Fluorescent tracking identifies tumor migratory dendritic cells as critical for cross-presentation in the lymph node following radiation therapy induced maturation
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ABSTRACT:
PROVIDER: PRJNA828105 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18811400.fastq.gz | Fastqsanger.gz | |||
| SRR18811401.fastq.gz | Fastqsanger.gz | |||
| SRR18811402.fastq.gz | Fastqsanger.gz | |||
| SRR18811403.fastq.gz | Fastqsanger.gz | |||
| SRR18811404.fastq.gz | Fastqsanger.gz |
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Project description:Antigen presenting dendritic cells (DCs) and monocytes capture and transport antigens from barrier tissues for presentation to antigen-specific T cells in the draining-lymph nodes (LNs). While DCs enter LNs through afferent lymphatics in a CCR7-dependent manner, how exactly antigen-carrying monocytes reach LNs is less clear since monocytes do not express CCR7 and can also enter LNs via the bloodstream. In steady state, and following injection of several PAMPs, scRNA-seq data on LN mononuclear phagocytes identified LN resident versus migratory type 1 and type 2 conventional (c)DCs despite downregulation of DC subset-defining transcripts, such as Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a on migratory cDCs. Migratory cDCs gained expression of transcripts controlling cellular migration such as Ccr7, Ccl17, Ccl22, and Ccl5, while migratory monocytes expressed Ccr5 without Ccr7. Using two tracking methods and a gating strategy that clearly distinguishes migratory CD88hiCD26lo monocytes from CD88-CD26hi cDCs, we found that both captured antigens in the lung and migrated to lung-draining LNs. Using global and mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice, we found that CCR5+ monocytes follow CCL5-secreting migratory cDCs to reach the draining LN via lymphatic vessels. In a model of asthma, such recruited monocytes regulated the induction of type 2 immunity. Overall, our data suggest that CCL5-secreting migratory cDCs lay down the chemokine trail for CCR5+ antigen-presenting monocytes to reach draining lymph nodes and regulate adaptive immunity.
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Project description:Molecular tracking devices quantify antigen distribution and archiving in the lymph node.
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Project description:During immune responses, T cells differentiate into subsets with different functions and migratory properties. Here we characterize migratory behavior of endogenous αβ CD8+ and γδ T cells in lymph nodes by long-term tracking following in vivo photoconversion. We identified subsets of γδ T cells with distinct circulation kinetics that closely mirrored migratory subsets of αβ CD8+ T cells. Notably, αβ CD8+ and γδ T cells both comprised resident populations which stayed in lymph nodes for 4 weeks without circulation or proliferation. Furthermore, in contrast to the common conception, we observed that central memory αβ CD8+ T cells circulate with slower kinetics than naïve cells. Our results show that, similar to αβ T cells, γδ T cells can acquire distinct migratory properties during their development and differentiation and reveal unexpected intricacies of T cell migratory patterns.
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Project description:PurposeTo determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection.Materials and methodsThe animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc)-labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test.ResultsIn vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L±0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6×10(3) counts·sec(-1)·?W(-1) per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P=.005 to P=.042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation.ConclusionThe multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging.
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