concentration used appeared to have little effect on the latter binding phase, consistent with the hypothesis that these PR-K^b molecules contain a p_M and that the rate of OVAp binding is determined by the off-rate of this p_M. The fact that the rate of OVAp binding to unstable PR-K^b molecules also became independent of OVAp concentration at high OVAp concentrations suggests that this binding rate might also be determined by the off-rate of a peptide, in this case p_L, from unstable PR-K^b molecules (see Discussion).

Rate of PR-K^b generation

We next measured how fast new PR-K^b molecules accumulated on the cell surface. A zero baseline of PR-K^b expression was established by prepulsing Con A blasts overnight with VSVp and then washing the cells three times. PR-K^b generation was monitored using OVAp and FITC-labeled 25D1.16 mAb. The measurements were fit to the equation (t) = /k*(1 - exp(-k*t)), where is the number of PR-K^b exported to the cell surface per unit of time, and k is the exponential decay constant for those that have reached the surface. Note that the ratio /k defines the plateau value reached. The fitting parameters are summarized in Table IV. The mean values of two independent experiments presented in Fig. 6 depict the kinetics of PR-K^b generation at 37°C. There was a steady increase in PR-K^b expression within 4 h of incubation, followed by a plateau of 17,700 PR-K^b molecules per cell (Fig. 6, Table IV). The plateau value was a bit higher than the number of surface PR-K^b on unmanipulated cells (12,700 PR-K^b molecules per cell; Table II, no. 7, A₁+A₂).

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Generation of PR-Kb on spleen cells. The accumulation of surface PR-Kb on Con A blasts was studied by prepulsing day 1 Con A blasts with VSVp (1 µg/ml) overnight, and then reculturing the cells at 37°C for various lengths of time (as indicated). At each time point, the expression of surface PR-Kb was examined by pulsing cells with OVAp (1 µg/ml) for 45 min and then staining the OVAp-Kb complexes with 25D1.16 mAb, as described in Fig. 3A. The data were plotted as the mean values (n = 2) of the number of 25D1.16 binding sites per cell against time. The results of curve fitting are summarized in Table IV.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Quasi-stable K^b appeared to be associated with p_M or p_H because K^b associated with p_H (e.g., OVAp-K^b) were more stable and had a longer t_1/2 (Fig. 3A and Table II, no. 3; t_1/2 = 45 ± 3 h) than the MHC-I associated with p_M or p_L (12, 39)_. We assume that when cells are prepulsed with a high concentration of p_H overnight, prebound p_M and p_L dissociate during the incubation and are replaced by p_H. Thus, prepulsing cells with p_H increases the percentage of p_H-MHC-I and decreases the amount of p_M/p_L-MHC-I on the cell surface. Note that unstable K^b molecules were not detectable on cells prepulsed with p_H when export of newly synthesized MHC-I was blocked (Fig. 3). This implies that the vast majority of unstable MHC-I molecules found on the cell surface are exported from the cell interior in this state rather than being produced from more stable MHC-I molecules already on the cell surface.

Unstable MHC-I molecules must form the bulk of newly produced MHC-I molecules arriving on the cell surface. Approximately one-half of K^b molecules on the surface of a Con A blast are unstable with a t_1/2 of 1 h; the remainder are quasi-stable with a t_1/2 of 20 h. To maintain this proportion, there must be 20 unstable molecules arriving on the cell surface for each newly produced quasi-stable molecule, a fact that does not appear to have been explicitly realized previously. However, Degen and Williams (40) made observations in exact agreement with the above numbers. They performed an [³⁵S]methionine pulse-chase experiment in which they monitored several different newly produced MHC-I molecules (including K^b) detectable with mAb against conformational epitopes of the heterotrimeric molecule and noted a 50% loss of molecules from maximum values in 1 h of incubation (see their Fig. 9). In this study, we have used an Ab that is specific for K^b-OVAp complexes (33) as a tool to measure numbers of PR-K^b. These can be inferred by measuring the number of K^b-OVAp complexes after pulsing with a sufficiently high concentration of OVAp. As seen for total MHC-I, we found that there were two subpopulations differing significantly in half-life, referred to here as unstable and quasi-stable. The unstable subpopulation made up about three-fourths of all PR-K^b and had a t_1/2 of 0.6 ± 0.2 h; the quasi-stable subpopulation made up the remaining one-fourth and had a t_1/2 of 4.0 ± 1 h (Fig. 4 and Table II, no. 7). Binding of exogenous peptide to a heterotrimeric MHC-I is determined by the off-rate of the prebound peptide (10, 12); therefore, a heterotrimeric K^b molecule is truly PR only after the prebound peptide has dissociated and before the molecule decays. Consequently, the PR-K^b quasi-stable populations are probably K^b bound with intermediate affinity (p_M) peptide. The unstable PR-K^b subpopulation may contain no peptide or peptides of extremely low affinity in their binding groove. See below for an argument that they must contain peptide bound with extremely low affinity.

