Anti-PD-L1 × anti-CD3 bispecific T-cell engager-armed T cells can overcome immunosuppression and redirect T cells to kill breast cancer cells expressing PD-L1.

T cell-based immunotherapy has transformed cancer treatment. Nonetheless, T cell antitumor activity can be inhibited by an immune checkpoint molecule expressed on cancer cells, program death ligand 1 (PD-L1), which interacts with the PD-1 on T cells. We generated αPD-L1 × αCD3 bispecific T-cell engager-armed T cells (BATs) to prevent PD-L1/PD-1 interaction and hence to redirect T cells to kill cancer cells. αPD-L1 × αCD3 bispecific T-cell engagers (BTEs) were produced from Chinese hamster ovary (CHO) cells to arm human primary T cells. Flow cytometry was used to investigate BTE binding to BATs. The cytotoxicity of BATs against PD-L1-expressing breast cancer (BC) cell lines was assessed in 2-dimensional (2D) and 3-dimensional (3D) culture models. The binding stability of BTE on BATs and their efficacy after cryopreservation were also examined. The CHO cell BTE expression yield was 3.34 mg/ml. The binding ability on T cells reached 91.02 ± 4.2 %. BATs specifically lysed PD-L1-expressing BC cells, with 56.4 ± 15.3 % HCC70 cells and 70.67 ± 15.6 % MDA-MB-231 cells lysed at a 10:1 effector-to-target ratio. BATs showed slight, nonsignificant lysis of PD-L1-negative BC cells, MCF-7, and T47D. Moreover, BATs significantly disrupted MDA-MB-231 3D spheroids expressing PD-L1 after 48 and 72 h of coculture. Cryopreserved BATs maintained BTE binding stability, cell viability, and anticancer activity, comparable to fresh BATs. αPD-L1 × αCD3 BATs induced the cytolysis of PD-L1-expressing BC cells in 2D and 3D coculture assays. BATs can be prepared and preserved, facilitating their use and transportation. This study demonstrates the potential of αPD-L1 × αCD3 BATs in treating cancers with positive PD-L1 expression.

High prevalence of Wilms tumor 1 expression in multiple myeloma and plasmacytoma: A cohort of 142 Asian patients' samples.

Wilms tumor 1 (WT1) is a promising target antigen for cancer immunotherapy. However, WT1 protein expression and its clinical correlation in multiple myeloma (MM) patients are still limited. We, therefore, investigated WT1 expression in 142 bone marrow and plasmacytoma samples of MM patients at different stages of the disease by immunohistochemistry. The correlations between WT1 expression and clinical parameters or treatment outcomes were evaluated. The overall positive rate of WT1 expression was 91.5%; this high prevalence was found in both bone marrow and plasmacytoma samples, regardless of the disease status. Cytoplasmic WT1 expression was correlated with high serum free light chain ratio at presentation. However, no significant association between WT1 expression and treatment outcome was observed. This study confirms the high prevalence of WT1 expression in an Asian cohort of MM, encouraging the development of immunotherapy targeting WT1 in MM patients, particularly in those with extramedullary plasmacytoma or relapsed disease.

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells.

BACKGROUND AIMS: Cholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA. METHODS: To overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28. RESULTS: Initially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8+ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed. CONCLUSION: Taken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.

Anti-CD19 chimeric antigen receptor T cells secreting anti-PD-L1 single-chain variable fragment attenuate PD-L1 mediated T cell inhibition.

Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19+ target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19+/PD-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19+/PD-L1high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma.

Alpha-mangostin inhibits viral replication and suppresses nuclear factor kappa B (NF-κB)-mediated inflammation in dengue virus infection.

