doi:10.1182/blood-2006-11-058750Prepublished online July 12, 2007;2007 110: 2931-2939   

 Christoph Salat, Christian Bogner, Arndt Borkhardt, Hans-Jochem Kolb and Angela M. KrackhardtChristine Hennard, Christina Kuttler, Joachim W. Ellwart, Bernhard Frankenberger, Elfriede Nößner, Ingrid G. Schuster, Dirk H. Busch, Elfriede Eppinger, Elisabeth Kremmer, Slavoljub Milosevic, malignancieshave potent antitumor activity against hematologic and other Allorestricted T cells with specificity for the FMNL1-derived peptide PP2

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IMMUNOBIOLOGY

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2have potent antitumor activity against hematologic and other malignanciesIngrid G. Schuster,1 Dirk H. Busch,2 Elfriede Eppinger,1 Elisabeth Kremmer,1 Slavoljub Milosevic,1 Christine Hennard,1

Christina Kuttler,3 Joachim W. Ellwart,1 Bernhard Frankenberger,1 Elfriede Noßner,1 Christoph Salat,4 Christian Bogner,5

Arndt Borkhardt,6 Hans-Jochem Kolb,7 and Angela M. Krackhardt1

1Institute of Molecular Immunology, Forschungszeutrum fur Umwelt und Gesundheit (GSF)–National Research Center for Environment and Health,Munich; 2Clinical Cooperation Group Immunmonitoring, GSF–National Research Center for Environment and Health and Institute of Microbiology,Technische Universitat, Munich; 3Institute of Biomathematics and Biometry, GSF–National Research Center for Environment and Health, Neuherberg;4Hamato-Onkologische Schwerpunkt-Praxis, Munich; 5Medizinische Klinik III, Innere Medizin mit Schwerpunkt Hamatologie und Onkologie, TechnischeUniversitat, Munich; 6Klinik fur Kinder-Onkologie, -Hamatologie und -Immunologie, Heinrich-Heine-Universitat, Dusseldorf; and 7Clinical Cooperation GroupHamatopoetische Zelltransplantation, GSF–National Research Center for Environment and Health and Medizinische Klinik III mit SchwerpunktHamatologie und Onkologie, Ludwig-Maximilians-Universitat, Munich, Germany

Cell-based immunotherapy in settings ofallogeneic stem cell transplantation ordonor leukocyte infusion has curativepotential, especially in hematologic malig-nancies. However, this approach is se-verely restricted due to graft-versus-hostdisease (GvHD). This limitation may beovercome if target antigens are molecu-larly defined and effector cells are specifi-cally selected. We chose formin-relatedprotein in leukocytes 1 (FMNL1) as atarget antigen after intensive investiga-tion of its expression profile at the mRNAand protein levels. Here, we confirm re-

stricted expression in peripheral bloodmononuclear cells (PBMCs) from healthydonors but also observe overexpressionin different leukemias and aberrant ex-pression in transformed cell lines derivedfrom solid tumors. We isolated allo-restricted T-cell clones expressing asingle defined TCR recognizing a particu-lar HLA-A2–presented peptide derivedfrom FMNL1. This T-cell clone showedpotent antitumor activity against lym-phoma and renal cell carcinoma cell lines,Epstein-Barr virus (EBV)–transformedB cells, and primary tumor samples de-

rived from patients with chronic lympho-cytic leukemia (CLL), whereas nontrans-formed cells with the exception ofactivated B cells were only marginallyrecognized. Allorestricted TCRs withspecificity for naturally presented FMNL1-derived epitopes may represent promis-ing reagents for the development of adop-tive therapies in lymphoma and othermalignant diseases. (Blood. 2007;110:2931-2939)

© 2007 by The American Society of Hematology

Introduction

Although it is controversially discussed whether adaptive immuneresponses in tumor-bearing hosts play a role in controlling growthand recurrence of human tumors,1,2 T cells can be converted tohighly efficient killers of tumors. Donor leukocyte infusions (DLIs)are responsible for a graft-versus-leukemia effect (GvL) afterallogeneic stem-cell transplantation (SCT), representing a therapeu-tic option with curative potential in different diseases at advancedstages.3 Although GvL responses have also been shown forlow-grade lymphomas, improvement of long-term survival afterallogeneic transplantation has not been demonstrated in thesepatients.4-8 This can be attributed primarily to the high treatment-associated mortality due to transfer of T cells recognizing alloge-neic minor histocompatibility (MHC) antigens, thereby causingpotentially life-threatening graft-versus-host disease (GvHD). Forbroader applicability of this therapeutic option, it is thereforeessential to reduce the risk of detrimental GvHD.

One approach to gain a tumor-specific effect while significantlyreducing the risk of GvHD is to generate allorestricted T cells withspecificity for epitopes derived from tumor-associated antigens (TAAs).9

Such allorestricted peptide-specific T cells may display high avidity

toward MHC-presented TAAs, because they have not been negativelyselected in the thymus. Moreover, they can be isolated by tetramers andcloned by limiting dilution to identify the specific TCR responsible fortumor-selective killing.10 The isolation of a TCR with defined specificityfor TAAs facilitates genetic TCR transfer into T-cell lines,11-14 allowingmajor expansion of tumor-specific T cells.

