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The Journal of Immunology, 2003, 170: 4515-4523.
Copyright © 2003 by The American Association of Immunologists

Correction of Defects Responsible for Impaired Qa-2 Class Ib MHC Expression on Melanoma Cells Protects Mice from Tumor Growth 1

Eugene Y. Chiang, Maile Henson and Iwona Stroynowski2

Center for Immunology, Departments of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One of the principal mechanisms of tumor immune evasion is alteration of class I MHC expression. We have identified defects contributing to down-regulation of class I MHC expression in the widely studied murine B16 melanoma and its variants B16F1, B16F10, BL6-2, BL6-8 and B78H1. Transcription of the nonclassical class I MHC genes Q8 and Q9 (Qa-2 Ags) has been switched off in the entire panel of melanoma lines, suggesting that this event occurred early during tumor progression. B78H1, unlike B16F1 and B16F10 sublines, is also selectively devoid of TAP2 and low molecular weight protein 7 as well as classical class I MHC Kb and Db transcripts. Cotransfection of B78H1 with TAP2 and class I H chain genes is sufficient to reconstitute surface expression of exogenously delivered class I MHC without concomitant re-expression of endogenous {beta}2-microglobulin-associated class I. The serological absence of endogenous class Ia and Ib at the surface of TAP2-negative as well as TAP2-transfected B78H1 makes this system a suitable model for studying the properties of isolated class I proteins in tumors. We used this system to demonstrate that B78H1 cells genetically manipulated to re-express Q9 Ag have reduced tumor potential in syngeneic B6 mice compared with TAP2-transfected parental melanoma. Both NK cells and CTLs appear to collaborate in restraining growth of Q9-positive tumors. The results implicate Qa-2 in antitumor responses and illustrate the utility of the B78H1 system for identifying in vivo interactions between class I MHC molecules of interest and immune cells of innate and/or adaptive immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The B16 melanoma is one of the best characterized mouse tumors. It has been used extensively to study antitumor T cell and NK cell immune responses and as an experimental model for T cell-based immunization and vaccination strategies (1, 2, 3, 4, 5, 6). The primary B16 tumor arose spontaneously in the skin of a C57BL/6 mouse in 1954. Since its isolation, B16 has been maintained in vitro as an unstable, mixed population of cells with heterogeneous phenotypes. In later years, the parental B16 was used to establish a variety of melanoma sublines with distinct phenotypes. These include amelanotic, low metastatic variant B78H1 (7, 8, 9) and pigmented B16F1/B16F10 sublines (10) distinguishable by their low/high metastatic potentials. The highly metastatic B16F10 gave rise to highly invasive variants, BL6-2 and BL6-8, that were selected for their ability to penetrate bladder wall in vitro (11). All the B16-derived melanoma variants have low immunogenicity and express little or no detectable class I MHC Ags (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Serological studies demonstrated that the display of classical class I (class Ia) MHC and the relative levels of the individual Kb or Db cell surface MHC proteins vary among B16 sublines as does their ability to up-regulate class I MHC by IFN-{gamma}.

Despite wide use of B16 variants in investigations of class I MHC-dependent immune anti-tumor responses, the knowledge of the mechanisms underlying class I impairments in B16 variants is still fragmentary. A recent report by Seliger et al. (12) attributed deficient expression of Kb and Db in B16F1 and B16F10 cells to the transcriptional and translational down-regulation of multiple components of the class I Ag presentation pathway. Reduction/loss of transcription was reported for several gene products active in delivery of peptides to class I: the TAP1 subunit of the transporter associated with Ag processing, the chaperone tapasin; and multiple components of the proteasome degradation system, including the low molecular weight proteins (LMP) 3LMP2, LMP7, and LMP10 and proteasome activators PA28{alpha} and PA28{beta}. In B16F1 and B16F10, the transcriptional deficiencies and the surface expression of endogenously synthesized class I Ags were restored in the presence of IFN-{gamma}.

In contrast to B16F1 and B16F10, the class I deficiency of B78H1 is not alleviated by IFN-{gamma} and is known to involve a transcriptional blockade of endogenous Kb and Db loci (15). Here we identify additional defects responsible for the class I-negative phenotype of this melanoma. These include deficient transcription of multiple known class Ib genes, including Q8 and Q9 genes encoding Qa-2 Ags (17). The Qa-2 family is intriguing because of its numerous similarities to class Ia (i.e., primary acid sequence (18), three-dimensional crystal structure (19), peptide repertoire (20), ability to function as alloantigens (21)).

In addition, we find that B78H1, unlike B16F1, B16F10, BL6-2, and BL6-8 cells, is also selectively devoid of TAP2 and LMP7 transcripts. Transfection of constitutive promoter-driven class I in combination with TAP2 genes is sufficient to attain high surface expression of the exogenously introduced class I Ag without re-expression of endogenous {beta}2-microglobulin ({beta}2m)-associated class I molecules.

Although down-regulation of MHC class Ia is an effective mechanism for tumor escape from immunosurveillance, the role of nonclassical class I (class Ib) MHC molecules has been poorly characterized. Class Ib molecules are characterized by low polymorphism, thus allowing them to present nearly identical repertoires of peptide ligands in different mouse haplotypes. Class Ib Ags are also expressed at lower levels than class Ia, and many are distributed in a tissue-specific manner (17, 22). Despite these differences, several class Ib molecules have been demonstrated to have important functions in the immune system. For example, in the mouse, Qa-1 and H2-M3 are recognized by CD8+ CTLs (23, 24, 25, 26); Qa-1, Qa-2, and CD1d inhibit NK cell-mediated cytotoxicity (8, 27, 28, 29, 30). Nevertheless, immune functions for the majority of the class Ib molecules still remain ill defined.

