Preprocalcitonin signal generates a cytotoxic T lymphocyte-defined tumor epitope processed by a proteasome-independent pathway

Faten El Hage†, Vincent Stroobant‡, Isabelle Vergnon†, Jean-Franc¸ois Baurain§, Hamid Echchakir†¶, Vladimir Lazarʈ, Salem Chouaib†, Pierre G. Coulie§††, and Fathia Mami-Chouaib†,††‡‡

†Institut National de la Sante´et de la Recherche me´dicale, Unite´753, Laboratoire Immunologie des Tumeurs humaines: Interaction effecteurs cytotoxiques-syste`me tumoral, Institut Fe´de´ ratif de Recherche-54, Institut Gustave Roussy, F-94805 Villejuif, France; ‡Ludwig Institute for Cancer Research, Brussels Branch, B-1200 Brussels, Belgium; §Universite´Catholique de Louvain and de Duve Institute, B-1200 Brussels, Belgium; and ʈUnite´deGe´ nomique Fonctionnelle, Institut Fe´de´ ratif de Recherche-54, Institut Gustave Roussy, F-94805 Villejuif, France

Communicated by Jean Dausset, Centre d’Étude du Polymorphisme Humain, Paris, France, May 16, 2008 (received for review November 7, 2007) We identified an antigen recognized on a human non-small-cell Most antigenic recognized by CD8 T cells originate lung carcinoma by a cytotoxic T lymphocyte clone derived from from degradation in proteasomes of intracellular mature pro- autologous tumor-infiltrating lymphocytes. The antigenic peptide teins and their transport, by the transporter associated with is presented by HLA-A2 and is encoded by the CALCA gene, which antigen processing (TAP) from the cytosol into the endoplasmic codes for and for the ␣-calcitonin gene-related peptide. reticulum (ER) (for review, see ref. 13). The resulting peptides The peptide is derived from the carboxy-terminal region of the of 9 to 10 amino acids bind MHC class I (MHC-I) molecules and preprocalcitonin signal peptide and is processed independently of are then conveyed to the cell surface. An increasing number of proteasomes and the transporter associated with antigen process- epitopes recognized by tumor-reactive T cells has been reported ing. Processing occurs within the of all to result from nonclassical mechanisms acting at the transcrip- tumoral and normal cells tested, including dendritic cells, and it tion, splicing, or translational levels (for review, see ref. 14). It involves and the , signal peptide is noteworthy that several tumor epitopes are poorly processed peptidase. The CALCA gene is overexpressed in medullary thyroid by dendritic cells (DCs), which are unique in their capacity to carcinomas and in several lung carcinomas compared with normal process Ags and to prime CD8 T cells, but which constitutively express immunoproteasomes (15, 16). In this article, we identi- tissues, leading to recognition by the T cell clone. This new epitope fied an antigenic peptide recognized on a human LCC by an is, therefore, a promising candidate for cancer immunotherapy. autologous CTL clone. This epitope is derived from the carboxy (C)-terminal region of the calcitonin (CT) precursor signal ͉ ͉ antigen processing signal peptidase sequence and is processed by a proteasome-independent path- way involving signal peptidase (SP) and signal peptide peptidase he analysis of tumor-reactive cytotoxic T lymphocytes (SPP). T(CTLs) derived from patients with various solid tumors had led to promising new treatments for malignant diseases, by either Results expanding the T cells in vitro before transferring them with IL-2 A CTL Clone Recognizing Autologous Lung Carcinoma Cells. Patient into patients (1) or identifying their target antigens (Ags), which Heu is a now disease-free lung cancer patient 12 years after can then be used in therapeutic vaccines. A large number of resection of the primary tumor. LCC cell line IGR-Heu was tumor-associated Ags recognized by CTLs has been identified derived from a tumor resected from the patient in 1996. Mono- mainly in malignant melanoma. Unfortunately, clinical studies nuclear cells infiltrating the primary tumor were isolated and indicate that, despite an increase