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Clinical Cancer Research Vol. 12, 4949-4957, August 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Peptidome from Renal Cell Carcinoma Contains Antigens Recognized by CD4+ T Cells and Shared among Tumors of Different Histology

Elena Tassi1,3,6, Valeria Facchinetti1,3, Samantha Seresini1,3,6, Anna Borri5,6, Giacomo Dell'Antonio4, Claudio Garavaglia2,3,6, Giulia Casorati2,3,6 and Maria Pia Protti1,3,6

Authors' Affiliations: 1 Tumor Immunology Unit, DIBIT, 2 Experimental Immunology Unit, 3 Cancer Immunotherapy and Gene Therapy Program, 4 Pathology Unit, 5 Medicine Unit, and 6 Department of Oncology, Scientific Institute H. San Raffaele, Milan, Italy

Requests for reprints: Maria Pia Protti, Tumor Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, DIBIT, Scientific Institute H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy. Phone: 39-02-2643-4185; Fax: 39-02-2643-4786; E-mail: m.protti{at}hsr.it.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Renal cell carcinoma (RCC) is considered immunogenic; nonetheless, rare tumor-associated antigens have been identified or are expressed in RCC. Peptidome (i.e., the total content of natural peptides of whole cells) from other tumors, such as melanoma, has proved to be immunogenic. The aims of this study were to determine whether peptidome from RCC is immunogenic and whether it contains tumor peptides shared among allogenic RCCs.

Experimental Design: Autologous dendritic cells pulsed with RCC peptidome were used to activate in vitro CD4+ T cells from healthy donors and a metastatic RCC patient. CD4+ T-cell polyclonal lines and clones were characterized for tumor cell recognition by proliferation assay, killing activity, and cytokine secretion.

Results: CD4+ T-cell lines and clones recognized HLA-DR-matched allogenic RCC and, for the patient, the autologous tumor. RCC-reactive CD4+ T cells showed a heterogeneous Th1 or Th0/Th2 pattern of cytokine secretion. Moreover, RCC-reactive CD4+ T cells recognized also melanoma, colon carcinoma, cervical carcinoma, pancreas carcinoma, lung carcinoma, gastric carcinoma, and lymphoma cells but not autologous T-cell blasts.

Conclusions: Our results show that (a) the RCC peptidome contain antigens recognized by CD4+ T cells and (b) shared among tumors of different histology and (c) it induces both Th1-type and Th2/Th0-type immune responses. These data support the use of the peptidome from allogenic RCC for specific immunotherapy in RCC and possibly in other neoplastic diseases. Moreover, the CD4+ T-cell clones generated here are useful tools for tumor antigen identification.

Renal cell carcinoma (RCC) is a relatively rare disease, representing 2% to 3% of all malignancies; nonetheless, 20% to 30% of patients have metastatic disease at diagnosis, ~30% develop recurrent disease after radical nephrectomy, and conventional therapies, such as chemotherapy and radiotherapy, are not effective (1, 2). Thus, RCC remains a therapeutic challenge.

The immune system is believed to play an important role in the control of RCC growth because of (a) clinical evidence of late relapses after nephrectomy and prolonged stabilization of the disease in the absence of systemic treatment (1, 2), (b) the occurrence of spontaneous regression of metastatic lesions (3), (c) the presence of tumor-infiltrating lymphocytes in the lesions composed primarily of natural killer and T lymphocytes (4–6), and (d) durable, albeit infrequent, responses with interleukin (IL)-2 and IFN-{alpha} therapy (7, 8). Therefore, RCC may be considered a target of innovative therapies, such as immunotherapy.

Induction of tumor-specific adaptive immune responses is the main goal of active immunotherapy. Tumor-specific CD8+ T cells are considered the main effectors in tumor rejection, but increasing evidence points to a fundamental role for CD4+ T cells in the induction, maintenance, and effector functions of productive antitumor immunity (9, 10).

Several tumor-associated antigens (TAA), suitable for cancer immunotherapy, have been identified, among which shared tumor-specific antigens belonging to the cancer testis family expressed on a variety of solid tumors (reviewed in ref. 11). However, these tumor-specific antigens are not primarily expressed by RCC. Instead, TAAs recognized by RCC-reactive CD8+ T cells are either overexpressed normal antigens (12–18) or resulting from splicing aberration (19, 20) or unique (21, 22). Even less is known about antigen specificity of RCC-reactive CD4+ T cells, but also in this case, they seem to recognize overexpressed RCC candidate antigens (23, 24).

Alternative strategies in RCC vaccine trials used undefined antigens derived from total tumor cells with the goal of eliciting T-cell responses against several unknown antigens expressed by the tumor; such strategies include the use of inactivated tumor cells, gene-modified tumor cells, dendritic cells expressing antigens derived from RCC lysates, or tumor RNA (reviewed in ref. 25). These approaches were found feasible with limited toxicities and some clinical efficacies (25): possible drawback being the difficulty in obtaining sufficient material for vaccine preparation.

