Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

Shannon M Walsh,Johnathon Schafer,Brian C Ware,Jay R Hesselberth,Beth Ann Jiron Tamburini,Thu A Doan,Erin D Lucas,Ryan M Sheridan,Rui Fu,Matthew A Burchill

doi:10.7554/elife.62781

Abstract

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

Highlights

Depending on the route of infection, vaccination mode, and ability of antigens to traffic, different dendritic cell (DC) subsets are required to initiate T cell priming
Using a vaccine formulation that elicits robust cell-mediated immunity comprised of antigen, a Toll-like receptor (TLR) ago[75] nist, and an agonistic CD40 antibody (TLR/ CD40 vaccination) or viral infection[15, 16, 17, 18, 19, 20, 76 21, 22, 23, 24, 25, 26], we discovered that antigens were durably retained in the lymph node[12, 13, 14]
To quantify the dissemination and uptake of antigen in the draining lymph node (LN) after vaccination, we developed a vaccination strategy to measure antigen levels using single-cell mRNA sequencing

Summary

INTRODUCTION

Depending on the route of infection, vaccination mode, and ability of antigens to traffic, different dendritic cell (DC) subsets are required to initiate T cell priming. Most adaptive immune responses require antigen processing and presentation by conventional dendritic cells in either the draining lymph node or at the site of infection or vaccination (migratory cutaneous or 70 dermal DCs) 5. Antigen storage was dependent on the presence of a TLR agonist (e.g. polyI:C alone (TLR3/MDA5/RIGI or Pam3cys (TLR1/2)+ CD40), and occurred with antigen conjugated to 79 a TLR agonist (e.g. 3M019 (TLR7)) 14 We named this process “antigen archiving” and showed it 80 is important to poise memory T cells for future antigenic encounters[14]. Detection of antigen in the lymph node and other tissues has relied on flow cytometric analysis using cell surface markers, restricting analysis to specific cell types To address these limitations and better understand antigen archiving, we developed a new approach to track an antigen-phosphorothioate DNA. We outline the tissue distribution in vivo of this antigen-DNA conjugate by utilizing the conjugated phosphorothioate DNA as an adjuvant and tracking device

RESULTS

DISCUSSION

MATERIALS AND METHODS