Role of Antigen-Processing Machinery in the In Vitro Resistance of Squamous Cell Carcinoma of the Head and Neck Cells to Recognition by CTL

Andrés López-Albaitero, Jayakar V. Nayak, Takeshi Ogino, Avinash Machandia, William Gooding, Albert B. DeLeo, Soldano Ferrone and Robert L. Ferris
J Immunol March 15, 2006, 176 (6) 3402-3409; DOI: https://doi.org/10.4049/jimmunol.176.6.3402

Abstract

Squamous cell carcinoma of the head and neck (SCCHN) cells are poorly recognized in vitro by CTL despite expressing the restricting HLA class I allele and the targeted tumor Ag (TA). Several lines of evidence indicate that the lack of SCCHN cell recognition by CTL reflects defects in targeted TA peptide presentation by HLA class I Ag to CTL because of Ag-processing machinery (APM) dysfunction. First, lack of recognition of SCCHN cells by CTL is associated with marked down-regulation of the IFN-γ-inducible APM components low-m.w. protein 2, TAP1, TAP2, and tapasin. Second, SCCHN cell recognition by CTL is restored by pulsing cells with exogenous targeted TA peptide. Third, the restoration of CTL recognition following incubation of SCCHN cells with IFN-γ is associated with a significant (p = 0.001) up-regulation of the APM components TAP1, TAP2, and tapasin. Lastly, and most conclusively, SCCHN cell recognition by CTL is restored by transfection with wild-type TAP1 cDNA. Our findings may explain the association between APM component down-regulation and poor clinical course of the disease in SCCHN. Furthermore, the regulatory nature of the APM defects in SCCHN cells suggests that intralesional administration of IFN-γ may have a beneficial effect on the clinical course of the disease and on T cell-based immunotherapy of SCCHN by restoring SCCHN cell recognition by CTL.

In recent years, enthusiasm for the application of T cell-based immunotherapy for the treatment of malignant diseases has resulted in the enrollment of an increasing number of patients in clinical trials (1). Contrary to expectations, the results of these clinical investigations have been rather disappointing, because no significant correlation has been found between clinical response and tumor Ag (TA)3-specific immune response (2). In most patients, the disease continues to progress or recurs despite the induction or augmentation of a TA-specific CTL immune response.

Several immune escape mechanisms have been shown to underlie the lack of correlation between immune and clinical responses. They include T cell and dendritic cell dysfunction (3, 4, 5), HLA class I Ag structural and functional abnormalities, and/or targeted TA loss and mutations (6). One condition that remains unexplained is the lack of recognition of malignant cells by functional HLA class I Ag-restricted, TA-specific CTL despite the expression of the restricting HLA class I allospecificity and of the targeted TA. This condition appears to occur frequently with squamous cell carcinoma of head and neck (SCCHN) cells (7, 8). Because of the crucial role played by Ag-processing machinery (APM) components in the generation, transport, and loading of Ag-derived peptides on HLA class I H chain-β2-microglobulin (β2m) complexes, APM dysfunction leads to defects in the presentation of TA peptides by HLA class I Ags to CTL. Therefore, in the present investigation, we have used SCCHN cell lines to test the hypothesis that their resistance to CTL recognition is caused by functional APM abnormalities.

In our studies, we have taken advantage of a panel of APM component-specific mAb (9, 10) and a recently developed methodology (11) to measure APM component levels (10) in SCCHN cells under basal conditions and following incubation with IFN-γ. As model TA, we have used HER2, a membrane-bound “self” Ag, and MAGE-3, a cytoplasmic cancer-testis Ag (12). Because factors regulating expression, processing, and presentation of these two TA are likely to be different, our data may be applicable to different types of TA.

Materials and Methods

Cell lines

The SCCHN cell lines SCC-4 (13), SCC-90 (14), PCI-13, PCI-30, PCI-4A, PCI-4B, PCI-6A, PCI-6B, PCI-15A, PCI-15B, PCI-37A, and PCI-37B have been characterized and described previously (15). PCI cells denoted “A” and “B” were derived from autologous primary and metastatic tumors, respectively. The CD80 (B7.1) transfected PCI-13 cell line, a gift from Dr. T. L. Whiteside (University of Pittsburgh Cancer Institute), has been previously described (16). Both the melanoma cell line MEL-526 and the breast carcinoma cell line MCF-7 have been described and characterized elsewhere (17, 18). All tumor cell lines were cultured in DMEM supplemented with 8% FBS (Mediatech), 2% l-glutamine, and 1% penicillin/streptomycin (Invitrogen Life Technologies). The mutant T2 cell line, which does not express low-m.w. protein 2 (LMP2), LMP7, TAP1, and TAP2 gene products (19), was maintained in serum-free medium AIM V (Invitrogen Life Technologies). All cell lines and cultures were monitored for mycoplasma infection at monthly intervals.

Cytokines

GM-CSF, IL-1β, IL-4, and TNF-α were purchased from R&D Systems. IFN-γ was purchased from InterMune.

