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[Cancer Research 59, 4642-4650, September 1, 1999]
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[Cancer Research 59, 4642-4650, September 15, 1999]
© 1999 American Association for Cancer Research

Immunology

Small Cell Lung Carcinomas Express Shared and Private Tumor Antigens Presented by HLA-A1 or HLA-A21

Koichi Yamazaki, Gina Spruill, Joseph Rhoderick, Julie Spielman, Niramol Savaraj and Eckhard R. Podack2

Departments of Microbiology and Immunology [K. Y., G. S., J. R., J. S., E. R. P.] and Medicine [N. S.], University of Miami School of Medicine, Miami, Florida 33101


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Evaluation of Results and...
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Evaluation of Results and...
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor progression in vivo requires evasion of an effective immune response. Tumors have evolved various strategies for immune avoidance (1 , 2) . Immunosuppressive tumors, such as gliomas, produce or stimulate production of factors including transforming growth factor ß (3) and IL3 -10 (4) that suppress or deviate a cellular cytotoxic immune response to the tumor. Immunogenic tumors represented by melanomas and renal cell carcinomas provoke an immune response that, however, does not proceed to tumor cell lysis, in part because of the expression of death ligands, such as Fas-L (5) or other death ligands (6 , 7) . Another large group of tumors that includes lung carcinomas avoid an immune response by reducing their immunogenicity through the down-regulation of MHC class I molecules (8, 9, 10) or by blockade of antigen processing or peptide transport for MHC class I loading (9, 10, 11) . Individual tumors may use several components of these strategies concurrently.

Costimulatory molecules for T-cell activation, such as B7.1 and B7.2, are highly expressed on activated APCs. Transfection of B7.1 or B7.2 and expression in tumor cells has been shown to increase their immunogenicity and mediate their in vivo rejection in experimental animals (12 , 13) as well as facilitate CTL generation against melanoma in vitro (14 , 15) .

A number of tumor antigens have been identified for immunogenic tumors such as melanoma (16, 17, 18, 19, 20, 21, 22, 23) , but little is known about tumor antigens in low or nonimmunogenic tumors, such as lung tumors, and their MHC presentation (24, 25, 26) . To begin to identify tumor antigens on this large group of human tumors, it is necessary to devise methods to render these tumors antigenic and to define MHC restriction elements for tumor peptides. MHC restriction of CTL responses to tumor antigens in humans has been studied extensively in melanoma, and many of the identified tumor antigens can be presented by HLA-A2 and HLA-A1 (17, 18, 19, 20, 21 , 27, 28, 29, 30) . The frequency of HLA haplotype expression is thought to reflect their use for directing T-cell responses to viral and tumor-derived peptide recognition (31) . HLA-A1 and HLA-A2, expressed with high frequency in the Caucasian population, therefore, were chosen in this study to survey the presence of tumor antigens on SCLC cells and their restriction by HLA-A1 or HLA-A2.

CD8+ CTL responses to tumors are thought to be important for tumor rejection. MHC class I-restricted, peptide-specific CD8+ CTLs are also required for the discovery and analysis of tumor antigens (17, 18, 19 , 21, 22, 23 , 29) . However, CD8+ CTL activation is a complex process, usually involving at least three cell types in addition to the tumor cells, i.e., APC, CD4+ helper T cells, and CD8+ CTLp (32, 33, 34) . The role of the APC is to present peptide via MHC classes I and II and to provide appropriate costimuli such as B7 (32 , 35 , 36) , whereas CD4+ helper T cells have recently been shown to induce B7 expression on APCs through CD40-Ligand/CD40 interaction (37, 38, 39, 40) . CD4+ helper T cells, in addition, produce IL-2 and other cytokines that facilitate CD8+ CTL expansion. The complexity of CD8+ CTL induction is further compounded by the potential of APCs to induce tumor-specific tolerance (41) and of CD4+ T cells to be suppressive (42 , 43) . We therefore sought to define a less complex system for the generation of MHC class I-restricted CD8+ CTLs specific for non- or low immunogenic tumors. As described in this report using transfected SCLC cells and purified CD8+ T cells without additional cells in primary stimulation, we were able to generate SCLC-specific CTLs and to demonstrate the presence of shared antigens that can be restricted by HLA-A1 and HLA-A2.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Evaluation of Results and...
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, cDNAs, and Transfection
Human SCLC cell lines (SCLC#2 and SCLC#7) and human lung adenocarcinoma cell lines (AD#100 and AD#101) were established as described (44) . The human melanoma cell line, MEL#113, was obtained from Dr. Levy of the Department of Microbiology and Immunology. K562 was provided by American Type Culture Collection. All cells were cultured in IMDMEM medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated FCS (Life Technologies, Inc.). Lung carcinoma cell lines and melanoma cell lines were maintained as monolayer cultures and passaged by short trypsinization with 0.05% trypsin plus EDTA (Life Technologies, Inc.) as required.

