Evaluating frequency and quality of pathogen-specific T cells

Abstract

It is generally accepted that enumeration and characterization of antigen-specific T cells provide essential information about potency of the immune response. Here, we report a new technique to determine the frequency and potency of antigen-specific CD8 T cells. The assay measures changes of intracellular Ca²⁺ in real time by fluorescent microscopy in individual CD8 T cells responding to cognate peptides. The T cells form continuous monolayer, enabling the cells to present the peptides to each other. This approach allows us to evaluate the kinetics of intracellular Ca²⁺ signalling that characterizes the quality of T cell response. We demonstrate the usefulness of the assay examining the frequency and quality of cytomegalovirus-specific CD8 T cells from healthy donor and patient after haploidentical stem cell transplantation. The new assay has a potential to provide essential information determining the status of the immune system, disease morbidity, potency of therapeutic intervention and vaccine efficacy.

Figure 1. Schematic presentation of CaFlux assay.

T cells form monolayers on the glass bottom of the well covered with antibodies against T-cell's surface receptors. A peptide antigen is injected into the well. The peptide molecules bind to MHC proteins expressed on the T-cell surface to form recognizable pMHC ligands. Each T cells can serve as a target cells for neighbouring T cells, but only rare antigen-specific T cell is expected to recognize and respond to defined antigenic peptide used in the assay. Recognition events induce rise in the level of intracellular Ca²⁺. To detect Ca²⁺ influx, the T cells are labelled with Ca²⁺ sensitive fluorophore. The changes in fluorescent intensity in individual cells before and after peptide addition are measured with fluorescent microscopy to identify the frequency of responding T cells.

Figure 2. Detection of responding T cells in the monolayer.

Cloned CD8⁺ T cells CER43 labelled with Fluo-4 were attached to a glass bottom of 96-well plates covered with poly-L-lysine via non-blocking anti-LFA-1 antibodies to form a monolayer ensuring direct contact of T cells with each other (a,b). Addition of a strong agonist peptide GILGFVFTL (GL9) to the monolayer of the labelled T cells induced intracellular Ca²⁺ flux resulting in the time-dependent increase of intracellular fluorescence intensity that reaches maximum in 2–3 min (c,d). Addition of non-stimulatory peptide ILKEPVHGV (IV9) did not lead to the increased of intracellular fluorescence (d). Data are representative of nine independent experiments. Images (b) and (c) are taken at time 0 and 220 s after the stimulation, respectively. The results shown on d are based on the analysis of 4,500 T cells. Scale bars, 50 μm.

Figure 3. Strength of stimulation influences the kinetics of Ca²⁺ signalling in CTL.

(a) Cytotoxic activity by human 68A62 cell line against HLA-A2⁺ JY target cells is shown. Differences in concentration of strong ILKEPVHGV (IV9) and weak (IV9-A7 and IV9-A4) agonist peptides that are required to achieve the same extent of specific target cells lysis by 68A62 cells are marked by arrows. (b) Difference in the kinetics of calcium accumulation in responding 68A62 CTL stimulated with strong (IV9) and weak (IV9-A7 and IV9-A4) agonist peptides at a high peptide concentration (10⁻⁴ M) is shown. The data are representative from two to five independent experiments. The results shown on b are based on the analysis of 2,000 T cells.

Figure 4. Breadth of tested peptides and the sensitivity of CaFlux assay.

(a) The dynamics and magnitude of calcium response by CD8⁺ T cells CER43 stimulated by strong agonist peptide GILGFVFTL (GL9) at various concentrations. (b) The dependence of the sensitivity of calcium response in CD8 T cells CER43 induced by GL9 peptide ligand diluted in a mixture of non-stimulatory peptides (see ‘Methods' section for details) at total concentration 10⁻⁶ M and indicated ratios. The data are representative of 2 independent experiments and are based on the analysis of ∼5,000 cells.

Figure 5. Kinetics of calcium response of CMV-specific CD8+ T cells.

CMV-specific CD8+ T cells from a healthy donor (a) and a patient after haploidentical stem cell transplantation (b) revealed different kinetics of Ca²⁺ signalling on antigen stimulation. CD8+ T cells were purified from PBMC by negative magnetic sorting and purified CD8 T cells were labelled with Fluo-4 and immobilized on a glass surface as described in ‘Methods' section. The T cells from healthy donor were stimulated by CMV-derived peptide NLVPMATV at 10⁻⁴ M while the T cells from the patient were triggered with ProMix CMV peptide pool at 9 × 10⁻⁵ M. The images of responding cells are taken at 720 s after the stimulation. The data shown are representative of 5 (a) and 2 (b) independent experiments and are based on the analysis of 4,600 and 4,200 T cells, correspondingly. Scale bars, 50 μm.