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The Journal of Immunology, 2004, 173: 3707-3715.
Copyright © 2004 by The American Association of Immunologists

Differential Requirement for Tapasin in the Presentation of Leader- and Insulin-Derived Peptide Antigens to Qa-1b-Restricted CTLs1

LiQi Li2,*, Barbara A. Sullivan3,{dagger}, Carla J. Aldrich{ddagger}, Mark J. Soloski§, James Forman, Andres G. Grandea, III4,*, Peter E. Jensen{dagger} and Luc Van Kaer5,*

* Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; {dagger} Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322; {ddagger} Department of Microbiology and Immunology, Indiana University School of Medicine, Evansville Center, Evansville, IN 47712; § Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class I molecules present peptides derived from cytosolic proteins to class I-restricted CTL. The stable assembly of MHC class I molecules with peptides takes place in the endoplasmic reticulum (ER)6 and is controlled by a variety of accessory proteins, including chaperones with general housekeeping functions and factors with dedicated roles in class I assembly (1, 2, 3). Shortly after their synthesis, class I H chains bind with the lectin-like chaperone calnexin. Class I H chains then bind with {beta}2-microglobulin ({beta}2m), with release of calnexin, and assembly of H chain/{beta}2m heterodimers into the class I peptide-loading complex. This complex includes the lectin-like chaperone calreticulin, the thioloxidoreductase ERp57, the two subunits of TAP, and TAP-associated glycoprotein (tapasin). Like calnexin, calreticulin and ERp57 participate in the assembly of a variety of glycoproteins in the ER, whereas TAP and tapasin are dedicated to the assembly of class I molecules with peptides. The peptide-loading complex retains class I molecules in a peptide-receptive conformation until they assemble with high-affinity peptides, which induces their dissociation from the loading complex and export from the ER (4).

Tapasin is a 428-aa, proline-rich transmembrane protein with an ER retention signal in its cytoplasmic domain (5). Interestingly, the membrane-proximal region of tapasin consists of an Ig-like domain with sequence similarity to the Ig-like domain of MHC class I H chains, suggesting that tapasin may in fact be an MHC class I-like protein (5, 6). Tapasin forms a physical link between the class I H chain and TAP within the loading complex (7, 8), and also interacts with ERp57 (9). Although the precise role of tapasin in the quality control of class I assembly remains unclear, several functions have been proposed (10, 11, 12, 13, 14). Because tapasin brings class I close to the source of peptides (i.e., TAP), it has been suggested that tapasin enhances the speed by which peptides are loaded onto class I molecules. Although attractive, this idea has been called into question by studies with a truncated form of tapasin, which fails to interact with TAP but retains its interaction with class I, yet fully restores class I surface expression when introduced in tapasin-deficient cells (15). However, it is clear that the interaction of tapasin with TAP plays some role in Ag presentation, because tapasin mutants that fail to interact with class I H chains but retain their interaction with TAP clearly influence peptide acquisition by class I molecules (16). In addition, other studies have shown that tapasin deficiency reduces peptide translocation into the ER (15, 17, 18), that tapasin stabilizes TAP (18), and that tapasin is required for efficient peptide binding to TAP (19). These findings indicate that tapasin promotes peptide supply into the ER lumen, which may be critical in situations when the generation of high-affinity peptides in the cytosol is limiting. Studies with soluble tapasin that does not promote TAP association further suggested that tapasin stabilizes empty H chain/{beta}2m heterodimers within the peptide-loading complex (15, 20, 21), which is supported by studies of class I folding in cell lysates (22). This aspect of tapasin function may be mediated by its interaction with ERp57 (9). Reconstitution experiments of mouse class I molecules in insect cells (23) or human tapasin-deficient cell lines (24), and studies with tapasin-deficient mice (17, 25) have provided evidence for a role of tapasin in ER retention of peptide-receptive class I molecules. This possibility is consistent with the finding that tapasin interacts with coat protein I-coated vesicles (26), which ferry cargo molecules from the Golgi to the ER. Finally, there is significant evidence that tapasin can modulate the peptide repertoire of class I molecules, either by promoting the replacement of low-affinity peptides with high-affinity peptides (i.e., peptide editor function) (27, 28), or by broadening the class I-bound peptide repertoire (i.e., peptide facilitator function) (29). It has proven difficult to determine the relative importance of each of these proposed tapasin functions.

