.2024 Jul 19;10(29):eadm8660.

doi: 10.1126/sciadv.adm8660. Epub 2024 Jul 19.

Targeting conserved TIM3⁺VISTA⁺ tumor-associated macrophages overcomes resistance to cancer immunotherapy

Isaure Vanmeerbeek¹, Stefan Naulaerts¹, Jenny Sprooten¹, Raquel S Laureano¹, Jannes Govaerts¹, Rosa Trotta^{2 3}, Samantha Pretto^{2 3}, Shikang Zhao^{2 3}, Sarah Trusso Cafarello^{2 3}, Joren Verelst^{2 3}, Maarten Jacquemyn⁴, Martyna Pociupany¹, Louis Boon⁵, Susan M Schlenner⁶, Sabine Tejpar⁷, Dirk Daelemans⁴, Massimiliano Mazzone^{2 3}, Abhishek D Garg¹

Affiliations

¹ Laboratory of Cell Stress and Immunity, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
² Laboratory of Tumour Inflammation and Angiogenesis, VIB Center for Cancer Biology, Leuven, Belgium.
³ Laboratory of Tumour Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium.
⁴ Laboratory of Virology and Chemotherapy, Department of Microbiology, Immunology and Transplantation, KU Leuven, Rega Institute, Leuven, Belgium.
⁵ JJP Biologics, Warsaw, Poland.
⁶ Laboratory of Adaptive Immunity, Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium.
⁷ Laboratory for Molecular Digestive Oncology, Department of Oncology, KU Leuven, Leuven, Belgium.

PMID:39028818
PMCID: PMC11259173
DOI: 10.1126/sciadv.adm8660

Targeting conserved TIM3⁺VISTA⁺ tumor-associated macrophages overcomes resistance to cancer immunotherapy

Isaure Vanmeerbeek et al. Sci Adv.2024.

.2024 Jul 19;10(29):eadm8660.

doi: 10.1126/sciadv.adm8660. Epub 2024 Jul 19.

Authors

Affiliations

¹ Laboratory of Cell Stress and Immunity, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
² Laboratory of Tumour Inflammation and Angiogenesis, VIB Center for Cancer Biology, Leuven, Belgium.
³ Laboratory of Tumour Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium.
⁴ Laboratory of Virology and Chemotherapy, Department of Microbiology, Immunology and Transplantation, KU Leuven, Rega Institute, Leuven, Belgium.
⁵ JJP Biologics, Warsaw, Poland.
⁶ Laboratory of Adaptive Immunity, Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium.
⁷ Laboratory for Molecular Digestive Oncology, Department of Oncology, KU Leuven, Leuven, Belgium.

PMID:39028818
PMCID: PMC11259173
DOI: 10.1126/sciadv.adm8660

Abstract

Despite the success of immunotherapy, overcoming immunoresistance in cancer remains challenging. We identified a unique niche of tumor-associated macrophages (TAMs), coexpressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA), that dominated human and mouse tumors resistant to most of the currently used immunotherapies. TIM3⁺VISTA⁺ TAMs were sustained by IL-4-enriching tumors with low (neo)antigenic and T cell-depleted features. TIM3⁺VISTA⁺ TAMs showed an anti-inflammatory and protumorigenic phenotype coupled with inability to sense type I interferon (IFN). This was established with cancer cells succumbing to immunogenic cell death (ICD). Dying cancer cells not only triggered autocrine type I IFNs but also exposed HMGB1/VISTA that engaged TIM3/VISTA on TAMs to suppress paracrine IFN-responses. Accordingly, TIM3/VISTA blockade synergized with paclitaxel, an ICD-inducing chemotherapy, to repolarize TIM3⁺VISTA⁺ TAMs to proinflammatory TAMs that killed cancer cells via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. We propose targeting TIM3⁺VISTA⁺ TAMs to overcome immunoresistant tumors.

