Beclin1 Binds to Enterovirus 71 3D Protein to Promote the Virus Replication.

Enterovirus 71 (EV71) is the main pathogen causing hand-foot-mouth disease (HFMD) in infants and children, which can also lead to severe neurological diseases and even death. Therefore, understanding the replication mechanism of EV71 is of great significance for the prevention and control of EV71-induced diseases. Beclin1 (BECN1, a mammalian homologue of ATG6 in yeast) is an important core protein for the initiation and the normal process of autophagy in cells. In addition to its involvement in autophagy, Beclin1 has also been reported to play an important role in cancer and innate immune signaling pathways. However, the role of Beclin1 in EV71 replication remains elusive. Here, we primarily found that Beclin1 facilitates EV71 replication in human rhabdomyosarcoma (RD) cells and the autophagy was actually induced, but Beclin1 was not significantly affected at either mRNA level or protein level during early EV71 infection. Further studies discovered that Beclin1 could interacts with EV71 non-structural protein 3D mainly through its evolutionary conserved domain (ECD) and coiled-coiled domain (CCD), thus promoting the replication of EV71 in human rhabdomyosarcoma (RD) cells and human astroglioma (U251) cells. Collectively, we reveal a novel regulatory mechanism associated with Beclin1 to promote EV71 replication, thus providing a potential therapeutic target for the prevention and control of EV71-associated diseases.

Single-nucleotide variants in human CD81 influence hepatitis C virus infection of hepatoma cells.

An estimated number of 71 million people are living with chronic hepatitis C virus (HCV) infection worldwide and 400,000 annual deaths are related to the infection. HCV entry into the hepatocytes is complex and involves several host factors. The tetraspanin human CD81 (hCD81) is one of the four essential entry factors and is composed of one large extracellular loop, one small extracellular loop, four transmembrane domains, one intracellular loop and two intracellular tails. The large extracellular loop interacts with the E2 glycoprotein of HCV. Regions outside the large extracellular loop (backbone) of hCD81 have a critical role in post-binding entry steps and determine susceptibility of hepatocytes to HCV. Here, we investigated the effect of five non-synonymous single-nucleotide variants in the backbone of hCD81 on HCV susceptibility. We generated cell lines that stably express the hCD81 variants and infected the cells using HCV pseudoparticles and cell culture-derived HCV. Our results show that all the tested hCD81 variants support HCV pseudoparticle entry with similar efficiency as wild-type hCD81. In contrast, variants A54V, V211M and M220I are less supportive to cell culture-derived HCV infection. This altered susceptibility is HCV genotype dependent and specifically affected the cell entry step. Our findings identify three hCD81 genetic variants that are impaired in their function as HCV host factors for specific viral genotypes. This study provides additional evidence that genetic host variation contributes to inter-individual differences in HCV infection and outcome.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES: The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS: To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS: Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS: This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

Detection of Lactate Dehydrogenase Elevating Virus in a Mouse Vivarium Using an Exhaust Air Dust Health Monitoring Program.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.

Absence of bovine leukemia virus proviral DNA in Japanese human blood cell lines and human cancer cell lines.

Bovine leukemia virus (BLV) infects cattle worldwide and causes B-cell lymphoma in cattle. BLV has been identified in human breast and lung cancer and in blood, but the association of BLV and human cancer is controversial. In this study, we investigated the existence of BLV in 145 Japanese human blood cell lines and 54 human cancer cell lines, using a new highly sensitive PCR assay that can amplify even one copy of BLV using LTR primers different from those in previous studies on BLV provirus in breast cancer. All samples were found negative for BLV provirus, suggesting that BLV is unlikely to infect humans.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin.

BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.

Anti-tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells.

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first-line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti-tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase-dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra-tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus-induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.

The BeWo cell line derived from a human placental choriocarcinoma is permissive for respiratory syncytial virus infection.

The respiratory syncytial virus (RSV) is the main pathogen associated with upper respiratory tract infections during early childhood. Vertical transmission of this virus has been suggested in humans, based on observations recorded during animal studies that revealed an association of RSV with persistent structural and functional changes in the developing lungs of the offspring. However, human placentas have not yet been evaluated for susceptibility to RSV infection. In this study, we examined the capacity of RSV to infect a human trophoblast model, the BeWo cell line. Our results suggest that BeWo cells are susceptible to RSV infection since they allow RNA viral replication, viral protein translation, leading to the production of infectious RSV particles. In this report, we demonstrate that a human placenta model system, consisting of BeWo cells, is permissive to RSV infection. Thus, the BeWo cell line may represent a useful model for studies that aim to characterize the events of a possible RSV infection at the human maternal-fetal interface.