Studies in relevant tumor systems may help in elucidating novel roles for class Ib Ags. Here, we used the class I-deficient B78H1 melanoma as a model to evaluate the hypothesis that early loss of Qa-2 expression observed in B16 melanoma variants may have contributed to the advantage of the selective growth of the tumor in vivo. TAP2-expressing B78H1 tumor cells transfected with the canonical Q9 isoform of Qa-2 were injected s.c. into syngeneic mice to determine whether Qa-2 was capable of eliciting a protective immune response. Strikingly, mice challenged with Qa-2-positive B78H1 resisted the tumor cells compared with mice challenged with class I-negative B78H1. In vivo depletion studies indicated that both NK cells and CD8+ cells are prominent effector populations in the rejection response. These findings suggest that loss of Qa-2 class Ib MHC expression may indeed confer a selective advantage to tumors, an observation visualized here in a tumor system devoid of other surface-expressed class I MHC Ags.


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

C57BL/6 (abbreviated B6) mice were maintained in the Microbiology animal colony at University of Texas Southwestern Medical Center (Dallas, TX). Adult mice 8–10 wk old were used for all experiments.

Cell lines and cell culture

Melan-a2, an immortal differentiated melanocyte line derived from skin of B6 mice (31), was generously provided by Dr. V. J. Hearing (National Cancer Institute, National Institutes of Health, Bethesda, MD) and was cultured in RPMI 1640 supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA), 10 U/ml penicillin/10 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO), 200 nM 12-O-tetradecanoylphorbol acetate (Sigma-Aldrich), and 2-ME (Sigma-Aldrich). B16 and the variants B16F1 and B16F10 (10) were obtained from S. Ostrand-Rosenberg (University of Maryland Baltimore County, Baltimore, MD). The variants BL6-2 and BL6-8 (14) were obtained from E. Gorelik (University of Pittsburgh Medical Center, Pittsburgh, PA). The variant B78H1 (7) was provided by H. I. Levitsky (the Johns Hopkins School of Medicine, Baltimore, MD). GM-CSF-transduced B78H1 cells (15) were provided by S. Ostrand-Rosenberg. B16 and all variants were cultured in 50% DMEM, 50% RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin. B78H1 was additionally supplemented with 1 mM sodium pyruvate (Life Technologies, Gaithersburg, MD) and 0.1 mM nonessential amino acids (Life Technologies). B78H1 transfectants expressing cell surface Q9 (designated Q9.A7), Q9 in conjunction with TAP2 (Q9TAP.C1 and Q9TAP.11 clones), Kb in conjunction with TAP2 (clone MJ1-2.9 designated here as H1KbTAP), Db in conjunction with TAP2 (clone MJ1-3.4 designated here as H1DbTAP) and empty vector (vector) have been previously described (8); GM-CSF-transduced B78H1 expressing both Q9 and TAP2 (GMQ9TAP.13 and GMQ9TAP.17 clones) or TAP2 alone have also been previously described (8). GM-CSF-transduced B78H1 transfected with both Kb and TAP2 (clone MJ1-5.7, designated here as GMKbTAP) was generated using similar techniques by M. Henson. Briefly, GMKbTAP was generated by cotransfection with Kb/pcDNA3.1 and TAP2/pcDNA1neo using the FuGENE 6 transfection reagent according to manufacturer’s recommendations (Boehringer Mannheim, Indianapolis, IN) and subsequent selection using medium supplemented with 800 µg/ml G418 (Life Technologies). Clones were generated by single-cell sort of bulk transfections. B78H1 transfectants expressing CMV promoter-driven Kb alone (clone MJ7-2.24 designated here as H1Kb) and Db alone (clone MJ7-3.8 designated here as H1Db) were generated by M. Henson in a similar manner. All clones were monitored by flow cytometry and RT-PCR for presence of class I and TAP2 expression and maintained in medium supplemented with 400 µg/ml G418. All cell lines were grown at 37°C and 5% CO2. To maintain Mycoplasma-free conditions, all cell lines were periodically cultured in medium supplemented with 10 µg/ml CellGro ciprofloxacin HCl (Mediatech, Herndon, VA). For IFN-{gamma} induction, cells were treated with 20 U/ml recombinant mouse IFN-{gamma} (Sigma-Aldrich) for 3 days.

Antibodies

The following Abs were used in this study: anti-Qa-2 mAb 31-1-2 (BD PharMingen, San Diego, CA), anti-Kb mAb Y3 (32), anti-Db mAbs MC86 (33) and 28-14-8 (34), anti-Qa-1b mAb 6A8.6F10.1A6 (BD PharMingen), and anti-{beta}2m mAb S19.8 (35). FITC-conjugated goat anti-mouse IgG (FITC-GAM; Cappell, Durham, NC) was used as the secondary Ab. PE-conjugated anti-NK1.1 mAb PK136 and PE-conjugated DX5 mAb were purchased from BD PharMingen. Anti-CD8{alpha} mAb 53-6.7 and anti-CD4 mAb GK1.5 used for in vitro blocking assays were purchased from BD PharMingen. Anti-CD8 mAb 2.43 used for in vivo depletions was generously provided by M. Bennett (University of Texas Southwestern Medical Center); anti-asialo-GM1 was purchased from Wako Pure Chemical Industries (Richmond, VA).