HLA-A0101 (A1) and HLA-A0201 (A2) cDNAs were kindly provided by Dr. Peter Parham (Stanford University, Palo Alto, CA). They were cloned into the eukaryotic expression vector, pBCMGSNeo (45) , or a new double expression vector, B45NeoCM, constructed from B45Neo (46) to express two genes by two different promoters, the cytomegalovirus promoter and the mouse metallothionein promoter.4 Human B7.1 cDNA was generated by reverse transcription-PCR with primers that amplified the cDNA between the ATG codon of the leader peptide and the termination codon (47) . The PCR product was cloned into the eukaryotic expression vector, pBCMGHis (48, 49, 50) , or the double expression vector, B45NeoCM. Human ICAM-1 was kindly provided by Dr. Lewis L. Lanier (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA) in a hygromycin-selectable expression vector, SRa296-hygro. The cDNAs were used to transfect SCLC#2, SCLC#7, AD#100, AD#101, and MEL#113 by Lipofectin (Life Technologies, Inc.). Transfected cells were selected with 1 mg/ml of G418 (Life Technologies, Inc.) for pBCMGSNeo or B45NeoCM, with 5–10 mM of L-Histidinol (Sigma Chemical Co., St. Louis, MO) for pBCMGHis, or with 800 µg/ml of hygromycin B (Calbiochem-Novabiochem Corporation, La Jolla, CA) for SRa296-hygro for at least 2 weeks. Cells highly expressing HLA-A1, HLA-A2, B7, or ICAM-1 were sorted on a FACStar flow cytometer (Becton Dickinson, Mountainview, CA) and expanded to cell lines.

Antibodies
Mouse anti-human CD8 monoclonal antibody OKT8 (IgG2a) was purified from hybridoma supernatants, followed by protein G affinity chromatography. Monoclonal antibodies against HLA-A1, A36 and HLA-A2, A24 (IgM) were purchased from One Lambda (Canoga Park, CA) and used diluted at optimal concentration. Phycoerythrin-labeled anti-BB1/B7 antibody (B7-PE) was purchased from Becton Dickinson. Biotin-labeled anti-human CD54 (ICAM-1-Biotin) was purchased from Southern Biotechnology Associates (Birmingham, AL). Secondary antibodies for fluorescence-activated cell sorting included FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-mouse IgM (Boehringer Mannheim, Indianapolis, IN) at a dilution of 1:40 and streptavidin-PerCP (Becton Dickinson) at a dilution of 1:10. Microbeads conjugated with goat anti-mouse IgG (Miltenyi Biotec, Sunnyvale, CA) were used as secondary reagent for magnetic cell sorting.

Immunofluorescence Phenotyping of MHC Class I Molecules, B7.1 and ICAM-1
For surface expression of HLA-A1 and HLA-A2, 5 x 105 tumor cells were incubated with optimal concentrations of anti-HLA-A1, A36 or anti-HLA-A2, A24 monoclonal antibody or isotype-matched control antibody for 30 min at 4°C, followed by incubation with FITC-conjugated goat anti-mouse IgM for 15 min at 4°C. For surface expression of B7.1, 5 x 105 tumor cells were incubated with optimal concentrations of B7-PE or isotype-matched control antibody for 30 min at 4°C. For surface expression of ICAM-1, 5 x 105 tumor cells were incubated with optimal concentrations of ICAM-1-Biotin or isotype-matched control antibody for 30 min at 4°C, followed by incubation with streptavidin-PerCP for 15 min at 4°C. The cells were washed, resuspended in PBA [PBS supplemented with 0.5% BSA (Sigma, St. Louis, MO) and 5 mM EDTA (Sigma), pH 7.2], and analyzed by a Becton Dickinson FACScan flow cytometer. For double or triple staining of HLA-A1 or HLA-A2, B7, and ICAM-1, cells were stained in the order of HLA-A1 or HLA-A2, B7, and ICAM-1.

HLA Typing
HLA typing of tumor cells or of PBLs from normal healthy volunteers was done in the Division of Transplantation Surgery, University of Miami (51) .

Isolation of CD8+ T Cells
PBMCs were obtained from peripheral blood from two normal healthy volunteers. Heparinized samples were diluted with PBS, overlaid onto Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden), and centrifuged at 1400 rpm for 30 min. The lymphocyte-rich layer at the interface was then washed three times with PBS and resuspended in PBS to stain for isolation of CD8+ T cells. CD8+ T cells were obtained by magnetic cell sorting (52) . Briefly, PBMCs were labeled with mouse anti-human CD8 monoclonal antibody OKT8 for 15 min at 4°C, followed by incubation with microbead-conjugated goat anti-mouse IgG for 15 min at 4°C in PBA. Cells were immediately applied to a MACS separation column type AS (Miltenyi Biotec), which was placed in the separator. The quality of the separation was subsequently determined by flow cytometry after labeling with FITC-conjugated goat anti-mouse IgG. It is important to obtain highly purified CD8+ T cells and eliminate dimly CD8+-positive NK cells by using low concentrations of anti-CD8 antibody (1:4000) to avoid overgrowth of cultures by NK-type cells.