We showed previously that the peptidome [i.e., the total content of natural tumor peptides (NTP) obtained by acid extract of whole melanoma cells] contains shared TAAs and that autologous dendritic cells pulsed with the peptidome from allogenic melanoma are able to activate CD8+ and CD4+ T-cell immunity against the autologous tumor in melanoma patients (26–28).7 A pilot clinical trial in metastatic melanoma patients based on this approach is currently under way.

The use of peptidome from allogenic tumor cells specifically addressed the necessity for regulatory requirements of ready-to-use "standardized" products prepared in good manufacturing procedure conditions and for immunologic monitoring of a source of common TAAs.

In the present study, we aimed at verifying whether the same approach used for melanoma could be applied to RCC immunotherapy. In particular, we focused on CD4+ T cells and specifically asked whether peptidome from RCC is immunogenic in vitro and whether it contains tumor peptides shared among allogenic RCCs. These data, along with the demonstration that allogenic RCC peptidome can activate T cells specific for the autologous tumor in RCC patients, are a fundamental prerequisite for the application of this immunotherapeutic approach in the clinic.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Subjects and cells. Peripheral blood mononuclear cells (PBMC) were obtained from two normal donors and one RCC patient (ND1, ND2, and Pt3). The patient had a nonmetastatic clear cell carcinoma at diagnosis; at the time of the blood drawn, the patient had two small lung metastases treated with IFN-{alpha}. The normal donors were not on medication that could have altered their immune status. The informed consent was obtained from all subjects before blood sampling. RCC lines Ben-TC, Bi-TC, Bor-TC, Bos-TC, Ca-TC, Cr-TC, Gu-TC, Ge-TC, Gr-TC, Pr-TC, Ra-TC, Sa-TC, So-TC, Tr-TC, and Vi-TC, melanoma cell line MD-TC, and pancreatic adenocarcinoma line Ber-TC were established in our laboratory. RCC cell line MR196 was the generous gift of C. Traversari (MolMed, S.p.A., Milan, Italy). Melanoma HT144 and SK-Mel 24, cervical carcinoma HeLa, C-4I, and MS751, Burkitt's lymphoma Daudi, gastric carcinoma Kato III, colon carcinoma HT-29, and human erythroleukemia K562 cell lines were from the American Type Culture Collection (Rockville, MD). Pancreatic adenocarcinoma HuP-T3, CFPAC-1, A8184, HPAF, and Paca44 were kindly provided by L. Piemonti (DIBIT, Milan, Italy). Colon carcinoma SW480 cell line was from Interlab Cell Line Collection (CBA, Genoa, Italy). EBV-transformed lymphoblastoid cell lines (LCL) from the donors were established in our laboratory. Cell lines were cultured in RPMI 1640 or DMEM (Life Technologies, Inc., Grand Island, NY) containing 2 mmol/L L-glutamine, 100 units/mL penicillin, 50 mg/mL streptomycin (BioWhittaker, Walkersville, MD), and 10% FCS [tissue culture medium (TCM); Life Technologies, Inc., Paisley, Scotland]. Autologous phytohemagglutinin (PHA)-activated T cells (PHA blasts) were obtained by PBMC activation in TCM containing 10% heat-inactivated pooled autologous or human serum (BioWhittaker), 1 µg/mL PHA (Sigma-Aldrich, Milan, Italy), and IL-2 (600 IU/mL; Proleukin, Chiron, Siena, Italy) for 1 week followed by weekly restimulation with irradiated allogenic EBV-LCLs (1:1 ratio) as feeder cells in the presence of the same dose of IL-2 and PHA. The HLA types of the donors and the cells used in this study, identified by molecular typing, are reported in Table 1 .


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  		Table 1. HLA-DR type of donors and cells used in this study

 
RCC peptidome purification and generation of dendritic cells. Peptidome (i.e., NTPs with molecular weight of 100-5,000 kDa) was obtained by acid treatment of Gr-TC cells as described in refs. 26, 27 with minor modification. Briefly, tumor cells were expanded to reach 5 x 109. Cells were lysed by cycles of freezing and thawing in the presence of trifluoroacetic acid at pH 2.1, and peptides were retrieved after supernatant centrifugation, dialysis (Spectra/Por DispoDialyzer, cellulose ester membrane, molecular weight cutoff 100; Spectrum Europe B.V., Breda, the Netherlands), and ultrafiltration on centrifugal filter units (Centricon Plus-20 with Biomax-PB membrane, molecular weight cutoff 5000; Millipore S.p.A., Vimodrone, Italy). Dendritic cells were generated as described previously (27). Briefly, PBMCs (obtained from 50-100 mL of peripheral blood) were suspended in TCM and plated at the concentration of 15 x 106/mL in six-well plates for 2 hours at 37°C. Nonadherent cells were then removed, and adherent cells were cultured for 7 days with TCM (1% human serum) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF; 800 units/mL; Mielogen, Schering-Plough, Milan, Italy) and IL-4 (500 units/mL; Schering-Plough). Half of the supernatant was replenished with fresh TCM and cytokines every other day. At day 7, dendritic cells were retrieved and incubated with the NTPs (75-100 µg/mL) for 1 hour at 37°C before tumor necrosis factor-{alpha} (1,000 units/mL) was added; after 5 to 7 hours, the medium was removed and fresh TCM containing NTPs (50 µg/mL) was added. NTP-pulsed dendritic cells were then used for the flow cytometry analysis and CD4+ T-cell activation.