in the frequency of antitumor stimulated with irradiated IGR-Heu tumor cells, irradiated CD8 T cells, the efficacy of current therapeutic vaccines remains autologous EBV-transformed B cells, and IL-2. Responder limited (2). Current studies are focusing on a better understand- lymphocytes were cloned by limiting dilution. Several tumor- specific CTL clones were obtained and classified into three ing of the mechanisms of rare tumor regressions observed (3, 4), ␤ the activation state of anti-vaccine CD8 T cells, and their groups on the basis of their TCRV usage (5). We previously capacity to migrate to the tumor site. reported that the first two groups of clones recognized an antigenic peptide encoded by a mutated ␣-actinin-4 gene (9, 17). Much less is known about the antigenicity and susceptibility to Here, we analyze the third group of clones, including Heu161, CTL attack of human lung tumors. Most of these tumors are which expresses a V␤3-J␤1.2 TCR. CTL Heu161 lysed the non-small-cell lung carcinomas (NSCLCs), a large group that autologous tumor cell line, but not autologous EBV-B cells or includes squamous-cell, adeno-cell, and large-cell (LCC) carci- the NK-target K562 (Fig. 1A). The recognition of IGR-Heu by nomas. NSCLCs can be infiltrated by T cell antigen receptor the CTL clone was inhibited by anti-HLA-A2 mAb (9). (TCR) ␣/␤ T cells (5). The identified T cell target Ags include peptides encoded by the HER2/neu protooncogene (6), which is Identification of the Gene Encoding the Ag Recognized by Heu161 CTL. overexpressed in many lung tumors, and by several genes that A cDNA library from IGR-Heu cells was cloned into expression were found to contain a point mutation in tumor cells compared plasmid pCEP4 (9) and divided into 264 pools of Ϸ100 recom-

with autologous normal cells. These mutated genes include IMMUNOLOGY elongation factor 2 (7), malic enzyme (8), ␣-actinin-4 (9), and NFYC (10). In addition, several cancer/germ-line genes are Author contributions: S.C., P.G.C., and F.M.-C. designed research; F.E.H., I.V., and H.E. expressed in NSCLCs (11, 12), which should lead to the presence performed research; J.-F.B. and V.L. contributed new reagents/analytic tools; F.E.H. and V.S. of tumor-specific Ags at the surface of cancer cells. However, analyzed data; and P.G.C. and F.M.-C. wrote the paper. spontaneous T cell responses against MAGE-type Ags have not The authors declare no conflict of interest. been observed in lung cancer patients thus far. Therefore, ¶Present address: Nokad SA, 91058 Evry, France. identification of new lung cancer Ags, in particular those shared ††P.G.C. and F.M.-C. contributed equally to this work. by tumors of several patients, would help the design and immu- ‡‡To whom correspondence should be addressed. E-mail: [email protected]. nological monitoring of vaccination strategies in lung cancer. © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802753105 PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10119–10124 Downloaded by guest on September 28, 2021 Fig. 1. CTL clone Heu161 recognizes an Ag expressed by IGR-Heu autologous tumor cells. (A) Cytotoxic activity of CTL Heu161 toward tumor cells IGR-Heu, 51 autologous Heu-EBV B cells, and K562. Cytotoxicity was measured by Cr- Fig. 2. Identification of the gene segment encoding the epitope recognized release assay at indicated E/T ratios. (B) Identification of a cDNA clone encod- by Heu161 CTL. (A) Representation of cDNA 150 compared to the CT and ing the Ag recognized by the CTL clone. Heu161 (3,000 cells) was stimulated ␣-CGRP gene and transcripts. Numbered boxes represent exons. Arrows indi- for 24 h by 293-EBNA (30,000 cells) cotransfected with vectors pCEP4 contain- cate forward (O) and reverse (R) primers used in RT-PCR. (B) Minigenes used to ing cDNA clone 150 and pcDNA3.1 containing HLA-A2. Control stimulator cells identify the region coding for the antigenic peptide. A series of truncated included IGR-Heu and 293-EBNA