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Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens

Effective immune responses depend on efficient antigen uptake in the periphery, transport of those antigens to, and presentation in draining lymph nodes (LNs). These processes have been studied intensively using stable fluorescent proteins (FPs) as model antigens. To date, ZsGreen is the only FP tha...

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Autores principales: Yi, Rulan, Chen, Emily, Roberts, Edward W., Krummel, Matthew F., Serwas, Nina Kathrin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9384993/
https://www.ncbi.nlm.nih.gov/pubmed/35976946
http://dx.doi.org/10.1371/journal.pone.0272857
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author Yi, Rulan
Chen, Emily
Roberts, Edward W.
Krummel, Matthew F.
Serwas, Nina Kathrin
author_facet Yi, Rulan
Chen, Emily
Roberts, Edward W.
Krummel, Matthew F.
Serwas, Nina Kathrin
author_sort Yi, Rulan
collection PubMed
description Effective immune responses depend on efficient antigen uptake in the periphery, transport of those antigens to, and presentation in draining lymph nodes (LNs). These processes have been studied intensively using stable fluorescent proteins (FPs) as model antigens. To date, ZsGreen is the only FP that can be tracked efficiently towards LNs, hence, it is difficult to compare studies using alternated tracking proteins. Here, we systematically compared six different FPs. We included ZsGreen, ZsYellow, DsRed, AsRed, mCherry, and mRFP based on sequence homology and/or origin species, and generated FP-expressing tumor cell lines. Stability of fluorescent signal was assessed in vitro over time, across different pH environments, and in vivo through FP antigen uptake and transfer to immune cells isolated from tumors and tumor-draining LNs. ZsGreen could be detected in high percentages of all analyzed tumor-infiltrating immune cells, with highest amounts in tumor-associated macrophages (TAMs) and type 2 conventional dendritic cells (cDC2s). ZsYellow, AsRed, and DsRed followed a similar pattern, but percentages of FP-containing immune cells in the tumor were lower than for ZsGreen. Strikingly, mRFP and mCherry demonstrated a ‘non-canonical’ antigen uptake pattern where percentages of FP-positive tumor-infiltrating immune cells were highest for cDC1s not TAMs and cDC2s despite comparable stabilities and localization of all FPs. Analysis of antigen-containing cells in the LN was hindered by intracellular degradation of FPs. Only ZsGreen could be efficiently tracked to the LN, though some signal was measurable for ZsYellow and DsRed. In summary, we find that detection of antigen uptake and distribution is subject to variabilities related to fluorophore nature. Future experiments need to consider that these processes might be impacted by protein expression, stability, or other unknown factors. Thus, our data sheds light on potential under-appreciated mechanisms regulating antigen transfer and highlights potential uses and necessary caveats to interpretation based on FP use.
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spelling pubmed-93849932022-08-18 Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens Yi, Rulan Chen, Emily Roberts, Edward W. Krummel, Matthew F. Serwas, Nina Kathrin PLoS One Research Article Effective immune responses depend on efficient antigen uptake in the periphery, transport of those antigens to, and presentation in draining lymph nodes (LNs). These processes have been studied intensively using stable fluorescent proteins (FPs) as model antigens. To date, ZsGreen is the only FP that can be tracked efficiently towards LNs, hence, it is difficult to compare studies using alternated tracking proteins. Here, we systematically compared six different FPs. We included ZsGreen, ZsYellow, DsRed, AsRed, mCherry, and mRFP based on sequence homology and/or origin species, and generated FP-expressing tumor cell lines. Stability of fluorescent signal was assessed in vitro over time, across different pH environments, and in vivo through FP antigen uptake and transfer to immune cells isolated from tumors and tumor-draining LNs. ZsGreen could be detected in high percentages of all analyzed tumor-infiltrating immune cells, with highest amounts in tumor-associated macrophages (TAMs) and type 2 conventional dendritic cells (cDC2s). ZsYellow, AsRed, and DsRed followed a similar pattern, but percentages of FP-containing immune cells in the tumor were lower than for ZsGreen. Strikingly, mRFP and mCherry demonstrated a ‘non-canonical’ antigen uptake pattern where percentages of FP-positive tumor-infiltrating immune cells were highest for cDC1s not TAMs and cDC2s despite comparable stabilities and localization of all FPs. Analysis of antigen-containing cells in the LN was hindered by intracellular degradation of FPs. Only ZsGreen could be efficiently tracked to the LN, though some signal was measurable for ZsYellow and DsRed. In summary, we find that detection of antigen uptake and distribution is subject to variabilities related to fluorophore nature. Future experiments need to consider that these processes might be impacted by protein expression, stability, or other unknown factors. Thus, our data sheds light on potential under-appreciated mechanisms regulating antigen transfer and highlights potential uses and necessary caveats to interpretation based on FP use. Public Library of Science 2022-08-17 /pmc/articles/PMC9384993/ /pubmed/35976946 http://dx.doi.org/10.1371/journal.pone.0272857 Text en © 2022 Yi et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Yi, Rulan
Chen, Emily
Roberts, Edward W.
Krummel, Matthew F.
Serwas, Nina Kathrin
Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens
title Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens
title_full Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens
title_fullStr Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens
title_full_unstemmed Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens
title_short Impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: A comparison of different fluorescent proteins as model antigens
title_sort impact of protein identity on tumor-associated antigen uptake into infiltrating immune cells: a comparison of different fluorescent proteins as model antigens
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9384993/
https://www.ncbi.nlm.nih.gov/pubmed/35976946
http://dx.doi.org/10.1371/journal.pone.0272857
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