In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Summary

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Abstract

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8⁺ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp100_25-33 and OVA_257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

Introduction

Multiple protocols exist to assess the cytolytic (CTL) potential of a CD8⁺ or CD4⁺ T-cell. This assessment can be readily done in vitro under controlled conditions^1,2,3. In addition, infectious disease models, such as LCMV, have classically examined CTL function through the use of differentially CFSE (5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester) labeled target cells where the CFSE^hi-labeled cells are pulsed with a peptide and CFSE^lo-labeled target cells are left unpulsed. The cells are then injected at a 1:1 r....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas MD Anderson Cancer Center.

1. Preparation of Peptide for the Vaccine

1. Dissolve the lyophilized peptide with phosphate-buffered saline (PBS) to the appropriate concentration and vortex for 30 s.
   NOTE: For hgp100_25-33, the final concentration is 1 mg/mL and for OVA_257-264, the final concentration is 0.5 mg/mL. Reconstitute peptide prior to injection. Do not store after reconstitution.

2. Isolation ....

Results

Prior to injection of CFSE-labeled target cells, the 1:1 cell mixture is run on a flow cytometer to determine the baseline frequencies of both the CFSE^hi and CFSE^lo target cells. Figure 1A shows the gating strategy to detect changes in the CFSE populations, an initial gate is made using FSC and SSC parameters. The total CFSE-positive cells are then subgated prior to assessing changes in frequency, as this population is r.......

Discussion

While this protocol is straightforward, there are a few critical steps that must be carefully performed. The covax priming following injection of the antigen-specific T-cell being tested is necessary to see any killing of the pulsed targets. While it is possible that the water-based covax vaccination creates an acute inflammatory condition, for the chronic inflammatory phase, replacing the short-lived water-based formulation with a slow-antigen release oil-based approach may produce a better outcome⁷

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 to 12 week old female C57BL/6 mice	Charles River	027	C57 Black 6 mice
OT-1 6-12 week old female mice	Jackson Labs	003831
hgp10025–33	CPCScientific	834139	KVPRNQDWL
OVA 257–264	CPCScientific	MISC-012	SIINFEKL
Imiquimod cream 5%	Fougera	51672-4145-6	Aldara Cream
CD40-specific mAb	BioXcell	BE0016-2	clone FGK4.5
rhIL-2 protein	Hoffman LaRoche Inc	136	Recombinant human IL-2 protein
70% isopropyl alcohol Prep	Kendall	S-17474
PBS	Life Technologies	10010-023	Phosphate Buffered Saline
FBS	Life Technologies	26140-079	fetal bovine serum
RBC lysis buffer	Life Technologies	A10492-01	red blood cell lysis buffer
RPMI 1640 Media	Life Technologies	11875119
L-Glutamine	Life Technologies	25030081
Pen/Strep	Life Technologies	15140122	penicillin/streptomycin
CFSE	Life Technologies	C34554	5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester
Bovine serum albumin (BSA)	Sigma	A4503
1.5 mL MCT graduated natural	Fisher	05-408-129	microcentrifuge tube
70% ethanol	Fisher	BP8201500	EtOH
Trypan blue solution, 0.4%	Life Technologies	15250-061
Hemocytometer	Fisher	267110
27 gauge needle	BD	305109
1 mL syringe	BD	309659