Feline infectious peritonitis, caused by feline infectious peritonitis virus [FIPV], is a nearly universally fatal disease of cats. Despite over three decades of research into the virus, there are still no approved treatments for FIP although some antivirals have shown promise. Thus, novel approaches are needed to treat this devastating disease. An alternative to antiviral therapy is to deliver immunotherapy, which can take the form of immunomodulatory drugs, therapeutic antibodies, or cell-based immunotherapies. No immunotherapies have been developed for clinical FIP. However, in the prior funding for this project, we made significant progress towards the development of a cell-based immunotherapy using chimeric antigen receptor (CAR)-engineered immune cells. CARs are comprised of two main components: a single chain antibody fragment (ScFv) and a signaling domain from an immune costimulatory receptor. We designed, created, and tested a CAR specific for the FIPV spike protein, which is found on the surface of infected cells and is thus a viable immunotherapeutic target. Our anti-FIPV-spike-CAR was the first in-feline for CARs, and the first in any veterinary species for a CAR against a viral target. During the initial funding period, we cloned an FIPV anti-spike antibody sequence, generated an ScFV of that antibody, developed an FIPV-spike-CAR construct using that ScFV, confirmed expression, functionality, and specificity of the FIPV-spike-CAR construct, and developed the procedures to isolate, enrich, expand, and genetically engineer primary feline T cells. We have also published a review article on the use of CARs for veterinary medicine. We provide additional data demonstrating the potential of in vitro transcribed mRNA delivery into primary feline T cells, which will avoid the use of lentivirus for delivery of the CAR into T cells. We also show that we can culture feline T cells in cytokine cocktails to drive CD4 T cells down different polarization paths. This is important because CD4 cells promote the growth and function of CD8 cells, especially Th1 CD4 T cells that produce interferon gamma. We propose a short-term (one year) project with the following specific aims 1) optimize the non-viral mRNA-based genetic engineering of primary feline T cells, and 2) determine the role of CD4 T cells in CAR-CD8-T cell cytotoxicity on target cells. This grant will support the graduate student, James Cockey, as he completes this project and the PhD component of his DVM/PhD training. The specific components of this short-term project, in the context of the overall project, will lay the foundation for further clinical development of CAR-T therapy for FIP as well as develop the tools and procedures necessary to expand feline CAR-T therapy to other diseases such as lymphoma.