LTBR acts as a novel immune checkpoint of tumor-associated macrophages for cancer immunotherapy

Abstract

Tumor-associated macrophages (TAMs) greatly contribute to immune checkpoint inhibitor (ICI) resistance of cancer. However, its underlying mechanisms and whether TAMs can be promising targets to overcome ICI resistance remain to be unveiled. Through integrative analysis of immune multiomics data and single-cell RNA-seq data (iMOS) in lung adenocarcinoma (LUAD), lymphotoxin β receptor (LTBR) is identified as a potential immune checkpoint of TAMs, whose high expression, duplication, and low methylation are correlated with unfavorable prognosis. Immunofluorescence staining shows that the infiltration of LTBR⁺ TAMs is associated with LUAD stages, immunotherapy failure, and poor prognosis. Mechanistically, LTΒR maintains immunosuppressive activity and M2 phenotype of TAMs by noncanonical nuclear factor kappa B and Wnt/β-catenin signaling pathways. Macrophage-specific knockout of LTBR hinders tumor growth and prolongs survival time via blocking TAM immunosuppressive activity and M2 phenotype. Moreover, TAM-targeted delivery of LTΒR small interfering RNA improves the therapeutic effect of ICI via reversing TAM-mediated immunosuppression, such as boosting cytotoxic CD8⁺ T cells and inhibiting granulocytic myeloid-derived suppressor cells infiltration. Taken together, we bring forth an immune checkpoint discovery pipeline iMOS, identify LTBR as a novel immune checkpoint of TAMs, and propose a new immunotherapy strategy by targeting LTBR⁺ TAMs.

Keywords: CD8+ T cells; LTBR; immune checkpoint; myeloid derived suppressor cells; tumor‐associated macrophages.

Figure 1

Integrative analysis of immune multiomics data and single‐cell RNA (scRNA)‐seq data (iMOS) identifies lymphotoxin β receptor (LTBR) as a potential immune checkpoint of tumor‐associated macrophages (TAMs). (A) The workflow of iMOS was displayed. (B) Based on lung adenocarcinoma (LUAD) cohorts from GEO database, credible immune‐related prognostic genes (cIPG) were screened out, including credible immune‐related beneficial genes (cIBG) and harmful genes (cIHG), p < 0.05 by the log‐rank test. Heatmaps showed cIPG expressions and survival times of LUAD cases. (C) The histogram showed pan‐cancer survival analysis of LTBR, dark blue column indicating that higher expression of the gene correlates with shorter survival (p < 0.05), red column indicating that higher expression of the gene correlates with longer survival (p < 0.05), while light blue indicating no significance by log‐rank test. (D) Waterfall plot presented the mutation distribution of the top 10 mutated genes from cIPG in LUAD. (E) Bubble plot represented the profile between the expression and copy number variation (CNV) level of cIPG. (F) The association between LTBR expression and its duplication was tested via Pearson correlation analysis. (G) Kaplan–Meier plot showed the duplication of LTBR was associated with poor survival in LUAD. (H) Bubble plot represented the profile between the expression and methylation level of cIPG. (I) The heatmap displayed the binding frequence and location of eight methylation probes in LUAD patients. (J) The relative level of LTBR in primary LUAD tissues (n = 515) and normal lung tissues (n = 59) was analyzed via The Cancer Genome Atlas (TCGA) database. (K) The relative protein level of LTΒR in LUAD and adjacent tissues was measured via western blot analysis (n = 6). (L) The relative level of LTBR in different T stages was analyzed via TCGA database. (M and N) By analyzing human LUAD scRNA‐seq data, Uniform Manifold Approximation and Projection plots (M) and histogram (N) showed the expression distribution of LTBR among immune cells. (O) Spatial scRNA‐seq analysis showed the spatial colocalization between TAMs and LTBR in LUAD. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 using unpaired Student's t test (J, K) or one‐way analysis of variance with Tukey's multiple comparison test (L). DC, dendritic cells; GEO, genomics expression omnibus; IL, interleukin; NK cells, natural killer cells; SNV, single‐nucleotide variation.