The choice of an appropriate target antigen is of fundamentalimportance for the success of an antitumor immunotherapeuticapproach. So far, there is no universal antigen that serves as a targetantigen in a broad range of malignancies. Many tumor-specificantigens are derived from aberrant expression of cell type–specificproteins and are expressed only in a restricted number of tumortypes, limiting the tumors that can be treated. However, even thepresence of a suitable antigen does not guarantee effective antigenrecognition, because tumor cells evade immune recognition bydown-regulation of the specific target or by inhibition of MHCclass I–restricted antigen presentation. Thus, the level of epitopepresentation in the context of classical or nonclassical MHCmolecules is probably the most important predictor for tumor-specific recognition.15,16 Selecting specificities for a number of

Submitted November 28, 2006; accepted July 9, 2007. Prepublished online asBlood First Edition paper, July 12, 2007; DOI 10.1182/blood-2006-11-058750.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2007 by The American Society of Hematology

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defined epitopes of different target antigens may therefore be offundamental importance for the development of effective therapiesto treat a broad variety of tumors and to reduce the risk of evasionfrom immune-mediated destruction.

We previously identified 14 novel lymphoma-associated antigensusing the SEREX approach in chronic lymphocytic leukemia (CLL).17

One of them, KW13, is homologous to the formin-related protein inleukocytes 1 (FMNL1). FMNL1 belongs to a conserved family offormin-related proteins, which are present in all eukaryotes and regulateactin dynamics.18 Human FMNL1 mRNA has been shown to beselectively expressed in peripheral blood mononuclear cells (PBMCs)and overexpressed in CLL samples as well as in malignant cell lines.17,19

Recently, Gomez et al identified FMNL1 to play a role in centrosomepolarity and cytotoxicity of T cells.20

Here, we demonstrate expression of FMNL1 in differentsubtypes of blood-derived cells and aberrant expression in tumorcells, making FMNL1 an attractive target antigen for allorestrictedimmune responses. In addition, we identified a candidate peptideepitope derived from FMNL1 that is specifically recognized byallorestricted cytotoxic T-cell clones that all display an identicalTCR. This T-cell clone, originating from an HLA-A2� donor,specifically recognized HLA-A2� tumor cell lines derived fromlymphomas and other malignant cells as well as primary tumormaterial specimens from HLA-A2� CLL patients. We observedhigh peptide specificity and restriction to HLA-A2. Several HLA-A2subtypes have been recognized, whereas cross-reactivity againstHLA-A2� allotypes has been observed in only 2 of 16 virus-transformed lymphoblastoid cell lines (LCLs). Altogether, theselected T-cell receptor may be a suitable tool for the developmentof allorestricted target-specific therapies in lymphomas and othermalignant diseases.

Materials and methods

Cells and cell lines

PBMCs from healthy donors as well as patients with CLL, T- and B-cellacute lymphoblastic leukemia (T-ALL and B-ALL, respectively), and acutemyeloid leukemia (AML) were collected with donors’ and patients’informed consent in accordance with the Declaration of Helsinki and afterapproval by the institutional review board of Ludwig-Maximilians-Universitat, Munich, Germany. Patients had diagnosis of CLL, ALL, andAML by morphology, flow cytometric analysis, and cytogenetics. PBMCswere obtained by density gradient centrifugation on Ficoll/Hypaque(Biochrom, Berlin, Germany). T cells and other PBMC subpopulationsfrom healthy donors were isolated by negative or positive magnetic beaddepletion (Invitrogen, Karlsruhe, Germany), and high purity was confirmedby flow cytometric analysis.

Peptide-pulsed T2 cells (ATCC CRL-1992; American Type CultureCollection, Manassas, VA) were used for priming and restimulation ofHLA-A2� T cells. The following cell lines were used for analysis ofFMNL1 expression and as targets for FMNL1-specific T cells: HLA-A2�;Epstein-Barr virus–negative (EBV�) lymphoma cell lines BJ18 and DG75(kindly provided by J. Mautner); HLA-A2� lymphoma cell lines Daudi(ATCC CCL-213) and Raji (ATCC CCL86); chronic myelogenous leuke-mia cell line K562 (ATCC CCL-243); HLA-A2� renal cell carcinomasRCC26 and RCC5321; HLA-A2� renal cell carcinoma KT187 (DKFZ,Heidelberg, Germany); HLA-A2� 143 TK� lung fibroblasts (kindlyprovided by R. Mocikat); 293 HEK embryonal kidney cells (ATCCCRL-1573); HLA-A2� and HLA-A2� EBV-transformed B-cell lines; anddifferent homozygous EBV-transformed B-cell lines.22 The human B-celllines C1R untransfected and transfected with HLA-A*0201, HLA-A*3303,and HLA-A*6601 (kindly provided by S. Stevanovic)23 were used foranalysis of cross-reactivity.

Unspecific stimulation of PBMCs was induced by IL-2 (ChironVaccines International, Marburg, Germany) and OKT3 (ATCC) for 3 days.CLL and normal B cells were stimulated by CD40 ligand–transfectedNIH3T3 fibroblasts.24

Real-time polymerase chain reactionfor FMNL1 expression

To evaluate quantitative mRNA expression of FMNL1, total RNA wasextracted from primary tumor cell material, normal PBMCs, and celllines,25 and cDNA was synthesized by Superscript II reverse transcriptase(Invitrogen) following the manufacturer’s instructions. Detection of FMNL1mRNA was conducted using the Light Cycler PCR Master Mix (Roche,Grenzach-Wyhlen, Germany). Exon-overlapping primers for FMNL1 detec-tion were used (5�-CAAGAACCCCAGAACCAAGGCTCTGG and 3�-CTGCAGGTCGTACGCCTCAATGGC) resulting in a 299-bp amplicon.The amplicon was detected by fluorescence (530 nm) using a double-stranded (ds) DNA binding dye, SYBR Green I with the Light CyclerInstrument (Roche). 18S rRNA was processed as a control for relativequantification. RNA from normal tissue pooled from different donors(Clontech, Palo Alto, CA) was used for determination of FMNL1 expres-sion in nontransformed tissues. Relative quantitative expression wascalculated using the delta-delta Ct (cycle threshold) method.26 Skeletalmuscle was used as reference tissue because it showed the highest Ct fordetection of FMNL1.

