A non-depleting PC61 antibody allows residual CD25 function that maintains regulatory T cell-mediated immune homeostasis

Abstract IL-2 is a critical regulator of immune homeostasis through its role in the maintenance and function of regulatory T cells (TR) and its stimulatory capacity on effector cells. Most TR constitutively express the high affinity IL-2 receptor component CD25, which increases their sensitivity to IL-2 and allows them to outcompete NK cells and memory T cells for access to IL-2 in vivo. Historically, the blocking CD25 antibody clone PC61 has been used to examine the role of CD25 in IL-2 signaling in mice. However, interpretation of results has been difficult due to uncertainty of whether the observed in vivo effects are mediated by CD25 blockade or TR depletion. We used an engineered isoform of PC61 (PC61N297Q) that cannot deplete CD25-expressing cells to specifically explore how functional inhibition of CD25 impacts TR mediated immune homeostasis. In vivo treatment of B6 mice for one week with IL-2 neutralizing antibodies (αIL-2) reduced TR frequency and number by half in lymphoid tissues, resulting in immune dysregulation that included enhanced DC activation. By contrast, treatment with PC61N297Q did not decrease TR abundance compared to controls. Although PC61N297Q reduced TR sensitivity to IL-2 by about 100-fold compared to control TR, this was not sufficient to erase their selective advantage over effector cells for IL-2 access in vivo, as normal levels of pSTAT5 were maintained in TR stained directly ex vivo from mice treated with PC61N297Q. Residual IL-2 signaling in PC61N297Q treated TR was CD25 dependent, since it could be blocked with an antibody that directly binds IL-2 at the CD25 binding site. Thus, we conclude that reducing the CD25 function on PC61N297Q treated TR is not sufficient to alter immune homeostasis.