Studies regarding the role of tapasin in class I assembly have been confounded by the diversity of peptides that can bind with classical class I (class Ia) molecules. Therefore, we have focused on a nonclassical MHC class I (class Ib) molecule, Qa-1, the product of the T23 gene (30). Qa-1 is relatively nonpolymorphic and predominantly binds with a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of class Ia molecules (31, 32, 33). Despite its localization in a signal peptide, presentation of Qdm is TAP dependent (32, 34, 35), presumably because after cleavage by signal peptidase, signal peptides become substrates for signal peptide peptidase, and are released in the cytosol (36). The complex between Qa-1 and Qdm can be recognized by TCRs expressed by CTL (37, 38) as well as by CD94/NKG2 receptors expressed by NK and NK T cells (39, 40, 41). Although Qdm has an ideal sequence for binding with Qa-1b (39), other peptides can bind Qa-1b (42, 43, 44, 45, 46, 47). For example, Qa-1b molecules can present a peptide derived from the insulin B chain to T cells (42). In contrast to Qdm, however, presentation of insulin is independent of TAP expression and involves an exogenous pathway (42). Before the present study, it was unclear whether tapasin played a role in Qa-1b-restricted Ag presentation.

The unique properties of the Qa-1b Ag presentation system provided us with an opportunity to evaluate tapasin function. We found that tapasin links Qa-1b molecules to TAP. In the absence of tapasin, most Qa-1b molecules were retained in the ER, but egress of these molecules to the cell surface was partially rescued by culture of cells at reduced temperature (26°C). Despite these profound effects on Qa-1b trafficking, tapasin deficiency had only modest effects on Qa-1b cell surface expression. Presentation of endogenous Qdm peptides to Qa-1b-restricted, alloreactive CTL required tapasin expression, whereas presentation of insulin to Qa-1b-restricted, insulin-specific CTL was tapasin independent. Nevertheless, positive selection of Qa-1b-restricted, insulin-specific CD8+ T cells in TCR transgenic, tapasin-deficient mice was significantly impaired. Collectively, our findings indicate that tapasin is dispensable for retention of empty Qa-1b molecules in the ER and plays a critical role for quality control of Qa-1b assembly with Qdm but not insulin peptides. Our findings are also consistent with the proposed peptide-editing function of tapasin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Eight- to 10-wk-old C57BL/6J (B6) and {beta}2m–/– (B6.129P2-B2mtm1Unc) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TAP1–/– mice and Tapasin–/– (Tpn–/–) mice have been described previously (17, 48). 6C5 TCR transgenic mice have been described (49) and were crossed with TAP–/– and Tpn–/– mice to generate 6C5+.TAP–/– and 6C5+.Tpn–/– mice. Transgenic mice were identified by staining peripheral blood with anti-CD4, -CD8, and -V{alpha}3.2 Abs, followed by flow cytometry. TAP and tapasin mutations were identified by PCR. All mice were bred and maintained in the animal facility at Vanderbilt University School of Medicine.

Immunoprecipitations and pulse-chase analysis

For immunoprecipitation experiments, Con A (Calbiochem, La Jolla, CA)-induced T cell lymphoblasts were radiolabeled with 500 µCi of [35S]methionine/cysteine mix (PerkinElmer Life Sciences, Boston, MA) for 1 h at 37°C. Cells were lysed in 50 mM Tris (pH 7.5), 150 mM NaCl (TBS), 1% digitonin (Calbiochem), 1 mM PMSF (Sigma-Aldrich, St. Louis, MO), and 2 µg/ml leupeptin (Sigma-Aldrich), and cleared of debris by centrifugation. Proteins were precipitated by antisera directed against tapasin (50) or TAP1 (obtained from Dr. J. Monaco, University of Cincinnati, Cincinnati, OH). Immunoprecipitates were collected using protein A-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ). For secondary precipitations, the primary precipitates were boiled in 1% Nonidet P-40 plus 0.2% SDS in TBS, and the solution was increased in volume with 1% Nonidet P-40 in TBS, to dilute the SDS to a concentration of 0.02%. Free H2-Kb H chains were isolated with anti-X8, a conformation-independent antiserum that reacts against the K locus exon 8 product (obtained from Dr. S. Joyce, Vanderbilt University, Nashville, TN), and Qa-1b H chains were precipitated with a mAb 6A8.6F10.1A6 specific for peptide 161–179 of Qa-1b (BD Pharmingen, San Diego, CA). Precipitates were resolved by SDS-PAGE and visualized by fluorography/autoradiography or phospor imaging (Amersham Pharmacia Biotech). In some experiments, bands were quantified by ImageQuant software (Amersham Pharmacia Biotech).

Pulse-chase assays were performed as described (48). Briefly, Con A blasts were pulsed for 10 min with [35S]methionine/cysteine, and then chased at either 26 or 37°C with culture medium containing 1 mM cold methionine and 1 mM cold cysteine. Cell aliquots from each time point were lysed in 1% Nonidet P-40 in TBS, containing 1 mM PMSF and 2 µg/ml leupeptin. Class I molecules were then precipitated with H2-Kb-specific Y3 mAb (American Type Culture Collection, Manassas, VA) or Qa-1b-specific 6A8.6F10.1A6 mAb. Where indicated, the immunoprecipitates were treated with endoglycosidase H (Endo H; New England Biolabs, Beverly, MA) for 1 h at 37°C before resolving samples by SDS-PAGE.

