Int. J. Cancer: 100, 441– 444 (2002) © 2002 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
RENAL CELL CARCINOMA–ASSOCIATED ANTIGEN G250 ENCODES A NATURALLY PROCESSED EPITOPE PRESENTED BY HUMAN LEUKOCYTE ANTIGEN-DR MOLECULES TO CD4⫹ T LYMPHOCYTES Joost L.M. VISSERS1, I. Jolanda M. DE VRIES1, Linda P.H. ENGELEN1, Nicole M. SCHARENBORG1, Janneke MOLKENBOER2, Carl G. FIGDOR1, Egbert OOSTERWIJK2 and Gosse J. ADEMA1* 1 Tumor Immunology Laboratory, University Medical Center Nijmegen St. Radboud, Nijmegen, the Netherlands 2 Department of Urology, University Medical Center Nijmegen St. Radboud, Nijmegen, the Netherlands We previously identified an HLA-A2.1-restricted epitope within the RCC-associated antigen G250 that is recognized by CTLs. Using DCs of healthy individuals, which were loaded with overlapping 20 mer G250-derived peptides, we here report the induction of CD4ⴙ T cells that recognize the G250 peptide of amino acids 249 –268. Moreover, naturally processed G250 protein is readily recognized by these G250specific CD4ⴙ T cells in the context of HLA-DR molecules. Interestingly, peptide G250:249 –268 overlaps the previously identified HLA-A2.1-restricted G250 epitope recognized by CTLs. These data and the high prevalence of G250 in RCC patients make peptide G250:249 –268 a potential target in peptide-based vaccines to induce both CD4ⴙ and CD8ⴙ Tcell responses in patients. © 2002 Wiley-Liss, Inc. Key words: G250; renal cell carcinoma; MN/CA IX; T-helper cell; human leukocyte antigen class II
Immunotherapy of cancer has progressed substantially with the identification of tumor-associated antigens and the subsequent finding that immune responses can be induced against these antigens. Next to CTLs, the importance of T-helper cells in antitumor immunity has been clearly demonstrated in several murine tumor models.1–3 Ossendorp et al.4 demonstrated that CD4⫹ T-helper cells are needed for optimal induction of antitumor-specific CTLs, most likely by activating professional APCs. In addition, T-helper cells participate in the effector phase of the immune response by recruiting and activating macrophages and eosinophils.3,5 Therefore, vaccines designed to treat cancer should elicit both CD4⫹ and CD8⫹ T-cell responses, preferentially to epitopes derived from tumor-associated antigens. RCC, the most frequent renal tumor, is relatively immunogenic in humans. In a fair number of RCC patients, spontaneous partial or complete remissions have been observed and immunotherapies have increased the reactivity of the immune system against RCC.6 Identification of potential target antigens might result in RCCspecific vaccine strategies. In contrast to melanoma, a very limited number of RCC-associated antigens have been identified. Moreover, the shared tumor antigens characterized in melanoma, which are also expressed in a variety of other tumors, are not expressed in RCCs. Previously, we demonstrated that the RCC-associated antigen G250 (also called MN/CA IX)7,8 encodes an HLA-A2.1restricted epitope from amino acids 254 through 262, which can be recognized by CTLs.9 The high prevalence of G250 in RCCs makes it a potential target for immunotherapy. The suggestion that G250 might not be the optimal target for antibody-mediated immunotherapy10 makes the identification of T-cell epitopes within G250 highly important. We investigated whether the G250 antigen, besides a CTL response, also can mount a T-helper response. Using computer-aided prediction programs and DCs loaded with synthetic G250-derived peptides, we induced HLA-DR-restricted T-helper cells against the G250-derived peptide from amino acids 249 –268 that also recognize naturally processed G250 protein.
