**2.4 Cytokines and transcription factors mediate Th1/Th2 cross-regulation**

In humans and mice, multiple lines of evidence support that Th1 differentiation inhibits Th2 differentiation, and vice versa [120, 121]. For example, *in vitro* experiments reveal that IFNγ inhibits Th2 differentiation whereas IL-4 suppresses Th1 differentiation [122–124]. In addition, studies using knockout mice and retroviral-transduced CD4+ T cells demonstrate that T-bet blocks Th2 differentiation by inhibiting the transcription of genes associated with Th2 cytokine production [54, 125]. Similarly, GATA-3 prevents Th1 differentiation by suppressing the transcription of genes associated with Th1 cytokines, and interfering with Th1 promoting transcription factors [126, 127]. Collectively, these findings confirm that Th1 and Th2 transcription factors and cytokines cross-regulate each other, ensuring that CD4+ T cells differentiate into either Th1 or Th2 cells. In cattle, however, most of the differentiated clones represent a "hybrid" that co-expresses both IFNγ and IL-4 in the same cell (explained in detail in Section 3) [22, 128]. While it is clear in mice and humans that T-bet and GATA-3 are the transcription factors that regulate expression of IFNγ and IL-4 respectively, at this moment, it is unclear if this is equally true for cattle. In addition, we do not know if the coproduction of both Th1 and Th2 cytokines in the hybrid bovine clones corresponds to the co-expression of both transcriptional factors. Therefore, further research is needed to understand the underpinning regulatory mechanism of hybrid clone differentiation in cattle.