The half-life distribution of MHC-I molecules on the cell surface will depend upon their immediate past history. After pulsing overnight with p_H and adding BFA, no PR-MHC-I and no unstable MHC-I molecules were observed. All were converted to quasi-stable molecules or lost during the peptide pulse, and no more were generated because of the BFA block (Fig. 3). On unmanipulated Con A blasts, approximately one-fourth of PR-K^b belonged to the quasi-stable population. After an overnight incubation with a p_H followed by a 6-h incubation in the absence of peptide, unstable PR-MHC-I were rapidly regenerated. However, the number of quasi-stable PR-MHC-I was down at least 5-fold (Fig. 4). We hypothesize that this is because 6 h was not sufficient time to allow them to regenerate to equilibrium value.

We examined the kinetics of OVAp binding to PR-K^b on cells for which the various forms of MHC-I were in equilibrium (Fig. 5, Table III). OVAp binding was biphasic with a rapid initial phase corresponding to binding to unstable PR-K^b followed by a much slower increase corresponding to binding to quasi-stable PR-K^b. The rate of binding to quasi-stable PR-K^b was independent of OVAp concentration, consistent with the rate being determined by the off-rate of p_M from quasi-stable PR-K^b molecules. The initial rapid binding rate was also constant for high OVAp concentrations, with 11 min being required to reach half-maximum value for concentrations of 100 and 1000 ng/ml. However, the rate fell for lower OVAp concentrations, with 29 min being required to reach half-maximum value at a concentration of 1 ng/ml. If the OVAp were binding directly to an "empty" K^b molecule, then the binding rate should have continued to increase with increasing OVAp concentration. Instead, there appeared to be an intermediate state with a t_1/2 of 11 min to which OVAp could not bind. We hypothesize that this is K^b containing a p_L that dissociates with a mean t_1/2 of 11 min, after which OVAp can bind. At high OVAp concentrations this is essentially instantaneous; at lower concentrations the binding rate starts to become dependent on the OVAp concentration. A corollary to this hypothesis is that there are few truly "empty" K^b molecules on the cell surface implying that most/all PR-K^b molecules arrive on the cell surface containing a p_L. This is in agreement with evidence that a freshly synthesized MHC-I molecule is only released from the ER for export to the cell surface after being stabilized by binding a peptide (35, 41, 42, 43, 44). Note also that the cell line RMA contains many more PR-MHC-I molecules than its TAP-deficient variant RMA-S (14), favoring the hypothesis that peptide must be present in the ER for the formation of PR-MHC-I.

If most PR-K^b molecules actually contain a p_L, this implies that truly empty K^b molecules will decay very rapidly unless reoccupied by nearby peptide. If p_L dissociates to form a potentially p_H-receptive empty K^b complex with a t_1/2 of just 11 min, then how can one explain that the t_1/2 of unstable PR-K^b molecules in the absence of p_H was measured to be 0.5 h (Table II, no. 8)? We hypothesize that the original p_L or another nearby p_L has a high probability of being rebound to the empty K^b molecule before it denatures. To test this, we created conditions in which the concentration of exogenous p_L should be reduced. Cells were incubated in CM containing dialyzed FCS and protease inhibitors vs regular nondialyzed FCS. The lifetime of PR-K^b molecules was significantly shorter under the first set of conditions (data not shown).

We measured that 10% (12,600 binding sites for 25D1.16 mAb per cell, Fig. 4, t = 0) of surface K^b (124,000 binding sites for AF6-88.5 mAb per cell) are peptide receptive. This is in agreement with the measurements of Christinck et al. (8) and Luscher et al. (11) who showed that 10% of total D^b on EL4 cells are peptide receptive. Approximately three-fourths of PR-K^b (9500 molecules) are in the unstable population of K^b. Unstable K^b make up 50% of the total K^b population, i.e., 62,000 molecules, so that PR molecules appear to make up no more than one-sixth of all unstable K^b! Is there something different about the remaining five-sixths that prevents them from being peptide receptive? Or do they disappear before having a chance to pick up a p_H? A possible explanation for this apparent anomaly involves the phenomenon of MHC-I recycling.

T cells activated with Con A or in an MLR spontaneously internalize their MHC-I molecules into endosome-like vesicles with a t_1/2 of 1 h and recycle them from these vesicles to the cell surface with a t_1/2 of 20 min (24, 26, 27). MHC-I containing a p_H can be recycled to the cell surface still containing the p_H (25). However, in the vesicles they are exposed to a pH of 5.6, probably sufficiently low to denature unstable MHC-I. Denatured material is then routed to lysosomes. Endocytosis is via clathrin-coated pits (45) and depends upon a motif present in the cytoplasmic tail of the MHC-I molecule (46). Other cells can also recycle MHC-I. Thus, EBV-transformed B cells internalize MHC-I with a t_1/2 of 35 min and return them to the cell surface with a t_1/2 of 2–3 min (47). We hypothesize that unstable MHC-I lose their p_L in the endosome, denature, and are routed to the lysosomal pathway. This would provide a convenient mechanism for eliminating the large number of MHC-I molecules that reach the cell surface carrying a p_L. We hypothesize that the one-sixth of unstable MHC-I molecules that can be detected as peptide receptive is that fraction that binds an exogenous p_H before either undergoing spontaneous decay or being endocytosed and degraded.