MATERIAL AND METHODS
Proteins, lysates and peptides G250 protein (⬎95% pure) and rhodopsin protein (75% pure) were purified from Spodoptera frugiperda (Sf9) cells (ATCC, Rockville, MD) infected with G250 baculovirus and rhodopsin baculovirus, respectively, as described.8,11 Peptides were synthesized by Fmoc chemistry using a multiple-peptide synthesizer. (J.W. Dryfhout, Leiden University Medical Center, Leiden, The Netherlands). As determined by reversed-phase HPLC, peptides were ⬎90% pure. Induction of CD4⫹ T cells using peptide-loaded DCs At day – 8, PBMCs of healthy individuals were separated using Percoll-density centrifugation and allowed to adhere for 1 hr at 37°C in RPMI-1640 (Life Technologies, Grand Island, NY) enriched with 2% human serum in 75 cm2 tissue culture flasks (Costar, Badhoevedorp, the Netherlands). Adherent monocytes were cultured in X-VIVO 15 medium (BioWhittaker, Walkersville, MD) enriched with 1% autologous serum in the presence of IL-4 (500 U/ml) and GM-CSF (800 U/ml; both from ScheringPlough, Amstelveen, the Netherlands) for 6 days. Fresh cytokinecontaining culture medium was added at day –5. At day –2, immature DCs were stimulated with 10 ng/ml TNF-␣ (Bender, Vienna, Austria) and 10 g/ml prostaglandin E2 (Sigma, St. Louis, MO). Based on SYFPEITHI and TEPITOPE, 2 MHC class II– restricted epitope prediction software programs (kindly performed by Dr. S. Stevanovic),12–14 14 G250-derived peptides were selected (Table I). At day –1, 10 g/ml of each G250-derived peptide were added to the DCs (4 or 5 peptides/DC pool). At day 0, peptide-loaded mature DCs were loaded again with 10 g/ml of each G250-derived peptide at 37°C for 4 hr. Peptide-loaded DCs (5 ⫻ 104/well) were cocultured with 5 ⫻ 105 enriched autologous Abbreviations: APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBV, Epstein-Barr virus; GM-CSF, granulocytemacrophage colony-stimulating factor; HLA, human leukocyte antigen; IFN, interferon; MAb, monoclonal antibody; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cell; RCC, renal cell carcinoma; TNF, tumor necrosis factor. Grant sponsor: Dutch Cancer Society; Grant number: AZN/KUN 961362. *Correspondence to: Department of Tumor Immunology, University Medical Center Nijmegen St. Radboud, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, the Netherlands. Fax: ⫹31-24-3540339. E-mail:
[email protected] Received 29 January 2002; Revised 23 April 2002; Accepted 29 April 2002 DOI 10.1002/ijc.10518 Published online 13 June 2002 in Wiley InterScience (www.interscience. wiley.com).
442
VISSERS ET AL.
TABLE I – G250-DERIVED PEPTIDES (20 MER) COVERING PREDICTED HLA CLASS II– BINDING PEPTIDES Group
I
II
III
1
Amino acid sequence1
Position
G250:146–165 G250:154–173 G250:162–181 G250:170–189 G250:178–197 G250:399–418 G250:407–426 G250:415–434 G250:423–442 G250:105–124 G250:241–260 G250:249–268 G250:336–355 G250:344–363
GDPPWPRVSPACAGRFQSPV SPACAGRFQSPVDIRPQLAA QSPVDIRPQLAAFCPALRPL QLAAFCPALRPLELLGFQLP LRPLELLGFQLPPLPELRLR AAEPVQLNSCLAAGDILALV SCLAAGDILALVFGLLFAVT LALVFGLLFAVTSVAFLVQM FAVTSVAFLVQMRRQHRRGT EGSLKLEDLPTVEAPGDPQE VEGHRFPAEIHVVHLSTAFA EIHVVHLSTAFARVDEALGR AQGVIWTVFNQTVMLSAKQL FNQTVMLSAKQLHTLSDTLW
Overlapping amino acids are depicted in bold.