Propagation of CD4+ T-cell lines and clones. CD4+ T cells were purified by positive magnetic selection after incubation with beads coated with anti-CD4 antibody (Miltenyi Biotec. S.r.l., Bergisch Gladbach, Germany) following the manufacturer's instructions. CD4+ T cells were admixed with NTP-pulsed dendritic cells at a 10:1 or 20:1 ratio and cultured in TCM. At day 7, activated cells were separated by a Percoll gradient (29) and expanded in IL-2 (20 IU/mL) containing TCM for 7 to 10 days. CD4+ T cells were then weekly restimulated with NTP-pulsed autologous dendritic cells or PBMCs. CD4+ T-cell clones were obtained by limiting dilution of polyclonal T-cell lines after two to four rounds of stimulation with the antigen as described in ref. 27.

Flow cytometry and monoclonal antibodies used. Cytofluorimetric analyses were done on a FACStarPlus (Becton Dickinson, Sunnyvale, CA). The following monoclonal antibodies were used: anti-DR-FITC, anti-CD4-PE, anti-CD8-FITC, anti-CD14-PE, anti-CD83-FITC (Becton Dickinson), anti-CD1a-FITC (Serotec, Oxford, United Kingdom), anti-CD40, anti-CD80, anti-CD86 (Calbiochem, San Diego, CA), anti-CCR7 (PharMingen, Becton Dickinson), and anti-HLA class I (W6/32). FITC-rabbit anti-mouse immunoglobulin antibody (DAKO A/S, Glostrup, Denmark) was used as second-step reagent in indirect immunofluorescence staining.

CD4+ T-cell stimulation assays (proliferation and ELISA). CD4+ T cells were cultured in triplicate in 96 U-bottomed plates in the presence of irradiated tumor cells or control cells at a 1:3 ratio. To allow the expression of MHC class II molecules, tumor cells, which do not constitutively express HLA-DR (see Table 1), were cultured for 48 hours in the presence of IFN-{gamma} (1,000 units/mL; R&D System, Minneapolis, MN). At day 2, half of the supernatant from each well was removed for cytokine detection and the plates were pulsed for 16 hours with [3H]TdR (1 µCi/well, 6.7 Ci/mol; Amersham Corp., Milan, Italy). The cells were collected with a FilterMate Universal Harvester (Packard, Wellesley, MA) in specific plates (UniFilter GF/C, Packard), and the thymidine incorporated was measured in a liquid scintillation counter (TopCount NXT, Packard). Release of IFN-{gamma}, IL-5, and GM-CSF was determined by commercially available kits (BioSource, Camarillo, CA; Mabtech AB, Stockholm, Sweden) following the manufacturer's instructions. Th1-Th2 (IFN-{gamma}, tumor necrosis factor-{alpha}, IL-10, IL-4, IL-5, and IL-2) release of cytokines was also determined using the Cytometric Bead Array, Human Th1-Th2 Cytokines kit (Becton Dickinson).

Cytotoxicity assay. CD4+ T cells were tested for specific lytic activity in a standard 4-hour 51Cr release assay as described in ref. 30. Target used is reported in the figures. To allow the expression of MHC class II molecules, tumor cells, which do not constitutively express HLA-DR (see Table 1), were treated with IFN-{gamma} as described above. The percentage of specific 51Cr release of triplicates was calculated as follows: [(average experimental cpm – average spontaneous cpm) / (average maximum cpm – average spontaneous cpm)] x 100. 51Cr release of target cells alone (spontaneous release) was always <25% of maximal 51Cr release (target cells in 0.25 mol/L HCl). In cold target competition assays, unlabeled target cells (cold targets) were seeded at 100:1 ratio of cold-to-hot target cells. Effector CD4+ T cells and 51Cr-labeled target cells (hot targets) were then added, and cytotoxicity was assessed as described above. Percentage inhibition was calculated as follows: [(% specific lysis without cold target – % specific lysis with cold target) / (% specific lysis without cold target)] x 100.