Flow cytometry

Single-cell suspensions were prepared from freshly isolated thymi, inguinal lymph nodes, and spleens. Cells were incubated with mAbs for 30 min at 4°C. Cells were washed twice, and analyzed by flow cytometry. Unconjugated, FITC-, PE-, or PerCP-conjugated Abs specific for murine CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.72), B220 (RA3-6B2), H2-Kb (Y-3), Qa-1b (6A8.6F10.1A6), or V{alpha}3.2 (RR3-16) (all from BD Pharmingen) were used in various combinations for flow cytometric analysis. For evaluation of Qa-1b staining, biotinylated goat anti-mouse IgG was developed with PE-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA). Data acquisition was performed using a FACSCalibur instrument (BD Biosciences, San Diego, CA). Electronic gates were set on lymphocytes based on their forward- and side-scatter profiles. Fluorescence profiles were analyzed using CellQuest software (BD Biosciences).

Cytotoxicity assays

The anti-Qa-1b-specific CTL clones were generated from B6.Tlaa (H-2b, Qa-1a) anti-B6 (H-2b, Qa-1b) or B10.BR (H-2k, Qa-1a) anti-C3H/HeJ (H-2k, Qa-1b) mixed lymphocyte cultures (32, 51). Cytotoxic activity was measured in standard 4-h 51Cr release assays (48). Briefly, Con A-activated lymphoblast target cells were labeled with Na251CrO4 (0.1 mCi/106 cells; Amersham Pharmacia Biotech) at 37°C for 1 h, and incubated with or without 100 µM Qdm peptide (sequence, AMAPRTLLL; Biosynthesis, Lewisville, TX) during labeling. After extensive washing, cells were incubated with effectors at different E:T ratios in triplicate for 4 h at 37°C. Supernatants were harvested, and radioactivity was determined using a gamma counter (PerkinElmer Life Sciences). Results are presented as percentage of specific lysis: ((sample release – spontaneous release)/(maximal release – spontaneous release)) x 100.

For evaluation of Qa-1b-restricted Ag presentation of insulin peptides to 6C5 T cells, splenocytes from 6C5 transgenic mice were activated at 3 x 106 cells/ml with 100 µg/ml beef insulin (Invitrogen Life Technologies, Carlsbad, CA) and 3 ng/ml rIL-2 (R&D Systems, Minneapolis, MN) for 5 days. Cells were then used as effectors to measure lysis of Con A-activated, beef insulin (300 µg/ml)-prepulsed target cells from wild-type, Tpn–/–, TAP–/–, and {beta}2m–/– mice.

Proliferation and IFN-{gamma} production assays

Splenocytes (2 x 105/well) were irradiated (3500 rad) and incubated with Qa-1b-restricted CTL clones (2 x 105/well) in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-ME, 2 mM glutamine, antibiotics, and 10 mM HEPES. In some cultures, Qdm peptide (100 µM) was also added. Cells were cocultured for different time periods (16–72 h). For proliferation assays, 1 µCi of [3H]thymidine (PerkinElmer Life Sciences) was added to the wells, and after an additional 8 h of culture, cells were collected with a cell harvester (Tomtec, Orange, CT), and [3H]thymidine incorporation was measured with a betaplate reader (Wallac, Gaithersburg, MD). IFN-{gamma} production in the culture supernatants was determined by ELISA, as described (52). Purified and biotinylated Ab pairs and standards were purchased from BD Pharmingen. For detection, streptavidin-HRP conjugate (Zymed Laboratories, San Francisco, CA) was used in conjunction with the substrate 3,3',5,5'-tetramethylbenzidine (DakoCytomation, Carpinteria, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tapasin bridges Qa-1b to TAP within the class I peptide-loading complex

To examine the association of Qa-1b with ER-resident chaperones that are involved in the assembly of class Ia molecules with peptides, we performed coimmunoprecipitation experiments with metabolically labeled Con A blasts from wild-type, Tpn–/–, and TAP–/– mice (Fig. 1). Lysates were first precipitated with Abs directed against tapasin or TAP, precipitates were then solubilized, and samples were reprecipitated with either anti-Qa-1b or anti-H2-Kb Abs. Qa-1b and H2-Kb H chains were detected in the anti-tapasin precipitates from wild-type cells, indicating physical association of Qa-1b with tapasin. In addition, comparable levels of Qa-1b H chains were present in lysates from TAP–/– mice, indicating that, akin to class Ia H chains, Qa-1b binds with tapasin in the absence of a functional TAP peptide transporter. Likewise, Qa-1b and H2-Kb H chains were detected in the anti-TAP precipitates from wild-type cells, indicating physical association between Qa-1b and TAP as well as H2-Kb and TAP. However, this association was absent in cells from Tpn–/– and TAP–/– mice, indicating that association of Qa-1b with TAP requires tapasin expression. Thus, Qa-1b associates with the same ER-resident proteins that are required for the assembly of class Ia molecules, and tapasin links Qa-1b to TAP within the loading complex.