Severe dengue virus (DENV) infection results from viral replication and dysregulated host immune response, which trigger massive cytokine production/cytokine storm. The result is severe vascular leakage, hemorrhagic diathesis, and organ dysfunction. Subsequent to previously proposing that an ideal drug for treatment of DENV infection should efficiently inhibit both virus production and cytokine storm, we discovered that α-mangostin (α-MG) from the pericarp of the mangosteen fruit could inhibit both DENV infection and cytokine/chemokine production. In this study, we investigated the molecular mechanisms underlying the antiviral and anti-inflammatory effects of α-MG. Time-of-drug-addition and time-of-drug-elimination studies suggested that α-MG inhibits the replication step of the DENV life cycle. α-MG inhibited polymerization activity of RNA-dependent RNA polymerase (RdRp) with IC50 values of 16.50 µM and significantly reduced viral RNA and protein syntheses, and virion production. Antiviral and cytokine/chemokine gene expression profiles of α-MG-treated DENV-2-infected cells were investigated by polymerase chain reaction array. α-MG suppressed the expression of 37 antiviral and cytokine/chemokine genes that relate to the NF-κB signaling pathway. Immunofluorescence and immunoblot analyses revealed that α-MG inhibits NF-κB nuclear translocation in DENV-2-infected cells in association with reduced RANTES, IP-10, TNF-α, and IL-6 production. These results suggest α-MG as a potential treatment for DENV infection.

Mesothelin­specific T cell cytotoxicity against triple negative breast cancer is enhanced by 40s ribosomal protein subunit 3­treated self­differentiated dendritic cells.

Triple negative breast cancer (TNBC) lacks targeted treatment resulting in poor prognosis. Targeting overexpressing mesothelin (MSLN) using MSLN­specific T cells is an attractive treatment approach and the aim of the present study. The expression of MSLN in human TNBC paraffin sections was analyzed by immunohistochemistry. Lentiviral vector harbored granulocyte­macrophage colony stimulating factor (GM­CSF), interleukin­4 (IL­4) and MSLN cDNAs was constructed to generate self­differentiated myeloid­derived antigen­presenting­cells reactive against tumor expressing MSLN dendritic cell (MSLN­SmartDC) for MSLN­specific T cell activation. The results showed high MSLN in 32.8% of all breast cancer subtypes and 57% in TNBC. High MSLN was significantly associated with TNBC subtype and the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. MSLN­SmartDC exhibited comparable phenotype to DC generated by exogenous cytokine treatment and an addition of 40s ribosomal protein subunit 3 (RPS3), a toll­like receptor 4 ligand, enhanced DC maturation and function by upregulation of CD40, CD80 and CD83 expressions and IL­12p70 secretion. MSLN­specific CD8+CD69+ IFN­Î³+ T cells were detected in T cells activated by both MSLN­SmartDC and RPS3­MSLN­SmartDC. MSLN­specific T cells activated by these DCs showed more specific killing capability against naturally expressed MSLN­HCC70 and artificially MSLN­overexpressing MDA­MB­231 compared with parental MDA­MB­231 in both two dimensional (2D)­ and 3D­culture systems. In conclusion, the results demonstrated the efficacy of MSLN­SmartDC to promote MSLN­specific T cells response against TNBC and RPS3 can enhance the cytolytic activity of these T cells providing an alternative treatment approach for patients with TNBC.

Combination gemcitabine and PD-L1xCD3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a lethal cancer with rapid progression and poor survival. Novel and more effective therapies than those currently available are, therefore, urgently needed. Our research group previously reported the combination of gemcitabine and cytotoxic T lymphocytes to be more effective than single-agent treatment for the elimination of CCA cells. However, gemcitabine treatment of CCA cells upregulates the expression of an immune checkpoint protein (programmed death-ligand 1 [PD-L1]) that consequently inhibits the cytotoxicity of T lymphocytes. To overcome this challenge and take advantage of PD-L1 upregulation upon gemcitabine treatment, we generated recombinant PD-L1xCD3 bispecific T cell engagers (BiTEs) to simultaneously block PD-1/PD-L1 signaling and recruit T lymphocytes to eliminate CCA cells. Two recombinant PD-L1xCD3 BiTEs (mBiTE and sBiTE contain anti-PD-L1 scFv region from atezolizumab and from a published sequence, respectively) were able to specifically bind to both CD3 on T lymphocytes, and to PD-L1 overexpressed after gemcitabine treatment on CCA (KKU213A, KKU055, and KKU100) cells. mBiTE and sBiTE significantly enhanced T lymphocyte cytotoxicity against CCA cells, especially after gemcitabine treatment, and their magnitudes of cytotoxicity were positively associated with the levels of PD-L1 expression. Our findings suggest combination gemcitabine and PD-L1xCD3 BiTE as a potential alternative therapy for CCA.

Cytotoxic T Cells Activated by Self-differentiated Monocyte-derived Dendritic Cells Against Multiple Myeloma Cells.