T-cell receptor Vß usage and heteroduplex analysis. Vß expression was determined with the IOTest Beta Mark kit (Beckman Coulter, Fullerton, CA) for flow cytometric analysis of the T-cell receptor (TCR) Vß repertoire of human T lymphocytes following the manufacturers' instructions. To determine the expression of TCR Vß chains not included in the kit (Vß6 and Vß21), PCR analysis was also done according to published protocols (31). Briefly, total RNA was extracted from 5 x 105 cells of CD4+ T-cell lines and clones, reverse transcribed into cDNA, and amplified by PCR using Vß6, Vß21, and a Cß downstream-specific oligonucleotide. PCR products (10 µL) were visualized on a 1.5% agarose gel. To verify whether clones bearing the same Vß (Vß5.1, Vß6, Vß13.6, Vß20, and Vß21) are indeed the same clones, we did the PCR heteroduplex analysis. In this case, 20 µL of the Vß-Cß PCR products from each clone were mixed with the same amount of PCR product amplified from a carrier DNA (a plasmid with a Vß cDNA having the same Vß and Cß sequence of the T-cell clone but a different N-region) and subjected to heteroduplex formation (31). Each T-cell clone bearing a different N-region will give a heteroduplex with the carrier because of a different mobility on a 12% acrylamide gel. This analysis allows the identification of T-cell clones bearing the same TCR Vß sequence.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
CD4+ T cells stimulated with RCC peptidome-pulsed dendritic cells recognize allogenic HLA-DR-matched RCCs and the autologous tumor. NTPs were purified from Gr-TC cells (undifferentiated papillary RCC); CD4+ T cells were obtained from two normal donors (ND1 and ND2) and a metastatic RCC patient (Pt3). ND2 shared the DR*01 allele with Gr-TC, whereas ND1 and Pt3 and Gr-TC did not have any HLA-DR allele in common (Table 1). Polyclonal cells were obtained after two to four rounds of in vitro stimulation of purified CD4+ T cells with NTP-pulsed autologous dendritic cells. CD4+ T-cell recognition of RCC cells was tested by microproliferation assays, IFN-{gamma} release, and cytotoxic activity (Table 2 ). Polyclonal line from ND1 recognized RCC cells matched at DRß1*08 (Ca-TC), DRß1*11 (Ben-TC, Bi-TC, MR196, and Pr-TC), and DRß3*02 (Ben-TC, MR196, Pr-TC, Ge-TC, and Ra-TC), whereas they did not recognize the HLA-DR-unrelated Gr-TC cells. Polyclonal line from ND2 recognized RCC cells matched at DRß1*01 (Gr-TC and Ge-TC) and DRß1*11 (Bi-TC, MR196, and Pr-TC), whereas they did not recognize the HLA-DR-unrelated Bos-TC and Ca-TC cells. Polyclonal line from Pt3 recognized RCC cells matched at DRß1*04 (Ca-TC) and DRß3*01 (Bor-TC and Bos-TC), whereas they did not recognize cells matched only at DRß1*13 (Ge-TC, Sa-TC, and So-TC) and the HLA-DR-unrelated Cr-TC cells. Most importantly, polyclonal line from Pt3 strongly recognized the autologous tumor (Table 2). The HLA class II restriction for all polyclonal lines was further confirmed by blocking experiments with an anti-MHC class II monoclonal antibody (data not shown) and the lack of recognition of RCC cells that had not been treated with IFN-{gamma} for induction of MHC class II molecules (data not shown).


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  		Table 2. Recognition of allogenic HLA-DR-matched RCC and the autologous tumor by polyclonal CD4+ T-cell lines from the healthy donors and the patient

 
Characterization of RCC-reactive CD4+ T cells. To further characterize the repertoire and cytokine profile of RCC-reactive CD4+ T cells, polyclonal lines from the normal donors were cloned by limiting dilution: 25 clones from ND1 and 14 clones from ND2 were further characterized. CD4+ T-cell clones were cultured in the presence of RCC cells sharing each of the restricting alleles of the polyclonal lines and, after 2 days, 3H incorporation tested. Figure 1 shows the results of the HLA-DR restriction for one representative clone for each restriction. From ND1 (Fig. 1A), we obtained 9 DRß1*08-restricted clones (represented by clone 14), 8 DRß1*11 (represented by clone 38), and 8 DRß3*02 (represented by clone 44); from ND2 (Fig. 1B), we obtained 10 DRß1*01-restricted clones (represented by clone 2) and 4 DRß1*11 (represented by clone 18). The study of the TCR Vß usage and the PCR heteroduplex analysis in the presence of a Vß carrier DNA (see Materials and Methods), which determines if clones expressing the same Vß are indeed the same clones, revealed the existence of a more restricted number of independent clones. In fact, T-cell clones derived from the same donor bearing the same Vßs gave an identical heteroduplex mobility pattern on acrylamide gel, indicating that they were indeed the same clones (data not shown). In summary, we obtained one DRß1*08-restricted clone (bearing the Vß21 chain), four DRß1*01-restricted clones (bearing the Vß5.1, Vß8, Vß20, and Vß21.3 chains), and six DRß1*11-restricted clones [three from ND1 (bearing the Vß6, Vß17, and Vß22 chains) and three from ND2 (bearing the Vß2, Vß17, and Vß22 chains)]. Moreover, we found that polyclonal line from Pt3 contained a small percentage (20%) of clones expressing TCRs carrying the Vß5.1, Vß2, Vß12, Vß17, and Vß22 chains, whereas the remaining 80% expressed the Vß6 and the Vß21 chains, suggesting that RCC-reactive CD4+ T cells from Pt3 are oligoclonal.


