In Vitro Generation of Influenza-Virus Specific T Cells for Adoptive Immunotherapy,

Abstract 4040

Influenza viruses cause fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived adaptive immunity to influenza and the potential for generating such cells for clinical use was investigated. The inactivated influenza vaccine (Fluvax, CSL Australia) was used as the antigen source. Only reagents and culture media approved for clinical manufacture were used. Monocyte-derived dendritic cells (MoDC) pulsed with Fluvax were used to stimulate autologous PBMC at a responder to stimulator ratio of 10:1. On Day 7, a second stimulation was performed. 20U/ml IL-2 was added from Day 7 and 50U/ml IL-2 from Day 14 onwards. Media exchanges were performed as required using fresh medium containing IL-2.

Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n=7). Cultures were mainly T cells (mean 92.9%) with effector and central memory phenotypes. CD4 cells dominated (mean 78.1%) with a lower percentage of CD8 cells (mean 14.9%). Following expansion, CD154 was expressed on 8.1% of CD4 cells (<1% in unstimulated cells). Both CD4 (mean 26.3%) and CD8 (mean 3.2%) cells produced one or more of the Th1 cytokines IFNg, TNFa and/or IL-2 when restimulated with Fluvax antigen pulsed MoDC. Most responding CD4 cells produced all three cytokines simultaneously, while CD8 cells primarily produced IFNg alone. A mean of 11.1% CD4 and 9.1% of CD8 cells mobilized CD107 on the cell surface when restimulated with Fluvax. Th1 cytokines were produced in response to stimulation with antigens derived from individual H1N1, H3N2 and Brisbane virus strains by the CD4 subset in all of 6 cases and CD8 subset in 2 of 6 cases. In addition, both CD4 and CD8 cells proliferated on restimulation with Fluvax or antigen from H1N1, H3N2 or Brisbane strains.

In conclusion, we demonstrate a clinically applicable method that yields high numbers of highly reactive T cells specific for influenza viruses including the H1N1 strain. Cells express a phenotype of activated antigen specific cells capable of B cell helper function. We propose that reconstructing host immunity through adoptive transfer of donor derived influenza virus specific T cells possibly combined with early post-transplant influenza vaccination will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows allogeneic stem cell transplant.