In Vitro Analyses of T Cell Effector Differentiation

In vitro culture is an important complement, or substitute, to in vivo approaches in order to study T cell effector differentiation. Here, we describe culture conditions that generate specific effector cell types by exposing naïve T cells to appropriate cytokine signals.

1. Introduction

Effective T cell function is required for protection from invading pathogens. T cell effector differentiation is determined by several signals, notably from the innate immune cells, including (1) stimulation through the T cell receptor (TCR), (2) co-stimulation through the CD28 costimulatory molecule and (3) cytokine exposure that induces acquisition of specific T cell differentiation. Studies exploring how naïve T cells differentiate into fully functional effector cells often require assessing the effector potential of these T cells; protocols for such analyses are presented in this chapter.

During in vivo immune responses, effector T cell differentiation occurs in secondary lymphoid organs or tissues, and is driven by architecturally constrained interactions between T cells, antigen-presenting cells (APC)s and other immune cells. Early stages of an immune response involve small cell numbers of antigen-specific T cells, which may be difficult to identify and purify. Thus, it can be advantageous to reproduce the conditions that promote T cell effector differentiation in vitro. In vitro differentiation allows acquisition of a large number of cells, e.g., for biochemical or gene expression studies. In addition, in vitro studies allow for tighter control of cytokines and other stimuli offered to T cells. These in vitro cultures typically include two distinct stimuli. First, a ligand for the T cell antigen receptor complex is required to promote expression of specific cytokine receptors (e.g., for IL-2) and cell proliferation. This can be a specific peptide antigen if using cells of defined specificity (e.g., carrying a TCR transgene); in many instances however, antibodies against TCR or CD3 are used to mimic antigen stimuli and trigger TCR signaling. In both cases, TCR stimulation needs to be accompanied by engagement of CD28 costimulatory molecules. The second series of ligands is intended to direct cytokine gene expression; it includes cytokines and anti-cytokine antibodies to neutralize the effect of unwanted cytokines. Appropriate combinations of these reagents typically “polarize” T cell differentiation into specific effector fates: for CD4 cells, these generally include Th1 [1], Th2 [1 – 3], Th17, and inducible (i) T regulatory (Treg); and for CD8 + cytotoxic T (Tc) cells (see Table 1 and refs. 2, 4, 5). While it is possible to activate highly purified T cells with the latter reagent combinations, using separately purified APCs as “feeder” cells for the differentiating effector T cells results in greater survival for most effector types [6].

Table 1

Effector differentiation in vitro

During in vitro culture, antigenic stimulation (or its surrogate) and cytokines drive cell proliferation, which typically starts within 24–36 h after stimulation and continues for 3 or 4 days. Expression of cytokine genes, and cytokine production (evaluated by ELISA or intracellular cytokine staining) is detected within 3–5 days of stimulation, depending on the type of cytokine. Similar kinetics are observed for fate-determining transcription factors.

Although the choice of the starting T cell population is typically dictated by the specific application, special emphasis must be placed on separating naïve from antigen-experienced (generally referred to as “memory”) cells obtained from peripheral lymphoid organs. In laboratory mice housed under specific pathogen-free conditions, most spleen and lymph node T cells are “naïve.” That is, they are directly derived from thymic precursors without having encountered the antigen that their TCR specifically reacts against and therefore, they do not express effector (e.g., cytokine) genes. A simple conceptual example of a naive cell is a lymphocyte carrying a TCR directed against a virus-derived peptide in a host that has not been in contact with that particular virus. In contrast, upon infection or immunization with the cognate antigen, these newly “antigen-experienced” cells proliferate, and acquire effector properties or differentiate into memory cells. In unmanipulated laboratory mice, cells exhibiting marks of antigen experience are typically reactive against commensal and environmental antigens.

Regardless of whether they are effector or memory, antigen-experienced cells have two properties not shared by naïve cells. They are generally “preprogrammed” to produce specific cytokines (most antigen-experienced cells in a mouse spleen make IFNγ), and they produce these cytokines quickly, within hours of antigen receptor triggering. In contrast, naïve cells typically do not produce effector cytokines until they have undergone multiple rounds of proliferation, and their effector differentiation is heavily influenced by the surrounding cytokine milieu ( Table 2 ). Thus, in activation cultures, the cytokines produced by antigen-experienced cells have the potential to skew, often towards IFNγ production, the effector differentiation of the naïve cells. To avoid this potential bias, it is generally advisable to purify naïve cells for in vitro stimulation cultures. This is all the more necessary when such cells are prepared from inflammatory contexts, in which the frequency of effector or memory cells is much higher. For both CD4 + and CD8 + mouse T cells, CD44 is the most commonly used marker to distinguish naïve (CD44 lo ) from antigen experienced (CD44 hi ) subsets.

Table 2

Effector differentiation in vivo

Here we delineate protocols designed to evaluate the effector potential of naïve T cells exposed to conditions that partially mimic in vivo antigen stimulation. These protocols are designed to differentiate naïve T cells into CD4 + iTregs and CD4 + and CD8 + T effector cells. Because of the versatility of in vitro cultures, the choice of appropriate controls is crucial to establish sound conclusions. We have provided suggestions in Subheading 4 in this regard.

The protocols described below use antibody (anti-CD3)-mediated TCR triggering as a surrogate for antigen stimulation, APCs (either dendritic cells or T cell-depleted splenocytes), and mixes of cytokines and anti-cytokine antibodies appropriate for promoting the differentiation of Th1, Th2, or Th17 CD4 + effectors, Tc CD8 + effectors, or CD4 + iTreg cells. We also provide an alternate protocol that eliminates the APCs.