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The Journal of Immunology, 1998, 160: 4305-4312.
Copyright © 1998 by The American Association of Immunologists

TAP-Independent MHC Class I Peptide Antigen Presentation to Alloreactive CTL Is Enhanced by Target Cell Incubation at Subphysiologic Temperatures1

Yan Shi2,*,{ddagger}, Kelly D. Smith3,*,{dagger} and Charles T. Lutz4,*,{ddagger},§

Departments of * Pathology and {dagger} Microbiology and {ddagger} Graduate Program in Immunology and § Molecular Biology, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the peptide dependency of a group of CD8+ anti-HLA-B7 alloreactive CTL. The CTL killed target cells after acid denaturation of more than 98% of target cell surface peptide/MHC class I complexes. The CTL also killed TAP- HLA-B7-transfected T2 (T2B7) cells. The killing was enhanced by target cell incubation at 26°C. Despite these findings, which suggested peptide-independent allorecognition, CTL-mediated cytolysis was reduced or abolished by several point mutations affecting the HLA-B7 peptide-binding groove. Acid denaturation of HLA complexes on T2B7 cells prohibited CTL recognition. CTL recognition was restored by T2B7 cell incubation with ß2-microglobulin and a single HPLC fraction containing peptides extracted from TAP+HLA-B7+ cells, but not by any of a panel of 17 synthetic HLA-B7-binding peptides. These findings indicated that CTL allorecognition was peptide specific. Sensitizing peptide was extracted from T2B7 cells only after incubation at 26°C. The amount of peptide detected in TAP+ cells was at least 10-fold and 100-fold greater than that detected in TAP- cells incubated at 26°C and at 37°C, respectively. TAP-independent peptide epitope presentation was sensitive to treatment with brefeldin A, but not sensitive to treatment with chloroquine, consistent with an endogenous peptide source. We propose that subphysiologic temperature incubation can enhance peptide/MHC class I presentation in the total absence of TAP function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MHC class I molecules are composed of heavy chain, ß2-microglobulin (ß2m),5 and peptide, and are assembled in the endoplasmic reticulum (ER) and transported to the cell surface for CD8+ T cell recognition. TAP-1/TAP-2 heterodimers translocate MHC class I-binding peptides from the cytosol into the ER (1). TAP-2-deficient mouse RMA-S cells and TAP- human 721.174 and T2 cells express little cell surface MHC class I and fail to present most viral and other MHC class I-associated peptides (1). Furthermore, viral inhibitors of TAP function cause low MHC class I cell surface expression and poor peptide Ag presentation (2, 3). Mouse MHC class I molecules on TAP-deficient cells can be efficiently loaded with exogenous synthetic peptides (4, 5). For these reasons, MHC class I molecules on TAP-deficient cells appear to be "empty" or devoid of tightly bound peptides. Subphysiologic temperature incubation significantly enhances MHC class I expression on TAP-deficient cells, especially for mouse MHC class I molecules (4). This is widely believed to be due to the low temperature stabilization of empty MHC class I molecules. Indeed, incubating TAP-deficient cells with anti-MHC mAbs or exogenous synthetic peptides in the cold stabilizes MHC class I molecules after the incubation temperature is raised to 37°C (4, 5, 6, 7, 8). Without MHC-binding mAbs or exogenous peptides, surface MHC class I molecules on TAP-deficient cells are thermolabile.

Despite their obvious defects in MHC class I Ag processing, TAP-deficient cells do express cell surface class I-bound peptides. In certain instances, CD8+ T cells recognize TAP-deficient cells that express viral proteins (9, 10, 11, 12, 13, 14) or MHC class I epitopes expressed by transfected minigene constructs (15, 16). Sequence analysis has revealed that HLA-A2-bound peptides in TAP- cells are derived from protein signal sequences (17, 18). Peptides that are eluted from HLA-B7 molecules on transfected TAP- T2 cells have a typical HLA-B7-binding sequence motif; the ratio of peptide to HLA-B7 heavy chain, measured spectrophotometrically, is the same in TAP- and TAP+ cells (19). However, little or no radioactively labeled peptide has been eluted from most MHC class I molecules on metabolically labeled TAP- cells (18, 19). This indicates that TAP- cells process MHC-bound peptides by a slow and inefficient route (19).

TAP-independent MHC class I Ag processing and presentation, and the induction of empty MHC class I molecules by subphysiologic temperature incubation are relevant to CD8+ T cell recognition of alloantigen. TAP-deficient target cells have been used to assess whether T cells recognize allogeneic MHC class I molecules in a peptide-independent or peptide-nonspecific fashion. Many alloreactive CTL do not kill TAP-deficient cells unless the target cells have been incubated with specific cell extracts or HPLC fractions from TAP+ APC lysates, providing convincing evidence that many alloreactive CTL are peptide specific (20). In a few cases, the allopeptides have been identified (21, 22, 23, 24). In contrast, some alloreactive CTL kill TAP-deficient target cells in the absence of exogenous peptides (25, 26). CTL recognition of TAP-deficient cells has been cited to support peptide-independent MHC allorecognition.

