Induction of Human Cytotoxic T LymphocytesSpecific for Prostate-Specific Antigen

Bao-Hua Xue, Yong Zhang, Jeffrey A. Sosman, and David J. Peace*

Division of Hematology/Oncology, Department of Medicine, Loyola University MedicalCenter, Maywood, Illinois

ABSTRACT: Prostate-specific antigen (PSA), a tissue-specific protein expressed by mostadenocarcinomas of the prostate, might be a useful target for T-cell-mediated immunother-apy of prostate cancers. The current study examined whether it is possible to elicit humancytotoxic T lymphocytes (CTL) with specificity for PSA. A synthetic nonamer peptide, cor-responding to residues 146–154 of PSA and containing a canonical HLA-A2-binding motif,was shown to stabilize the expression of HLA-A2 on the T2 antigen-processing mutant cellline. Repeated in vitro stimulation of peripheral blood lymphocytes from a normal HLA-A2+ donor induced CTL with specificity for the PSA 146-154 peptide. The peptide-inducedCTL expressed the CD4− CD8+ cell surface phenotype and were restricted by HLA-A2. Alarge portion of patients with prostate cancer express the HLA-A2 phenotype, implying thatmany prostate cancers might be targeted by HLA-A2-restricted CTL with specificity for thePSA 146–154 epitope. Prostate 30:73–78, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: tumor antigen; in vitro immunization; immunotherapy; class I majorhistocompatibility complex; HLA-A2

INTRODUCTION

Prostate cancer is the most common malignancyand the second most common cause of cancer-relatedmortality in U.S. males [1]. The majority of prostatecancers are not curable by available methods of treat-ment. Immunotherapy regimens might improve thesurvival of patients with advanced locoregional andmetastatic tumors. Most prostatic cancers express tis-sue-specific proteins which potentially can selectivelytarget malignant prostatic cells for lysis by cytotoxic Tlymphocytes (CTL). Prostate-specific antigen (PSA), a34 kD kallikrein-like serine protease, which is exclu-sively produced by ductal and acinar epithelial cellsof the prostate gland in males [2–5], is detected innearly all adenocarcinomas of the prostate [6]. Theo-retically, it might be possible to elicit PSA-specificCTL, capable of lysing prostate cancer cells, for use intumor therapy.

CTL recognize processed peptide antigens pre-sented in association with class I MHC molecules [7].All endogenous cellular proteins potentially can bedegraded, bound as peptides to class I MHC, andpresented at the cell surface for recognition by class I

MHC-restricted CTL [8]. The ability of a peptide toelicit CTL restricted by a particular class I MHC mol-ecule is contingent on the ability of the peptide tobind to the class I MHC molecule. Allele-specific pep-tide sequence motifs have been identified for variousclass I MHC molecules which facilitate the identifica-tion of candidate-binding peptides [9,10]. In vitro im-munization of unprimed T cells with peptides thatcontain defined class I MHC-binding motifs has beenused to elicit CTL restricted by the relevant class IMHC molecule [11–15]. The most prevalent class IMHC allele in the U.S. population is HLA-A2 [16].

In the current study, the amino acid sequence ofPSA was examined for peptide motifs likely to bind toHLA-A2. A synthetic peptide homologue of an iden-tified nonamer motif within PSA was tested for theability to bind to HLA-A2 and to elicit HLA-A2-re-stricted CTL by in vitro immunization.

*Correspondence to: D. J. Peace, Division of Hematology/Oncology,Loyola University Medical Center, 2160 S. First Ave., May-wood, IL 60153.Received 6 June 1995; Accepted 19 July 1995

The Prostate 30:73–78 (1997)

© 1997 Wiley-Liss, Inc.

