Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells

Abstract

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.

Fig. 1

Culture and phenotypical analysis of CD40-activated B cells. (a) Absolute amount of CD19⁺ and CD3⁺ T cells as measured in the CD40-B cell cultures of donors A–E. All cultures were started from 72 × 10⁶ (donors A–D) or from 36 × 10⁶ (donor E) density gradient-isolated PBMC. (b) Phenotypical analysis of CD40-B cells compared to naive B cells as found in a total PBMC population using flow cytometric analysis. All dot plots and histograms shown are based on the viable CD45⁺CD19⁺ cells present. Thin lines on histograms represent naive peripheral B cells, bold lines represent CD40-B cells. Results shown are representative of three individual experiments.

Fig. 2

Peptide-pulsed CD40-activated B cells can act as alternative antigen-presenting cells. (a) IFN-γ production of PBMC primed with CMV pp65 mRNA-electroporated mature DC after restimulation with unloaded or with CMV pp65 peptide-pulsed T2 or autologous CD40-B cells. (b) IFN-γ production of PBMC primed with influenza M1 peptide-pulsed CD40-B cells after restimulation with unloaded or with influenza M1 peptide-pulsed T2 or autologous CD40-B cells. (c) IFN-γ production of PBMC primed with influenza HA peptide-pulsed CD40-B cells after restimulation with unloaded or with influenza HA peptide-pulsed autologous CD40-B cells. (d) IFN-γ production of PBMC primed with influenza HA peptide-pulsed CD40-B cells after restimulation with unloaded or with influenza HA peptide-pulsed allogeneic EBV-LCL. Graphs represent results from IFN-γ ELISA assays (error bars indicate standard deviation) and are representative of two individual experiments.

Fig. 3

CD40-activated B cells can be mRNA-electroporated with high efficiency and viability. (a) Viability of non-electroporated and EGFP mRNA-electroporated CD40-B cells as determined by ethidium bromide (EtBr) staining (dot plots). Flow cytometric analysis of EGFP expression in CD40-activated B cells; thin lines represent mock-electroporated CD40-B cells, bold lines represent EGFP mRNA-electroporated CD40-B cells (histogram). (b) Viability of not electroporated and EGFP mRNA-electroporated mature DC as determined by ethidium bromide staining (dot plots). Flow cytometric analysis of EGFP expression in mature DC; thin lines represent mock-electroporated mature DC, bold lines represent EGFP mRNA-electroporated mature DC (histogram). Data are representative of five individual experiments.

Fig. 4

CD40-activated B cells transfected with mRNA coding for viral antigens can induce viral-specific T cell responses. (Top row) IFN-γ release of PBMC from donors A–C primed with CMV pp65 mRNA-electroporated autologous CD40-B cells after restimulation with CMV pp65 peptide-pulsed (T2/CMV) or with unloaded T2 cells (T2). (Bottom row) IFN-γ release of PBMC from donors C–E primed with influenza M1 matrix mRNA-electroporated autologous CD40-B cells after restimulation with influenza M1 peptide-pulsed (T2/M1) or with unloaded T2 cells (T2). Graphs represent results from the IFN-γ ELISA assays (error bars indicate standard deviation).

Fig. 5

IFN-γ secretion by antigen-specific CD8⁺ T cells after ex vivo induction of viral antigen-specific cellular immune responses by mRNA-electroporated autologous CD40-activated B cells. Antigen-specific cellular immune responses against influenza M1 and/or CMV pp65 after restimulation of primed PBMC with T2 cells (left dot plots) or with T2 cells pulsed with the relevant peptide (right dot plots). Donors A, B and F were typed CMV seropositive. The figures represent the percentage viable IFN-γ positive CD3⁺CD8⁺ T cells.