Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation

Abstract

Background aims: Hematopoietic stem cell transplant (HSCT) is the treatment of choice for a proportion of patients with hematologic malignancies as well as for non-malignant diseases. However, viral infections, particularly Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus (Ad), remain problematic after transplant despite the use of antiviral drugs. We have shown that cytotoxic T lymphocytes (CTL) generated against CMV-pp65, EBV and Ad antigens in a single culture are capable of controlling infections with all three viruses after HSCT. Although pp65-specific CTL have proved efficacious for the control of CMV infection, several reports highlight the importance of targeting additional CMV antigens.

Methods: To expand multivirus-specific T cells with activity against both CMV-pp65 and CMV-IE-1, peripheral blood mononuclear cells (PBMC) were transduced with the adenoviral vector (Ad5f35-IE-1-I-pp65). After 9-12 days the CTL were restimulated with autologous EBV-transformed B cells transduced with the same Ad vector.

Results: After 18 days in culture nine CTL lines expanded from less than 1.5 × 10(7) PBMC to a mean of 6.1 × 10(7) T cells that recognized CMV antigens pp65 [median 273 spot-forming cells (SFC), range 47-995] and IE-1 (median 154 SFC, range 11-505), the Ad antigens hexon (median 153 SFC, range 26-465) and penton (median 37 SFC, range 1-353), as well as EBV lymphoblastoid cell lines (median 55 SFC, range 9-301). Importantly, the T cells recognized at least two antigens per virus and lysed virus peptide-pulsed targets.

Conclusions: CTL that target at least two antigens each of CMV, EBV and Ad should have clinical benefit with broad coverage of all three viruses and enhanced control of CMV infections compared with current protocols.

Figure 1

Expression of CMV-IE-1 and CMV-pp65, T-cell expansion and T-cell phenotype. (a) An intracellular stain of HEK 293T cells transfected with the pSHUTTLE-IE-1-I-pp65 plasmid and stained with anti-IE-1 or anti-pp65. (b) FLY-RD18 cells were transduced with the Ad5f35-IE-1-I-pp65 vector, harvested, and run on a sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gel. Two identical blots were then probed with anti-IE-1 or anti-pp65 antibodies. (c) Expansion of nine CTL lines to day 18. (d) Reactivity of CTL lines to surface markers for CD3, CD4, CD8, CD16, CD56, CD45RA and CD62L. (e) Terminally differentiated effector memory, central memory and naive T-cell populations after gating on CD3⁺ and NLVPMVATV (NLV⁺) cells.

Figure 2

Virus-specific reactivity of generated T cells by ELISPOT and intracellular cytokine staining. (a) Virus-specific activity of nine CTL lines as determined by IFN-γ ELISPOT assay in response to direct stimulation with CMV-pp65, CMV-IE-1, Ad-hexon, Ad-penton and autologous EBV LCL. For each T-cell line, mean values of duplicate or triplicate experiments are reported and error bars indicate the standard error. (b) Virus-specific activity of resulting T cells from one representative donor as measured by intracellular cytokine staining of IFN-γ after pulsing overnight with CMV-pp65, CMV-IE-1, Ad-hexon or Ad-penton pepmix. (c) A panel of pepmixes against EBV antigens (EBNA-3a, b, and c, as well as LMP1, LMP2 and BZLF-1) was tested against resulting T cells. Data are from one representative T-cell line. For each T-cell line, mean values of duplicate or triplicate experiments are reported and error bars indicate the standard deviation.

Figure 3

Epitope mapping of CMV-IE-1, CMV-pp65 and Ad-hexon. (a, b) Ad5f35-IE-1-I-pp65 T cells were screened by ELISPOT against 11 pools of hexon peptides, 21 pools of pp65 peptides and 20 pools of IE-1 peptides (all of which contain 20-mer peptides overlapping by 15 amino acids) to identify which peptides for each antigen were recognized by each T-cell line. Two representative donors are shown. Results are expressed as the number of SFC/100 000 cells. (c) Where available, identified epitopes were confirmed by pentamer analysis for HLA-A1-, HLA-A2-, HLA-A24-, HLA-B7- and HLA-B8-restricted epitopes. Indicated is the percentage of pentamer-positive cells within the CD8⁺ population. Two representative donors are shown.

Figure 4

Cytolytic function of Ad5f35-IE-1-I-pp65 T cells. (a, b) ⁵¹Cr release after 4 h of co-incubation of autologous EBV LCL with CMV-IE-1, CMV-pp65 and Ad-hexon/penton pepmix or EBV LCL alone with Ad5f35-IE-1-I-pp65 T cells. The data are the percentage of lysis at E:T ratios of 40:1, 20:1, 10:1, and 5:1. (c) ⁵¹Cr release after 6 h of incubating Ad5f35-IE-I-I-pp65 T cells with allogeneic HLA-A2 + CMV AD169-infected or uninfected fibroblasts. Error bars indicate the standard deviation of the triplicates.