Nur77-Tempo mice reveal T cell steady state antigen recognition

Abstract

In lymphocytes, Nr4a gene expression is specifically regulated by antigen receptor signalling, making them ideal targets for use as distal T cell receptor (TCR) reporters. Nr4a3-Timer of cell kinetics and activity (Tocky) mice are a groundbreaking tool to report TCR driven Nr4a3 expression using Fluorescent Timer protein (FT). FT undergoes a time-dependent shift in its emission spectrum following translation, allowing for the temporal reporting of transcriptional events. Our recent work suggested that Nr4a1/ Nur77 may be a more sensitive gene to distal TCR signals compared to Nr4a3, so we therefore generated Nur77-Timer-rapidly-expressed-in-lymphocytes (Tempo) mice that express FT under the regulation of Nur77. We validated the ability of Nur77-Tempo mice to report TCR and B cell receptor (BCR) signals and investigated the signals regulating Nur77-FT expression. We found that Nur77-FT was sensitive to low strength TCR signals, and its brightness was graded in response to TCR signal strength. Nur77-FT detected positive selection signals in the thymus, and analysis of FT expression revealed that positive selection signals are often persistent in nature, with most thymic Treg expressing FT Blue. We found that active TCR signals in the spleen are low frequency, but CD69 + lymphoid T cells are enriched for FT Blue +Red + T cells, suggesting frequent TCR signalling. In non-lymphoid tissue, we saw a dissociation of FT protein from CD69 expression, indicating that tissue residency is not associated with tonic TCR signals. Nur77-Tempo mice, therefore, combine the temporal dynamics from the Tocky innovation with increased sensitivity of Nr4a1 to lower TCR signal strengths.

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronicTrypanosoma bruceiinfection

“…To further resolve whether T cell activation occurs in situ , we used the newly reported Nur77 Tempo mice, a novel murine reporter line in which the expression of a fluorescent timer (FT) protein is driven by Nur77 expression 64,65 . This model enabled the discrimination of newly activated (FT blue + ), persistent (FT blue + red + ), and arrested (FT red + ) T cells based on MHC-dependent TCR engagement 64,65 . We observed a significant increase in the frequency of newly activated and persistent CD4 + T cells, and a concomitant reduction in the frequency of arrested CD4 + T cells in the meninges in response to infection compared to naïve controls ( Figure 5L and 5M ), indicating that most CD4 + T cells are actively partaking in the local immune response, likely via antigenic presentation.…”

“…To further resolve whether T cell activation occurs in situ, we used the newly reported Nur77 Tempo mice, a novel murine reporter line in which the expression of a fluorescent timer (FT) protein is driven by Nur77 expression 64,65 . This model enabled the discrimination of newly activated (FT blue + ), persistent (FT blue + red + ), and arrested (FT red + ) T cells based on MHC-dependent TCR engagement 64,65 . We observed a significant increase in the frequency of newly…”

Salmonellacancer therapy metabolically disrupts tumours at the collateral cost of T cell immunity

“…Three strains of mice were used in this study: (i) C57BL/6 (Charles River, UK), (ii) Nr4a3-Timer ("Tocky") (Bending et al, 2018a(Bending et al, , 2018b crossed with Great-Smart ifng-YFP reporter (Price et al, 2012), and (iii) Nr4a1-Tempo mice (Elliot et al, 2022). Males For use of Nr4a3-Tocky mice please contact Dr Masahiro Ono (m.ono@imperial.ac.uk).…”

“…Three strains of mice were used in this study: (i) C57BL/6 (Charles River, UK), (ii) Nr4a3 -Timer (“Tocky”) (Bending et al, 2018a, 2018b) crossed with Great-Smart ifng -YFP reporter (Price et al, 2012), and (iii) Nr4a1-Tempo mice (Elliot et al, 2022). Males and females aged 6-32 weeks were used throughout the experiments, with age matching wherever possible.…”

“…Nr4a3-Timer ("Tocky") (Bending et al, 2018a(Bending et al, , 2018b crossed with Great-Smart ifng-YFP reporter (Price et al, 2012), and (iii) Nr4a1-Tempo mice (Elliot et al, 2022) (Porwollik et al, 2014) was transduced into an aroA deleted STm UF20 strain (SR-11x3181 (Gulig and Doyle, 1993)) using P22 phage-mediated transduction (Schmieger, 1972). Both strains were grown in LB broth (Merck) overnight to saturation, followed by 1:20 sub-culture the following day until an OD600 of 0.7 was reached, indicating logarithmic growth.…”