In this report, we present an analysis of TAP-independent allorecognition of HLA-B7. Previously, we characterized D7 and a group of related cloned CD8+ T cells (D7-like CTL) that are specific for EBV EBNA-3c presented by HLA-B37. Here we show that D7-like CTL cross-react with allogeneic HLA-B7 molecules that are expressed by both TAP+ and TAP- cells. We demonstrate that D7-like CTL are highly specific for a cellular peptide that is expressed on TAP+ cells. Using these cells, we investigate the hypothesis that TAP-independent MHC class I peptide Ag presentation is enhanced at subphysiologic temperatures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

D7-like CTL clones and the control peptide-specific, HLA-B7-alloreactive KOR-18 CTL clone have been described (27, 28, 29, 30). TAP- T2 cells; HLA-A-, -B-, and -C-negative 721.221 cells; and lymphoblastoid cell lines (LCL) have been described (27, 28, 29, 30). JB LCL was gift of Dr. A. McMichael, Oxford University, U.K. Mouse EL4 cells transfected with the HLA-A2 gene (EL4A2) or the HLA-B7 gene (EL4B7) were gifts of Dr. V. Engelhard, University of Virginia. The cell lines were maintained in culture medium (RPMI 1640, Life Technologies, Gaithersburg, MD; with 10% supplemented calf serum, HyClone, Logan, UT). For the serum-free cell culture, JB LCL and EL4B7 cells were grown in AIM-V medium (Life Technologies) for 2 wk. T2 and 721.221 cells were transfected with various HLA genes in the pHeBo vector and maintained in culture medium supplemented with 300 µg/ml of hygromycin B (Calbiochem, San Diego, CA) as described (19, 29, 30, 31). The HLA-B7/Aw68 chimera was constructed by subcloning an {alpha}3 exon-containing BglII-SalI fragment from an HLA-Aw68 genomic clone (a gift of Dr. P. Parham, Stanford University) into a similar site in an HLA-B7 genomic clone in the pHeBo vector. Expression of transfected gene products in target cells lines was confirmed by flow cytometry using W6/32 mAb, as described (19, 29, 30, 31).

Ag presentation inhibitors

Brefeldin A (BFA; Sigma, St. Louis, MO) was dissolved in methanol at 20 mg/ml and diluted in culture medium to a final concentration of 1 µg/ml. Chloroquine (Sigma) was dissolved in culture medium immediately before assays and used at 20 µM. EDTA (Sigma) was used at a final concentration of 1 mM. For addition 1 h after initiation of the 51Cr release assay, 50 µl of 5 mM EDTA (in an aqueous solution of 140 mM NaCl and 15 mM HEPES) was added to 200 µl of assay mixture.

Cell extraction and HPLC separation

HLA-B7+ JY and T2B7 cells (109) were grown to a density of 106 cells/ml with less than 5% cell death. Some batches had been incubated at 26°C for 18 to 20 h before extraction. Cells were washed once with PBS and lysed in 0.1% trifluoroacetic acid (Fisher, Itasca, IL) in HPLC-grade water (Fisher) by vigorous vortexing for 1 min. Another 15 ml of 1% trifluoroacetic acid in water was added to the lysates. The lysates were kept on ice for 30 min with brief vortexing every 5 min. After centrifuging the lysates at 28,000 rpm at 4°C on a Beckman L8-80 M centrifuge (Beckman, Fullerton, CA) with a type 28 rotor, the supernatants were lyophilized. The lyophilates were dissolved in 0.15% trifluoroacetic acid in HPLC-grade water and centrifuged through Centricon 10 filters (Amicon, Beverly, MA) at 5000 x g for at least 4 h. The filtrates were again lyophilized and dissolved in 1 ml of 0.15% trifluoroacetic acid. The suspensions were subjected to HPLC separation as previously described (19).

