In FY FY24 we continued to focus the immunotoxicology research program within the Division of Translation Toxicology (DTT) on in vitro approaches. A major effort involved investigating how interindividual susceptibility factors and environmental risk factors impact the response to viral infection, using an in vitro human whole blood culture system. Endpoints include lymphocytotoxicity, cytokine release, and Natural Killer cell activity. We have screened >200 individual samples to date and in FY24 developed statistical models to analyze the in vitro data to examine how intrinsic factors such as age, gender and ethnicity influence the response of peripheral blood leukocytes to influenza and SARS-CoV-2 antigens. Preliminary results demonstrate an association between sex and NK activity with males having higher NK activity in peripheral blood than females. Immunophenotyping data are being analyzed to determine if this effect is due to males having higher abundance of NK cells or more active NK cells than females. A second phase of this study is investigating the responses to these antigens following in vitro exposure to known immunotoxicants, including polyaromatic hydrocarbons (PAC)s and how exposure to these environmental agents may affect susceptibility to viral infection. The PAC data will be referenced against mouse in vivo data previously collected in the PAC-MAP mixtures studies and serve to anchor human relevance and strengthen the data that can be generated using the in vitro system. As proof of concept, whole blood cultures were unstimulated or stimulated with anti-CD3/CD28 or viral peptide pools in the presence of dexamethasone, a known immunosuppressive drug that will serve as a positive control in future studies. In FY24, in vitro studies with the PAC and known immune suppressant B(a)P were conducted. Whole blood was used to test the effects of B(a)P on cytokine production following stimulation with viral peptide pools or anti-CD3/CD28. Solubility experiments indicated that the maximum concentration that could be tested in the system was a final concentration of 1.6 mg/mL or 6.3 mM. In preliminary studies treatment of the whole blood culture with B(a)P did not influence anti-CD3/CD28 stimulated cytokine production. This was not surprising as the immunosuppressive effects of B(a)P have been associated with reactive metabolites of the parent compound that are produced in vivo. A metabolic system, S9 liver fraction, was then added to the whole blood cultures to metabolize the B(a)P into reactive metabolites. Treatment of the in vitro whole blood system with B(a)P in the presence of S9 resulted in potent concentration related immunosuppression of cytokine production stimulated by anti-CD3/CD28 or SARS-CoV-2 antigen pool. B(a)P was also tested for impacts on T-cell activation using the whole blood in vitro system. Memory T-cell activation was induced using a positive control CytoStim™ which activates the majority of effector CD4+ and CD8+ T-c...