Naito, MasayasuHainz, UrsulaBurkhardt, Ute E.Fu, BuyinAhove, DeborahStevenson, Kristen E.Rajasagi, MohiniZhu, BaogongAlonso, AnselmoWitten, ElizabethMatsuoka, Ken-ichiNeuberg, DonnaDuke-Cohan, JonathanWu, CatherineFreeman, Gordon2013-10-162012Naito, Masayasu, Ursula Hainz, Ute E. Burkhardt, Buyin Fu, Deborah Ahove, Kristen E. Stevenson, Mohini Rajasagi, et al. 2012. Cd40l-tri, a novel formulation of recombinant human cd40l that effectively activates b cells. Cancer Immunology, Immunotherapy 62(2): 347-357.0340-7004http://nrs.harvard.edu/urn-3:HUL.InstRepos:11179849CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1331-4) contains supplementary material, which is available to authorized users.en-USCD40CD40LB lymphocytesImmunotherapyAntigen-presenting cellJournal Article2013-10-1610.1007/s00262-012-1331-4