CD4⫹ T cells [depleted for CD8⫹ and CD56⫹ cells by magnetic sorting (Dynal, Oslo, Norway)] in X-VIVO 15 medium supplemented with 1% autologous serum in the presence of 1,000 U/ml IL-6 (Novartis, Basel, Switzerland) and 10 ng/ml IL-12 (R&D Systems, Abingdon, UK). At days 7 and 14, responder T cells were restimulated with peptide-loaded immature DCs (37°C, 4 hr) and 20 IU/ml IL-2 (Chiron, Berkeley, CA). At days 21 and 35, bulk T cells were tested for peptide specificity in both a proliferation assay and IFN-␥ secretion assay with peptide-loaded autologous EBV-B cells as stimulator cells. Additionally, the percentage of CD4⫹ T cells in these T-cell cultures was established by indirect immunofluorescence using mouse antihuman CD4 MAb RIV-715 and FITC-labeled goat antimouse secondary antibodies (Zymed, San Francisco, CA) followed by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA). Every 7 days, T cells were alternately given 20 IU/ml IL-2 or restimulated with peptideloaded EBV-B cells (37°C, 4 hr) and 20 IU/ml IL-2. IFN-␥ release assay and proliferation assay Autologous APCs were loaded with G250-derived peptides at 37°C for 4 hr. Antigen-loaded APCs were irradiated (5,500 rad) and plated in 96-well round-bottomed plates (Costar) at 3.5 ⫻ 105 cells/well in X-VIVO 15 medium enriched with 1% autologous serum. Bulk T cells were added at 5 ⫻ 105 cells/well. For HLA¨ 22 blocking experiments, antibodies B7/21 (anti-HLA-DP), TY (anti-HLA-DQ) and L243 (anti-HLA-DR) (antibodies kindly provided by Dr. G. Pawelec) were added to each well with an end concentration of 25% (v/v); at this concentration, proliferation could completely be blocked. To test whether T-cell cultures were able to release IFN-␥ upon antigen-specific stimulation, the supernatants of these cultures were harvested after 16 hr. Subsequently, the amount of IFN-␥ in the supernatants was determined using an IFN-␥-specific sandwich ELISA. Proliferation of responder T cells was determined after 72 hr of culture by pulsing the cells for another 16 hr with 1 Ci/well [3H]TdR (Amersham, Aylesbury, UK). Thymidine incorporation was measured using a liquid scintillation counter (LKB Wallac, UK). RESULTS
Induction of a G250-derived, peptide-specific T-helper response Antitumor reactivity of CTLs is enhanced by antigen-specific T-helper responses. To investigate the potential of the RCC-associated antigen G250 to induce a CD4⫹ T-cell response, we selected 14 G250-derived peptides based on 2 prediction software programs, SYFPEITHI and TEPITOPE.12–14 The selected 20 mer peptides were located in regions of the G250 protein that contained a high density of the predicted binding motifs for HLA-DR1, HLA-DR3, HLA-DR4 and HLA-DR11. For the induction of anti-
FIGURE 1 – T-helper cell lines I and III specifically proliferate in response to G250-derived peptides. Autologous EBV-B cells loaded with an irrelevant pool (open columns) and the relevant pool (closed columns) of G250-derived peptides were used as stimulator cells in a proliferation assay. From 1 donor, 3 CD4⫹ T-cell cultures, induced against autologous DCs loaded with 1 of the 3 groups of G250-derived peptides, were used as responder cells.
G250 T-helper cells, we used professional antigen-presenting DCs from healthy individuals. Per donor, 3 pools of DCs were loaded separately with 1 of the 3 groups, each containing 4 or 5 G250derived peptides (Table I). Peptide-loaded DCs were cocultured with autologous CD4⫹ T cells. As shown in Figure 1, we obtained T-cell lines that specifically proliferated upon stimulation with autologous EBV-B cells loaded with G250-derived peptides from groups I and III. In contrast, out of 8 healthy donors, no peptidespecific T-cell proliferation was obtained against peptides of group II. Since the T-cell line raised against G250-derived peptides of group III exhibited the highest proliferative response and could be expanded most efficiently, these T cells were subjected to further analysis. To test which G250-derived peptides of group III were recognized by the T-helper cell line III, autologous EBV-B cells loaded with each G250-derived peptide of group III separately were used as stimulator cells in a proliferation assay. Figure 2a shows that this T-cell culture (⬎98% CD4⫹) specifically recognized peptide G250:249 –268 and none of the other G250-derived peptides. Subsequently, the MHC class II molecule by which peptide G250: 249 –268 is presented to T cells was examined. Figure 2b shows that peptide G250:249 –268 specifically induced secretion of IFN-␥ by T-helper cell line III and that IFN-␥ production was blocked with an antibody against HLA-DR (L243) but not by ¨ 22). These antibodies against HLA-DP (B7/21) or HLA-DQ (TY results show that recognition of peptide G250:249 –268 by Thelper cell line III is HLA-DR-restricted. G250:249 –268-specific T-helper cell line recognizes naturally processed G250 To determine whether G250-derived peptide 249 –268 is naturally processed and presented, autologous DCs were loaded with purified G250 protein (5 g/ml) and used to stimulate CD4⫹ T-helper cell line III. For this purpose, baculovirus-produced G250 protein was used and recombinant rhodopsin, produced in the same baculovirus system, included as a negative control protein. T cells specifically proliferated (Fig. 3a) and specifically secreted IFN-␥ (Fig. 3b) upon interaction with autologous DCs loaded with G250 protein but not upon interaction with autologous DCs loaded with rhodopsin protein. Since the fraction of rhodopsin protein contains 25% baculovirus proteins, the observed proliferation and IFN-␥ secretion are G250-specific. As shown in Figure 3a, recognition of naturally processed G250 could be blocked by anti-HLA-DR antibodies but not by anti-HLA-DP and anti-HLA-DQ antibodies.
