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BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). β-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAFV600E-driven tumors have been speculated to rely on Wnt/β-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of β-catenin in BrafV600E-driven thyroid cancer in a transgenic mouse model. In BrafV600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of β-catenin was observed in thyroid tumors. In BrafV600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFβ pathways and loss of epithelial–mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual β-catenin/KDM4A inhibitor PKF118–310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo. These findings indicate that β-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/β-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Background Monocytes play an important role in immune and inflammatory diseases and monocyte subsets are predictors of disease in certain conditions. Expression of the chemokine receptors, CCR2 and CX3CR1 on monocyte subsets relates to their function and can be used in their characterization. Our objective was to determine whether CD14, CD16, CCR2 and CX3CR1 on monocyte subsets are potential indicators of asthma severity. Methods Blood samples were collected from Saudi Arabian patients with asthma and normal healthy individuals. Six-color flow-cytometry phenotypic analysis was used to identify human blood monocyte subsets, based on their expression of CD14 and CD16 following CD45 gating. Expression of CCR2 and CX3CR1 was analysed on classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) subsets and correlated with disease severity. Results We demonstrated a significant increase in percentage of total CD45-positive monocytes in the blood of patients with severe asthma, but the proportion of the individual monocyte subsets was not significantly changed when patients with mild, moderate and severe asthma were compared with healthy individuals. CD16 expression (mean fluorescence intensity, MFI) was decreased on intermediate and non-classical subsets in patients with severe asthma compared to healthy controls. CX3CR1 expression was also lower, with a lower percentage of cells expressing CX3CR1 in the non-classical CD14+CD16++ subset in all patients with asthma and this was inversely related to the percentage of cells expressing CCR2. Conclusions CCR2 expression on monocytes indicated a tendency toward more phagocytic monocytes in patients with asthma. The differential expression of CD16, CX3CR1 and CCR2 on monocyte subsets in peripheral blood indicates modulation of the inflammatory response and suggests a role for monocytes in asthma pathogenesis.

Recent years have witnessed major progress in development of novel therapeutic agents such as chemotherapy, targeted therapy and immune checkpoint inhibitors for breast cancer. However, cancer-related death remains high especially in triple-negative breast cancer (TNBC) due limited therapeutic options. Development of targeted therapies for TNBC requires better understanding of biology and signaling networks that promote disease progression. Fascin, an actin bundling protein, was identified as a key regulator of many signaling pathways that contribute to breast cancer progression. Herein, fascin ShRNA was used to generate stable fascin knockdown (FSCN1KD) in the MDA-MB-231 TNBC cell line and then were subjected to comprehensive mRNA and miRNA transcriptome analysis. We identified 129 upregulated and 114 downregulated mRNA transcripts, while 14 miRNAs were differentially expressed in FSCN1KD. Ingenuity pathway analysis (IPA) was used to predict the impact of differentially expressed transcripts on signaling pathways and functional categories and to construct miRNA-mRNA regulatory networks in the context of FSCN1 knockdown. Compared to FSCN1KD, fascin-positive (FSCN1CON) breast cancer cells showed enrichment in genes promoting cellular proliferation, migration, survival, DNA replication and repair. Expression of FSCN1high (identified in BRCA dataset from TCGA) in conjunction with elevated expression of the top 10 upregulated or decreased expression of the top 10 downregulated genes (identified in our FSCN1CON vs. FSCN1KD) correlates with worst survival outcome. Taken together, these data confirmed fascin’s role in promoting TNBC progression, and identified a novel opportunity for therapeutic interventions via targeting those FSCN1-related transcripts.

Breast cancer remains the second cause of tumor‐related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem‐like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin‐bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, β1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin‐mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self‐renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co‐expression and short disease‐free as well as overall survival in chemo‐treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self‐renewability and chemoresistance strongly encourages for dual targeting of fascin‐ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root., What's new? Residual cancer stem cells (CSCs) have the ability to regrow tumors and to metastasize to distant organs, resulting in disease relapse and increased cancer mortality. In breast cancer, CSC populations are enriched by aberrant expression of the actin‐bundling protein fascin, induction of which is also associated with chemoresistance and metastasis. In this study, fascin was found to upregulate β1 integrin (ITGB1) expression, an effect that proved critical to breast cancer cell adhesion and self‐renewal. Coexpression of fascin and ITGB1 was associated with decreased survival in chemotherapy‐treated breast cancer patients. The findings identify the fascin‐ITGB1 axis as a potential therapeutic target.