FMNL1 cloning, protein expression, monoclonal antibodyproduction, and Western blot

FMNL1 cDNA was amplified by polymerase chain reaction (PCR) andcloned into a modified pTris-His–tagged vector (Invitrogen) resulting in anadditional N-terminal His-Tag. XLI (Stratagene, La Jolla, CA) and Top10E coli bacteria (Invitrogen) were used for transformation and proteinexpression. Purification of the His-tagged protein from supernatants oflysed bacteria was performed by incubation with nickel-NTA-agarose(Qiagen, Hilden, Germany) overnight, followed by imidazole elution anddialysis against PBS over 24 hours. The identity of the purified FMNL1protein was verified by mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF]) (Figure S1A, available on theBlood website; see the Supplemental Materials link at the top of the onlinearticle).

For eukaryotic expression, FMNL1 cDNA was amplified by PCR andcloned into an eukaryotic pCMV-His–tagged expression vector (Invitrogen).

A peptide comprising the following amino acids “KKEAAAQEA-GADT” of the C-terminal end of the FH2 domain of FMNL1 wassynthesized and coupled to KLH or OVA (PSL, Heidelberg, Germany).After immunization of Lou/C rats, myeloma cell line P3X63-Ag8.653(ATCC) was fused to rat immune spleen cells.27 Bound monoclonalantibodies were detected with biotinylated mouse anti–rat IgG subclass-specific antibodies (TIB 170, TIB 173, TIB 174 [ATCC]; R2c, GSF) in aperoxidase reaction (horseradish peroxidase avidin D; Vector, Burlingame,CA). An irrelevant peptide coupled to OVA was used as a negative control.Hybridoma supernatants (dilution, 1:10) were screened by Western blotanalysis for reactivity against the recombinant FMNL1 protein using asecondary peroxidase-conjugated goat antirat antibody at 1:2000 (JacksonImmunoResearch, West Grove, PA). Proteins were detected by chemilumi-nescence. Anti-Xpress antibody (Invitrogen) and anti-His antibody (Invitro-gen) were used for size comparison of bands detected by supernatantscontaining anti-FMNL1–specific antibodies. Several specific antibodieswere identified, and the antibody 6F2 (rat IgG2b subclass) was used furtherin this study (Figure S1B). Actin served as loading control, using a rabbitantihuman actin antibody (Sigma, St Louis, MO) and a secondary antirabbitantibody (Pharmacia, North Peapack, NJ). Prepared cell lysates of cell linesand primary tumor samples were used for FMNL1 protein detection.

Peptides

FMNL1-derived peptides were selected using different prediction algo-rithms for immunogenic epitopes including binding to HLA-A2 (genotype

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A*0201), TAP transportation, and cleavage by the immune proteasome(Table S1).28-32 We chose the following FMNL1-derived peptides forpulsing of antigen-presenting cells: FMNL1-PP1 (VLLEYLAFA), FMNL1-PP2 (RLPERMTTL), FMNL1-PP6 (CVNEIALSL), and FMNL1-PP8(TLLHYLVKV). The peptide HDAC6 (residues 862-871: RLAERMTTR)derived from histone deacetylase 6 (HDAC6) with high homology toFMNL1-PP2, the HLA-A2–restricted influenza-associated peptide Flu(MP58: GILGFVFTL), the HLA-A2–binding Her2-derived peptide 369(residues 369-378: KIFGSLAFL), the HLA-A2–binding tyrosinase-derivedpeptide 369 (residues 369-378: YMNGTMSQV), the predicted BCR-derived peptide BCR (residues 205-214: GLQGTYQDV), and the HLA-B35–binding cytomegalovirus-derived peptide IPS (IPSINVHHY)33 wereused as control antigens. Peptides were synthesized by standard fluorenyl-methoxycarbonyl (Fmoc) synthesis (Metabion, Martinsried, Germany;Biosyntan, Berlin, Germany). Purity was more than 90% as determined byreverse-phase high-performance liquid chromatography (RP-HPLC) andverified by mass spectrometry. Lyophilized peptides were dissolved inDMSO (Sigma) for 2-mM stock solutions.

Peptide binding to HLA-A2

A peptide competition assay was used to assess binding of syntheticpeptides derived from FMNL1.34 T2 cells were prepulsed with the specificFMNL1-derived peptide (100 �M) followed by pulsing with the tyrosinase-derived peptide 369 (1 �M). The tyrosinase 369-specific T-cell clone IVSB(kindly provided by T. Wolfel)35 was assayed at various E/T ratios for lyticactivity in response to peptide- and nonpeptide-prepulsed T2 targets in a4-hour 51Cr-release assay as previously described (Figure S2).17 TheHLA-A2–binding Flu peptide served as a positive control and the HLA-B35–binding cytomegalovirus-derived IPS peptide, as negative control forbinding competition.