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Figures

**Fig. 1..HAVCR2⁺*VSIR*⁺ TAMs are specifically associated with immunoresistant human tumors.**
(A) Overview of our computational framework for single-cell transcriptome mapping based on existing datasets with patient-derived tumor tissue (created withBioRender.com). (B andC) Dot plots of scRNA-seq data from patients with skin cutaneous melanoma (n = 32) responding or not responding to PD1/CTLA4 blockade. Size of dots represents number of cells, and color intensity represents expression levels of indicated genes in single-cell profiles of (B) T cells and (C) myeloid cells. (D) Density plots of scRNA-seq data from 14 treatment-naive patients across four different types of cancer (lung, endometrial, and colorectal adenocarcinoma and clear cell renal cell carcinoma) showingHAVCR2 andVSIR expression in indicated immune cell types. T_reg, regulatory T cell; T_em, effector memory T cell. (E) Dot plots of 33 scRNA-seq datasets including eight cancer types (non–small cell lung, head and neck, colorectal, stomach, breast, and pancreas cancer, hepatocellular carcinoma, and lymphoma) (n = 45). Size of dots represents number of cells, and color intensity represents expression levels of indicated genes in single-cell profiles in indicated immune cell types. mreg, mature regulatory. (F) Density plots of scRNA-seq data from 23 patients with primary colorectal cancer (CRC) [4 microsatellite instable (MSI) high and 19 microsatellite stable (MSS)] including 10 matched normal adjacent tissues, showingHAVCR2 andVSIR expression inSPP1⁺ TAMs and S100A8/9^HIGH TAMs. (G) Aggregated scores for representative pathway terms based on a broader differential pathway enrichment analysis betweenHAVCR2⁺*VSIR*⁺TAMs versusHAVCR2^NEGVSIR^NEGTAMs in dataset from (F). Full differential pathway enrichment analysis in fig. S1 (C and D). AP1, activating protein 1.

**Fig. 2.. TIM3⁺VISTA⁺ TAM signature shows prognostic and predictive impact in patients with cancer.**
(A) Coexpression of indicated genes’ correlation (Jaccard’s index) among themselves in scRNA-seq profiles of 2,489,382 MΦs from 1679 scRNA-seq studies of human origin (pan-disease or pan-tissue). (B) Automated cell ontology analyses run on an existing database of ~300 scRNA-seq human datasets interrogated using a genetic signature ofHAVCR2⁺*VSIR*⁺TAMs derived from Fig. 1F. Final datasets were selected as “hits” based on a high Jaccard’s index threshold of 0.7. (C) Overview of the high-powered bulk tumor transcriptome and high-resolution scRNA-seq mapping pipeline that was used for prognostic and predictive validation of our biomarker (created withBioRender.com). (D) Radar plot showing the correlation of immune-deconvoluted MΦ fractions with the scRNA-seq–validatedHAVCR2⁺*VSIR*⁺ TAM signature in TCGA datasets consisting of six pan-cancer immune landscape classes (C1,n = 2067; C2,n = 2424, C3,n = 2349; C4,n = 1142, C5,n = 387; C6,n = 180) (for more details, see fig. S2, A and B, and table S1). (E) Hazard ratios (HRs) for the impact ofHAVCR2⁺*VSIR*⁺ TAM signature on overall survival (OS) of patients with cancer in TCGA datasets consisting of six pan-cancer immune landscape classes (C1,n = 2067; C2,n = 2424, C3,n = 2349; C4,n = 1142, C5,n = 387; C6,n = 180) (for more details, see fig. S2, A and B, and table S1). (F andG) Kaplan-Meier curve of patients with cancer spanning five cancer types (skin cutaneous melanoma, bladder cancer, kidney cancer, glioblastoma, and stomach adenocarcinoma), where tumors were transcriptome-profiled before anti-PD(L)1 and/or anti-CTLA4 antibody treatments. Kaplan-Meier curve shows (F) OS (n = 152) and (G) progression-free survival (PFS) (n = 129) ofHAVCR2⁺*VSIR*⁺TAM signature^HIGH versusHAVCR2⁺*VSIR*⁺TAM signature^LOW patients (statistical autocutoff for expression; log-rank test forP value).

