# Identification of human leucocyte antigen (HLA)-A\*0201-restricted cytotoxic T lymphocyte epitopes derived from HLA-DOβ as a novel target for multiple myeloma

Yoon Joong Kang, Wanyong Zeng, Weihua Song, Bruce Reinhold, Jaewon Choi, Vladimir Brusic, Takuto Yamashita, Aditya Munshi, Cheng Li, Stephane Minvielle, et al.

► To cite this version:

Yoon Joong Kang, Wanyong Zeng, Weihua Song, Bruce Reinhold, Jaewon Choi, et al. Identification of human leucocyte antigen (HLA)-A\*0201-restricted cytotoxic T lymphocyte epitopes derived from HLA-DOβ as a novel target for multiple myeloma. British Journal of Haematology, 2014, 163 (3), pp.343 - 351. 10.1111/bjh.12544. hal-04984331

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Br J Haematol. 2013 November; 163(3): 343-351. doi:10.1111/bjh.12544.

# Identification of HLA-A\*0201-Restricted Cytotoxic T Lymphocyte Epitopes Derived from HLA-DOβ as a Novel Target for Multiple Myeloma

Yoon Joong Kang<sup>1,2,4</sup>, Wanyong Zeng<sup>1,2</sup>, Weihua Song<sup>1</sup>, Bruce Reinhold<sup>1,2</sup>, Jaewon Choi<sup>2</sup>, Vladimir Brusic<sup>1,2</sup>, Takuto Yamashita<sup>5</sup>, Aditya Munshi<sup>1</sup>, Cheng Li<sup>3</sup>, Stephane Minvielle<sup>6</sup>, Kenneth C. Anderson<sup>1</sup>, Nikhil Munshi<sup>1</sup>, Ellis L. Reinherz<sup>1,2</sup>, and Tetsuro Sasada<sup>1,2,7</sup>

<sup>1</sup>Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

<sup>2</sup>Cancer Vaccine Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

<sup>3</sup>Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

<sup>4</sup>Department of Biomedical Science, Jungwon University, Chungcheongbuk-do 367-805, South Korea

<sup>5</sup>Biostatistics Centre, Kurume University Graduate School of Medicine, Kurume, Fukuoka 830-0011, Japan

<sup>6</sup>INSERM UMR 892, CNRS UMR 6299, University of Nantes, Nantes, France

<sup>7</sup>Department of Immunology and Immunotherapy, Kurume University School of Medicine, Kurume, Fukuoka 830-0011, Japan

## Summary

Despite the recent development of effective therapeutic agents against multiple myeloma (MM), new therapeutic approaches, including immunotherapies, remain to be developed. Here we identified novel human leucocyte antigen (HLA)-A\*0201 (HLAA2)-restricted cytotoxic T lymphocyte (CTL) epitopes from a B cell specific molecule HLA-DOβ (DOB) as a potential target for MM. By DNA microarray analysis, the *HLADOB* expression in MM cells was significantly higher than that in normal plasma cells. Twenty-five peptides were predicted to bind to HLA-A2 from the amino acid sequence of HLA-DOB. When screened for the immunogenicity in HLA-A2-transgenic mice immunized with *HLA-DOB* cDNA, 4 peptides were substantially immunogenic. By mass spectrometry analysis of peptides eluted from HLA-A2-immunoprecipitates of MM cell

<footnote>Correspondence: Tetsuro Sasada, Department of Immunology and Immunotherapy, Kurume University School of Medicine, Kurume, Fukuoka 830-0011, Japan; Tel: +81-942-31-7551; Fax: +81-942-31-7699; tsasada@med.kurume-u.ac.jp.</footnote>

<footnote>Author contributions</footnote>

<footnote>Y.J.K. and T.S. designed and performed research, analysed data, and wrote the paper; W.Z., W.S., B.R., J.C., V.B., and S.M. designed and performed research and analysed data; T.Y., A.M., and C.L. analysed data; K.C.A., N.M. and E.L.R designed the research, and critically evaluated and edited the paper.</footnote>

<footnote>**Competing interests:** The authors have no competing interests.</footnote>
lines, only two epitopes, HLA-DOB232-240 (FLLGLIFLL) and HLA-DOB185-193 (VMLEMTPEL), were confirmed for their physical presence on cell surface. When healthy donor blood was repeatedly stimulated *in vitro* with these two peptides and assessed by antigen-specific γ-interferon secretion, HLA-DOB232-240 was more immunogenic than HLA-DOB185-193. Additionally, the HLA-DOB232-240-specific CTLs, but not the HLA-DOB185-193-specific CTLs, displayed a major histocompatibility complex class I-restricted reactivity against MM cell lines expressing both HLA-A2 and HLA-DOB. Taken together, based on the physical presence on tumour cell surface and high immunogenicity, HLA-DOB232-240 might be useful for developing a novel immunotherapy against MM.

