LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems
Joseph F. Grosso, Cristin C. Kelleher, Timothy J. Harris, Charles H. Maris, Edward L. Hipkiss, Angelo De Marzo, Robert Anders, George Netto, Derese Getnet, Tullia C. Bruno, Monica V. Goldberg, Drew M. Pardoll, Charles G. Drake
Joseph F. Grosso, Cristin C. Kelleher, Timothy J. Harris, Charles H. Maris, Edward L. Hipkiss, Angelo De Marzo, Robert Anders, George Netto, Derese Getnet, Tullia C. Bruno, Monica V. Goldberg, Drew M. Pardoll, Charles G. Drake
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Research Article Oncology

LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems

Abstract

Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4+ regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8+ T cells using 2 murine systems. Naive CD8+ T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8+ T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8+ T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8+ T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8+ T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8+ T cells and suggest that LAG-3 blockade may be a potential cancer treatment.

Authors

Joseph F. Grosso, Cristin C. Kelleher, Timothy J. Harris, Charles H. Maris, Edward L. Hipkiss, Angelo De Marzo, Robert Anders, George Netto, Derese Getnet, Tullia C. Bruno, Monica V. Goldberg, Drew M. Pardoll, Charles G. Drake

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Figure 2

LAG-3 blockade enhances the accumulation of clone 4 CD8+ effectors within HA-producing prostates of ProHA × TRAMP mice.

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LAG-3 blockade enhances the accumulation of clone 4 CD8+ effectors withi...
(A) In vivo expression of LAG-3 on clonotypic CD8+ T cells. We transferred 2 × 106 Thy1.1+ clone 4 CD8+ cells into VV-HA–injected ProHA × TRAMP mice. Seven days later, prostates were harvested and single-cell suspensions were gated on CD8+ and Thy1.1+ cells and analyzed for expression of LAG-3. Both surface (left) and intracellular staining (middle and right) were performed for detection of LAG-3. Blue line, LAG-3; red line, rat IgG1 isotype; black line, LAG-3–/– cells. (B) αLAG-3 enhances the accumulation of clonotypic cells in prostates. We transferred 106 LAG-3+/+ or LAG-3–/–CD8+Thy1.1+ clone 4 CD8+ cells into ProHA × TRAMP mice. Mice were given VV-HA, VV-HA plus αLAG-3, or nothing at the time of transfer. Mice receiving αLAG-3 were given another dose 3 days later. Seven days after transfer, prostates and livers were collected and homogenized, and single-cell suspensions were analyzed for IFN-γ by intracellular staining after stimulation in vitro in the presence of HA peptide plus monensin for 5 hours. Prostates were analyzed for (B) percentage of CD8+Thy1.1+ and (C) percentage of IFN-γ+ clonotypic cells. Absolute numbers of pooled prostates to determine (D) prostate-derived clonotypic cells and (E) IFN-γ+ clonotypic cells are shown. Error bars are absent because pooling was necessary to count CD8+ cells from prostate tissue. The results are representative of at least 4 experiments. (F) Enhanced accumulation of clone 4 CD8+ cells using αLAG-3 is specific for organs expressing cognate antigen. Livers from ProHA × TRAMP mice do not express the HA antigen (data not shown) and fail to attract and promote division of HA-specific CD8+ T cells. Data from 1 of at least 3 individual experiments are shown. PerCP, peridinin chlorophyll protein.