Statistical analyses were done by Student’s t-test. From an immunologic standpoint, complete blood counts and Fluorescence-activated cell sorting analysis of immune cell subpopulations were found to be unchanged. Furthermore, functional analysis based on mixed lymphocyte response and CD3-induced proliferation assays of CD4 cells demonstrated no significant differences. In addition, similar to previous studies with hormonal therapies, enzalutamide treatment was associated with increased thymic weights and increased T-cell excision circles, consistent with the production of new T-cells from the thymus. (Figure 7) This series of experiments (publication pending) provides evidence that enzalutamide likely will not diminish the quantity or functionality of immune cells and may even enhance thymic production of naïve T-cells, allowing for potential immune stimulation by a therapeutic cancer vaccine such as PSA-TRICOM. Figure 7. Enzalutamide mediates an enlargement of thymus and an increase in T-cell Receptor Excision Circles (TREC) levels. (A). Male C57BL/6 mice (n=7) were not treated (open circles) or treated with Enzalutamide (closed circles) for 14 days. On day 15, mice were sacrificed, and their thymus harvested and weighed. (7B). Enzalutamide significantly increases T-cell Excision Circle (TREC) levels in male mice. Male 17 Short Title: Enzalutamide Vaccine in mCRPC Version Date: 03/29/2022 C57BL/6 mice (n=7) were not treated (open circles) or treated with Enzalutamide (closed circles) for 14 days. On day 15, 100ng of DNA from blood was collected and TREC levels were quantified by RT-PCR in triplicate.