**Fig. 3.. TIM3⁺VISTA⁺ TAMs have an anti-inflammatory phenotype and dominantly enrich in an immunoresistant murine tumor.**
(A) Basal immunophenotyping of LLC and MC38 tumors, showing percentages of CD8⁺ T cells, CD4⁺ T cells, and TAMs (n = 5). See fig. S3B for the violin plots and statistics. (B andC) Tumor volume curve of (B) LLC and (C) MC38 tumor–bearing mice treated with anti-PD1/PD-L1/CTLA4 antibody or phosphate-buffered saline (PBS) [n = 4; Kruskal-Wallis test corrected for false discovery rate (FDR)]. (D toG) KDE plots showing TAM populations in LLC and MC38 tumors from flow cytometry data of CD45⁺ cell fraction obtained from LLC and MC38 tumors. KDE plots were made on the basis of the Uniform Manifold Approximation and Projections (UMAPs) shown in fig. S3 (E and F). (H) Percentage of TIM3⁺VISTA⁺ TAMs in LLC and MC38 tumors (n = 9; Mann-Whitney test). (I) Different markers used in Fig. 2 (D to G) for LLC and MC38 tumor samples. Size of dots represents fraction of cells and color intensity represents mean expression levels in each group. (J andK) CD45⁺ cell fraction from LLC and MC38 tumors. (J) Ratio of M1-like (MHCII^HIGHCSF1R^LOW) TAMs to M2-like (CSF1R^HIGHMHCII^LOW) TAMs and (K) ratio of M1-like (MHCII^HIGHCD206^LOW) TAMs to M2-like (CD206^HIGHMHCII^LOW) TAMs of TIM3⁺VISTA⁺ TAMs (n = 6; Mann-Whitney test). (L) UMAP of scRNA-seq data from LLC tumors. (M) scRNA-seq LLC tumor tissue dataset, showingVsir andHavcr2 in different immune cell subsets. (N) Percentage of TIM3⁺VISTA⁺ on CD8⁺ and CD4⁺ T cells, TAMs, and DCs—conventional DC 1 (cDC1) and cDC2, monocytic DC (moDC), and plasmacytoid DC (pDC)—in LLC tumors (n = 4; Kruskal-Wallis test corrected for FDR, comparison to TAMs). (O) pHrodo Green⁺ TIM3⁺VISTA⁺ CD11b⁺ F4/80⁺ or TIM3⁻VISTA⁻ CD11b⁺ F4/80⁺ in cocultures of BMDMs or TAMs from LLC or MC38 tumors, cocultured with pHrodo labeledE. coli bioparticles (n = 4; Kruskal-Wallis test corrected for FDR).N number represents number of biological repeats or number of independent animals used.

**Fig. 4.. Antitumor synergism between TIM3/VISTA blockade and ICD depends on MΦs.**
(A andB) Tumor volume curve of (A) LLC tumors and (B) MC38 tumors treated with CDDP or PBS with anti-TIM3/VISTA antibody (LLC,n = 8; MC38,n = 4; Kruskal-Wallis test corrected for FDR). (C andD) Tumor volume curve of (C) LLC tumors and (D) MC38 tumors treated with PTX or PBS with anti-TIM3/VISTA antibody (LLC,n = 10; MC38,n = 4; Kruskal-Wallis test corrected for FDR). (E toG) CD45⁺ cell fraction obtained from (C). (E) Percentage of TIM3⁺VISTA⁺ TAMs (n = 8; Kruskal-Wallis test corrected for FDR). (F) Ratio of M1-like (MHCII^HIGHCSF1R^LOW) TAMs to M2-like (CSF1R^HIGHMHCII^LOW) TAMs (n = 8; Kruskal-Wallis test corrected for FDR). (G) Percentage of CD8⁺ T cells (n = 8; Kruskal-Wallis test corrected for FDR). (H andI) Tumor volume curve of LLC tumors treated with PTX or PBS and/or anti-TIM3/VISTA antibody (H) combined with clodronate liposomes (n = 9; Kruskal-Wallis test corrected for FDR, comparison to PBS) and (I) combined with anti-CD8 antibody (n = 8; Kruskal-Wallis test corrected for FDR, comparison to PBS). (J andK) Lysate from CD45⁻ cell fraction obtained from (C). (J) Protein levels of different analytes. Incalculable values are depicted as gray (n = 4). (K) Pro–caspase 9, cleaved caspase 9, and actin Western blot. Ratio of quantification of pixel intensity is represented in a violin plot (n = 4; Kruskal-Wallis test corrected for FDR.) (L) Percentages of cancer cell death after coincubation with TAMs with different inhibitors [n = 4; two-way analysis of variance (ANOVA) corrected for FDR]. (M) Overview of a CRISPR-Cas9–mediatedVsir andHavcr2 knockout in CSF1R⁺ cells (MΦs) (created withBioRender.com). WT, wild-type. (N toP) LLC tumor–bearing TIM3⁻VISTA⁻ MΦ or control knockout MΦ mice, treated with PTX or PBS. (N) Percentages of CSF1R⁺ and CSF1R⁻ TIM3⁺VISTA⁺ TAMs (n = 5; Kruskal-Wallis test corrected for FDR). (O) Percentages of TIM3⁺VISTA⁺ TAMs (n = 5; Kruskal-Wallis test corrected for FDR). (P) Tumor volume curve (control knockout PTX,n = 8; others,n = 6; Kruskal-Wallis test corrected for FDR).

