Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA

Abstract

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.

Fig. 1

Efficient RNA transfer of IVT RNA into antigen presenting cells. a PBMCs obtained by Ficoll density centrifugation, whole blood leukocytes generated by erythrocyte lysis, and purified monocytes were electroporated with 20 μg eGFP RNA. Cells were cultured for 7 h and stained with anti CD14 antibody. Transfection efficiency was measured by flow cytometry. The numbers given refer to the transfection efficacy of the CD14⁺ population. This experiment is representative for six independent experiments. b PBMCs generated via Ficoll density centrifugation were electroporated with 20 μg eGFP RNA. The eGFP expression in the eGFP⁺ and CD14⁺ monocyte population was followed (black histogram) from 30 min to 24 h. PBMCs electroporated with control RNA were used as negative control (white histogram)

Fig. 2

Inability of pp65 RNA to stimulate antigen-specific CD4⁺ lymphocytes in ex-vivo assays. PBMCs from healthy HCMV⁺ donors were tested in CFC for CD8⁺ (a) and CD4⁺ (b) reactivity against pp65 peptide pool (1.75 μg/ml; black bars), and pp65 RNA (10 μg each; gray bars). Each bar represents the percentage of IFN(secreting CD8⁺ or CD4⁺ lymphocytes after subtraction of background IFN(secretion against irrelevant control RNA encoding NY-ESO-I. CD8⁺ (a) and CD4⁺ (b) lymphocytes (1 × 10⁵/well) from a healthy HCMV⁺ donor were stimulated in overnight IFNγ-ELISPOT with purified autologous monocytes (4 × 10⁴/well) either transfected with 20 μg IVT mRNA or loaded with the HCMV pp65 peptide pool. Each bar represents the mean spot number of triplicates + SD. This result is representative for two independent experiments

Fig. 3

Simultaneous stimulation of CD4⁺ and CD8⁺ T-cells using IVT RNA of MHC fusion constructs. a Schematic diagram of the plasmid templates for in vitro transcription of RNA. b CD8⁺ and CD4⁺ T lymphocytes (1 × 10⁵/well) from a healthy HCMV⁺ donor were stimulated in overnight IFNγ-ELISPOT with purified autologous monocytes (4 × 10⁴/well) transfected with 20 μg IVT RNA. Each bar represents the mean spot number of triplicates + SD. This result is representative for three independent experiments. c The frequency of antigen-specific IFNγ secreting CD4⁺ and CD8⁺ T lymphocytes was measured in six healthy HCMV sero-positive (filled circles) and three HCMV sero-negative (open circles) donors by CFC. PBMCs were either loaded with pp65 peptide pool (1.75 μg/ml) or transfected with 10 μg pp65 or pp65-MHCI RNA. The background IFNγ reactivity against NY-ESO-I-MHCI RNA as irrelevant control was subtracted. Data points obtained with the PBMCs of the same donor are connected by a dotted line. The bar represents the mean frequency of antigen-specific T-cells. d Dot plots of a CFC from a representative donor are shown. The numbers represent the percentage of IFNγ secreting CD8⁺ or CD4⁺ T lymphocytes. e Frequencies of CD8⁺ IFNγ secreting lymphocytes were compared using either bulk populations obtained by Ficoll gradient purification or cells after erythrocyte lysis of whole blood. Cells were tested after electroporation with 10 μg of IVT RNA or loading with pp65 peptide pool (1.75 μg/ml). This result is representative for two independent experiments. f The frequency of CD8⁺ lymphocytes secreting IFNγ in PBMCs obtained from a healthy HCMV⁺ donor was measured in three independent runs

Fig. 4

Simultaneous detection of multiple T-cell epitopes using HCMV pp65-MHCI RNA. a PBMCs from a healthy HCMV sero-positive HLA-A*0201⁺ donor were tested in CFC for CD8⁺ reactivity against the control peptide, pp65 peptide 495–503, NY-ESO-I-MHCI RNA as an irrelevant control, and pp65-MHCI RNA (10 μg each). Numbers represent the percentage of IFNγ secreting cells. Data are representative for three independent experiments. b CD8⁺ and CD4⁺ lymphocytes of a HCMV⁺ healthy donor were co-cultivated with autologous DCs loaded with four different sub pools of overlapping pp65 peptides (each peptide 1.75 μg/ml; see schematic drawing). c After expansion for 7 days each effector cell population (4 × 10⁴/well) was tested in IFN(ELISPOT on autologous DCs (3 × 10⁴/well) either loaded with the whole pp65 peptide pool, a pool of irrelevant peptides spanning the WT1 protein sequence (1.75 μg/ml), or transfected with pp65-MHCI RNA (20 μg). Each bar represents the mean spot number of duplicates. Due to the lack of adequate discrimination spot numbers were only quantified to a maximum of 800 per well