Susceptibility of epithelial tumour cell lines to hyperthermia.

BACKGROUND: Human skin or mucosa exposes cells to both an internal and exogeneous thermal environment and the cells survive within a certain range of temperature. Exogeneous hyperthermia has been applied for the treatment of various types of cancers, fungal disease, and warts. OBJECTIVES: To determine whether different cellular components in the skin adapt to hyperthermic conditions differentially and further elucidate the mechanisms involved. MATERIALS & METHODS: Cell lines derived from normal and tumour epithelial cells were treated with hyperthermic conditions and tested for viability (using an MTS assay), apoptosis (using a FITC-conjugated annexin V apoptosis detection kit), and changes in intracellular calcium (using a calcium-sensitive fluorescent single-wavelength dye, Fluo-4 AM). RESULTS: Thermo-resistance of different cell types was different when cells were subjected to heat at 45̊C for 30 minutes. Stronger effects of hyperthermia were noted on cell viability and apoptosis in epidermal cells relative to their malignant counterparts, except for cell lines harbouring human papillomavirus (HPV). Hyperthermia had a much greater effect on cell viability and apoptosis in a HPV-negative cell line compared to HPV-positive cell lines. We further found that hyperthermia treatment resulted in a strong calcium influx which led to apoptotic cells. However, no obvious increase in apoptosis was observed in cells treated with the CRAC channel selective inhibitor, BTP2, before application of hyperthermia in all cell types, except three cervical cell lines harbouring HPV. CONCLUSION: We propose that hyperthermia results in a CRAC-related strong calcium influx which induces apoptosis, with the exception of HPV-positive cells.

[Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines].

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

RSV Growth and Quantification by Microtitration and qRT-PCR Assays.

Defective interfering viral particles have been reported as important determinants of the course of viral infection, and they can markedly temper the virulence of the infection. Here, we describe a simple method, based on limiting dilution, for the removal of defective interfering particles from RSV. This method results in a high-titer viral preparation from both HEp-2 and Vero cell lines. We evaluated two concentrations of sucrose to stabilize the virus preparation, and demonstrate that RSV is stable when prepared and stored in 25 % sucrose at -152 °C. In addition, this chapter describes some commonly used methods of RSV titration, detection using microtitration and quantitative real-time RT-PCR, and the use of immunostaining for antigenic characterization.

Quantification of RSV Infectious Particles by Plaque Assay and Immunostaining Assay.

One of the most commonly used approaches for determining the quantity of infectious RSV particles in a given sample is the plaque assay. RSV infectious particles can be quantified by various direct and indirect methods. Here, we explain two simple methods for RSV titration: plaque assay and immunostaining assay.

Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

Geneticin Stabilizes the Open Conformation of the 5' Region of Hepatitis C Virus RNA and Inhibits Viral Replication.

The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.

The effects of oncolytic reovirus in canine lymphoma cell lines.

Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus.

Cysteine-rich secretory protein 3 inhibits hepatitis C virus at the initial phase of infection.

Hepatitis C virus (HCV) affects 2-3% of the global population. Approximately one-quarter of acute infections cause chronic hepatitis that leads to liver cirrhosis or hepatocellular carcinoma. The major obstacle of current research is the extremely narrow host tropism of HCV. A single HCV strain can replicate in the Huh7 human hepatoma cell line. Huh7 cells can be adapted under selective pressure in vitro to identify host factors that influence viral replication. Here, we extended this strategy to the in vivo condition and generated a series of cell lines by multiple rounds of adaptation in immunocompromised mice. Adaptation increased the cellular resistance to HCV infection. Microarray analyses revealed that the expression levels of several genes were associated with HCV resistance. Notably, up-regulation of the mRNA encoding cysteine-rich secretory protein 3 (CRISP3), a glycoprotein with unknown function that is secreted from multiple exocrine glands, was correlated with HCV resistance. The presence of CRISP3 in the culture medium limited HCV replication at the early phase of infection.

[Monitoring of enterovirus circulation in Irkutsk region].