Establishment of CTL Lines in Microcultures
Various numbers (103, 3 x 103, and 104) of highly purified, NK (CD8+dim)-depleted, CD8+ T cells were mixed in 96 round-bottomed microwells (Costar, Cambridge, MA) with 300 tumor cells, irradiated with 12,000 rads from a Cobalt source as stimulator cells in 160 µl of complete medium prepared from IMDMEM medium supplemented with 10% FCS, 50 µM of ß-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA), and 50 µg/ml of gentamicin (Life Technologies, Inc.), and incubated at 37°C in 5% CO2. IL-2 (Hoffman-La Roche, Nutley, NJ), at a final concentration of 25 IU/ml, and IL-4 (Genzyme, Cambridge, MA), at a final concentration of 50 IU/ml, were added on day 5 (final volume, 200 µl). The microcultures were restimulated on days 8 and 15 by replacing 100 µl of fresh complete medium containing IL-2 (50 IU/ml), IL-4 (100 IU/ml), 300 irradiated tumor cells, and 105 allogeneic or autologous PBLs, irradiated with 4000 rads, as feeder cells. Proliferating cells were transferred into 96 flat-bottomed microwells (Costar) on days 14 to 19. When the number of the cells reached approximately 3 x 105/well, they were transferred into 24-well plates (Costar). On day 20, cytotoxic activity of the cells was tested against wild-type, HLA-matched, and HLA-mismatched tumor cells and K562 by 4-h Cr-release assays. Long-term culture of CTLs was achieved by repeating restimulation as above.

CTL Cloning
Expanded CTL lines were seeded at 3, 1, and 0.3 cells/well in 200 µl of complete medium containing IL-2 (25 IU/ml), IL-4 (50 IU/ml), 300 irradiated tumor cells, and 105 irradiated allogeneic PBMCs in 96 round-bottomed microwells. The microcultures were restimulated on days 8 and 15 by replacing 100 µl of fresh complete medium containing IL-2 (50 IU/ml), IL-4 (100 IU/ml), 300 irradiated tumor cells, or 105 irradiated allogeneic PBMCs. In the third week, cytotoxic activity of the cells was tested against wild-type, HLA-matched, and HLA-mismatched SCLC cells and K562 by Cr-release assays.

Cytotoxicity Assays
Target tumor cells were labeled with Na251CrO4, and 100-µl aliquots containing 5 x 103 labeled tumor cells were distributed into 96 round-bottomed microwells containing 100 µl of medium alone or 5 x 104 CTLs in medium to result in an E:T ratio of 10:1. To analyze MHC class I specificity of tumor-specific CTLs, at least 1.5 x 105 effector cells were required. The plates were then centrifuged for 30 s at 200 x g and incubated for 4 h at 37°C in 5% CO2. The plates were centrifuged again for 5 min at 200 x g, and 100 µl of supernatant were collected and counted in a gamma counter (LKB-Wallac RiaGamma; Wallac Oy, Finland). The percentage of 51Cr-specific release was calculated as follows:

where ER was the observed experimental 51Cr release, SR was the spontaneous release measured by incubation of 5 x 103 labeled cells in 200 µl of medium alone, and MR was the maximum release obtained by adding 100 µl of 2% Triton X-100 (Sigma) to 100 µl of the target cells. The spontaneous release of most target cells ranged between 10 and 15% of the maximum release.


   Evaluation of Results and CTLp Frequency
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Evaluation of Results and...
RESULTS
 DISCUSSION
 REFERENCES
 
Specific Lysis.
Values of specific lysis exceeding three times the SD of the spontaneous release were considered significant lysis in the whole study. For example, if spontaneous lysis was 10% with 3% SD, then specific lysis by CTL had to be >19% to be considered as significant. In most experiments, specific lysis exceeded spontaneous lysis by 10% or more.

MHC Restriction of Lysis and Specificity.
Differences in lysis observed with different tumor targets, i.e., wild-type, A1-, or A2-transfected, were evaluated using the Student t test. We defined a 30% difference in the specific lysis of different targets as significant. For example, A1-restricted CTLs are expected to lyse A1-transfected targets at least 1.3 times better than the A2 or wild-type targets. Experiments were set up in triplicate, and a significant difference in the lysis (P < 0.05) of different target cells was observed in most experiments.

Allogeneic and Promiscuous CTLs Are Defined as CTLs That Lyse Wild-Type and Transfected SCLC Cells Equally Well and at Least 10% Above Spontaneous Release.
Nonspecific or noncytotoxic cells are those that lyse only K562 or none of the target cells used in the cytotoxicity assay.