A remaining puzzle in our data is that the t_1/2 of the K^b-OVAp complex on the cell surface was 45 h in the presence of BFA but only 14 h in its absence (Fig. 3 and Table II, lines 3 and 5). How is the presence of BFA producing such a large increase in the t_1/2 of K^b-OVAp complexes? BFA acts by preventing formation of coat protein I-coated vesicles required for transport of molecules through the Golgi (48, 49). As reviewed in Klausner et al. (50), this leads to the collapse of the Golgi into the ER, resulting in a complete block in transport of molecules from the ER to the cell surface. It seems unlikely that this block would affect the t_1/2 of K^b-OVAp complexes already on the cell surface. BFA also causes endosomes to fuse with the trans-Golgi into a single large structure. However, this fusion does not affect the recycling time (t_1/2 of 1 h) of cell surface molecules such as the transferrin receptor although there are subtle effects on lysosomal pathways. The prolongation of the t_1/2 of K^b-OVAp complexes could be one such subtle effect. Details of these pathways remain shrouded in ignorance and controversy (51). An unrelated possibility involves the observation that T cells (and B cells) continuously shed MHC molecules. These are not shed as individual molecules but are highly enriched in lipid vesicles (52). Shedding appears to be an active process as it is down-regulated by exposure to IFN- (53). BFA might somehow hinder/block this shedding process. Clearly, there is still much to be learned about the expression, structure, and stability of MHC-I.

It has been recently shown (54, 55) that an exogenous p_H can be transported to the ER where it can bind to MHC-I molecules. The free peptide is taken up into endosome-like vesicles. Blockers of endocytosis can prevent uptake, but addition of BFA does not. However, addition of BFA should prevent any p_H-MHC-I molecules synthesized in this way from reaching the cell surface and thus not affect the major conclusions reached here. Furthermore, Abdel Motal et al. (25) compared endosomal uptake of free exogenous peptide with endosomal uptake of the same peptide bound to cell surface MHC-I and, in comparison, could not detect uptake of free peptide.

It has been widely assumed that the functional MHC-I conformation is the peptide-associated heterotrimer. However, previous work from this laboratory suggests that NK cells express inhibitory receptor(s) recognizing the PR form of MHC-I (1, 2) and not the stable heterotrimer. We have recently found that this ability may be important in combating certain virus infections.⁵ Whereas T cells recognize the peptide residues presented in the peptide binding groove of MHC-I as well as part of the MHC-I molecule itself (56), NK cells seem to "see" conformationally specific MHC-I (57). The prototypic mouse NK inhibitory receptor, Ly49A, was shown to recognize MHC-I associated with peptide (58, 59). Analysis of the crystal structure of the extracellular domain of Ly49A and its ligand, D^d, suggests that the peptide is not directly involved in the recognition (57). As confirmed in a more recent structure-function study (60), it is the conformation of D^d, shaped by peptide binding, that is important for Ly49A recognition. Furthermore, another NK inhibitory receptor, Ly49C, seemed to recognize PR-K^b and not K^b associated with a p_H; binding of Ly49C to K^b was prevented by loading surface PR-K^b with a p_H (2). The important physiological roles of MHC-I in both NK and T recognition highlight the necessity of studying the structural conformations and stability of cell surface MHC-I.

	Acknowledgments

 
We thank Dr. David Williams and Dr. Michelle Letarte for their insightful reviews of this manuscript.

	Footnotes

 
¹ This work was supported by research grants (to R.G.M.) from the National Cancer Institute of Canada (NCIC). R.C.S. was a research student of the NCIC. NCIC is supported with funds provided by the Canadian Cancer Society.

² Current address: Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095-1662.

³ Address correspondence and reprint requests to Dr. Richard G. Miller, Ontario Cancer Institute, 610 University Avenue, Room 9-305, Toronto, Ontario, Canada, M5G 2M9. E-mail address: miller{at}oci.utoronto.ca

⁴ Abbreviations used in this paper: MHC-I, MHC class I; ₂m, ₂-microglobulin; ABC, Ab binding capacity; BFA, brefeldin A; CM, complete medium; Con A blasts, Con A-activated lymphoblasts; ER, endoplasmic reticulum; Flu-NP, a D^b-restricted epitope of influenza nucleoprotein (ASNENMETM); OVAp, a K^b-restricted epitope of chicken OVA (SIINFEKL); p, peptide; p_L, low affinity peptide; p_H, high affinity peptide; PR, peptide receptive; VSV, vesicular stomatitis virus; VSVp, a K^b-restricted epitope of VSV NP (RGYVYQGL); Y-Flu-NP, a D^b restricted peptide (YSNENMETM); p_M, medium affinity peptide; NP, nuclear protein.

⁵ R.C. Su, F. L. Graham, and R. G. Miller. A novel mechanism for recognition of adenovirus 5 infected cells by natural killer cells. Submitted for publication.

Received for publication March 8, 2001. Accepted for publication August 22, 2001.

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