Transcription of the Antisense Long Non-Coding RNA, SUPPRESSOR OF FEMINIZATION, Represses Expression of the Female-Promoting Gene FEMALE GAMETOPHYTE MYB in the Liverwort Marchantia polymorpha.

Sexual differentiation is a fundamental process in the life cycles of land plants, ensuring successful sexual reproduction and thereby contributing to species diversity and survival. In the dioicous liverwort Marchantia polymorpha, this process is governed by an autosomal sex-differentiation locus comprising FEMALE GAMETOPHYTE MYB (FGMYB), a female-promoting gene, and SUPPRESSOR OF FEMINIZATION (SUF), an antisense strand-encoded long non-coding RNA (lncRNA). SUF is specifically transcribed in male plants and suppresses the expression of FGMYB, leading to male differentiation. However, the molecular mechanisms underlying this process remain elusive. Here, we show that SUF acts through its transcription to suppress FGMYB expression. Transgene complementation analysis using CRISPR/Cas9D10A-based large-deletion mutants identified a genomic region sufficient for the sex differentiation switch function in the FGMYB-SUF locus. Inserting a transcriptional terminator sequence into the SUF-transcribed region resulted in the loss of SUF function and allowed expression of FGMYB in genetically male plants, leading to conversion of the sex phenotype from male to female. Partial deletions of SUF had no obvious impact on its function. Replacement of the FGMYB sequence with that of an unrelated gene did not affect the ability of SUF transcription to suppress sense-strand expression. Taken together, our findings suggest that the process of SUF transcription, rather than the resulting transcripts, is required for controlling sex differentiation in M. polymorpha.

Conserved CKI1-mediated signaling is required for female germline specification in Marchantia polymorpha.

In land plants, gametes derive from a small number of dedicated haploid cells.1 In angiosperms, one central cell and one egg cell are differentiated in the embryo sac as female gametes for double fertilization, while in non-flowering plants, only one egg cell is generated in the female sexual organ, called the archegonium.2,3 The central cell specification of Arabidopsis thaliana is controlled by the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1), which is a two-component signaling (TCS) activator sharing downstream regulatory components with the cytokinin signaling pathway.4,5,6,7 Our phylogenetic analysis suggested that CKI1 orthologs broadly exist in land plants. However, the role of CKI1 in non-flowering plants remains unclear. Here, we found that the sole CKI1 ortholog in the liverwort Marchantia polymorpha, MpCKI1, which functions through conserved downstream TCS components, regulates the female germline specification for egg cell development in the archegonium. In M. polymorpha, the archegonium develops three-dimensionally from a single cell accumulating MpBONOBO (MpBNB), a master regulator for germline initiation and differentiation.8 We visualized female germline specification by capturing the distribution pattern of MpBNB in discrete stages of early archegonium development, and found that MpBNB accumulation is restricted to female germline cells. MpCKI1 is required for the proper MpBNB accumulation in the female germline, and is critical for the asymmetric cell divisions that specify the female germline cells. These results suggest that CKI1-mediated TCS originated during early land plant evolution and participates in female germ cell specification in deeply diverged plant lineages.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution.

Holliday junction recognition protein as a prognostic biomarker and therapeutic target for oral cancer.

Since oral cancer (OC) is highly malignant and the efficacy of standard treatments is limited, the development of new therapeutics is urgently awaited. To identify potential molecular targets for new OC diagnosis and therapies, we screened oncoantigens by gene expression profile and focused on Holliday junction recognition protein (HJURP), a mammalian centromere­specific chaperone. HJURP was found to be highly expressed in the majority of OC cell lines and tissues as compared to normal oral epithelial cells. Tissue microarray analysis confirmed that HJURP was expressed in 103 (67.8%) of 152 OC tissue specimens, but expression in normal oral tissues was limited. Positive HJURP expression was significantly correlated with shorter overall survival (P=0.003). Depletion of HJURP by small­interfering RNAs dramatically inhibited the growth of OC cells by inhibition of cell cycle progression and induced senescence of OC cells. In addition, inhibition of the interaction between HJURP and CENP­A significantly suppressed the growth of OC cells. These results indicate that HJURP is a potential prognostic biomarker, and targeting HJURP and its molecular pathway presents a new strategy for the development of treatments against OC.