transfected with cDNA 150 or HLA-A2 alone. constructs were cotransfected into 293-EBNA cells with HLA-A2. The corre- ␤ The concentration of TNF released in medium was measured. Data are sponding encoded sequences are shown. Recognition by Heu161 was assessed representative of five independent experiments. by using TNF␤ assay as in Fig. 1B. Statistical analyses were performed by using a Mann–Whitney U test (P Ͻ 0.01). Data are representative of three indepen- dent experiments. binant clones. DNA prepared from each pool was transfected into 293-EBNA cells, together with an HLA-A*0201 construct. CTL Heu161 was added to the transfectants, and then TNF␤ was efficient than the 11-mer by a factor of Ϸ3 (Fig. 3B). We measured. A large proportion (85 of 264) of cDNA pools proved concluded that the optimal peptide recognized by Heu161 was positive, suggesting that a surprisingly high frequency of Ϸ0.4% VLLQAGSLHA or ppCT16–25. It contains one of the consensus of cDNA clones encoded the Ag. One pool of cDNA was HLA-A2 peptide-binding motifs, Leu, Ile, or Met in position 2, subcloned, and a cDNA clone named 150 was isolated (Fig. 1B). but fails to contain the peptide-binding motif, Leu or Val in cDNA 150 was 956 bp long and contained a polyadenylation position 10, and has therefore a moderate ability to bind to signal and a poly(A) tail. Its sequence corresponded to that of HLA-A2 (data not shown). This peptide corresponds exactly to gene CALCA, which codes for both the calcium-lowering hor- the C-terminal part of the ppCT signal peptide (19). mone CT and the CT gene-related peptide ␣ (␣-CGRP). A primary transcript is spliced into either CT or ␣-CGRP mRNA Processing of the Antigenic Peptide. This localization of the peptide through tissue-specific alternative RNA processing (18). cDNA in the suggested that it could be processed in the ER 150 contains the complete CT coding sequence, spanning exons independently of proteasomes and TAP. To examine the in- 2, 3, and 4 of gene CALCA (Fig. 2A). However, its 5Ј end differs volvement of proteasomes, IGR-Heu cells were treated with from that of the CT cDNA sequences present in databanks by the specific proteasome inhibitor epoxomicin (Fig. 4A). Epoxomicin presence of an intronic sequence of 213 nucleotides.

Identification of the Antigenic Peptide. The region coding for the antigenic peptide was identified with truncated cDNA fragments cloned into expression plasmids and cotransfected with the HLA-A2 construct into 293-EBNA cells. As shown in Fig. 2B,a fragment encoding the first 41 residues of preproCT (ppCT) transferred the expression of the Ag, whereas a fragment encoding the first 35 residues did not. We then prepared a series of CT cDNA fragments truncated at their 5Ј end and engineered to contain an initiation codon and a Kozak consensus sequence. Screening with the CTL clone indicated that the antigenic peptide was contained within residues 9–47 (Fig. 2B). Further trimming narrowed down the peptide-encoding region to resi- dues 9–38 (Fig. 2B). Among a set of overlapping peptides Fig. 3. Identification of the peptide recognized by clone Heu161. (A) CTL covering this region, two were recognized by clone Heu161, stimulation with purified synthetic peptides. Peptides were loaded on allo- VLLQAGSLHA and LVLLQAGSLHA, which are identical, geneic HLA-A2 MZ2-MEL.3.1 melanoma cells for 1 h at room temperature before addition of Heu161 at 1/10 E/T ratio. TNF␤ release was measured 24 h but with an additional Leu in the latter peptide. As shown in Fig. later. (B) Cytotoxicity of Heu161 toward peptide-pulsed cells. 51Cr-labeled 3A, both peptides sensitized HLA-A2 melanoma cells to recog- Heu-EBV B cells were incubated over 1 h with the indicated concentrations of nition by Heu161, with half-maximal effects obtained with Ϸ10 peptides before addition of CTL at 10:1 E/T ratio. Data are representative of nM of peptide. In a lysis assay, the decamer was slightly more four independent experiments.