Cancer stem cells (CSCs), a rare population of tumor cells with high self-renewability potential, have gained increasing attention due to their contribution to chemoresistance and metastasis. We have previously demonstrated a critical role for the actin-bundling protein (fascin) in mediating breast cancer chemoresistance through activation of Focal Adhesion Kinase (FAK). The latter is known to trigger the β-catenin signaling pathway. Whether fascin activation of FAK would ultimately triggers β-catenin signaling pathway has not been elucidated. Here, we assessed the effect of fascin manipulation in breast cancer cells on triggering β-catenin downstream targets and its dependence on FAK. Gain and loss of fascin expression showed its direct effect on the constitutive expression of β-catenin downstream targets and enhancement of self-renewability. In addition, fascin was essential for Glycogen Synthase Kinase 3 beta (GSK-3β) inhibitor-mediated inducible expression and function of the β-catenin downstream targets. Importantly, fascin-mediated constitutive and inducible expression of β-catenin downstream targets as well as its subsequent effect on CSC function was at least partially FAK dependent. To assess the clinical relevance of the in vitro findings we evaluated the consequence of fascin, FAK and β-catenin downstream target co-expression on the outcome of breast cancer patient survival. Patients with co-expression of fascinhigh and FAKhigh or high β-catenin downstream targets showed the worst survival outcome, while in fascinlow patients co-expression of FAKhigh or high β-catenin targets had less significant effect on the survival. Altogether, our data demonstrated the critical role of fascin-mediated β-catenin activation and its dependence on intact FAK signaling to promote breast CSC function. These findings suggest that targeting of fascin-FAK-β-catenin axis may provide a novel therapeutic approach for eradication of breast cancer from the root.

The T-cell inhibitory molecule PD-L1 is expressed on a fraction of breast cancer cells. The distribution of PD-L1 on the different subpopulations of breast cancer cells is not well-defined. Our aim was to study the expression level of PD-L1 on breast cancer stem-like (CSC-like) cells and their differentiated-like counterparts. We used multi-parametric flow cytometry to measure PD-L1 expression in different subpopulations of breast cancer cells. Pathway inhibitors, quantitative immunofluorescence, cell sorting, and western blot were used to investigate the underlying mechanism of PD-L1 upregulation in CSC-like cells. Specifically, PD-L1 was overexpressed up to three folds on breast CSC-like cells compared with more differentiated-like cancer cells. Functional in vitro and in vivo assays show higher stemness of PD-L1hi as compared with PD-L1lo cells. Among different pathways examined, PD-L1 expression on CSCs was partly dependant on Notch, and/or PI3K/AKT pathway activation. The effect of Notch inhibitors on PD-L1 overexpression in CSCs was completely abrogated upon mTOR knockdown. Specific knockdown of different Notch receptors shows Notch3 as a mediator for PD-L1 overexpression on CSCs and important for maintaining their stemness. Indeed, Notch3 was found to be overexpressed on PD-L1hi cells and specific knockdown of Notch3 abolished the effect of notch inhibitors and ligands on PD-L1 expression as well as mTOR activation. Our data demonstrated that overexpression of PD-L1 on CSCs is partly mediated by the notch pathway through Notch3/mTOR axis. We propose that these findings will help in a better design of anti-PD-L1 combination therapies to treat breast cancer effectively.

The expression of PD‐L1 in breast cancer is associated with estrogen receptor negativity, chemoresistance and epithelial‐to‐mesenchymal transition (EMT), all of which are common features of a highly tumorigenic subpopulation of cancer cells termed cancer stem cells (CSCs). Hitherto, the expression and intrinsic role of PD‐L1 in the dynamics of breast CSCs has not been investigated. To address this issue, we used transcriptomic datasets, proteomics and several in vitro and in vivo assays. Expression profiling of a large breast cancer dataset (530 patients) showed statistically significant correlation (p, What's new? Cancer cells that express the T‐cell inhibitory molecule programmed death‐ligand 1 (PD‐L1) readily escape immune attack. In addition, PD‐L1 expression contributes to chemoresistance and is associated with epithelial‐to‐mesenchymal transition, a process that generates cancer stem cells (CSCs). This study shows that in breast cancer, PD‐L1 expression further plays a direct part in maintaining CSC stemness. In breast cancer cells, PD‐L1 expression sustained stemness factors OCT‐4A and Nanog, via a PI3K/AKT‐dependent pathway, and promoted expression of the stemness controlling factor BMI1, independent of PI3K/AKT. Targeting PD‐L1 could help advance breast cancer therapy, owing to impacts on the pool of breast CSCs.