Figure 1
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  		Fig. 1. HLA-DR restriction of CD4+ T-cell clones obtained by limiting dilution of polyclonal cell lines from the normal donors. CD4+ T cells were tested in 2-day microproliferation assays in the presence of HLA-DR-matched or HLA-DR-unmatched RCC cell lines. A, CD4+ T-cell clones (14, 38, and 44) from ND1. B, CD4+ T-cell clones (2 and 18) from ND2. HLA-DR alleles expressed by RCC lines shared with the donor are reported in the figure. White and gray columns, blanks (i.e., the basal level of proliferation of CD4+ T cells alone and RCC alone); black columns, proliferation of CD4+ T cells plus tumor cells. Columns, mean of triplicate determination and are representative of several experiments; bars, SD.

 
To determine the pattern of cytokines secreted, CD4+ T cells were challenged in vitro with RCC cell lines known to induce proliferation (see Fig. 1) and then culture supernatant was tested for IFN-{gamma}, tumor necrosis factor-{alpha}, IL-5, IL-10, IL-4, and IL-2 release (Fig. 2 ). DRß1*08-restricted clones from ND1 (Fig. 2A, left) and DRß1*01-restricted clones from ND2 (Fig. 2B, left) produced all cytokines with the exception of IL-10; DRß1*11-restricted clones from both donors (Fig. 2A, middle and Fig. 2B, right) and DRß3*02 clones from ND1 (Fig. 2A, right) secreted IFN-{gamma}, tumor necrosis factor-{alpha}, and IL-2; polyclonal line from Pt3 challenged with the autologous tumor-secreted IFN-{gamma} and IL-5 (Fig. 2C). Furthermore, the analysis of cytokine secretion by CD4+ T-cell clones with the same HLA-DR restriction but bearing different TCR Vß chains showed a similar pattern of cytokine secretion (data not shown).


Figure 2
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  		Fig. 2. Cytokine release profile. A and B, CD4+ T-cell clones were cultured in the presence of HLA-DR-matched RCCs. After 2 days of culture, supernatant was removed and tested for cytokine release by cytometric bead array. Clones from ND1 (A) were tested in the presence of Ca-TC (clone 14), Pr-TC (clone 21), and Ge-TC (clone 44), respectively. Clones from ND2 (B) were tested in the presence of Ge-TC (clone 2) and Pr-TC (clone 18). C, polyclonal cell line from Pt3 was tested in the presence of the autologous tumor (Gu-TC). Black columns, CD4+ T cells plus tumor cells; gray columns, tumor cells alone; white columns, CD4+ T cells alone.

 
RCC-reactive CD4+ T cells recognize TAAs shared among tumors of different histology. RCC-reactive CD4+ T cells were then challenged with a panel of HLA-DR-matched tumor cells of different histology and tested for cytokines or 51Cr release assays (Fig. 3 ). All clones (Fig. 3A and B) and polyclonal CD4+ T-cell line (Fig. 3C) strongly recognized the RCC-positive control and, with differences among different CD4+ T cells depending on the availability of HLA-DR-matched tumor cells, also pancreatic carcinoma, cervical carcinoma, melanoma, colon carcinoma, gastric carcinoma, lung carcinoma, and the autologous LCLs but not the autologous PHA blasts or Daudi cells used as negative controls. HLA class II expression of tumor cells, after IFN-{gamma} treatment, and of PHA blasts was verified by flow cytometry (data not shown).


Figure 3
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  		Fig. 3. RCC-reactive CD4+ T cells recognize tumors of different histology. A, clones obtained from ND1 representative for each HLA-DR restriction. B, clones obtained from ND2 representative for each HLA-DR restriction. C, polyclonal line from Pt3. CD4+ T cells were tested either in cytokine (IL-5, IFN-{gamma}, and GM-CSF) release assays after challenge with the tumors (A-B) or in a 51Cr release assay (C). Cytokine release assays. Black columns, CD4+ T cells plus tumor cells or CD4+ T cells alone; white columns, tumor cells alone. Tumor cells tested are indicated in the figure and described in Table 1. Columns, mean of triplicate determination and are representative of several experiments for each clone; bars, SD.

 
Recognition of the autologous LCLs is tumor specific. Surprisingly, RCC-reactive CD4+ T cells strongly recognized the autologous LCLs (Fig. 3); to verify whether this recognition was tumor specific, we did 51Cr release assays, in which the level of unspecific cytotoxic activity was determined by testing the lysis of Daudi and K562 cells. As shown in Fig. 4 , clones restricted at DRß1*01, DRß1*08, DRß1*11, and the polyclonal line from Pt3 killed both the positive control RCC cells and the autologous LCLs, whereas they did not recognize Daudi cells (Fig. 4A). On the contrary, DRß3*02-restricted CD4+ T cells, along with the positive tumor and the autologous LCLs, strongly recognized Daudi cells but not K562 cells (Fig. 4B), suggesting that Daudi cell recognition could be specific. Indeed, Daudi cells express the DRß3*02 allele (Table 1) and were recognized by DRß3*02-restricted clones also in microproliferation and cytokine release assays (Fig. 4C). Moreover, cold target competition assays showed that the lytic activity of CD4+ T-cell clones (a representative clone from Fig. 4A is shown) against RCC cells was inhibited by the addition of autologous cold LCLs but not of Daudi cells (Fig. 4D), showing that CD4+ T cells recognize the same epitope on RCC and LCLs. Collectively, these data support a specific recognition of the autologous LCLs, which indeed may be regarded as in vitro–induced Burkitt's lymphoma cells.