10120 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802753105 El Hage et al. Downloaded by guest on September 28, 2021 Fig. 4. Processing of the ppCT16–25 peptide is proteasome- and TAP- independent. (A) IGR-Heu cells were incubated in the presence or absence of the proteasome inhibitor epoxomicin (10 ␮M), and then Heu161 cells were added at 1/10 E/T ratio. The autologous Heu127 clone was included as a positive control. TNF␤ released in medium after 24 h of culture was measured. (B) 293-EBNA cells were cotransfected with pCEP4 containing either cDNA 150 (Upper) or the mutated ␣-actinin-4 cDNA (Lower), with HLA-A2 construct, and with various amounts of vector pBJi-neo containing IPC47 cDNA. Heu161 (Upper) or Heu127 (Lower) were then added at 1/10 E/T ratio. TNF␤ released after 24 h of culture was measured. Controls included 293-EBNA cells trans- fected with HLA-A2 or pBJi-neo-IPC47 alone and incubation of transfectants with either ppCT or ␣-actinin-4 peptides. Data correspond to one of four independent experiments.

had no effect on recognition by anti-ppCT CTL (Fig. 4A). In contrast, it strongly inhibited stimulation of another autologous CTL clone, Heu127, which recognizes a mutated ␣-actinin-4 peptide (9). This finding was expected because ␣-actinin-4 is a cytosolic protein that is degraded, at least in part, in proteasomes (20). These results suggest that the processing of the ppCT16–25 peptide does not require proteasomal activity. The involvement of TAP was tested by cotransfecting into 293-EBNA cells Fig. 5. Processing of the ppCT16–25 epitope involves SP and SPP. (A) Process- constructs coding for the antigenic peptide, HLA-A2, and the ing of the ppCT16–25 epitope is SP-dependent. IGR-Heu cells were incubated immediate-early protein ICP47 of herpes simplex virus type 1, with the SP inhibitor DCI (250 ␮M) before addition of anti-ppCT (Left)or which binds to and inhibits human TAP (21). As shown in Fig. anti-␣-actinin-4 (Right) CTL. (B) Processing of the ppCT16–25 epitope involves 4B, cotransfecting ICP47 had no detectable effect on recognition SPP. Analysis of SPP mRNA expression by real-time RT-PCR analysis. Total RNA of the transfectants by anti-ppCT CTL, whereas it strongly extracted from IGR-Heu, electroporated or not with siRNA targeting SPP inhibited that by the anti-␣-actinin-4 CTL. These results strongly (siRNA-S1 and siRNA-S2), was reverse-transcribed and quantified by TaqMan. (C) Effect of SPP knockdown on tumor cell recognition. (Left) Lytic activity of suggest that the processing of the ppCT16–25 epitope is TAP- Heu161 and Heu127 against IGR-Heu, electroporated or not with siRNA-S1 or independent. control siRNA, determined by 51Cr-release assay at 10/1 E/T ratio. (Right) Because the C terminus of antigenic peptide VLLQAGSLHA Cytotoxicity of Heu161 against IGR-Heu, electroporated or not with siRNA-S1, corresponded to the C terminus of the ppCT signal sequence siRNA-S2, or control siRNA, determined by 51Cr-release assay at indicated E/T (19), it was expected to be generated by type I SP, which cuts off ratios. (D) Production of IFN␥ by Heu161 and Heu127 clones stimulated with signal peptides from secretory on the luminal side of the tumor cells electroporated or not with siRNA-S1 or control siRNA. (E) The ER membrane (22). SP involvement was tested by using the ppCT16–25 peptide is located at the C terminus of the signal sequence of the CT serine protease inhibitor dichloroisocoumarin (DCI) (23). Re- hormone precursor. The optimal peptide recognized by Heu161 is boxed. Arrows indicate the SP and the approximate SPP cleavage sites. The n, h, and markably, preincubation of IGR-Heu cells with DCI rendered c regions in the ppCT signal peptide were predicted by using SignalP 3.0

