Peripheral blood mononuclear cells of breast cancer patients can be reprogrammed to enhance anti-HER-2/neu reactivity and overcome myeloid-derived suppressor cells [poster abstract]

Barriers limiting the efficacy of adoptive cellular therapy (ACT) for breast cancer patients include immune suppression mediated by myeloid-derived suppressor cells (MDSC) and a low frequency of tumor-reactive memory T cells (Tm). Recently, we developed an ex vivo protocol to reprogram tumor-reactive murine splenocytes; these cells were found to be resistant to MDSC suppression and protected FVBN202 mice from tumor challenge. Here, we evaluated the clinical applicability of reprogramming tumor-sensitized PBMCs isolated from patients with early stage breast cancer by treatment with bryostatin 1 and ionomycin (B/I) combined with IL-2, IL-7 and IL-15. Our data demonstrate that reprogrammed cells are enriched with Tm cells (n=5; p=0.006), as well as activated CD56+(n=6; p=0.003) and CD161+ (n=4; p=0.02) NKT cells, and demonstrate expansion in total cell numbers (n=16; p=0.003) compared to baseline cells. Reprogrammed PBMCs displayed enhanced HER-2/neu-specific IFN-γ producing immune responses (n=6; p=0.04); non-reprogrammed control PBMC IFN-γ production was not significant (n=6; p=0.4). Furthermore, high-throughput sequencing analysis of the T cell receptor (TcR) Vβ in one patient demonstrated clonal expansion of specific TcR VJ recombination events resulting from cellular reprogramming, suggestive of an enriched frequency of specific tumor antigen-primed T cell clones. Interestingly, reprogrammed T cells were resistant to autologous CD33+ CD11b+ HLA-DRlo/- MDSCs, as determined by further enhanced HER-2/neu-specific IFN-γ secretion in the presence of MDSCs (n=6; p=0.03). Activated CD161+ NKT cells comprising 3% or greater of total reprogrammed cells rendered T cells resistant to MDSCs (n=3; p=0.02). Upregulation of NKG2D expression on CD161+(n=5; p=0.0006) and CD56+ (n=5; p=0.04) NKT cells resulted from cellular reprogramming. Therefore, NKG2D signaling was blocked using anti-NKG2D blocking antibody in our co-culture system, resulting in the abrogation of resistance to MDSCs as determined by blunted IFN-γ secretion (n=3; p=0.04). Finally, the phenotype of MDSCs after co-culture with reprogrammed PBMC was examined; we observed downregulation of CD11b expression (n=3; p=0.02) concomitant with HLA-DR upregulation on MDSCs (n=3; p=0.001); suggestive of induced maturation of MDSCs into Dendritic Cells (DC). The results of our study offer the following strategies to improve ACT of breast cancer: i) inclusion of activated NKT cells in ACT to overcome MDSC suppression by inducing MDSC maturation into DCs, and ii) PBMC reprogramming to enrich the frequency of tumor-reactive Tm cells

CD44 Expression in Oro-Pharyngeal Carcinoma Tissues and Cell Lines

Expression of CD44, a transmembrane hyaluronan-binding glycoprotein, is variably considered to have prognostic significance for different cancers, including oral squamous cell carcinoma. Although unclear at present, tissue-specific expression of particular isoforms of CD44 might underlie the different outcomes in currently available studies. We mined public transcriptomics databases for gene expression data on CD44, and analyzed normal, immortalized and tumour-derived human cell lines for splice variants of CD44 at both the transcript and protein levels. Bioinformatics readouts, from a total of more than 15,000 analyses, implied an increased CD44 expression in head and neck cancer, including increased expression levels relative to many normal and tumor tissue types. Also, meta-analysis of over 260 cell lines and over 4,000 tissue specimens of diverse origins indicated lower CD44 expression levels in cell lines compared to tissue. With minor exceptions, reverse transcribed polymerase chain reaction identified expression of the four main isoforms of CD44 in normal oral keratinocytes, transformed lines termed DT and HaCaT, and a series of paired primary and metastasis-derived cell lines from oral or pharyngeal carcinomas termed HN4/HN12, HN22/HN8 and HN30/HN31. Immunocytochemistry, Western blotting and flow cytometric assessments all confirmed the isoform expression pattern at the protein level. Overall, bioinformatic processing of large numbers of global gene expression analyses demonstrated elevated CD44 expression in head and neck cancer relative to other cancer types, and that the application of standard cell culture protocols might decrease CD44 expression. Additionally, the results show that the many variant CD44 exons are not fundamentally deregulated in a diverse range of cultured normal and transformed keratinocyte lines

Competitive Tendering In The Netherlands: Central Planning Or Functional Specifications?

Institute of Transport and Logistics Studies. Faculty of Economics and Business. The University of Sydne