Cell surface MHC class I denaturation, reconstitution, and cytotoxicity assay

51Cr-labeled HLA-B7+ cells were washed with HBSS (Cancer Center, University of Iowa), and incubated for 90 s with acid-stripping medium (0.3 M glycine-HCl and 1% BSA in water, pH 2.4) at cell densities no higher than 2 x 107 cells/ml. Culture medium (100 x vol) was added to neutralize pH. Cells were washed three times and suspended in assay medium (RPMI 1640 with 5% supplemented calf serum) at 106/ml. Completeness of acid stripping was assessed by flow cytometry, as described above. In some experiments, cell suspensions (50 µl) were mixed with equal volumes of each HPLC peptide fraction in the presence of 10 µg/ml of human ß2m (Calbiochem). The mixtures were shaken at room temperature for 2 h and the volumes were expanded to 1 ml with the assay medium before the 51Cr release assay. HLA-B7-binding synthetic peptides were mixed at a final concentration of 10-5 M with ß2m (10 µg/ml) and 51Cr-labeled acid-stripped T2B7 cells (5 x 104 cells in 1 ml) and were shaken at room temperature for 2 h. For all the cytotoxicity assays, peptide incubation mixtures were transferred to 96-well plates at 100 µl/well. An equal volume of effector cells was added to each well. 51Cr release assays were performed as previously described (31), at the E:T ratios indicated. Assays were 5 h long, except where otherwise indicated. Relative lysis used to estimate the effects of HLA-B7 mutations on CTL and was calculated as the percent specific lysis of 721.221 cells transfected with HLA-B7 variant genes divided by the percent specific lysis of 721.221 cells transfected with the parental HLA-B7 gene at the same E:T ratio.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EBV-specific CTL recognize allogeneic HLA-B7 molecules

D7 and the other CD8+ T cell clones used in this study (D7-like CTL) specifically recognize an EBV EBNA-3c peptide (amino acids 285-293) that is presented by autologous HLA-B37 class I molecules (28). D7-like CTL also killed HLA-B7+, but not HLA-B7- allogeneic LCL (Table IGo). D7-like CTL did not kill mouse EL4 target cells or human HLA-A-, -B-, or -C-negative 721.221 target cells. Recognition of both mouse and human target cells was achieved by transfection with the HLA-B7 gene, but not with the HLA-A2 gene, confirming cross-reactivity with allogeneic HLA-B7 molecules (Table IGo). As expected, the anti-HLA class I mAbs, MB40.5 and W6/32, blocked the alloreactive CTL killing, confirming MHC class I specificity (data not shown). To test the CD8 dependence of the allorecognition, we transfected 721.221 cells with a chimeric HLA gene encoding the {alpha}1 and {alpha}2 domains of HLA-B7, and the {alpha}3, transmembrane, and cytoplasmic domains of HLA-Aw68. The HLA-Aw68 molecule has an {alpha}3 mutation that disrupts CD8{alpha} subunit binding and efficient recognition by many CTL (32). D7-like CTL equivalently lysed 721.221 cells transfected with either unmutated HLA-B7 or the B7/Aw68 chimera (data not shown). CD8 dependency can be overcome by high ligand density and high TCR affinity (33, 34). This suggests that the specific antigenic determinant that was recognized by D7-like CTL on HLA-B7+ cells was of high affinity or was highly expressed.


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  		Table I. D7-like CTL recognize allogeneic HLA-B7 molecules1

 
D7 CTL kill target cells that express minimal HLA-B7-bound peptide

To address whether HLA-B7 recognition by D7-like CTL is peptide-dependent, we acid-treated target cells to denature HLA class I complexes (35). Treatment of HLA-B7-transfected 721.221 (B7.221) cells with HCl/glycine (pH 2.4) medium removed 98.5% of cell surface trimeric HLA/ß2m/peptide complexes, as assessed by the binding of conformation-sensitive W6/32 mAb (Fig. 1GoA). Because HLA-B7 is the only HLA-A, -B, or -C molecule expressed by B7.221 cells, this result indicates that acid denaturation removed the great majority of cell surface peptide/HLA-B7 complexes. Acid stripping minimally decreased the cytolytic activity by D7 CTL and control alloreactive KOR-18 CTL (Fig. 1GoB). KOR-18 CTL recognize allogeneic HLA-B7 molecules in a peptide-dependent manner (27, 29). We propose that KOR-18 recognition of acid-stripped B7.221 cells was due to target cell reexpression of peptide/HLA-B7 complexes during the 3-h 51Cr release assay. To test this hypothesis, we used two treatments to minimize new HLA expression following acid stripping. EDTA chelates Ca2+ and Mg2+, prevents cell-cell contact, and prohibits both perforin-mediated and Fas-mediated killing (36). EDTA treatment throughout the assay completely blocked CTL-mediated killing (Fig. 1GoB). EDTA added 1 h after the assay began did not reduce the cytolysis by either D7 or KOR-18 CTL (Fig. 1GoB). This indicates that both CTL caused considerable target cell damage in the first hour of the assay. BFA disrupts the Golgi complex and halts expression of HLA complexes (37). BFA treatment alone did not inhibit CTL-mediated killing (data not shown). Acid stripping followed by the addition of EDTA after 1 h, acid stripping and continuous BFA treatment, or the combination of treatments severely diminished allorecognition by the peptide-specific KOR-18 CTL (Fig. 1GoB). This indicates that acid stripping of the target cells efficiently removed cell surface peptide/HLA-B7 complexes that are recognized by KOR-18 CTL. However, the same treatments did not significantly diminish killing by D7 CTL (Fig. 1GoB), indicating that D7 CTL recognized target cells after acid denaturation of 98.5% of surface HLA-B7 molecules. Thus D7 CTL may recognize extremely low levels of a specific peptide/HLA-B7 complex, a peptide/HLA-B7 complex that is resistant to acid denaturation, or "empty" HLA-B7 molecules that are stabilized by serum ß2m after acid denaturation.



