Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Summary

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Abstract

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8⁺ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for T_CD8 “immunodominance” in this powerful system. Accordingly, the bulk of the T Ag-specific T_CD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor T_CD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape T_CD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Introduction

Conventional CD8⁺ T cells (T_CD8) play important parts in anticancer immune surveillance. They primarily function in the capacity of cytolytic T lymphocytes (CTLs) that recognize tumor-specific or -associated peptide antigens (Ags) displayed within the closed cleft of major histocompatibility complex (MHC) class I molecules. Fully armed CTLs utilize their cytotoxic arsenal to destroy malignant cells. Anticancer T_CD8 can be detected in the circulation or even inside primary and metastatic masses of many cancer patients and tumor-bearing animals. However, they are often anergic or exhausted and fail to eradicate cancer. Therefore, many im....

Protocol

The experiments described here follow animal use protocols approved by institutional entities and adhered to established national guidelines.

1. Inoculation of C57BL/6 Mice with T Ag-expressing Tumor Cells

1. Grow the SV40-transformed fibrosarcoma cell line C57SV (or a similar T Ag⁺ adherent cell line) in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L D-glucose and L-glutamine (1x) and supplemented with 1 mM sodium pyruvate and 10% heat-inactivated fetal bovine serum (FBS) in tissue culture-treated flasks at 37 °C in humidified atmosphere containing 10% CO₂.
2. Once the cells be....

Results

The goal of the experiment whose results are depicted in Figure 1 was to determine whether the presence and functions of nTreg cells shape or alter the immunodominance hierarchy of T Ag-specific T_CD8. C57BL/6 mice were injected i.p. with PBS or with 0.5 mg of an anti-CD25 mAb (clone PC-61.5.3 [PC61]) four days before they received 2 x 10⁷ C57SV tumor cells i.p. In separate experiments, a rat IgG1 isotype control was used in lieu of PBS. Successful nTreg cell depletion b.......

Discussion

CFSE-based in vivo cytotoxicity assays offer several advantages over traditional killing assays such as radioactive chromium (⁵¹Cr) release and colorimetric lactate dehydrogenase (LDH) release assays. First, they permit the monitoring of CTL function within an architecturally intact secondary lymphoid organ.

Second, the specific killing of target cells in in vivo cytotoxicity assays reflects the absolute number of Ag-specific T_CD8, which is usually, but not always, a func.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA (1X)	Thermo Fisher Scientific	25200-056
ACK Lysing Buffer	Thermo Fisher Scientific	A1049201
Anti-mouse CD25 (clone PC-61.5.3)	Bio X Cell	BE0012
Anti-mouse PD-1 (clone RMP1-14)	Bio X Cell	BE0146
CFSE	Thermo Fisher Scientific	C34554
DMEM (1X)	Thermo Fisher Scientific	11965-092
Fetal bovine serum (FBS)	Wisent Bioproducts	080-150	Heat-inactivate prior to use
GlutaMAX (100X)	Thermo Fisher Scientific	35050-061
HEPES (1M)	Thermo Fisher Scientific	15630080	10 mM final concentration
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher Scientific	11140-050
Penicillin/Streptomycin	Sigma-Aldrich	P0781	Stock is 100X
Rat IgG1 (clone KLH/G1-2-2)	SouthernBiotech	0116-01	Isotype control
Rat IgG1 (clone HRPN)	Bio X Cell	BE0088	Isotype control
Rat IgG1 (clone TNP6A7)	Bio X Cell	BP0290	Isotype control
Rat IgG2a (clone 2A3)	Bio X Cell	BP0089	Isotype control
RPMI 1640 (1X)	Thermo Fisher Scientific	11875-093
Sodium Pyruvate (100 mM)	Thermo Fisher Scientific	11360-070	1 mM final concentration