## Keywords

multiple myeloma; HLA-DOβ; cytotoxic T lymphocyte; T cell epitope; DNA microarray

## Introduction

Multiple myeloma (MM) remains difficult to cure with currently available treatment options (Kumar 2010, Laubach, *et al* 2011, Palumbo, *et al* 2011). Therefore, new treatment strategies aiming at prolonging survival and improving quality of life remain to be developed. Immunotherapy might be a promising intervention to maintain a long-lasting control of minimal residual disease or to even eradicate disseminated malignant cells (Prabhala and Munshi 2007, Pratt, *et al* 2007). Currently, many immunotherapeutic approaches in development for MM employ whole tumour cell lysates or tumour-specific idiotypic proteins to generate patient-specific vaccines (Curti, *et al* 2007, Rosenblatt, *et al* 2011, Röllig, *et al* 2011, Yi, *et al* 2010, Zahradova, *et al* 2012). However, these approaches are very labour-intensive and expensive, making their general applicability difficult. Therefore, off-the-shelf immunotherapies, such as peptide-based vaccines, remain to be developed for treating patients more efficiently. Indeed, peptide-based vaccines would offer several advantages over individualized vaccines due to higher safety and lower cost, as well as easier immune monitoring to clarify the levels of specific immune responses sufficient to eradicate malignant cells in patients (Purcell, *et al* 2007).

Cytotoxic T lymphocyte (CTL) epitopes for targeting MM cells have been identified from a variety of tumour antigens, such as MUC1, CYP1B1, PRAME, WT1, Sp17, HM1.24, XBP1, SOX2, CD138, and CS-1 (Bae, *et al* 2012, Bae, *et al* 2011, Brossart, *et al* 2001, Chiriva-Internati, *et al* 2002, Jalili, *et al* 2005, Kessler, *et al* 2001, Lotz, *et al* 2005, Maecker, *et al* 2005, Oka, *et al* 2008, Spisek, *et al* 2007). However, most of these epitopes have been identified from peptide candidates that were predicted to bind human leucocyte antigen (HLA) molecules, solely by their immunogenicity using *in vitro* cultured T cells. Therefore, it is questionable whether they are naturally processed and presented by malignant cells, even though they are immunogenic *in vitro* (Kessler and Melief 2007, Singh-Jasuja, *et al* 2004). Here we identified novel HLAA\*0201 (HLA-A2)-restricted CTL epitopes, which were naturally processed and presented by MM cells, from a B cell-specific molecule HLA-DOβ (DOB) (Denzin, *et al* 2005, Naruse, *et al* 2002). In particular, given its
strong immunogenicity for human T cells, one of the identified CTL epitopes, HLA-DOB<sub>232-240</sub>, could provide a promising immunotherapeutic approach for targeting MM.

# Methods

## Peptides and cell lines

HLA-DOB-derived peptides and a control peptide, modified Mart1<sub>26-35</sub> epitope (M26, ELAGIGILTV), were provided by Proimmune (Oxford, UK) or Mimotope (Melbourne, Australia) at purities of higher than 90%. An erythroleukaemia cell line K562, a TAP-deficient cell line T2, and MM cell lines, U266, IM-9, MC/CAR, and RPMI8226, were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), and OPM2 was kindly provided by Dr. Leif Bergsagel (Mayo Clinic, Tucson, AZ, USA). K562 cells stably transfected with HLA-A2 (K562-A2) was established as a target for immune assays (Anderson, et al 2011). Cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Lonza, Walkersville, MD), 100 µg/ml streptomycin, and 100 IU/ml penicillin (Mediatech, Herndon, VA). Expression of HLA-DOB and HLA-A2 in MM cell lines were examined by flow cytometry (FC500; Beckman-Coulter, Miami, FL) with the CXP software (Beckman-Coulter) after staining with anti-HLA-DOB (B01P mouse polyclonal; Abnova, Taiwan) and anti-HLA-A2 (BB7.2 mouse monoclonal; BD Biosciences, San Jose, CA) antibodies (Abs), respectively.

## DNA microarray analysis

The DNA microarray data (GeneChip® Human Exon 1.0 ST Array; Affymetrix, Santa Clara, CA) of CD138<sup>+</sup> myeloma cells from 170 newly diagnosed MM patients and plasma cells (PCs) from 6 normal donors were quality controlled and normalized with the aroma.affymetrix package. The gene expression level was estimated with a probe level model (PLM) (unpublished observations). The raw data for expression profiling and the CEL files can be found at the website Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE39754. The statistical significance of differences among the groups was assessed using the Kruskal-Wallis test.

