Blockade of PD-1/PD-L1 promotes adoptive T-Cell immunotherapy in a tolerogenic environment

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The coinhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PDL1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcmlike cells. © 2015 Blake et al.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Steady-state dendritic cells expressing cognate antigen terminate memory CD8+ T-cell responses

Antigen stimulation of naive T cells in conjunction with strong costimulatory signals elicits the generation of effector and memory populations. Such terminal differentiation transforms naive T cells capable of differentiating along several terminal pathways in response to pertinent environmental cues into cells that have lost developmental plasticity and exhibit heightened responsiveness. Because these cells exhibit little or no need for the strong costimulatory signals required for full activation of naive T cells, it is generally considered memory and effector T cells are released from the capacity to be inactivated. Here, we show that steadystate dendritic cells constitutively presenting an endogenously expressed antigen inactivate fully differentiated memory and effector CD8+ T cells in vivo through deletion and inactivation. These findings indicate that fully differentiated effector and memory T cells exhibit a previously unappreciated level of plasticity and provide insight into how memory and effector T-cell populations may be regulated. © 2008 by The American Society of Hematology.

Chemotherapy pretreatment sensitizes solid tumor-derived cell lines to Vα24+ NKT cell-mediated cytotoxicity

There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human Vα24 +/Vβ11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination

Multipotent neural stem cells (NSCs) provide a model to investigate neurogenesis and develop mechanisms of cell transplantation. In order to define improved markers of stemness and lineage specificity, we examined self-renewal and multi-lineage markers during long-term expansion and under neuronal and astrocyte differentiating conditions in human ESC-derived NSCs (hNSC H9 cells). In addition, with proteoglycans ubiquitous to the neural niche, we also examined heparan sulfate proteoglycans (HSPGs) and their regulatory enzymes. Our results demonstrate that hNSC H9 cells maintain self-renewal and multipotent capacity during extended culture and express HS biosynthesis enzymes and several HSPG core protein syndecans (SDCs) and glypicans (GPCs) at a high level. In addition, hNSC H9 cells exhibit high neuronal and a restricted glial differentiative potential with lineage differentiation significantly increasing expression of many HS biosynthesis enzymes. Furthermore, neuronal differentiation of the cells upregulated SDC4, GPC1, GPC2, GPC3 and GPC6 expression with increased GPC4 expression observed under astrocyte culture conditions. Finally, downregulation of selected HSPG core proteins altered hNSC H9 cell lineage potential. These findings demonstrate an involvement for HSPGs in mediating hNSC maintenance and lineage commitment and their potential use as novel markers of hNSC and neural cell lineage specification.

Associations of serum perfluoroalkyl acid levels with T-helper cell-specific cytokines in children: By gender and asthma status

Perfluoroalkyl acids (PFAAs) are a group of common chemicals that ubiquitously exist in wildlife and humans. Experimental data suggest that they may alter T-lymphocyte functioning in situ by preferentially enhancing the development of T-helper 2 (TH2)- and inhibiting TH1-lymphocyte development and might increase allergic inflammation, but few human studies have been conducted. To evaluate the association between serum PFAAs concentrations and T-lymphocyte-related immunological markers of asthma in children, and further to assess whether gender modified this association, 231 asthmatic children and 225 non-asthmatic control children from Northern Taiwan were recruited into the Genetic and Biomarker study for Childhood Asthma. Serum concentrations of ten PFAAs and levels of TH1 [interferon (IFN)-γ, interleukin (IL)-2] and TH2 (IL-4 and IL-5) cytokines were measured. The results showed that asthmatics had significantly higher serum PFAAs concentrations compared with the healthy controls. When stratified by gender, a greater number of significant associations between PFAAs and asthma outcomeswere found in males than in females. Among males, adjusted odds ratios for asthma among those with the highest versus lowest quartile of PFAAs exposure ranged from 2.59 (95% CI: 1.14, 5.87) for the perfluorobutanesulfonate (PFBS) to 4.38 (95% CI: 2.02, 9.50) for perfluorooctanesulfonate (PFOS); and serum PFAAs were associated positively with TH2 cytokines and inversely with TH1 cytokines among male asthmatics. Among females, no significant associations between PFAAs and TH2 cytokines could be detected. In conclusion, increased serum PFAAs levels may promote TH cell dysregulation and alter the availability of key TH1 and TH2 cytokines, ultimately contributing to the development of asthma that may differentially impact males to a greater degree than females. These results have potential relevance in asthma prevention.

Ratios of T-cell immune-effectors with tumour associated macrophages and PD-1/PD-L1 axis immune-checkpoint molecules, as prognosticators in diffuse large B cell lymphoma: A population-based study

Background Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured despite initial R-CHOP. Although the prognostic importance of the tumour microenvironment (TME) is established, the optimal strategy to quantify it is unknown. Methods The relationship between immune-effector and inhibitory (checkpoint) genes was assessed by NanoString™ in 252 paraffin-embedded DLBCL tissues. A model to quantify net anti-tumoural immunity as an outcome predictor was tested in 158 R-CHOP treated patients, and validated in tissue/blood from two independent R-CHOP treated cohorts of 233 and 140 patients respectively. Findings T and NK-cell immune-effector molecule expression correlated with tumour associated macrophage and PD-1/PD-L1 axis markers consistent with malignant B-cells triggering a dynamic checkpoint response to adapt to and evade immune-surveillance. A tree-based survival model was performed to test if immune-effector to checkpoint ratios were prognostic. The CD4*CD8:(CD163/CD68)*PD-L1 ratio was better able to stratify overall survival than any single or combination of immune markers, distinguishing groups with disparate 4-year survivals (92% versus 47%). The immune ratio was independent of and added to the revised international prognostic index (R-IPI) and cell-of-origin (COO). Tissue findings were validated in 233 DLBCL R-CHOP treated patients. Furthermore, within the blood of 140 R-CHOP treated patients immune-effector:checkpoint ratios were associated with differential interim-PET/CT+ve/-ve expression.

