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.
The initial antigenic activation of T cells involves localization of the T-cell receptor to lipid rafts, of which gangliosides are major components. To understand the role of lipid rafts in the activation and differentiation of CD4+ and CD8+ T cells, it is necessary to dissect the ganglioside composition in each respective T-cell subset. In the present study, we used the B subunits of type-II heat-labile enterotoxins (LT-IIa-B5, LT-IIb-B5 and LT-IIc-B5), which possess defined ganglioside-binding specificities, as molecular probes to dissect the membrane ganglioside composition of human T cell subsets. Peripheral blood mononuclear cells isolated from leukocyte reduction filters by Ficoll density gradient centrifugation were stained with recombinant LT-IIa-B5, LT-IIb-B5, mutant LT-IIb-B5(T13I) or LT-IIc-B5 and with monoclonal antibodies to CD4, CD8, CD27, CD45RA, CD45RO and CD62L followed by flow cytometric analysis. Our results show that ligands for LT-IIa (gangliosides GD1b, GM1, and GT-1b), LT-IIb (gangliosides GD1a, GT1b and GM2), and LT-IIc (gangliosides GM1, GM2, and GM3) are increasingly expressed during differentiation from naïve (CD4+ or CD8+CD45RO-CD27+) to memory (CD4+ or CD8+CD45RO+CD27+) T cells, and that discrete subsets of central memory (CD4+ or CD8+CD45RA-CD62L+), effector memory (CD4+ or CD8+CD45RA-CD62L-), and terminally differentiated effector memory (CD4+ or CD8+CD45RO+CD27-) T cells express the highest ganglioside levels, particularly of LT-IIb-ligands. On the other hand, mutant LT-IIb-B5(T13I), which had negligible binding in vitro to gangliosides GD1a, GT1b, and GM2, exhibited no detectable binding to naive or central memory T cells, but significantly bound subsets of terminally differentiated effector memory (CD4+ and CD8+) T cells. Our results suggest an important role for gangliosides GD1b and GM1 in naive T-cell responses, while central memory, effector memory, and terminally differentiated effector memory T-cell responses may be more dependent upon gangliosides GD1a and GT1b. Furthermore, the lymphocyte-binding activities of LT-IIb-B5(T13I) confirm that its parental B-pentameter essentially recognizes gangliosides GD1a, GT1b, and GM2 on T cells and suggest that LT-IIb-B5 (T13I) may bind to one or more unknown receptor(s) expressed on subsets of terminally differentiated effector memory (CD4+ and CD8+) T cells. Citation Format: Taylor A. Johnson, Madison K. Ritter, Mary-Peyton A. Knapp, Robert O. Rainer, Nathalie D. King-Lyons, Terry D. Connell, Sergio Arce. Differentiation of human naive T cells to various effector memory cell subtypes correlates with increased binding of the B subunit of Type-IIb heat-labile enterotoxin (LT-IIb-B5) to its cognate ganglioside receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A25.
Gangliosides are glycosphingolipids that play important regulatory roles in cell signaling. Their patterns of expression during human lymphoid cell development and differentiation, however are not not known. Here, we used the ganglioside-binding B subunits of E. coli enterotoxins (LT-IIa-B, LT-IIb-B and LT-IIc-B) as molecular probes to dissect the membrane ganglioside composition of human CD8+ T cells and NK cells, two important players in the anti-tumor response. Human normal PBMC isolated from leukoreduction filters by Ficoll density gradient centrifugation were stained with recombinant LT-IIa-B, LT-IIb-B or LT-IIc-B, and with monoclonal antibodies to CD3, CD8, CD16, CD19, CD27, CD62L, CD45RA, CD45RO and CD56. Using FACS analysis, we found that ganglioside ligands for LT-IIb-B (avidity: GD1a>GT1b>GM2/GM3) and LT-IIc-B (avidity: GM1>GM2 >GM3/GD1a) were increasingly expressed during transitions from naïve T cells, either (CD8+CD45RA+CD62L+) or (CD8+CD45RO-CD27+), to memory T cells of both central memory (CD8+CD45RA-CD62L+) and effector memory (CD8+CD45RA-CD62L-) phenotypes and from memory T cells (CD8+CD45RO+CD27+) to terminally differentiated effector memory (CD8+CD45RO+CD27-) T cells, with expression of ganglioside ligands for LT-IIb-B exhibiting the most significant increase. Patterns of ganglioside expression in the NK cell subsets were remarkably similar to those observed in the CD8+ T cell subsets: Ganglioside ligands for LT-IIb-B and LT-IIc-B were increasingly expressed during transitions from circulating naïve (CD16dimCD56bright) to effector (CD16+CD56dim) NK cells and from effector NK cells to activated mature (CD16+CD56-) NK cells, with expression of ganglioside ligands for LT-IIb-B exhibiting the most significant increase. No major changes in expression of ganglioside ligands for LT-IIa-B (avidity: GD1b>GM1>GT1b) were detected across either CD8+ T cell or NK cell populations. Taken together these results demonstrate dynamic changes in the patterns of ganglioside expression throughout CD8+ T cell and NK cell differentiation. The unique and shared pattern suggests the existence of cell signaling similarities during development and function of these distinct cell types, and this relationship could prove invaluable in the development of novel cancer immunotherapies. Citation Format: Madison K. Ritter, Mary-Peyton A. Knapp, Taylor A. Johnson, Natalie D. King-Lyons, Lorrie Mandell, Jennifer T. Grier, Terry D. Connell, Sergio Arce. Dynamic changes in patterns of ganglioside expression characterize maturation of cytotoxic T cells and natural killer cells into effector cells and suggest a close relationship between the two cell types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4525.
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