RNA isolation, cDNA synthesis, and RT-PCR

Total RNA was isolated using the RNA STAT-60 method (Tel-Test, Friendswood, TX), first strand cDNA was synthesized using the Life Technologies SuperScript II synthesis kit, and PCR was performed using the HotStarTaq DNA polymerase system (Qiagen, Valencia, CA) as previously described (8). Briefly, PCR was performed as follows: 0.5 to 2.0 µl cDNA were added to a total PCR mixture of 25 µl containing 2.5 µl of PCR buffer containing Tris-Cl, KCl, (NH4)2SO4, and MgCl2, 5 µl of Q-Solution, 0.5 µl of 10 mM dNTPs, 1.0 µl of 20 µM upstream primer, 1.0 µl of 20 µM downstream primer, 0.625 U of HotStarTaq DNA polymerase, and diethylpyrocarbonate-treated sterile H2O. Primers used in this study are listed in Table I. All primers were specific for the analyzed genes by sequencing of the PCR products (this study and M. Chen and I. Stroynowski, unpublished observation). The mixture was placed in a GeneAmp PCR System 9700 thermal cycler (Perkin-Elmer, Foster City, CA) and heated to 94°C for 15 min followed by 35 cycles at the settings of 94°C for 1 min for denaturation, 55°C for 1 min for annealing, and 72°C for 1 min for extension, followed by a final incubation at 72°C for 7 min. The PCR products were analyzed on a 1.0% agarose gel stained with ethidium bromide.


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  		Table I. Primers used to detect MHC class I and Ag-processing/ presentation pathway gene productsa

 
Flow cytometry

1 x 106 cells were washed once in staining buffer (PBS with 1% FCS and 0.1% sodium azide) and pelleted in a 1-ml polystyrene conical tube. A saturating amount of primary Ab was added to the cell pellet in a volume of 100 µl, vortexed, and incubated on ice for 15 min. Excess unbound Ab was removed by washing the suspension once with staining buffer. A saturating dilution of FITC-labeled secondary Ab was added in a final volume of 100 µl and incubated on ice for 15 min. The samples were washed twice, resuspended in 300 µl of staining buffer, and filtered through 35-µm pore size nylon mesh. Cells (1 x 104) were collected on a FACScan flow cytometer (BD Biosciences, San Jose, CA), and data were analyzed using CellQuest version 3.1f software (BD Biosciences).

Peptide-induced MHC stabilization assays

Peptide-binding assays were performed by growing cells at 37°C to ~70% confluency. Cells were counted and plated onto 6-well plates at 2 x 106 cells/well in 2 ml of medium. Cells were cultured in the presence or absence of peptide and incubated overnight at 26°C. Peptides were present at a final concentration of 50 µg/ml. After 16–20 h, cells were transferred to 37°C for 1 h. Cells were then collected and analyzed by flow cytometry. Peptides used were Kb-specific peptides OVA (OVA257–264 SIINFEKL; Ref.38) and vesicular stomatitis virus (VSV; VSV52–64 RGYVYQGL; Ref.39).

In vivo tumor growth and protection assays

Cells for injection were harvested from in vitro culture and washed three times in serum-free HBSS. Live tumor cells (1 x 105) were injected s.c. in a volume of 200 µl into the right rear flank. Mice were monitored daily. Mice were considered tumor bearing when the tumor was palpable and measured at least 3 x 3 mm. NK cell-depleted mice were generated by i.p. injection of anti-asialo-GM1 (0.2 ml of a 1/20 dilution) at 2 days before, 1 day before, and day of tumor inoculation, followed by twice weekly injections. CD8-depleted mice were generated by i.p. injection of anti-CD8 mAb 2.43 (0.25 mg/mouse) following the same schedule as for NK depletion. Depletion of lymphocyte subsets was assessed in depleted control mice by flow cytometry on the day of tumor challenge and 1 wk thereafter by double staining with anti-CD8 mAb and either anti-NK1.1 mAb or DX5 mAb. At these time points, >95% depletion of the appropriate subset was achieved, with other subsets unaffected. The number of mice used in each experiment is indicated in the corresponding figure legend, and each experiment was repeated at least once. Data are represented as Kaplan-Meier plots.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
B78H1 is deficient in expression of class Ia and class Ib MHC Ags

Although the class Ia MHC Ags are present on most somatic tissues, their relative expression levels differ drastically in a cell-dependent manner (40). This is further exaggerated for class Ib MHC, which often display tissue-specific expression patterns (22). Thus, to assess whether a tumor cell has down-regulated class I, it is necessary to compare it directly with a parental cell type. Melanomas, including B16 tumor cells, are descendants of transformed, neural crest-derived melanocytes that normally reside in the skin. We examined surface expression and transcriptional activity of class Ia Ags Kb and Db in nontransformed, B6 mouse-derived melanocyte line Melan-a2, in B16 primary melanoma, and in the B78H1 variant of B16 (Fig. 1). Under normal, noninduced conditions, Melan-a2 and B16 cell lines displayed very low levels of class Ia. In contrast, Kb and Db were serologically undetectable in B78H1. IFN-{gamma}, a cytokine that enhances transcription of class I H chains; {beta}2m, and many components of the class I Ag presentation pathway, strongly up-regulated Kb and Db in Melan-a2 and B16, but not in B78H1. Other variants of B16 (B16F1, B16F10, BL6-2, and BL6-8) had a phenotype similar to that of B16 (Refs.12 and 14 and data not shown). This suggests that B78H1 suffered mutations that distinguish it from the parental melanocytic lineage as well as from other B16 derivatives. Consistent with this interpretation is the finding that B78H1 lacks RT-PCR-detectable constitutive expression of Kb and Db transcripts (Fig. 1B and Ref.15), despite the presence of intact class Ia genomic DNA (determined by PCR analysis, data not shown). In contrast, Melan-a2, B16, and its other variants transcribe class Ia H chains constitutively (Fig. 1B and data not shown). Because all three cell lines are positive for {beta}2m transcripts (Fig. 2B), severe class I deficiency in B78H1 can be primarily attributed to stable suppression of class Ia H chain transcription, whereas low surface Kb and Db expression on Melan-a2 and B16 lines may stem from other factors limiting class I display under constitutive but not IFN-{gamma}-induced conditions.



