The inhibitory receptor TIM-3 limits activation of the cGAS-STING pathway in intra-tumoral dendritic cells by suppressing extracellular DNA uptake

Abstract

Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1⁺ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.

Keywords: CD103(+) cDC1; CXCL10; CXCL9; DNA; STING; TIM-3; XCR1(+) cDC1; cGAS; dendritic cells; type I interferon.

Figure 1.. TIM-3 blockade induces a type I IFN response in splenic cDC1s.

(A) Intracellular flow cytometric detection of CXCL9 in splenic cDCs following a 2–6 hr incubation with tumor cell debris generated by heat shock (HS), either in the presence of a rat IgG_2a isotype control or the RMT3–23 TIM-3 blocking antibody (αTIM-3). n=3 technical replicates, with one of three independent experiments shown. (B) Significant (Z<0.05) gene expression changes in splenic CD8α⁺ cDC1 following stimulation with HS and αTIM-3, compared to HS and IgG_2a. n=6 biological replicates, data compiled from two independent experiments. (C) Pathway analysis of significantly (p<0.05) altered genes from B. (D) Intracellular flow cytometric detection of CXCL9 in splenic cDCs following a 6 hr stimulation with 10 ng/ml IFN-γ, IFN-α, or IFN-β. n=3 technical replicates, with one of four independent experiments shown. (E) Intracellular flow cytometric detection of CXCL9 in splenic cDCs following a 6 hr stimulation with HS, αTIM-3, or αIFNAR1. n=3 technical replicates, with one of three independent experiments shown. For A, D and E data reflect the mean; significance determined by an unpaired t test (A) or one-way ANOVA (D, E) and shown as *p<0.05, **p <0.01, ***p<0.001. See also Figure S1.

Figure 2.. CXCL9 expression is dependent upon extracellular DNA and STING

(A-F) Intracellular flow cytometric detection of CXCL9 in splenic cDCs following a 6 hr stimulation with supernatant generated by heat shock (HS). (A) Splenic cDCs incubated with HS ± αTIM-3, with DNase (50 U/ml) or RNase (10 μg/ml) added to the supernatant 15 min prior to stimulation as indicated. (B) Splenic cDCs stimulated with 10 μg/ml of 2’3’-cGAMP or 3’3’-cGAMP in the presence or absence of αIFNAR1. (C) C57BL6/J or STING-deficient CD8α⁺ cDCs incubated with HS ± αTIM-3. (D) C57BL6/J, MyD88-deficient, TRIF-deficient, or MAVS-deficient CD8α⁺ cDCs incubated with HS ± αTIM-3. (E) C57BL6/J or Cgas-deficient CD8α⁺ cDCs incubated with HS ± αTIM-3. (F) CD8α⁺ cDCs stimulated with HS generated using PyMT cells deficient in Sting or Cgas. (G) Intracellular flow cytometric detection of CXCL9 in iCD103⁺ BMDCs incubated for 24 hr with HS ± αTIM-3. (H) Western blot of nuclear pIRF3 or pTBK1 in iCD103⁺ BMDC lysate following a 3 hr incubation with HS ± αTIM-3. The STING agonist DMXAA was used as a positive control. Nuclear p84, as well as total IRF3, TBK1, vinculin and β-actin were used as loading controls. For A-G, data reflect technical replicates and the mean, with one of three (A, C, G, H) or one of two (B, D, E, F) representative experiments shown. Significance was determined by a one-way ANOVA (A-E, G) and is shown as ***p<0.001. See also Figure S2.

Figure 3.. STING expression by cDC1s is required for efficacy of αTIM-3/PTX

(A) Treatment schematic for creation of BM chimeras, PyMT tumor implantation, and treatment with paclitaxel (PTX) and αTIM-3. (B-F) Relative tumor volume in chimeric C57BL/6J animals reconstituted with BM from wild type (WT) C57BL/6J mice or Sting-deficient (B), Cgas-deficient (C), Trif-deficient (D), Myd88-deficient (E), or Mavs-deficient (F) animals. For A-F, data reflect the mean ± SEM, with n=6–10 mice per group, and one of two representative experiments shown. (G) Relative tumor volume in mixed BM chimeric animals after the administration of DT to deplete Xcr1-DTR⁺ cDC1s just prior to treatment with PTX ± αTIM-3. Data reflect the mean ± SEM, with n=8–10 mice per group, merged from two independent experiments. (H) Percentage of cDC1s within tumors from F, using flow cytometry to distinguish Xcr1-DTR⁺ cDC1s by expression of Venus. Data reflect the mean ± SEM, with 4–5 mice per group. Significance was determined by a two-way (A-G) or one-way (H) ANOVA and is shown as *p<0.05, ***p<0.001. See also Figure S3.

Figure 4.. TIM-3 suppresses endocytosis of extracellular DNA by cDCs

(A) Intracellular flow cytometric detection of tumor cell DNA (EdU-labeled) within splenic cDCs after a 2 hr incubation with HS ± αTIM-3. DNase (50 U/ml) was added at the start of incubation to prevent uptake, or after 90 min to demonstrate intracellular localization. Data reflect the mean of 3 technical replicates, with one of three representative experiments shown. Significance was determined by one-way ANOVA and is shown as *p<0.05, **p <0.01, ***p<0.001. (B) Same as A, but using image cytometry to detect intracellular localization of tumor cell-derived DNA. Images are representative of three independent experiments. (C) Stacked confocal microscopy images displaying EdU-labeled exogenous DNA (red), MHCII (blue) and GAPDH (green) in iCD103⁺ BMDCs treated with HS ± αTIM-3 for 2 hrs. DNase was added for the final 15 min to digest remaining extracellular DNA. Two representative images from one of 4 independent experiments are shown. Analysis of 9 images per group is shown on the right, quantifying the detection of EdU within 20 individual cells (top) and the percent of EdU colocalized with GAPDH (bottom). Data reflects the mean, with significance determined by an unpaired t test and shown as **p <0.01, ***p<0.001. (D) Impact of Ciliobrevin D (Cilio. D) on EdU-labelled DNA uptake or phagocytosis of pHrodo Deep Red E. coli BioParticles by FLT-3L BMDCs. (E) Impact of Dynasore on EdU-labelled DNA uptake or endocytosis of pHrodo Red Transferrin by FLT-3L BMDCs. For D-E, data reflect the mean ± SD, significance determined by an unpaired t test and shown as ***p<0.001, with one of three independent experiments shown. See also Figure S4.