BACKGROUND/AIM: B cell maturation antigen (BCMA) is an ideal target for adoptive T cell therapy of multiple myeloma (MM). In this study, we evaluated self-differentiated monocyte-derived dendritic cells expressing BCMA (SD-DC-BCMA) to activate T cells for killing MM cells. MATERIALS AND METHODS: Lentivirus-modified SD-DC-BCMA harboring tri-cistronic cDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and BCMA was generated. Cytotoxicity of T cells activated by SD-DC-BCMA against MM cells was evaluated. RESULTS: T cells activated by SD-DC-BCMA exhibited a dose-dependent cytotoxicity against BCMA-expressing MM cells and produced high IFN-γ levels, compared to inactivated T cells or control T cells. A significantly higher killing ability of T cells activated by SD-DC-BCMA was further demonstrated in BCMA-overexpressing cells when compared with BCMA-negative cells. CONCLUSION: The potency of SD-DC-BCMA to activate T cells for antigen-specific cancer killing provides a framework for therapeutic application of adoptive T cell therapy in MM.

Antitumor activity of T cells secreting αCD133-αCD3 bispecific T-cell engager against cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a lethal cancer of bile duct epithelial cells with a high mortality rate and limited therapeutic options. An effective treatment is, therefore, urgently needed to improve treatment outcomes for these patients. To develop a new therapeutic option, we engineered T cells secreting αCD133-αCD3 bispecific T-cell engager and evaluated their antitumor effects against CD133-expressing CCA cells. The cDNA encoding αCD133-αCD3 bispecific T-cell engager (αCD133-αCD3-ENG) was cloned into pCDH lentiviral construct and its expression was tested in Lenti-X 293T cells. T cells from healthy donors were then transduced with engineered lentiviruses to create T cells secreting αCD133-αCD3 engager to evaluate their antitumor activities. The average transduction efficiency into T cells was approximately 60.03±21.65%. In the co-culture system containing T cells secreting αCD133-αCD3 engager (as effector cells) and mWasabi-luciferase-expressing CCA cells (KKU-100 and KKU-213A; as target cells), the effector T cells exhibited significantly higher cytolytic activities against the target CCA cells (49.0±9.76% and 64.10±13.18%, respectively) than those observed against the untransduced T cells (10.97±10.65%; p = 0.0103 and 9.80±11.05%; p = 0.0054) at an effector-to-target ratio of 5:1. In addition, the secreted αCD133-αCD3 engager significantly redirected both transduced T cells and bystander T cells to kill the target CCA cells (up to 73.20±1.68%; p<0.05). Moreover, the transduced and bystander T cells could kill the target CCA spheroids at a rate approximately 5-fold higher than that of the no treatment control condition (p = 0.0011). Our findings demonstrate proof-of-principle that T cells secreting αCD133-αCD3 engager can be an alternative approach to treating CD133-positive CCA, and they pave the way for future in vivo study and clinical trials.

Phylogenetic molecular evolution and recombination analysis of complete genome of human parechovirus in Thailand.

Human parechovirus (HPeV), which is a member of the Picornavirus group of viruses, is a pathogen that is reported to be associated with manifestations that include respiratory tract involvement, gastroenteritis, sepsis-like symptom, and central nervous system complication. Until now, nineteen genotypes have been identified. The lack of proofreading property of viral RNA-dependent RNA polymerase (RdRp) together with recombination among the intra- and inter-genotypes of the virus results in high diversity. However, data specific to the molecular evolutionary perspective of the complete genome of HPeV remains limited. This study aimed to analyze the phylogenetic, molecular evolution, and recombination characteristics of the complete genome of HPeV strains isolated in Thailand during 2009-2012. Fifty-eight samples that were previously confirmed to be HPeV positive and then evaluated for genotyping were subjected to complete genome amplification to generate ten overlapping PCR fragments using a set of in-house designed primers. The same position of the viral genome was read in triplicate using direct Sanger sequencing. All samples were classified into the same previously defined genotypes in both whole-genome and VP1 phylogenic tree. However, sample B1091/HPeV14/2011 exhibited discordant grouping between whole-genome and VP1 on the phylogenetic tree. Bootscan analysis revealed that B1091/HPeV14/2011 inherited from two genotypic viruses, including VP1 from HPeV14, and the rest of the genome from HPeV1B. The results of this study provide important insights into the molecular evolution of and recombination in the viral genome of HPeV that will improve and accelerate our ability to develop treatment and prophylactic strategies in the future.

