Location: Foreign Animal Disease Research
Project Number: 3022-32000-064-043-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2024
End Date: Jul 31, 2026
Objective:
Determine immune cells draining to lymph nodes using single cell RNA sequencing. Commercial inactivated Foot and Mouth Disease (FMD) vaccines induce immune protection that usually last only 6 months even after a secondary vaccination. Understanding the immune cells draining to lymph nodes is the first critical step to improve the vaccine efficacy. The objective of this Agreement is to characterize immune cells draining to lymph nodes and to identify factors affecting the drainage using single cell RNA sequencing.
Approach:
Immune cells draining to lymph nodes will be collected from cannulated afferent lymphatic vessels. Cannulation will be performed on (1) sutured afferent lymphatic vessels and (2) pseudo-afferent lymphatic vessels. In the first study, the cooperator will purchase three cattle and provide husbandry for the animals to be utilized for this study. The surgical procedures of the cannulation including application for IACUC approval will be developed according to the publication (Lin Y, Xue J, Liao S. Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice. J Vis Exp. 2020 May 14;(159). doi: 10.3791/61178. PMID: 32478754.). Indocyanine green (ICG) and/or isosulfane blue (ISB) will be injected subcutaneously facilitate identifying afferent lymphatic vessels of pre-scapular and pre-femoral lymph nodes as reported (Balaya V, Guani B, Pache B, Durand YG, Bonsang-Kitzis H, Ngô C, Mathevet P, Lécuru F. Sentinel lymph node in cervical cancer: time to move forward. Chin Clin Oncol. 2021, 10(2):18.). Before the surgery, commercially available vaccines and/or adjuvants will be injected to determine the changes in draining cells. After the cannulation, the lymph will be collected into a plastic sterile containers (T25 tissue culture flask) containing heparin for 2 hours. The animals will be euthanized after sample collection. The lymph collection bottles will be stored in ice and shipped to ARS laboratory for further processing (scRNA-seq) performed by personnel in ARS. In the second study, the cooperator will purchase and provide husbandry for three cattle to be utilized for this study. The surgical procedures of the cannulation including application for IACUC approval will be developed according to the publication (Hope JC, Howard CJ, Prentice H, Charleston B. Isolation and purification of afferent lymph dendritic cells that drain the skin of cattle. Nat Protoc. 2006;1(2):982-7.). After the cannulation, the lymph will be collected into a plastic sterile containers (T75 tissue culture flask) containing heparin. The lymph collection bottle will be changed twice daily in the morning and afternoon. After the change, the bottles will be stored in ice and shipped to the ARS laboratory for further processing (scRNA-seq) performed by personnel in ARS. The animals will be euthanized 3 weeks after first lymph collection or earlier when the collection is not feasible. During the 3-week collection period, commercially available vaccines and/or adjuvants will be injected to determine the changes in draining cells. The scRNA-seq data will be analyzed by ARS PI to determine the immune cell types and the numbers of cell types. The transcriptomic data of the immune cells will be deposited in the NCBI GEO database.