MATERIALS AND METHODS

Reagents

Synthetic peptides corresponding to PSA residues146-154 and human immunodeficiency virus-reversetranscriptase (HIV-RT) residues 476-484 were syn-thesized and purified by Research Genetics Corp.(Huntsville, AL) [9,17]. Peptides were dissolved insterile water at 2 mg/ml and stored at 4°C. Anti-HLA-A2 monoclonal antibody was produced as su-pernatant from the BB7.2 hybridoma (American TypeCulture Collection, Rockville, MD). FITC-conjugatedgoat anti-mouse IgG F(ab8)2 fragment was purchasedfrom Sigma Chemical Co. (St. Louis, MO). Humanrecombinant interleukin-2 (rIL-2) was a gift from Hoff-man La Roche, Inc. (Nutley, NJ). FITC-conjugatedanti-CD8 and phycoerythrin-conjugated anti-CD4monoclonal antibodies were purchased from BectonDickinson Co. (San Jose, CA).

Cell Lines

Human tumor cell lines SW480, Capan-1, A549,and PANC-1 were obtained from the American TypeCulture Collection. Sublines of the DU-145 and PC-3prostate cancer cell lines were provided by Dr. G.Wilding (University of Wisconsin, Madison, WI). Themarmoset Epstein Barr virus-producing cell lineMCU-V5 and human lymphoma cell lines K562 andDaudi were provided by Dr. T. Ellis (Loyola Univer-sity, Maywood, IL). T2 is a peptide transport-defi-cient B-lymphoblastoid × T-lymphoblastoid fusioncell line that was generated by Dr. P. Cresswell [18].Human LCL lines were established by incubating 2 ×106 peripheral blood mononuclear cells (PBMC) in 1ml RPMI 1640/10% fetal bovine serum plus 1 ml su-pernatant from the MCU-V5 cell line. Fresh culturemedium was added weekly to stable transformants.

Peptide-Binding Assay

The antigen-processing defective cell line, T2, wasused to assay peptide binding to HLA-A2. T2 cells(5 × 105) were incubated with peptide dissolved inRPMI 1640/10% fetal bovine serum at the designatedconcentration for 18 hr at 37°C. HLA-A2 expressionwas measured by indirect immunofluorescence usingthe murine monoclonal antibody from the BB7.2 hy-bridoma as the first step and FITC-conjugated goatanti-mouse IgG F(ab8)2 fragment as the second step.All incubations were performed at 4°C for 45 min.Cells were analyzed for fluorescence at 495 nm by aFACScan flow cytometer (Becton Dickinson Co.).Fluorescence ratios were determined by dividing themean fluorescence of T2 cells incubated with peptide

by the mean fluorescence of cells incubated withoutpeptide.

In Vitro Immunization of CTL

Peripheral blood (50 ml) was collected in a heparin-ized syringe from a healthy HLA-A2+ donor afterinformed consent. The HLA-A2 phenotype was de-termined by immunofluorescent analysis as de-scribed above. PBMC were separated over Ficoll bygradient centrifugation, washed twice with Hank’sbalanced salt solution (HBSS), and resuspended inRPMI 1640 supplemented with 10% autologous heat-inactivated plasma. Aliquots of cells were used di-rectly or mixed with dimethyl sulfoxide (10%) andfrozen for later use. Cells were plated in 24-well flat-bottom plates (Costar, Cambridge, MA; 5 × 106 cells/well) with PSA 146-154 peptide (20 mg/ml) in a finalvolume of 1.5 ml/well. On day 3, rIL-2 (10 U/ml) wasadded. Seven days later cells were restimulated withpeptide in the presence of irradiated autologousPBMC (2 × 106 cells/well irradiated 2,500 rad) andadditional rIL-2 (10 U/ml). Cells were restimulated inthis manner for eight cycles. In subsequent cycles, cellswere restimulated with peptide and rIL-2 in thepresence of irradiated autologous LCL cells (2,500rad).

Chromium Release Assays

T-cell lines were tested for specific lytic activity instandard 4-hr 51Cr release assays. Target cells wereprepared by incubating cells with Na51CrO3 for 1 hr.Where indicated, target cells were further incubatedfor 2 hr with designated peptide (20 mg/ml) prior touse in chromium release assays. Effector cells wereplated in triplicate with target cells (1 × 104 cells/well)at designated effector:target (E:T) ratios, in 96-wellround-bottom plates (Costar)—final volume 200 ml/well. Supernatant (100 ml) recovered after 4 hr of in-cubation was assayed for gamma irradiation. The per-cent specific lysis was calculated as previouslydescribed [19].