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  		FIGURE 1. Classical class I MHC Ag expression by B78H1 melanoma. A, Flow cytometry analysis was performed on B78H1, B16, and Melan-a2 cell lines under uninduced conditions (thin line) or after IFN-{gamma} stimulation (thick lines). Cells were stained for Kb with mAb Y3 or Db with mAb MC86, followed by secondary Ab FITC-GAM. Shaded histograms represent background staining with FITC-GAM alone. B, RT-PCR analysis of class Ia MHC gene transcriptional activity. Cells were cultured in the absence (-) or presence (+) of IFN-{gamma}, RNA was extracted and cDNA was synthesized. cDNA derived from Melan-a2 cells was used as a positive control. {beta}-Actin was included as a control for RNA and cDNA integrity in each sample. Reagents and conditions used for RT-PCR were optimized to detect low copy numbers of transcribed genes and primers yielded PCR products of expected size. The trace amounts of PCR products detected for Kb and Db in IFN-{gamma}-treated B78H1 cDNA may be specific or may represent cross-reactive class I species.

 


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  		FIGURE 2. Expression of the class Ib MHC Ag Qa-2 by B16 primary melanoma and derivatives. A, Flow cytometry analysis was performed on the indicated cell lines under resting conditions (thin line) or after IFN-{gamma} stimulation (thick lines). Cells were stained for {beta}2m with mAb S19.8 or Qa-2 with mAb 1-1-2, followed by secondary Ab FITC-GAM. Shaded histograms represent background staining with FITC-GAM alone. B, RT-PCR analysis of Qa-2 and {beta}2m gene transcription. Cells were cultured in the absence (-) or presence (+) of IFN-{gamma}. cDNA derived from B6 splenocytes and the Melan-a2 cell line were used as a positive controls. Q8 and Q9 primers detect mRNA transcribed by Qa-2-encoding Q6/Q8 and Q7/Q9 gene pairs, respectively. {beta}-Actin was included as a control for RNA and cDNA integrity in each sample.

 
To address whether suppression of class I MHC in B16 melanomas affects class Ib MHC loci, we first stained all tumor variants and control melanocyte line with mAb against {beta}2m (Fig. 2A, left). Because this mAb detects all {beta}2m-associated surface class I, any discrepancies between Kb and Db surface phenotype and staining with anti-{beta}2m should be indicative of class Ib presence. Under constitutive conditions, all cell lines were either {beta}2m-negative (B78H1, B16F1, BL6-2, BL6-8), or very low (B16, B16F10, Melan-a2). IFN-{gamma} treatment resulted in up-regulation of {beta}2m staining in all cells in which class Ia were induced. The B78H1 variant remained {beta}2m surface negative, indicating that it does not express serologically detectable {beta}2m-associated class Ia or class Ib molecules.

To verify that melanoma cells down-regulated surface expression of the most abundant of the ubiquitously distributed class Ib molecules, the Qa-2 family of Ags (17, 22), the cell lines were stained with anti-Qa-2 mAb. We previously reported that Melan-a2 becomes serologically Qa-2 positive in the presence of IFN-{gamma} and that this property is lost in B16 tumor due to a transcriptional block affecting Qa-2 genes Q8 and Q9 (8). In agreement with this interpretation, we found that the Q9 locus is present in B78H1 genomic DNA (PCR analysis not shown). Here we demonstrate by flow cytometry (Fig. 2A, right) and RT-PCR (Fig. 2B) that the Qa-2 expression defect is present in all melanoma variants. This suggests that the Q8/Q9 transcriptional lesion(s) occurred early during the development of the parental B16 tumor. Therefore, this mutational event appears independent of the subsequent Kb and Db losses and other alterations (see below) that distinguish B78H1 from other B16 variants.

Three other well-characterized, ubiquitously distributed class Ib were predicted to be expressed in melanocytic lines: Qa-1 encoded by T23 (41), T22 (42) and M3 (43). The transcripts of all three were detectable by sensitive RT-PCR (Fig. 3), but only T22 was abundantly transcribed. Given these results, we cannot formally exclude a possibility that some class Ib protein products are present at the cell surface at levels or in forms undetectable by staining with anti-{beta}2m mAb. It is unlikely, however, that the canonical form of Qa-1 is expressed on the surface of B78H1, because flow cytometry staining for Qa-1 was negative even in the presence of endogenous Qdm-peptide loading pathway (41) established after cotransfection with Dd and TAP2 (data not shown). Consistent with this, Griffiths et al. (9) found no Qa-1 chain by Western blotting or surface staining on untransfected B78H1.