## Prediction of HLA-A2-binding peptides derived from HLA-DOB

Based on our previous comprehensive analysis of prediction accuracy of various servers (Lin, et al 2008), three servers, IEDB ANN (http://tools.immuneepitope.org/analyze/html/mhcque.html), NetMHC ANN (http://www.cbs.dtu.dk/services/NetMHC), and MHC-I Multiple Matrix (http://atom.research.microsoft.com/hlabinding/hlabinding.aspx), were employed to predict 9-mer HLA-A2 binding peptides, and two servers, IEDB ARB (http://tools.immuneepitope.org/analyze/html/mhcque.html) and MHC-I Multiple Matrix, were used to predict 10-mer HLA-A2 binding peptides.

## HLA class I stabilization assay

The actual binding of predicted peptides to HLA-A2 molecules was evaluated by T2 cell surface HLA class I stabilization assay. Briefly, T2 cells were cultured for 18 h at 37°C in serum-free IMDM medium (Life Technologies, Rockville, MD) in the presence of peptides
(40 µg/ml). At 0, 3, 6 and 24 h after washing, the cells were stained with anti-HLA-A2 Ab (BB7.2), followed by analysis with flow cytometry. The binding capability of each peptide to HLA-A2 molecules was evaluated by the increase in mean fluorescence intensity (MFI) of the HLA-A2 expression, as follows: MFI increase = (MFI with a given peptide – MFI without a peptide)/(MFI without a peptide). The modified Mart1<sub>26-35</sub> epitope (M26) was used as a positive control. Assays were performed in triplicate, and mean values were used for calculation.

## Generation of antigen-specific T cells

Peripheral blood mononuclear cells (PBMCs) from HLA-A2<sup>+</sup> healthy donors were obtained with written informed consent under the Institutional Review Board approval at the Dana-Farber Cancer Institute. PBMCs were purified using Ficoll-Paque density centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden), and HLA-A2 expression was confirmed by flow cytometry with anti-HLA-A2 Ab (BB7.2). Mature dendritic cells (DCs) were used to stimulate autologous CD8<sup>+</sup> T cells, which were purified with anti-CD8 bead (Miltenyi Biotec, Auburn, CA). Mature DCs were generated from monocytes isolated from PBMCs, as previously described (Anderson, et al 2011). CD8<sup>+</sup> T cells (2 x 10<sup>6</sup> cells/well in a 24-well plate) were first primed with autologous mature DCs pulsed with peptides (10 µg/ml) in RPMI 1640 medium supplemented with 10% human AB serum (Cellgro, Herndon, VA), 2 mM glutamine (Cellgro), 10 mM HEPES (Cellgro), and 15 µg/ml gentamycin (Invitrogen) in the presence of 10 ng/ml of interleukin (IL)7 (Endogen Inc., Woburn, MA) on day 0 and 20 iu/ml of IL2 (Chiron Corp., Emeryville, CA) on days 1 and 4. Re-stimulation of T cells was performed with autologous mature DCs pulsed with the same peptides (2 µg/ml) every week. Antigen-specific T cells were enriched by sorting with PE-conjugated peptide/HLA-A2 tetramer (NIH Tetramer Facility; Emory University, Atlanta, GA) in combination with anti-PE Micro-Beads (Miltenyi Biotec), and then cloned by limiting dilution using irradiated feeder cells (allogeneic PBMCs and Epstein-Barr virus-transformed lymphoblastoid cell lines) in the presence of 1% phytohaemagglutinin (Invitrogen) and 100 iu/ml IL2 (Chiron Corp). Antigen-specificity of the T cell clones was determined by γ-interferon (IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay and/or peptide/HLA-A2 tetramer staining.

## Immune assays

To examine the cytotoxicity of T cells, europium release assay was performed, as previously described (Anderson, et al 2011). Specific europium release was measured by a multiplate reader (Victor3 multilabel counter model 1420; PerkinElmer, Waltham, MA, USA). For blocking the HLA class I molecules, anti-HLA class I Ab (clone W6/32) or isotype control IgG2a Ab (10 µg/ml; both from eBioscience, San Diego, CA) were used. All experiments were performed in triplicate and mean values were used for calculation.

For IFN-γ ELISPOT assay with human T cells, the indicated numbers of T cells were cultured for 24 h at 37°C with the T2, K562-A2, or MM cells (5 × 10<sup>4</sup> cells/well) loaded without or with control or specific peptides (5 µ/ml) in 96-well ELISPOT plate (MultiScreen HTS; Millipore, Bedford, MA) coated with anti-human IFN-γ Ab (MABTECH, Cincinnati, OH). After washing, the spots were developed with biotin-conjugated anti-human IFN-γ Ab
(MABTECH), streptavidin-alkaline phosphatase (MABTECH), and 5-bromo-4-chloro-3-indolyl-phosphate/nitro-blue tetrazolium (BCIP/NBT) substrate (Promega, Madison, WI), and then counted by ELISPOT reader (CTL Technologies, Cleveland, OH).

