Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid–immunoglobulin complexes in humans
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
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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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 5

Stimulation of IL-8 expression by CML66-associated ICs requires both nucleic acid and protein in (A) HEK cells, transfected with vector (HEK-neo), TLR9, and TLR9/CD32 (mean results ± SEM from 3 separate experiments), and in (B) normal monocytes (mean results ± SEM from 5 experiments).

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Stimulation of IL-8 expression by CML66-associated ICs requires both nuc...
ICs (black bars) were isolated from K562 lysates by immunoprecipitation with anti-CML66 mAbs or isotype control mAbs (anti-actin and anti-CD3), isolated with Protein G–coated magnetic beads. IL-8 expression was measured by qRT-PCR of HEK cell–derived or monocyte-derived total RNA after a 3-hour incubation with ICs. (C) CML66 IC-bound DNA/RNA was isolated by phenol-chloroform extraction from K562 lysate immunoprecipitated with anti-CML66 antibody (compared with control anti-actin antibody) and Protein G sepharose beads. The isolated nucleic acid was visualized on 1.0% agarose gel with ethidium bromide staining.