Asthma is a chronic inflammatory disorder associated with airway hyper-responsiveness. Although a number of studies have investigated asthma at the molecular level, the molecular immune signatures associated with asthma severity or with the response to corticosteroids are still being unraveled. The present study integrated four asthma-related gene expression datasets from the Gene Expression Omnibus and identified immune-gene signatures associated with asthma development, severity, or response to treatment. Normal and mild asthmatic patients clustered separately from the severe asthma group, suggesting substantial progression-related changes in gene expression. Pathway analysis of up-regulated severe asthma-related genes identified multiple cellular processes, such as polymorphism, T-cell development, and transforming growth factor-β signaling. Comparing gene expression profiles of bronchoalveolar lavage cells in response to corticosteroid treatment, showed substantial reductions in genes related to the inflammatory response, including tumor necrosis factor signaling in the corticosteroid sensitive versus resistant patients, suggesting a defective immune response to corticosteroids. The data highlight the multifactorial nature of asthma, but revealed no significant overlap with the gene expression profiles from different datasets interrogated in current studies. The presented profile suggests that genes involved in asthma progression are different from those involved in the response to corticosteroids and this could affect the clinical management of different groups of patients with asthma.

The Wilms tumor antigen 1 (WT1) antigen is over-expressed in human leukemias, making it an attractive target for immunotherapy. Most WT1-specific Cytotoxic T Lymphocytes (CTLs) described so far displayed low avidity, limiting its function. To improve the immunogenicity of CTL epitopes, we replaced the first-amino-acid of two known immunogenic WT1-peptides (126 and 187) with a tyrosine. This modification enhances 126Y analogue-binding ability, triggers significant number of IFN-gamma-producing T cells (P = 0.0003), induces CTL that cross-react with the wild-type peptide, exerts a significant lytic activity against peptide-loaded-targets (P = 0.0006) and HLA-A0201-matched-leukemic cells (P = 0.0014). These data support peptide modification as a feasible approach for the development of a leukemia-vaccine.

We previously identified follicular dendritic cell secreted protein (FDC-SP), a small secreted protein of unknown function expressed in human tonsillar germinal centers (GC). To assess potential in vivo activities of FDC-SP, transgenic mice were generated to constitutively express FDC-SP in lymphoid tissues. FDC-SP transgenic mice show relatively normal development of immune cell populations, with the exception of a small increase in mature follicular B cells, and normal lymphoid tissue architecture. Upon immunization with a T-dependent Ag, FDC-SP transgenic mice were capable of producing an Ag-specific Ab; however, the titers of Ag-specific IgG2a and IgE were significantly reduced. GC responses after immunization were markedly diminished, with transgenic mice showing decreased numbers and sizes of GCs but normal development of follicular dendritic cell networks and normal positioning of GCs. FDC-SP transgenic mice also showed reduced production of Ag-specific IgG3 Ab after immunization with a type II T-independent Ag, suggesting that the FDC-SP can also regulate the induction of B cell responses outside the GC. Purified FDC-SP transgenic B cells function normally in vitro, with the exception of blunted chemotaxis responses to CXCL12 and CXCL13. FDC-SP can induce the chemotaxis of CD40-stimulated nontransgenic B cells and can significantly enhance B cell migration in combination with chemokines, indicating that FDC-SP may function in part by regulating B cell chemotaxis. These results provide the first evidence for immunomodulatory activities of FDC-SP and implicate this molecule as a regulator of B cell responses.

The BCR serves to both signal cellular activation and enhance uptake and presentation of Ags by B cells; however, the intracellular signaling mechanisms linking the BCR to Ag presentation functions have been controversial. PI3Ks are critical signaling enzymes controlling many cellular processes, with the p110δ isoform playing a critical role in BCR signaling. In this study, we used pharmacological and genetic approaches to evaluate the role of p110δ signaling in Ag presentation by primary B lymphocytes. It was found that activation of allogeneic T cells is significantly reduced when B cells are pretreated with global PI3K inhibitors, but was intact when p110δ signaling was specifically inactivated. In contrast, inactivation of p110δ significantly impaired the ability of B cells to activate T cells in a BCR-mediated Ag uptake and presentation model. Prestimulation of p110δ-inactivated B cells with anti-CD40 or LPS could not rescue their BCR-mediated Ag presentation ability to normal levels. p110δ signaling was required for efficient presentation of either anti-Ig or protein Ag via a lysozyme-specific BCR. p110δ-inactivated B cells were able to internalize Ag normally, and no defects in association of Ag with lysosome-associated membrane protein 1+ late endosomes were observed; however, these cells were less effective in forming polarized conjugates with Ag-specific T cells. Our data demonstrate a role for p110δ signaling in B cell Ag presentation function, implicating 3-phosphoinositides and their targets in the latter stages of this process.

