staining and flow cytometry

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1 Detection of influenza virus-specific T cell responses by intracellular by cytokine intracellular staining cytokine staining and flow cytometry Detection of influenza virus-specific T cell responses and flow cytometry Key features: antigen-specific stimulation by infectious influenza virus or viral peptides; response frequencies per PBMC subset (e.g. CD4 T cells and/or CD8 T cells) WHITEpaper

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3 Detection of influenza virus-specific T cell responses by intracellular cytokine staining and flow cytometry Key features: antigen-specific stimulation by infectious influenza virus or viral peptides; response frequencies per PBMC subset (e.g. CD4 T cells and/or CD8 T cells) This paper describes the validation of the assay that is implemented at Viroclinics Biosciences for routine determinations of influenza A virus-specific T cell frequencies among CD4 and CD8 T cell subsets in human peripheral blood mononuclear cell (PBMC) samples. Application Detection of influenza virus-reactive T cell response levels during the course of vaccine trials or infection. PBMC obtained from multiple visits can be cryopreserved for parallel analysis. Method outline Cryopreserved PBMC samples are thawed, rested and stimulated by adding infectious influenza A virus or sets of overlapping peptides spanning viral proteins (e.g. nucleoprotein (NP) or membrane protein 1 (M1) (Figure 1). During the final 6 hours of incubation, a secretion inhibitor is added to facilitate intracellular accumulation of cellular proteins, e.g. interferon-gamma and IL-2, in activated cells. After staining of the cells with a viability marker and fluorochromeconjugated antibodies directed against CD3, CD4, CD8, IFNg, IL2 and/or CD69, the frequency of activated CD4 and CD8 T cells responding to the antigen stimulation by expressing IFNg and/or IL-2 is quantified by flow cytometry. Positive controls (staphylococcal enterotoxine B; SEB) and negative controls are included for each sample, and reference samples are included in each assay run to monitor between-run variability. Through-put Up to 24 samples and three viral antigen preparations per assay run; one assay run per week. Validation Validation of methods is performed to get insight in the possibilities and restrictions of a method and to demonstrate that it is suitable for its intended purpose. Here we describe the validation parameters specificity, linearity, range, repeatability, intermediate precision, detection limit, quantification limit and robustness for Viroclinics Biosciences CMI assay for detection of influenza virus-reactive T cells. Specificity PBMC obtained from human adults (n=4) showed positive results (i.e., response levels >0.05%) for CD4 and CD8 T cells in cultures stimulated by infection with a sucrose gradient purified infectious influenza A virus strain (Fig xi), or after adding sets of overlapping peptides spanning the virus NP and M1 protein sequence (Fig xii), or SEB as a positive control (Fig xiii). No responses were detected in medium control cultures or after adding allantoic fluid from uninfected eggs or sucrose. The latter two conditions were included to assess potential T cell reactivity to non-influenza virus components in the virus stock. Linearity SEB-stimulated PBMC were diluted in triplicate in unstimulated PBMC from the same donor using eight 3-fold dilutions. The linearity of the response curve (R2) was 0.8 or higher, the results of the relative standard deviation (percentage coefficient of variation; %CV) was < 35%. Day 1: Day 2: - Thaw PBMC - Add peptides/seb/ - Rest >3 hrs secretion inhibitor - 1x10^6 cells/0.1ml - Incubate 6 hr - Add virus at MOI~3 - Add surface stains and viability marker - Fix cells Day 3: - Permeabilize cells - Add intracellular stains - Analyse by flow cytometry Figure 1. Work-flow of the procedure for the detection of Influenza A virus-specific T cells. Figure adapted from reference 2. IFN-γ Range In the linearity experiment, SEB-response levels ranged from 0.01% 11.7% and % for the CD4 and CD8 T cell subsets, respectively. Because linearity was not lost at the high end of the range, these values most likely underestimate the actual range of the assay. The range may prove to be larger in future assays if PBMC populations with higher response levels are found. The range detected in the present experiments is adequate for detection of influenza A virus-specific T cell frequencies among healthy adults, as such frequencies have been reported to range between 0.05% and 10%.

4 Detection of influenza virus-specific T cell responses by intracellular cytokine staining and flow cytometry PBMC Subset %SEB-stimulated PBMC mean %IFNγ +(1) SD %CV (2) % IFNγ+ T cells CD3CD4 CD3CD8 R 2 = R 2 = CD3CD %SEB-stimulated PBMC Figure 2. Dilution of SEB-stimulated PBMC in un-stimulated PBMC from the same donor. CD69IFNγ+ T cells (means of triplicates ± 1 SD) are shown for CD3CD4 (open diamonds) and CD3CD8 (closed diamonds) PBMC subsets. Values are shown in Table 2. CD3CD (1) Mean of triplicates (medium control values subtracted) (2) Values > 35% are shown in boldface italics. Table 2 Repeatability PBMC obtained from healthy adult (n=4) were tested three times on a single day by one technician. All results that were above 0.05% responding T cells showed %CV <35% around the mean of three tests (Table 4). This was observed for PBMC obtained from all of the 4 tested donors and all of the tested antigens: RESVIR-9, NP-peptides, M1-peptides, and the control antigen SEB. %CD69 + IFN- γ+ among viable CD3 + CD4 + events %CD69 + IFN- γ+ among viable CD3 + CD8 + events Antigen Donor Well 1 (1) Well 2 Well 3 mean (2) SD %CV Well 1 Well 2 Well 3 mean SD %CV RESVIR-9 (1) average %CV range %CV ( ) ( ) NP average %CV range %CV ( ) ( ) M NA NA average %CV range %CV ( ) ( ) SEB average %CV range %CV ( ) ( ) (1) Each value represents the result of individual wells with medium control values subtracted. (2) Values > 0.08% are shown in boldface italics. Table 4. Repeatability of the VC Flu-ICS assay evaluated for CD69IFN-γ responses among CD4+ T cells and CD8+ T cells to influenza virus infection, CEF or SEB stimulation.