Characterization of Opa interacting protein 5 as a new biomarker and therapeutic target for oral cancer.

Oral cancer is a leading cause of cancer­related death worldwide. Current treatment for oral cancer includes surgery, radiotherapy, and chemotherapy; however, their effectiveness is still limited. To identify a new prognostic biomarker and therapeutic target for oral cancer, the Opa interacting protein 5 (OIP5), which plays an essential role in the proper segregation of chromosomes, was examined. Immunohistochemical staining using tissue microarrays indicated that OIP5 was expressed in 120 of 164 (73.2%) oral cancers but was minimally expressed in normal oral tissues. OIP5 expression was significantly associated with poor prognosis in patients with oral cancer. Overexpression of OIP5 enhanced the growth of oral cancer cells, whereas OIP5 knockdown using small interfering RNAs (siRNAs) significantly inhibited cell growth through cell cycle arrest at the G2/M phase. Suppression of OIP5 expression also induced senescence of oral cancer cells. Overall, the findings of the present study suggest that OIP5 may be a candidate prognostic biomarker and therapeutic target in oral cancer.

Identification of the sex-determining factor in the liverwort Marchantia polymorpha reveals unique evolution of sex chromosomes in a haploid system.

Sex determination is a central process for sexual reproduction and is often regulated by a sex determinant encoded on a sex chromosome. Rules that govern the evolution of sex chromosomes via specialization and degeneration following the evolution of a sex determinant have been well studied in diploid organisms. However, distinct predictions apply to sex chromosomes in organisms where sex is determined in the haploid phase of the life cycle: both sex chromosomes, female U and male V, are expected to maintain their gene functions, even though both are non-recombining. This is in contrast to the X-Y (or Z-W) asymmetry and Y (W) chromosome degeneration in XY (ZW) systems of diploids. Here, we provide evidence that sex chromosomes diverged early during the evolution of haploid liverworts and identify the sex determinant on the Marchantia polymorpha U chromosome. This gene, Feminizer, encodes a member of the plant-specific BASIC PENTACYSTEINE transcription factor family. It triggers female differentiation via regulation of the autosomal sex-determining locus of FEMALE GAMETOPHYTE MYB and SUPPRESSOR OF FEMINIZATION. Phylogenetic analyses of Feminizer and other sex chromosome genes indicate dimorphic sex chromosomes had already been established 430 mya in the ancestral liverwort. Feminizer also plays a role in reproductive induction that is shared with its gametolog on the V chromosome, suggesting an ancestral function, distinct from sex determination, was retained by the gametologs. This implies ancestral functions can be preserved after the acquisition of a sex determination mechanism during the evolution of a dominant haploid sex chromosome system.

Design principles of a minimal auxin response system.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

Generative Cell Specification Requires Transcription Factors Evolutionarily Conserved in Land Plants.

Land plants differentiate germ cells in the haploid gametophyte. In flowering plants, a generative cell is specified as a precursor that subsequently divides into two sperm cells in the developing male gametophyte, pollen. Generative cell specification requires cell-cycle control and microtubule-dependent nuclear relocation (reviewed in [1-3]). However, the generative cell fate determinant and its evolutionary origin are still unknown. In bryophytes, gametophytes produce eggs and sperm in multicellular reproductive organs called archegonia and antheridia, respectively, or collectively called gametangia. Given the monophyletic origin of land plants [4-6], evolutionarily conserved mechanisms may play key roles in these diverse reproductive processes. Here, we showed that a single member of the subfamily VIIIa of basic helix-loop-helix (bHLH) transcription factors in the liverwort Marchantia polymorpha primarily accumulated in the initial cells and controlled their development into gametangia. We then demonstrated that an Arabidopsis thaliana VIIIa bHLH transiently accumulated in the smaller daughter cell after an asymmetric division of the meiosis-derived microspore and was required for generative cell specification redundantly with its paralog. Furthermore, these A. thaliana VIIIa bHLHs were functionally replaceable by the M. polymorpha VIIIa bHLH. These findings suggest the VIIIa bHLH proteins as core regulators for reproductive development, including germ cell differentiation, since an early stage of land plant evolution.

Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice.

Understanding the genetic basis of reproductive barriers between species has been a central issue in evolutionary biology. The S1 locus in rice causes hybrid sterility and is a major reproductive barrier between two rice species, Oryza sativa and Oryza glaberrima The O. glaberrima-derived allele (denoted S1g) on the S1 locus causes preferential abortion of gametes with its allelic alternative (denoted S1s) in S1g/S1s heterozygotes. Here, we used mutagenesis and screening of fertile hybrid plants to isolate a mutant with an allele, S1mut, which does not confer sterility in the S1mut/S1g and S1mut/S1s hybrids. We found that the causal mutation of the S1mut allele was a deletion in the peptidase-coding gene (denoted "SSP") in the S1 locus of O. glaberrima No orthologous genes of SSP were found in the O. sativa genome. Transformation experiments indicated that the introduction of SSP in carriers of the S1s allele did not induce sterility. In S1mut/S1s heterozygotes, the insertion of SSP led to sterility, suggesting that SSP complemented the loss of the functional phenotype of the mutant and that multiple factors are involved in the phenomenon. The polymorphisms caused by the lineage-specific acquisition or loss of the SSP gene were implicated in the generation of hybrid sterility. Our results demonstrated that artificial disruption of a single gene for the reproductive barrier creates a "neutral" allele, which facilitates interspecific hybridization for breeding programs.

Characterization of KIF11 as a novel prognostic biomarker and therapeutic target for oral cancer.

Oral cancer has a high mortality rate, and its incidence is increasing gradually worldwide. As the effectiveness of standard treatments is still limited, the development of new therapeutic strategies is eagerly awaited. Kinesin family member 11 (KIF11) is a motor protein required for establishing a bipolar spindle in cell division. The role of KIF11 in oral cancer is unclear. Therefore, the present study aimed to assess the role of KIF11 in oral cancer and evaluate its role as a prognostic biomarker and therapeutic target for treating oral cancer. Immunohistochemical analysis demonstrated that KIF11 was expressed in 64 of 99 (64.6%) oral cancer tissues but not in healthy oral epithelia. Strong KIF11 expression was significantly associated with poor prognosis among oral cancer patients (P=0.034), and multivariate analysis confirmed its independent prognostic value. In addition, inhibition of KIF11 expression by transfection of siRNAs into oral cancer cells or treatment of cells with a KIF11 inhibitor significantly suppressed cell proliferation, probably through G2/M arrest and subsequent induction of apoptosis. These results suggest that KIF11 could be a potential prognostic biomarker and therapeutic target for oral cancer.

Cell division cycle associated 1 as a novel prognostic biomarker and therapeutic target for oral cancer.

Oral cavity carcinoma (OCC) is one of the most common causes of cancer-related death worldwide and has poor clinical outcome after standard therapies. Therefore, new prognostic biomarkers and therapeutic targets for OCC are urgently needed. We selected cell division cycle associated 1 (CDCA1) as a candidate OCC biomarker. Immunohistochemical analysis confirmed that CDCA1 protein was expressed in 67 of 99 OCC tissues (67.7%), but not in healthy oral epithelia. CDCA1 expression was significantly associated with poor prognosis in OCC patients (P=0.0244). Knockdown of CDCA1 by siRNAs significantly increased apoptosis of tumor cells. These data suggest that CDCA1 represents a novel prognostic biomarker and therapeutic target for OCC.

