<p>The cues that cause B cells to adopt a regulatory phenotype are unknown. We previously reported on the regulatory B-T axis in mouse fibrosarcoma. This work demonstrates the presence of TGF-β-generating Bregs in the sarcoma microenvironment as well as variable gene expression depending on TGF-β levels. Breg depletion reduced tumor-evoked Bregs and progression of WEHI-164 fibrosarcoma, which was reversed by adoptive transfer of B cells. Tumor-derived PGE2 acting via EP4/STAT3 signaling is a key driver of tBreg formation, ultimately resulting in increased Tregs. Only the TGF-β⁺ tBregs caused T cell suppression. PGE2 activated STAT3 in a non-canonical manner since pre-treatment of B cells with JNK and NF-κB inhibitors completely restored tBreg-induced T cell suppression. The COX-2 inhibitor NS-398 prevented the generation of tumor-evoked Bregs, resulting in a lower tumor burden, fewer Tregs, and improved T cell responses. TGF-β-producing Bregs were identified in sarcomas and other cancers with elevated PGE2. The B cells from PTGS2^hi cancers displayed unique Breg signatures when compared to PTGS2^lo cancers. TCGA analysis showed that the Breg signature was higher in patients with high PTGS2 levels, which was also associated with a poor survival rate. In summary, we found that the tumor-derived PGE2 activates non-canonical MAPK/STAT3 in B cells, leading to the formation of TGF-β-generating Breg cells. PTGS2^hi Breg^hi patients were associated with a poor survival rate, while the COX-2 inhibitor NS-398 prevented tBreg generation and reduced tumor burden. This could lead to the development of novel strategies for reducing tBreg production in cancer.</p>