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  		FIGURE 1. D7 CTL killing of TAP+HLA-B7+ cells is resistant to acid stripping. A, B7.221 cells were either untreated (dotted line) or acid stripped (broken line) as described in Materials and Methods, and immediately stained on ice with W6/32 mAb followed by FITC-conjugated goat anti-mouse IgG. Untransfected parental 721.221 cells were also stained (continuous line). B, D7 CTL (filled bars) and peptide-specific alloreactive CTL clone KOR-18 (open bars) were used at an E:T ratio of 5:1 with B7.221 target cells in a 3-h 51Cr release assay. Cells were either untreated (NONE) or were treated with EDTA (1 mM), either throughout the 51Cr release assay (E) or beginning at 1 h after initiation of the assay (E1h). Other B7.221 target cells were acid stripped (AS) as described in Materials and Methods. Acid-stripped target cells received no further treatment, were incubated continuously in 1 µg/ml of BFA in assay medium both before and during the 51Cr release assay (B), were exposed to EDTA 1 h after initiation of the 51Cr release assay (E1h), or received a combination of treatments, as indicated.

 
HLA-B7 peptide-binding groove point mutations abolish recognition by D7 CTL

To further examine whether D7 CTL recognized peptide/HLA-B7 complexes, we used an indirect approach. HLA-B7 variants with point mutations affecting the peptide-binding groove were transfected into 721.221 cells. The expression of the HLA-B7 variants was comparable by mAb staining and each transfectant had been shown to be killed well by at least one HLA-B7-specific alloreactive CTL (29, 30, 35). Therefore, all variants had relatively unaltered tertiary structure. As predicted, D7 CTL recognition was abrogated by several point mutations in solvent-accessible residues (Fig. 2Go). Importantly, D7 CTL recognition also was abolished by multiple point mutations in the HLA-B7 peptide-binding groove (Fig. 2Go) that affected pockets A, B, C, D, and E (see Fig. 2Go legend). The HLA-B7 A pocket preferentially binds peptide P1 arginine and alanine, the B pocket binds P2 proline, and the D pocket binds P3 arginine (38). Four point mutations in the peptide-binding groove pockets B (residue 67), F (residue 116), and A (residues 163 and 171), did not significantly affect D7 CTL recognition (Fig. 2Go). Lack of peptide-binding groove mutation effects has been interpreted to support peptide-independent allorecognition (26). However, a panel of peptide-specific allogeneic CTL clones (29, 30) that were studied in our laboratory, like D7 CTL, were variably affected by peptide-binding groove mutations.



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  		FIGURE 2. HLA-B7 peptide-binding groove variants affect recognition by D7 CTL. Shown is a top view of the HLA class I molecule. Filled residues denote relative cytolysis (see Materials and Methods) of the variant transfectant at less than 20% of the unmutated HLA-B7 transfectant, stippled residues denote 20 to 70% relative cytolysis, and open residues denote no significant decrease in cytolysis. The E45A mutation and the amino acids substituted at other residues have been described (29, 35). Cell surface expression of each variant transfectant was found to be within twofold of the unmutated HLA-B7 transfectant. D7 CTL recognition was reduced by mutations in the following peptide-binding pockets: residue 9 (pockets B, C); 45 (B); 63 (A, B); 66 (A, B); 70 (B, C); 73 (C); 74 (C); 97 (C, E); 99 (A, B, D); 152 (E); 155 (D); 156 (D, E); and 167 (A).

 
Expression of the D7 CTL epitope on TAP- T2 cells is dependent on temperature and on HLA-B7-bound peptide

MHC class I/peptides complexes are poorly expressed by TAP-deficient cells. Therefore, we investigated the ability of D7-like CTL to kill TAP- T2 cells transfected with the HLA-B7 gene (T2B7). We also tested two HLA-B7 variants (E45A and S97R) that were not recognized by D7 CTL when expressed on TAP+ 721.221 cells. Unlike many mutants, these two HLA-B7 variants are expressed at levels equivalent to or higher than unmutated HLA-B7 on transfected T2 cells (19). D7 and D5 CTL killed T2B7 cells (Fig. 3Go). Overnight T2B7 target cell culture at 26°C increased recognition by D7 and D5 CTL equivalent to that of TAP+ B7.221 target cells (Fig. 3Go). However, D7 and D5 CTL did not kill T2 cells that were transfected with either the E45A or the S97R HLA-B7 variant genes, even after target cell incubation at 26°C (Fig. 3Go and data not shown). These data indicate that D7 CTL allorecognition is sensitive to changes in HLA-B7 peptide-binding groove residues, even on TAP- target cells.