An oncofetal antigen, IMP-3-derived long peptides induce immune responses of both helper T cells and CTLs.

Insulin-like growth factor II mRNA-binding protein 3 (IMP-3), an oncofetal antigen identified using genome-wide cDNA microarray analyses, is overexpressed in several malignancies. IMP-3-derived cytotoxic T lymphocyte (CTL) epitopes have been used for peptide-based immunotherapies against various cancers. In addition to CTLs, induction of tumor-associated antigen (TAA)-specific helper T (Th) cells is crucial for establishment of effective antitumor immunity. In this study, we aimed to identify IMP-3-derived long peptides (IMP-3-LPs) carrying CTL and promiscuous Th-cell epitopes for use in cancer immunotherapy. IMP-3-derived Th-cell epitopes that bind to multiple HLA-class II molecules were predicted by in silico analysis, and their immunogenicity was determined by utilizing human T cells. We identified two highly immunogenic IMP-3-LPs presented by multiple HLA-class II molecules. One of the IMP-3-LPs encompassed two CTL epitopes that have been used for peptide-vaccine immunotherapy in ongoing clinical trials. IMP-3-LPs-specific Th cells responded to autologous dendritic cells (DCs) loaded with the recombinant IMP-3 proteins, suggesting that these s (LPs) can be naturally processed and presented. The IMP-3-LPs and specific Th cells augmented the expansion of IMP-3-specific CTLs, which was further enhanced by programmed cell death-1 (PD-1) blockade. In addition, IMP-3-LP encapsulated in liposomes was efficiently cross-presented in vitro, and this LP successfully cross-primed CTLs in HLA-A2 transgenic mice (Tgm) in vivo. Furthermore, one of the IMP-3-LPs induced IMP-3-specific Th cells from peripheral blood mononuclear cells (PBMCs) of head-and-neck malignant tumor (HNMT) patients. These findings suggest the potential usefulness of IMP-3-LPs in propagating both Th cells and CTLs and may have implications for IMP-3-LPs-based cancer immunotherapy.

Sentinel lymph node biopsy reduces the incidence of secondary neck metastasis in patients with oral squamous cell carcinoma.

It has recently been established that sentinel node biopsy (SNB) is an applicable and feasible procedure for the prediction of neck lymph node status in patients with early oral squamous cell carcinoma (OSCC) who are clinically negative for neck metastasis (cN0). The aim of this study was to retrospectively compare excision followed by watchful waiting with excision and SNB, in order to determine the effectiveness of SNB. A total of 125 patients with cN0 early OSCC were divided into two groups, namely the excision alone (n=78) and excision with SNB (n=47) groups. The clinical data of these two groups between 2006 and 2013 were analyzed. In the excision with SNB group, the negative predictive value and false-negative rate of SNB were 94% (30/32) and 18% (2/11), respectively. Secondary neck metastasis, also known as delayed neck metastasis, occurred in 24.2% of the patients in the excision alone group and 4.9% of the patients in the excision with SNB group. The 5-year overall survival (OS) rates were 84.0 and 97.5% in the excision alone and excision with SNB groups, respectively. Significant differences were found in the rate of secondary neck metastasis and OS between the two groups. SNB may be effective in the detection of occult neck lymph node metastasis, with a reduction in the incidence of secondary neck metastasis and improvements in the 5-year OS in patients with early-stage (stage I/II) oral cancer.

A clinical trial of multiple peptides vaccination for advanced head and neck cancer patients induced immune responses and prolonged OS.

A phase II clinical trial for head and neck squamous cell cancer (HNSCC) patients using multiple peptides vaccine derived from tumor-associated antigens (TAAs) was performed. The therapy was well tolerated and overall survival was statistically significantly longer in vaccinated patients, and the patients exhibiting cytotoxic T lymphocytes (CTL) responses to multiple peptides exhibited better prognosis.

Cancer immunotherapy using novel tumor-associated antigenic peptides identified by genome-wide cDNA microarray analyses.