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  		FIGURE 3. MHC class Ib gene expression in B78H1 variant of B16 melanoma. B78H1 as well as Melan-a2 and B16 cells were cultured in the absence or presence of IFN-{gamma} (as indicated), and then RNA was extracted and cDNA was synthesized. Primers were designed to specifically amplify characterized class Ib genes encoded within the Q, T, and M regions of the MHC. Because many class Ib genes are expressed in a tissue-restricted manner, positive control cDNAs were synthesized from various tissues. Liver-specific Q10 (44 ) and gut-specific Q1 (45 ), Q2 (46 ), and T3 (47 ) transcripts were not detectable by RT-PCR in melanocytic lineage cells. {beta}-Actin was included as a control for RNA and cDNA integrity in each sample.

 
These results identify B78H1 as a class Ia/class Ib-deficient cell line that has stably extinguished transcription of Kb, Db, and Qa-2.

Defects in class I Ag presentation pathway in B78H1 melanoma

In our earlier studies, we used B78H1 as a recipient for expression of the Qa-2 Ag Q9 (8). While characterizing the first set of Q9 transfectants, we discovered that IFN-{gamma} treatment is required for appreciable surface expression of this TAP-dependent MHC Ag. This prompted us to re-examine the factors that limit exogenous class I expression in uninduced B78H1.

The steady state mRNAs from B78H1, B16, and Melan-a2 were probed for transcriptional activity of several components of the class I Ag presentation pathway by RT-PCR (Fig. 4). Remarkably, in the uninduced state, B78H1 clearly displays abrogation of transcription of TAP2 subunit and LMP7 proteasome component genes as compared with B16 and Melan-a2. In addition, we observed an apparent decrease in transcript intensity of TAP1 and LMP2. The conditions of our RT-PCR experiments were set to optimize detection of very rare transcripts and to screen for absence vs presence of mRNA, rather than to quantitate different levels of clearly detectable products. Treatment with IFN-{gamma} corrected all observed transcriptional deficiencies to the level observed in Melan-a2 and B16 control cells.



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  		FIGURE 4. Ag-processing and presentation pathway component expression in B78H1. RT-PCR analysis was performed on untreated and IFN-{gamma}-treated B78H1 and B16 melanoma cells. Melan-a2 was included as a positive control cell line. Primers used are listed in Table I. RT-PCR was performed at least five times, and similar expression patterns were observed in all experiments. Conditions were optimized to detect low copy numbers. Although relative differences in expression levels could be ascertained, the PCR conditions were not designed to be quantitative.

 
The TAP2 deficiency detected in B78H1 is reminiscent of the well-characterized RMA-S T cell mutant cell line (48, 49). To determine whether B78H1 behaves like a TAP2 mutant similar to RMA-S, we examined the effects of cold induction (overnight incubation at 26°C, followed by an additional 1 h of incubation at 26°C or 37°C, as indicated in the legend to Fig. 5) and peptide stabilization on Kb expression on the cell surface of Kb-transfected B78H1. Under normal culture conditions at 37°C (without cold induction) untransfected parental B78H1 cells do not express detectable surface Kb, Db, and {beta}2m at (Fig. 1, 2), and cold induction does not alter this phenotype (data not shown). Kb-transfected B78H1 cells (H1Kb) express moderate levels of Kb when grown continuously at 37°C (Fig. 5A). Cold induction leads to a >2-fold increase in Kb expression on H1Kb (Fig. 5A), roughly equivalent to the increased Kb expression observed for RMA-S cells (Fig. 5C). This demonstrates that class I expression on B78H1 cells can be up-regulated by low temperature to a degree similar to that on RMA-S. To determine whether Kb molecules expressed on B78H1 cells are receptive to exogenous peptide loading, cells were incubated overnight at 26°C in the presence of saturating amounts of the known Kb-specific peptides OVA and VSV (Fig. 5A). Peptide stabilization was assessed after shifting cells to 37°C for 1 h. As seen in Fig. 5A, H1Kb cells express ~10-fold higher levels of Kb after cold induction in the presence of specific peptides. After transfer to 37°C, expression levels remained high (~8- to 9-fold higher than at 37°C without peptide), showing that nearly all of the Kb/peptide complexes were stable. RMA-S cells cultured in the presence of Kb-specific peptides displayed comparable increases in Kb expression (Fig. 5C).



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  		FIGURE 5. Class I MHC surface expression on B78H1 is reconstituted by exogenous peptide stabilization. Peptide-induced stabilization of surface-expressed Kb molecules was measured by incubating Kb-transfected B78H1 (A), Kb/TAP2 cotransfected B78H1 (B) or RMA-S (C) cells overnight at 26°C in the absence or presence of 50 µg/ml indicated Kb-specific peptides followed by an additional 1 h of incubation at either 26°C (open bars) or 37°C (shaded bars). Kb cell surface expression levels determined by flow cytometry (recorded as mean fluorescence intensities) were expressed as a percentage of Kb expression on cells after overnight incubation at 37°C in the absence of peptides. Data shown are mean ± SE. Ten experiments were performed for H1Kb, and four experiments were performed for H1KbTAP and RMA-S.

 
Similar experiments were also performed with Kb-transfected B78H1 cells that had the TAP2 defect corrected by TAP2 gene transfection (H1KbTAP; Fig. 5B). Cold induction in the absence of exogenous peptides had minimal impact on Kb expression levels on H1KbTAP cells, as expected for Kb molecules that are filled with endogenously synthesized and loaded peptides. The effect of exogenous peptides on up-regulation of Kb surface levels was also small (~2-fold), supporting the notion that most Kb on H1KbTAP cells are already peptide filled.

