T-cell responses to multiple antigens presented by RNA-transfected APCs: a possible immunomonitoring tool

Abstract

The increasingly deeper understanding of how the immune system recognizes and destroys tumors promises to enable the development of new approaches for gene therapy and immunotherapy. However, a treatment that induces safe and potentially beneficial antitumor responses is expected to require stepwise refinements. As part of this challenge, assays are needed to measure specific antitumor immune responses in patients. This becomes problematic because most tumors express unknown tumor antigens and it is often difficult to obtain sufficient amounts of viable tumor material for in vitro assays. Recently it was demonstrated that RNA derived from tumor cells stimulated T cells in an antigen-specific manner. These studies have formed the basis for the development of dendritic cell vaccines that express tumor antigens following translation of tumor RNA. Therefore, it occurred to us that antigen-presenting cells transfected with total tumor RNA might also be valuable in monitoring the antitumor responses induced in patients who participate in clinical trials. To test this hypothesis, we developed a model in which Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines were used as a source of RNA. Since this RNA encodes for known EBV antigens, it was possible to determine whether the expected responses were observed. Our results show for the first time that T cells primed to APC transfected with RNA isolated from EBV-infected lymphocytes exhibited a fine specificity that enabled them to recognize individual EBV antigens.

Fig. 1.

IFN-γ production by T cells primed with autologous APCs transfected with allogeneic total RNA. PBMCs (A) were either cocultured with media (NONE), irradiated (x), autologous PBMCs (Ax), autologous PBMCs transfected with allogeneic RNA from LCL-B (A:Ax ^RNA-B ), or with the allogeneic LCLs (LCL-Bx) for 10 days. These responder cells were then restimulated with either media alone, autologous PBMCs (Ax) or LCL (LCL-Bx) cells for 20 h in an ELISpot assay. The data is representative of several experiments using six donors

Fig. 2.

IFN-γ production by T cells primed with autologous APCs transfected with autologous total RNA. PBMCs (A) were either cocultured with media (NONE), irradiated (x), autologous PBMCs (Ax), autologous PBMCs transfected with autologous RNA from LCL-A (A:Ax ^RNA-A ), or with the autologous LCLs (LCL-Ax) for 10 days. These responder cells were then restimulated with either media alone, autologous PBMCs (Ax) or LCL (LCL-Ax) cells for 72 h in a proliferation assay detecting ³H-thymidine uptake. The data is representative of three experiments

Fig. 3.

Cytolytic assays for CTLs generated by LCL-RNA–transfected PBMCs. CTLs that were generated from PBMCs (A) by using autologous PBMCs transfected with LCL-derived RNA (A:Ax ^RNA-B ) or LCLs from which the RNA was obtained (A:LCL-Bx) were tested in a 4-h chromium assay. The targets cells included allogeneic LCLs (B) and untransfected, autologous PBMCs as controls (A). Effector to target cell ratios used ranged from 5:1 to 40:1. The data points were calculated using the mean value of triplicate cultures and the results represent the mean standard deviation

Fig. 4.

IFN-γ production by T cells primed with autologous APCs pulsed with allogeneic amplified mRNA. PBMCs (A) were cocultured with media (NONE), irradiated, autologous PBMCs (Ax), autologous PBMCs transfected with allogeneic LCL-RNA (A:Ax ^RNA-B ), autologous PBMCs transfected with amplified allogeneic LCL-RNA (A:Ax ^aRNA-B ), or the allogeneic LCLs (LCL-Bx) for 10 days. These responder cells were restimulated with either media alone, irradiated, autologous PBMCs (Ax), or LCL (LCL-Bx) cells for 20 h in an ELISpot assay. The data is representative of three experiments

Fig. 5.

Recognition of individual EBV antigens by T cells generated after stimulation with LCL-RNA–pulsed APCs. PBMCs were initially primed with irradiated, autologous PBMCs transfected with allogeneic LCL-RNA (A:Ax ^RNA-B ) or with allogeneic LCL (LCL-Bx) cells for 10 days. Autologous non-T cells were infected with recombinant vaccinia virus (rVV) expressing single EBV antigens or control rVV and cocultured with the indicated responders at a ratio of 1:10 for 20 h in an ELISpot assay

Fig. 6A–D.

CD4⁺ T cells preferentially proliferate in response to APCs pulsed with LCL-RNA. CFSE-labeled PBMCs, cocultured with irradiated, autologous PBMCs transfected with LCL-RNA (A, B) or irradiated LCL (C, D) cells for 10 days and then restimulated with LCL cells for another 3 days, were sampled at 3, 5, 7, and 10 days during the primary stimulation and 3 days after restimulation (3*). The cells were labeled with fluorochrome-conjugated isotype controls or mAbs—CD3-PerCp, CD8-APC, and CD4-PE—and analyzed by four-color immunofluorescence. The histograms shown were gated on CD3⁺ and CD4⁺ or CD8⁺ as indicated. Data are representative of three independent experiments

Fig. 7.

RNA-pulsed APCs blocked for MHC class II do not effectively stimulate T-cell responses in PBMCs. PBMCs were cultured with autologous PBMCs transfected with allogeneic LCL-RNA for 10 days in the presence of isotype-matched IgG, anti–HLA class I monoclonal antibody, W6/32, or anti–HLA-DR, anti–HLA-DP, anti–HLA-DQ monoclonal antibody, CR3/43. The primed responder cells were then cocultured with either media alone; irradiated, autologous PBMCs; or LCL cells in a proliferation assay detecting ³H-thymidine uptake

Fig. 8.

CD4⁺ T-cell–depleted PBMCs respond weakly to RNA-pulsed APCs. PBMCs or CD8⁺ or CD4⁺ T-cell–depleted PBMCs were cocultured with autologous PBMCs transfected with allogeneic LCL-derived RNA. After 10 days, responders were harvested and restimulated with either media alone; irradiated, autologous PBMCs (Ax); or LCLs (LCL-Bx) in an ELISpot assay