Phosphoinositide 3-kinases (PI3K) regulate immune activation via their roles in signal transduction of multiple classes of receptors. Here, we examined the effect of genetic inactivation of the hemopoietic cell-restricted PI3K isoform p110delta on systemic cytokine and chemokine responses and allergic airway inflammation. We found that type 2 cytokine responses (IL-4, IL-5 and IL-13) are significantly decreased in p110delta mutants, whereas type 1 cytokine responses (IFN-gamma and CXCL10) were robust. Elevated IFN-gamma production during the primary response to ovalbumin (OVA) was associated with reduced production of the regulatory cytokine IL-10. IFN-gamma and IL-10 production normalized after secondary OVA immunization; however, type 2 cytokine production was persistently reduced. Type 2 cytokine-dependent airway inflammation elicited by intranasal challenge with OVA was dramatically reduced, with reduced levels of eosinophil recruitment and mucus production observed in the lungs. Induction of respiratory hyper-responsiveness to inhaled methacholine, a hallmark of asthma, was markedly attenuated in p110delta-inactivated mice. Adoptive transfer of OVA-primed splenocytes from normal but not p110delta-inactivated mice could induce airway eosinophilia in naive, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease.

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4+ T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4+ T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.

Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcγ receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

Background: A major therapeutic challenge for breast cancer is the ability of cancer cells to evade killing of conventional chemotherapeutic agents. We have recently reported the actin-bundling protein (fascin) as a major regulator of breast cancer metastasis and survival. Methods: Survival of breast cancer patients that received chemotherapy and xenograft tumour model was used to assess the effect of chemotherapy on fascin-positive and -negative breast cancer cells. Molecular and cellular assays were used to gain in-depth understanding of the relationship between fascin and chemoresistance. Results: We showed a significant correlation between fascin expression and shorter survival in breast cancer patients who received chemotherapy. In xenograft experiments, fascin-positive cancer cells displayed significantly more resistance to chemotherapy-mediated apoptotic cell death than fascin-negative counterparts. This increased chemoresistance was at least partially mediated through PI3K/Akt signalling, and was paralleled by increased FAK phosphorylation, enhanced expression of the inhibitor of apoptosis proteins (XIAP and Livin) and suppression of the proapoptotic markers (caspase 9, caspase 3 and PARP). Conclusions: This is the first report to demonstrate fascin involvement in breast cancer chemotherapeutic resistance, supporting the development of fascin-targeting drugs for better treatment of chemoresistance breast cancer.

The binding of a T cell to an APC results in T cell actin cytoskeletal rearrangement leading to the formation of an immunological synapse. The APC cytoskeleton has been thought to play a passive role in this process. In this study, we demonstrate that dendritic cells (DC), unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells. DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting T cells. This study provides compelling evidence that the APC cytoskeleton plays an active role in the immunological synapse and may explain the unique ability of DC to activate resting T cells.

B cell Ag receptors function in both signaling activation of Ag-specific cells and in collecting specific Ag for presentation to T lymphocytes. Signaling via PI3K is required for BCR-mediated activation and Ag presentation functions; however, the relevant downstream targets of PI3K in B cells are incompletely defined. In this study, we have investigated the roles of the PI3K effector molecule Bam32/DAPP1 in BCR signaling and BCR-mediated Ag presentation functions. In mouse primary B cells, Bam32 was required for efficient activation of the GTPase Rac1 and downstream signaling to JNK, but not activation of BLNK, phospholipase C γ2, or calcium responses. Consistent with a role of this adaptor in Rac-mediated cytoskeletal rearrangement, Bam32 was required for BCR-induced cell adhesion and spreading responses on ICAM-1 or fibronectin-coated surfaces. The function of Bam32 in promoting Rac activation and adhesion required tyrosine 139, a known site of phosphorylation by Lyn kinase. After BCR crosslinking by Ag, Bam32-deficient B cells are able to carry out the initial steps of Ag endocytosis and processing, but show diminished ability to form Ag-specific conjugates with T cells and polarize F-actin at the B-T interface. As a result, Bam32-deficient B cells were unable to efficiently activate Ag-specific T cells. Together, these results indicate that Bam32 serves to integrate PI3K and Src kinase signaling to promote Rac-dependent B cell adhesive interactions important for Ag presentation function.