To test whether the block in class I surface expression observed in B78H1 could be overcome by transfection of selected Ag-presenting genes, we selected a series of clones transcribing constitutive promoter-driven Kb or Db genes. Each of these class Ia H chain-positive cells was cotransfected with constitutive promoter-driven TAP2 gene (H1KbTAP clone MJ1-2.9 and H1DbTAP clone MJ1-3.4; Ref.7). In each case, the presence of TAP2 enhanced expression of exogenous class Ia by a significant 2- to 5-fold factor, as judged by flow cytometry (data not shown). The magnitudes of increased class I MHC surface levels in the Kb/TAP2 and Db/TAP2 B78H1 clones are lower than that previously reported for TAP2-transfected RMA-S cells (nearly 10-fold increase for Kb, >10-fold increase for Db; Ref.48). Therefore, it is likely that restoration of TAP2 does not fully rescue the ability of B78H1 to express class I MHC. This provides support for our findings that additional defects in B78H1 contribute to its class I-negative phenotype.

Culture of H1Kb and H1Db transfectants in the presence of IFN-{gamma} resulted in increased surface expression levels of Kb and Db, respectively, corroborating the RT-PCR data demonstrating that this cytokine restores the Ag-processing pathway (data not shown). Similar effects were observed for CMV promoter-driven Q9 introduced into TAP2-deficient B78H1 (8).

Surface-expressed Q9 protects mice from melanoma

Early loss of Qa-2 expression in B16-derived melanomas and other in vivo selected tumors led us to hypothesize that Qa-2, like some classical class I MHC, restricts tumor outgrowth and protects hosts from malignancies. To test this hypothesis, we took advantage of the B78H1 system, engineered a panel of class I-positive and -negative cells, and followed their growth in vivo. The experimental system was modeled on studies by Levitsky et al. (15), who demonstrated Kb-mediated rejection of Kb-transfected, GM-CSF-transduced B78H1 tumors in syngeneic B6 mice.

Age- and sex-matched groups of B6 mice were injected s.c. in the hind flank with 1 x 105 live, GM-CSF-transduced tumor cells transfected with empty vector alone, TAP2 plasmid alone, or a combination of class I (Kb or Q9) and TAP2 constructs. We challenged nonimmunized mice with live tumor cells because the experiments were designed to evaluate contributions of both innate (NK cells) and adaptive (T cells) immunity to tumor immunosurveillance. The onset of tumor growth was recorded as the day a palpable solid tumor mass, defined by caliper measurements of 3 x 3 mm, was detected at the site of injection. The outgrowth of class I-negative B78H1 was consistently detectable ~3 wk postinjection with mice becoming moribund ~3 wk afterward. The critical tumor load of 1 x 105 cells was chosen on the basis of preliminary experiments with titered B78H1 cells (data not shown). The lethal burden and the time course of B78H1 tumor growth in our studies recapitulate published data (15).

Remarkably, mice injected with live GM-CSF transduced B78H1 cells transfected with both Q9 and TAP2 (GMQ9TAP.17) were able to resist, or fully reject, the tumor challenge (Fig. 6). The control empty vector-transfected (H1 vector) and TAP2-transfected GM-CSF-transduced (GMTAP) B78H1 tumors grew in syngeneic mice and became detectable at day 20, with all mice (10 of 10) having detectable tumor masses by 4 wk (Fig. 6A) and all mice dying by day 60 (Fig. 6B). Approximately one-half of the animals challenged with GMQ9TAP.17 cells were protected from tumor outgrowth, and a delayed onset was seen in the remainder (Fig. 6A). The survival curve reflects a similar pattern, with life span extended in those animals that had tumor growth and survival of all animals that were tumor free (Fig. 6B). The protective effectswere not specific to the GMQ9TAP.17 clone. A second, independently derived GM-CSF-transduced Q9/TAP-transfected B78H1 clone, GMQ9TAP.13, yielded similar results (Fig. 6). Both clones expressed similar levels of peptide-loaded Q9 as defined by flow cytometry analysis (data not shown).



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  		FIGURE 6. Protective effect of Q9 expression on B78H1 tumor challenge. Q9-mediated protection against B78H1 is more potent than that provided by Kb. Syngeneic B6 mice were given a s.c. challenge of 1 x 105 live class I MHC-transfected or control B78H1 melanoma cells. Ten mice were used in each group. A, Tumor take in mice challenged with Q9-transfected B78H1 (GMQ9TAP.17 and GMQ9TAP.13) was compared with tumor take in mice challenged with Kb-transfected B78H1 (GMKbTAP). Tukey multiple comparison of rank means was performed to determine statistically significant differences between groups. Both the GMQ9TAP.17 and GMQ9TAP.13 groups were statistically different from the GMTAP and vector groups at the 0.05 significance level. B, Survival curves were generated by recording the day animals challenged with various B78H1 melanoma transfectants were considered moribund.

 
The in vivo response to Q9-expressing tumor was also compared with the response against a control MHC class Ia+ tumor challenge (Fig. 6). Live GM-CSF-transduced B78H1 cells transfected with Kb (GMKbTAP) injected into the hind flank of B6 mice were able to generate a protective response, but this response was weaker than that invoked by Q9. Two of 10 mice challenged with live Kb-expressing tumor were tumor free after 35 days (Fig. 6), whereas more than one-half of the mice challenged with the Q9-expressing clones were tumor free. In the previously published study (15), Kb offered stronger protection than observed here. The experimental protocols differed in that Levitsky et al. (15) included a potentiating preimmunization step. In contrast, our approach followed direct outgrowth of live tumor, without preimmunization with irradiated, Kb-expressing cells.