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  		FIGURE 1. Tapasin bridges TAP and Qa-1b in the peptide-loading complex. Antisera specific for mouse tapasin or TAP1 were used to immunoprecipitate proteins from digitonin lysates of metabolically labeled spleen Con A-induced lymphoblasts from wild-type (Wt), Tpn–/–, or TAP–/– mice. Precipitates were boiled in 0.2% SDS and diluted 10-fold with 1% Nonidet P-40 buffer. H2-Kb H chains (HC) were immunoprecipitated with an antiserum (X8) directed against the K-locus exon 8 products. Qa-1b HC were precipitated with the mAb 6A8.6F10.1A6 specific for peptide 161–179. One representative experiment of two is shown.

 
Impaired Qa-1b maturation in Tpn–/– cells

In cells from TAP-deficient mice, the intracellular transport of class I H2-Kb and -Db molecules is significantly impaired (48), whereas in cells from tapasin-deficient mice, the intracellular transport of class Ia molecules proceeds at a normal rate (17). To evaluate the role of tapasin and TAP for the maturation of Qa-1b molecules and their transport to the cell surface, we performed pulse-chase experiments with Con A blasts from wild-type, Tpn–/–, and TAP–/– mice. The extent of intracellular transport was estimated by treating immunoprecipitates with Endo H, which cleaves sugar moieties from class I H chains before, but not after, receiving glycan modifications in the medial Golgi. Fig. 2 compares the maturation and intracellular transport of H2-Kb (A) and Qa-1b (B) molecules in wild-type, Tpn–/–, and TAP–/– cells. In wild-type cells, most H2-Kb H chains became resistant to Endo H digestion within 30 min after their synthesis, whereas most Qa-1b H chains required 1–2 h to become Endo H resistant. In addition, a significant proportion of Qa-1b molecules from wild-type cells failed to be immunoprecipitated at the later chase points, suggesting significant degradation in post-Golgi compartments. This result is consistent with the finding that Qa-1b/peptide complexes at the cell surface are remarkably unstable (53). Consistent with prior studies (17, 48), most H2-Kb molecules were retained within the ER of TAP-deficient cells, but exited the ER of tapasin-deficient cells at a normal rate (Fig. 2A). In sharp contrast, acquisition of Endo H resistance by Qa-1b H chains in both TAP- and tapasin-deficient cells was substantially delayed (Fig. 2B), indicating impaired maturation and transport of Qa-1b in both of these cell types.



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  		FIGURE 2. Maturation of H2-Kb and Qa-1b glycoproteins in cells from wild-type, tapasin-, and TAP-deficient mice. Con A blasts from wild-type (Wt), Tpn–/–, and TAP–/– mice were pulse-labeled with [35S]methionine/cysteine for 10 min and chased at 37°C for the indicated times. Cell aliquots from each time point were lysed in Nonidet P-40 buffer. Class I molecules were precipitated with either the H2-Kb-specific Y3 mAb (A) or the Qa-1b-specific 6A8.6F10.1A6 mAb (B). Samples were then divided into two portions. One half was untreated, and the other half was treated with Endo H, followed by SDS-PAGE, fluorography, and autoradiography. The positions of Endo Hr and Endo Hs H chains are indicated by arrowheads. Endo Hr forms of H2-Kb and Qa-1b were quantified by ImageQuant software (C). Data are shown as absolute band intensities minus background on an arbitrary scale. One representative experiment of three is shown.

 
In the case of murine class Ia molecules, only peptide-filled and fully conformed class I heterodimers transit from the ER to the cell surface (54, 55, 56). In the absence of peptide supply, class Ia molecules are retained in the ER and targeted for ER-associated degradation (57, 58). However, such unstable class Ia molecules can be stabilized, at least in part, by reducing the culture temperature, which facilitates their transport to the cell surface, albeit at a reduced rate (48, 54). We therefore compared Qa-1b and H2-Kb maturation in wild-type and mutant cells cultured at 37 and 26°C. As expected, reducing the temperature resulted in delayed H2-Kb maturation in wild-type cells, but induced substantial conversion of Endo H-sensitive H2-Kb H chains to Endo H-resistant forms in TAP–/– cells (Fig. 3, A and B). In wild-type cells, temperature reduction similarly resulted in a slight delay in Qa-1b maturation (Fig. 3, C and D). In TAP- and tapasin-deficient cells, reduced temperature promoted the conversion of Endo H-sensitive Qa-1b H chains to Endo H-resistant forms. These findings indicate that temperature reduction facilitates egress of Qa-1b molecules in TAP- and tapasin-deficient cells to the cell surface.