RESULTS

PSA Protein Contains Putative NonamerHLA-A2-Binding Motifs

Naturally processed peptides that bind to HLA-A2with high affinity are predominantly nine aminoacids in length with dominant anchor residues con-sisting of leucine or methionine at position 2 and va-line at position 9 [9]. The primary amino acid se-quence of PSA [17] contains two nonamer segmentswith appropriately aligned dominant anchor residuesat positions 2 and 9 (Table I). The two nonamers each

74 Xue et al.

contain putative secondary anchor residues, accord-ing to the model proposed by Falk et al. [9] (Table I).However, one of the identified nonamers, PSA 151-159, contains a negatively charged aspartic acid atposition 1 which is rarely found in peptides with highaffinity for HLA-A2 [20]. The other nonamer, PSA146–154, does not contain residues that are predictedto inhibit binding to HLA-A2 and shares additionalconsensus residues with peptides that exhibit highaffinity for HLA-A2 (Table I) [20]. Consequently, thePSA 146–154 peptide was selected for further study.

PSA 146–154 Binds to HLA-A2 With High Affinity

The PSA 146–154 peptide was synthesized andtested for the ability to stabilize the expression ofHLA-A2 on the T2-processing mutant cell line. T2cells normally express low levels of HLA-A2, becausethey lack transporter proteins necessary for class IMHC presentation of most endogenously generatedpeptides. When T2 cells are incubated with exoge-nous peptides capable of binding and stabilizingHLA-A2, increased numbers of HLA-A2/peptidecomplexes are generated that are detectable on thecell surface by immunofluorescence [21,22]. The levelof HLA-A2 expression on T2 cells incubated with orwithout PSA 146–154 peptide was measured. ThePSA 146–154 peptide induced increased expression ofHLA-A2 in a concentration-dependent fashion (Fig.1). Peptide concentrations greater than 20 mg/ml con-sistently induced more than a threefold increase inHLA-A2 expression. The expression of HLA-A2 wasenhanced to a much greater degree with the PSA146–154 peptide than with an equivalent concentra-tion of a defined HLA-A2-restricted CTL epitope de-rived from HIV-RT (Fig. 2) [9,23,24].

CTL Specific for PSA 146–154 Peptide can beGenerated by Primary In Vitro Immunization

Recent studies have demonstrated that class IMHC-restricted human CTL can be elicited in vitro by

immunization of peripheral blood lymphocytes withpeptides known to bind to the restricting class I MHCmolecule [12–15]. Experiments were performed to de-termine whether the PSA 146–154 peptide is able toelicit HLA-A2-restricted human CTL in vitro. PBMCfrom an HLA-A2+ donor were stimulated in vitrowith the PSA 146–154 peptide. After repeated cyclesof stimulation, a CTL line was established that ex-pressed the CD4− CD8+ cell surface phenotype(94%) and that specifically lysed T2 cells treated withthe sensitizing PSA 146–154 peptide (Fig. 3). Targetcells that were pretreated with the HIV-RT 476-484peptide or that were not pretreated with any peptidewere not lysed (Fig. 3). The PSA 146–154 peptide-induced CTL lysed targets treated with the sensitiz-ing peptide in a concentration-dependent fashion(Fig. 4). Specific lysis was evident at peptide concen-trations as low as 2 ng/ml. The cytolytic activity of thePSA-specific CTL line was HLA-A2 restricted. ThePSA 146–154 peptide-induced CTL exclusively lysedvarious PSA 146–154 peptide-treated targets that ex-pressed HLA-A2, but not targets that did not expressHLA-A2 (Table II). Neither the NK-sensitive targetcell line, K562, nor the LAK-sensitive target cell line,Daudi, was lysed by the PSA 146–154 peptide-in-duced CTL (Table II).