Antibodies

HLA-A, -B, -C Ag-specific mAb W6/32; HLA-A2 and -A68 Ag-specific mAb BB7.2; β2m)-specific mAb L368; HLA-DR, -DQ, and -DP-specific mAb LGII-612.14; δ-specific mAb SY-4; MB-1-specific mAb SJJ-3; LMP2-specific mAb SY-1; LMP7-specific mAb SY-3; LMP10-specific mAb TO-7; calnexin-specific mAb TO-5; calreticulin-specific mAb TO-11; ERp57-specific mAb TO-2; and tapasin-specific mAb TO-3 were developed and characterized as described (9, 10, 20, 21, 22). The TAP1-specific mAb NOB-1 and the TAP2-specific mAb NOB-2 are secreted by hybridomas derived from the fusion of murine myeloma cells P3-X63-Ag8.653 with splenocytes from BALB/c mice immunized with partial-length TAP1 recombinant protein (aa 434–735) and a keyhole limpet hemocyanin-conjugated TAP1 peptide (aa 717–735) and with partial-length TAP2 recombinant protein (aa 316–703), respectively. The specificity of the mAb was assessed by their reactivity with molecules of a size corresponding to the immunizing TAP1 and TAP2 when tested in Western blotting with a lysate of lymphoid cells that express TAP1 and TAP2 and by the lack of reactivity with a lysate of the T2 cell line, which does not express these molecules (19, 23). The human ICAM-1-specific mAb CL203.4 was developed and characterized as described (24). Purified human HER2-specific mouse mAb was purchased from BD Biosciences.

Recombinant vaccinia viruses expressing APM components

Recombinant vaccinia virus (rVV) vectors expressing the APM components TAP1, TAP1/2, and tapasin were generous gifts of Dr. Jonathan Yewdell (National Institutes of Health, Bethesda, MD), and vSC8 containing the irrelevant gene lacZ was a generous gift of Dr. Bernard Moss (National Institutes of Health, Bethesda, MD). These rVV have been described elsewhere (25, 26, 27). Briefly, the rVV were propagated in thymidine kinase-deficient human 143B osteosarcoma cells and titrated for optimal dose of infection. To effectively induce the expression of APM components, the appropriate titer of virus (1 × 107 viral particles) was added to the flask growing the specific cell line to be used as a target. Cell lines were plated at a concentration of 1 × 106 cells per T75 flask (Corning Glass). Following a 24-h incubation at 37°C, each rVV was added in suspension in opti-DMEM medium while the flask sat at 37°C on a rocking machine. Experiments were conducted 8 h after the initial infection.

Quantitative RT-PCR

Quantification of MAGE-3 expression was performed as described (28). RNA was extracted from cell lines harvested when at 70% confluence, using Trizol (Invitrogen Life Technologies) and the RNeasy kit (Qiagen). The purified RNA was resuspended in RNA secure solution (Ambion) and cleared of DNA. A one-step reverse transcriptase reaction was performed using 500 and 2000 ng/μl of the RNA with random hexamer primers and Superscript II (Invitrogen Life Technologies) as described previously (29). Quantitative RT-PCR was then conducted on the Applied Biosystems 7700 Sequence Detection Instrument, using a previously validated assay (28). Expression of the target gene (MAGE-3) relative to that of β-glucuronidase (GUS; an endogenous control gene) was calculated using the ΔCT (cycle time) method described previously: relative expression = 2−ΔCT, where ΔCT = CT(MAGE) − CT(GUS) (29).

Peptides and tetramers

The HER2369–377 (KIFGSLAFL (30)) and MAGE-3271–279 (FLWGPRALV (31)) peptides were synthesized by the University of Pittsburgh Peptide Synthesis facility, using F-moc technology. The Influenza Matrix58–66 peptide (GILGFVFTL (32)) was purchased from Commonwealth Biotechnologies. The lyophilized peptides were resuspended at 1 mg/ml in DMSO for use. HLA-A∗0201-MAGE-3271–279 (referred to as HLA-A2-MAGE) tetramer was synthesized by the National Institutes of Health Tetramer Facility (Emory University, Atlanta, GA) and used according to their instructions after careful titration.

Flow cytometry

Cell surface and intracytoplasmic staining of cells was performed as described previously (11). Mouse IgG1 mAb (DakoCytomation) staining was set at a mean fluorescence intensity of 5 on the FL1 channel of an EPICS XL cytometer (Beckman Coulter) for each of the cell lines (using separate controls for stimulated and unstimulated cells). The flow cytometer was calibrated daily with fluorescent beads to enable comparison among experiments; all samples were run using identical settings to collect ≥10,000 gated events. Analyses were performed using EXPO32 ADC software (Beckman Coulter). HLA class I and class II Ag and APM component expression was determined based on mean fluorescence intensity as well as % positive cells (shifting into a defined flow cytometry gate) and was expressed as mean ± SD of the results obtained in experiments repeated at least three times.