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  		FIGURE 3. Intracellular transport of H2-Kb and Qa-1b glycoproteins at 37 and 26°C. Con A blasts from the indicated mice were subjected to pulse-chase experiments at either 37 or 26°C. Aliquots of cells from the indicated individual time points were lysed in Nonidet P-40 buffer. H2-Kb molecules were precipitated with Y3 mAb (A), and Qa-1b molecules were immunoprecipitated with 6A8.6F10.1A6 mAb (C). Immunoprecipitates were then treated with Endo H, resolved by SDS-PAGE, and analyzed by autoradiography (A and C) or phosphor imaging (B and D). The positions of Endo Hr and Endo Hs H chains are indicated by arrowheads. Endo Hr forms of H2-Kb (B) and Qa-1b (D) were quantified by ImageQuant software. Data are shown as absolute band intensities minus background, using an arbitrary scale. One representative experiment of two is shown.

 
Impact of tapasin deficiency on Qa-1b cell surface expression

We compared the levels of H2-Kb and Qa-1b surface expression on cells from wild-type, Tpn–/–, TAP–/–, and {beta}2m–/– mice. Because Qa-1b surface expression levels, compared with class Ia expression levels, on unmanipulated cells are very low (Fig. 4, top panels), we evaluated Qa-1b surface expression levels on Con A-induced lymphoblasts. As expected (10, 11, 48), H2-Kb expression was substantially reduced on both Tpn–/– and TAP–/– cells (Fig. 4, left panels). However, Qa-1b surface expression on Tpn–/– and TAP–/– cells, compared with wild-type cells, was only modestly decreased, and there was no detectable Qa-1b expression on {beta}2m–/– cells over background (Fig. 4, right panels). In most experiments, Qa-1b expression on Tpn–/– cells was slightly lower than on TAP–/– cells (Fig. 4, right panels; data not shown), for reasons that are unclear.



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  		FIGURE 4. Effects of tapasin deficiency on Qa-1b cell surface expression. PBLs or splenic Con A-induced lymphoblasts from wild-type (bold line), Tpn–/– (thin line), TAP–/– (dotted line), and {beta}2m–/– (dashed line) mice were stained with anti-H2-Kb-FITC, anti-Qa-1b-FITC mAb (for peripheral blood), or anti-Qa-1b mAb followed by biotinylated goat anti-mouse IgG and revealed by PE-conjugated streptavidin (for spleen lymphoblasts). The filled histogram represents a negative control (C) without addition of Ab. One representative of six experiments is shown.

 
Impaired recognition of Tpn–/– cells by Qdm-specific, Qa-1b-restricted alloreactive CTL

To determine whether the reduced cell surface expression on Tpn–/– cells is functionally relevant, we evaluated the reactivity of a panel of Qa-1b-restricted alloreactive CTL clones against cells from Tpn–/– mice. For all clones, wild-type target cells were efficiently recognized, and none of the clones lysed target cells from {beta}2m-deficient mice, either in the absence or presence of exogenous Qdm peptide (Fig. 5). In addition, in the absence of exogenous peptides, none of the CTL clones recognized TAP–/– target cells to a significant extent, which is consistent with prior studies (32, 47). However, lysis of TAP–/– cells was restored by addition of exogenous Qdm peptides to the culture medium. Recognition of Tpn–/– target cells was significantly impaired, but to a lesser extent than TAP–/– cells. Recognition of Tpn–/– cells by each of these clones was reconstituted by addition of exogenous Qdm peptides to the culture medium. Similar results were obtained when proliferation or IFN-{gamma} production was used as a readout of CTL reactivity (data not shown).



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  		FIGURE 5. In vitro Ag presentation assays to Qa-1b-restricted alloreactive CTL. Con A blasts from wild-type (wt), Tpn–/–, TAP–/–, and {beta}2m–/– mice were labeled with 51Cr and incubated with or without 100 µM Qdm peptide at 37°C for 1 h. Cells were then used as targets for the indicated Qa-1b-restricted alloreactive CTL clones, at the indicated E:T ratios. Supernatants were harvested, and radioactivity was determined using a gamma counter. Percentage of specific lysis was calculated as described in Materials and Methods. Results represent the mean from triplicate cultures. Measurements of T cell proliferation and IFN-{gamma} production were performed in parallel (not shown). Results are representative of at least two independent experiments for each clone.