Cancer stem cells in oesophageal squamous cell carcinoma: Identification, prognostic and treatment perspectives

Cancer stem cells (CSCs) are a vital subpopulation of cells to target for the treatment of cancers. In oesophageal squamous cell carcinoma (ESCC), there are several markers such as CD44, ALDH, Pygo2, MAML1, Twist1, Musashi1, Side population (SP), CD271 and CD90 that have been proposed to identify the cancer stem cells in individual cancer masses. It has also been demonstrated that stem cell markers like ALDH1, HIWI, Oct3/4, ABCG2, SOX2, SALL4, BMI-1, NANOG, CD133 and podoplanin are associated with patient's prognosis, pathological stages, cancer recurrence and therapy resistance. Finding new cancer stem cell targets or designing drugs to manipulate the known molecular targets in CSCs could be useful for improvements in clinical outcomes of the disease. To conclude, data suggest that CSCs in oesophageal squamous cell carcinoma are related to resistance to therapy and poor prognosis of patients with ESCC. Therefore, innovative insights into CSC biology and CSC-targeted therapies will help to achieve more effective management of patients with oesophageal squamous cell carcinoma.

Electrochemical properties and intermediate-temperature fuel cell performance of dense yttrium-doped barium zirconate with calcium addition

Although BaZr 0.8Y 0.2O 3-δ(BZY) possesses large bulk proton conductivity and excellent chemical stability, its poor sinterability and grain boundaries block proton conduction. In this work, the effect of Ca as a co-dopant and as a sintering aid (as CaO), on the sinterability, proton conductivity, and fuel cell performance of BZY was investigated. The addition of 4 mol% CaO significantly improved the BZY sinterability: BZY pellets with densities of 92.7% and 97.5% with respect to the theoretical density were obtained after sintering at 1500°C and 1600°C, respectively. The improved BZY sinterability by CaO addition resulted also in a large proton conductivity; at 600°C, the total conductivity of BZY-CaO was 2.14 × 10 -3 S/cm, in wet Ar. Anode-supported fuel cells with 25 μm-thick BZY-CaO electrolyte membranes were fabricated by a dual-layer co-firing technique. The peak power density of the fuel cell with a BZY-Ni/BZY-4CaO/BZY-LSCF (La 0.6Sr 0.4Fe 0.8Co 0.2O 3-δ) configuration was 141 mW/cm 2 at 700°C, several times larger than the reported values of BZY electrolyte membrane fuel cells sintered with the addition of CuO or ZnO, demonstrating promising features for practical fuel cell applications.

Lowering grain boundary resistance of BaZr0.8Y0.2O3−δ with LiNO3 sintering-aid improves proton conductivity for fuel cell operation

A novel sintering additive based on LiNO3 was used to overcome the drawbacks of poor sinterability and low grain boundary conductivity in BaZr0.8Y0.2O3-δ (BZY20) protonic conductors. The Li-additive totally evaporated during the sintering process at 1600°C for 6 h, which led to highly dense BZY20 pellets (96.5% of the theoretical value). The proton conductivity values of BZY20 with Li sintering-aid were significantly larger than the values reported for BZY sintered with other metal oxides, due to the fast proton transport in the "clean" grain boundaries and grain interior. The total conductivity of BZY20-Li in wet Ar was 4.45 × 10-3 S cm-1 at 600°C. Based on the improved sinterability, anode-supported fuel cells with 25 μm-thick BZY20-Li electrolyte membranes were fabricated by a co-firing technique. The peak power density obtained at 700°C for a BZY-Ni/BZY20-Li/La0.6Sr0.4Co0.2Fe 0.8O3-δ (LSCF)-BZY cell was 53 mW cm-2, which is significantly larger than the values reported for fuel cells using electrolytes made of BZY sintered with the addition of ZnO and CuO, confirming the advantage of using Li as a sintering aid.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

Promotion of a cancer-like phenotype, through chronic exposure to inflammatory cytokines and hypoxia in a bronchial epithelial cell line model

Globally, lung cancer accounts for approximately 20% of all cancer related deaths. Five-year survival is poor and rates have remained unchanged for the past four decades. There is an urgent need to identify markers of lung carcinogenesis and new targets for therapy. Given the recent successes of immune modulators in cancer therapy and the improved understanding of immune evasion by tumours, we sought to determine the carcinogenic impact of chronic TNF-α and IL-1β exposure in a normal bronchial epithelial cell line model. Following three months of culture in a chronic inflammatory environment under conditions of normoxia and hypoxia (0.5% oxygen), normal cells developed a number of key genotypic and phenotypic alterations. Important cellular features such as the proliferative, adhesive and invasive capacity of the normal cells were significantly amplified. In addition, gene expression profiles were altered in pathways associated with apoptosis, angiogenesis and invasion. The data generated in this study provides support that TNF-α, IL-1β and hypoxia promotes a neoplastic phenotype in normal bronchial epithelial cells. In turn these mediators may be of benefit for biomarker and/or immune-therapy target studies. This project provides an important inflammatory in vitro model for further immuno-oncology studies in the lung cancer setting.

Characterisation of novel primary tumour derived epithelial prostate cancer cell lines

Current translational and basic prostate cancer research is limited by the number of cell lines that truly reflect the spectrum of disease progression, with most commonly used cell lines being derived from metastatic lesions. There are essentially no prostate cancer cell lines derived from primary tumours or localised disease in wide use.