Chimeric Antigen Receptor T Cells Targeting Integrin αvß6 Expressed on Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvß6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvß6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvß6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvß6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvß6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvß6 expression and short survival time (p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvß6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvß6-positive CCA cells (p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvß6-positive CCA tumor spheroids (p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvß6-positive CCA.

Anti-mucin 1 chimeric antigen receptor T cells for adoptive T cell therapy of cholangiocarcinoma.

Current treatments for cholangiocarcinoma (CCA) are largely unsuccessful due to late diagnosis at advanced stage, leading to high mortality rate. Consequently, improved therapeutic approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a newly potential therapy that can recognize specific surface antigen without major histocompatibility complex (MHC) restriction. Mucin 1 (MUC1) is an attractive candidate antigen as it is highly expressed and associated with poor prognosis and survival in CCA. We, therefore, set forth to create the fourth-generation CAR (CAR4) construct containing anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ and evaluate anti-MUC1-CAR4 T cells in CCA models. Compared to untransduced T cells, anti-MUC1-CAR4 T cells produced increased levels of TNF-α, IFN-γ and granzyme B when exposed to MUC1-expressing KKU-100 and KKU-213A CCA cells (all p < 0.05). Anti-MUC1-CAR4 T cells demonstrated specific killing activity against KKU-100 (45.88 ± 7.45%, p < 0.05) and KKU-213A cells (66.03 ± 3.14%, p < 0.001) at an effector to target ratio of 5:1, but demonstrated negligible cytolytic activity against immortal cholangiocytes. Furthermore, the anti-MUC1-CAR4 T cells could effectively disrupt KKU-213A spheroids. These activities of anti-MUC1-CAR4 T cells supports the development of this approach as an adoptive T cell therapeutic strategy for CCA.

Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains.

Second-generation anti-CD19-chimeric antigen receptor T cells (anti-CD19-CAR2 T cells) are effective for treating B-cell malignancies; however, anti-CD19-CAR2 T cells can induce human anti-mouse immune responses because anti-CD19 single-chain variable fragment (scFv) in the CAR molecules is derived from a murine FMC63 (mFMC63) monoclonal antibody. Consequently, the persistence of mFMC63-CAR2 T cells and their therapeutic efficiency in patients are decreased, which results in tumor relapse. In an attempt to remedy this shortcoming, we generated a new anti-CD19-CAR T cells containing fully human anti-CD19 scFv (Hu1E7-CAR4 T cells) to pre-clinically evaluate and compare with mFMC63-CAR4 T cells. The human anti-CD19 scFv (Hu1E7) was isolated from a human scFv phage display library and fused to the hinge region of CD8α, the transmembrane domain of CD28, three intracellular costimulatory domains (CD28, 4-1BB, and CD27), and a CD3ζ signaling domain (28BB27ζ). Compared to mFMC63-CAR2 T cells (BBζ) and mFMC63-CAR3 (BB27ζ), the mFMC63-CAR4 T cells (28BB27ζ) exerted superior anti-tumor activity against Raji (CD19+) target cell. The Hu1E7-CAR4 and mFMC63-CAR4 T cells demonstrated comparable cytotoxicity and proliferation. Interestingly, compared to mFMC63-CAR4 T cells, the Hu1E7-CAR4 T cells secreted lower levels of cytokines (IFN-γ and TNF-α), which may be due to the lower binding affinity of Hu1E7-CAR4 T cells. These findings demonstrated the successfulness in creation of a new CAR T cells containing a novel fully human-derived scFv specific to CD19+ cancer cells. In vivo studies are needed to further compare the anti-tumor efficacy and safety of Hu1E7-CAR4 T cells and mFMC63-CAR4 T cells.

Anti-tumour effect of the fourth-generation chimeric antigen receptor T cells targeting CD133 against cholangiocarcinoma cells.