Tetramers and antibodies

Tetramers were synthesized as previously reported.36 Specific tetramers wereused for the following peptides:A2-FMNL1-PP1,A2-FMNL1-PP2,A2-FMNL1-PP6, and A2-FMNL1-PP8 as well as the control tetramers detecting T cellsspecific for cytomegalovirus (CMV) pp65, A2-pp65, and B7-pp65.37 Tetramer-binding assays were performed essentially as described38 and analyzed by flowcytometry. The following antibodies were used to characterize PBMC-derivedcells, primary tumor cells, and malignant cell lines: anti–CD3-FITC (UCHT1;BD, Heidelberg, Germany), anti–CD4-FITC (RPA-T4; BD), anti–CD8-FITC (V5T-HIT8a; BD), anti–CD8-PE and -APC (RPA-T8; BD), anti–CD5-FITC(BL1a; Beckman Coulter, Krefeld, Germany), anti–CD19-FITC and -PE (HIB19;BD), anti–CD14-PE (M5E2; BD), anti–CD56-PE (B159; BD), anti–HLA-A2-FITC (BB7.2; ATCC), anti–HLA-A2 unlabeled (BB7.2; ATCC), and anti–��-TCR-FITC (T10B9.1A-31; BD). The anti–HLA-A2 antibody HB54 (ATCC)was used for blocking experiments. A V�14-specific antibody (Dako, Hamburg,Germany) was used for TCR staining of the isolated T-cell clone.

CTLs

Cytotoxic T lymphocyte (CTL) lines were generated using peptide-pulsedT2 cells for specific stimulation. T2 cells were pulsed with specific peptides(10 �M) and used for CTL priming and restimulation at a stimulator–effector cell ratio � 1:10. Cytokines were added as follows: IL-2(50 U/mL) (Chiron Vaccines International), IL-7 (10 ng/mL; Peprotech,London, United Kingdom), and IL-15 (10 ng/m; Peprotech). Peptide-specific T cells were detected by flow cytometry using PE-conjugatedpeptide-presenting HLA-A2� tetramers and sorted by a high-performancecell sorter (MoFlo; Dako). Sorted cells were nonspecifically restimulatedusing pooled allogeneic irradiated PBMCs together with OKT3, IL-2, IL-7,and IL-15. Sorted cells were cultured after limiting dilution in 96-wellplates. T cells were investigated for their cytotoxic potential by 4-hour51Cr-release assays. For cold target assays, T cells were exposed concur-rently to 51Cr-labeled and unlabeled targets in different ratios.

BioPlex analysis and ELISA

For cytokine detection, effector and target cells were incubated at a ratio of 1:5 or1:2 for 24 hours. Supernatants were collected and stored at �20°C until analysis.

The presence of cytokines was analyzed using standard enzyme-linked immu-nosorbent assay (ELISA) for IFN-� (BD) following the recommendations of themanufacturer and the BioPlex system (as described by de Jager et al39; Bio-Rad,Munich, Germany). For BioPlex analysis, the following cytokines were investi-gated: IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulatingfactor (GM-CSF), IFN-�, and TNF-�.

TCR analysis

PCR analysis of expressed TCR chains was performed as previouslydescribed.40 Total RNA from T-cell clones and lines was extractedaccording to the manufacturer’s recommendation (Trizol reagent; Invitro-gen). cDNA was synthesized using superscript II reverse transcriptase(Invitrogen) and oligo dT primers. Subfamily-specific TCR-PCR wasperformed using 34 V� and 37 V� primers followed by gel isolation(NucleoSpin; Macherey-Nagel, Duren, Germany) and direct DNA sequenc-ing of the amplified products. The T-cell receptor nomenclature was usedaccording to the WHO-IUIS nomenclature subcommittee on TCR designa-tion.41 In addition to the single V gene segment-specific primer approach,2 degenerate primers covering 98% of the 55 V� region gene segmentswere used for analysis of TCR �-chains.42

Results

Expression of FMNL1 in normal and malignant tissue

FMNL1 is a formin-related protein in leukocytes that has been shown tobe selectively expressed at the mRNA level in PBMCs, thymus, andspleen of healthy donors and overexpressed in CLL samples andmalignant cell lines.17 We confirmed and extended these data byquantitative RT-PCR and Western blot using a monoclonal antibodydirected against a FMNL1-derived C-terminal peptide. Here, we showdetailed data of FMNL1 expression in normal and malignant tissues(Figure 1A-E). In normal tissue, FMNL1 mRNA is almost exclusivelyexpressed in PBMCs and homing tissues of lymphocytes and myeloidcells, such as bone marrow and thymus (Figure 1A). We then investi-gated the expression of FMNL1 mRNA in PBMCs more in detail. Wetested the expression in PBMCs stimulated with IL-2 and OKT3;PBMC subpopulations including CD19� cells and CD19� cells acti-vated with CD40 ligand; and CD4�, CD8�, CD14�, and mature DCs aswell as CD34� cells (Figure 1B), demonstrating the highest expressionof FMNL1 mRNAin stimulated PBMCs and CD19 cells, whereas otherpopulations showed slightly lower expression. In comparison withnormal PBMCs, FMNL1 mRNA is overexpressed in malignant cells inmore than 60% of CLL samples tested and also in acute B- andT-lymphoblastic leukemia cells (Figure 1C). Furthermore, FMNL1protein expression correlates largely to the mRNA expression data inhealthy and malignant tissues as determined by Western blot analysis(Figure 1D,E). Unstimulated and stimulated PBMCs as well as PBMCsubpopulations showed distinct expression of FMNL1 protein. Therewas no expression of FMNL1 protein in lung fibroblasts and nontrans-fected 293 HEK cells (Figure 1D) as well as 293 HEK cells transfectedwith the control vector (Figure S1B). High protein expression wasobserved in native malignant cells from patients with lymphatic as wellas myeloid leukemias and EBV-transformed B cells. Moreover, FMNL1protein expression has been also detected in lymphoma-derived celllines (BJ18, DG75), and aberrant protein expression was found in renalcell carcinoma lines (RCC26, RCC53) compared with nontransfectedembryonal kidney cells (293 HEK; Figure 1E).