# Results

## Identification of a potential target molecule for MM by DNA microarray analysis

In order to identify genes that were over-expressed in MM cells as potential targets for immunotherapy, we first identified 53 genes, whose expressions in CD138⁺ MM cells were more than 20 times higher than those in bone marrow PCs from normal donors by DNA microarray analysis in a smaller dataset (data not shown). Among them, *HLA-DOB*, which has been reported to be selectively expressed in B-lineage cells (Denzin, *et al* 2005, Naruse, *et al* 2002), was selected for further study to identify novel antigenic CTL epitopes. We further examined RNA expression levels in CD138⁺ cells from MM patients (n = 170) and bone marrow PCs from normal donors (n = 6) by the DNA microarray analysis, and observed that *HLA-DOB* levels were more than 2 standard deviations above the mean for normal bone marrow PCs in 155 of 170 MM samples (Fig 1). From the entire amino acid sequence of HLA-DOB, 25 peptides (9-mer or 10-mer) were predicted to bind to HLA-A2 with higher probability by the prediction servers (Table I).

## Screening of HLA-DOB-derived peptides for the immunogenicity in HLA-A2-transgenic AAD mice

We examined the immunogenicity of each of the HLA-DOB-derived peptides by using HLA-A2-transgenic mice, AAD. The peptide epitopes presented and recognized by mouse T cells in the context of the HLA-A2/H2-D<sup>d</sup> class I molecules in these mice have been reported to be almost the same as those in HLA-A2<sup>+</sup> humans (Mullins, *et al* 2001, Newberg, *et al* 1996, Peng, *et al* 2006). To identify immunogenic epitopes, AAD mice were immunized three times with a plasmid containing *HLA-DOB* cDNA for inducing antigen-specific immune responses. Two weeks after the last immunization, splenocytes were analysed *ex vivo* for IFN-γ production in response to each of the predicted peptides. As shown in Fig S1, DNA immunization successfully induced T cells specific to 4 of the 25 peptides; HLA-DOB<sub>232-240</sub> (FLLGLIFL.L), HLA-DOB<sub>185-193</sub> (VMLEMTPEL), HLADOB<sub>224-233</sub> (KMLSGIAAFL), and HLA-DOB<sub>184-193</sub> (VVMLEMTPEL). As we used the DNA vaccination method for immunization, the identified peptides were most likely naturally processed and presented in complex with the HLA-A2/H2-D<sup>d</sup> class I molecules *in vivo* in the AAD mice.

The binding of the identified peptides to the HLA-A2 molecule was further confirmed by the cell surface HLA class I stabilization assay with the HLA-A2-positive TAP-deficient lymphoma cell line T2. As illustrated in Fig 2, all of the 4 identified peptides showed substantial binding to the HLA-A2 molecule. The binding capability of HLA-DOB<sub>232-240</sub>, HLA-DOB<sub>185-193</sub>, and HLA-DOB<sub>184-193</sub> to HLA-A2 was stronger than that of HLA-DOB<sub>224-233</sub>.
## Physical identification of HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub> on the cell surface of MM cell lines by mass-spectrometry (MS)

We next examined whether the peptides identified by the *in vivo* immunogenicity in the AAD mouse system were actually expressed in complex with the HLA-A2 molecules on cell surface of MM cells. Three MM cell lines, U266, IM-9, and MC/CAR, which were positive for both HLA-DOB and HLA-A2 (data not shown), were employed. HLA-A2-binding peptides were acid-released from the HLA-A2/peptide complexes, which were affinity-purified from these MM cell lines using the anti-HLA-A2 Ab BB7.2, and then analysed by nanospray ionization and MS³ ion fragmentation (Reinhold, *et al* 2010, Riemer, *et al* 2010). HLA-DOB<sub>185-193</sub> could be easily identified by this analysis in the peptides recovered from all of these 3 cell lines. In addition, HLA-DOB<sub>232-240</sub> was detected in IM-9 and MC/CAR cells, but not in U266 cells. Representative data of IM-9 cells are given in Fig S2 with detection evident by the high peak at the “0” offset on the X-axis. In contrast, the physical existence of other two peptides, HLA-DOB<sub>224-233</sub> and HLA-DOB<sub>184-193</sub>, on MM cell surface was not confirmed by the nanospray ionization and MS³ ion fragmentation (data not shown).

## Immunogenicity of HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub> in CD8 T cells from human peripheral blood

To determine the immunogenicity of the HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub> peptides in humans, CD8<sup>+</sup> T cells from 5 different HLA-A2<sup>+</sup> healthy donors were repeatedly stimulated with autologous mature DCs pulsed with the synthetic peptides. After 4 rounds of *in vitro* stimulation, CD8<sup>+</sup> T cell lines secreting IFN-γ in response to HLA-DOB<sub>232-240</sub> or HLA-DOB<sub>185-193</sub> could be established in 4 of 5 or 1 of 5 donors, respectively (Fig 3). This result suggested that HLA-DOB<sub>232-240</sub> was more immunogenic than HLA-DOB<sub>185-193</sub> in human CD8<sup>+</sup> T cells.