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  		FIGURE 3. D7-like CTL do not recognize T2 cells transfected with a peptide-binding groove HLA-B7 mutant. T2B7 cells and T2 cells that were transfected with the E45A HLA-B7 variant were incubated at 37°C or 26°C overnight before the 51Cr release assay, as indicated (Temp). B7.221 cells were cultured continuously at 37°C. D5 and D7 CTL clones were used at an E:T ratio of 10:1.

 
To remove peptide/HLA-B7 complexes from the TAP- target cells, we acid stripped T2B7 cells. As above, D7 and D5 CTL recognition was enhanced by a 26°C overnight incubation of T2B7 cells (Fig. 4Go). D7 and D5 CTL did not kill acid-stripped T2B7 cells (Fig. 4Go). Peptide processing by TAP- T2B7 cells is slow (19), obviating the need for BFA to prevent peptide/HLA-B7 reexpression in the 5-h 51Cr release assay (Fig. 4Go).



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  		FIGURE 4. D7-like CTL recognition of T2B7 cells is sensitive to acid stripping. B7.221 target cells were used without treatment. T2B7 target cells were incubated as indicated at 26°C (Low Temp) or at 37°C overnight, acid stripping was done immediately before the 51Cr release assay, and BFA was used at 1 µg/ml. CTL clones D5 and D7 were used at an E:T ratio of 10:1.

 
D7 CTL recognize a specific HLA-B7-bound peptide presented by TAP+ and TAP- cells

It has been proposed that some alloreactive T cells are peptide dependent but not peptide specific (39, 40). Therefore, we tested acid-stripped T2B7 cells that had been pulsed with human ß2m and a panel of 17 synthetic HLA-B7-binding peptides. D7 (Table IIGo) and D5 (data not shown) CTL did not kill the target cells pulsed with a high concentration (10-5 M) of these synthetic peptides. Therefore, D7-like CTL do not promiscuously recognize peptide/HLA-B7 complexes or an HLA-B7 structure that is stabilized by peptide binding.


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  		Table II. D7-like CTL do not promiscuously recognize peptide-stabilized HLA-B7 molecules1

 
We next investigated the cellular HLA-B7-bound peptides that are recognized by D7-like CTL. To avoid potential loss of low affinity peptides during HLA-B7 purification, we extracted TAP+ HLA-B7+ JY cells and HPLC-separated peptides as outlined in Materials and Methods. Acid-stripped T2B7 target cells were incubated with human ß2m and individual HPLC fractions. A single HPLC fraction (fraction 27) sensitized target cells for lysis by D7 CTL (Fig. 5GoA), consistent with the specific recognition of a single HLA-B7-bound peptide. Sensitizing peptide also was eluted from B7.221 cells (data not shown). Little or no D7 CTL lysis was induced by a similarly prepared extract from T2B7 cells that had been cultured at 37°C (Fig. 5GoA). However, considerable targeting activity, also present in HPLC fraction 27, was extracted from T2B7 cells after they had been cultured overnight at 26°C (Fig. 5GoA). This indicates that expression of the HLA-B7-bound peptide epitope was enhanced by subphysiologic temperature incubation. We quantitated the abundance of sensitizing peptide in each extract by dilution of HPLC fraction 27 (Fig. 5GoB). Even without dilution, sensitizing activity in the T2B7 cell (37°C) extract was not detectable. Fraction 27 of the T2B7 cell (26°C) extract sensitized target cells for lysis without dilution, but failed to do so after a 10-fold dilution (Fig. 5GoB). In contrast, fraction 27 of the JY cell extract sensitized target cells for lysis after a 100-fold dilution. Activity was above background after a 1000-fold dilution (Fig. 5GoB). These data indicate that TAP+ JY cells contained at least 100-fold more sensitizing peptide than did TAP- T2B7 cells incubated at 37°C and at least 10-fold more sensitizing peptide than did T2B7 cells incubated at 26°C.



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  		FIGURE 5. T2B7 cell peptide presentation is significantly enhanced by subphysiologic temperature incubation. Low m.w. molecules were acid extracted from TAP- T2B7 cells that had been incubated at 37°C (T2B7) or 26°C (T2B7/26*C), or from TAP+ HLA-B7+ JY cells (JY), as described in Materials and Methods. Extracts were HPLC fractionated and mixed with ß2m and 51Cr-labeled acid-stripped T2B7 target cells as described in Materials and Methods. D7 CTL were tested at an E:T ratio of 10:1. A, Test of HPLC fractions 19 to 30. B, HPLC fraction 27 was diluted in PBS before mixing with ß2m and acid-stripped T2B7 target cells.

