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 immunotherapeutic modalities are designed to increase anticancer T_CD8 frequencies and to restore and boost their functions.

Tumor proteins harbor many peptides, some of which can be immunogenic and potentially immunoprotective. However, quantifiable T_CD8 responses are elicited with varying magnitudes against few peptides only. This creates an “immunodominance hierarchy” among T_CD8 clones¹. Accordingly, immunodominant (ID) T_CD8 occupy prominent hierarchical ranks, which is commonly judged by their abundance. In contrast, T_CD8 cells whose T cell receptor (TCR) is specific for subdominant (SD) epitopes occur in lower frequencies. We and others have identified some of the factors that dictate or shape immunodominance in T_CD8 responses. These include, among others, the mode of Ag presentation to naïve T_CD8 (i.e., direct presentation, cross-presentation, cross-dressing)^2,3,4, the type of Ag-presenting cells (APCs) participating in T_CD8 activation⁵, the abundance and stability of protein Ags^6,7 and the efficiency and kinetics of their degradation by proteasomes^7,8, the relative selectivity of transporter associated with Ag processing (TAP) for peptides⁹, the affinity of liberated peptides for MHC I molecules^9,10, the presence, precursor frequencies and TCR diversity of cognate T_CD8 in T cell pools^11,12,13, cross-competition among T cells for access to APCs^14,15, and the fratricidal capacity of T_CD8 clones¹⁶. In addition, T_CD8 immunodominance is subjected to immunoregulatory mechanisms mediated by several suppressor cell types such as naturally occurring regulatory T (nTreg) cells¹⁷, the cell surface co-inhibitory molecule programmed death-1 (PD-1)¹⁶, and certain intracellular enzymes such as indoleamine 2,3-dioxygenase (IDO)¹⁸ and the mammalian target of rapamycin (mTOR)¹⁹. It is important to note, however, that the above factors do not always fully account for immunodominance.

Apart from the basic biology of T_CD8 immunodominance, the examination of this intriguing phenomenon has important implications in cancer immunology and immunotherapy. First, an ID status does not necessarily confer upon a given T_CD8 clone the ability to prevent tumor initiation or progression²⁰. Whether and how ID and SD T_CD8 contribute to antitumor immunity may be dependent upon the type and the extent of malignancy and the experimental system employed. Second, it is thought that ID T_CD8 clones may be ‘too visible’ to the immune system and consequently more prone to central and/or peripheral tolerance mechanisms^16,21. Third, heterogeneic tumors may contain neoplastic cells that avoid detection by many, if not most, CTLs by displaying only a narrow spectrum of peptide:MHC complexes. Under these circumstances, T_CD8 responses of insufficient breadth are likely to afford such tumor cells a survival advantage, thus potentiating their outgrowth²². It is for the above reasons that many view immunodominance as a hurdle to successful T_CD8-based vaccination and therapies against cancer.

Inoculation of C57BL/6 mice with simian virus 40 (SV40)-transformed cells that express large tumor Ag (T Ag) provides a powerful preclinical system to study T_CD8 immunodominance. This model offers several benefits. First, the peptide epitopes of this clinically relevant oncoprotein are well-characterized in this mouse strain²³ (Table 1). Second, T Ag epitopes, which are called sites I, II/III, IV, and V, trigger T_CD8 responses that are consistently arranged in the following hierarchical order: site IV >> site I ≥ site II/III >> site V. Accordingly, site IV-specific T_CD8 mount the most robust response to T Ag. In contrast, sites I and II/III are subdominant, and site V-specific T_CD8 are least abundant and usually only detectable in the absence of responsiveness to other epitopes^23,24. Third, the T Ag⁺ tumor cell line utilized in the protocol described herein, namely C57SV fibrosarcoma cells, and those used in our previous investigations^{16,17,18,19,25,26}, are transformed with subgenomic SV40 fragments²⁵. Therefore, they are unable to assemble and release SV40 virions that could potentially infect host APCs. In addition, C57SV cells are devoid of classic costimulatory molecules such as CD80 (B7-1), CD86 (B7-2), and CD137 ligand (4-1BBL)¹⁶. The above attributes make these lines ideal for examination of in vivo T_CD8 activation via cross-priming. Cross-priming is a major pathway in inducing T_CD8 responses, especially those launched against tumor cells of non-hematopoietic origin that fail to directly prime naïve T cells²⁵.

Antitumor T_CD8 frequencies and/or functions can be monitored by MHC I tetramer staining, intracellular staining for effector cytokines (e.g., interferon [IFN]-γ) or lytic molecules (e.g., perforin), enzyme-linked immunospot (ELISpot) assays and ex vivo cytotoxicity assays. Since their inception in the 1990s^27,28, carboxyfluorescein succinimidyl ester (CFSE)-based in vivo killing assays have enabled evaluation of cytotoxic responses mediated by antiviral CTLs^29,30,31, antitumor CTLs^16,32, natural killer (NK) cells³³, glycolipid-reactive invariant natural killer T (iNKT) cells³⁴, and preexisting and de novo donor-specific alloantibodies²⁶. Therefore, their applications can be of interest to a wide readership, including but not limited to investigators working in the areas of tumor immunology and immunotherapy, anti-pathogen immunity, and preventative and therapeutic vaccine design.  

To assess cell-mediated cytotoxicity in typical scenarios, two populations of naïve splenocytes that display either an irrelevant Ag or a cognate Ag(s) are labeled with two different doses of CFSE, mixed in equal numbers and injected into naïve (control) or killer cell-harboring mice. The presence/absence of each target population is then examined by flow cytometry.                                             

We have optimized and employed in vivo killing assays in our studies on immunodominance in both antiviral and antitumor T_CD8 responses^12,16,17. Here, we provide a detailed protocol for the simultaneous assessment of ID and SD T_CD8 responses to T Ag epitopes, which can be readily adopted for similar investigations in other experimental systems. We also provide representative results demonstrating that nTreg cell depletion and PD-1 blockade can selectively enhance ID T_CD8– and SD T_CD8-induced cytotoxicity, respectively. At the end, we will discuss multiple advantages of in vivo killing assays as well as some of their inherent limitations.

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 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…

Representative 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 TCD8. 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

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