Stimulation by means of dendritic cells followed by Epstein–Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses

Abstract

SummaryAdoptive immunotherapy with in vitro expanded antigen-specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocytederived dendritic cells (DC) as antigen-presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp-specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2-5¥) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC-DC or autologous PPC-pulsed BLCL (PPC-BLCL). Lines from the DC/BLCL arm demonstrated Asp f16-specific cytotoxicity earlier and to a higher degree than lines generated with PPC-DC alone. The DC/BLCLprimed lines showed a higher frequency of Asp f16-specific interferon (IFN)-g producing cells but an identical effector cell phenotype and peptide specificity compared to PPC-DC-only-primed lines. Tumour necrosis factor (TNF)-a, but not IL-10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16-specific T cell responses and that a culture approach using initial priming with PPC-DC followed by PPC-BLCL may be a more effective method to generate Asp f16-specific T cell lines and requires less starting blood than priming with PPC-DC alone.

Generation of cytotoxic T-cell lines using overlapping pentadecapeptides derived from conserved regions of the adenovirus hexon protein

“…Consistent with our experience with Aspergillus (Zhu et al, 2007), optimal results were obtained by switching to BLCLs as APCs after initial use of DCs. BLCLs had the further advantage of reducing requirements for large numbers of DCs that can be limiting as the line expands in numbers.…”

“…Similar to CMV (Trivedi et al, 2005) and Aspergillus (Ramadan et al, 2004(Ramadan et al, , 2005aZhu et al, 2007), the use of protein-spanning overlapping pools of pentadecapeptides proved highly effective at priming T-helper 1 cellular immune responses to targeted conserved regions of Ad hexon. Consistent with our experience with Aspergillus (Zhu et al, 2007), optimal results were obtained by switching to BLCLs as APCs after initial use of DCs.…”

“…Unless otherwise indicated, monocyte-depleted peripheral blood lymphocytes were stimulated three times at 7-10 day intervals using immature DCs, as described previously (Ramadan et al, 2004), then subsequently stimulated with Ad PP-pulsed autologous BLCLs as (Zhu et al, 2007). Cultures were initiated in X-VIVO 15/5 % AB serum (BioWhittaker) and 500 pg IL-12 ml 21 , at a ratio of 1 : 10 DCs : T cells.…”

“…Subsequent testing used 51 Cr-release assays or ELISPOT assays for IFN-c production, as described previously (Zhu et al, 2007).…”

“…Based on our previous studies using a similar overlapping pentadecapeptide approach (Zhu et al, 2007), we retested the six donors along with three additional donors, but switched the lines to Ad PP-pulsed autologous BLCLs as APCs after the third DC stimulation. After a single use of Ad PP-pulsed BLCLs, eight of the nine lines had measurable CTL activity that increased further with additional rounds of stimulation (Fig.…”