Figure 5.. Uptake of extracellular DNA by cDCs is HMGB1-dependent

(A) Intracellular flow cytometric detection of CXCL9 in CD8α⁺ splenic cDC1s following a 6 hr stimulation with tumor debris generated by heat shock (HS) or irradiation (IR). αTIM-3 and a neutralizing antibody against HMGB1 were used as indicated. (B) Intracellular flow cytometric detection of tumor cell DNA (EdU-labeled) within splenic cDCs after a 2 hr incubation with HS, αTIM-3, or αHMGB1. (C) Flow cytometry detection of synthetic, rhodamine-labelled B-DNA in iCD103⁺ BMDCs after a 2 hr incubation in the presence or absence of αTIM-3. HMGB1 was admixed with B-DNA at a 1:1 ratio (w/w) for 15 min prior to the incubation as indicated. For A-C, data reflect the mean of 3 technical replicates, with one of two (A) or three (B, C) representative experiments shown. Significance was determined by one-way ANOVA and is shown as ***p<0.001. (D) Relative volume of PyMT tumors in C57BL/6J animals treated with PTX, αTIM-3, or αHMGB1. Data reflect the mean ± SEM, with n=7–10 per group, and one of two representative experiments shown. Significance was determined by two-way ANOVA and is shown as ***p<0.001. See also Figure S5.

Figure 6.. Galectin-9 regulates TIM-3 clustering and function

(A) Relative tumor volume in mice bearing orthotopic PyMT tumors treated with PTX and IgG_2a, αTIM-3, or αGalectin-9 (αGal-9). Treatment was initiated when tumors reached ~100 mm³. Data reflect the mean ± SEM, with n=11–12 mice per group compiled from 2 separate experiments. Significance was determined by two-way ANOVA and is shown as *p<0.05. (B) Intracellular flow cytometric detection of CXCL9 in splenic CD8α⁺ cDC1s following a 6 hr incubated with HS ± αTIM-3 or αGal-9. Data reflects the mean of 3 technical replicates, with one of two experiments shown. Significance determined by one-way ANOVA and is shown as ***p<0.001. (C) Representative histograms displaying surface expression of galectin-9 on splenic or tumor cDCs, as determined by flow cytometry. One of two experiments is shown. (D) Intracellular flow cytometric detection of tumor cell DNA (EdU-labeled) within splenic cDCs after a 2 hr incubation with HS, αTIM-3, or αGal-9 (clone RG9–1). Data reflect the mean of 3 technical replicates, with one of three experiments is shown. Significance was determined by one-way ANOVA and is shown as *p<0.05; ***p<0.001. (E) Surface expression of TIM-3 or galectin-9 on iCD103⁺ BMDCs, either untreated (black) or treated with 2 μg/ml recombinant murine galectin-9 (rmGal-9) for 30 min (red). Data reflect the mean of 3 technical replicates, with one of three experiments is shown. Significance was determined by t test and is shown as **p<0.01. (F) Stacked confocal microscopy images displaying TIM-3 (green) and DAPI (blue) in iCD103⁺ BMDCs, either untreated or treated with rmGal-9 ± αGal-9 (RG9–1) for 30 min. Analysis of images is shown to the right, quantifying the number of TIM-3 clusters per cell and shown as the average per field of view (FOV) from one of three experiments. Significance was determined by one-way ANOVA and is shown as ***p<0.001. See also Figure S6.

Figure 7.. TIM-3 blockade increases DNA uptake and chemokine expression by human cDCs

(A-C) Intracellular flow cytometric detection of EdU-labelled DNA in human peripheral blood cDCs isolated by negative selection from healthy donors. cDCs were incubated with MDA-MB-231 cellular debris generated by heat shock (HS) and TIM-3 blocking antibodies for 2 hr as indicated. (A) Representative density plots for CD141⁺ cDC1s. (B) Percentage of EdU positive CD141⁺ cDC1s. (C) Percentage of EdU positive CD1c⁺ cDC2s. (D) Significant (p<0.05) gene expression changes in peripheral blood CD141⁺ cDC1 following stimulation with HS and αTIM-3 for 24 hrs, compared to HS and IgG₁. n=5 biological replicates, data compiled from two independent experiments. (E) Pathway analysis of significantly altered genes from D, showing Process Networks with a false discovery rate (FDR) > 4, and Pathway Maps with a FDR > 5. (F-H) Intracellular flow cytometric detection of CXCL10 in human peripheral blood cDCs following a 24 hr incubation with HS ± αTIM-3. (F) Representative density plots for CD141⁺ cDC1s. (G) Percentage of CXCL10 positive CD141⁺ cDC1s. (H) Percentage of CXCL10 positive CD1c⁺ cDC2s. Data reflect the mean of 2 technical replicates from 6 individual donors. Data compiled from three independent experiments. Significance was determined by a ratio paired t test and is shown as *p<0.05, **p <0.01, ***p<0.001. See also Figure S7.