Regulatory rebound in Interleukin-12-treated tumors is driven by uncommitted peripheral Treg (TUM9P.1012)

Interleukin-12 (IL-12) promotes a rapid reversal of immune suppression in the tumor microenvironment. However the adjuvant activity of IL-12 is short-lived due to T-regulatory cell (Treg) re-infiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes (TDLN) revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the post-treatment rebound was driven by the CD4+ CD25+ Foxp3+ Neuropilin-1(Nrp-1)low peripheral Treg (pTreg) resulting in a 5-fold increase in the pTreg to CD4+ CD25+ Foxp3+ Nrp-1high thymic Treg (tTreg) ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region (TSDR), CTLA-4 exon 2 and GITR exon 5; were phenotypically unstable; and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg (iTreg)-like phenotype. In vitro culture of post-therapy Treg populations under TH1-promoting conditions resulted in the production of IFNg by pTreg but not by tTreg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified Indoleamine 2,3 dioxygenase (IDO)-positive Dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.