Cell type-specific transcriptome analysis reveals a major role for Zeb1 and miR-200b in mouse inner ear morphogenesis.

Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type-specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants.

FcgammaRIIIa polymorphisms and cetuximab induced cytotoxicity in squamous cell carcinoma of the head and neck.

PURPOSE: The interaction of Fc fragments of antibodies with the Fcgamma receptors is an essential checkpoint in antibody-dependent cellular cytotoxicity (ADCC). Specific polymorphisms at position 158 enhance FcgammaRIIIa affinity for IgG1 and are associated with improved clinical outcome in lymphoma patients treated with IgG1 anti-CD20 antibody. The role of ADCC in the therapeutic effects of the alpha-epidermal growth factor receptor (EGFR) mAb, cetuximab, in patients with squamous cell carcinoma of the head and neck (SCCHN) is poorly defined. We employed three SCCHN cell lines to test two hypotheses: (1) SCCHN is susceptible to cetuximab-mediated ADCC, (2) efficacy of ADCC is associated with polymorphisms at position 158 of FcgammaRIIIa. EXPERIMENTAL DESIGN: FcgammaRIIIa-158 polymorphisms were determined for healthy donors, and their purified NK cells were used as effector cells against three SCCHN cell lines in ADCC assays. Cytotoxicity levels were compared for each polymorphism class. Proliferation and cell cycle assays were done to examine the direct effects of cetuximab. RESULTS: Our results indicate that SCCHN is susceptible to cetuximab-mediated ADCC in vitro. NK cytotoxic efficiency correlates with donor 158-polymorphisms in FcgammaRIIIa. Overall cytotoxicity was greatest for individuals having a single V allele when compared to homozygous F/F individuals; the cumulative percent cytotoxicity for each polymorphism among the cell lines was 58.2% V/V, 50.6% V/F, and 26.1% F/F (P < 0.001). Additionally, the presence of a V allele correlated with superior natural cytotoxicity against NK sensitive targets. CONCLUSION: These data have both prognostic and therapeutic relevance and support the design of a prospective trial to determine the influence of FcgammaRIIIa polymorphisms on the clinical outcome of patients with SCCHN treated with alpha-EGFR mAbs.

Epitope mapping of a chimeric CD137 mAb: a necessary step for assessing the biologic relevance of non-human primate models.

Antibody based manipulation of the CD137 (4-1BB) co-signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti-human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti-CD137 mAb (4B4-1), recognized activated PBMCs from both human and non-human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species ( approximately 95% identical), with the extracellular domain differing by only 9-10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co-stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes.

CD44 is a marker for the outer pillar cells in the early postnatal mouse inner ear.

Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters' cells in the Fgfr3 ( P244R/ ) (+) mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.