DISCUSSION

The current study demonstrated that it is possibleto generate human CTL with specificity for PSA, adifferentiation antigen which is expressed by nearlyall prostate cancers. The identified PSA epitope wasrecognized by CTL restricted to the HLA-A2 class IMHC determinant. Approximately 40% of patients

TABLE I. PSA Protein Contains Putative NonamericHLA-A2 Binding Motifs*

Amino acid position

PSA peptide 1 2 3 4 5 6 7 8 9

PSA 146–154 K L Q C V D L H VPSA 151-159 D L H V I S N D V

*Dominant HLA-A2 anchor residues as described by Falk et al., [9]are shown in boldface. Putative secondary anchor residues areunderlined [9,20]. Fig. 1. PSA 146–154 peptide stabilizes HLA-A2 in a concentra-

tion-dependent fashion. T2 cells were incubated overnight with theindicated concentration of PSA 146–154 peptide, then analyzed forthe expression of HLA-A2 as described in Materials and Methods.Data represent the ratio of the mean fluorescent intensity of T2cells incubated with peptide/mean fluorescent intensity of cellsincubated without peptide. The results are representative of twoseparate experiments.

PSA-Specific Cytotoxic T Lymphocytes 75

with prostate cancer exhibit the HLA-A2 phenotype[25] and, thus, potentially can display processed PSA146–154 epitopes in association with HLA-A2 mole-cules on the surface of their tumors. The results in-dicate that HLA-A2+ patients with prostate cancermight be amenable to specific T-cell immunotherapytargeted to the PSA 146–154 epitope. Other PSAepitopes restricted to alternative class I MHC deter-minants also may exist, such that it might be possibleto elicit autologous PSA-specific CTL for the treat-ment of most patients bearing tumors that expressPSA.

The identification of candidate tumor antigens thatcan be recognized by class I MHC-restricted CTL hasbeen greatly facilitated by the elucidation of class IMHC consensus-binding motifs. The PSA 146–154peptide was selected for examination, because it ex-hibited features often found in peptides that bind toHLA-A2 with high affinity. The peptide had an ideallength of nine amino acids with dominant HLA-A2anchor residues at positions 2 and 9 [9]. The peptide

also contained putative secondary anchor residues,notably lysine at position 1 and cysteine at position 4,common to peptides with the highest affinities forHLA-A2 [9,20]. Importantly, the peptide did not con-tain any residues known to impede or prevent pep-tide binding to HLA-A2 [20,26]. Although the predic-tive power of available consensus motifs issubstantial, not all peptides with discernible motifsexhibit significant binding. Empirical testing remainsan important and necessary step in the evaluation ofthe potential for any candidate peptide to bind toclass I MHC molecules and to serve as an epitope forclass I MHC-restricted CTL.

Several methods to assess the binding affinity ofpeptides for class I MHC molecules have been re-

Fig. 3. PSA 146–154 peptide-induced CTL specifically lyse tar-gets treated with the sensitizing peptide. 51Cr-labeled T2 cellswere incubated with PSA 146–154 peptide (closed circle), HIV-RT476-484 peptide (open circle), or no peptide (closed square) asdescribed in Materials and Methods, then used as targets in 4-hr51Cr release assays. Data represent the percent specific lysis oftargets by PSA 146–154 peptide-induced CTL at the indicated E:Tratios. The results are representative of three separate experi-ments.

Fig. 4. PSA 146–154 peptide-induced CTL lyse peptide-treatedtargets in a concentration-dependent fashion. 51Cr-labeled T2 cellswere incubated for 2 hr with the indicated concentration of PSA146–154 peptide, then used in 4-hr 51Cr release assays at an E:Tratio of 3:1. The results are representative of three separate ex-periments.

Fig. 2. PSA 146–154 peptide stabilizes HLA-A2 more effectivelythan HIV-RT 476-484 peptide. T2 cells were incubated overnightwith the indicated peptide (20 µg/ml), then analyzed for the ex-pression of HLA-A2. Data represent the relative fluorescent in-tensity of T2 cells incubated with no peptide vs. PSA 146–154peptide (A) or HIV-RT 476-484 peptide (B). The amino acidsequence of the HIV-RT 476-484 peptide is ILKEPVHGV.

