Appnote on Cell Analysis of T Cells

Abstract

T cells are an essential part of the immune response, capable of recognizing and eliminating foreign pathogens and tumor cells. However, tight control of the T cell response is necessary to prevent autoimmunity and immunopathology. A 16-color T cell immunophenotyping panel was developed for the Agilent NovoCyte Advanteon to simultaneously analyze T cell differentiation, activation, exhaustion, and functional output through degranulation and cytokine expression. This flow cytometry panel provides comprehensive analysis of T cell function in an immune response.

Introduction

The role of the immune system is to fight off pathogens while limiting autoimmunity. T cells are an important part of the adaptive immune response, capable of recognizing specific antigens and resolving infections. In a normal immune response, T cells are activated to rapidly divide and destroy all infected or cancerous cells. Activation through the T cell receptor (TCR) and costimulatory receptors must be balanced with inhibitory receptors such as programmed cell death protein 1 (PD-1), lymphocyte-activation gene 3 (LAG-3), and T cell immunoglobulin and mucin-domain containing-3 (TIM-3).This is needed to prevent the development of autoimmunity and immunopathology.

When stimulation of T cells is sustained, there is a progressive increase in the expression of inhibitory receptors and a decrease in T cell effector function and proliferative capacity (T cell

exhaustion). This can be seen during a chronic infection or cancer. Inhibitory receptors have become a focus of cancer immunotherapy research because reversing inhibitory receptor

expression through checkpoint inhibitor blockade has restored T cell function and improved tumor immunity in the clinic. The severity of T cell exhaustion correlates heavily with the co-expression of multiple inhibitory receptors, making combination therapy promising because

multiple inhibitory receptors are targeted, demonstrating an increase in tumor immunity. Analysis of T cell inhibitory receptor expression alone does not provide a comprehensive view of T cell

state. Measuring additional markers for T cell differentiation, activation, previous

antigen exposure, degranulation, and cytokine production provides a more comprehensive understanding.

Studies have identified inherent differences in inhibitory receptor expression among naive, effector, and memory T cell subsets. A 16-color immunophenotyping panel was designed to run on the NovoCyte Advanteon (Table 1) to examine CD4 and CD8 T cell differentiation and activation state, and measure functional T cell responses. Using the NovoCyte Advanteon’s high capacity for multiparameter analysis, these data demonstrate a comprehensive look at the kinetic changes that occur in T cell subsets and function after stimulation.

In depth analysis of T cell state after TCR stimulation

To fully examine the T cell response to TCR activation, we looked at differentiation, activation, and inhibitor markers as well as degranulation and cytokine production in CD4 and CD8 T cells (Table 1). Isolated peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3, anti-CD28 antibodies, and interleukin-2 (IL-2) to

induce T cell activation. Six hours before flow cytometric analysis, cytokine secretion was blocked with a protein transport inhibitor. Samples were measured initially after two and  four days in culture, and again on day 5 after being restimulated. Figure 1 demonstrates a representative gating strategy for the day 4 sample. First, dead cells were excluded using

viability dye followed by examining the frequency of CD4 and CD8 T cells within the total CD3 T cell population. Next, T cell differentiation was assessed by determining the relative frequency of T cell subsets based on surface expression of CD45RA and CCR7. T cells

are separated into Naive‑like T naive-like (CD45RA+/CCR7+), central memory T (CD45RA-/CCR7+), effector memory CM EM (CD45RA-/CCR7-), and CD45RA+

effector memory T EMRA (CD45RA+/ CCR7–). Furthermore, T

naive-like are separated into true (T memory T cells (T naive) and T stem‑cell SCM) by the expression of CD95 on T SCM.

Next, the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 was examined as well as activation markers CD25 and CD127. These markers provide additional information on the activation state of the T cells. Finally, production of cytokines IL-2, tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) were measured as well as degranulation by expression of CD107α, which demarcates cytolytic T cells. All gates were determined with the use of fluorescence minus one (FMO) controls (Figure 1D). Four days after stimulation, most cells had undergone T cell activation, and there were no longer any T populations. A large upregulation in CD25 expression and downregulation of CD127 was seen in T cells, also consistent with highly activated T cells. Both CD4 and CD8 T cells had upregulated inhibitory receptor expression, although not all cells had positive expression. naive cells in both the CD4 and CD8 T cell Cytokine production of TNFα and IFNγ was also present while only minimal IL-2 production was observed. Most T cells were also CD107α positive, showing that they had cytolytic potential. This high-parameter staining panel with the NovoCyte Advanteon allows extensive analysis of T cells after stimulation. Next, kinetic changes in the parameters measured were examined in T cells after TCR activation.