**Fig. 5.. TIM3 and VISTA inhibit TAM-intrinsic ISG responses, thereby blunting ICD.**
(A) Volcano plot of gene expression alterations between MC38 (red) and LLC tumors (green). (B) Mean fluorescence intensity (MFI) of TIM3 on VISTA⁺ BMDMs (MΦ) stimulated with different cytokines (IL-4/IL-13/IL-18,n = 3; others,n = 4; Kruskal-Wallis test corrected for FDR). (C toF) IL-4 and IL-13 secretion in tumor lysate. Ratio of absolute values with blank was taken and ratio to total TAMs. (C and D) IL-4 (C) and IL-13 (D) production in LLC and MC38 tumors (n = 4; Mann-Whitney test). (E and F) IL-4 (E) and IL-13 (F) production in LLC and LLC-OVA tumors (n = 4; Mann-Whitney test). (G) Percentages of CD8⁺ T cells, CD4⁺ T cells, and TAMs (n = 4). See fig. S13D for the violin plot and statistics. (H) Percentage of TIM3⁺VISTA⁺CSF1R⁺ TAMs in LLC and LLC-OVA tumors (n = 5, Mann-Whitney test). (I) Tumor volume curves of LLC-OVA tumors (n = 4; Kruskal-Wallis test corrected for FDR). (J) IFN-β secretion of LLC cancer cells alone and their cocultures with BMDMs, with or without blockade with indicated antibodies (Abs) (n = 4; Kruskal-Wallis test corrected for FDR). (K) Quantitative polymerase chain reaction (qPCR) of ISG expression (n = 3). (L andM) IFN/ISG response in J774 MΦs with blockade of isotype or (L) anti-TIM3 and (M) anti-VISTA antibody and their cocultures with LLC [(M) PTX,n = 4; others,n = 3; Kruskal-Wallis test corrected for FDR]. (N toQ) LLC alone and their cocultures with MΦ with blockade of indicated antibodies. (N and O) IFN-β secretion in (N) MΦ blocked with isotype/anti-TIM3 antibody or (O) isotype/anti-VISTA antibody (n = 3; Kruskal-Wallis test). (P and Q) IFN/ISG response in J774 MΦs (P) blocked with isotype/anti-TIM3 antibody or (Q) isotype/anti-VISTA antibody [TIM3 LLC untreated (UT),n = 3; others,n = 4; VISTA LLC PTX,n = 4; others,n = 3; Kruskal-Wallis test corrected for FDR]. (R) Tumor volume curves of LLC tumors inifnar1^−/− mice (n = 8; Kruskal-Wallis test corrected for FDR).

