PurposeVitreous seeding remains the primary reason for treatment failure in eyes with retinoblastoma (Rb). Systemic and intra-arterial chemotherapy, each with its own inherent set of complications, have improved salvage rates for eyes with advanced disease, but the location and biology of vitreous seeds present a fundamental challenge in developing treatments with minimal toxicity and risk. The aim of this study was to target the platelet-derived growth factor (PDGF)– PDGF-receptor β (PDGFRβ) signaling pathway and investigate its role in the growth of Rb seeds, apoptotic activity, and invasive potential.MethodsWe performed ex vivo analyses on vitreous samples from Rb patients that underwent enucleation and from patient-derived xenografts. These samples were evaluated by quantitative PCR, immunohistochemistry, and ELISA. The effects of disruption of the PDGF-PDGFRβ signaling pathway, both by pharmacologic and genomic knockdown approaches, were evaluated in vitro by cell proliferation and apoptotic assays, quantitative PCR analyses, Western blotting, flow cytometry, and imaging flow cytometry. A three-dimensional cell culture system was generated for in-depth study of Rb seeds.ResultsOur results demonstrated that PDGFRβ signaling is active in the vitreous of Rb patients and patient-derived xenografts, sustaining growth and survival in an AKT-, MDM2-, and NF-κB-dependent manner. The novel three-dimensional cell culture system mimics Rb seeds, as the in vitro generated spheroids have similar morphologic features to Rb seeds and mimicked their natural physiology.ConclusionsTargeting the PDGFRβ pathway in vitro reduces Rb cell growth, survival, and invasiveness and could augment current therapies. This represents a novel signaling pathway for potential targeted therapy to further improve ocular survival in advanced Rb.
Gangliosides are glycosphingolipids that play important regulatory roles in cell signaling and differentiation. To investigate their expression patterns during lymphoid cell development, human PBMC were stained with recombinant B subunits of LT-IIa, LT-IIb, or LT-IIc and with select mAb. FACS analysis showed that ganglioside ligands for LT-IIaB, LT-IIbB, and LT-IIcB were increasingly expressed during transition from naïve to memory to effector memory CD4+ T cells, with ganglioside ligands for LT-IIbB exhibiting the greatest increase. A similar trend was observed in CD8+ T cell subsets, except no major changes in the expression of LT-IIaB ligands were detected. Patterns of ganglioside expression in NK cell subsets were remarkably similar to those observed in the CD8+ T cells: The ganglioside ligands for LT-IIbB and LT-IIcB were increasingly expressed during transition from tissue-resident to effector to differentiated/exhausted NK cells, with ganglioside ligands for LT-IIbB exhibiting the greatest increase and ligands for LT-IIaB showing little change across subsets. Finally, expression of LT-IIaB- and LT-IIcB-binding gangliosides increased during transition from naïve to memory to antibody-secreting B cells, with ganglioside ligands for LT-IIaB exhibiting the greatest increase and ligands for LT-IIbB showing little change across subsets. Our results demonstrated the existence of dynamic changes in the patterns of gangliosides expressed throughout lymphoid cell development and differentiation. The distinct patterns suggest the existence of cell signaling differences between and within the distinct lymphoid cell lineages and a close developmental and functional relationship between CD8+ T cells and NK cells.
There is an urgent need for development of new adjuvants to target and drive immune responses in cancer immunotherapies. The E. coli type-II heat-labile enterotoxins (HLEs) LT-IIa, LT-IIb and LT-IIc exhibit strong adjuvant properties with varying capacities to elicit immune responses in mice. The immunomodulatory capabilities of these HLEs depend upon their interactions with specific ganglioside receptors that may vary in expression across immune cell types. Little is known, however, about the lymphoid cells that interact with HLEs in humans. To bridge this gap, human normal PBMCs, isolated by Ficoll density gradient centrifugation, were stained with recombinant B subunits of LT-IIa, LT-IIb, or LT-IIc, and mAbs to CD4, CD8, CD19, CD27, CD38 and CD45RO. Using FACS analysis, we found that LT-IIa, LT-IIb and LT-IIc increasingly bound to CD4+ T cells as the cells transitioned from naive (CD4+CD45RO-CD27+) to memory (CD4+CD45RO+CD27+) cells and from memory cells to effector memory (CD4+CD45RO+CD27-) cells, with LT-IIb exhibiting the most significant increase in binding. Likewise, LT-IIb and LT-IIc increasingly bound to CD8+ T cells as the cells transitioned from naive (CD8+CD45RO-CD27+) to memory (CD8+CD45RO+CD27+) cells and from memory cells to effector memory (CD8+CD45RO+CD27-) cells, with the greatest increase observed for LT-IIb. No major changes in LT-IIa binding were detected during CD8+ T cell differentiation. Finally, LT-IIa and LT-IIc increasingly bound to CD19+ B cells as the cells transitioned from naive (CD19+CD27-) to memory (CD19+CD27+) cells and from memory cells to antibody-secreting (CD19+/-CD27++CD38++) plasma cells, with LT-IIa exhibiting the most significantly increased binding activity. No major changes in LT-IIb binding were observed during B cell differentiation. Taken together, these results suggest that the ganglioside receptors for HLEs (e.g., GD1a, GD1b and GM1) are differentially expressed throughout B and T cell development/differentiation and that binding of HLEs to the various lymphocyte subsets is likely to elicit distinct cellular events associated with immune modulation or immune responsiveness. Understanding of these HLE binding patterns may also prove useful in targeting additional immunomodulating drugs to specific T or B cell subpopulations. Ultimately, characterization of immunological responses to these enterotoxins will lead to a greater understanding of their potential use in targeted cancer immunotherapies for treatment of a wide variety of conditions. Citation Format: Mary-Peyton A. Knapp, Taylor A. Johnson, Madison K. Ritter, Robert O. Rainer, Steven E. Fiester, Jennifer T. Grier, Terry D. Connell, Sergio Arce. Differential binding of E. coli enterotoxins LT-IIa, LT-IIb and LT-IIc to human B and T cell subsets identifies a potential use as adjuvants in cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO024.
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