This panel was developed, optimized, and validated for assessment of CD4+ and CD8+ T-cell responses to various peptide pools for antigens of interest in cryopreserved peripheral blood mononuclear cells (PBMC) from adult humans (Table 1). The panel has been used to evaluate HIV- and TB-specific responses to candidate vaccines for these pathogens, although the panel can be used with peptide pools for any proteins. The panel has not been tested with freshly-isolated PBMC or with whole blood.
The focus of this panel is functional T-cell characterization and therefore only a minimum number of phenotyping markers are included to identify the CD4+ and CD8+ T cell lineages (CD3, CD4, CD8). This allows for a large number of functional markers, which include functions common to both CD4+ and CD8+ T cells and also functions more likely to be associated with a single T-cell lineage (Table 2; see supporting information for further details on our panel development strategy). IFN-γ, IL-2, and TNF-α are considered key cytokines for both lineages and are commonly examined in intracellular cytokine staining (ICS) assays (1); thus, these were given high priority. MIP-1β is a chemokine that can be produced by both CD4+ and CD8+ T cells and has been shown to be useful in identifying polyfunctional T cells (2). It has relatively higher background when examined individually in terms of cells staining in the unstimulated control condition, and thus we mainly examine it in the context of coexpression with other functional markers. A fifth function commonly included when examining polyfunctionality is CD107a (3), a marker of degranulation, which may be a surrogate for cytotoxic potential. Although traditionally considered a function of cytolytic CD8+ T cells, some CD4+ T cells can degranulate.
We included two additional markers of interest for CD4+ T cells: IL-4 (a representative Th2 cytokine) and CD40 ligand (CD40L, also known as CD154). For IL-4, a bright fluorochrome was chosen since IL-4-producing cells are difficult to detect due to the low intensity of staining. Cells expressing CD40L interact with B cells expressing CD40; CD40L thus likely identifies T cells (mainly CD4+ T cells) that can provide help to B cells (4). CD40L is expressed on the surface of cells and can be detected either by surface staining in a co-culture assay where the fluorochrome-conjugated antibody is included during the ex vivo antigen stimulation (4), or through intracellular staining. We used the latter method since the CD40L coculture assay is incompatible with use of Brefeldin A, which is essential for the most sensitive detection of some cytokines, in particular TNF-α. In situations where sensitivity is not as critical, monensin alone can be used to allow for coculture surface staining of CD40L. Note that both Brefeldin A and monensin were used in our assay, since monensin was required for the CD107a coculture assay.
Finally, a viability marker is considered essential for any assay identifying cells present at low frequency, and CD14 was included to exclude monocytes to improve the specificity of the assay. This could have been included in the same channel as the viability marker, but we chose to use another unused channel. Figure 1 shows an example staining profile for PBMC stimulated with Staphylococcal enterotoxin B.
Similarity to Published OMIPs
OMIP-001 (5), OMIP-008 (6) and OMIP-009 (7), since these OMIPs examine antigen-specific human T cells by ICS. Our OMIP is focused on multiple functions rather than a combination of functions and memory markers (OMIP-001, OMIP-009) and includes a larger variety of functions compared with all these OMIPs. Additionally, our panel was developed for use in a good clinical laboratory practices (GCLP) setting and has been validated.1, 2
Table 1. Summary table for application of OMIP-014
T-cell cytokine production and function after in vitro stimulation
OMIP-001, OMIP-008, OMIP-009
Table 2. Reagents used for OMIP-014 (see Online Table 2 for additional details)
The authors thank the James B. Pendleton Charitable Trust for their generous equipment donation. The authors thank the laboratory technicians who performed the assays and Stephen Voght for help with editing.