Recent genome-wide cDNA microarray analysis of gene expression profiles in comprehensive tumor types coupled with isolation of cancer tissues by laser-microbeam microdissection have revealed ideal tumor-associated antigens (TAAs) that are frequently overexpressed in various cancers including head and neck squamous cell cancer (HNSCC) and lung cancer, but not in most normal tissues except for testis, placenta, and fetal organs. Preclinical studies using HLA-transgenic mice and human T cells in vitro showed that TAA-derived CTL-epitope short peptides (SPs) are highly immunogenic and induce HLA-A2 or -A24-restricted CTLs. Based on the accumulated evidence, we carried out a phase II clinical trial of the TAA-SP vaccine in advanced 37 HNSCC patients. This study showed a significant induction of TAA-specific CTLs in the majority of patients without serious adverse effects. Importantly, clinical responses including a complete response were observed in this study. Another phase II clinical trial of therapeutic TAA-SP vaccine, designed to evaluate the ability of prevention of recurrence, is ongoing in HNSCC patients who have received curative operations. Further studies in human preclinical studies and in vivo studies using HLA class I transgenic mice showed TAA-derived long peptides (TAA-LPs) have the capacity to induce not only promiscuous HLA class II-restricted CD4(+) T helper type 1 cells but also tumor-specific CTLs through a cross-presentation mechanism. Moreover, we observed an augmentation of TAA-LP-specific T helper type 1 cell responses and tumor antigen-spreading in HNSCC patients vaccinated with TAA-SPs. This accumulated evidence suggests that therapeutic TAA-SPs and LPs vaccines may provide a promising cancer immunotherapy.

The effects of phytochrome-mediated light signals on the developmental acquisition of photoperiod sensitivity in rice.

Plants commonly rely on photoperiodism to control flowering time. Rice development before floral initiation is divided into two successive phases: the basic vegetative growth phase (BVP, photoperiod-insensitive phase) and the photoperiod-sensitive phase (PSP). The mechanism responsible for the transition of rice plants into their photoperiod-sensitive state remains elusive. Here, we show that se13, a mutation detected in the extremely early flowering mutant X61 is a nonsense mutant gene of OsHY2, which encodes phytochromobilin (PΦB) synthase, as evidenced by spectrometric and photomorphogenic analyses. We demonstrated that some flowering time and circadian clock genes harbor different expression profiles in BVP as opposed to PSP, and that this phenomenon is chiefly caused by different phytochrome-mediated light signal requirements: in BVP, phytochrome-mediated light signals directly suppress Ehd2, while in PSP, phytochrome-mediated light signals activate Hd1 and Ghd7 expression through the circadian clock genes' expression. These findings indicate that light receptivity through the phytochromes is different between two distinct developmental phases corresponding to the BVP and PSP in the rice flowering process. Our results suggest that these differences might be involved in the acquisition of photoperiod sensitivity in rice.

Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS.

PURPOSE: The peptides derived from ideal cancer-testis antigens, including LY6K, CDCA1, and IMP3 (identified using genome-wide cDNA microarray analyses), were used in immunotherapy for head and neck squamous cell cancer (HNSCC). In this trial, we analyzed the immune response to and safety and efficacy of vaccine therapy. EXPERIMENTAL DESIGN: A total of 37 patients with advanced HNSCC were enrolled in this trial of peptide vaccine therapy, and the OS, PFS, and immunologic response were evaluated using enzyme-linked ImmunoSpot (ELISPOT) and pentamer assays. The peptides were subcutaneously administered weekly with IFA. The primary endpoints were evaluated on the basis of differences between HLA-A*2402-positive [A24(+)] patients treated with peptide vaccine therapy and -negative [A24(-)] patients treated without peptide vaccine therapy among those with advanced HNSCC. RESULTS: Our cancer vaccine therapy was well tolerated. The OS of the A24(+) vaccinated group (n = 37) was statistically significantly longer than that of the A24(-) group (n = 18) and median survival time (MST) was 4.9 versus 3.5 months, respectively; P < 0.05. One of the patients exhibited a complete response. In the A24(+) vaccinated group, the ELISPOT assay identified LY6K-, CDCA1-, and IMP3-specific CTL responses in 85.7%, 64.3%, and 42.9% of the patients, respectively. The patients showing LY6K- and CDCA1-specific CTL responses demonstrated a longer OS than those without CTL induction. Moreover, the patients exhibiting CTL induction for multiple peptides demonstrated better clinical responses. CONCLUSIONS: The immune response induced by this vaccine may improve the prognosis of patients with advanced HNSCC.