Isolation of FMNL1-specific T cells

Several prediction algorithms, including binding to HLA-A2, TAPtransportation, and proteasomal cleavage, were used for selection

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of FMNL1-derived peptides (Table S1). Binding to HLA-A2 wasinvestigated by a peptide-competition assay (Figure S2), and4 peptides were selected for stimulation assays (FMNL1-PP1,FMNL1-PP2, FMNL1-PP6, and FMNL1-PP8). Peptide-pulsed T2cells were then used for priming and restimulation of T cells. After2 stimulations, specific T cells were detected by tetramers only atvery low numbers (0.01%-0.1%; Figure 2A) and could not beenriched in bulk cultures by additional stimulations (data notshown). However, T cells with specificity toward one peptide,FMNL1-PP2, could be enriched up to 45% by tetramer sorting(Figure 2A) and were subsequently cloned by limiting dilution(Figure 2A). T cells positive for the tetramer A2-FMNL1-PP2 didnot react with A2-pp65– or B7-pp65–specific tetramers (Figure2A). To investigate functional specificity of T-cell lines and clones,we performed a 51Cr-release assay to detect peptide-specific killing.Peptide-specific killing was observed in 10 T-cell clones that havebeen cloned from the primary sorted cell line showing cytotoxicityagainst peptide FMNL1-PP2 (Figure 2B) but not against thecontrol peptide Flu loaded on T2 cells. We also detected cytokinesecretion of IL-2, GM-CSF, IFN-�, and TNF-� in culture superna-

tants of clone SK22 stimulated with T2 cells pulsed with thespecific peptide but not T2 cells pulsed with the control peptide Flu(Figure 2C), representative of 9 other clones.

TCR analysis of FMNL1-PP2–specific T-cell clones

To test the clonal origin of the different T-cell clones, we performed aTCR repertoire of all peptide-specific clones. Thirty-four single V alphagene segment–specific primers were used for the �-repertoire revealingonly one single in-frame V�14 chain (Figure S3A) for all clones tested(n � 10). Sequencing of gel-purified bands revealed one sequenceidentical in all clones tested. For the TCR �-chain repertoire, 2 cloneswere tested using 37 single V-beta segment–specific primers (FigureS3B); for the other clones, degenerative primers were applied (data notshown). Sequencing of all gel-purified bands revealed only one singlein-frame sequence coding for a specific V�14 chain, identical in allclones tested (n � 10). We additionally stained T cells with a V�14-specific antibody revealing a single V�14� population (99%) (FigureS3C). These data demonstrate that all clones derived from the sameclonal T cell.

Figure 1. Expression of FMNL1 in normal and malignant tissue. (A) Relative quantitative mRNA expression of FMNL1 in different tissues pooled from different healthydonors was measured using quantitative real-time PCR. The relative quantitative expression compared with skeletal muscle was calculated using the delta-delta Ct method.(B) Relative quantitative mRNA expression of FMNL1 in stimulated PBMCs and PBMC-derived subpopulations is shown. Subpopulations were isolated by negative (CD4,CD8, CD14, CD19) and positive (CD34) magnetic bead depletion. Monocyte-derived dendritic cells (DCs) were isolated by adhesion, cultured in presence of IL-4 and GM-CSF,and matured with IL1�, IL-6, TNF-�, and prostaglandin E2. For activation of PBMCs, cells were incubated for 3 days using IL-2 and OKT3; B-cell activation was induced byincubation with CD40L-transfected NIH3T3 cells for at least 3 weeks. Error bars indicate the standard deviation of different samples tested. (C) Relative quantitative mRNAexpression of FMNL1 in CLL samples and tumor cells derived from patients with acute B- and T-ALL as well as AML is shown in comparison with normal PBMCs. (D) FMNL1protein expression in PBMC-derived cells, lung fibroblasts, and nontransfected 293 HEK cells by Western blot analysis. Total protein (50 �g) was used for sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE). 293 HEK cells transfected with FMNL1 were used as positive control. Probing with antiactin antibody served asloading control. (E) FMNL1 protein expression in native tumor cells and transformed cell lines by Western blot analysis. Total protein of native leukemic cells (50 �g) and totalprotein of lysed cell lines (100 �g) was used for SDS-PAGE. 293 HEK cells transfected with FMNL1 were used as positive control. Probing with antiactin antibody served asloading control.