AIM: Monitoring of circulation of enteroviruses (EVI) in Irkutsk Region and study of regional specter of circulating enteroviruses. MATERIALS AND METHODS: 1419 samples from patients with suspected EVI, contact in foci ofenterovirus infection, acute intestine infections and 964 samples of sewage water were studied in total. In 2011 isolation of viral agents from 97 samples positive on enterovirus by RT-PCR from patients with preliminary EVI diagnosis and 5 samples of sewage water of Irkutsk city was carried out. Transplantable line of human rhabdomyosarcoma RD cell culture was used for isolation of enteroviruses. Infection of cells and 2 serial passages of the studied material were carried out. The isolates were typed in neutralization reaction (NR) with a set of 32 diagnostic type-specific immune sera against viral poliomyelitis I-III; Coxsackie B1-6; Coxsackie A2, A4, A7, A9, A10; ECHO 68 - 71; ECHO 2, 4, 7, 8, 9, 12, 16, 20, 25, 26, 27, 29, 31, 33. RESULTS: In 2011 circulation of enterovirus serotypes that were previously absent on the territory of the region was established: ECHO 68, ECHO 70, ECHO 71. These strains were isolated from patients, circulation of ECHO 70 serotype was established also in samples of sewage water. The analysis of enterovirus landscape carried out showed the possibility of complication of epidemic situation on the territory of the region due to change of serovariants of causative agents of non-polioenterovirus infections and detection ofepidemically significant enteroviruses - ECHO 68, 70 and 71 serotypes. CONCLUSION: Determination of specter ofenterovirus serotypes, detection of serotypes that had not previously circulated in Irkutsk Region allows to prognose epidemic situation on morbidity of enterovirus infections and timely develop and make decisions for ensuring epidemiologic welfare of the population.

Therapeutic effect of oncolytic adenovirus expressing relaxin in radioresistant oral squamous cell carcinoma.

Radioresistance is one of the main determinants of treatment outcome in oral squamous cell carcinoma (OSCC), and treatment of radioresistant OSCC is difficult due to cross resistance to other conventional treatments. We aimed to identify whether genetically modified oncolytic adenovirus expressing relaxin (RLX), which affects collagen metabolism, can effectively inhibit growth of the radioresistant OSCC. Therapeutic effect of oncolytic adenovirus was compared between radiosensitive and radioresistant OSCC cell lines in vitro and in vivo, and spread of adenovirus throughout the tumor mass was verified by immunohistochemistry (IHC). Oncolytic adenovirus effectively killed cancer cells and there was no significant difference in the cytotoxic effect between radiosensitive and radioresistant OSCC cell lines. In animal experiments, the adenovirus significantly reduced the size of tumor, and there was no significant difference between radiosensitive and radioresistant OSCC. In IHC, RLX expressing adenovirus showed better proliferation and eliminated collagens more effectively compared to RLX nonexpressing adenovirus. These findings suggested that genetically modified oncolytic adenovirus can effectively inhibit growth of the radioresistant OSCC and might be a new therapeutic option in radioresistant OSCC.

Cytotoxicity of lymphocytes activated by superantigen toxic-shock-syndrome toxin-1 against colorectal cancer LoVo cells.

Toxic-shock-syndrome toxin-1 (TSST-1), a superantigen, can stimulate T cell activation and be used for immunotherapy. In this study, we employed the carcinoembryonic antigen (CEA)-positive LoVo cells to test whether retrovirus-mediated TSST-1 expression could activate human T cells and promote cytotoxicity against tumor cells. We first generated plasmids of pLEGFP-N1-5HRE-CEAp-TSST-1-linker-CD80TM containing a fusion gene of the CEA promoter, 5 copies of the hypoxia-response elements (HRE) as an enhancer, the fragments for TSST-1, and the transmembrane domain of CD80 (CD80TM) and control pLEGFP-N1-5HRE-CEAp (without TSST-1) and generated retroviruses of 5HCTC and 5HC, respectively. After infection with 5HC and 5HCTC retroviruses to establish cell lines, the high levels of TSST-1 expression were observed on the membrane and cytoplasm of the 5HCTC-infected LoVo cells, particularly culture under a hypoxic condition, but not on CEA(-) HeLa cells. Furthermore, the TSST-1-expressing LoVo cell lysates, but not the control cell lysates, stimulated human T cell proliferation, and the co-culture of the TSST-1-expressing LoVo, but not control cells, with human peripheral blood mononuclear cells (PBMC) induced a high frequency of TNF-α- and IL-2-secreting T cells in vitro, particularly under hypoxic conditions. More importantly, co-culture of the TSST-1-expressing LoVo cells, particularly under hypoxic conditions, but not control cells, with different numbers of PBMC promoted potent cytotoxicity against LoVo cells in a dose-dependent manner in vitro. These data provide proof of the principle that selective induction of TSST1 expression in CEA(+) colorectal cancer (CRC) cells activates T cells that destroy tumor cells, particularly under a hypoxic condition. Therefore, our findings may aid in the design of new immunotherapy for the intervention of CRC at clinic.