76 Xue et al.

ported [14,20–23]. The T2-binding assay, used in thecurrent study, measures the ability of peptides to sta-bilize ‘‘empty’’ class I MHC molecules on the surfaceof viable cells. The assay reflects the dynamic equi-librium that is established between peptide and classI MHC molecules in living cells and, thus, may beparticularly appropriate for the assessment of immu-nologically relevant peptides. PSA 146–154 peptidebound to and stabilized HLA-A2 with exceptionallyhigh affinity in the T2-binding assay. The PSA pep-tide was much more effective than HIV-RT 476-484,a well-characterized HLA-A2-restricted CTL epitopethat shares the same dominant leucine 2/valine 9 an-chor motif [24]. The observed difference in bindingaffinities of these two peptides underscores the im-portance of nondominant residues in determiningthe relative binding affinity of peptides for class IMHC molecules. In general, the relative binding af-finity of a peptide for class I MHC corresponds to theability of the peptide to elicit specific class I MHC-restricted CTL [27]. However, high-affinity bindingbetween peptide and class I MHC does not ensurethat a particular peptide/MHC complex will be immu-nogenic. The T-cell repertoire may lack or deleteT-cell clones bearing receptors of the appropriatespecificity. The present results have established thatthe human T-cell repertoire includes CTL with recep-tors that specifically recognize PSA 146–154 peptidein association with HLA-A2.

Our findings have potential therapeutic ramifica-tions. Autologous PSA-specific T cells elicited in vitro

might be expanded to large numbers, then adoptivelytransferred in vivo in order to confer an overwhelm-ing specific immune response in tumor-bearing pa-tients. The clinical feasibility of the adoptive transferof specific T-cell immunity has been previously dem-onstrated [28]. Vaccine-based strategies to directlyelicit PSA-specific CTL in vivo also might be feasibleand therapeutically effective. Ongoing studies are ex-amining the conditions wherein HLA-A2-restrictedCTL with specificity for the PSA 146–154 peptide canrecognize and lyse HLA-A2+ prostate cancer cellsthat express PSA protein. Tumors utilize variousstrategies to limit class I MHC-restricted presentationof antigenic epitopes. Malignant cells can reduce theformation and display of class I MHC/peptide com-plexes by decreasing the expression of class I MHCheavy chains or the expression of components of theantigen-processing and transport pathway. Both ofthese mechanisms of attenuated class I MHC expres-sion have been detected in prostate cancer lines [29].In some circumstances, interferon gamma can restoreclass I MHC assembly and expression by prostatecancer cells [29]. Tumors also can reduce the expres-sion of antigenic proteins to evade immunologicalrecognition. The expression of PSA is often attenu-ated in anaplastic and poorly differentiated prostatecancers, but may remain inducible by androgens [30].Ultimately, the coordinated use of biological responsemodifiers and androgens to promote antigen expres-sion and presentation may be able to render prostatecancers most susceptible to specific T-cell immuno-therapy.

Tumor treatment strategies based on the inductionof PSA-specific T-cell immune responses carry therisk of provoking autoimmune toxicity in normal tis-sues. Presumably, toxicity mediated by PSA-specificCTL should be limited to tissues that endogenouslyexpress PSA protein. The risks of autoimmune injurymay be completely obviated in patients who undergoradical prostatectomy prior to immunotherapy withPSA-specific CTL. In such patients, PSA should serveas a de facto tumor-specific marker for residual cancercells. PSA-specific CTL also might be used, without arisk of autoimmune toxicity, to treat females with tu-mors that aberrantly express PSA. Females do notnormally express PSA, however, approximately 30%of adenocarcinomas of the breast express PSA protein[31].

ACKNOWLEDGMENTS

The authors thank Drs. R. I. Fisher and R. Flaniganfor reviewing the manuscript and P. Simms of theLoyola University FACS facility for her expert tech-nical assistance.