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Functional activity and avidity of the isolatedFMNL1-PP2–specific T-cell clone

We next investigated the functional activity of the isolated cloneand detected activity of the T-cell clone down to an effector-targetratio of 0.8:1 (Figure 3A). We examined the functional avidity ofthe T-cell clone by titrating the peptide concentration on target cellsnecessary for peptide-specific killing. Half-maximal lysis of peptide-pulsed T2 cells was observed with a peptide concentration of0.1 �M (Figure 3B) representative of all clones tested (n � 8) andcorresponding to an intermediate functional avidity.

Detection of natural target cells by the FMNL1-PP2-specificT-cell clone

To investigate whether the FMNL1-PP2–specific T-cell clone alsodetects natural targets, different tumor targets, including lymphomacell lines, solid tumor cell lines, as well as native leukemia cells,EBV-transformed B cells, and nontransformed cells were tested.The FMNL1-PP2–specific T-cell clone specifically lysed HLA-A*0201� targets such as EBV-negative lymphoma cell lines, BJ18and DG75, and HLA-A*0201� renal cell carcinoma cell lineRCC26 (Figure 4A). In contrast, the HLA-A2� lymphoma cell lineRaji and the HLA-A2� renal cell carcinoma cell line KT187 werenot recognized (Figure 4A). In addition, HLA-A*0201� EBV-transformed cell lines were lysed (Figure 4B). Moreover, 4 of 5CD40L-activated HLA-A2� CLL cell samples were recognized, asseen by IFN-� release, whereas there was no IFN-� release inresponse to 3 of 3 CD40L-activated HLA-A2� CLL cell samples(Figure 4C).

In healthy tissue, we observed only minor killing of activatedHLA-A2�–activated PBMCs, whereas CD40L-activated HLA-A2� CD19� cells were well recognized (Figure 4D). HLA-A2�

PBMCs and HLA-A2� CD19� cells were not recognized. Similarresults were obtained using PBMC-derived cells from different

HLA-A2� and HLA-A2� donors. HLA-A2� cultured lung fibro-blasts and HLA-A2� embryonal kidney cells (293 HEK) were nottargeted (Figure 4D).

Specificity, TCR dependency, and cross-reactivityof the FMNL1-PP2–specific T-cell clone

To evaluate the specificity of the FMNL1-PP2–specific T-cell clonemore in detail, we used T2 cells as target cells pulsed with a numberof different peptides. Cytotoxicity of peptide-pulsed T2 cells wasdetected only against FMNL1-PP2–pulsed T2 cells. In contrast, T2cells pulsed with a set of irrelevant peptides including the highlyhomologous peptide RLAERMTTR derived from histone-deacety-lase 6 (HDAC6) were not recognized (Figure 5A). To furtherexclude NK- or non-MHC–restricted killing, K562 and Daudi cellswere used as targets in a 51Cr-cytotoxicity assay, neither of whichwas recognized by FMNL1-PP2–specific T-cell clone (Figure 5B).Specific killing of peptide-pulsed T2 cells could be inhibited byadding a monoclonal blocking antibody against HLA-A2 (Figure

Figure 3. The FMNL1-PP2–specific T-cell clone shows high functional activityand an intermediate functional avidity. (A) T2 cells pulsed with FMNL1-PP2(10 �M) were used as targets for the FMNL1-PP2–specific T-cell clone at differenteffector-target ratios in a 51Cr-release assay. Error bars indicate the standarddeviation of tested duplicates. (B) T2 cells were pulsed with FMNL1-PP2 at differentconcentrations and used as targets for the FMNL1-PP2–specific T-cell clone in a51Cr-release assay at an effector-target ratio of 7.5:1. Tyrosinase- and Her2/neu-derived peptides were used as negative controls. Error bars indicate the standarddeviation of tested duplicates.

Figure 2. Isolation of allorestricted FMNL1-specific T cells. (A) FMNL1-PP2–specific CD8� T cells in bulk cultures after 2 stimulations with peptide-pulsed T2 cells, in sortedT-cell lines and in T cells cloned by limiting dilution, were detected by flow cytometry using anti-CD8 monoclonal antibodies and FMNL1-PP2–specific HLA-A2 tetramers. Thenumbers in the fluorescence-activated cell sorting (FACS) plot represent percentage of cells in that quadrant. As control, staining with HLA-A2 and HLA-B7 pp65-specifictetramers was performed. (B) Peptide recognition of cloned T cells was investigated using T2 cells pulsed with FMNL1-PP2 (f) and T2 cells pulsed with Flu (� ) by 51Cr-releaseassay at an effector-target ratio of 7.5:1. Error bars indicate standard deviation of tested duplicates. (C) Cytokine secretion of the FMNL1-PP2–specific T-cell clone SK22 wasinvestigated in response to T2 cells pulsed with the peptide FMNL1-PP2 (f) and pulsed with the Flu peptide (�) at an effector-target ratio of 1:5. Culture supernatants wereharvested after 24 hours and assessed by BioPlex multicytokine analysis.

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5C). In addition, recognition of natural targets could be alsoinhibited by addition of T2 cells pulsed with FMNL1-PP2 as coldtargets but not pulsed with Flu (Figure 5D).

As alloreactive T cells may recognize different epitopes,43 wetested 26 different EBV-transformed cell lines with different HLAallotypes. Several HLA-A2 subtypes were recognized (Figure 6A),whereas HLA-A2–negative samples (Table S2) were only recog-nized in 2 of 16 samples (Figure 6B). One of them (LCL-23) hadthe allotype HLA-A*6802 also belonging to the HLA-A2 super-type family.44 The other restriction element was HLA-A*3303(LCL-14), and restriction to HLA-A*3303 was confirmed usingC1R cells transfected with HLA-A*3303 (Figure S4).

