Multiparametric flow cytometry to characterize vaccine-induced polyfunctional T cell responses and T cell/NK cell exhaustion and memory phenotypes in mouse immuno-oncology models

Abstract

Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4+ and CD8+ T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models.

CBL0137 and NKG2A blockade: a novel immuno-oncology combination therapy for Myc-overexpressing triple-negative breast cancers

“…Local induction of ICD not only increases tumor cell immunogenicity, but also promotes the release of soluble mediators such as ATP, HMGB1 and other endogenous danger-associated molecular patterns (DAMPs) that trigger pro-in ammatory responses and promote recruitment and activation of immune cells [46]. To investigate the in vivo immunomodulatory effects of CBL0137 on the tumor microenvironment, we performed Opal multi-plex immunohistochemistry (IHC) and multi-parametric ow cytometry on tumors collected 10 days after the last CBL0137 treatment and characterized in ltration, activation and exhaustion pro les of T and NK cells in treated and untreated tumors [33]. Opal analysis revealed that CBL0137 reduced the relative number of CD8 + T cells and NK (NCR1 + ) cells per 1000 nuclei with no signi cant change in the proportion of total CD4 + T cells and Treg (CD4 + CD25 + FoxP3 + ) cells (Fig.…”

“…Cells were then pelleted and resuspended in ACK buffer to remove erythrocytes. Cells were then washed, resuspended in PBS and stained for surface and intracellular markers as previously described [33]. The list of the antibodies and reagents used for FACS pro ling are indicated in Table S4.…”