Generation of HLA class I Ag-restricted, TA-specific CTL by in vitro stimulation

HLA-A2-HER2369–377-specific and HLA-A2-MAGE-3/6271–279-specific CTL, referred to as HER2-specific and MAGE-specific CTL, respectively, were generated from PBMC of HLA-A2+ healthy donors or of HLA-A2+ SCCHN patients as described (7, 33), with the following modifications. Monocytes were separated with a Percoll (Pharmacia) gradient, selected by plastic adherence for 2 h at 37°C, and cultured for 6 days in AIM-V medium supplemented with 10% human AB serum (Mediatech), GM-CSF (100 ng/ml), and IL-4 (100 ng/ml). The resulting immature dendritic cells were matured for 48 h at 37°C in medium supplemented with IFN-α (1000 IU/ml), IFN-γ (1000 IU/ml), IL-1β (25 ng/ml), and TNF-α (50 ng/ml); TA-derived peptide was added at a final concentration of 1 μM 2 h before the end of the incubation. CD8+ PBMC obtained by negative selection using CD19, CD14, and CD4 MACS beads (Miltenyi Biotec) were resuspended in AIM-V medium supplemented with 2% human AB serum, 7% FBS, IL-2 (100 ng/ml), and IL-7 (25 ng/ml). CTL were expanded by multiple rounds of stimulation every 2 wk using peptide-pulsed T2 cells irradiated at 50 Gy as described (7). Once specificity and activity were confirmed using IFN-γ ELISPOT assays, CTL aliquots were stored for later use at −80°C in IMDM containing 10% DMSO and 10% FBS. Unless otherwise specified, all experiments were performed with CTL obtained from HLA-A2+ SCCHN patient PBMC.

ELISPOT assays

IFN-γ ELISPOT assays were performed in triplicate as described previously (7). Plates were read using a Vision ELISPOT reader (Zeiss). Background was considered as the number of spots secreted by CD8+ T cells alone. In all experiments, T2 cells pulsed with an irrelevant peptide and with the targeted TA peptide (1 μg/ml AIM V medium for 2 h at room temperature (RT)) were used as negative and positive controls, respectively. PMA/ionomycin-treated CTL were used as a control for CTL secretion of IFN-γ. To determine the HLA class I Ag restriction of the CTL reactivity, target cells were incubated with either HLA-A, -B, -C Ag-specific mAb W6/32; HLA-A2, -A68 Ag-specific mAb BB7.2; or HLA-DR Ag-specific mAb L243 (final concentration 10 μg/ml) for 30 min at 37°C before addition of effector CTL.

Statistical analysis

T cell reactivity as measured by the ELISPOT assay was considered positive if the number of spots in test wells was significantly higher than that in background wells when using a one-tailed permutation test for α≤ 0.05. Association of APM component levels with HLA class I and class II Ag levels was explored using nonparametric regression. The coefficient of determination (R2) was used to evaluate the strength of the association of APM component levels with HLA class I and class II Ag levels. A test of significance of the regression was conducted as if the analyzed cell lines were random samples of SCCHN cell lines.

Results

Identification of SCCHN cell lines that express HLA-A2 Ag as well as HER2 and/or MAGE-3

Screening of 12 SCCHN cell lines to identify those that express HLA-A2 Ag as well as HER2 and/or MAGE-3 resulted in the identification of four HLA-A2+ SCCHN cell lines expressing one or both of the TA (data not shown). Of these, SCC-4 and SCC-90 cells express HER2 only, PCI-13 cells express MAGE-3 only, and PCI-30 cells express both TA. The levels of HLA-A2, HER2, or MAGE-3 expression in the SCCHN cell lines were similar to those in MEL-526 cells (MAGE-3+) derived from a melanoma lesion and in MCF-7 cells (HER2+) derived from a breast adenocarcinoma lesion (Fig. 1⇓). The latter two cell lines were used as target controls in some of the experiments performed.

FIGURE 1.
FIGURE 1.

Similar HLA-A2 Ag expression by SCCHN PCI-30 cells, breast carcinoma MCF-7 cells, and melanoma MEL-526 cells and lack of modulation by pulsing with TA peptide. A, PCI-30 cells (thin line), MCF-7 cells (dotted line), and MEL-526 cells (thick line) were stained with HLA-A2, -A68 Ag-specific FITC-conjugated mAb BB7.2 and analyzed by flow cytometry. B, Control PCI-30 cells (solid line) and PCI-30 cells pulsed with exogenous MAGE-3217–279 peptide (10 μg/ml AIM V medium for 1 h at RT) (dotted line) were stained with HLA-A2, -A68 Ag-specific FITC-conjugated mAb BB7.2 and analyzed by flow cytometry.

Generation of HLA-A2+ Ag-restricted, HER2- or MAGE-specific CTL

HLA-A2-HER2-specific (Fig. 2⇓A) and HLA-A2-MAGE-specific (Fig. 2⇓B) CTL, respectively, were generated from PBMC of HLA-A2+ normal donors and SCCHN patients by in vitro stimulation. As shown in Fig. 2⇓ (white bars), these CTL recognize T2 cells pulsed with either HER2369–377 or MAGE-3271–279. The recognition is specific because these CTL did not recognize unpulsed T2 cells or T2 cells pulsed with the irrelevant peptide Influenza Matrix58–66.

