Agonistic anti-CD40 antibody treatment converts resident regulatory T cells into activated type 1 effectors within the tumor microenvironment.

In pancreatic ductal adenocarcinoma (PDAC), agonistic anti-CD40 (αCD40) reduces frequencies of intratumoral regulatory T (Treg) cells despite a lack of CD40 expression on Treg cells. Here, we leveraged spatiotemporal imaging and lineage tracing approaches to examine intratumoral Treg cell fate in a mouse model of PDAC, where immune checkpoint blockade (ICB) (αPD-1 + αCTLA-4) combined with αCD40 controls tumor growth. Intratumoral Foxp3+ Treg cell numbers collapsed upon treatment, dependent on CD40-activated dendritic cells (DCs) and induction of interleukin (IL)-12 and interferon (IFN)-γ. This reduction corresponded with cellular alterations; Treg cells acquired an "ExTreg" phenotype characterized by loss of Foxp3 expression and acquisition of T helper 1 (Th1)-like features (Tbet+IFN-γ+). αCD40 promoted a spatially reorganized tumor microenvironment (TME), with Cxcr3⁺ Treg and ExTreg cells localized to the tumor periphery with Cxcl9-expressing DCs. Through in situ analyses of T cell receptor (TCR) signaling, we found that ExTreg cells had the highest antigen-driven activation among tumor-infiltrating T cells. Reprogramming of intratumoral Treg cells into Th1-like effectors reveals plasticity and an anti-tumor capacity of these cells.