Figure 4
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  		Fig. 4. Recognition of autologous LCLs by RCC-reactive CD4+ T cells is tumor specific. A and B, CD4+ T-cell clones representative of each HLA-DR restriction and polyclonal cell line from Pt3 were tested in 51Cr release assays against the positive control RCCs, the autologous LCLs, Daudi, and K562 cells. Points, mean of triplicate determination and are representative of at least three experiments for each clone; bars, SD. C, DRß3*02-restricted clones were tested in microproliferation assays and GM-CSF release assay after challenge with the RCC-positive control Daudi and the autologous PHA blasts as negative control. D, cold target competition assays. Left, cytolytic activity of representative DRß1*08-restricted clone 14 against 51Cr-labeled RCC cells (Ca-TC), autologous LCLs, and Daudi cells alone (open symbols) or in the presence of fixed amount (100:1 ratio) of cold targets (closed symbols); right, results are expressed as percentage inhibition (at 20:1 E:T ratio).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The lack of clinically relevant TAAs in RCC and the need for regulatory requirements of "standardized" cellular products for their use in cancer immunotherapy protocols prompted us to verify whether the RCC peptidome, which can be obtained in good manufacturing procedure conditions from a certified master cell bank, is immunogenic and contains shared TAA among allogenic RCCs.

We show here that RCC-reactive CD4+ T cells are obtained by stimulation with peptides derived from an allogenic RCC when presented by autologous dendritic cells and that they recognize an antigen(s) shared among allogenic RCCs but also several tumors of different histology comprising pancreatic adenocarcinoma, cervical cancer, colon and gastric cancer, lung cancer, melanoma, and hematologic malignancies. Importantly, CD4+ T cells from a metastatic RCC patient stimulated with peptides from a HLA-unrelated RCC line strongly recognized and killed the autologous tumor, establishing the independence of HLA-class II matching in a clinical setting.

The RCC-reactive CD4+ T cells obtained in this study recognize a very broadly expressed antigen(s). Due to the ubiquitous expression, we first excluded that CD4+ T cells recognized processed peptides derived from the FCS present in the TCM used to grow the tumor cells recognized. To this aim, RCC cells and LCLs were cultured for 4 to 7 days in human serum and PHA blasts in FCS, respectively, before testing. Recognition of RCC cells and LCLs and lack of recognition of PHA blasts by RCC-reactive CD4+ T cells did not change in these different culture conditions (data not shown). Next, we evaluated the possibility that CD4+ T cells recognized a known RCC TAA. The RAGE-1 (18), PRAME (15), G250 (13, 24), RU2 (17), and RU2AS (17) antigens were excluded based on their pattern of expression on the tumor cells recognized as tested by reverse transcription-PCR (data not shown). The EphA2 (23), FGF-5 (19), and M-CSF (20) were excluded from the expression data reported in the literature. Recognition of the oncofetal antigen (32) was excluded by experiments, in which RCC-reactive CD4+ T cells, challenged with recombinant oncofetal antigen (the generous gift of Dr. J.H. Coggin, University of South Alabama College of Medicine, Mobile, AL) loaded autologous PBMC or dendritic cells, failed to specifically proliferate or release cytokines (data not shown).

The similar pattern of broad recognition by the different RCC-reactive CD4+ T-cell clones may indicate that they recognize either different or the same epitope(s) of a common or different ubiquitous predominant particularly immunogenic protein(s). The presence into the RCC peptidome of one or more broadly expressed antigens could be also explained with bias related to the peptide purification procedure; indeed, as an example, hydrophilic proteins should be advantaged, whereas hydrophobic proteins may be more easily lost. Immunodominance of shared TAAs as immunogens for the activation of T cells in RCC was also shown in clinical studies that used undefined RCC antigens as cancer vaccines (32–35).