G250-DERIVED EPITOPE PRESENTED BY HLA CLASS II
443
FIGURE 3 – Naturally processed G250 is recognized by T-helper cell line III. Peptide G250:249 –268-specific T-helper cells were tested for their ability to proliferate (a) and to secrete IFN-␥ (b) in response to autologous DCs loaded with rhodopsin protein (containing 25% baculovirus proteins) or autologous DCs loaded with G250 protein (⬎95% pure). One of 3 representative experiments is shown. MHC class II restriction was examined by blocking G250-specific proliferation with anti-HLA-DR antibody (L243) and both anti-HLA-DP ¨ 22) (a). One of 2 representative exper(B7/21) and anti-HLA-DQ (TY iments is shown.
FIGURE 2 – T-helper cell line III recognizes peptide G250:249 –268 in the context of HLA-DR. (a) The G250-derived peptide recognized by T-helper cell line III was examined using a proliferation assay in which EBV-B cells were loaded with each peptide of pool III, separately. (b) To identify the MHC class II molecule by which peptide G250:249 –268 is presented to T-helper cell line III, peptide-specific IFN-␥ production was measured in the presence or absence of blocking antibodies against HLA-DR (L243), HLA-DP (B7/21) and ¨ 22). HLA-DQ (TY
These data demonstrate that peptide 249 –268 is naturally processed from the G250 protein and presented by HLA-DR. DISCUSSION
To induce strong antitumor immunity, both a CTL and a Thelper response are essential.16 Here, we describe the identification of an MHC class II–restricted peptide derived from the RCCassociated antigen G250 that is able to induce a T-helper response against G250. T-helper cells induced against the G250-derived peptide of amino acid 249 –268 (EIHVVHLSTAFARVDEALGR) recognize naturally processed G250 in the context of HLA-DR. RCC belongs, together with melanoma, to the small group of immunogenic tumors. In RCC patients, partial or complete remission has been observed and additional immunotherapies have increased the immune reaction against RCC.6 The presence of CD4⫹ lymphocytes as well as CD8⫹ lymphocytes in RCC supports the idea that the immune system is triggered in these tumors. So far, only a low number of RCC-associated antigens recognized by CD8⫹ CTLs have been characterized, including RAGE-1, Her-2/neu, G250 and SART3.9,17–19 Epitopes that can be recognized by T-helper cells have been identified within Her-2/neu and RAGE-1 (expressed in 30% and 2% of primary RCCs, respectively).20,21 Using 2 prediction software programs, SYFPEITHI and TEPITOPE,12–14 the G250 protein was screened for the presence
of binding motifs for HLA-DR1, HLA-DR3, HLA-DR4 and HLADR11. Subsequently, 14 G250-derived peptides were synthesized, which covered the regions of the G250 protein that contained a high density of predicted MHC class II epitopes. Using DCs loaded with 20 mer G250-derived peptides, we demonstrated that G250-specific CD4⫹ T-helper cells can be induced against peptide G250:249 –268 in vitro. Subsequently, the peptide-specific T cells specifically proliferated and secreted IFN-␥ in response to DCs loaded with baculovirus-produced G250 protein. Moreover, using anti-HLA-DR antibodies, these responses could be blocked. These data demonstrate that peptide G250:249 –268 contains an epitope that is naturally processed and presented in the context of HLADR. Tso et al.22 used a baculovirus-produced G250 –GM-CSF fusion protein to stimulate PBMCs of RCC patients: responder CD8⫹ and CD4⫹ T cells recognized autologous RCCs, demonstrating that baculovirus-produced G250 is processed and presented by APCs as endogenously expressed G250 is by RCCs. At the site of the RCC, T-helper stimulation can occur by MHC class II⫹ tumor cells or local APCs presenting G250⫹ tumor cell debris. Additionally, T-helper cells do not always have to act directly on tumor cells. Induction of a T-helper response can induce protection against MHC class II– tumor cells in mice.4 Peptide G250:249 –268 is situated in a region of the G250 protein that contains several predicted MHC class II binding motifs. Within this peptide, the epitope EIHVVHLSTAFARVD is predicted to bind to HLA-DR1, HLA-DR4 and HLA-DR11 (HLADR4 is expressed by T-helper cell line III). Peptide G250:241–260 (VEGHRFPAEIHVVHLSTAFA) does not contain this full-length epitope and is not recognized by T-helper cell line III. This finding further supports the idea that the epitope of amino acid 249 –263 contains the essential MHC class II binding and T-cell receptor recognition sequence. The prediction that peptide G250:249 –268 binds to several HLA-DR molecules together with the knowledge that T-helper peptides are usually promiscuous in binding to MHC class II molecules suggests that peptide G250:249 –268 could serve as a T helper– based vaccine, which is more widely applicable. Interestingly, the G250-derived peptide of amino acid 249 –268, which is able to induce T-helper cells, also overlaps the previously identified HLA-A2.1-restricted CTL epitope of amino acid 254 – 262 (HLSTAFARV).9 Overlapping CD4⫹ and CD8⫹ T-cell epitopes have been described in experimental models of influenza,
444
VISSERS ET AL.