them resistant to lysis by the anti-ppCT CTL (Fig. 5A). The same software. Data are representative of three independent experiments. IMMUNOLOGY treatment had only a moderate effect on recognition by the Heu127 clone (Fig. 5A), and this outcome probably resulted from a slight decrease in MHC-I expression on DCI-treated IGR-Heu with two distinct siRNA. siRNA-S1 and siRNA-S2 tumor cells (data not shown). These results are compatible with specifically inhibit SPP expression at both RNA (Fig. 5B) and involvement of SP in processing the ppCT16–25 peptide. After protein (data not shown) levels. The down-regulation of SPP cleavage by SP, some of the signal sequences inserted in the ER resulted in a strong decrease in the sensitivity of the tumor cells membrane in a type II or loop-like orientation can be further to lysis by the anti-ppCT, but not by the anti-␣-actinin-4 CTL cleaved by the intramembrane protease SPP (reviewed in ref. (Fig. 5C). Similar inhibition was observed when tumor cells were 24). We therefore specifically knock down SPP expression in used to stimulate production of IFN␥ by CTL (Fig. 5D). Partial

El Hage et al. PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10121 Downloaded by guest on September 28, 2021 Table 1. Expression of the CT transcript in lung tumors Table 2. Relative expression of CT transcript in tumor cell lines and samples Variable Tumor samples Tumor cell lines Relative expression NSCLC Variable Histological type of CT transcript SCC 7/122 0/3 ADC 10/61 0/7 Tumor cell lines LCC 2/8 1/5 IGR-Heu LCC 191.34 Undifferentiated carcinomas 1/3 — DMS53 SCLC 116.97 SCLC 3/5 4/23 TT MTC 259.57 Neuroendocrine tumors 3/6 — Tumor samples Bronchioalveolar tumors 1/4 — NSCLC 1 (Heu-T) LCC 14.93 CT was tested by RT-PCR. 2 LCC 0.02 3 SCC 0.28 inhibition of Heu161 reactivity correlates with partial inhibition 4 SCC 0.82 of SPP protein expression in siRNA-treated IGR-Heu cells. This 5 SCC 0.11 finding may be due to the relative stability of SPP homodimers 6 SCC 0.17 in ER membrane (25). Together, these results indicate that the 7 SCC 0.02 8 ADC 19.43 ppCT16–25 peptide was most likely processed by SP and SPP within the ER (Fig. 5E). 9 ADC 4.86 10 ADC 0.02 11 ADC 0.00 Expression of the CT Gene Product in Tumor Samples. Expression of the CT transcript was tested in a panel of lung carcinoma samples 12 ADC 12.82 13 ADC 9.92 and cell lines by RT-PCR. Twenty-seven of 209 tumor samples 14 ADC 29.65 and 5 of 38 cell lines were positive (Table 1). Quantitative gene 15 ADC 1.00 expression analysis of the CT transcript was then carried out on 16 ADC 7.26 some of the positive samples (Table 2). Levels of CT gene 17 Undifferentiated 7.57 expression in the three cell lines tested, namely, LCC IGR-Heu, 18 Undifferentiated 13.64 SCLC DMS53, and medullary thyroid carcinoma (MTC) TT, SCLC were at least 100-fold higher than those found in normal human 19 SCLC 0.14 thyroid. It is noteworthy that the level of expression observed in 20 Neuroendocrine 2.27 the lung carcinoma cell lines was similar to that observed in the 22 Neuroendocrine 0.74 MTC cell line (Table 2). High levels of expression of the CT 23 Bronchioalveolar 0.02 transcript also were detected in the tumor of patient Heu Normal tissues (Heu-T) and in several lung