Costimulatory Molecules.
To compare the costimulatory efficacy of MHC class I molecules with B7.1 and ICAM-1, 104 CD8+ T cells were stimulated and restimulated with 300 irradiated wild-type or transfected SCLC cells. More than 150 wells for each condition were set up in 20 experiments with CD8+ T cells from A1- and A2-positive volunteers. The significance of the difference between using different costimulators was evaluated on the basis of the Student t test.

CTLp Analysis.
For CTLp analysis, the percentage of negative wells in cytotoxicity assays was plotted on a logarithmic scale against the number of responder CD8+ T cells plated at the beginning of the experiment on a linear scale (53 , 54) . Linear regression analysis of number of responder cells/well against log percentage of negative wells was then performed, and the frequency of CTLp was determined on the basis of the Poisson distribution (53) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Evaluation of Results and...
 RESULTS
DISCUSSION
 REFERENCES
 
Partial MHC Class I Matching of Stimulator Tumor Cells and Responder CD8+ T Cells Generates Tumor-specific, MHC Class I-restricted CTLs.
HLA-A1 and HLA-A2 are frequently expressed MHC alleles in the Caucasian population and are effective in presenting tumor peptides to CD8+ T cells. A1 and A2, therefore, are ideal molecules to test the presentation of tumor peptides in tumors with as yet undefined tumor antigens and restriction elements. To affect optimal CTL stimulation by tumor cells, high expression of matched HLA and costimulatory molecules may be necessary, especially for poorly immunogenic tumors such as lung tumors.

SCLC tumor cells frequently show low expression of MHC class I molecules. This was observed also for the cell lines used in this study; HLA-A2 was detected in SCLC#7 at a very low level, whereas HLA-A1 was not detected in any of the tumor cell lines used in this study. Expression of high levels of HLA-A1 or HLA-A2 on tumor cells was achieved by transfection of the tumor cells with HLA-A1 or HLA-A2 cDNA.

Singly transfected tumor cells expressing HLA-A1 or HLA-A2 or B7.1 (CD80) or ICAM-1 (CD54), double transfectants expressing HLA-A1 and B7.1, HLA-A2 and B7.1, HLA-A1 and ICAM-1, or HLA-A2 and ICAM-1, and triple transfectants expressing HLA-A1, B7.1, and ICAM-1, or HLA-A2, B7.1, and ICAM-1 were prepared from the two SCLC cell lines, SCLC#2 and SCLC#7. The transfectants were used for both purposes, as stimulator cells for mixed CD8+ T-cell tumor cell cultures and as target cells for cytotoxicity assays. Fig. 1Citation shows expression levels of the transfected cDNAs HLA-A1, HLA-A2, B7.1, and ICAM-1 on SCLC#2 and SCLC#7 by flow cytometry. Neither wild-type SCLC#2 nor SCLC#7 expresses HLA-A1. SCLC#7 is HLA-A2 positive by HLA typing, but its expression level of HLA-A2 was much lower than that of HLA-A2 on PBLs of a healthy volunteer (not shown). After transfection of tumor cells with HLA-A1 or HLA-A2, their expression levels increased to the levels found on PBLs. Wild-type SCLC#2 expressed very low levels of ICAM-1 but no B7.1, whereas wild-type SCLC#7 expressed neither B7.1 nor ICAM-1. However, after transfection with B7.1 or ICAM-1, their expression levels increased, as shown in Fig. 1Citation . Double and triple transfectants were made by transfection and sorting for the positive population and then transfecting with the next cDNA. Almost 100% of the cells expressed HLA-A1 or HLA-A2, B7.1, and/or ICAM-1 in each double or triple transfectant, as determined by two- and three-color flow cytometric analysis (not shown). HLA-A2 expression levels were very similar on single, double, or triple transfectants of SCLC#7 (Fig. 1)Citation . However, HLA-A1 expression levels on the triple transfectant SCLC#2-HLA-A1-B7-ICAM-1 was somewhat lower than on the other SCLC#2 transfectants, despite selection pressure. We were unable to generate stable HLA-A1 expressing double or triple transfectants with SCLC#7. After initial HLA-A1 expression, the cells invariably lost their expression within two to three weeks after transfection, even when sorted for high-level HLA-A1 expression and maintained under selection pressure. Similarly, HLA-A2 expression on double and triple transfectants of SCLC#2 was unstable for reasons that are not clear. Therefore, the series of SCLC#2-HLA-A1 transfectants were used as stimulators to generate HLA-A1-restricted CTLs and the series of SCLC#7-HLA-A2 transfectants as stimulators to generate HLA-A2-restricted CTLs.