TABLE II. PSA 146–154 Peptide-Induced CTL SelectivelyLyse HLA-A2+ Targets Incubated With the PSA

146–154 Peptide*

TumorHLA-A2.1phenotype

Lysis (%)

No peptide Peptide

T2 + 6 66SW480 + 9 38Capan-1 + 7 35A549 - 1 0PANC-1 - -2 -3PC-3 - -2 -3DU-145 - -5 -5K562 - 4 3Daudi - 5 8

*HLA-A2 expression of various tumor cell lines was determined byindirect immunofluorescent staining with the BB7.2 monoclonalantibody. Four-hour 51Cr release assays were performed at an E:Tratio of 10:1. Indicated targets were preincubated with PSA 146–154 peptide (20 mg/ml) for 2 hr. The results are representative oftwo separate experiments.

PSA-Specific Cytotoxic T Lymphocytes 77

REFERENCES

1. Boring CC, Squires TS, Tong T, Montgomery S: Cancerstatistics, 1994. CA 43:7–26, 1994.

2. Li TS, Beling CG: Isolation and characterization of twospecific antigens of human seminal plasma. Fertil Steril24:134–144, 1973.

3. Watt KWK, Lee PJ, M’TimKulu T, Chan WP, Loor R:Human prostate-specific antigen: Structural and func-tional similarity with gene proteases. Proc Natl AcadSci USA 83:3166–3170, 1986.

4. Hara M, Koyanagi Y, Inoue T, Fukuyama T: Somephysico-chemical characteristics of gamma-seminopro-tein, an antigenic component for human seminalplasma. Jpn J Legal Med 25:322–324, 1971.

5. Oesterling JE: Prostate-specific antigen: A critical as-sessment of the most useful tumor marker for adeno-carcinoma of the prostate. J Urol 145:907–923, 1991.

6. Ford TF, Butcher DN, Masters JRW, Parkinson MC:Immunocytochemical localization of prostate-specificantigen: Specificity and application to clinical practice.Br J Urol 57:50–55, 1985.

7. Townsend A, Bodmer H: Antigen recognition by classI-restricted T lymphocytes. Annu Rev Immunol 7:601–624, 1989.

8. Grey HM, Sette A, Buus S: How T cells see antigen. SciAm 261:56–64, 1989.

9. Falk K, Rotzschke O, Stevanovic S, Jung G, Ram-mensee HG: Allele-specific motifs revealed by sequenc-ing of self-peptides eluted from MHC molecules. Na-ture 351:290–296, 1991.

10. Kubo RT, Sette A, Grey HM, Appella E, Sakaguchi K,Zhu N-Z, Arnott D, Sherman N, Shabanowitz J, MichelH, Bodnar W, Davis TA, Hunt DF: Definition of specificpeptide motifs for four major HLA-A alleles. J Immunol152:3913–3924, 1994.

11. Carbone F, Moore M, Sheil J, Bevan M: Induction ofcytotoxic T lymphocytes by primary in vitro stimulationwith peptides. J Exp Med 167:1767–1779, 1988.

12. Disis ML, Smith JW, Murphy AE, Chen W, CheeverMA: In vitro generation of human cytolytic T-cells spe-cific for peptides derived from the HER-2/neu protoon-cogene protein. Cancer Res 54:1071–1076, 1994.

13. Houbiers JGA, Nijman HW, Burg SH van der, Drijf-hout JW, Kenmans P, Velde CJH van der, Brand A,Momburg F, Kast WM, Melief CJM: In vitro inductionof human cytotoxic T lymphocytes responses againstpeptides of mutant and wild-type p53. Eur J Immunol23:2072–2077, 1993.

14. Celis E, Tsai V, Crimi C, DeMars R, Wentworth PA,Chestnut RW, Grey HM, Sette A, Serra HM: Inductionof anti-tumor cytotoxic T lymphocytes in normal hu-mans using primary cultures and synthetic peptideepitopes. Proc Natl Acad Sci USA 91:2105–2109, 1994.

15. Macatonia SE, Patterson S, Knight SC: Primary prolif-erative and cytotoxic T-cell responses to HIV induced invitro by human dendritic cells. Immunology 74:399–406, 1991.