p53, HIV, ras and heat-shock protein 60.23–26 Besides G250, the tumor-specific antigens mucin-1 and prostate-specific antigen also contain peptides with both MHC class I and class II motifs.27–29 Such long peptides that contain both class I and class II motifs could be promising tools to treat cancer patients. In this report, we demonstrated that the G250-derived peptide of amino acids 249 –268 induces CD4⫹ T-helper cells, which recognize naturally processed and presented G250 protein. Together with the indication that anti-G250 antibodies can be detected in serum of RCC patients (data not shown) and the evidence that G250 also encodes an HLA-A2.1-restricted epitope from amino
acids 254 –262, which can be recognized by CTLs, these data implicate that the RCC-associated antigen G250 is a potential target for T cell– based anti-RCC immunotherapy.
ACKNOWLEDGEMENTS
We thank Dr. W.J. de Grip for providing baculovirus-produced rhodopsin. We thank Drs. H. Rammensee and S. Stevanovic for the search for MHC class II– binding motifs within G250 and Dr. G. Pawelec for providing the anti-class II antibodies.
REFERENCES
1. 2. 3. 4.
5. 6.
7.
8.
9.
10.
11.
12. 13.
14.
15.
⫹
Schild HJ, Kyewski B, von Hoegen P, Schirrmacher V. CD4 helper T cells are required for resistance to a highly metastatic murine tumor. Eur J Immunol 1987;17:1863– 6. Romerdahl CA, Kripke ML. Role of helper T-lymphocytes in rejection of UV-induced murine skin cancers. Cancer Res 1988;48: 2325– 8. Hung K, Hayashi R, Lafond-Walker A, Lowenstein C, Pardoll D, Levitsky H. The central role of CD4(⫹) T cells in the antitumor immune response. J Exp Med 1998;188:2357– 68. Ossendorp F, Mengede E, Camps M, Filius R, Melief CJ. Specific T-helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J Exp Med 1998;187:693–702. Greenberg PD. Adoptive T cell therapy of tumors: mechanisms operative in the recognition and elimination of tumor cells. Adv Immunol 1991;49:281–355. Vissers JLM, De Vries IJM, Oosterwijk E, Figdor CG, Adema GJ. Gp100 and G250: Towards specific immunotherapy employing dendritic cells in melanoma and renal cell carcinoma. In: Kast WM, ed. Peptide-based cancer vaccines. Austin, TX: Eurekah, 2000. 200 –14. Opavsky R, Pastorekova S, Zelnik V, Gibadulinova A, Stanbridge EJ, Zavada J, Kettmann R, Pastorek J. Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. Genomics 1996;33:480 –7. Grabmaier K, Vissers JLM, De Weijert MCA, Oosterwijk-Wakka JC, Van Bokhoven A, Brakenhoff RH, Noessner E, Mulders PA, Merkx G, Figdor CG, Adema GJ, Oosterwijk E. Molecular cloning and immunogenicity of renal cell carcinoma–associated antigen G250. Int J Cancer 2000;85:865–70. Vissers JLM, De Vries IJM, Schreurs MWJ, Engelen LPH, Oosterwijk E, Figdor C, Adema GJ. The renal cell carcinoma–associated antigen G250 encodes a human leukocyte antigen (HLA)-A2.1–restricted epitope recognized by cytotoxic T lymphocytes. Cancer Res 1999;59:5554 –9. Stadick H, Stockmeyer B, Kuhn R, Schrott KM, Kalden JR, Glennie MJ, van de Winkel JGJ, Gramatzki M, Valerius T, Elsasser D. Epidermal growth factor receptor and G250: useful target antigens for antibody mediated cellular cytotoxicity against renal cell carcinoma? J Urol 2002;167:707–12. Janssen JJ, Bovee-Geurts PH, Merkx M, DeGrip WJ. Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry. J Biol Chem 1995;270:11222–9. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 1999;50:213–9. de Lalla C, Sturniolo T, Abbruzzese L, Hammer J, Sidoli A, Sinigaglia F, Panina-Bordignon P. Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction. J Immunol 1999;163:1725–9. Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat Biotechnol 1999;17:555– 61. Leerling MF, Vaessen LM, Reubsaet CH, Weimar W, Ettekoven H, Marsman FR, Kreeftenberg JG. Quality control of anti human CD3 and CD4 monoclonal antibodies. Dev Biol Stand 1990;71:191–200.
