Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid–immunoglobulin complexes in humans
Yun Lin, … , Terry K. Means, Catherine J. Wu
Yun Lin, … , Terry K. Means, Catherine J. Wu
Published March 14, 2011
Citation Information: J Clin Invest. 2011;121(4):1574-1584. https://doi.org/10.1172/JCI44581.
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Research Article Immunology

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.

Authors

Yun Lin, Li Zhang, Ann X. Cai, Mark Lee, Wandi Zhang, Donna Neuberg, Christine M. Canning, Robert J. Soiffer, Edwin P. Alyea, Jerome Ritz, Nir Hacohen, Terry K. Means, Catherine J. Wu

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Figure 4

Post-DLI plasma stimulates TLRs that respond to nucleic acid.

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Post-DLI plasma stimulates TLRs that respond to nucleic acid. (A) Stimul...
(A) Stimulation of IL-8 expression from monocytes is dependent on presence of DNA, RNA, and protein in the plasma. This figure summarizes results of 5 experiments (mean ± SEM). (B) Plasma from CML patients responsive to DLI therapy stimulates IL-8 production from HEK cells that are stably transfected with TLR8 and TLR9 (shaded), but not with TLR3 or TLR4. Negative controls were HEK transfectants stimulated with plasma from normal adult volunteers. Positive controls were HEK transfectants stimulated with TLR agonists. (C) Post-DLI plasma or ICs derived from post-DLI plasma were tested against control, TLR8-expressing, and TLR8/CD32-expressing transfectants. Cell lines were also tested against the known TLR8 agonist R848. Data shown represent mean values ± SEM. (D) Post-DLI plasma was tested against control, TLR9-expressing, and TLR9/CD32-expressing transfectants. DNase abrogates TLR9 stimulatory activity, while purification of IC enhances immunostimulatory activity. Cell lines were also tested against the known TLR9 agonist CpG. Data shown represent mean values ± SEM. (E). Plasma nucleic acid from DLI responders and a normal volunteer were extracted using phenol-chloroform and visualized by ethidium bromide-staining of a 1.0% agarose gel. ICs from the same subjects were isolated using Protein G beads. IC-associated nucleic acid was visualized by GelRed on a 1.0 % agarose gel following heat disassociation from beads (65°C × 5 minutes).