Current treatment of cholangiocarcinoma (CCA) - a lethal bile duct cancer - is ineffective because the disease is usually diagnosed at late and advanced stage. Thus, a novel therapeutic modality is urgently required. Fourth-generation chimeric antigen receptor (CAR4) T cells was created to target CD133, a well-known cancer stem cell marker, that is highly expressed and associates with cancer progression. The anti-CD133-CAR4 T cells showed high efficacy against CD133-expressing CCA cells. Tumour cell lysis occurred in a dose- and CD133 antigen-dependent manner, and significantly higher, up to 57.59% ± 9.62 at effector to target ratio of 5:1 in a CCA cell line - KKU-213A cells, compared to mock control (p = 0.008). Similarly, significant IFN-γ (p = 0.011) and TNF-α (p = 0.002) upregulation was observed upon tumour treatment. The effectiveness of our anti-CD133-CAR4 T cells will be beneficial not only for CD133-expressing CCA, but also for other CD133-expressing tumours. This study may guide future in vivo study and clinical trials.

Breast cancer stem cell RNA-pulsed dendritic cells enhance tumor cell killing by effector T cells.

Cancer stem cells (CSCs) underpin the resistance of breast cancer (BC) cells to therapy. Dendritic cell (DC)-based treatment is efficacious and safe, but the efficiency of this technique for targeting CSCs in BC treatment requires further investigation. The present study aimed to investigate the ability of DCs pulsed with breast CSC antigens to activate effector lymphocytes for killing BC cells. CD44+/CD24- CSCs were isolated from BCA55-121, an in-house patient-derived BC cell line, and acquisition of stemness properties was confirmed by upregulated expression of OCT4A and a superior proliferative capacity in colony formation assays compared with whole population of BCA55-121 (BCA55-121-WP). DCs were differentiated from monocytes from peripheral blood of healthy donors and pulsed with CSC total RNA. Maturation of the CSC RNA-pulsed DCs was confirmed by increased expression of CD11c, CD40, CD83, CD86 and HLA-DR, as well as reduced CD14 expression compared with monocytes. Total lymphocytes co-cultured with CSC RNA-pulsed DCs were analyzed by flow cytometry for markers including CD3, CD4, CD8, CD16 and CD56. The results revealed that the co-cultures contained mostly cytotoxic CD8+ T lymphocytes followed by CD4+ T lymphocytes and smaller populations of natural killer (NK) and NKT cells. ELISA was used to measure IFN-γ production, and it was revealed that activated CD4+ and CD8+ lymphocytes produced more IFN-γ compared with naïve T cells, suggesting that CD8+ T cells were effector T cells. CSC RNA was a more efficient antigen source compared with RNA from mixed BC cells for activating tumor antigen-specific killing by T cells. These CSC-specific effector T cells significantly induced BC cell apoptosis at a 20:1 effector T cell:tumor cell ratio. Of note, the breast CSCs cultures demonstrated resistance to effector T cell killing, which was in part due to increased expression of programmed death ligand 1 in the CSC population. The present study highlights the potential use of CSC RNA for priming DCs in modulating an anticancer immune response against BC.

Cytolytic Activity of Effector T-lymphocytes Against Hepatocellular Carcinoma is Improved by Dendritic Cells Pulsed with Pooled Tumor Antigens.

Cellular immunotherapy is a promising new therapeutic approach for hepatocellular carcinoma (HCC), which has a high recurrence rate, irrespective of the treatment administered. In this study, we attempted to improve the cytolytic activity of effector T-lymphocytes against HCC. T-lymphocytes were activated by monocyte-derived dendritic cells (DCs) pulsed with cell lysate or RNA prepared from HCC cell lines. Monocytes were activated for differentiation into DCs by treatment with the IL4 and GM-CSF. DCs were pulsed with cell lysate or RNA prepared from a single cell line or combinations of two or three HCC cell lines, and then co-cultured with autologous T-lymphocytes with the intent of creating specific cytotoxicity. We discovered that DCs pulsed with total RNA effectuated greater T-lymphocyte function than DCs pulsed with total cell lysate, as evidenced by greater cytolytic activities against HCC target cells. The percentage of Huh7, HepG2, and SNU449 cell apoptosis at effector:target ratio of 10:1 was 42.6 ± 4.5% (p = 0.01), 33.6 ± 3.1% (p = 0.007), and 21.4 ± 1.4% (p < 0.001), respectively. DCs pulsed with pools of antigens prepared from three cell lines improved the cytolytic function of effector T-lymphocytes by approximately two-fold (p < 0.001), which suggests that this approach be further developed and applied for adoptive transfer treatment of HCC.