We further characterized these T cells specific to the HLA-DOB-derived peptides. We first established a T cell clone, 232-27, from a CTL line specific to HLA-DOB<sub>232-240</sub> by limiting-dilution method. The clone 232-27 showed IFN-γ secretion in response to the HLA-DOB<sub>232-240</sub> peptide in an antigen-specific manner (Fig 4A). Its clonality (> 95%) was also verified by the HLA-A2/HLA-DOB<sub>232-240</sub> tetramer staining determined by flow cytometry (data not shown). The reactivity of the clone 232-27 was assessed against HLA-DOB<sup>+</sup>/HLA-A2<sup>+</sup> MM cell lines, U266, IM-9, and MC/CAR, as well as HLADOB<sup>+</sup>/HLA-A2<sup>-</sup> lines, RPMI8226 and OPM2, as negative controls. As shown in Fig 4B, the clone 232-27 showed an apparent IFN-γ production in response to IM-9 and MC/CAR cells. This clone also produced less, but a still substantial amount of IFN-γ in response to U266, whereas it showed no response against HLA-DOB<sup>+</sup>/HLA-A2<sup>-</sup> MM cell lines. Of note, U266 cells pulsed with the synthetic HLA-DOB<sub>232-240</sub> peptide clearly enhanced IFN-γ secretion from the clone 232-27, suggesting that the cell surface expression of HLA-A2/HLA-DOB<sub>232-240</sub> in U266 cells may not be high enough to fully stimulate cytokine production from this T cell clone. Consistent with this finding, the cytotoxicity assay showed that cytotoxic activity of the clone 232-27 against IM-9 and MC/CAR cells were substantially higher than that against U266 cells (Fig 4C). The cytotoxicity was significantly inhibited by incubation with anti-
HLA-class I Ab (clone W6/32), but not by an isotype control Ab, confirming that this
response was major histocompatibility complex (MHC) class I-restricted (Fig 4D).

We also established antigen-specific T cell clones from an HLA-DOB<sub>185-193</sub>- specific CTL line by the limiting dilution method. As shown in Fig 5A, T cell clone 185-275 showed IFN-γ secretion in response to the synthetic HLA-DOB<sub>185-193</sub> peptide. However, this clone showed a marginal reactivity against IM-9 cells, but almost no responses against U266 or HLA-DOB<sup>+</sup>/HLA-A2<sup>-</sup> lines, as determined by IFN-γ ELISPOT (Fig 5B). In addition, it showed no cytotoxicity against HLA-DOB<sup>+</sup>/HLA-A2<sup>+</sup> MM cell lines, IM-9 or U266 (Fig 5C), although the cell surface display of HLA-DOB<sub>185-193</sub> peptide on these MM cells was confirmed by MS analysis (Fig S2). Given that the clone 185-275 revealed a clear reactivity against IM-9 and U266 cells after treatment with the synthetic HLA-DOB<sub>185-193</sub> peptide (Fig 5B), the avidity of T cell receptor (TCR) of this clone might be too low to respond to the copy number of naturally processed peptides arrayed on these MM cell lines, therefore requiring exogenous peptide addition. Although we established 5 other different HLA-DOB<sub>185-193</sub>-specific T cell clones, they showed similar results (data not shown).

Discussion

Despite the development of novel therapeutic agents against MM, this disease is still
difficult to cure with currently available treatment options (Kumar 2010, Laubach, et al
2011, Palumbo, et al 2011). In the current study, we identified two HLA-A2-restricted CTL
epitopes, HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub>, which were naturally processed and
presented on MM cell surface. In particular, the HLA-DOB<sub>232-240</sub> epitope was highly
immunogenic in human CD8<sup>+</sup> T cells, suggesting that this epitope might be useful for
immunotherapies against MM patients.

