Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

Summary

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

Abstract

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Introduction

Advances in immunology and nanomaterials have led to an abundance of potential new therapeutic strategies for applications in biomedicine, including drug delivery and immunostimulation. Optimization of the administration route is a vital aspect affecting the efficacy of immunostimulatory agents. An immunostimulatory nanoparticle (INP) consisting of DNA is a newly developed nano-immune adjuvant self-assembled by microphase separation because of the amphiphilic structure of lipid-DNA¹. Therefore, protocols for INP involving administration of the material¹ in vivo via different routes, and three procedures for....

Protocol

All experimental procedures including animal handling, sacrifice, and organ isolation were performed in strict accordance with the rules of the International Animal Care and Use Committee at Shanghai Public Health Clinical Center and Asan Medical Center. The study protocol was approved by the respective committees on the Ethics of Animal Experiments at Shanghai Public Health Clinical Center (Mouse Protocol Number: SYXK-2010-0098) and Asan Medical Center (2016-02-168).

1. Preparation of Material

NOTE: A nano-adjuvant, INP, comprised of a self-assembled lipid-DNA carrier, namely U4T, and CpG-motif containing oligonuc....

Results

To evaluate appropriate injection routes of INP for lymphatic tissue DC activation, the DC population as lineage was defined as ^-CD11c⁺ cells in the spleen, iLN, and mLN and analyzed the expression levels of co-stimulatory molecules. Treatment of INP by subcutaneous (s.c.) and intravenous (i.v.) injection promoted substantial increases in CD40, CD80, and CD86 expression in the spleen and iLN DCs (Figure 2B and 2.......

Discussion

Many advances in nanotechnology and immunology have been achieved through therapeutic research of drug delivery and immunostimulation. Careful selection of the injection method is known to be important for immunostimulation, which was the focus of the present study.

Different injection routes were evaluated for a naturally non-toxic and biodegradable DNA-based material, INP (immunostimulatory nanoparticle), to determine which route yielded the best outcome. This approach is also relevant for d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
phosphate buffer saline	Corning	21-040-CVR	Washing organs
(PBS, pH 7.4)
isoflurane solution	Aesica Queenborough limited	26675-46-7	Anesthesia process
Tuberculin 1mL syringe -	Junglim	N/A	Injection
50 mL conical tube	S.P.L	50050	Anesthesia process
1mL Insulin Syringe	(BD Ultra-FineTM­II)_short needle	324826	Intramuscular Injection
DMEM High Glucose	Hyclone	SH30081.01	Storing organs
Histopaque	Sigma-Aldrich	10771	FACS analysis
Ethyl alcohol anhydrous 99.5 %	Daejung	4022-4110	Disinfectant
Equipments
FineCycler C100 (Thermocycler)	Ssufine	-	Anealing
Centrifuge			Centrifuge
FACS tube	FALCON	2052	FACS analysis
Automated High-performance Flow Cytometer	BD (USA), FACSVerse	-	FACS analysis