Inhibition of IL-10 and TGF-ß receptors on dendritic cells enhances activation of effector T-cells to kill cholangiocarcinoma cells.

Tumor escapes host immune responses by producing immunosuppressive cytokines, such as IL-10 and TGF-ß, secreted into the tumor microenvironment. These cytokines play important roles in the suppression of dendritic cell (DC) function, leading to decreased immune responses of the effector CD4+ and CD8+ T cells. To improve DC functions and enhance cytolytic activity of activated effector T-cells, we suppressed the effect of these cytokines on DCs by using specific neutralizing antibodies that inhibit IL-10 and TGF-ß receptors. Monocyte-derived DCs generated in vitro showed up-regulation of MHC (HLA-DR) and co-stimulatory molecules (CD40 and CD86). The IL-10 and TGF-ß receptors were expressed and localized on cell membrane of DCs, as shown by Western blot analysis and immunofluorescence staining, whereas the IL-10 and TGF-ß ligands were detected in the culture supernatants of DCs and cholangiocarcinoma (CCA) cell line, respectively. Inhibition of the IL-10 and TGF-ß receptors on DCs by specific neutralizing antibodies significantly increased level of IFN-γ and enhanced cytolytic activity of the DC-activated effector T-cells against CCA cell line. These results indicate that the IL-10 and TGF-ß receptors are the targets for inhibition to increase DC functions and enhance cytolytic activity of the DC-activated effector T-cells against CCA cells. Thus, inhibition of the IL-10 and TGF-ß receptors on DCs is crucial in the preparation of DC-activated effector T cells for adoptive T-cell therapy.

The prevalence and genotype diversity of Human Rotavirus A circulating in Thailand, 2011-2014.

Human rotavirus A (RVA) is the major infectious virus causing acute watery diarrhea in children, especially those younger than 5 years of age, and is a major public health problem in Thailand. Outbreaks of this virus have been reported worldwide. Besides the common genotypes, unusual genotypes providing evidence of inter-species transmission have also been described. Therefore, the aim of this study was to investigate the prevalence and genotypes of RVA in Thailand. A total of 688 samples were collected from children who were hospitalized with acute diarrhea in Chumphae Hospital in Khon Kaen and Chulalongkorn Hospital in Bangkok. RVA was detected using one-step RT-PCR and the genotypes were evaluated by sequencing. Overall, 204 of the 688 samples (30%) were positive for RVA. Nine genotypes were identified: three common in humans (G1P[8] [53%], G2P[4] [18%], G3P[8] [12%]), one feline-like (G3P[9] [1%]), four porcine-like (G4P[6] [0.5%], G5P[6] [0.5%], G9P[8] [0.5%], G12P[6] [1.5%]), and one bovine-like (G8P[8] [13%]). The variation in virus genotypes and the animal-like genotypes detected in this study suggested that a high diversity of RVA types is circulating in the Thai population. Therefore, continuous molecular epidemiological monitoring of RVA is essential and has implications for the national vaccination program.

PARVOVIRUS B19 INFECTION PRESENTING AS ACUTE HEPATITIS AND TRANSIENT ANEMIA IN A PREVIOUSLY HEALTHY CHILD.

Acute hepatitis has been noted as a manifestation of parvovirus B19 infection in children and adults, although its pathogenesis remains unclear. In this report, we present a case of an 11-year old Thai boy with parvovirus B19-associated acute hepatitis who presented with jaundice, hepatomegaly and transient aplastic crisis. Our report underscores the need to consider parvovirus B19 as the causative pathogen in patients with increased liver enzymes, jaundice and anemia.

Human norovirus genogroup II recombinants in Thailand, 2009-2014.

Norovirus (NoV) is a major cause of nonbacterial acute gastroenteritis worldwide. New strains emerge partly due to viral recombination. In Thailand, there is a lack of data on NoV recombinants among clinical isolates. We screened stool samples from pediatric diarrheal patients for norovirus by RT-PCR and found GII.4 to be the most prevalent genotype. Phylogenetic and SimPlot analyses detected seven intra-genogroup recombinant strains: three GII.21/GII.3, two GII.12/GII.3, and two GII.12/GII.1 recombinants. Maximum chi-square analysis indicated that all had similar breakpoints near the ORF1/ORF2 junction (p < 0.001), either slightly upstream within the C-terminus of RdRp or downstream within the N-terminal domain of VP1.