Ab affinity maturation within germinal centers (GCs) requires weeks to complete. Several signaling pathways in B cells have been shown to be required for initiation of the GC response; however, the signaling checkpoints controlling progression and eventual dissolution of the GC reaction are poorly understood. The adaptor protein Bam32/DAPP1 was originally isolated from human GCs and functions downstream of phosphoinositide 3-kinase enzymes, which are known to have critical roles in B cell activation and GC responses. In this study we identify a unique role of Bam32/DAPP1 in promoting GC progression. Bam32-deficient mice show normal GC initiation, but premature GC dissolution after immunization with protein Ag in alum or low doses of sheep red blood cells. Adoptive transfer studies confirmed that Bam32-deficient B cells have an intrinsic impairment in the ability to mount sustained GC responses. Bam32 deficiency was also associated with impaired Ab affinity maturation. Proliferation of Bam32-deficient GC B cells was not compromised; however, these cells show impaired switch to IgG1 and increased apoptosis in situ. GCs formed by Bam32-deficient B cells contain fewer T cells, indicating that Bam32 is required for B cell–dependent T cell accumulation within established GCs. Exogenous CD40 ligand restored GC B cell numbers and switch to IgG1, indicating that Bam32-deficient B cells are competent to respond to CD40 stimulation when ligand is available. These data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival.

Maturation of dendritic cells (DC) is critical to their development into potent APCs. Upon maturation, DC up-regulate the expression of MHC class II as well as costimulatory and adhesion molecules, all of which are important in Ag presentation. In addition, they undergo structural changes characterized by the expression of numerous long dendrites. Fascin is an actin-bundling protein that has been reported to be important for the development of dendrites. In this study, we evaluated fascin expression and function during DC maturation into potent APC. In vitro, treatment of bone marrow-derived DC (BM-DC) with GM-CSF resulted in increased levels of fascin expression. This increase correlated directly with an increase in MHC class II and B7-2 expression. Fascin expression was decreased by the addition of TGF-β and increased by the addition TNF-α to the culture. These cytokines suppress or enhance DC maturation, respectively. Increased levels of fascin expression were found to correlate with increased APC activity in a one-way MLR. Specific inhibition of fascin expression, using antisense oligonucleotides, markedly reduced this APC allostimulatory activity. These data demonstrate that fascin expression correlates with DC maturation into APC, and it plays a significant role in the ability of DC to function as APC. This observation is the first evidence linking fascin-mediated dendrite formation with the APC activity of DC.

Introduction B7-H1 (PD-L1, CD274) is a T cell inhibitory molecule expressed in many types of cancer, leading to immune escape of tumor cells. Indeed, in previous reports we have shown an association of B7-H1 expression with high-risk breast cancer patients. Methods In the current study, we used immunohistochemistry, immunofluorescence and Western blot techniques to investigate the effect of neoadjuvant chemotherapy on the expression of B7-H1 in breast cancer cells. Results Among tested chemotherapeutic agents, doxorubicin was the most effective in downregulating cell surface expression of B7-H1 in vitro. These results were validated in vivo in a xenograft mouse model, as well as in murine heart tissue known to constitutively express B7-H1. The doxorubicin-dependent cell surface downregulation of B7-H1 was accompanied by an upregulation of B7-H1 in the nucleus. This re-distribution of B7-H1 was concurrent with a similar translocation of phosphorylated AKT to the nucleus. Inhibition of the PI3K/AKT pathway abrogated the doxorubicin-mediated nuclear up-regulation of B7-H1, suggesting an involvement of PI3K/AKT pathway in the nuclear up-regulation of B7-H1. Interestingly, siRNA knock down of B7-H1 lead to an increase in spontaneous apoptosis, as well as doxorubicin-induced apoptosis, which indicates an anti-apoptotic role for B7-H1 in breast cancer cells. The novel discovery of B7-H1 expression in the nuclei of breast cancer cells suggests that B7-H1 has functions other than inhibition of T cells. Conclusions Our findings explain the previously reported immunomodulatory effect of anthracyclines on cancer cells, and provide a link between immunoresistance and chemoresistance. Finally these results suggest the use of dual combinatorial agents to inhibit B7-H1 beside chemotherapy, in breast cancer patients.

Background The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. Methods We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. Results Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p

Meeting abstracts The Wilms Tumour Antigen 1 (WT1) has been shown to be expressed at low levels in some normal cells. Therefore, many of the potential CTL epitopes against this antigen may be absent or suboptimal. To this end, different groups introduced modifications in the sequence of the anchor