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  		Fig. 1. Analysis of wild-type and transfected SCLC#2 (A) and SCLC#7 (B) by flow cytometry. Tumor cells were stained with anti-HLA-A1 or anti-HLA-A2 antibody, followed by FITC-conjugated goat anti-mouse IgM and anti-B7-PE or by anti-ICAM-1-Biotin and streptavidin-PerCP and analyzed by flow cytometry. Black histograms, samples stained with specific antibodies; white histograms, samples stained with isotype immunoglobulin control. The specificity of the antibody is to the antigen noted inside the box; the transfectant used is noted above the box.

 
Highly purified, HLA-A1-positive CD8+ T cells were stimulated with irradiated wild-type SCLC#2, SCLC#2-B7, SCLC#2-HLA-A1, or SCLC#2-HLA-A1-B7. During primary culture, only tumor cells and highly purified CD8+bright T cells were present. The presence of other cells including CD8+dim during primary culture diminished tumor specificity and HLA-A1 restriction while increasing nonspecific proliferation. After 7 and 14 days, the proliferating CD8+ T cells were restimulated with the same transfectants used originally, together with irradiated autologous or allogeneic PBLs. During secondary and tertiary stimulation, feeder cells were required to enhance proliferation of tumor-specific CTLs. Cytotoxic activity toward several target cells was tested in triplicate on the 20th day by Cr-release assay at an E:T ratio of 10:1. Wells stimulated with SCLC#2-HLA-A1 or SCLC#2-HLA-A1-B7 proliferated sufficiently to generate enough cells for Cr-release assays, but wells containing wild-type SCLC#2 as stimulators frequently did not have enough cells for assay. To assess the specificity of the CTLs generated, their cytotoxicity was assessed routinely with four targets: against the HLA-A1-transfected, matched tumor cells used as stimulator; against mock-transfected, wild-type tumors, against the mismatched HLA-A2 transfectants; and against the NK target K562.

The stimulation of A1-matched CD8+ T cells with HLA-A1-transfected SCLCs is expected to produce HLA-A1-restricted CTLs, if HLA-A1 can serve as a restricting element for tumor peptides and if CD8+ CTLp with appropriate specificity are present. Because allogeneic responses to unmatched HLA alleles will also occur in this system, it is critical to distinguish A1 (or A2 when applicable) restricted lysis from allogeneic or other CTL responses. This is particularly important because the microcultures may have several clonal specificities in each well, including the desired A1 (or A2)-restricted CTLs, allogeneic CTLs, or promiscuous CTLs. The assay system is designed to distinguish between HLA-A1 (or A2)-restricted CTL responses and other allogeneic or nonspecific responses by analyzing CTL activity against the four targets as noted above. Three types of specificities were observed in the analysis of CTL microcultures and were defined as follows. HLA-A1 (or A2)-restricted CTLs are required to lyse HLA-matched SCLC cells at least 1.3 times better than wild-type SCLC cells or HLA-mismatched SCLC cells (Fig. 2Citation , arrows). Allogeneic or promiscuous CTLs are defined as CTLs that lyse wild-type and HLA-transfected SCLC cells equally well. Nonspecific or noncytotoxic cells are those that either lyse only K562 or none of the target cells used in the cytotoxicity assay. In Fig. 2Citation , the results of a representative experiment are shown comparing the effects of stimulator cell transfection with A1 and B7 on the specificity of the CD8+ CTL response. When wild-type SCLCs were used as stimulators, only three of six wells proliferated sufficiently to allow analysis. Of the three CTL microcultures generated with wild-type SCLCs, none fulfilled the criteria for A1 restriction (Fig. 2Citation , left panel). A1 transfection increased CTL expansion during culture, and one of six wells (Fig. 2Citation , middle panel, arrow) lysed A1-transfected targets with higher specificity than other targets. Double transfection of SCLCs with A1 and B7 further increased cytotoxicity and frequency of A1 restriction (arrows).



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  		Fig. 2. Specificity of HLA-A1-positive CD8 CTLs after stimulation with mock-transfected or HLA-A1/B7.1-transfected tumor cells. Six wells were used for each tumor stimulator, SCLC# 2 (mock), SCLC#2-A1, and SCLC#2-A1-B7.1 for stimulation of CD8 T cells as described. Cytotoxicity was examined on day 20 of culture. The E:T ratio was 10:1 and in 4-h assays against the four targets indicated at the abscissa of the graph, SCLC#2 (mock), SCLC#2-A1, SCLC#2-A2, and K562. The specific cytotoxicity of individual wells is plotted. Arrows, wells that fulfill criteria for MHC restriction by A1, defined as 1.3-fold or higher lysis in comparison with mock-transfected or A2-transfected SCLCs.