FIGURE 2.
FIGURE 2.

In vitro recognition of human breast carcinoma MCF-7 cells and human melanoma MEL-526 cells by HLA-A2 Ag-restricted, TA-specific CTL. HER2+, HLA-A2+ MCF-7 cells (▪), and MAGE-3+, HLA-A2+ MEL-526 cells (▦) were incubated for 18 h at 37°C with HER2-specific CTL (A) and with MAGE-3-specific CTL (B), respectively. IFN-γ ELISPOT assays were performed at an E:T ratio of 10:1. T2 cells (□) either untreated or pulsed with the targeted TA peptide or with the irrelevant peptide Influenza Matrix58–66 (Flu58–66; 10 μg/ml AIM V medium for 2 h at RT) were used as controls. PMA/ionomycin-treated CTL were used as a control for maximal CTL secretion of IFN-γ. Background was considered as the number of spots secreted by CD8+ T cells alone. HLA class I Ag restriction of CTL reactivity was tested by incubating target cells with HLA-A2, -A68 Ag-specific mAb BB7.2 (final concentration, 10 μg/ml) for 30 min at 37°C before the addition of effector CTL. HLA-DR Ag-specific mAb L243 was used as a control.

Additional experiments showed that these CTL recognize tumor cells with the appropriate antigenic phenotype. The recognition is TA specific, because the HER2 CTL did not recognize HER2− MEL-526 cells, and the MAGE-specific CTL did not recognize MAGE-3− MCF-7 cells (Fig. 2⇑). IFN-γ ELISPOT reactivity was HLA-A2 Ag restricted, because IFN-γ spots were reduced to background levels when target cells were pretreated with the HLA-A2, -A68 Ag-specific mAb BB7.2, but were not affected when target cells were pretreated with the HLA-DR Ag-specific mAb L243 (Fig. 2⇑). Peptide titration assays, using CTL derived from PBMC of either HLA-A2+ healthy individuals or SCCHN patients, indicated a half-maximal CTL reactivity of ∼50 ng/ml peptide. Tetramer-based flow cytometry showed that ∼10% of the CTL cultures generated from both sets of donors were stained positive in the CD3+CD8+tet+ gate (A. Lopez-Albaitero and R. L. Ferris, unpublished data). Because the results obtained with the CTL from normal donors and SCCHN patients were superimposable, the CTL used for the remainder of the studies were derived from the PBMC of one SCCHN patient.

Lack of CTL recognition of SCCHN cells despite HLA-A2 Ag and targeted TA expression

The HLA-A2+ SCCHN cell lines SCC-4, SCC-90, PCI-13, and PCI-30 were not recognized by HER2-specific CTL or by MAGE-specific CTL, even when the E:T ratios were increased to 10:1. Figs. 3⇓ and 4⇓ show representative data using the SCCHN cell line PCI-30, which expresses both HER2 and MAGE-3. CTL recognition of PCI-30 cells, as measured by IFN-γ ELISPOT or 51Cr release (Fig. 5⇓), was observed only after addition of IFN-γ (100 IU/ml for 72 h at 37°C).

FIGURE 3.
FIGURE 3.

CTL recognition of HER2+ SCCHN PCI-30 cells following pulsing with HER2369–377 peptide or incubation with IFN-γ. PCI-30 cells pulsed with exogenous HER2369–377 peptide (10 μg/ml AIM V medium for 1 h at RT) or incubated with IFN-γ (100 IU/ml for 72 h at 37°C) were incubated for 18 h at 37°C with HER2-specific CTL at an E:T ratio of 10:1. IFN-γ ELISPOT assays were performed in triplicate. Untreated PCI-30 cells were used as controls. HLA class I Ag restriction of CTL recognition was tested by incubating target cells with HLA-A2, -A68 Ag-specific mAb BB7.2 (final concentration 10 μg/ml) for 30 min at 37°C before addition of effector CTL. HLA-DR Ag-specific mAb L243 was used as a control.

FIGURE 4.
FIGURE 4.

CTL recognition of MAGE-3+ SCCHN PCI-30 cells following pulsing with MAGE-3271–279 peptide or incubation with IFN-γ. PCI-30 cells pulsed with exogenous MAGE-3271–279 peptide (10 μg/ml AIM V medium for 1 h at RT) or incubated with IFN-γ (100 IU/ml for 72 h at 37°C) were incubated for 18 h at 37°C with MAGE-specific CTL at an E:T ratio of 10:1. IFN-γ ELISPOT assays were performed in triplicate. Untreated PCI-30 cells were used as controls. HLA class I Ag restriction of CTL recognition was tested by incubating target cells with HLA-A2, -A68 Ag-specific mAb BB7.2 (final concentration 10 μg/ml) for 30 min at 37°C before the addition of effector CTL. HLA-DR Ag-specific mAb L243 was used as a control.

