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 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.

[1]  O. Boyman,et al.  CCR7-guided neutrophil redirection to skin-draining lymph nodes regulates cutaneous inflammation and infection , 2022, Science Immunology.

[2]  J. Yewdell,et al.  A few good peptides: MHC class I-based cancer immunosurveillance and immunoevasion , 2020, Nature Reviews Immunology.

[3]  Zhaowu Ma,et al.  Dendritic cell biology and its role in tumor immunotherapy , 2020, Journal of Hematology & Oncology.

[4]  En Cai,et al.  Visualizing Synaptic Transfer of Tumor Antigens among Dendritic Cells. , 2020, Cancer cell.

[5]  Talley J. Lambert,et al.  FPbase: a community-editable fluorescent protein database , 2019, Nature Methods.

[6]  Michael J. Shannon,et al.  Fluorescent Proteins for Investigating Biological Events in Acidic Environments , 2018, International journal of molecular sciences.

[7]  J. Borst,et al.  Subcellular Localization of Antigen in Keratinocytes Dictates Delivery of CD4+ T-cell Help for the CTL Response upon Therapeutic DNA Vaccination into the Skin , 2018, Cancer Immunology Research.

[8]  J. Solheim Faculty Opinions recommendation of Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in melanoma. , 2016 .

[9]  T. Kaisho,et al.  Critical Role for CD103(+)/CD141(+) Dendritic Cells Bearing CCR7 for Tumor Antigen Trafficking and Priming of T Cell Immunity in Melanoma. , 2016, Cancer cell.

[10]  F. Ginhoux,et al.  Expansion and Activation of CD103(+) Dendritic Cell Progenitors at the Tumor Site Enhances Tumor Responses to Therapeutic PD-L1 and BRAF Inhibition. , 2016, Immunity.

[11]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[12]  L. Fiette,et al.  Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes. , 2005, Blood.

[13]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[14]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[16]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.