Discussion

Substantial numbers of TAAs recognized by T cells have beenidentified and described.45 However, most of these antigens areexpressed in selected tissues, and universal applicability of anyantigen has not been proved so far. Moreover, it is likely that avariety of antigens needs to be targeted to reduce the risk of tumorescape. Thus, the identification of novel target antigens is offundamental importance for the development of effective immuno-therapies for various types of malignancies.

FMNL1 is a formin-related protein in leukocytes and belongs tothe family of diaphanous-related formins (DRFs).46 Most recently,FMNL1 has been shown to play a role in polarization of T cells.20

We demonstrate expression of FMNL1 also in other PBMC-derived cells such as healthy B cells, monocytes, and dendriticcells, as well as overexpression in leukemic cells of different originand aberrant expression in tumor cell lines. Whereas the function ofFMNL1 needs to be further elucidated, formins have been sug-gested to represent a family of attractive drug targets for thetreatment of actin-dependent processes such as inflammation,metastasis, and invasive diseases.46

As most tumor-associated antigens are self-antigens andtherefore are not recognized by autologous T cells, we used theallorestricted approach to generate T cells with specificity for

epitopes derived from FMNL1.9 Such allorestricted peptide-specific T cells might display high avidity toward MHC-presented TAAs, because they have not been negatively selectedin the thymus.47 In this study, we isolated a peptide-specificallorestricted T-cell clone recognizing the peptide FMNL1-PP2derived from FMNL1. The isolated T-cell clone showed peptide-specific functional activity at very low effector-target ratios.Functional avidity for peptide-pulsed T2 cells was intermediate(half-maximal lysis of 100 nM) which is in common with thefunctional avidity observed for many foreign antigens.43 How-ever, the reduced capacity of FMNL1-PP2 to bind to HLA-A2that we repeatedly observed in the peptide-competition assay incomparison with other tested peptides may have an impact onthe functional avidity of this clone. Nevertheless, the isolatedT-cell clone showed potent cytotoxic activity against cell linesderived from several malignant tissues as well as primary tumormaterial, suggesting that the selected peptide is not only anaturally presented epitope but also an attractive tumor-associated target antigen. T cells specifically killed malignantcell lines derived from lymphomas and renal cell carcinomaaberrantly expressing FMNL1. The T-cell clone also specificallyrecognized FMNL1-expressing EBV-transformed B cells andtumor cells from CLL patients after stimulation with CD40L. Inhealthy donors, activated B cells were best recognized, whereasactivated PBMCs were killed to a lesser extent. No cytotoxicactivity was detected against lung fibroblasts and embryonalkidney cells not expressing FMNL1. The isolated T-cell cloneshowed high peptide specificity as it did not recognize a num-ber of peptides including the highly homologous peptideRLAERMTTR derived from HDAC6. HLA-A2 restriction wasconfirmed using a blocking antibody against HLA-A2 onpeptide-pulsed T2 cells. Investigating cross-reactivity, we ob-served a broader recognition of LCLs with different subtypes ofHLA-A2. This is in common with previous reports demonstrat-ing that a majority of peptides binding to the subtype ofHLA-A*0201 may cross-react with other alleles belonging tothe A2-like supertype and that HLA-A*0201–restricted CTLsare able to recognize TAAs in the context of different HLA-A2

Figure 4. Specific recognition of natural targets bythe FMNL1-PP2–specific T-cell clone. The FMNL1-PP2–specific T-cell clone was tested against the HLA-A2�

lymphoma cell lines BJ18 and DG75; the HLA-A2�

lymphoma cell line Raji; the HLA-A2� renal cell carci-noma cell line RCC26; the HLA-A2� renal cell carcinomacell line KT187 (A); and HLA-A2� EBV-transformed celllines (B), in a 51Cr-release assay at an effector-targetratio of 7.5:1. Error bars indicate the standard deviation oftested duplicates. Results shown are representative of atleast 3 experiments. (C) IFN-� release was investigatedby ELISA to test the FMNL1-PP2–specific T-cell cloneagainst CD40L-activated CLL cells (HLA-A2� and HLA-A2�) at an effector-target ratio of 1:2. Error bars indicatethe standard deviation of tested triplicates. (D) PBMCs(HLA-A2� and HLA-A2�) activated with IL-2 and OKT3,CD19� B cells (HLA-A2� and HLA-A2�) stimulatedwith CD40L, as well as HLA-A2� lung fibroblasts andHLA-A2� embryonal kidney cells (293 HEK) were usedas target cells for the FMNL1-PP2–specific T-cell clone ina 51Cr-release assay at an effector-target ratio of 7.5:1.Error bars indicate the standard deviation of tested dupli-cates. Results shown are representative of 5 experiments.