Based on gene expression analysis by the DNA microarray, we identified 53 potential MM-associated molecules, which showed >20-fold expression in MM cells compared to bone marrow PCs from normal donors. Among these proteins over-expressed in MM cells, HLA-DOB, a MHC class II-related molecule, was selected for further analysis to identify antigenic CTL epitopes, because this molecule was reported to be exclusively expressed in B-lineage cells and a subset of thymic medullary epithelium (Denzin, et al 2005). In addition, the *HLA-DOB* gene has a limited number of allelic variants in contrast to other MHC class II-related molecules (Naruse, et al 2002). Furthermore, HLA-DO, which is composed of HLA-DOα and HLA-DOB, has been shown to impair MHC class II presentation through inhibition of HLA-DM in B cells (Denzin, et al 2005). Thus, even if HLA-DOB expression is down-regulated by the immune escape mechanism after targeting this molecule, the skewed balance between intracellular HLA-DM and HLA-DO expression would ultimately induce up-regulation of MHC class II-restricted presentation of tumour antigens, which might lead to an enhanced anti-tumour immunity (Chamuleau, et al 2006). In view of these properties of HLA-DOB, we considered that this molecule might be a good target for immunotherapy against MM. Of note, other B cell-specific molecules, such as CD19 and CD20, have also been reported to be appropriate targets for treating B cell malignancies (Maloney, et al 1997, Porter, et al 2011).
The identification method of appropriate CTL epitopes for cancer immunotherapy remains controversial (Kessler and Melief 2007, Singh-Jasuja, et al 2004). Currently, many CTL epitopes have been identified from HLA-binding peptide candidates solely by examining indirect immunological surrogates (killing, proliferation, cytokine production, etc.) *in vitro* with cultured T cells. However, it is questionable whether the CTL epitopes identified by this approach are actually 'real' antigens that are naturally processed and presented on tumour cell surface, even though they are immunogenic *in vitro* (Kessler and Melief 2007, Singh-Jasuja, et al 2004). Therefore, although a large number of tumour antigens and epitopes have been already described (Bae, et al 2011, Brossart, et al 2001, Chiriva-Internati, et al 2002, Jalili, et al 2005, Kessler, et al 2001, Lotz, et al 2005, Maecker, et al 2005, Oka, et al 2008, Spisek, et al 2007), it may be possible that many of them cannot direct the host immune system toward malignant cells because they are not present on the tumour cell surface. Hence, in the current study, we employed more reliable approaches to identify 'real' antigenic epitopes from HLA-DOB. The HLA-A2 transgenic AAD mice that were vaccinated *in vivo* with *HLA-DOB* DNA showed substantial immune responses to 4 of the 25 predicted HLA-A2-binding epitopes, suggesting that these epitopes may be naturally processed and presented *in vivo* in AAD mice. In addition, two of them were physically detected on the cell surface by MS analysis of peptide pools extracted from the HLA-A2/ peptide complexes on MM cells. The reason why the remaining two epitopes that were identified in AAD mice were not verified by the MS analysis might be due to a difference of the machinery in processing and/or presentation of endogenous proteins between mice and humans, as previously reported (Street, et al 2002). Alternatively, in principle, it might be explained by insufficient sensitivity of detection using the MS method that we employed. The latter appears less likely given the documented detection threshold demonstrated by other peptides assessed with this Poisson detection approach (Reinhold, et al 2010, Riemer, et al 2010).

In order to induce effective anti-tumour immune responses, tumour antigens should be sufficiently immunogenic to stimulate antigen-specific T-cells well. When CD8+ T cells from healthy HLA-A2+ donors were stimulated with the antigen peptide-pulsed autologous DCs, T cell lines specific to the HLA-DOB<sub>232-240</sub> epitope were easily generated in 4 of 5 donors. In addition, the HLA-DOB<sub>232-240</sub>-specific T cell lines/clones demonstrated antigen-specific reactivity against HLA-A2+, but not HLA-A2-, MM cell lines. Notably, the reactivity of the HLA-DOB<sub>232-240</sub>-specific T cell clones against IM-9 and MC/CAR cells were substantially higher than that against U266 cells, although U266 cells pulsed with the synthetic HLA-DOB<sub>232-240</sub> peptide showed clearly enhanced reactivity. This result was consistent with the finding that HLA-DOB<sub>232-240</sub> was detected only in IM-9 and MC/CAR cells, but not in U266 cells. These results suggest that the cell surface expression of HLA-A2/HLA-DOB<sub>232-240</sub> in U266 cells may not be high enough to fully stimulate antigen-specific T cell clone. Since the expression levels of HLA-DOB protein determined by flow cytometric analysis were similar among the HLA-A2+ MM cell lines, IM-9, MC/CAR, and U266 (data not shown), the difference in cell surface expression of HLA-A2/HLA-DOB<sub>232-240</sub> may be explained by the different efficiency of peptide processing of HLA-DOB<sub>232-240</sub> among these MM cell lines tested. To further confirm the suitability of this
epitope as a therapeutic target, its expression on MM cells derived from patients remains to be examined.

In contrast, the HLA-DOB<sub>185-193</sub> epitope seemed to be less immunogenic, as HLA-DOB<sub>185-193</sub>-specific T cells could be generated in only one of 5 different normal donors after repeated stimulation with the antigen peptide-pulsed autologous DCs. In addition, the established HLA-DOB<sub>185-193</sub>-specific T cell line/clones did not show apparent cytotoxic reactivity against the tumour cell lines examined, possibly due to low avidity of their TCR. Although not shown, we tried several other methods for stimulating CD8<sup>+</sup> T cells by using different types of antigen-presenting cells (APCs), such as CD40L-activated autologous B cells and K562-based artificial APCs (Anderson, et al 2011, Schultze, et al 1997). Notably, these other APC systems also successfully generated T cells specific to HLA-DOB<sub>232-2403</sub>, but not to HLA-DOB<sub>185-193</sub>. Although not proved, the lower immunogenicity of the HLA-DOB<sub>185-193</sub> epitope might be explained by deletion of high-avidity T cells due to central and/or peripheral tolerance.

