Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4(+) lymphocytes with a Th2 cytokine profile

The concept that IL-4 is the primary signal for Th2 lymphocyte differentiat ion has recently been put in doubt by studies in which the production of Th 2-associated cytokines was detected in mice deficient in IL-4 synthesis or IL-4R triggering. In this study, we formally demonstrate by single cell ana lysis that CD4(+) lymphocytes with a classical Th2 phenotype (IL-4(+),IL-5( +), IFN-gamma(-), IL-2(-)) develop in significant numbers in helminth-infec ted mice deficient in either IL-4R alpha-chain or Stat6, While an expanded population of Th1 (IL-4(-), IL-5(-), IFN-gamma(+), IL-2(+)) lymphocytes was observed in the same animals, surprisingly, cells with a mixed Th0 cytokin e pattern were rare. The cytokine production phenotypes of the Th1 and Th2 subpopulations generated in infected Stat6-deficient mice were unaffected b y in vitro neutralization of endogenous IL-4 or IFN-gamma, Nevertheless, wh ile addition of exogenous rIL-12 resulted in transitory IFN-gamma productio n by Th2 lymphocytes from both wild-type and Stat6-deficient mice, IL-4 syn thesis was preserved in the former, but temporarily ablated in the latter c ells. Importantly, IL-4(+) IFN-gamma(-) and IL-4(-) IFN-gamma(+) population s similar to those arising in helminth-infected Stat6;deficient mice could also be generated in vitro by repetitive polyclonal stimulation of CD4(+)CD 62L(high) lymphocytes from uninfected mice of the same strain. Together, th e results of these single cell analysis experiments demonstrate that n-4R/S tat6 signaling, while influencing the final frequency of Th2 lymphocytes, i s not essential for Th2 cell development, and suggest that this pathway has a previously unrecognized function in stabilizing Th2 populations once the y have emerged.