16. Baur MP, Danilovs JA: Population analysis of HLA-A,B, C, Dr and other genetic markers. In PI Terasaki (ed):‘‘Histocompatibility Testing.’’ Berlin: Springer-Verlag,1980, p 955.

17. Lundwall A, Lilja H: Molecular cloning of human pros-tate specific antigen cDNA. FEBS Lett 214:317–322,1987.

18. Salter RD, Cresswell P: Impaired assembly and trans-port of HLA-A and -B antigens in a TxB cell hybrid.EMBO J 5:943–949, 1986.

19. Peace DJ, Kern DE, Schultz KR, Greenberg PD,Cheever MA: Interleukin-4-induced lymphokine acti-vated killer cells: Lytic activity is mediated by pheno-typically distinct NK-like and T cell-like large granularlymphocytes. J Immunol 140:3679–3685, 1988.

20. Ruppert J, Sidney J, Celis E, Kubo RT, Grey HM, SetteA: Prominent role of secondary anchor residues in pep-tide binding to HLA-A2.1 molecules. Cell 74:929–937,1993.

21. Townsend A, Rothbard J, Frances M, Gotch G, Baha-dur J, Wrath D, McMichael A: The epitopes of influ-enza nucleoproteins recognized by cytotoxic lympho-cytes can be defined with short synthetic peptides. Cell44:959–968, 1986.

22. Kast W, Brandt R, Drijfhout J, Melief C: Human leuko-cyte antigen-A2.1 restricted candidate cytotoxic T lym-phocyte epitopes of human papillomavirus type 16 E6and E7 proteins identified by using the processing-de-fective human cell line T2. J Immunother 14:115–120,1993.

23. Parker K, Bednarek M, Hull L, Utz U, Cunningham B,Zweerink H, Biddison W, Coligan J: Sequence motifsimportant for peptide binding to the human MHC classI molecule HLA-A2. J Immunol 148:3580–3587, 1992.

24. Tsomides TJ, Walker BD, Eisen HN: An optimal viralpeptide recognized by CD8+ T cells binds very tightlyto the restricting class I major histocompatibility com-plex protein on intact cells but not to the purified classI protein. Proc Natl Acad Sci USA 88:11276–11280, 1991.

25. Akdas A, Turkeri L, Alican Y, Simsek F, Akoglu T:Human leukocyte antigen subtype analysis in patientswith advanced adenocarcinoma of the prostate. Pros-tate 24:111–113, 1994.

26. Parker KC, Bednarel MA, Coligan JE: Scheme for rank-ing potential HLA-A2 binding peptides based on inde-pendent binding of individual peptide side-chains. JImmunol 152:163–175, 1994.

27. Stauss HJ, Davies H, Sadovnikova E, Chain B, Horo-witz N, Sinclair C: Induction of cytotoxic T lympho-cytes with peptides in vitro: Identification of candidateT-cell epitopes in human papilloma virus. Proc NatlAcad Sci USA 89:7871–7875, 1992.

28. Riddell SR, Watanabe KS, Goodrich IM, Li CR, AghaME, Greenberg PD: Restoration of viral immunity inimmunodeficient humans by the adoptive transfer of Tcell clones. Science 257:238–241, 1992.

29. Sanda MG, Restifo NP, Walsh JC, Kawakami Y, NelsonWG, Pardoll DM, Simons JW: Molecular characteriza-tion of defective antigen processing in human prostatecancer. J Natl Cancer Inst 87:280–285, 1995.

30. Montgomery BT, Young CY-F, Bilhartz DL, AndrewsPE, Prescott JL, Thompson NF, Tindall DJ: Hormonalregulation of prostate-specific antigen (PSA) glycopro-tein in the human prostatic adenocarcinoma cell line,LNCaP. Prostate 21:63–73, 1992.

31. Monne M, Croce CM, Yu H, Diamandis EP: Molecularcharacterization of prostate-specific antigen messengerRNA expressed in breast tumors. Cancer Res 54:6344–6347, 1994.

78 Xue et al.