 
D7 CTL recognize an HLA-B7-bound peptide that is derived from an endogenous cellular protein in TAP+ and TAP- cells

The observations that were presented above indicated that D7-like CTL recognition of T2 cells was peptide specific. We thus wanted to analyze the TAP-independent MHC class I peptide presentation pathway. We considered the possibility that the peptide was derived from calf serum present in cell culture media. HLA-B7+TAP+JB LCL and EL4B7 cells were cultured in serum-free AIM-V medium for 2 wk. T2B7 target cells died when cultured in AIM-V medium and were not tested. Traces of putative retained calf serum peptides were removed from JB LCL and EL4B7 cells by acid stripping. The acid-treated cells were further incubated in serum-free medium for 1 day and tested with D7 CTL in serum-free medium. D7 CTL did not show any decrease in killing of these target cells (data not shown), suggesting that the peptide recognized by D7 CTL is not derived from a calf serum protein.

To test whether the HLA-B7-bound peptide on T2B7 cells was from an exogenous source, we incubated acid-stripped B7.221 cells and T2B7 cells overnight in chloroquine and tested the killing of these target cells by D7 CTL. Chloroquine inhibits endosomal compartment acidification and prevents exogenous Ag presentation. For comparison, we used BFA, which disrupts the Golgi complex. As expected, acid-stripped B7.221 cells were sensitive to D7 CTL killing in the presence of either inhibitor during the overnight incubation (Fig. 6Go). The killing of acid-stripped B7.221 cells in the presence of BFA was somewhat lower. This might be due to gradual loss of residual peptide/HLA-B7 complexes from acid-stripped B7.221 cells or to increased B7.221 target cell death observed after overnight culture in BFA at 37°C (data not shown). B7.221 target cells incubated for 6 h after acid stripping in the presence of BFA were killed well (data not shown). Overnight incubation of acid-stripped T2B7 cells with BFA at 26°C was not toxic and prevented the recognition by D7 CTL (Fig. 6Go). Overnight treatment with chloroquine did not prohibit D7 CTL recognition. These data are consistent with the hypothesis that D7 and similar CTL recognized a peptide epitope that was derived from an endogenous cellular protein in TAP- cells.



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  		FIGURE 6. Presentation of the specific peptide on TAP- cells is inhibited by BFA, but not by chloroquine. Target cells were B7.221 (A) and T2B7 (B). Some cells were acid stripped either 24 h (Prior AS) or immediately (Imm AS) before the 51Cr release assay. Twenty-four hours before the assay, B7.221 and T2B7 target cells were incubated at 37°C and 26°C, respectively. BFA (1 µg/ml) and chloroquine (20 µM) inhibitors were added as indicated during the incubation and throughout the cytotoxicity assay. D7 CTL were used at an E:T ratio of 5:1. EDTA (1 mM) was added to each cytotoxicity assay mixture 1 h after initiation of the 51Cr release assay to prevent CTL recognition of newly regenerated cell surface peptide/MHC class I complexes. In this assay, BFA blocked the control KOR-18 CTL peptide epitope regeneration after acid stripping (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TAP molecules are required for efficient MHC class I peptide Ag presentation. In the absence of functional TAP heterodimers, cell surface MHC class I expression is low and presentation of endogenous cellular or viral Ags is poor. Nonetheless, TAP-deficient cell presentation of MHC class I molecule-bound peptides to CTL has been documented. We investigated EBV-specific CTL that cross-react with allogeneic HLA-B7 on TAP+ and TAP- cells in a peptide-specific fashion. Targeting activity that was extracted from TAP- T2B7 cells and from TAP+ B7.221 cells migrated in the same HPLC fraction, suggesting a single HLA-B7-bound peptide. At least 100-fold more targeting activity was extracted from TAP+ B7.221 cells grown at 37°C than from T2B7 cells grown at 37°C. This is consistent with the greater efficiency of TAP-dependent than TAP-independent peptide Ag presentation. Despite differences in presentation efficiency, it is clear that D7-like CTL recognize a peptide epitope in a TAP-independent fashion. TAP-independent presentation of alloreactive CTL has been observed in other systems (29, 41, 42).

