In FY 22 we continued to focus the immunotoxicology research program on in vitro approaches. A major effort involved investigating how interindividual susceptibility factors and environmental risk factors such as exposure to polyaromatic hydrocarbons impact the response to viral infection (SARS-CoV-2 and Influenza), using an in vitro human whole blood culture system. Endpoints include lymphocytotoxicity, cytokine release, and Natural Killer cell activity. In FY22, BRT performed these assays for 200 blood samples purchased from commercial sources. The data are currently being analyzed prior to reporting and publication. Efforts in FY22 to add to this toolbox of in vitro approaches resulted in the development of a flow cytometry-based assay to measure T-cell activation following in vitro stimulation with viral antigens. BRT continues to expand the toolbox and is currently testing this culture system to facilitate interrogation of humoral antibody mediated immunity to viral antigens. We have begun preliminary in vitro studies with an immunosuppressive compound that will serve as a positive control for the in vitro PAC-MAP studies. The data from these studies will be referenced against mouse in vivo data previously collected under this contract and serve to anchor human relevance and establish scientific confidence in the data that can be generated using the in vitro system. This in vitro toolbox will be critically important to identify chemicals that have the potential to modulate immune function in humans and will position NTP at the cutting edge of testing in this field.