 
Unimpaired presentation of insulin by Tpn–/– cells to insulin peptide-specific, Qa-1b-restricted CTL

Next, we tested whether Tpn–/– cells can present beef insulin to Qa-1b-restricted CTL. For this purpose, we used short-term CTL cultures from 6C5 TCR transgenic mice, which express a TCR consisting of V{alpha}3.2-J{alpha}36 TCR{alpha} and V{beta}5.1-D{beta}2-J{beta}2.6 TCR{beta} chains from a T cell hybridoma (6C5) with specificity for the insulin B chain presented by Qa-1b (49). 6C5 transgenic T cells were then used as effectors to lyse Con A-activated wild-type, Tpn–/–, TAP–/–, or {beta}2m–/– lymphoblasts in the absence or presence of beef insulin, in a standard 4-h 51Cr release assay. Results showed that, consistent with prior studies, 6C5 CTL lyse beef insulin-treated wild-type and TAP-deficient cells at equal efficiency (42), whereas control {beta}2m-deficient cells are resistant to lysis by 6C5 CTL (Fig. 6). Cells from Tpn–/– mice were able to present beef insulin to 6C5 CTL with equal efficiency as cells from wild-type and TAP–/– mice. These findings demonstrate that soluble insulin is presented by Qa-1b to 6C5 cells in a TAP- and tapasin-independent mechanism.



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  		FIGURE 6. Ag-processing requirements for the presentation of insulin Ags to Qa-1b-restricted, insulin peptide-specific T cells. Con A blasts from wild-type (wt), TAP–/–, Tpn–/–, and {beta}2m–/– mice were labeled with 51Cr and pulsed or not pulsed with 300 µg/ml beef insulin (BINS), and used as target cells for short-term CTLs from 6C5 transgenic mice generated by in vitro stimulation with BINS for 5 days. Effector and target cells were mixed at the indicated E:T ratios and incubated for 4 h in triplicate. Specific 51Cr release was measured as described in Materials and Methods. Results are representative of two independent experiments with short-term CTL lines from a total of five individual animals.

 
Impaired selection of insulin-specific, Qa-1b-restricted CD8+ T cells in TCR transgenic, tapasin-deficient mice

Positive selection of 6C5 T cells in TCR transgenic mice requires Qa-1b and TAP but not Qdm expression (49). To investigate the requirement of tapasin for the positive selection of Qa-1b-restricted T cells, we crossed 6C5 TCR transgenic mice onto the Tpn–/– and TAP–/– backgrounds. We analyzed the prevalence of CD4+ and CD8+ T cells in the thymus, lymph nodes, and spleen, and the prevalence of CD8+V{alpha}3.2+ cells in the lymph nodes and spleen. For the control transgenic mice (6C5.Tpn+/– and 6C5.TAP+/–) we observed, as previously reported (49), a bias toward CD8+ T cell development (Fig. 7A), due to positive selection of CD8+V{alpha}3.2+ T cells (B). Profiles for 6C5.Tpn–/– mice were clearly distinct from those of control transgenic mice, and similar to the profiles observed for 6C5.TAP–/– mice (Fig. 7). Thymocytes from 6C5.Tpn–/– and 6C5.TAP–/– mice had a relatively large population of double-positive cells, expressing high levels of CD4 and CD8, whereas few single-positive CD8+ cells were observed (Fig. 7A). In the lymph nodes and spleen, we observed for both 6C5.Tpn–/– and 6C5.TAP–/– mice, a reduced bias toward CD8+ T cell development, compared with 6C5.Tpn+/– or 6C5.TAP+/– mice (Fig. 7A). In addition, numbers of V{alpha}3.2+CD8+ T cells in lymph node and spleen of 6C5.Tpn–/– and 6C5.TAP–/– mice were substantially reduced, compared with transgenic wild-type mice (Fig. 7B and data not shown), indicating impaired positive selection of transgenic T cells in the former animals. We also observed an increase in the prevalence of CD4+V{alpha}3.2+ T cells in peripheral lymphoid organs of 6C5.Tpn–/– and 6C5.TAP–/– mice, compared with wild-type transgenic mice (see data for lymph nodes in Fig. 7B). This probably reflects enhanced positive selection of MHC class II-restricted CD4+V{alpha}3.2+ T cells coexpressing endogenous TCR {alpha}- or {beta}-chains. When compared with 6C5.TAP–/– mice, we consistently observed in 6C5.Tpn–/– mice a slight increase in the positive selection of CD8+ transgenic T cells (Fig. 7 and data not shown). Collectively, our data indicate that, although tapasin-deficient cells are fully competent to present insulin Ags to Qa-1b-restricted 6C5 cells, tapasin expression is important for the efficient positive selection of 6C5 cells in transgenic mice.