At least four potential mechanisms can explain TAP-independent MHC class I Ag presentation. First, Ags may be derived from extracellular sources. Extracellular peptides or proteins may be digested by serum proteases and then bind to cell surface MHC class I molecules (43). Ags from phagocytosed bacteria may be "regurgitated" and gain access to cell surface MHC class I molecules (44). Particulate Ags may be phagocytosed, processed in endosomal compartments, and presented to MHC class I molecules in a BFA-independent fashion (45). Second, the TAP-1 protein may form homodimers in cells that lack functional TAP-2 proteins. In cells with a defect in TAP gene expression, introducing a rat TAP-1 cDNA restored presentation of a viral epitope (46). A vesicular stomatitis virus peptide epitope is presented well by infected TAP-deficient RMA-S cells, but not by infected, H2Kb-transfected TAP- T2 cells (10). Third, some peptides that are generated in the ER can directly bind nascent MHC class I molecules, without the need for TAP transport. Peptides from protein signal sequences have been isolated from HLA-A2 class I molecules in TAP- T2 cells (17, 18). In some cases, signal sequences can direct the cell surface presentation of MHC class I epitopes in TAP- cells (15). Fourth, cytosolic peptides may be transported into the ER by a TAP-independent mechanism. The proteins that mediate this transport are not known. Some viral epitopes (9, 10, 11, 12, 13, 14) and peptides encoded by plasmid minigenes (15, 16) are presented in TAP-deficient cells, including TAP- T2 cells. Common features of TAP-independent MHC class I peptide Ag transport are slow and inefficient peptide processing. In addition, the peptides that are presented have been generated in high quantity in the cytoplasm, often from viral or plasmid genes.

It is unlikely that the HLA-B7-bound epitope that is recognized by D7-like CTL is derived from an exogenous protein or processed by serum proteases. T2 cells are not known to be phagocytic, and acid stripping and overnight chloroquine incubation did not prevent recognition of T2B7 cells. This is inconsistent with uptake of exogenous Ag via an endosomal route. Long-term target cell growth and assay in serum-free media combined with acid stripping to remove residual peptide/MHC complexes did not diminish recognition of B7.221 cells by D7 CTL. In regard to the second proposed TAP-independent MHC class I Ag presentation mechanism, T2B7 cells completely lack both TAP-1 and TAP-2 proteins, ruling out potential TAP-1 homodimer function, as suggested for mouse RMA-S cells.

Our data do not exclude the possibility that the HLA-B7-bound peptide epitope is derived from a protein signal sequence, even though the peptide epitope was presented more efficiently in TAP+ cells than in TAP- cells. Some signal sequences are not processed for MHC class I binding in the ER and must be exported to the cytoplasm for further processing and subsequent TAP-mediated importation into the ER (22, 47). Signal sequence peptide processing may be inefficient in the ER. Our peptide is presented at a low level in the absence of TAP and at higher levels in the presence of TAP. However, peptide synthesis is slower at subphysiologic temperatures, and it is expected that generation of HLA-binding signal sequence peptides also would be reduced.

We speculate that the peptide epitope is transported from the cytoplasm into the ER efficiently by TAP and inefficiently by a TAP-independent mechanism. The relatively inefficient putative TAP-independent transport is consistent with the delayed and relatively feeble presentation of viral (9, 10, 11, 12, 13, 14) and plasmid minigene (15, 16) epitopes by TAP-deficient cells. Slow and relatively inefficient transport of peptides into the ER by a TAP-independent mechanism also is consistent with the observation that HLA-B7-bound peptides are poorly labeled by 3H-labeled amino acids, but are detectable spectrophotometrically in T2B7 cells (19).

Mouse MHC class I molecules on the surface of TAP-deficient cells may be largely empty or devoid of tightly bound peptides. The low level of surface MHC class I molecules is greatly enhanced by overnight incubation of TAP-deficient cells at subphysiologic temperatures. These molecules are unstable and gradually disappear upon raising the temperature to 37°C, unless the TAP-deficient cells are incubated with MHC class I-binding mAb or peptides (4, 5, 6, 7, 8). Subphysiologic temperatures stabilize empty MHC class I molecules in vitro and in vivo. However, incubation at subphysiologic temperatures induces relatively little expression of most human MHC class I molecules on either human TAP- T2 cells or mouse TAP-deficient RMA-S cells (5, and data not shown).

D7-like CTL killed TAP- T2B7 cells and recognition was greatly enhanced by overnight target cell incubation at 26°C. Enhanced CTL recognition correlated with enhanced expression of CTL targeting activity at 26°C. To our knowledge, the current study is the first to quantitate TAP-independent MHC class I-bound peptide expression at different temperatures. Subphysiologic temperature-enhanced peptide presentation has been reported for mouse TAP-2-deficient RMA-S cells (41). Our results show the same phenomenon may be observed in the total absence of TAP proteins.

Because very little is known about the mechanism of putative TAP-independent peptide transport, it is unclear why expression of the peptide epitope is enhanced at subphysiologic temperatures. However, several mechanisms can be envisioned. Decreased temperature may increase formation of trimeric peptide/ß2m/HLA-B7 complexes in the ER. Subphysiologic temperatures may increase TAP-independent peptide transport, stabilize ß2m/HLA-B7 heterodimers, or slow HLA-B7 heavy chain, ß2m, or peptide degradation. It is known that unbound peptides are rapidly exported from the ER into the cytoplasm (48). The ER peptide export mechanism may be temperature sensitive. Alternatively, it is possible that slow turnover of cell surface peptide/MHC complexes at subphysiologic temperatures may account for enhanced expression of the peptide epitope.