16. Toes RE, Ossendorp F , Offringa R, Melief CJ. CD4 T cells and their role in antitumor immune responses. J Exp Med 1999;189:753– 6. 17. Gaugler B, Brouwenstijn N, Vantomme V, Szikora JP, Van der Spek CW, Patard JJ, Boon T, Schrier P, Van den Eynde BJ. A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma. Immunogenetics 1996;44:323–30. 18. Brossart P, Stuhler G, Flad T, Stevanovic S, Rammensee HG, Kanz L, Brugger W. Her-2/neu-derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes. Cancer Res 1998;58:732– 6. 19. Kawagoe N, Shintaku I, Yutani S, Etoh H, Matuoka K, Noda S, Itoh K. Expression of the sart3 tumor rejection antigen in renal cell carcinoma. J Urol 2000;164:2090 –5. 20. Kobayashi H, Wood M, Song Y, Appella E, Celis E. Defining promiscuous MHC class II helper T-cell epitopes for the HER2/neu tumor antigen. Cancer Res 2000;60:5228 –36. 21. Stassar MJ, Raddrizzani L, Hammer J, Zoller M. T-helper cell-response to MHC class II-binding peptides of the renal cell carcinoma-associated antigen RAGE-1. Immunobiology 2001;203:743–55. 22. Tso CL, Zisman A, Pantuck A, Calilliw R, Hernandez JM, Paik S, Nguyen D, Gitlitz B, Shintaku PI, de Kernion J, Figlin R, Belldegrun A. Induction of G250-targeted and T-cell-mediated antitumor activity against renal cell carcinoma using a chimeric fusion protein consisting of G250 and granulocyte/monocyte-colony stimulating factor. Cancer Res 2001;61:7925–33. 23. Carreno BM, Turner RV, Biddison WE, Coligan JE. Overlapping epitopes that are recognized by CD8⫹ HLA class I–restricted and CD4 – class II–restricted cytotoxic T lymphocytes are contained within an influenza nucleoprotein peptide. J Immunol 1992;148: 894 –9. 24. Noguchi Y, Chen YT, Old LJ. A mouse mutant p53 product recognized by CD4⫹ and CD8⫹ T cells. Proc Natl Acad Sci USA 1994; 91:3171–5. 25. Takeshita T, Takahashi H, Kozlowski S, Ahlers JD, Pendleton CD, Moore RL, Nakagawa Y, Yokomuro K, Fox BS, Margulies DH. Molecular analysis of the same HIV peptide functionally binding to both a class I and a class II MHC molecule. J Immunol 1995;154: 1973– 86. 26. Mertz AK, Wu P, Sturniolo T, Stoll D, Rudwaleit M, Lauster R, Braun J, Sieper J. Multispecific CD4⫹ T cell response to a single 12-mer epitope of the immunodominant heat-shock protein 60 of Yersinia enterocolitica in Yersinia-triggered reactive arthritis: overlap with the B27-restricted CD8 epitope, functional properties, and epitope presentation by multiple DR alleles. J Immunol 2000;164: 1529 –37. 27. Domenech N, Henderson RA, Finn OJ. Identification of an HLAA11-restricted epitope from the tandem repeat domain of the epithelial tumor antigen mucin. J Immunol 1995;155:4766 –74. 28. Hiltbold EM, Ciborowski P, Finn OJ. Naturally processed class II epitope from the tumor antigen MUC1 primes human CD4⫹ T cells. Cancer Res 1998;58:5066 –70. 29. Corman JM, Sercarz EE, Nanda NK. Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells. Clin Exp Immunol 1998;114:166 –72.
