Allogeneic hematopoietic progenitor cell transplantation (HPCT) is the optimal treatment for several hematological malignancies, aplasia or other disorders of the hematopoietic or immune system. Post HPCT, patients undergo a prolonged period of immune incompetence during which they are highly susceptible to viral and fungal infections. Epstein Barr Virus (EBV), Adenovirus (Ad) and Aspergillus fumigates are among the most common and lethal infections during this period. We have described the use of overlapping pools of pentadecapeptides pulsed onto monocyte-derived dendritic cells (DC) to generate cytolytic T-cell (CTL) lines specific for an A. fumigatus-derived antigen, Asp f16 (
Ramadan et. al., 2005, J Clin Immunol 139:257
; Ramadan et. al., 200 J Clin Immunol 140:81). We have further demonstrated in 5 donors, the generation of CTL lines recognizing peptides contained within the conserved region of the Adenovirus hexon protein using a similar approach (Keever-Taylor, et. al, Cytotherapy 8, Suppl 1, 122). To reduce the need for large numbers of DC to prime and expand the CTL cultures, we modified our procedures to use peptide pool (pp)-pulsed EBV-transformed B lymphoblastoid cell lines (BLCL) as antigen presenting cells (APC) following 2–3 rounds of initial priming with pp-DC. This approach results in lines that are predominately reactive to Asp f16 or Ad hexon, and which are moderately reactive to EBV. We expanded our studies to determine the feasibility of generating CTL lines that recognize all three infectious agents. Peripheral blood lymphocytes (PBL) from donor RD0309 were primed weekly ×3 with autologous DC (10:1 PBL to DC ratio) pulsed with a pool of both Asp f16 (104 individual peptides) and Ad hexon (105 individual peptides) pentadecapeptides, then switched to Asp/Ad-pulsed BLCL as APC. The cultures were screened one week following restimulation for lysis of non-pulsed (np-BLCL) and Asp f16-pp-pulsed (Asp-BLCL) and Ad hex-pp-pulsed (Ad-BLCL) BLCL targets. Weak reactivity to Ad-BLCL was detected after 3 primings with DC alone that increased following one round of Asp/Ad-BLCL priming. Asp f16-specific CTL activity was not clearly detected until one week after 3DC plus 3BLCL primings, at which time there was also clear reactivity to np-BLCL targets (26.4% np-BLCL, 37% Asp-BLCL, and 53.9% Ad-BLCL at 50:1), indicating CTL activity to all three infectious agents. With an additional priming, Aspergillus specific reactivity exceeded that towards Adenovirus and EBV-specific reactivity declined. These data show that a pool of overlapping peptides from two different antigens presented on DC followed by BLCL as APC can result in multi-specific cell lines that can be potentially used in adoptive immunotherapy protocols for the treatment or prevention of common opportunistic infections following HPCT.
Disclosure: No relevant conflicts of interest to declare.
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