Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid–immunoglobulin complexes in humans

Abstract

Donor lymphocyte infusion (DLI), whereby donor mononuclear cells are infused into patients, is one of the few effective immunotherapeutic strategies that generate long-lasting tumor remissions. We previously demonstrated that chronic myelogenous leukemia (CML) patients treated with DLI develop high-titer plasma antibodies specific for CML-associated antigens, the majority of which have been reported to bind nucleic acids These observations led us to predict that circulating antibody-antigen complexes in DLI-responsive patients carry nucleic acids that can engage innate immune sensors. Consistent with this, we report here that post-DLI plasma from 5 CML patients that responded to DLI treatment induced massive upregulation of MIP-1α, IP-10, and IFN-α in normal blood mononuclear cells. Importantly, this was not observed with plasma obtained before DLI and from DLI nonresponders and imatinib-treated patients. This endogenous immunostimulatory activity required nucleic acid and protein for its adjuvant effect and activated antigen-presenting cells through the RNA and DNA sensors TLR8 and TLR9. Presence of the immunoglobulin Fc receptor CD32 enhanced cellular responses, suggesting that immunoglobulins associate with this activity. Finally, a TLR-induced expression signature was detectable in post-DLI but not pre-DLI blood, consistent with an active circulating TLR8/9-stimulating factor. We have therefore demonstrated that effective tumor immunity correlates with the presence of endogenous nucleic acid-immunoglobulin complexes in patient plasma, thus providing a putative mechanism for the induction of potent antigen-specific immunity against malignant cells.

Autoantibody Induction by DNA-Containing Immune Complexes Requires HMGB1 with the TLR2/MicroRNA-155 Pathway

“…Less than 0.01 U/mg endotoxin was present in any of the DNA samples, based on a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). Isolation of ICs and treatment of plasma with nucleases and proteases were performed as previously described (20). Circulating ICs were isolated from patient plasma using the IgG Spin Purification Kit (Pierce), according to the manufacturer's instructions.…”

“…Briefly, SLE plasma was added into the spin columns containing the purification support, and IgG was eluted by centrifuge. The presence of DNA was confirmed on a 1% agarose gel, as previously described (20). To test the role of DNA in the stimulatory activity of test plasma, 20 ml patient plasma or ICs were treated with DNase I (Ambion) for 30 min at room temperature before stimulation of cells.…”

Reversal of in situ T-cell exhaustion during effective human antileukemia responses to donor lymphocyte infusion

“…[15][16][17] We recently demonstrated that CD8-depleted (CD4 1 ) DLI for CML rapidly expanded preexisting marrow-resident CD8 1 T cells specific for CML66, an immunogenic antigen expressed on myeloid progenitor cells. 18 To explore the hypothesis that marrow-infiltrating immune populations contribute to effective DLI responses, we examined a cohort of CML patients treated primarily with CD4…”

Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation

“…[15][16][17][18] Cellular localization may also function to enhance immunogenicity. Recent studies suggest that proteins complexed with nucleic acid are immunostimulatory, 40 providing a plausible mechanism of immunogenicity for the nuclear protein PIM1. Expression of DAPK2 generates apoptosis, 24 and apoptotic blebs have been reported to possess enriched expression of immunogenic Ags.…”