Chicken peripheral blood lymphocyte response to ALV-J infection assessed by single-cell RNA sequencing

Chicken peripheral blood lymphocytes (PBLs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Previously, we found that viremia caused by avian leukosis virus subgroup J (ALV-J) was eliminated by 21 days post infection (DPI), accompanied by increased CD8+ T cell ratio in PBLs and low antibody levels. Here we performed single-cell RNA sequencing (scRNA-seq) of PBLs in ALV-J infected and control chickens at 21 DPI to determine chicken PBL subsets and their specific molecular and cellular characteristics, before and after viral infection. Eight cell clusters and their potential marker genes were identified in chicken PBLs. T cell populations (clusters 6 and 7) had the strongest response to ALV-J infection at 21 DPI, based on detection of the largest number of differentially expressed genes (DEGs). T cell populations of clusters 6 and 7 could be further divided into four subsets: activated CD4+ T cells (cluster A0), Th1-like cells (cluster A2), Th2-like cells (cluster A1), and cytotoxic CD8+ T cells. Hallmark genes for each T cell subset response to viral infection were initially identified. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection probably induced CD4+ T cell differentiation into Th1-like cells in which the most immune related DEGs were detected. With respect to the control group, ALV-J infection also had an obvious impact on PBL cell composition. B cells showed inconspicuous response and their numbers decreased in PBLs of the ALV-J infected chickens at 21 DPI. Percentages of cytotoxic Th1-like cells and CD8+ T cells were increased in the T cell population of PBLs from ALV-J infected chicken, which were potentially key mitigating factors against ALV-J infection. More importantly, our results provided a rich resource of gene expression profiles of chicken PBL subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.