 
The cumulative results of 480 wells analyzed against four targets in 20 similar experiments using A1 or A2 as restriction element for SCLCs and their statistical analyses are summarized in Fig. 3Citation . In the absence of HLA transfection, wild-type and B7.1-transfected tumor cells produced only allogeneic responses, lysing untransfected and A1- or A2-transfected SCLCs to a similar extent. HLA-A1-transfected stimulators increased the frequency of HLA-A1-restricted CTLs significantly (P < 0.05). B7 transfection of SCLCs, together with HLA, had two effects. It increased the frequency and level of cytotoxicity in all wells, and significantly, it increased the frequency of A1-restricted CTLs (P < 0.05). Similarly, SCLC#7-HLA-A2 stimulation helped to generate HLA-A2-restricted CTLs (P < 0.05 versus SCLC#7 and SCLC#7-B7). Double transfection of SCLC#7-HLA-A2-B7 increased the frequency of HLA-A2-restricted CTLs, compared with HLA-A2 transfection alone, although the value was not significant (P = 0.075).



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  		Fig. 3. Frequency of specificity of CD8 CTLs after stimulation with wild-type, B7.1-transfected, class I-transfected, or double transfected SCLCs. For stimulation in the left panel, SCLC#2 and HLA-A1 was used; in the right panel, SCLC#7 and HLA-A2. The data from 20 experiments are calculated, and the frequency of wells containing MHC-restricted CTLs versus nonrestricted CTLs is shown in dependence of the transfection of the stimulator tumor cell. Statistical significance is indicated by Ps. Culture conditions were as described in "Materials and Methods."

 
The criteria used to define MHC restriction used are conservative and are likely to underestimate the true frequency. For instance, CTLs that lyse wild-type and HLA-A1-transfected SCLCs equally well may contain both allogeneic CTLs and A1-restricted CTLs. They would, however, not be counted as A1 restricted by the definition used because the allogeneic specificity cannot be distinguished from A1 restriction without cloning.

To confirm specificity and HLA-A1 or HLA-A2 restriction of CTLs, single-cell cloning experiments using limiting dilution techniques were carried out. Expansion of clones from 3, 1, or 0.3 cells/well required 3 weeks of culture with periodic restimulation. All clones were analyzed for specificity on a panel of target cells as above. An example of one cloning experiment is shown in Fig. 4ACitation . Eight clones generated by limiting dilution and culture for 3 weeks were compared with the specificity of the uncloned parent A1-specific CTL line containing also lytic activity for K562. During the cloning period, cytotoxic activity decreased, but MHC class I-restricted specificity was increased. Thus, after cloning, few allogeneic or promiscuous CTL clones were present, despite their presence at the initiation of the cloning procedure. A large fraction of clones were HLA-A1-restricted CTLs with high specificity for HLA-A1 and variable cross-reactivity for HLA-A2 but low activity for K562 or wild-type SCLCs (Fig. 4B)Citation . The cumulative data of 48 clones generated from >10 A1-restricted lines indicate that an equal number of clones retained A1 specificity or lost their activity for any of the target cells tested, respectively. Only very few clones retained allogeneic specificity.



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  		Fig. 4. Frequency of specificity of CTL clones generated by limiting dilution analysis. A, one HLA-A1-restricted CTL line was diluted and seeded at 3, 1, and 0.3 cells/well. together with irradiated SCLC#2-HLA-A1 and irradiated allogeneic PBMCs in 96 round-bottomed microwells. The microcultures were restimulated on days 8 and 15. On day 20, all clones that proliferated were analyzed for specificity on the panel of target cells as indicated by Cr-release assays. B, cumulative frequency of HLA-A1-restricted CTLs, allogeneic CTLs, and noncytotoxic cells obtained from the analysis of 48 clones obtained in a similar way as in A.

 
The data suggest that continuous culture and restimulation of tumor-specific CTLs with HLA-matched SCLC cells for longer periods of time increased the specificity and frequency of MHC class I-restricted CTLs over that of allogeneic or promiscuous CTLs (Fig. 4)Citation . However, prolonged culture of CTLs also resulted in a gradual decrease of cytotoxicity and a decreased rate of proliferation (not shown).

Shared and Private HLA-A1- and HLA-A2-restricted Tumor Antigens Expressed by Two Unrelated SCLC Lines.
The development of immunological tumor therapy is facilitated by the presence of shared tumor antigens expressed by the majority of tumors of the same type obtained from different patients. It was therefore important to determine whether A1-restricted CTLs could lyse unrelated SCLC lines after transfection with A1. Two unrelated A1- and A2-transfected SCLC lines were used as targets. As additional controls, non-SCLC and melanoma cells were transfected with HLA-A1 and used as targets. Thirty-six of 47 individual, A1-restricted CTL lines (76.6%) generated with SCLC#2-HLA-A1 also lysed the unrelated SCLC#7-HLA-A1, indicating the presence of one or more shared peptide epitopes presented by A1 (Table 1)Citation . Eleven of the 47 CTL lines lysed only the A1-transfected SCLC#2 line that was used for CTL generation but not SCLC#2, suggesting that these CTLs recognized A1-restricted, private antigens not expressed by unrelated SCLCs. The reciprocal experiments showed comparable results in that 29 of 40 CTL lines (72.5%) lysed the two unrelated A1-transfected tumors. Fewer A2-restricted CTL lines were tested, but three of four A2-restricted, CTLs tested detected shared A2-restricted antigens in an unrelated line (Table 2)Citation . In contrast, only one of seven A1-restricted SCLC-specific CTL lines lysed other A1-transfected tumor types such as non-SCLCs and a melanoma (Table 3)Citation . Apparently, the majority of SCLC-specific CTLs recognize antigens not found on other tumors. These data also exclude the possibility that HLA-A1- or HLA-A2-restricted CTLs are specific for peptides derived from the bovine papilloma virus expression vector that was used for the expression of HLA-A1 and other cDNAs in all tumor lines used.