cancer samples (Table 2). Pool of human lung Lung 0.00 Next, we tested whether CTL Heu161 also could recognize Pool of human thyroid Thyroid 1.15 other HLA-A2 cells that overexpressed the CT gene. As shown in Fig. 6A, Heu161 efficiently lysed MTC cells TT. As expected, Quantitative RT-PCR analysis of CT transcript in tumor cell lines and sam- Heu161 did not lyse HLA-A2Ϫ DMS53 cells, but did recognize ples. Normalized copy numbers of CT transcript are shown. The values of CT Ͻ these cells after transfection with an HLA-A2 construct (Fig. transcript that are statistically elevated were shown in bold (P 0.0001 6B). Finally, mature DCs derived from blood monocytes of a according to Mann–Whitney U test). healthy HLA-A2 donor and transfected with the CT cDNA strongly activated Heu161 CTL (Fig. 6C). We conclude that pp␣-CGRP16–25 peptide. It is therefore likely that cells express- processing of the peptide ppCT16–25 occurs in all cells tested, ing the ␣-CGRP, but not the CT transcripts, also can be namely, NSCLC, SCLC, MTC, melanoma, 293 embryonic kid- recognized by CTL such as Heu161. ney cells, and DCs. Therefore, it would appear that all cells We have shown here that the signal sequence of the CT and expressing the CT transcript at high levels can be recognized by ␣-CGRP preprohormones contains an antigenic peptide that the CTL clone described here. can be specifically recognized by CTL on lung or MTC cells Discussion expressing the gene CALCA. Induction of CALCA gene expres- sion in other cell types, such as 293 or DC, also results in CTL CT and ␣-CGRP polypeptides are encoded by the same gene, recognition. On the basis of the expression profile of its encoding CALCA, which includes five introns and six exons (26). Exons 1, 2, 3, and 4 are joined to produce the CT mRNA in thyroid C cells, gene, the ppCT peptide is a neuroendocrine differentiation Ag. whereas exons 1, 2, 3, 5, and 6 form the ␣-CGRP mRNA in Several tissue differentiation Ags have been found to be recog- neuronal cells (27). Mature ␣-CGRP is an endogenous vasodi- nized by tumor-specific CTL on melanomas. They are encoded latory peptide widely distributed in the body. CT is a hormone by genes with melanocyte-specific expression, such as , MART1 primarily involved in protecting the skeleton during periods of Melan-A , Pmel17/gp100, TRP-1, and TRP-2. Other ex- ‘‘calcium stress’’ (28, 29). In humans, it is synthesized as a ppCT, amples include gene PSA in prostate and CEA in gut carcinomas. which includes a signal sequence of 25 amino acids, and proCT, The ppCT16–25 peptide is the first differentiation Ag recognized comprising an N-terminal region, CT (32 amino acids), and a by CTL in lung cancer, and it is a promising candidate for C-terminal peptide (26). CT was known to be produced at high immunotherapy. Patient Heu mounted a spontaneous CTL levels by MTC and, more surprisingly, by some lung carcinomas response to this Ag without clinical autoimmunity. Whether this (30, 31). Here, we confirmed, by using quantitative RT-PCR, remains true with very immunogenic vaccination modalities or that gene CALCA was expressed at high levels in several NSCLC adoptive transfer of a high number of specific T cells warrants and SCLC. It is noteworthy that CT and ␣-CGRP preprohor- careful examination. Relevant information may come from mones share their 75 N-terminal residues encoded by CALCA vaccination studies against MTC carried out with the CT exons 2 and 3 and that the peptide ppCT16–25 is also the polypeptide (32).