FIGURE 5.
FIGURE 5.

CTL-mediated lysis of MAGE-3+ SCCHN PCI-30 cells following incubation with IFN-γ. PCI-30 cells incubated with IFN-γ (100 IU/ml for 72 h at 37°C) were labeled with 51Cr and incubated for 4 h at 37°C with HLA-A2 Ag-restricted, MAGE-specific CTL at an E:T ratio of 10:1. 51Cr release assays were performed in triplicate. MEL-526 cells were used as controls. Specific lysis was calculated as (experimental 51Cr release − spontaneous 51Cr release)/(maximal 51Cr release − spontaneous 51Cr release). Background (spontaneous) 51Cr release was ∼20% in this assay.

The level of HLA-A2 Ag expression on PCI-30 cells under basal conditions is similar to that on MAGE-3+ MEL-526 cells and HER2+ MCF-7 cells (Fig. 5⇑A). These results argue against insufficient levels of HLA class I Ag expression as a major mechanism underlying the lack of SCCHN cell recognition by CTL.

The lack of functional abnormalities of HLA-A2 Ag as a mechanism underlying the inability of CTL to recognize SCCHN cells was conclusively proven by the recognition by CTL of the four SCCHN cell lines that had been pulsed with exogenous HER2369–377 or MAGE-3271–279 peptide (10 μg/ml for 1 h at 37°C). Representative results of experiments performed with the HLA-A2+ SCCHN cell line PCI-30, which expresses both HER2 and MAGE-3 are shown in Figs. 3⇑ and 4⇑, as part of the specificity analysis of HER2-specific and MAGE- specific CTL.

To determine whether the restoration of CTL recognition of SCCHN cells by pulsing with exogenous TA peptide reflected up-regulation of HLA class I Ag, we measured HLA class I Ag expression on the cell lines PCI-13, PCI-30, SCC-4, and SCC-90 following pulsing with HER2369–377 or MAGE-3271–279 peptide. As shown in Fig. 1⇑B, no change in HLA-A2 Ag expression was detected on PCI-30 cells pulsed with exogenous HER2369–377 or MAGE-3271–279 peptides. Similar results were obtained with the other three SCCHN cell lines (data not shown). These data indicate that HLA-A2 Ag is expressed on SCCHN cell lines at levels sufficient for recognition by CTL and is functionally capable of peptide presentation to CTL. Furthermore, the data we have presented raise the possibility that the lack of recognition of SCCHN cells by CTL is caused by defects in targeted TA presentation to CTL, perhaps due to APM dysfunction.

Role of APM dysfunction in the resistance of SCCHN cells to recognition by HLA class I Ag–TA peptide-specific CTL

To determine whether the lack of generation of the targeted HLA class I Ag–TA peptide complex in the SCCHN cell lines was caused by low expression of functional APM components, cell lines PCI-13, PCI-30, SCC-4, and SCC-90 were intracellularly stained with APM component-specific mAb and analyzed by flow cytometry (Fig. 6⇓). The immunoproteasome subunits LMP2, LMP7, and LMP10, the TAP subunits TAP1 and TAP2, and the chaperone tapasin were found to be expressed at low or barely detectable levels in the four SCCHN cell lines. To determine whether the level of APM components in SCCHN cell lines could be up-regulated by IFN-γ, SCCHN cells were incubated with IFN-γ (concentrations ranging from 100 IU/ml to 1000 IU/ml) for 72 h at 37°C. Incubation of SCCHN cells with IFN-γ (100 IU/ml) led to consistent up-regulation of APM components LMP2, TAP1, TAP2 and tapasin, as well as HLA-A2 Ag (Fig. 6⇓). In descending order of magnitude, IFN-γ selectively enhanced the expression of APM components, TAP1, tapasin, TAP2, and LMP2. Using nonlinear regression we found that enhanced expression of these APM components was significantly associated (p < 0.001) with the up-regulation of HLA class I surface molecules, as measured by intracytoplasmic and surface flow cytometry (Table I⇓). This finding is consistent with the critical role of these APM components in the HLA class I Ag-processing pathway. This association is specific, because no correlation was found between the expression of any of these four APM components and that of HLA-DR Ag. Interestingly, nonlinear components of the association were seen, e.g., between HLA class I Ag and TAP1 and LMP2 (not shown), suggesting attenuation of HLA class I Ag up-regulation at the highest levels of TAP1 and LMP2 expression induced by IFN-γ. Most importantly, in addition to APM component up-regulation, IFN-γ treatment restored recognition of the four SCCHN cell lines by HLA class I Ag-restricted, TA-specific CTL, as assessed with IFN-γ ELISPOT (Fig. 2⇑) and 51Cr release assays (Fig. 4⇑). Further support for this conclusion is the lack of detectable changes in HER2 and MAGE-3 expression by the four HLA-A2+ SCCHN cell lines following incubation with IFN-γ (100 IU/ml for 72 h at 37°C) (Fig. 7⇓).

FIGURE 6.
FIGURE 6.