NK cells and CD8+ T cells are involved in the rejection of Q9-transfected B78H1

The tumor growth experiments demonstrate that mice challenged with Q9-positive B78H1 melanoma (in conjunction with TAP2 and GM-CSF) are able to mount effective, significantly protective antitumor immune responses.

To determine what effector cell population(s) is involved in the Q9-mediated protective response, in vivo depletion of specific lymphocyte subsets was performed. In these studies, we focused on NK cells and CD8+ cells as the most likely candidates. Depletion was performed using either mAb asialo-GM1 to deplete NK1.1+ cells or mAb 2.43 to deplete CD8+ cells. Depletion of NK1.1+ cells resulted in nearly complete loss of protection against GMQ9TAP cells; depletion of CD8+ cells also led to a loss of protection, albeit not as substantial (Fig. 7A). The patterns observed in tumor take experiments were similarly reflected in survival curves, with NK1.1-depleted animals becoming moribund at about the same rate as animals challenged with control GMTAP cells (Fig. 7B). CD8 depletion appears to have an effect on survival rate, but again it is not as pronounced as that observed with NK1.1 depletion (Fig. 7B). These results demonstrate that both NK cells and CD8+ effectors collaborate to eliminate the Q9-positive B78H1 tumor challenge and that functional presence of both types of lymphocytes is required to bring about a significant delay of Q9-transfected tumor outgrowth.



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  		FIGURE 7. NK cells and CD8+ T cells are involved in protective immunity against Q9-expressing B78H1 melanoma. B6 mice were depleted in vivo of either NK cells ({triangleup}, {blacktriangleup}, using asialo-GM1 Ab) or CD8+ cells ({square}, {blacksquare}, using 2.43 Ab) before s.c. challenge with 1 x 105 live GMQ9TAP cells ({blacktriangleup}, {blacksquare}, •) or GMTAP cells ({triangleup}, {square}, {circ}). Tumor cells were also injected into intact ({circ}, •) animals. Ten animals were used in each group. Data are shown as Kaplan-Meier plots. A, Tumor growth in challenged mice was assessed by palpation as previously described. Tukey multiple comparison of rank means was performed to determine statistically significant differences between groups. Intact mice challenged with GMQ9TAP showed statistically significant differences in tumor growth compared with intact, NK-depleted, and CD8-depleted mice challenged with GMTAP; CD8-depleted mice challenged with GMQ9TAP showed differences compared with NK-depleted and CD8-depleted mice challenged with GMTAP; NK-depleted mice challenged with GMQ9TAP showed differences compared with NK-depleted mice challenged with GMTAP. Differences were statistically significant at the 0.05 significance level. B, Survival curves were generated by recording the day animals reached a moribund state resulting from tumor growth.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The B16 melanoma is one of the best studied models of murine malignancies. It has been used extensively to examine immunological properties of tumor cells and to test vaccination approaches for preventing tumor development (1, 2, 3, 4, 5, 6). We analyzed a series of related sublines of B16 melanoma descended from pre-existing variants of the primary B16 tumor. The established lines of the amelanotic B78H1, the pigmented family of B16F1, B16F10, BL6-2, and BL6-8 derivatives, as well as the primary B16 tumor, all express very low levels of class I Ags. However, the underlying defects have been studied thus far only in B16F1 and B16F10 (12). We compared expression of genes in the class I Ag presentation pathway in B78H1 with the phenotype of other B16 lines. Because class I expression is highly variable in different cell types (40), we used as our positive control a parental, differentiated, nonmalignant melanocyte line, Melan-a2.

Serological typing of surface class I MHC on untransformed cells and tumor cells revealed that they all display either very low or undetectable levels of {beta}2m-associated proteins. Hence, if the Melan-a2 is indeed representative of the normal melanocytes in the skin, the murine melanomas that develop in situ are already predisposed to have low class I expression and weak CTL Ag-presenting properties compared with other cells in the body. Treatment of the in vitro propagated cell lines with IFN-{gamma} led to up-regulation of class I associated with {beta}2m in all cells tested except B78H1. This result clearly establishes that B78H1 has undergone a mutational evolution distinct from other B16 cells. Indeed, the RT-PCR analysis confirmed this interpretation and indicated that transcription of H chain class I genes, Kb and Db, is extinguished in B78H1 but not in other B16 cells or Melan-a2.

To address whether additional Ag presentation mutations occurred in B78H1, we performed RT-PCR using primers specific for a panel of known genes controlling assembly of the classical class I MHC. Under noninduced conditions, B78H1 lacked detectable transcripts for TAP2 and LMP7, while transcribing other genes found also in Melan-a2 or B16 cells. TAP2 encodes an essential subunit of the peptide delivery system and its absence in mutant cells, such as RMA-S T cell tumor, leads to a drastic down-regulation of class I MHC on cell surfaces (49). LMP7 is a component of the immunoproteasome implicated in generation of class I-destined peptides (50), but its presence is not generally required to maintain class I cell surface expression (51). Presence or absence of LMP7 and other components of the immunoproteasome may nevertheless be pivotal for generation of specific viral/tumor epitopes (52).

We have demonstrated unequivocally that B78H1 differs from pigmented B16 variants and is unique in its pattern of class I Ag presentation pathway impairments. Cotransfection of class I H chains Kb, Db, or Dd along with TAP2 genes into B78H1, as well as IFN-{gamma} treatment of class I-transfected B78H1, reconstituted surface expression of exogenously introduced classical class I genes, thus confirming the RT-PCR findings.