Animal models have shown that both Th1-type and Th2-type cytokines producing CD4+ T cells are effective in antitumor immunity (9, 10). The analysis of cytokine production by RCC-reactive CD4+ T cells showed a heterogeneous pattern of cytokine secretion: Th0 (DRß1*01-restricted clones; Fig. 2B), Th1 (DRß1*11- and DRß3*02-restricted clones; Fig. 2A and B), and Th0/Th2 (DRß1*08-restricted clones and oligoclonal line from Pt3; Fig. 2A and C). Recently, RCC-reactive CD4+ T-cell clones with similar heterogeneous patterns of cytokine secretion (except for the IL-10 production) were obtained from the blood of a patient with advanced RCC vaccinated with irradiated autologous GM-CSF gene-transduced tumor cells (35). The authors hypothesized that the induction of both T helper phenotypes could be either related to the effects of GM-CSF on the differentiation of dendritic cells or dependent, when systemic antitumor immunity is induced, on the activation of both T helper effector functions. The selective differentiation of different T helper subtypes is established during priming and can be influenced by a variety of factors. Cytokine environment is believed to be the major variable influencing T helper development (reviewed in ref. 36), also depending on activation of different dendritic cell subsets (reviewed in ref. 37) and kinetic of dendritic cell activation (reviewed in ref. 38), but costimulatory signals (reviewed in ref. 39), the dose of the antigen (reviewed in ref. 39), the period of TCR engagement (reviewed in ref. 38), and selection for TCR affinity (40) have also been put forward as important factors in cytokine polarization. In our system, other factors, different from the cytokine milieu produced by peptide-loaded dendritic cells, seem to have influenced cytokine polarization of the clones. Indeed, the different clones derive from the same polyclonal population obtained by primary stimulation with NTP-loaded autologous dendritic cells. Different peptides with different TCR and MHC binding affinity may have directed the cytokine polarization. It is interesting to note that all clones with the same HLA-DR restriction even if bearing different TCR Vß chains had the same pattern of cytokine secretion, suggesting that the HLA-DR restriction molecules more than TCR Vß usage may have directed the fate of polarization of CD4+ T cells. Collectively, our results support the hypothesis that, also in the human system, activation of both T helper effector functions is implicated in antitumor immunity.

In addition, we show here that all clones exert strong cytolytic activity, adding plasticity in the effector function of the different CD4+ T-cell subsets. Indeed, although lytic effector functions have been attributed to Th1 CD4+ T cells (41), we show here that also Th0 and Th2 clones are endowed with strong killing activity. We are currently evaluating whether the different T helper subsets use different mechanisms of killing.

In conclusion, our data support the development of a "universal" ready-to-use vaccine based on allogenic RCC peptidome-loaded dendritic cells easily produced in good manufacturing procedure conditions, in RCC, and possibly in other neoplastic diseases. Furthermore, the CD4+ T-cell clones generated here are useful tools for tumor antigen identification.


   Acknowledgments
 
We thank all donors for giving their blood samples for these experiments and Matteo Bellone, Angelo Manfredi, and Catia Traversari for critical reading of the manuscript.


   Footnotes
 
Grant support: Cancer Research Institute Preclinical grant, Italian Association for Cancer Research, European Community (Dendritic Cells for Novel Immunotherapies), Fondazione Berlucchi, Fondazione CARIPLO, Compagnia di San Paolo, and Italian Ministry of Health.

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.

Note: E. Tassi and V. Facchinetti contributed equally to this work. Current address for V. Facchinetti: Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas.

7 L. De Monte, S. Seresini, G. Concogno, and M.P. Protti, unpublished data. Back

Received 4/24/06; revised 5/29/06; accepted 6/24/06.


   References
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 Abstract
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 References
 