Identification of immunogenic LY6K long peptide encompassing both CD4+ and CD8+ T-cell epitopes and eliciting CD4+ T-cell immunity in patients with malignant disease.

Identification of peptides that activate both tumor-specific helper T (Th) cells and cytotoxic T lymphocytes (CTLs) are important for the induction of effective antitumor immune responses. We focused on a long peptide (LP) derived from lymphocyte antigen 6 complex locus K (LY6K) encompassing both candidate Th epitopes and a known CTL epitope. Using IFNγ ELISPOT assays as a marker of activated T cells, we studied the immunogenicity and cross-priming potential of LY6K-LP, assaying human immune cell responses in vitro and immunologic activities in HLA-A24 transgenic mice in vivo. We identified LY6K172-191-LP as an effective immunogen spanning naturally processed epitopes recognized by T helper type 1 (Th1) cells and CTLs. LY6K-specific CTLs were induced through cross-presentation of LY6K172-191-LP in vitro and in vivo. In addition, LY6K172-191-LP enhanced induction of LY6K-specific CTLs among the peripheral blood mononuclear cells (PBMCs) of head-and-neck malignant tumor (HNMT) patients. LY6K172-191-LP-specific Th1 immunologic response following 1 week in vitro stimulation of PBMCs with LY6K172-191-LP were detected in 16 of 21 HNMT patients (76%) vaccinated with CTL-epitope peptides and 1 of 11 HNMT patients (9%) prior to vaccination, but not in 9 healthy donors. Our results are the first to demonstrate the presence of LY6K-specific Th1 cell responses in HNMT patients and underscore the possible utility of LY6K172-191-LP for the induction and propagation of both LY6K-specific Th1 cells and CTLs.

Identification of CDCA1-derived long peptides bearing both CD4+ and CD8+ T-cell epitopes: CDCA1-specific CD4+ T-cell immunity in cancer patients.

We recently identified a novel cancer-testis antigen, cell division cycle associated 1 (CDCA1) using genome-wide cDNA microarray analysis, and CDCA1-derived cytotoxic T lymphocyte (CTL)-epitopes. In this study, we attempted to identify CDCA1-derived long peptides (LPs) that induce both CD4+ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II-binding peptides with CDCA1-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate CDCA1-LPs encompassing both Th cell epitopes and CTL-epitopes. We studied the immunogenicity of CDCA1-LPs and the cross-priming potential of LPs bearing CTL-epitopes in both human in vitro and HLA-class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head-and-neck cancer (HNC) patients before and after vaccination with a CDCA1-derived CTL-epitope peptide using IFN-γ enzyme-linked immunospot assays. We identified two CDCA1-LPs, CDCA1(39­64)-LP and CDCA1(55­78)-LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1-specific CTLs were induced through cross-presentation of CDCA1-LPs in vitro and in vivo. In addition, CDCA1-specific Th cells enhanced induction of CDCA1-specific CTLs. Furthermore, significant frequencies of CDCA1-specific Th cell responses were detected after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1-LPs in HNC patients (CDCA1(39­64)-LP, 74%; CDCA1(55­78)-LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1-specific Th cell responses in HNC patients and underline the possible utility of CDCA1-LPs for propagation of both CDCA1-specific Th cells and CTLs.