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subtypes.44,48-50 However, FMNL1-PP2–specific T cells alsocross-reacted with 2 of 16 HLA-A2� LCLs. One of them islikely to be HLA-A*6802 as one other HLA-A*6802–positivesample has been recognized (data not shown) and this HLA-Aallele also belongs to the A2 supertype family.44 The otherrecognized HLA-A2� LCL was positive for HLA-A*3303.HLA-A*3303 could be confirmed as a highly specific restrictionelement because the T cells specifically lysed HLA-A*3303–

transfected C1R cells but showed no reactivity against otherHLA-A alleles belonging to the A3-like supertype such asHLA-A03, HLA-A31, or the genotype HLA-A*3301 expressedon other tested HLA-A2� LCLs (Table S2). The capacity ofalloreactive TCRs to bind to different MHC-peptide complexeshas been repeatedly shown. Specificity for the same peptidepresented by different MHC alleles as well as for differentpeptides presented by the same or different MHC alleles hasbeen described.51-56 It is intensively discussed if this capacity ofalloreactive T cells represents a state of degeneracy or polyspeci-ficity.43,57,58 However, it has been shown recently that themechanism of cross-reactivity can occur independently ofmolecular mimicry.43,59 Moreover, cross-reactivity has been alsoobserved for high-affinity TCRs and therefore might be ageneral property of T-cell recognition.60-62 This is certainly animportant issue for the development of immunotherapies usingallorestricted peptide-specific T cells. Although FMNL1-PP2peptide–specific T cells are highly specific and cross-reactivityhas been observed only exceptionally, further investigations invitro and in vivo are necessary to clarify if other peptides andMHC molecules might be recognized by FMNL1-PP2–specificT cells. Knowledge of the TCR chains responsible for peptide-specific killing will allow TCR transfer studies and extensiveinvestigation of the specific TCR using peptide libraries andmultiple MHC alleles. For clinical use, patients would need tobe carefully selected prior to therapeutic application.

In summary, we identified a novel antigenic peptide (FMNL1-PP2) derived from FMNL1 with high potential for the use as atarget antigen for immunotherapeutic purposes. HLA-A2–allo-restricted T cells with specificity for this peptide have beenisolated and characterized. These T cells preferentially killedEBV-transformed B cells as well as tumor cells derived fromlymphoma and renal cell carcinoma, suggesting that the specificTCR is a highly interesting tool for the development of adoptiveimmunotherapies in EBV-associated lymphoproliferative dis-eases, lymphoma, and renal cell carcinoma. We observed only

Figure 6. Cross-reactivity of the isolated FMNL1-PP2–specific T-cell clone.LCLs with different HLA-A2 subtypes (A) and HLA-A2� LCLs (B) were used as targetcells for FMNL1-PP2–specific T-cell clone in a 51Cr-release assay at an effector-target ratio of 7.5:1. Error bars indicate the standard deviation of tested duplicates.Corresponding results were obtained by IFN-� ELISA.

Figure 5. The FMNL1-PP2–specific T-cell clone shows peptide specificity andTCR dependency. (A) T2 cells were pulsed with a set of different peptides at 10 �Mand investigated as targets for the FMNL1-PP2–specific T-cell clone in a 51Cr-releaseassay at an effector-target ratio of 7.5:1. Error bars indicate the standard deviation oftested duplicates. Results shown are representative of 2 experiments. (B) K562 andDaudi cells were used as target cells for the FMNL1-PP2–specific T-cell clone in a51Cr-release assay at an effector-target ratio of 7.5:1. Error bars indicate the standarddeviation of tested duplicates. Results shown are representative of 2 experiments.(C) Recognition of T2 cells pulsed with FMNL1-PP2 was inhibited by the monoclonalanti–HLA-A2 antibody (HB54) investigated by ELISA (IFN-� release). Error barsindicate the standard deviation of tested duplicates. Results shown are representa-tive of 2 experiments. (D) The cytotoxicity of the isolated T-cell clone against RCC26could be inhibited by addition of cold T2 cells pulsed with FMNL1-PP2 but not pulsedwith Flu. Error bars indicate the standard deviation of tested duplicates.

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minor killing against nontransformed cells, with the exceptionof healthy B cells that were better recognized. Future studiesincluding additional testing of peptide libraries and differentMHC alleles, mutational TCR analyses, and in vivo experimentsevaluating the antitumor and autoimmune potential of trans-genic TCR-expressing T cells, as well as functional studies ofFMNL1 to clarify its functional role in healthy and malignanttissue, will provide the basis for the design of clinical studies toassess the role of this epitope and the corresponding TCR inimmunotherapy.

Acknowledgments

This work was supported by grants from Life Science–Stiftung andDeutsche Forschungsgemeinschaft (DFG) (KR-2305/2–1) toA.M.K.as well as DFG-SFB/TR36 (projects A1, A7, A8, and B3).

We thank D. J. Schendel, R. Mocikat, and T. Blankenstein forcritical reading of the paper; S. Stevanovic, J. Mautner, R. Mocikat,and T. Wolfel for providing cell lines and T cells; A. Moosmann for

providing the peptide IPS; and C. S. Falk and B. Mosetter forintroduction into BioPlex analysis.

Authorship

Contribution: I.G.S. performed research and analyzed data; D.H.B.contributed new agents; E.E. performed research; E.K. contributednew agents; S.M., C.H., C.K., J.W.E., B.F., and E.N. contributedanalytical tools; C.S., C.B., A.B., and H.-J.K. provided clinical dataand samples; and A.M.K. designed and performed research,analyzed data, and wrote the paper.

Conflict-of-interest disclosure: I.G.S., D.H.B., and A.M.K.are holding a patent application about the peptide sequenceof FMNL1-PP2 and the sequences of the FMNL1-PP2–specific TCR. All other authors declare no competing financialinterests.

Correspondence: Angela M. Krackhardt, Institute of Molecu-lar Immunology, GSF–National Research Center for Environ-ment and Health, Marchioninistr. 25, 81377 Munich, Germany;e-mail: angela.krackhardt@gsf.de.

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