Influenza virus infection continues to be a significant cause of morbidity and mortality amongst susceptible populations. Current immunoprophylaxis is directed towards the induction of neutralizing antibodies via the administration of an inactivated viral vaccine. While it is well established that neutralizing antibodies are necessary for the prevention of influenza infection, there is convincing evidence that cytotoxic T lymphocytes (CTL) play an important role in the recovery from influenza virus infection. An effective vaccine strategy should, therefore, induce both neutralizing antibodies and crossreactive CTL. However, our understanding of the processes involved in the induction of the cytotoxic T lymphocyte is limited. Therefore, it is the purpose of this project to identify the accessory signal requirement for influenza virus-specific human memory CTL. In order to minimize the effect of endogenous lymphokine production, the CTL will be generated in limiting dilution cultures containing 16,000 CD8+ cells and inactivated virus- infected stimulator cells. Under these conditions the generation of CTL activity is dependent upon the addition of exogenous lymphokines. Various highly purified, recombinant DNA-derived lymphokines will be added at specific times in the culture period in order to identify the lymphokine requirement for each stage in CTL generation. In addition to testing the lymphokines, various sub-populations of autologous T lymphocytes will be added to the cultures in order to determine which cells can provide the requisite signal for CTL development. Since the helper T lymphocyte (HTL) response to influenza has not been well characterized in regards to the lymphokines produced and the phenotype of the HTL, a limiting dilution analysis of influenza virus-specific HTL will also be performed. Particular emphasis will be placed on determining the frequency of specific lymphokine producing cells amongst the different T lymphocyte subpopulations. In addition to defining the role of specific lymphokines and lymphocyte subsets in CTL generation, we will also attempt to characterize a novel differentiation factor (DF) present in conditioned medium from mitogen-stimulated lymphocyte cultures. Our approach will be to generate a cDNA library from mRNA derived from mitogen-stimulated 4B4+ cells, the putative source of the DF. The cDNA introduced into COS-7 cells and their supernates assayed for DF activity using CTL precursor cells lines.
