Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

Abstract

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158-66 and CMVpp65495-503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-gamma (IFN-gamma), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-gamma mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

Figure 1

The kinetics of IFN-γ, IL-2, IL-4 and IL-10 transcription following FluM1_58–66 stimulation of three HLA-A*0201 donors. The standard concentration of 2 × 10⁵cells/200μl was ex vivo exposed to the peptide. At hour 3, IFN-γ transcript production precedes IL-2, IL-4 and IL-10. At hour 12 there is consistent production of T_h1 (IFN-γ, IL-2) cytokines. IL-4 and IL-10 transcript production increases above the baseline levels from 24 to 48 hours. For each donor and at each time point mRNA copy numbers were calculated relative to unstimulated cells after normalization by β-actin. The result represents the mean ± SEM of averages of combined values of three independent experiments carried out on each of the three donors.

Figure 2

IFN-γ transcript production by memory T lymphocytes following ex vivo FluM1_58–66, CMVpp65_495–503, and Mage12_170–178 peptide stimulation. In the period of 3 to 12 hours, IFN-γ cytokine production was inversely proportional to the time of exposure during FluM1_58–66 stimulation (panel A), maintained an average level during CMVpp65_495–503 stimulation (panel B) or was not produced during Mage12_170–178 exposure (panel C). The results are expressed in mRNA copy numbers calculated relative to unstimulated cells after normalization by β-actin and represent the expressed values from each donor tested. Panel D specifically shows the kinetics of a representative donor (donor B) after the different viral peptide exposures relative to the negative value (unstimutated cells).

Figure 3

Effect of cell concentration on the specific kinetics of IFN-γ transcript production during CMVpp65_495–503 peptide stimulation. Different concentrations of PBMCs from two CMV seropositive donors and one CMV seronegative donor were ex vivo peptide stimulated and analyzed at hour 3, 12 and 24 of sensitization for IFN-γ transcript production. For all donors tested, panels A, B and C show IFN-γ transcript production for each time of exposure (hour 3, 12 and 24, respectively) relative to cell concentration. Inversely, panels D, E and F show IFN-γ transcription for each quantity of stimulated cells relative to the three times of exposure only for the two seropositive donors. All results represent the mean ± SEM of three independent experiments carried out in triplicate.

Figure 4

Assessment of the IFN-γ protein release kinetics by ex vivo peptide-stimulated PBMCs. Two donors, one CMV seropositive and one CMV seronegative, were exposed at various durations of time to both FluM1_58–66 and CMVpp65_495–503 peptides and cell supernatants were analyzed for IFN-γ protein release (panel A and B). Panel C shows the protein release from both FluM1_58–66 and CMVpp65_495–503 2-week sensitized PBMCs from the CMV seropositive donors either after an 18-hour peptide re-exposure or after no peptide re-exposure. Results represent the mean ± SEM of three independent experiments carried out in triplicate.

Figure 5

IFN-γ Intracellular Staining (ICS) after the standard six hours peptide exposure. All donors were exposed to both FluM1_58–66 and CMVpp65_495–503 peptides and then analyzed for intracellular IFN-γ protein release. Panel A: FACS analysis was conducted on gated CD3 positive lymphocytes after selecting only viable cells (R1 and R2) and plotting CD8 positive (x-axis) against either mouse IgG₁ isotype-stained (y-axis, upper row) or human anti-IFN-γ-stained (y-axis, lower row) lymphocytes. The figure shows one representative experiment of three carried out on each donor. Panel B: IFN-γ ICS results in 3 different donors. The calculation of the percentage of antigen-specific T cells was performed based on the CD8 and IFN-γ dot plot data according to the following formula: Percentage _{Ag-specific T-cells} = Percentage _{Upper right quadrant} /(Percentage _{Upper right quadrant} + Percentage _{Lower right quadrant})* 100%.