APM component and HLA class I Ag up-regulation by IFN-γ in SCCHN cells. Following incubation with IFN-γ (100 IU/ml for 72 h at 37°C) (dotted line), permeabilized SCCHN cell lines PCI-13, PCI-30, SCC-4, and SCC-90 were incubated with LMP2-specific mAb SY-1, TAP1-specific mAb NOB-1, TAP2-specific mAb NOB-2, or tapasin-specific mAb TO-3 and stained with FITC-conjugated anti-mouse IgG xenoantibodies. Viable cells were surface stained with HLA-A2, -A68-specific FITC-conjugated mAb BB7.2. Cells were then analyzed by flow cytometry. Untreated cells (solid line) or cells stained only with FITC-conjugated anti-mouse IgG xenoantibodies (shaded histogram) were used as controls.

FIGURE 7.
FIGURE 7.

Lack of modulation by IFN-γ of TA expression by SCCHN PCI-30 cells, breast carcinoma MCF-7 cells, and melanoma MEL-526 cells. A, Control PCI-30 cells (thin line), PCI-30 cells incubated with IFN-γ (100 IU/ml for 72 h at 37°C) (thick line), and MCF-7 cells (dotted line) were incubated with HER2-specific mAb and stained with FITC-conjugated anti-mouse IgG xenoantibodies. Cells were then analyzed by flow cytometry. Cells stained only with FITC-conjugated anti-mouse IgG xenoantibodies (gray histogram) were used as controls. B, MAGE-3 mRNA was isolated from control MEL-526 cells, control PCI-30 cells, and PCI-30 cells incubated with IFN-γ (100 IU/ml for 72 h at 37°C). MAGE-3 expression was measured by quantitative RT-PCR, using an endogenous housekeeping gene (GUS) as an internal control. Relative MAGE-3 expression between cell lines was determined using the 2−ΔCt method.

View this table:
Table I.

Association of LMP2, TAP1, TAP2, and tapasin expression with HLA class I antigen expression by SCCHN cells incubated with IFN-γa

Restoration by APM component transfection of SCCHN cell recognition by CTL

To conclusively prove the role of certain APM component up-regulation in the restoration of CTL recognition of SCCHN cell lines and to identify the key component(s) in this restoration, we tested whether transfection of the four HLA-A2+ SCCHN cell lines with wild-type TAP1, TAP2, and/or tapasin cDNA could restore their recognition by CTL. Recognition of SCCHN cell lines by HER2- or MAGE-specific CTL was induced by transfection of SCCHN cells with rVV containing TAP1 and TAP2 genes (data not shown). Surprisingly, the same effect was obtained when SCCHN cells were transfected only with TAP1 cDNA (Fig. 8⇓).

FIGURE 8.
FIGURE 8.

CTL recognition of SCCHN PCI-13, PCI-30, SCC-4, and SCC-90 cells following transfection with wild-type APM component cDNA. Cells were transiently transfected with rVV encoding cDNA (multiplicity of infection (MOI) = 5) encoding wild-type TAP1 or tapasin for 8 h. Transfected cells were then incubated for 18 h at 37°C with MAGE-specific (A and B) or HER2-specific (C and D) CTL at an E:T ratio of 10:1. IFN-γ ELISPOT assays were performed in triplicate. HLA class I Ag restriction of CTL recognition was tested by incubating target cells with HLA-A2, -A68 Ag-specific mAb BB7.2 (final concentration 10 μg/ml) for 30 min at 37°C before addition of effector CTL. HLA-DR Ag-specific mAb L243 was used as a control.

In the transfected cells, TAP1 and TAP2 were expressed at the same level as in cells that were incubated with IFN-γ (100 IU/ml for 72 h at 37°C) (Fig. 9⇓, A–C). TAP1 and TAP2 up-regulation (Fig. 9⇓) and restoration of CTL recognition caused by transfection of SCCHN cells with TAP1 and/or TAP2 cDNA are specific, because neither effect was observed when cells were transfected with the control rVV (vSC8) containing the irrelevant gene insert lacZ. Transfection of SCCHN cell lines with VV-tapasin did not restore their susceptibility to recognition by HER2- or MAGE-specific CTL. The failure of wild-type tapasin transfection to restore CTL recognition of SCCHN cell lines was not due to insufficient levels of this APM component, because the transfection yielded higher levels of tapasin expression than those achieved when cells were incubated with IFN-γ (100 IU/ml for 72 h at 37°C) (Fig. 9⇓D). It is noteworthy that transfection of SCCHN cells with VV-TAP1 caused no detectable change in expression of HLA class I Ag, including the restricting element HLA-A2 Ag, or expression of other APM components (Fig. 10⇓). These results show that TAP1 plays a crucial role in the recognition of SCCHN cells by HLA class I Ag-restricted, TA-specific CTL.

FIGURE 9.
FIGURE 9.