  1. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma. N Engl J Med 1996;335:865–75.[Free Full Text]
  2. Whang YE, Godley PA. Renal cell carcinoma. Curr Opin Oncol 2003;15:213–6.[CrossRef][Medline]
  3. de Riese W, Goldenberg K, Allhoff E, et al. Metastatic renal cell carcinoma (RCC): spontaneous regression, long-term survival, and late recurrence. Int Urol Nephrol 1991;23:13–25.[Medline]
  4. Finke JH, Tubbs R, Connelly B, Pontes E, Montie J. Tumor-infiltrating lymphocytes in patients with renal-cell carcinoma. Ann N Y Acad Sci 1988;532:387–94.[Medline]
  5. Kowalczyk D, Skorupski W, Kwias Z, Nowak J. Flow cytometric analysis of tumour-infiltrating lymphocytes in patients with renal cell carcinoma. Br J Urol 1997;80:543–7.[Medline]
  6. Van den Hove LE, Van Gool SW, Van Poppel H, et al. Phenotype, cytokine production, and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma. Clin Exp Immunol 1997;109:501–9.[CrossRef][Medline]
  7. Margolin KA. Interleukin-2 in the treatment of renal cancer. Semin Oncol 2000;27:194–203.[Medline]
  8. Fossa SD. Interferon in metastatic renal cell carcinoma. Semin Oncol 2000;27:187–93.[Medline]
  9. Pardoll DM, Topalian SL. The role of CD4+ T cell responses in antitumor immunity. Curr Opin Immunol 1998;10:588–94.[CrossRef][Medline]
  10. 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.[Free Full Text]
  11. Van Der Bruggen P, Zhang Y, Chaux P, et al. Tumor-specific shared antigenic peptides recognized by human T cells. Immunol Rev 2002;188:51–64.[CrossRef][Medline]
  12. Schmidt SM, Schag K, Muller MR, et al. Induction of adipophilin-specific cytotoxic T lymphocytes using a novel HLA-A2-binding peptide that mediates tumor cell lysis. Cancer Res 2004;64:1164–70.[Abstract/Free Full Text]
  13. Vissers JL, De Vries IJ, Schreurs MW, et al. 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.[Abstract/Free Full Text]
  14. Ronsin C, Chung-Scott V, Poullion I, Aknouche N, Gaudin C, Triebel F. A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ. J Immunol 1999;163:483–90.[Abstract/Free Full Text]
  15. Ikeda H, Lethe B, Lehmann F, et al. Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 1997;6:199–208.[CrossRef][Medline]
  16. Morel S, Levy F, Burlet-Schiltz O, et al. Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells. Immunity 2000;12:107–17.[CrossRef][Medline]
  17. Van Den Eynde BJ, Gaugler B, Probst-Kepper M, et al. A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription. J Exp Med 1999;190:1793–800.[Abstract/Free Full Text]
  18. Gaugler B, Brouwenstijn N, Vantomme V, et al. A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma. Immunogenetics 1996;44:323–30.[CrossRef][Medline]
  19. Hanada K, Yewdell JW, Yang JC. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 2004;427:252–6.[CrossRef][Medline]
  20. Probst-Kepper M, Stroobant V, Kridel R, et al. An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes. J Exp Med 2001;193:1189–98.[Abstract/Free Full Text]
  21. Brandle D, Brasseur F, Weynants P, Boon T, Van den Eynde B. A mutated HLA-A2 molecule recognized by autologous cytotoxic T lymphocytes on a human renal cell carcinoma. J Exp Med 1996;183:2501–8.[Abstract/Free Full Text]
  22. Gaudin C, Kremer F, Angevin E, Scott V, Triebel F. A hsp70-2 mutation recognized by CTL on a human renal cell carcinoma. J Immunol 1999;162:1730–8.[Abstract/Free Full Text]
  23. Tatsumi T, Herrem CJ, Olson WC, et al. Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma. Cancer Res 2003;63:4481–9.[Abstract/Free Full Text]
  24. Vissers JL, De Vries IJ, Engelen LP, et al. Renal cell carcinoma-associated antigen G250 encodes a naturally processed epitope presented by human leukocyte antigen-DR molecules to CD4(+) T lymphocytes. Int J Cancer 2002;100:441–4.[CrossRef][Medline]
  25. Frankenberger B, Regn S, Geiger C, et al. Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells. World J Urol 2005;23:166–74.[CrossRef][Medline]
  26. Protti MP, Imro MA, Manfredi AA, et al. Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro. Cancer Res 1996;56:1210–3.[Abstract/Free Full Text]
  27. Imro MA, Manici S, Russo V, et al. Major histocompatibility complex class I restricted cytotoxic T cells specific for natural melanoma peptides recognize unidentified shared melanoma antigen(s). Cancer Res 1999;59:2287–91.[Abstract/Free Full Text]
  28. Imro MA, Manici S, Bellone M, et al. Natural melanoma peptides pulsed dendritic cells stimulate both CD8+ and CD4+ tumor specific T cells. J Leukoc Biol 1998;S2:88.
  29. Protti MP, Manfredi AA, Straub C, Wu XD, Howard JF, Jr., Conti-Tronconi BM. Use of synthetic peptides to establish anti-human acetylcholine receptor CD4+ cell lines from myasthenia gravis patients. J Immunol 1990;144:1711–20.[Abstract]
  30. Manici S, Sturniolo T, Imro MA, et al. Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11. J Exp Med 1999;189:871–6.[Abstract/Free Full Text]
  31. Wack A, Montagna D, Dellabona P, Casorati G. An improved PCR-heteroduplex method permits high sensitivity detection of clonal expansion in complex T cell population. J Immunol Methods 1996;196:181–92.[CrossRef][Medline]
  32. Holtl L, Zelle-Reiser C, Gander H, et al. Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous dendritic cells. Clin Cancer Res 2002;8:3369–76.[Abstract/Free Full Text]
  33. Su Z, Dannull J, Heiser A, et al. Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res 2003;63:2127–33.[Abstract/Free Full Text]
  34. Zhou X, Jun DY, Thomas AM, et al. Diverse CD8+ T-cell responses to renal cell carcinoma antigens in patients treated with an autologous granulocyte-macrophage colony-stimulating factor gene-transduced renal tumor cell vaccine. Cancer Res 2005;65:1079–88.[Abstract/Free Full Text]
  35. Mautner J, Jaffee EM, Pardoll DM. Tumor-specific CD4+ T cells from a patient with renal cell carcinoma recognize diverse shared antigens. Int J Cancer 2005;115:752–9.[CrossRef][Medline]
  36. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol 1994;12:635–73.[CrossRef][Medline]
  37. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells. Cell 2001;106:263–6.[CrossRef][Medline]
  38. Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of TH1, TH2, and nonpolarized T cells. Nat Immunol 2000;1:311–6.[CrossRef][Medline]
  39. Constant SL, Bottomly K. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 1997;15:297–322.[CrossRef][Medline]
  40. Boyton RJ, Altmann DM. Is selection for TCR affinity a factor in cytokine polarization? Trends Immunol 2002;23:526–9.[CrossRef][Medline]
  41. Appay V. The physiological role of cytotoxic CD4(+) T-cells: the holy grail? Clin Exp Immunol 2004;138:10–3.[CrossRef][Medline]



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