In conclusion, we identified two novel HLA-A2-restricted, HLA-DOB-derived epitopes, HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub>, which were naturally processed and presented on MM cells. In particular, HLA-DOB<sub>232-240</sub> could be promising as an antigenic epitope to target MM cells due to its stronger immunogenicity. Future studies will be required to investigate the clinical utility of the identified epitopes by using blood samples from MM patients.

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgments

We wish to thank Drs. Pedro A. Reche, Honghuang Lin, Mei Su, and Derin B Keskin (Dana-Farber Cancer Institute, Boston, MA) and Dr Herve Avet-Loiseau (Centre Hospitalier Universitaire, Nantes, France) for their technical help and helpful discussion. This study was supported by the DFCI Cancer Vaccine Center funding and a grant from the International Myeloma Foundation Japan (Aki Horinouchi Research Grant).

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<figure><img src="image_3.png" /><figcaption>Fig 1. HLA-DOB expression in MM cells</figcaption></figure>

Expression levels of *HLA-DOB* RNA were examined in CD138⁺ myeloma cells from newly diagnosed multiple myeloma (MM) patients (n = 170) and bone marrow plasma cells from normal donors (n = 6) by DNA microarray analysis. The expression levels of *HLA-DOB* RNA were compared between MM patients and normal donors. The statistical significance of differences between the groups was assessed using the Kruskal-Wallis test.
<figure><img src="image_4.png" /><figcaption>Fig 2. HLA-A2 binding capability of HLA-DOB-derived peptides</figcaption></figure>

Binding of the peptides, HLA-DOB<sub>232-240</sub>, HLA-DOB<sub>185-193</sub>, HLA-DOB<sub>224-233</sub>, and HLA-DOB<sub>184-193</sub>, to the HLA-A2 molecule was confirmed by the cell surface HLA class-I stabilization assay. T2 cells were cultured for 18 h at 37°C in the presence of peptides (40 µg/ml). At 0, 3, 6 and 24 h after washing, the cells were stained with anti-HLA-A2 Ab (BB7.2), followed by analysis with flow cytometry. The binding capability of each peptide to HLA-A2 molecule was calculated by the increase in mean fluorescence intensity (MFI) of HLA-A2 expression: MFI increase = (MFI with a given peptide – MFI without a peptide)/(MFI without a peptide). The modified Mart1<sub>26-35</sub> epitope (M26) was used as a positive control. A representative experiment of three separate experiments is shown.
<figure><img src="image_5.png" /><figcaption>Fig 3. Immunogenicity of HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub> in CD8<sup>+</sup> T cells from peripheral blood</figcaption></figure>

Immunogenicity of the HLA-DOB<sub>232-240</sub> and HLA-DOB<sub>185-193</sub> peptides was examined with peripheral blood from 5 different HLA-A2<sup>+</sup> healthy donors. CD8<sup>+</sup> T cells were first primed with autologous mature dendritic cells pulsed with synthetic peptides (10 µg/ml). After three more rounds of *in vitro* stimulation with the same peptides (2 µg/ml), T cells (5 × 10<sup>3</sup> cells/well) were examined for reactivity against T2 cells (5 × 10<sup>4</sup> cells/well) pulsed with 5 µg/ml of control M26 peptide (-) or specific peptide (+) by γ-interferon enzyme-linked immunosorbent spot assay. The cells were cultured in duplicate.
<figure><img src="image_6.png" /><figcaption>Fig 4. Functional characterization of an HLA-DOB<sub>232-240</sub>-specific T cell clone</figcaption></figure>

(A) An HLA-DOB<sub>232-240</sub>-specific T cell clone 232-27 (2 × 10<sup>2</sup> cells/well) was stimulated with T2 cells (5 × 10<sup>4</sup> cells/well) pre-treated for 2 h with the control (M26) or specific (HLA-DOB<sub>232-240</sub>) peptide (5 µg/ml), and γ-interferon (IFN-γ secretion was examined by enzyme-linked immunosorbent spot (ELISPOT) assay in duplicate. (B) The T cell clone 232-27 (1 × 10<sup>3</sup> cells/well) was stimulated with the HLA-DOB<sup>+</sup>/HLA-A2<sup>+</sup> MM cell lines, U266, IM-9 and MC/CAR, or the HLA-DOB<sup>+</sup>/HLA-A2<sup>-</sup> lines, RPMI8226 and OPM2, (1 × 10<sup>4</sup> cells/well) pretreated for 2 h with or without the specific peptide (HLA-DOB<sub>232-240</sub>, 5 µg/ml). IFN-γ secretion was examined by ELISPOT assay in duplicate. (C) The clone 232-27 was examined for cytotoxicity against target cells (5 × 10<sup>3</sup> cells/well), including T2 cells pretreated for 2 h with 5 µg/ml of the control M26 peptide (-) or specific peptide (+), U266, IM-9, MC/CAR, RPMI8226 or OPM2, at the indicated effector/target ratios. The specific lysis was calculated as follows: specific lysis (%) = [(test release- spontaneous release) / (maximal release - spontaneous release)] × 100. (D) The cytotoxicity against IM-9 cells was examined in the presence or absence of anti-HLA class I (clone W6/32) or isotype control IgG2a Ab (10 µg/ml). A representative experiment of three separate experiments is shown.
<figure><img src="image_7.png" /><figcaption>Fig 5. Functional characterization of an HLA-DOB<sub>185-193</sub>-specific T cell clone</figcaption></figure>

