Collagen Fragments Produced in Cancer Mediate T Cell Suppression Through Leukocyte-Associated Immunoglobulin-Like Receptor 1

Abstract

The tumor microenvironment (TME) is a complex structure comprised of tumor, immune and stromal cells, vasculature, and extracellular matrix (ECM). During tumor development, ECM homeostasis is dysregulated. Collagen remodeling by matrix metalloproteinases (MMPs) generates specific collagen fragments, that can be detected in the circulation of cancer patients and correlate with poor disease outcome. Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is an inhibitory collagen receptor expressed on immune cells in the TME and in the circulation. We hypothesized that in addition to ECM collagen, collagen fragments produced in cancer can mediate T cell immunosuppression through LAIR-1. Our analyses of TCGA datasets show that cancer patients with high tumor mRNA expression of MMPs, collagen I and LAIR-1 have worse overall survival. We show that in vitro generated MMP1 or MMP9 collagen I fragments bind to and trigger LAIR-1. Importantly, LAIR-1 triggering by collagen I fragments inhibits CD3 signaling and IFN-γ secretion in a T cell line. LAIR-2 is a soluble homologue of LAIR-1 with higher affinity for collagen and thereby acts as a decoy receptor. Fc fusion proteins of LAIR-2 have potential as cancer immunotherapeutic agents and are currently being tested in clinical trials. We demonstrate that collagen fragment-induced inhibition of T cell function could be reversed by LAIR-2 fusion proteins. Overall, we show that collagen fragments produced in cancer can mediate T cell suppression through LAIR-1, potentially contributing to systemic immune suppression. Blocking the interaction of LAIR-1 with collagen fragments could be an added benefit of LAIR-1-directed immunotherapy.

Keywords: ECM - extracellular matrix; LAIR-1; T cells; cancer; collagen; immunotherapy.

Figure 1

High MMP, collagen I and LAIR1 expression correlate to poor survival of cancer patients. (A, B) Kaplan-Meier plots of the overall-survival probability for the subgroups of patients. (A) Cancer patients from the TCGA database were stratified into MMP1^highCOL1A1^highLAIR1^high and MMP1^lowCOL1A1^lowLAIR1^low based on the top 25% and bottom 25% tumoral mRNA expression respectively. Similarly, patients were stratified into (B) MMP9^highCOL1A1^highLAIR1^high and MMP9^lowCOL1A1^lowLAIR1^low. Significant differences in Kaplan-Meier plots were evaluated using a logrank test and are indicated in the bottom left corner of each graph (all p < 0.0001).

Figure 2

MMP1 and MMP9 generated collagen I fragments bind to and activate LAIR-1. (A) Analysis of MMP1 and MMP9 generated collagen I fragments by SDS-PAGE and silver staining. Representative image of n = 3. (B, C) LAIR-1-Fc protein binding to mock treated, MMP1 treated and MMP9 treated collagen I. (B) LAIR-1-Fc ELISA based immune binding assay (n = 3 with technical duplicates). (C) LAIR-1-Fc fluorescence based immune binding assay. Representative images show immunofluorescence by the AF647-conjugated secondary antibody alone or by secondary antibody binding to LAIR-1-Fc, representative of n = 3. (D) Flow cytometry analysis of NFAT-GFP reporter cells, representing LAIR-1 ligation (n = 3 with technical duplicates). Anti-human-LAIR-1 and anti-mouse-CD3 are positive controls for reporter cell activation. The isotype antibody and PBS are negative controls. (E) Representative images by live-cell IncuCyte imaging upon stimulation with indicated concentrations of collagen and collagen fragments (n = 3). (F) Quantification of the total green integrated intensity in the images from the live-cell IncuCyte imaging over time (n = 3 with technical duplicates). Significant differences at t = 24 hours compared to WT reporter cells are indicated, tested using a two-way ANOVA with Tukey’s multiple comparison correction. In all panels symbols represent the mean and whiskers indicate the standard deviation.

Figure 3

Purified collagen I fragments are functional LAIR-1 ligands. (A) Analysis of purified full-length alpha chains and purified MMP1 generated collagen I fragments by SDS-PAGE and silver staining. (B) Flow cytometry analysis of NFAT-GFP reporter cells, representing LAIR-1 ligation (n = 2). Anti-human-LAIR-1, anti-mouse-CD3 and 2.5 μg/ml human collagen I are positive controls for reporter cell activation. The isotype antibody and PBS are negative controls. Statistically significant differences compared to WT reporter cells are indicated with * (tested using two-way ANOVA with Tukey’s multiple comparison correction).

Figure 4

MMP1 and MMP9 generated collagen I fragments inhibit T cell function through LAIR-1. (A) Flow cytometry analysis of parental (light grey) or full-length human LAIR-1 expressing (dark grey) NFAT-GFP CD3 reporter cells unstimulated, and stimulated with anti-mouse-CD3 in the presence of 5 μg/ml BSA, untreated collagen I, mock treated collagen I, MMP1 treated collagen I and MMP9 treated collagen I. (B) Inhibition of CD3-signaling-induced GFP expression by increasing concentrations of collagen or collagen fragments compared to BSA. (C) Inhibition of anti-CD3 induced IFN-γ secretion by increasing concentrations of collagen or collagen fragments compared to BSA. Significant differences compared to the parental cell line are indicated, tested using a two-way ANOVA with Sidak’s multiple comparison correction.

Figure 5

LAIR-2 fusion proteins block LAIR-1 activation by collagen I fragments. (A) LAIR-2-Fc and LAIR-2-Fc dead binding to 5 μg/ml coated mock treated, MMP1 treated and MMP9 treated collagen I by ELISA based immune binding assay (n = 3 with technical duplicates). Symbols represent the mean and whiskers indicate the standard deviation. (B) LAIR-2-Fc and LAIR-2-Fc dead binding by fluorescence based immune binding assay (representative of n = 3). (C) LAIR-1-Fc ELISA based immune binding assay after pre-treatment with LAIR-2-Fc and LAIR-2-Fc dead of 5 μg/ml mock treated, MMP1 treated and MMP9 treated collagen I (n = 3 with technical duplicates). (D) LAIR-1-Fc fluorescence based immune binding assay after pre-treatment with LAIR-2-Fc and LAIR-2-Fc dead (representative of n = 3). (E) Flow cytometry analysis of GFP+ reporter cells, representing LAIR-1 activation, after pre-treatment with LAIR-2 fusion proteins (n = 3 with technical duplicates). (F) Quantification of the total green integrated intensity in the images from the live-cell IncuCyte imaging at 24 hours upon stimulation with pre-treated 5 μg/ml coated, differently treated collagen I (n = 3 with technical duplicates). In (C, E, F) * indicates statistically significant differences compared to the respective isotype control (isotype for LAIR-2-Fc and isotype dead for LAIR-2-Fc dead). # indicates statistically significant differences compared to the respective 10 μg/ml pre-treatment. In all panels, significance is tested using a two-way ANOVA with Tukey’s multiple comparison correction.