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  		Table 1 Shared antigens presented by HLA-A1 in the two unrelated lines, SCLC#2 and SCLC#7

 

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  		Table 2 Shared antigens presented by HLA-A2 in two unrelated lines, SCLC#2 and SCLC#7

 

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  		Table 3 Infrequent antigen sharing between A1-transfected SCLC, non-small lung adenocarcinomas, and melanoma

 
MAGE 1 and MAGE 3 is expressed by several tumors, including melanoma and lung tumors, and their peptides are presented by A1 (18 , 29) . However the HLA-A1-restricted CTL line in Table 3Citation that lysed several A1-transfected tumors did not lyse MAGE-peptide pulsed A1-positive B cells (data not shown).

Role of B7.1 and ICAM-1 in CTL Generation.
ICAM-1 interaction with LFA-1 is thought to deliver an important first signal for T-cell activation, rendering T cells permissive to subsequent signaling by other molecules (55) . Because SCLC lines express very low to undetectable levels of ICAM-1, we determined the effect of ICAM-1 transfection on CTL generation of stimulator tumor cells together with B7.1 transfection (Fig. 5)Citation . ICAM-1 cotransfection with MHC class I had only a modest effect on the frequency of MHC class I-restricted CTL generation when compared with B7.1 plus MHC class I. When ICAM-1 was cotransfected in triple transfectants with MHC class I and B7.1, it reduced the frequency of CTL generation in comparison to the double transfectants of B7.1 and MHC class I, although the effect was not statistically significant. The effect of ICAM-1 expression, although detectable, did not seem limiting or essential in this system. In contrast, the costimulatory effect of B7.1 is significant if the signals derived from B7.1 are presented by the same cell as MHC class I (in cis). In the trans situation, when SCLC#2-HLA-A1 (or -A2) and SCLC#2-B7 were mixed and used as stimulators, the frequency of CTL generation was reduced in comparison with the effect of double transfectants of B7.1 and HLA-A1 (or HLA-A2) on the same cell (P < 0.01).



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  		Fig. 5. Effect of B7.1 and ICAM-1 transfection on the frequency in generation of HLA-restricted CTLs. A1 and A2 responder CD8 cells were stimulated with class I-matched tumor transfectants and with double and triple transfectants, as indicated on the abscissa of the graph. In addition, stimulation was carried out by mixing class I-transfected and B7.1-transfected cells with CD8 responder cells. To evaluate MHC restriction, all wells were analyzed against four targets at an E:T ratio of 10:1; the four targets were SCLC#2 (mock), SCLC#2-HLA-A1, SCLC#2-HLA-A2, and K562 (left panel) or SCLC#7 (mock), SCLC#7-HLA-A1, SCLC#7-HLA-A2, and K562 (right panel). Results are means of 12 experiments for HLA-A1 CTL response and eight experiments for HLA-A2 CTL response; bars, SD. Significance values were calculated according to the Student t test. Class I-restricted CTLs lyse the appropriate target at least 1.3 times more than mismatched or wild-type targets.

 
Analysis of CTLp Frequency in CD8+ T Cells of Normal Healthy Volunteer’s PBMCs.
MHC class I-restricted CTLp frequencies were measured by stimulation of T cells with tumors coexpressing HLA-A1 or HLA-A2 with B7.1. Poisson analysis of the number of responder cells generating specific CTLs (Fig. 6Citation ; Ref. 53 ) showed CD8+ CTLp frequencies of 0.45 x 10-4 and 0.38 x 10-4 in the HLA-A1 and HLA-A2 volunteers, respectively.



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  		Fig. 6. CTLp frequencies of CD8+ T cells of two healthy volunteers for SCLC#2-HLA-A1 and SCLC#7-HLA-A2.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Evaluation of Results and...
 RESULTS
 DISCUSSION
REFERENCES
 
Several tumor antigens have been identified in human tumors (16, 17, 18, 19, 20, 21, 22, 23) , allowing their evaluation in immunotherapy. However, most human tumors are poorly immunogenic and remain largely unexplored because of the difficulty of generating CTL responses against these tumors. It is generally accepted that CD8+T cells physiologically are activated by APCs with CD4 help in a pathway known as cross-priming or indirect antigen presentation. Indirect antigen presentation, however, is not easily replicated in vitro and does not lend itself to the discovery of MHC-restricted tumor antigens. It is, therefore, desirable to devise a method of direct antigen presentation that allows the exploration of the important field of tumor antigenicity in poorly immunogenic solid tumors.