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  		FIGURE 7. Positive selection of CD8 T cells expressing the 6C5 TCR is impaired in tapasin-deficient mice. 6C5 TCR transgenic mice were crossed with TAP–/– or Tpn–/– mice to generate 6C5+.Tpn+/–, 6C5+.Tpn–/–, 6C5+.TAP+/–, and 6C5+.TAP–/– mice. Cells from thymus lobes, inguinal lymph nodes, and spleens of these animals were stained with mAbs directed against CD4, CD8, and V{alpha}3.2. Flow cytometry data for expression of CD4 vs CD8 (A) and V{alpha}3.2 vs CD8 (B) are shown. Numbers represent the percentage of viable lymphocytes within the indicated quadrants. Data are representative of results for at least four mice within each experimental group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies provide the first evidence that tapasin plays a critical role in the quality control of Qa-1b molecules. We found that Qa-1b is physically linked to the TAP peptide transporter, and this association is disrupted in tapasin-deficient cells. Because Qa-1b directly interacts with tapasin (independently of TAP expression), these findings indicate that, akin to class Ia molecules, tapasin forms a physical link between Qa-1b and TAP. Tapasin-deficiency, as well as TAP-deficiency, resulted in the retention of a significant pool of Qa-1b molecules into the ER. This is most likely due to the dearth of available peptides in the ER that can sufficiently stabilize Qa-1b molecules in the absence of tapasin or TAP expression. Because conformation-dependent, Qa-1b-specific Abs are not available, we were unable to directly evaluate the stability of Qa-1b molecules in these cells. Nevertheless, reduction of the culture temperature during the pulse-chase experiments was able to induce transit of Qa-1b molecules from the ER to the trans-Golgi reticulum. In the case of class Ia molecules, it has been shown that temperature reduction stabilizes the class I structure and facilitates class I exit from the ER even in the absence of stably bound peptide (48, 54). Consequently, in the case of Qa-1b, the likely explanation for our findings is that, in the absence of tapasin or TAP, Qa-1b molecules in the ER fail to be stabilized by peptides of sufficient affinity to permit their transit to the cell surface, and that this maturation defect can be overcome by temperature reduction. Although we did not directly evaluate the fate of unstable Qa-1b molecules in the ER of tapasin- and TAP-deficient cells, it is likely that Qa-1b H chains are targeted for degradation by the ER-associated degradation pathway (57, 58).

Despite the significant defects in Qa-1b transport to the cell surface, tapasin- and TAP-deficient cells express a substantial amount of Qa-1b molecules at the cell surface, albeit at reduced levels compared with wild-type cells. Culture at reduced temperatures or addition of Qdm peptides had no significant effect on Qa-1b surface expression by these cells (data not shown). It remains unclear whether peptides are associated with the Qa-1b molecules expressed by tapasin- or TAP-deficient cells. Interestingly, however, empty (peptide-devoid) Qa-1b molecules are unusually stable, with a half-life of ~50 min compared with the half-life of ~10 min for empty H2-Kb molecules (53), raising the possibility that the Qa-1b molecules that reach the surface of tapasin- and TAP-deficient cells may be empty. Although Qa-1b transport to the cell surface is significantly impaired in these cells, we were able to detect the accumulation of some Endo Hr H chains in these cells. Whether these Qa-1b molecules exited the ER in the absence or presence of bound peptide remains an open question. In this context, it was interesting that thymic maturation of insulin B chain-specific, Qa-1b-restricted 6C5 T cells was dependent upon tapasin and TAP expression (Fig. 7; Ref.49), indicating that the Qa-1b molecules that reach the surface in the absence of tapasin or TAP expression are unable to induce efficient positive selection of 6C5 transgenic T cells.

Our findings indicate that the presentation of Qdm to alloreactive CTL is strictly dependent upon tapasin expression, whereas tapasin is dispensable for the presentation of insulin B chain to T cells. The differential requirements of these two Ags for distinct components of the Ag-processing machinery are remarkable. Qdm is the main peptide that is associated with Qa-1b molecules in wild-type cells (33) and is derived from the signal sequence of class Ia molecules (32). Accumulating evidence indicates that, after biosynthesis and translocation into the ER, the signal sequence of class Ia H chain is removed by signal peptidase and subsequently processed by signal peptide peptidase, which allows the release of the N-terminal signal peptide fragment into the cytosol (34, 35, 36). This fragment is then further processed by cytosolic proteases, translocated into the lumen of the ER by TAP, and loaded onto ER-resident Qa-1b molecules with the help of tapasin. Processing of the insulin B chain follows a very different path, involving an exogenous Ag presentation pathway that is sensitive to chloroquine (42). Presentation of this Ag is TAP independent (42) and, as shown here, tapasin independent as well. However, it remains unclear whether insulin-derived peptides are loaded onto Qa-1b molecules in endosomal compartments in a class I recycling pathway, or directly at the cell surface in a peptide regurgitation pathway. Nevertheless, presentation of the insulin B chain to 6C5 T cells appears mechanistically distinct from the predominant pathway involved in the presentation of exogenous Ags to class Ia-restricted CTL (59). The observation that TAP- and tapasin-deficient cells express significant amounts of peptide-receptive Qa-1b molecules at their cell surface suggests that the Qa-1b Ag presentation system is particularly effective for presenting exogenous Ags to T cells.