10122 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802753105 El Hage et al. Downloaded by guest on September 28, 2021 proteasomes and TAP (Fig. 4 A and B) and that therefore presumably occurs in the ER, and (iii) the effect of DCI, which is known to inhibit SP (Fig. 5A). That SP generates the C terminus of the antigenic peptide probably explains why mini- genes coding for peptides shorter than 9–38 did not confer antigenicity, although the antigenic peptide was much smaller (Fig. 2). We have probably defined the sequence that is necessary and sufficient to provide the conformation required for SP activity. Although it is well known from peptide elution ex- periments that MHC-I molecules can be loaded with peptides derived from leader sequences (34, 35) and that some of these peptides can be targeted by CTL (36, 37), little is known about the exact mechanisms of processing of these antigenic peptides. Our results are evidence for direct involvement of SP in this processing. After release from precursor proteins by cleavage with SP, some signal peptides with a type II orientation (i.e., those spanning the ER membrane with the n region exposed toward the cytosol and the c region facing the ER lumen) (24) can undergo intramembrane and be cleaved in the center of their h region by the presenilin-type aspartic protease SPP (38). Thereby, SPP promotes the release of signal peptide fragments from the ER membrane. After cleavage by SPP, signal peptide fragments can be released into either the cytoplasm, to be processed by the proteasome/TAP pathway, or the ER, where they follow TAP-independent processing (24). The substrate Fig. 6. Recognition of allogeneic cells overexpressing CT by Heu161 CTL. (A) spectrum of SPP is thus far limited to a variety of viral proteins, Cytotoxicity of CTL Heu161 against allogeneic MTC (TT) and SCLC (DMS53) cell such as hepatitis C virus (39) and GB virus (40), and signal lines. IGR-Heu cells were included as control. (B) Recognition of HLA-A2- transfected DMS53 by Heu161. DMS53 cells were transfected with HLA-A2 peptides, such as preprolactin (41) and human MHC-I (42). Our before addition of CTL clone at 1/10 E/T ratio. (C) Recognition of mature DC results strongly suggest that the ppCT signal peptide is another expressing CT. Monocytes were isolated from the blood of an HLA-A2 healthy substrate of SPP and that this protease directly processes the donor by using magnetic beads and cultured for 6 days in the presence of 100 ppCT16–25 CTL epitope. ng/ml rIL-4 and 250 ng/ml GM-CSF. After maturation by adding 20 ng/ml TNF␣ Thus far, the only known peptides loaded on HLA molecules for another 3 days, the DC (30,000 cells per well) were transfected with cDNA after processing by SPP are derived from MHC-I. Peptides clone 150 in pCEP4, and the amount of TNF␤ released by Heu161 (3,000 cells per well) was measured 24 h later. Data are representative of three indepen- processed by SPP from the N-terminal portion of the signal dent experiments. sequences of HLA-A, HLA-B, and HLA-C can be loaded onto nonclassical HLA-E molecules (42). This loading is required for HLA-E transfer to the cell surface, where these molecules can It is possible that gene CALCA is overexpressed in some bind the NK inhibitory receptors CD94/NKG2 and block NK neuroendocrine tumors compared with normal thyroid C cells, activity (43). It was shown that HLA-A, HLA-B, and HLA-C which would increase the tumoral selectivity of CTL such as peptides were released into the cytosol and required further Heu161. Our observation by using quantitative RT-PCR that processing by proteasomes and transfer into the ER through tumor cell lines express 100- to 200-fold higher levels of CALCA TAP before HLA-E loading (44). The CTL epitope identified in transcripts than normal thyroid tissue, which can be estimated to the present study derives from the C terminus of the ppCT signal contain Ϸ1% of CT-producing C cells, does not support the sequence (Fig. 5E). Therefore, it is probably released directly concept of overexpression in tumoral versus normal cells. How- into the ER and thus does not require proteasomes and TAP for ever, during the screening of the tumor cDNA library, we were its processing. The proteasome/TAP-independent Ag processing surprised by the high proportion of CT transcripts. Along the pathway leading to CTL recognition of tumor cells seems to same line, during our transfection experiments with cDNA clone operate in all of the cells we tested, including DC. It may lead to 150, we observed that a high level of gene expression was new Ag delivery strategies. required for recognition by Heu161. At this stage, and also considering the unusual mechanism of processing of the anti- Methods genic peptide the efficiency of which could be low, we favor the Cells and Functional Assays. The IGR-Heu cell line was derived from an LCC hypothesis that high levels of CALCA gene expression are indeed sample of patient Heu (9). The Heu161 clone was derived from autologous required for recognition by anti-ppCT16–25 CTL, but that such tumor-infiltrating lymphocytes (5). levels also might be present in some normal cells. Cytotoxic activity was measured by a conventional 4-h 51Cr-release assay (45). IGR-Heu, Heu-EBV, K562, TT, and DMS53 (European Collection of Cell

Another interesting aspect of the ppCT16–25 epitope lies in its IMMUNOLOGY processing. Like that of other proteins, the leader sequence of Cultures) cell lines were used as targets. TNF␤ was measured by using the ppCT is markedly hydrophobic. It mediates binding of the TNF-sensitive WEHI-164c13 cells (46). protein to the membrane of the ER, where nascent polypeptide Construction and Screening of the cDNA Library. The cDNA library from IGR-Heu precursors are processed (for review, see ref. 33). It is then tumor cells was constructed as described previously (9). Plasmid DNA was immediately cleaved by SP (22) at the Ala25–Ala26 site (19). extracted and cotransfected, together with the pcDNA3.1 ppCT16–25 peptide is at the C terminus of the leader sequence. (Invitrogen) containing an HLA-A*0201 cDNA, into 293-EBNA cells (30,000 Several results point to involvement of SP in its processing: (i) the cells per well; Invitrogen). After 24 h, Heu161 (3,000 cells per well) was added. exact match between the SP cleavage site and the peptide C After another 24 h, half of the medium was collected, and its TNF␤ content was terminus (Fig. 3A), (ii) the processing that is independent of measured.