In this report, the response of purified human CD8+ T cells to transfected SCLC cells matched at one MHC class I locus is analyzed. We demonstrate the presence of shared, HLA-A1- or HLA-A2-restricted antigens in addition to private, not shared antigens in two unrelated SCLC lines. HLA-A1 or HLA-A2 transfection and expression of tumors are critical for the generation of HLA-A1- or HLA-A2-restricted CTL responses. Single-cell cloning confirmed that microcultures contained the relevant MHC class I-restricted CTLs, along with allogeneic and promiscuous CTLs. Coulie et al. (56) and Mazzocchi et al. (54) showed that CTLp frequencies for autologous melanoma cells ranged from 1/720 to 1/33,000 in PBLs of melanoma patients. It is likely that, in some melanoma patients, expansion of CTLp takes place and may explain the high frequency in some patient samples. CTLp frequencies of 1/24,000 in our study are derived from healthy volunteers and represent the unexpanded, HLA-A1- or HLA-A2-restricted CD8+ CTLp repertoire for the two SCLC lines.

What is the nature of the tumor antigen recognized by the CTLs? About 75% of the MHC-restricted, SCLC-specific CTL lines recognize antigens shared by the two SCLC lines used, whereas the other 25% recognize private antigens. The molecular nature of the antigens is not known presently, and efforts are under way using these CTLs to define the antigen(s). By analogy to melanomas (17 , 20 , 21 , 27 , 28 , 57) , the most likely interpretation of our data would suggest that SCLCs express both shared and private tumor antigens. The shared antigens may represent unmutated embryonal antigens, whereas the private antigens may come from specific mutations.

For the generation of CTLs in the direct antigen stimulation mode, costimulation is very important. Suboptimal costimulation or culture conditions resulted in lower apparent CTLp frequencies and increased frequencies of allogeneic responses. Overgrowth of allogeneic CTLs is not a problem in the culture systems described. This is attributable largely to MHC class I transfection and high level expression by the tumor. Equally important is the omission of APCs and CD4 T cells in primary stimulation, reducing allogeneic stimulation. To obtain MHC-restricted CTLs, it was important to use highly purified CD8 cells and to exclude CD8dim NK cells. IFN-{gamma} (58) or IL-12 had no further effect on CTL generation in our system (data not shown), suggesting that class I expression is not limiting after transfection of the tumor with HLA. Limiting dilution cloning of CTLs confirmed the specificity that had been deduced.

B7.1 expression on the tumor cells, in conjunction with MHC class I, effectively bypassed the need for CD4+ helper T cells in the generation of CD8+ CTLs. The molecular mechanism of CD4+ helper T cells for the generation of CD8+ CTLs has recently been clarified. CD40-Ligand expressed by CD4+ helper T cells interacts with CD40 on APCs and up-regulates B7, which is used as costimulus for CD28 signaling of CD8+ T cells (37, 38, 39, 40) . In agreement with the proposed function of CD40-Ligand, our analysis suggests that overexpression of B7.1 and MHC class I on tumor cells is sufficient to activate CD8+ CTLs directly and to bypass the need for APCs.

Although this study focused on MHC class I-restricted CD8+ CTL responses to SCLCs, we have generated similar data also for HLA-A1 and B7.1-transfected non-SCLCs,5 and we suggest that this concept may be generalizable to other tumors. Our studies indicate that matching of MHC class I alleles between the transfected tumor cells and the responder CD8+ T cells is an important factor for generation of CTLs with defined MHC class I restriction. Although HLA-A1 and HLA-A2 are suitable for this purpose, other alleles could also be used and investigated in a similar strategy. Methods to identify tumor antigens rely on the generation of MHC class I-restricted CTLs. Using partial MHC class I matching and direct stimulation of purified CD8+ T cells opens a novel avenue in this effort.


   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 NIH Grants CA39201 and CA57904. Back

2 To whom requests for reprints should be addressed, at University of Miami School of Medicine, Department of Microbiology and Immunology, P. O. Box 016960 (R138), Miami, FL 33101. Back

3 The abbreviations used are: IL, interleukin; APC, antigen-presenting cell; SCLC, small cell lung carcinoma; CTLp, CTL precursor; ICAM, intercellular adhesion molecule; PBL, peripheral blood lymphocyte; PBMC, peripheral blood mononuclear cell; NK, natural killer. Back

4 Unpublished data. Back

5 Unpublished observations. Back

Received 4/15/99. Accepted 7/21/99.


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