Construction Of CD28 And 4-1BB Transgenic Pigs And Study On The Function Of Disease Resistance

T cell-mediated immunity plays a prominent role in eliminating intracellular pathogens.Both the engagement of the T cell receptor with the peptide-bound major histocompatibility complex and a costimulatory signal mediated by the interactions of CD28 or 4-1BB with its ligand are needed for optimal activation of naive T cells to initiate antigen-specific T cell responses.Costimulatory signals have a pivotal role in T cell function,as they not only determine the functional outcome of T cell receptor(TCR)signaling,but also reduce T cell activation thresholds.Whereas T cell will be anergy without costimulatory signal.Recently,great clinical successes have been achieved in the immunotherapy that targets T cell co-signaling such as CTLA-4 and PD-1.With the aim of improving immune response and disease resistance of the pig,in this study,we constructed two transgenic pig lines expressing an extra copy of CD28 and 4-1BB,respectively.We firstly constructed two transgenic vectors,which contained the coding sequences and regulatory elements of 4-1BB or CD28,respectively.Porcine fetal fibroblast cells were co-transfected with transgenic vector and CRISPR/Cas9 plasmid,followed by somatic cell nuclear transfer.Finally,five FO generation of CD28 transgenic and three FO 4-1BB transgenic piglets were obtained.PCR and Southern blot analyses showed that CD28 and 4-1BB were integrated into the first intron of Rosa26 gene with an extra copy.The results of Western blot and IFA revealed that introduced CD28 and 4-1BB could express on T cell surface stably.Real-time PCR analysis showed that the mRNA level of either CD28 or 4-1BB was higher in transgenic pigs than that of wild type pigs.Notably,all transgenic piglets developed normally.To further exploring the effect of CD28 and 4-1BB transgene on current commercial vaccine efficiency,we detected T cell mediated immune responses to PRRSV vaccination and challenge.Our results showed that CD28 transgene enhanced T cell activation by upregulating the phosphorylation of ERK1/2,while 4-1BB plays an important role in T cell proliferation.CD4 T cells were remarkably increased in vaccinated CD28 transgenic pigs and a similar response in CD8 T cells was elicited after challenge.In addition,the virus-neutralizing antibody production as well as interferon-y secretion,were enhanced in CD28 transgenic pigs.Whereas,4-1BB transgenic pigs upregulated Thl cytokines expression,including IL-2,TNF-?,and IFN-?,as well as enhanced specific cytotoxic T lymphocyte killing.To explore the infection resistance of CD28 transgenic pigs,we detected the adaptive immune responses to Toxoplasma gondii.The results showed that,although no significant difference in the anti-Toxoplasma immune responses between CD28 transgenic and wild type pigs,the parasite burdens in liver and muscle of transgenic pigs were obviously lower than wild type pigs.In summary,we obtained two lines of transgenic pigs that expressing an extra copy of CD28 or 4-1BB gene.Our findings reveal that CD28 and 4-1BB mediated costimulatory signals are necessary for effective T cell activation and also can serve as broad spectrum of disease-resistance genes to enhance the host immune response,providing a novel concept for farm animal breeding in disease resistance.