Up-regulation of APM component expression by SCCHN PCI-30 cells following transfection with wild-type APM component cDNA. PCI-30 cells were transiently transfected with rVV (MOI = 5) encoding wild-type TAP1 (A, solid line), wild-type TAP1/2 (B and C, solid line), or wild-type VV-tapasin (D, solid line). Permeabilized cells were incubated with TAP1-specific mAb NOB-1, TAP2-specific mAb NOB-2, or tapasin-specific mAb TO-3, and stained with FITC-conjugated anti-mouse IgG xenoantibodies. Cells were analyzed by flow cytometry. Untreated cells (shaded histogram) and cells incubated with IFN-γ (100 IU/ml for 72 h at 37°C) (dotted line) were used as controls.

FIGURE 10.
FIGURE 10.

Selective TAP1 up-regulation in SCCHN PCI-30 cells following transfection with VV-TAP1. PCI-30 cells were transiently transfected with rVV (MOI = 5) encoding wild-type TAP1 cDNA. Transfected cells (broken line) were surface stained with HLA-A2, -A68-specific FITC-labeled mAb BB7.2 (A) or incubated with HLA-A, -B, -C Ag-specific mAb W6/32 and stained with FITC-conjugated anti-mouse IgG xenoantibodies (B). Permeabilized transfected cells (broken line) were incubated with LMP2-specific mAb SY-1 (C), TAP1-specific mAb NOB-1 (D), TAP2-specific mAb NOB-2 (E), or tapasin-specific mAb TO-3 (F) and stained with FITC-conjugated anti-mouse IgG xenoantibodies. Cells were then analyzed by flow cytometry. Cells transfected with empty vector (solid line) and cells stained only with FITC-conjugated anti-mouse IgG xenoantibodies (shaded histogram) were used as controls.

Lack of a significant role of costimulatory and adhesion molecules in the restoration by IFN-γ of CTL recognition of SCCHN cells

Because IFN-γ modulates the expression of several molecules besides APM components and HLA class I Ag, we tested whether changes in costimulatory and adhesion molecules played a role in the restoration of recognition by CTL of SCCHN cells incubated with IFN-γ. The costimulatory molecules CD80 and CD86 were detected on the SCCHN cell lines PCI-13, PCI-30, SCC4, and SCC90 under basal conditions as well as following incubation with IFN-γ (100 IU or 1000 IU/ml for 72 h at 37°C) (data not shown). In addition, in the absence of IFN-γ treatment, CD80-transfected HLA-A2+, MAGE-3+ PCI-13 cells (16), like the control vector-transfected PCI-13 cells, were not recognized by MAGE-specific CTL in IFN-γ ELISPOT assays (data not shown). These results indicate that this costimulatory molecule does not play a major role, if any, in the induction of CTL recognition of SCCHN cells by IFN-γ treatment. Lastly, the adhesion molecule ICAM-1 is also unlikely to play a significant role in the CTL recognition of SCCHN cells we observed under these experimental conditions, because this molecule was found to be expressed at variable levels only on PCI-13 and SCC-4 cells, and its expression did not change consistently after incubation with IFN-γ (data not shown).

Discussion

It is the rule more than the exception that SCCHN cells are not recognized in vitro by HLA class I Ag-restricted, TA-specific CTL, although they express the restricting HLA class I allospecificity and the targeted TA. Taking advantage of a panel of newly developed APM component-specific mAb and of a recently developed technique (10), the present study has provided several lines of evidence that lack of SCCHN cell recognition by HLA class I Ag-restricted, TA-specific CTL is caused by functional defects in APM. First, the resistance of SCCHN cell lines to CTL recognition is associated with lack of expression or marked down-regulation in these cell lines of the IFN-γ-inducible APM components LMP2, TAP1, TAP2, and tapasin. These abnormalities in APM component expression appear to have functional significance, because defective peptide transport has been found in the SCCHN cell lines PCI-13 and PCI-15A (34). Second, CTL recognition of SCCHN cell lines by IFN-γ is associated with up-regulation of the down-regulated APM components, besides that of HLA class I Ag, including the restricting HLA-A2 allospecificity. Third, pulsing of SCCHN cells with the targeted TA-derived peptide leads to their recognition by HLA class I Ag-restricted, TA-specific CTL, without detectable changes in the expression level of the restricting HLA-A2 allospecificity. These results show that HLA class I Ag are expressed on SCCHN cells at a level sufficient for an efficient presentation of peptides to CTL and have no detectable abnormalities in their ability to present peptides to CTL. Lastly, and most conclusively, transfection of SCCHN cell lines with certain wild-type APM component cDNA restores the expression of the down-regulated APM components and the recognition of SCCHN cells by HLA class I Ag-restricted, TA-specific CTL without detectable changes in the expression of the restricting HLA-A2 allospecificities.

