Effective Graft-Versus-Leukemia Responses Are Associated with the Presence of Nucleic Acid-Immunoglobulin Complexes That Stimulate TLR8 and TLR9.
Authors: Lin Yun , Means Terry , Alyea Edwin P. , Canning Christine M. , Soiffer Robert J. , Ritz Jerome , Wu Catherine J. 
Published: October 2019

To elucidate the basis of the potent graft versus leukemia (GvL) immune response that is initiated with donor lymphocyte infusion (DLI), we hypothesized the existence of an endogenous adjuvant that enhances antigen-specific immunity. In patients with chronic myeloid leukemia (CML) who achieved molecular remission after DLI, we previously identified a panel of CML-associated antigens that are targets of antibodies present in post-DLI sera and are predicted to bind nucleic acids. Recent studies in SLE have identified immunostimulatory nucleic-acid antibody complexes in patient sera, which can stimulate Toll-like receptors (TLRs) in plasmacytoid dendritic cells (pDCs). To identify immunostimulatory factors associated with GvL, we studied 6 patients that responded to DLI without clinically significant GvHD. Consistent with our prediction that sera from DLI responders contain an immunostimulatory activity, we measured a 3 to 50-fold increase in the expression of MIP-1α, TNF-α, IP-10 and IFN-α transcripts in normal peripheral blood mononuclear cells (PBMC) induced by exposure to 5 of 6 post-DLI sera. No increase in cytokine/chemokine expression was observed upon exposure to sera from pre DLI sera from the same patients, from 3 of 3 DLI non-responders, nor from 3 of 3 CML patients who achieved molecular remission after imatinib treatment. To identify the cell type that responds to the immunostimulatory activity in post-DLI sera, we isolated B, T, NK cells, monocytes, myeloid DCs and pDCs, and cultured them with pre- or post-DLI responder sera. In contrast to published findings that SLE sera stimulate pDCs, we found that post DLI sera induced normal monocytes to express high levels of MIP-1α, IP-10, MCP, TNF-α, IFN-α, IL-6, IL-8 and IL-12. Pretreatment of post-DLI sera with DNase, RNase, papain or pepsin resulted in marked decrease in IL-8 induction, demonstrating that this endogenous immunostimulatory factor requires both nucleic acid and protein for its adjuvant activity. Four active post-DLI sera were then used to stimulate stable HEK transfectants expressing TLR2, 3, 4, 8 or 9. IL-8 expression increased only in the TLR8 and TLR9-expressing cells lines that are known to be responsive to RNA and DNA respectively. IL-8 expression was further induced by post-DLI sera in TLR9-expressing cell lines co-transfected with CD32, suggesting that internalization by FcR may enhance delivery of nucleic acids to endosomal TLR8/9. None of the TLR transfectants could be activated by pre-DLI sera from the same responder patients, nor by post-DLI sera from non-responders. Finally, sera from responders collected between 2 weeks to several months after DLI consistently activated TLR8 and TLR9 suggesting that endogenous TLR8/9 activation may contribute to the early immunologic events that lead to an effective DLI response. Ongoing studies are focused on precisely identifying the TLR8/TLR9 agonists in DLI responders, studying the immunologic effects of this endogenous adjuvant and determining the association of our previously identified CML antigens with nucleic acids that activate TLR8 and TLR9. In summary, we demonstrate for the first time that effective tumor immunity is correlated with the presence of endogenous adjuvant-immunoglobulin complexes in patient sera, and propose that these observations form the basis for novel tumor vaccine strategies.