Our findings are relevant to whether alloreactive T cells can recognize MHC class I molecules in a peptide-independent or peptide-nonspecific fashion. It is clear that many alloreactive CTL recognize complexes of specific peptides bound to allogeneic MHC class I molecules (20). However, it has been proposed that many other CTL recognize MHC epitopes that are independent of specific bound peptides (25, 26, 39, 40, 49). Many of these studies have utilized TAP-deficient cells. Enhanced alloantigen presentation at subphysiologic temperatures has been interpreted as evidence of peptide-independent allorecognition (27). Some of our results had initially suggested that D7-like CTL might recognize HLA-B7 in a peptide-independent manner. D7-like CTL recognized and killed TAP- T2B7 cells, and killing was enhanced by target cell incubation at subphysiologic temperatures. D7 and similar CTL killed TAP+HLA-B7+ target cells, even after >=98% of the peptide/HLA-B7 complexes had been removed from the cell surface and reexpression of new complexes was prohibited by BFA treatment. However, the suggestion that D7-like CTL allorecognition is independent of HLA-B7-bound peptide is not correct. In retrospect, it is clear that the ability of D7-like CTL to recognize acid-stripped target cells was due to the very high level of peptide epitope expression by TAP+ cells. CTL recognition exhibits a threshold pattern. Even when most peptide/MHC complexes are removed from target cells, CTL killing is efficient as long as the number of remaining peptide/MHC complexes are above threshold levels. For some T cells, this number can be very low, perhaps as few as one complex per cell (50). T2B7 cells grown at 26°C and 37°C contained at least 10-fold less and 100-fold less sensitizing peptide, respectively, than did B7.221 cells grown at 37°C, yet the T2B7 target cells were recognized and killed by D7-like CTL. The high abundance of the peptide epitope on TAP+ cells suggests that the peptide is produced in large quantities in the cell. In parallel with viral (9, 10, 11, 12, 13, 14) and plasmid minigene (15, 16) epitopes, this presents a plausible explanation of why the peptide is presented, albeit inefficiently, by TAP- T2B7 cells. As noted above, the reason for enhanced recognition of T2B7 cells after culture at subphysiologic temperatures was due to the unexpected enhanced expression of specific cell surface peptide/HLA-B7 complexes. Our results show that T cell recognition of allogeneic MHC class I molecules on living target cells may depend upon a tiny amount of specific MHC-bound peptide.

Previous studies (26, 51) have demonstrated allorecognition of purified, largely empty MHC class I molecules attached to plastic. These studies concluded that allorecognition can be peptide-independent and that some CTL may specifically recognize empty MHC class I molecules. In one of these studies (26), the alloreactive CTL also shared many properties of D7 CTL: recognition of acid-stripped target cells, lack of effect by some peptide-binding groove mutations, and recognition of TAP- cells. We were able to generate suitable targets for peptide targeting studies by acid-stripping TAP- T2B7 cells; we demonstrated that D7 and similar alloreactive CTL clones recognize specific peptides. This finding calls into question the physiologic significance of allorecognition of purified empty MHC class I molecules on plastic. Stimulation of alloreactive T cells by high density MHC on plastic surfaces may represent the inherent affinity of TCR for allogeneic MHC. CTL allorecognition of living cells may nonetheless involve specific peptides. Given that only tiny amounts of specific peptide are sufficient to stimulate some T cells, this possibility is difficult to exclude. Our results emphasize caution when interpreting apparently peptide-independent allorecognition.


   Acknowledgments
 
We thank Brian Mace for excellent technical support and Dr. John Harty for helpful discussions. HLA-B7-binding peptides were a kind gift of Dr. Victor H. Engelhard at the University of Virginia.


   Footnotes
 
1 This work was supported by Diabetes and Endocrinology Research Center Grant DK25295, National Institutes of Health Grant DE11139, and National Institutes of Health Medical Scientist Training Program Grant GM0737 to K.D.S. Back

2 Current address: Department of Pathology, University of Massachusetts Medical Center, 155 Lake Avenue N., Worchester, MA 01655. Back

3 Current address: Department of Pathology, University of Washington Medical Center, Seattle, WA 98195. Back

4 Address correspondence and reprint requests to Dr. Charles T. Lutz, University of Iowa, 153B MRC, Department of Pathology, Iowa City, IA 52242-1182. E-mail: Back

5 Abbreviations used in this paper: ß2m, ß2-microglobulin; B7.221, 721.221 cells transfected with the unmutated HLA-B7 gene; BFA, brefeldin A; ER, endoplasmic reticulum; LCL, lymphoblastoid cell line; T2B7, T2 cells transfected with the unmutated HLA-B7 gene. Back

Received for publication October 31, 1997. Accepted for publication January 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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