El Hage et al. PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10123 Downloaded by guest on September 28, 2021 Sequence Analysis and Localization of the Antigenic Peptide. cDNA clone 150 aca ttc aga agc agg a) (Fig. 2A) were used. PCR conditions were 5 min at 94°C, was sequenced as described previously (9). To identify the antigenic peptide- followed by 30 cycles consisting of 1 min at 94°C, 2 min at 63°C, 2 min at 72°C, encoding region, a panel of cDNA fragments was amplified from cDNA 150 by and a final elongation step of 10 min at 72°C. PCR. PCR products were cloned into expression plasmid pcDNA3.1 by using the Quantitative PCR analysis was performed by using the forward primer Eukaryotic TOPO TA cloning kit (Invitrogen) and then transferred into the 5Ј-atc ttg gtc ctg ttg cag gc located at the 5Ј end of exon 2 and the reverse Ј Ј pCEP4 expression vector to allow overexpression. primer 5 -tgg agc cct ctc tct ctt gct located at the 3 end of exon 3 of the CALCA gene. The Taq-man probe primer was Fam 5Ј-cct cct gct ggc tgc act ggt g-3Ј Tamra. The amount of RNA samples was normalized by the Chemical Reagents and RNAi. For proteasomes and SP inhibition, 106 tumor amplification of RNA 18S. PCR amplifications were performed as described cells were resuspended in media in the presence or absence of specific inhib- previously (47). itors. Briefly, cells were incubated for2hat37°C either with epoxomicin or DCI (Sigma), washed, resuspended in acid buffer, and then incubated for addi- ACKNOWLEDGMENTS. We thank S. Depelchin, C. Richon, and Dr. D. Grunen- tional3hinthepresence or absence of inhibitors. None of the inhibitors was wald for their help; Drs. K.-I. Hanada, D. Valmori, M. Ayyoub, C. Pinilla, P. toxic at the given concentrations. For SPP inhibition, we used siRNA targeting Romero, J. Riond, J.-E. Gairin, S. Stevanovic, and P. Van Endert for helpful human SPP, siRNA-S1 (5-GACAUGCCUGAAACAAUCAtt-3), and siRNA-S2 (5- discussions; and Dr. V. Braud for critical reading of the manuscript. This work UGAUUGUUUCAGGCAUGUCtg-3) (Ambion). Nontargeting siRNA was used as was supported by Institut National de la Sante´et de la Recherche me´dicale, a negative control as described previously (45). Institut Gustave Roussy, Association de la Recherche contre le Cancer, Ligue Nationale Franc¸aise de Recherche contre le Cancer, Fondation de France, and Cance´ropoˆle île de France and the Institut National du Cancer grants; and RT-PCR Analyses. RT-PCR were performed as described previously (47). For- Association de la Recherche contre le Cancer and Institut National du Cancer ward primer O (5Ј-ggt gtc atg ggc ttc caa aag t) and reverse primer R (5Ј-atc agc fellowships (to F.E.H.).

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