**Fig. 6.. Cancer cell–associated HMGB1 and VISTA engage TIM3 and VISTA on MΦs to suppress ISG responses.**
(A andB) MFI of (A) Ceacam1 and (B) Galectin-9 on LLC cells UT or treated for 48 hours with 100 μM PTX (n = 4; Mann-Whitney test). (C) Western blot showing extracellular released HMGB1 in LLC UT or treated for 48 hours with PTX supernatant. (D andE) MFI of (D) PSGL1 and (E) VISTA on LLC cells UT or treated for 48 hours with PTX (n = 4; Mann-Whitney test). (F andG) IFN/ISG response in J774 reporter MΦs with blockade with isotype antibodies or (F) anti-TIM3 antibody and their cocultures with CRISPR-Cas knockout LLC cancer cells for Adeno-associated virus serotype 1 (AAV1) empty (control),*LGALS9* (Galectin-9),*HMGB1* (HMGB1), andCEACAM1 (Ceacam1), UT or treated with either PTX and (G) anti-VISTA antibody and their cocultures with CRISPR-Cas knockout LLC cancer cells for AAV1 empty (control),*VSIR* (VISTA), andSELPLG (PSGL1), UT or treated with either PTX. Values are fold-changed to UT (n = 4; Kruskal-Wallis test corrected for FDR). (H) Tumor volume curves of LLCHMGB1^−/− tumors treated with PTX or PBS and/or combination with anti-TIM3 antibody (n = 8; Kruskal-Wallis test corrected for FDR). (I) Tumor volume curves of LLCVSIR^−/− tumors treated with PTX or PBS and/or combination with anti-VISTA antibody (n = 8; Kruskal-Wallis test corrected for FDR).N number represents the number of biological repeats or number of independent animals used.

**Fig. 7.. Targeting TIM3⁺VISTA⁺ TAM blunts immunoresistant and low neoantigen melanoma.**
(A toC) Basal immunophenotyping of (A) YUMM1.7, (B) YUMM1.7-OVA, and (C) YUMMER1.7 tumors showing percentages of CD8⁺ T cells, CD4⁺ T cells, and TAMs (n = 6). See fig. S14 (A to C) for the violin plots and statistics corresponding to these pie charts. (D) Flow cytometry analysis of CD45⁺ cell fraction obtained from subcutaneous YUMM1.7, YUMM1.7-OVA, and YUMMER1.7 tumors. Percentage of TIM3⁺VISTA⁺CSF1R⁺TAMs (YUMM1.7,n = 5; others,n = 6; Mann-Whitney test). (E toG) Tumor volume curve of (E) YUMM1.7, (F) YUMM1.7-OVA, and (G) YUMMER1.7 tumor–bearing mice treated with anti-PD1 antibody (n = 4; Kruskal-Wallis test corrected for FDR). (H toJ) Tumor volume curve of (H) YUMM1.7, (I) YUMM1.7-OVA, and (J) YUMMER1.7 tumor–bearing mice treated with PTX or PBS and/or combination with anti-TIM3 or anti-VISTA antibody (YUMM1.7,n = 8; others,n = 4; Kruskal-Wallis test corrected for FDR). (K toP) Flow cytometry analysis of CD45⁺ cell fraction obtained from subcutaneous YUMM1.7, YUMM1.7-OVA, and YUMMER1.7 tumors treated with PTX or PBS and/or combination with anti-TIM3 or anti-VISTA antibody. (K to M) Percentage of TIM3⁺VISTA⁺CSF1R⁺ TAMs, YUMM1.7 (K), YUMM1.7-OVA (L), and YUMMER1.7 (M) subcutaneous tumors (YUMM1.7,n = 8; others,n = 4; Kruskal-Wallis test corrected for FDR). (N to P) Percentage of CD3⁺CD8⁺ T cells of live cells in YUMM1.7 (N), YUMM1.7-OVA (O), and YUMMER1.7 (P) subcutaneous tumors (YUMM1.7,n = 8; others,n = 4; Kruskal-Wallis test corrected for FDR).N number represents the number of biological repeats or number of independent animals used.

**Fig. 8.. Study overview.**
Schematic overview of our study’s results and translational as well as clinical value (created withBioRender.com).

See this image and copyright information in PMC

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Targeting conserved TIM3⁺VISTA⁺ tumor-associated macrophages overcomes resistance to cancer immunotherapy

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Targeting conserved TIM3⁺VISTA⁺ tumor-associated macrophages overcomes resistance to cancer immunotherapy

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