Work

Multivirus-Specific CTl for Adoptive Transfer Using in Vitro Pepmix Stimulation

Year: 2011

Type: article

Abstract: We and others have shown that viral infections in allo-HSCT recipients can be prevented/treated by the adoptive transfer of virus-specific CTL. However, conventional CTL manufacture has taken > 3 months. We recently developed a protocol to rapidly generate CTLs (rCTLs) using DCs nucleofected with viral antigen-encoding DNA plasmids as APCs and expansion in a gas permeable bioreactor (G-Rex) in IL7+IL4, which reduces manufacturing time to 16 days. To further streamline production without affecting potency, we are now assessing whether multivirus CTL can be generated by directly stimulating donor PBMCs with clinically applicable pepmixes (15mer peptide libraries) spanning viral antigens. We isolated PBMCs from 4 healthy donors and generated rCTL using plasmid-nucleofected DCs or by directly stimulating bulk PBMCs using pepmixes spanning the same antigens encoded by the plasmids (EBV-EBNA1, LMP2, BZLF1; CMV-IE1, pp65 and Adv-Hexon, Penton) (pCTL). Subsequently the activated cells were expanded as outlined above. We compared the phenotype, proliferation, specificity and function of each cell population. There was no significant difference in the rate of expansion (rCTL vs pCTL, 8.6 vs 7.1 fold). Similarly, both were phenotypically indistinguishable, with a dominance of CD4 T cells (70.5 vs 74.2%) expressing CD62L (77 vs 61%). Both rCTL and pCTL were specific for the stimulating antigens as measured by IFNγ ELIspot with a median of 100 vs 55 EBNA1, 45 vs 47 LMP2, 55 vs 32 BZLF1, 195 vs 232 IE1, 3927 vs 4472 pp65, 525 vs 535 Hexon, 372 vs 782 Penton SFC/1x106 CTL. rCTL and pCTL lysed EBV (16 vs 19%), Adv (27 vs 34%) and CMV targets (42 vs 49% E:T 80:1). Finally, to evaluate whether stimulation using 15mer peptides, which contain all CD8 but possibly not all CD4 epitopes, results in loss of CD4+ specificity we generated pCTL using either 15-or 30mer peptides spanning a region of Hexon predominantly recognized by CD4+ T cells. Phenotypically the CTL were similar (CD4+ 56 vs 60%; CD8+ 21 vs 16%) and epitope specificity, as evaluated by stimulation with peptide pools, demonstrated slightly broader specificity using 15-over 30mer peptides with a mean of 10 vs 7 pools recognized. In summary, we can efficiently generate polyclonal multivirus CTLs by directly stimulating PBMCs with pepmixes. This approach is clinically applicable, requires a starting blood volume of only 15ml, and reduces manufacturing time to < 10 days, with a consequent reduction in cost and complexity.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2010.12.193