A striking feature of B78H1 is the absence of serologically detectable cell surface {beta}2m, even in IFN-{gamma}-conditioned medium, which restores transcription of TAP2 and LMP7 and up-regulates expression of all other genes in the class I Ag presentation pathway, with the exceptions of Kb and Db. This observation led us to examine whether B78H1 is devoid of class Ib Ags. The class Ib MHC represent a large family of {beta}2m-associated proteins that are structurally related to the class Ia MHC but are nonpolymorphic or oligomorphic (53). They are usually expressed at lower levels than the polymorphic class Ia and are frequently alternatively spliced. Little is known about class Ib Ag-presenting functions, although some class Ib are known to bind peptides or lipids and function as restriction elements for CD8+ or CD4+ T cells and/or ligands for NK cells (8, 23, 24, 25, 26, 27, 28, 29, 30). Recently, class Ib were postulated to play a role in tumor rejection (9, 37, 54).

One of the best characterized murine class Ib is ubiquitously expressed, TAP-dependent Qa-2 Ag that binds a wide peptide repertoire and with tertiary and quaternary structures that are similar to those of class Ia (19, 20, 37). We previously noted that expression of Q8 and Q9 genes encoding Qa-2 proteins are down-regulated in tumors and proposed that these molecules may be involved in immunosurveillance of transformed or stressed cells (37). Serological and RT-PCR experiments reported here demonstrate that the primary B16 tumor and all its variants are blocked in Q8 and Q9 expression, whereas the control melanocyte line is positive. These results suggest that extinction of Q8/Q9 transcription was an early event in the evolution of B16 tumor that may have preceded mutations in the class Ia Ag presentation pathway. If so, this lends credence to the possibility that Qa-2 proteins protect the host from cancers.

In addition to Qa-2, we tested B78H1 for transcription of three other ubiquitously expressed class Ib: M3, which presents prokaryotic N-formylmethionine peptides (43); Qa-1, which binds predominantly class I leader peptides and is a ligand for CD94/NKG2 receptors (27, 41); and peptide-independent, {gamma}{delta} T cell-recognized T22 (42). All three transcripts were detectable, suggesting that the apparent absence of these class Ib products on the cell surface is controlled at the posttranscriptional level.

The B78H1 mutations resulting in the stable loss of class Ia and Qa-2 expression and IFN-{gamma}-inducible TAP2 and LMP7 transcription have generated a new, versatile tumor model that can be exploited for future studies of class I Ag presentation and CTL/NK cell recognition. As expected from its class I-negative phenotype, B78H1 melanoma is sensitive to NK cells (8, 15), and introduction of peptide-loaded class I MHC offers a partial protection from NK cell-mediated killing (8). Transfectants with class I MHC expression also function as stimulators and targets for allogeneic CD8+ CTL (9, 15).

We used the B78H1 system as a tool to determine whether Qa-2-mediated antitumor immunity could be elicited in syngeneic mice inoculated with a lethal dose of B78H1 melanoma. Here we demonstrate that mice challenged with B78H1 modified to express Q9 in a functional TAP background are capable of rejecting the tumor cells. Significantly, the protection afforded against Q9-expressing melanoma was more pronounced than that against a class Ia molecule, Kb.

The protective response invoked by Q9 appears to involve both innate and adaptive immune effector cells. In vivo depletion studies indicated that CD8+ cells contribute to rejection of Q9-positive B78H1. Based on the known effects of class Ia MHC in tumor rejection, it could be speculated that these CD8+ effectors are likely to be CTLs. This finding is the subject of further investigation because, despite its capacity to present peptides and to serve as an allogeneic CTL target (55), Q9 has not been previously demonstrated to function as a restricting element for CTLs in pathogen infection or tumor models. Depletion of NK cells also abrogated the rejection response against Q9-transfected GM-CSF transduced B78H1 tumors. Interestingly, Q9 has been previously shown to protect B78H1 melanoma from lymphokine-activated killer cell-mediated lysis in vitro, but the protective effect of Q9 was only partial (8). Thus, it is likely that in vivo, a significant proportion of activated effector NK cells will still be cytolytic against Q9-transfected tumors and may participate in tumor rejection. Additionally, in this system, activated NK cells may exert immunomodulatory effects due to the production of cytokines and chemokines that promote generation of downstream T cell responses (56). Thus, depletion of NK cells achieved in our experiments not only might have eliminated a primary effector cell population capable of directly killing tumor cells but may also have incapacitated the ability to generate CTLs, effectively blocking the ability of the host to mount an adaptive immune response. Further studies are necessary to address the molecular mechanisms and pathways involved in the collaborative action of NK cells and CD8+ cells during Q9-mediated rejection of B78H1 melanoma.


   Acknowledgments
 
We thank Dr. Ming Chen for sharing his unpublished MHC class Ib-specific primers used in RT-PCR experiments. We also thank Dr. Suzanne Ostrand-Rosenberg for helpful discussions and for critically reading the manuscript and Dr. Thorbald van Hall for his expert advice on tumor studies.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants RO1 AI19624 and PO1 37818 and Grant EMF 1DSSO20401. Back

2 Address correspondence and reprint requests to Dr. Iwona Stroynowski, Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail address: iwona.stroynowski{at}utsouthwestern.edu Back

3 Abbreviations used in this paper: LMP, low molecular weight protein; class Ia, classical class I; class Ib, nonclassical class I; PA, proteasome activator; {beta}2m, {beta}2-microglobulin; GMTAP, TAP2-transfected GM-CSF-transduced; FITC-GAM, FITC-conjugated goat anti-mouse IgG. Back

Received for publication December 16, 2002. Accepted for publication February 27, 2003.


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