Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Summary

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Abstract

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

Introduction

Traditional vaccine development has mainly employed the empirical approach of trial-and-error. However, with the recent development of a wide array of biomaterials and discovery of molecular determinants of immune activation, it is now possible to rationally design vaccine formulations with biophysical and biochemical cues derived from pathogens^1,2. In particular, various particulate drug delivery platforms have been examined as vaccine carriers as they can be co-loaded with subunit antigens and immunostimulatory agents, protect vaccine components from degradation, and enhance their co-delivery to antigen presenting cells (APCs) residing in lymph nodes (LNs....

Protocol

All experiments described in this protocol were approved by the University Committee on Use and Care of Animals (UCUCA) at University of Michigan and performed according to the established policies and guidelines.

1. Synthesis and Characterization of ICMVs Co-loaded with Protein Antigen and Adjuvant Molecules

1. Mix 1:1 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] (MPB) in chloroform, keeping the total lipid amount at 1.26 µmol per batch (i.e., 500 µg of DOPC and 630 µg....

Results

The steps involved in the synthesis of ICMVs are illustrated in Figure 1⁶. Briefly, a lipid film containing any lipophilic drugs or fluorescent dyes is hydrated in the presence of hydrophilic drugs. Divalent cations, such as Ca²⁺, are added to drive fusion of anionic liposomes into multilamellar vesicles. Dithiol crosslinker, such as DTT, is added to “staple” maleimide-functionalized lipids on apposing lipid layers, and finally remaining external maleimide groups are quenched in a r.......

Discussion

The protocol provided in this article describes the synthesis and characterization of a new lipid-based nanoparticle system, termed ICMVs, and provides the process of validating effectiveness of nanoparticle-based vaccine formulations to induce antigen-specific CD8+ T cell responses. ICMV synthesis is completed in all aqueous condition, which is a major advantage compared with other commonly used polymeric nanoparticle systems (e.g., poly(lactide-co-glycolide) acid particles), which typically require organic sol.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (MPB)	Avanti Polar Lipids, INC.	870012
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)	Avanti Polar Lipids, INC.	850375
Monophosphoryl Lipid A (Synthetic) (PHAD™) (MPLA)	Avanti Polar Lipids, INC.	699800
20 ml glass vials	Wheaton	0334125D
Symphny Vacuum Oven	VWR	414004-580
Ovalbumin (OVA)	Worthington	3054
Bis-Tris Propane (BTP)	Fisher	BP2943
Q125 Sonicator (125 W/20 kHz)	Qsonica	Q125-110
Dithiothreitol (DTT)	Fisher	BP172
2 kDa Thiolated Polyethylene Glycol (PEG-SH)	Laysan Bio	MPEG-SH-2000-1g
Malvern ZetaSizer Nano ZSP	Malvern
ZetaSizer Cuvettes	Malvern	DTS1070
2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID)	Life Technologies	D-7757
Alexa Fluor 555-succinimidyl ester (AF555-NHS)	Life Technologies	A37571
Tissue-Tek OCT freezing medium	VWR	25608-930
Tissue Cryomolds	VWR	25608-922
3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging
C57BL/6 mice	Jackson	000664
Albino C57BL/6 mice	Jackson	000058
OT-1 C57BL/6 mice	Jackson	003831
70 μm nylon strainer	BD	352350
EasySep™ Mouse CD8+ T Cell Isolation Kit	StemCell	19853
IVIS® whole animal imaging system	Perkin Elmer
4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells
K2EDTA tubes	BD	365974
ACK lysis buffer	Life Technologies	A10492-01
Anti-CD16/32 Fc Block	Ebioscience	14-0161-86
H-2Kb OVA Tetramer	MBL	TS-5001-1C
Anti-CD8-APC	BD	553031
Anti-CD44-FITC	BD	553133
Anti-CD62L-PECy7	Ebioscience	25-0621-82
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)	SIGMA	D8417-10MG
CyAn Flow Cytometer	Beckman Coulter
FlowJo Software	FlowJo