Preincubation with either thymulin or IFN-γ can enhance NK activity. In addition, overnight in vitro pre-treatment with thymulin and IFN-γ increases NK activity further than either treatment alone. It has been hypothesized that thymulin increases the responsiveness of immune cells to IFN-γ by either increasing the expression of IFN-γR or by increasing the production and/or secretion of IFN-γ. The effects of thymulin on IFN-γ production and secretion were examined in this study. While an overnight incubation with the polyclonal activator Con A increased the number of cells positive for intracellular IFN-γ, a similar incubation with thymulin produced no change in the percentages of cells labeling positive for intracellular IFN-γ when compared to the media control cells. In addition, IFN-γ was not secreted by splenocytes following an overnight incubation with thymulin, but increased secretion was induced by Con A stimulation. Taken together, these results suggest that thymulin does not increase IFN-γ production or induce IFN-γ secretion by avian splenocytes.

The ability of thymulin to directly enhance NK cell-mediated cytotoxicity was examined. Specific cell population depletions were done in K and SLD chicken splenocyte preparations using anti-CD3, CD4, and CD8 monoclonal antibodies and secondary complement-fixing polyclonal antibodies. The remaining cells were incubated overnight with in vitro treatments of thymulin and IFN-gamma, either separately or together, followed by an assay for cytotoxicity. Although the control K-strain had higher overall NK cell-mediated cytotoxicity than the thymulin-deficient SLD-strain, the following trends were seen in both strains. Thymulin continued to enhance NK activity following CD4 or CD3 cell depletion, but not after CD8 or CD8 and CD4 cell depletion. Since avian NK cells express CD8 alpha, but not CD3 or CD4 on their surface, these results suggest that the ability of in vitro thymulin treatments to enhance NK activity is not mediated by T-cells but may be due to direct effects on NK cells.