Pigs lacking Natural Killer T cells have altered cellular responses to influenza

It is increasingly recognized that innate T cells such as natural killer T (NKT) cells, mucosal associated invariant T (MAIT) cells, and γδ T cells play an important role in shaping adaptive immune responses following influenza infection or vaccination. This is largely through the multiple cytokines these cells release upon activation, which have downstream effects on the scope and magnitude of virus-specific T and B cells, and antibodies which they form. Here, we examined the contribution of NKT cells using pigs, which are considered a highly translational model of human influenza A infection. CD1D-expressing and CD1D-deficient pigs that respectively possess and lack NKT cells, were infected with the swine influenza virus H3N2 A/Swine/Colorado/23619/1999 (CO99), with or without prior mucosal immunization with a recombinant H3N2 A/Swine/Texas/4199-2/1998 (TX98) modified live vaccine encoding a truncated NS1 protein (TX98 NS1Δ126). Vaccination reduced virus load and pulmonary pathology by similar amounts in both genotypes. However, NKT cell status had a significant impact on the underlying immune response: Contrary to the post vaccination period, virus-specific T cell expansion after infection was greater in CD1D-deficient than CD1D-expressing pigs, indicating that NKT cells play opposing roles in different phases of the immune response. NKT cell-deficient pigs also had reduced T cell cuffing around airways. Furthermore, paired single-cell and immune receptor profiling revealed altered gene expression and higher numbers of expanded T and B cell clones in the absence of NKT cells. Our newly established assay using porcine-specific γ and δ chain primers provided new insights into the TCR repertoire of various pulmonary γδ Τcell subsets. Overall, our results indicate a homeostatic role for NKT cells in regulating several important features of the influenza immune response, particularly virus-specific T cell dynamics.