5 Detection of influenza virus-specific T cell responses by intracellular cytokine staining and flow cytometry Intermediate precision PBMC obtained from healthy adults (n=4) were tested on three different days. Table 5 shows that %CV <35% for all results above 0.05% of CD4 and CD8 T cells. The acceptance criteria were also met for the NP-peptide stimulated PBMC in 3 out of the 4 tested individuals. Only the NP-peptide-specific CD8 T cell response in donor 2 PBMC showed a %CV of 36.5% for a mean result of 0.89%, which was demonstrated by performing the assay using PBMC with different response levels on three different days, with %CV below 35%. %CD69 + IFN- γ+ among viable CD3 + CD4 + events %CD69 + IFN- γ+ among viable CD3 + CD8 + events Antigen Donor Exp1 (1) Exp 2 Exp3 mean (2) SD %CV Exp 1 Exp 2 Exp3 mean SD %CV RESVIR average %CV range %CV ( ) ( ) NP average %CV range %CV ( ) ( ) M NA average %CV range %CV ( ) ( ) SEB average %CV range %CV ( ) ( ) (1) Values are means of triplicates with medium control values subtracted, except for Exp 2 NP and M1 values, which are based on duplicates. (2) Values > 0.08% are shown in boldface italics. Table 5. Intermediate precision of the VC Flu-ICS assay evaluated for CD69IFN-γ responses among CD4+ T cells and CD8+ T cells to influenza virus infection or to stimulation with NP-peptides, M1-peptides or SEB. Detection limit We determined the frequency of activated CD4 and CD8 T cells that produce IFN-γ in replicate negative control samples obtained from the healthy adults (n=4). The lower limit of detection, defined as 2 SD above the mean of replicate negative control samples (4), was always below 0,05%. Quantification limit The lower limit of quantification was defined as the lowest cytokine response for which the %CV remained below 35%. As shown in table 2, this was the case for response levels as low as 0.01% for CD4 T cells and 0.06% for CD8 T cells. Robustness Different sources and lots of serum for cryopreservation of PBMC were compared for their influence on the final results. Frequencies of CD4 T cells and CD8 T cells responding to Influenza A virus or a pool of HLA class-i restricted epitopes (CEF), including influenza A virus NP and M1 epitopes, were similar for the PBMC samples that had been cryopreserved in different serum batches (Figure y). This was observed for PBMC obtained from four different human adults, who showed different response levels to the tested antigens. Moreover, the different serum batches did not results in different levels of background responses. In conclusion, the variation in serum batches tested here did not impact the results.

6 Detection of influenza virus-specific T cell responses by intracellular cytokine staining and flow cytometry VC-ICS CD3 + CD4 + %CD69 + IFNγ CD3 + CD8 + FCS lot# CSJ0417 Donor 1 Donor 2 Donor 3 Donor 4 median Figure y. T cell response levels in PBMC obtained from healthy adults (n=4) that had been cryopreserved in different serum batches (FCS-10% DMSO) and subsequently shipped to Viroclinics Biosciences on dry ice. Open and closed symbols are duplicate measurements 0.01 med Flu CEF med Flu CEF 10 FCS lot# 47K3395 %CD69 + IFNγ Donor 1 Donor 2 Donor 3 Donor 4 median 0.01 med Flu CEF med Flu CEF References 1. Maino VC, Picker LJ. Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry 1998;34(5): BD-Biosciences. BD FastImmune CFC Handbook. Performance Characteristics of Antigen-Specific Cytokine Flow Cytometry (CFC) Assays. San Jose, CA, USA; He XS, Holmes TH, Zhang C, Mahmood K, Kemble GW, Lewis DB, Dekker CL, Greenberg HB, Arvin AM. Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines. J Virol 2006;80(23): Maecker HT, Hassler J, Payne JK, Summers A, Comatas K, Ghanayem M, Morse MA, Clay TM, Lyerly HK, Bhatia S and others. Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 2008;9:9. 5. Horton H, Thomas EP, Stucky JA, Frank I, Moodie Z, Huang Y, Chiu Y-L, McElrath MJ, De Rosa SC. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. Journal of Immunological Methods 2007;323(1):

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8 Detection of influenza virus-specific T cell responses by intracellular cytokine staining and flow cytometry Viroclinics Biosciences Rotterdam Science Tower info@viroclinics.com Tel Marconistraat AK Rotterdam The Netherlands WHITEpaper