Our studies provide significant insight into the role of tapasin for class I assembly. We found that the intracellular transport of Qa-1b to the surface of tapasin-deficient cells is substantially impaired, and comparable to Qa-1b trafficking in TAP-deficient cells. In this context, the behavior of Qa-1b in tapasin-deficient cells resembles that of human tapasin-dependent class I molecules expressed in the human tapasin-deficient cell line 721.220 (50, 60, 61). In sharp contrast, trafficking of H2-Kb and -Db molecules in tapasin-deficient cells is very similar to wild-type cells, whereas Kb and Db H chains are retained in the ER of TAP-deficient cells (Fig. 2; Refs. 17 and 48). The latter finding has been interpreted to suggest that one of the major functions of tapasin is to retain ill-folded class I molecules into the ER (17). However, it remains unclear whether the Kb and Db molecules that leave the ER of tapasin-deficient cells are truly devoid of peptides or loaded with low-affinity peptides. Because empty Qa-1b molecules, compared with empty Kb molecules, are relatively stable (53), the finding that Qa-1b H chains but not Kb H chains are retained in the ER of tapasin-deficient cells provides strong, albeit indirect, evidence that low-affinity peptides facilitate egress of Kb and Db to the surface of tapasin-deficient cells. Because Kb and Db molecules can bind with a wide variety of peptides of varying affinity, such low-affinity peptides that can partially stabilize the class I structure must be abundantly present in tapasin-deficient, but not in TAP-deficient, cells. In the case of Qa-1b molecules, which have a highly restricted peptide-binding specificity, it is likely that few peptides are available for stabilizing this molecule in the ER of both tapasin- and TAP-deficient cells, leading to their retention in the ER. These findings, therefore, suggest that truly empty class I molecules, at least in the case of Qa-1b, do not require interaction with tapasin for their retention in the ER. This conclusion is also supported by studies with the class Ib molecule H2-M3, which is retained in the ER of tapasin-deficient cells (62, 63). Collectively, these findings suggest that tapasin is dispensable for retention of truly empty class I molecules into the ER, and that class I molecules loaded with low-affinity peptides are the main substrates for tapasin function. Tapasin, by virtue of its association with coat protein I-coated vesicles (26), may be responsible for recycling back class I molecules that are either peptide-devoid or loaded with low-affinity peptides, until they are bound with high-affinity peptides. This conclusion is consistent with the proposed peptide-editor function of tapasin (10, 27, 28), but not with the idea that tapasin diversifies rather than optimizes the class I peptide repertoire (29).

Emerging evidence indicates that tapasin not only plays a critical role for the quality control of class Ia molecules but also for a variety of class Ib molecules, including Qa-1b, H2-M3 (62, 63), HLA-E (64), HLA-F (65), and HLA-G (61). Additional studies of these and other class Ib molecules will likely provide significant insight into the function of tapasin and other components of the class I peptide-loading complex.


   Acknowledgments
 
We thank Sebastian Joyce and John Monaco for providing critical reagents, and Jie Wei and Michele Nadaf for technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant HL68744 (to L.V.K.). Back

2 Current address: Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. Back

3 Current address: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92121. Back

4 Current address: Celltech R&D, Inc., Bothell, WA 98021. Back

5 Address correspondence and reprint requests to Dr. Luc Van Kaer, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Medical Center North, Room A-5321, Nashville, TN 37232. E-mail address: luc.van.kaer{at}vanderbilt.edu Back

6 Abbreviations used in this paper: ER, endoplasmic reticulum; {beta}2m, {beta}2-microglobulin; tapasin, TAP-associated glycoprotein; Qdm, Qa-1 determinant modifier; Endo H, endoglycosidase H. Back

Received for publication March 30, 2004. Accepted for publication June 16, 2004.


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V. Cirulli, J. Zalatan, M. McMaster, R. Prinsen, D. R. Salomon, C. Ricordi, B. E. Torbett, P. Meda, and L. Crisa
The Class I HLA Repertoire of Pancreatic Islets Comprises the Nonclassical Class Ib Antigen HLA-G.
Diabetes, May 1, 2006; 55(5): 1214 - 1222.
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J. Yan, V. V. Parekh, Y. Mendez-Fernandez, D. Olivares-Villagomez, S. Dragovic, T. Hill, D. C. Roopenian, S. Joyce, and L. Van Kaer
In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules
J. Exp. Med., March 20, 2006; 203(3): 647 - 659.
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