The transfection experiments with APM component cDNA allowed us to identify the APM component(s) that play(s) a key role in the restoration of CTL recognition of SCCHN cell lines. The results we have described indicate that TAP1 is the key APM component, because transfection of SCCHN cell lines with TAP1 cDNA was sufficient to restore target cell recognition by HLA class I Ag-restricted, TA-specific CTL. This unexpected finding is not unique to SCCHN cell lines, because similar results have been described in two mouse model systems and in a human renal carcinoma cell line. Like the SCCHN cell lines we have described, all of them appear to have abnormalities in APM component expression. Jefferies and his associates (35) have shown that introduction of the rat TAP1 in the vesicular stomatitis virus-infected lung carcinoma cells CMT.64 corrected the MHC class I deficiency, resulting in recognition of these cells by CTL in vitro, as well as in a decrease of tumor growth and incidence in vivo. Similarly Schreiber and his associates (36) have reported that overexpression of TAP1 in the IFN-γ-insensitive sarcoma clones RAD.gR28 and RAD.gR.30 with low TAP1 and MHC class I Ag expression resulted in their CD4- and CD8-dependent rejection when transplanted in immunocompetent mice, whereas the parental cell line grew progressively. Lastly, Seliger and her associates (37) have shown that following transfection with wild-type cDNA, the human renal carcinoma cell line MZ1257RC stimulated TNF-α and GM-CSF release from autologous TA-specific T cells, although did not become susceptible to lysis. The mechanism(s) underlying this discrepancy is not known at present. It is noteworthy that, at variance with our own results in SCCHN cell lines, in both the mouse model systems and the human renal carcinoma cell line analyzed, the evaluation of the APM component expression has been limited to a few components, has not been quantitative, and/or has been done at the mRNA level. In particular, no information is available about the level of TAP2 subunit expression in the TAP1-transfected tumor cell lines. Therefore, caution should be exercised in discussing the molecular mechanism(s) underlying restoration of CTL recognition of human cell lines and the two described mouse tumor cell models following transfection with TAP1. Nevertheless, we favor the possibility that, as described with a melanoma cell line by one of us (38), TAP1 expression stabilizes TAP2 mRNA and protein, leading to TAP expression, restoration of peptide transport to the endoplasmic reticulum (ER), and loading on β2m-HLA class I H chain complexes. In contrast, two lines of evidence argue against the possibility that the reconstitution of a TAP1- and TAP2-deficient cell line recognition by CTL following transfection only with wild-type TAP1 cDNA reflects translocation of peptides into the ER by a TAP1 homodimer. First, there is no evidence that transfection of T2 cells with only TAP1 restores Ag presentation, or for TAP1 homodimers in TAP-deficient cell lines following transfection with TAP1. It is likely that poorly functional TAP1 in SCCHN cells binds TAP2 with lower affinity. Thus, transfection with wild-type TAP1 may not necessarily up-regulate TAP2 expression, but rather may replace the endogenous TAP1 molecules due to enhanced binding to the endogenous TAP2.

Our in vitro findings have several implications. First, the lack of CTL recognition of SCCHN cells, despite the expression of the restricting HLA class I allospecificity and the TA recognized by CTL, suggests that detection of HLA class I Ag with Ab-based assays in malignant cells does not prove that these molecules present peptides to CTL. This mechanism may account for disease progression and/or recurrence in patients with malignant disease despite HLA class I Ag expression and the development and/or presence of a TA-specific CTL response (39). Second, the lack of correlation between HLA class I Ag expression by malignant cells and that of HLA class I Ag–peptide complexes emphasizes the need to test malignant lesions from patients treated with T cell-based immunotherapy not only for expression of HLA class I Ag but also and more importantly for expression of the targeted HLA class I Ag–peptide complex on malignant cells. Lastly, the resistance to CTL recognition of SCCHN cell lines with down-regulation of some APM components provides a potential mechanism for the association between APM component down-regulation, which is frequently found in SCCHN, and poor clinical course of the disease (34, 40). If our interpretation is correct, restoration of APM component expression in SCCHN lesions, which can be induced by intralesional administration of IFN-γ, is likely to have a positive impact on the clinical course of the disease as well as on the outcome of T cell-based immunotherapy. This possibility is supported by the improved immune recognition and reduced tumor incidence in mice when endogenous TAP1 expression is up-regulated upon IFN-γ treatment (41). Although deleterious side effects, such as toxicity, of this therapy have not been observed (42), inhibition of NK-mediated tumor recognition and lysis could result. Murine and human in vivo studies will be necessary to clarify these issues.

Disclosures

The authors have no financial conflict of interest.

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 a FAMRI Clinical Investigator Award and National Cancer Institute Grant R01CA110249.

  • ↵2 Address correspondence and reprint requests to Dr. Robert L. Ferris, The Hillman Cancer Center Research Pavilion, 5117 Centre Avenue, Room 1.19d, Pittsburgh, PA 15213. E-mail address: ferrisrl{at}upmc.edu

  • ↵3 Abbreviations used in this paper: TA, tumor Ag; SCCHN, squamous cell carcinoma of the head and neck; APM, Ag-processing machinery; LMP, low-m.w. protein; β2m, β2-microglobulin; VV, vaccinia virus; rVV, recombinant VV; RT, room temperature; ER, endoplasmic reticulum; MOI, multiplicity of infection.

  • Received October 27, 2004.
  • Accepted January 10, 2006.

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