(A) An HLA-DOB<sub>185-193</sub>-specific T cell clone 185-275 (1 × 10<sup>3</sup> cells/well) was stimulated with K562/HLA-A2 cells (5 × 10<sup>4</sup> cells/well) pretreated for 2 h with the control (M26) or specific (HLA-DOB<sub>185-193</sub>) peptide (5 µg/ml), and γ-interferon (IFN-γ secretion was examined by enzyme-linked immunosorbent spot (ELISPOT) assay in duplicate. (B) The T cell clone 185-275 (1 × 10<sup>3</sup> cells/well) was stimulated with U266, IM-9, RPMI8226 or OPM2 (1 × 10<sup>4</sup> cells/well), pretreated for 2 h with or without the specific peptide (HLA-DOB<sub>185-193</sub>, 5 µg/ml). IFN-γ secretion was examined by ELISPOT assay in duplicate. (C) The clone 185-275 was examined for cytotoxicity against target cells (5 × 10<sup>3</sup> cells/well), including T2 cells pretreated for 2 h with 5 µg/ml of the control M26 peptide (-) or specific peptide (+), U266, IM-9, RPMI8226 or OPM2, at the indicated effector/target ratios. The specific lysis was calculated. A representative experiment of three separate experiments is shown.
<caption>Table I</caption>

List of the HLA-DOB-derived peptides.

<table><thead><tr><td>Peptide number</td><td>Position</td><td>Number of amino acids</td><td>Sequence</td></tr></thead><tbody><tr><td>1</td><td>232-240</td><td>9</td><td>FLLGLIFLL</td></tr><tr><td>2</td><td>56-64</td><td>9</td><td>FIFNLEEYV</td></tr><tr><td>3</td><td>185-193</td><td>9</td><td>VMLEMTPEL</td></tr><tr><td>4</td><td>52-60</td><td>9</td><td>FVVRFIFNL</td></tr><tr><td>5</td><td>233-241</td><td>9</td><td>LLGLIFLLV</td></tr><tr><td>6</td><td>228-236</td><td>9</td><td>GIAFLLGL</td></tr><tr><td>7</td><td>235-243</td><td>9</td><td>GLIFLLVGI</td></tr><tr><td>8</td><td>11-19</td><td>9</td><td>ALLVNLTRL</td></tr><tr><td>9</td><td>71-79</td><td>9</td><td>GMFVALTKL</td></tr><tr><td>10</td><td>158-166</td><td>9</td><td>FLNGQEERA</td></tr><tr><td>11</td><td>199-207</td><td>9</td><td>CLVDHSSLL</td></tr><tr><td>12</td><td>232-241</td><td>10</td><td>FLLGLIFLLV</td></tr><tr><td>13</td><td>224-233</td><td>10</td><td>KMLSGIAAFL</td></tr><tr><td>14</td><td>184-193</td><td>10</td><td>VVMLEMTPEL</td></tr><tr><td>15</td><td>136-145</td><td>10</td><td>HQHNLLHCSV</td></tr><tr><td>16</td><td>220-229</td><td>10</td><td>YSWRKMLSGI</td></tr><tr><td>17</td><td>230-239</td><td>10</td><td>AAFLLGLIFL</td></tr><tr><td>18</td><td>175-184</td><td>10</td><td>RNGDWTFQTV</td></tr><tr><td>19</td><td>245-254</td><td>10</td><td>IQLRAKGYV</td></tr><tr><td>20</td><td>203-212</td><td>10</td><td>HSSLSPVSV</td></tr><tr><td>21</td><td>5-14</td><td>10</td><td>WVPWVVALLV</td></tr><tr><td>22</td><td>192-201</td><td>10</td><td>ELGHVYTCLV</td></tr><tr><td>23</td><td>92-101</td><td>10</td><td>DLLERSRQAV</td></tr><tr><td>24</td><td>228-237</td><td>10</td><td>GIAFLLGLI</td></tr><tr><td>25</td><td>126-135</td><td>10</td><td>TVYPERTPLL</td></tr></tbody></table>