Figure 2

Lymphotoxin β receptor (LTBR)⁺ tumor‐associated macrophages (TAMs) are associated with lung adenocarcinoma (LUAD) stages, immunotherapy failure, and clinical prognosis. (A) The immunofluorescence staining of LUAD tissue microarray was displayed. (B) The representative immunofluorescence staining of LUAD tissues from (A) was showed. (C, D) The number of LTΒR ⁺ TAMs in LUAD patients with different tumor stages (C) and pathological grades (D) was compared. (E) LUAD patients from (A) were divided into two groups: high infiltration of LTΒR ⁺ TAMs (high) and low infiltration of LTΒR ⁺ TAMs (low), and the correlation between LUAD survival and the infiltration of LTΒR ⁺ TAMs was analyzed by log‐rank test. (F) The correlation between LTBR expression and the infiltration of indicated immune cells was analyzed by using TIMER2.0 website. (G) In OAK and POPLAR immunotherapy cohorts, the relative level of LTBR in patients, with responses or without responses to atezolizumab treatment, was compared. (H) In OAK and POPLAR immunotherapy cohorts, patients administrated by atezolizumab were divided into two groups by LTBR expression: high expression (high) and low expression (low) group, whose survival analysis is analyzed by Kaplan–Meier method and tested by the log‐rank test. (I) Immunofluorescence staining was used to compare the number of LTΒR ⁺ TAMs in the immunotherapy responders and nonresponders of TD‐FOREKNOW cohort. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 using one‐way analysis of variance with Tukey's multiple comparison test (C, D) or unpaired Student's t test (I).

Figure 3

Lymphotoxin β receptor (LTBR) contributes to tumor‐associated macrophage (TAM) immunosuppressive activity and M2 phenotype. (A, B) RNA‐sequencing data of TAMs treated with control small interfering RNA (siRNA) (Ctrl) and LTBR siRNA (siLTBR) was utilized for gene set enrichment analysis of chemokines and chemokine receptors biogenesis (A) and T cell exhaustion (B). And the heatmaps showed the differentially expressed genes (p < 0.05 and fold change > 1.5) between Ctrl and siLTBR group. (C) The supernatant concentration of indicated chemokines and cytokines from TAMs treated with Ctrl and siLTBR was analyzed via enzyme‐linked immunosorbent assay array. (D) After transfection of siLTBR or Ctrl in TAMs, the protein level of ARG2, COX2, TGFβR1, and CSF2RB was tested by western blot analysis (n = 3). β‐actin was used as loading control. (E) The relative protein levels in (D) were compared (n = 3). (F, G) The mean fluorescence intensity of programmed cell death 1 ligand 1 (PDL1) (F) and mannose receptor (MR) (G) in TAMs treated with Ctrl and siLTBR was measured by a fluorescence‐activated cell sorter (FACS) (n = 3). (H, I) After activation of LTΒR in TAMs by agonistic LTΒR antibodies, the expression of genes involved in chemokines and chemokine receptor biogenesis (H) and T cell exhaustion (I) was measured by quantitative reverse transcription polymerase chain reaction (n = 3). (J) TAMs and sorted granulocytic myeloid‐derived suppressor cell (G‐MDSC) were co‐cultured in the transwell culture system, and the migration of G‐MDSC was observed by microscopy (n = 3). (K) TAMs transfected with siLTBR or Ctrl were co‐cultured with CFSE‐labelled allogeneic T cells for 72 h. The proliferation of T cells was determined by FACS (n = 3). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by paired Student's t test. AGR2, arginase 2; CFSE, carboxyfluorescein succinimidyl ester; COX2, cyclooxygenase 2; CSF2RB, colony stimulating factor 2 receptor subunit beta; IL, interleukin; PDL1, programmed cell death 1 ligand 1.

Figure 4

Lymphotoxin β receptor (LTBR) maintains tumor‐associated macrophages (TAMs) immunosuppressive behavior and M2 phenotype by noncanonical nuclear factor kappa B (NF‐κB) signalling and Wnt/β‐catenin signaling. (A, B) RNA‐sequencing data of TAMs treated with control small interfering RNA (siRNA) (Ctrl) and LTBR siRNA (siLTBR) was utilized for gene set enrichment analysis (GSEA) of noncanonical nuclear factor kappa B signaling (A) and Wnt/β‐catenin signaling (B). (C, D) After transfection of siLTBR or Ctrl in TAMs, the nuclear and cytoplasmic protein levels of indicated genes were detected by western blot analysis and then quantitatively compared (n = 3). (E, F) Chromatin immunoprecipitation (ChIP)‐seq data from the Cistrome Project were utilized to analyze potential binding sites of RELB, β‐catenin, and H3K4me3 on the promoter of genes involved in T cell exhaustion (E) and chemokines (F). (G, H) The binding of RELB (G) and β‐catenin (H) to the promoter of the indicated genes was analyzed via ChIP array (n = 3). (I) After activation of LTΒR by agonistic LTΒR antibodies, TAMs were transfected with RELB siRNA (siRELB), β‐catenin siRNA or control siRNA (Ctrl). Twenty‐four hours after transfection, the expression of indicated genes was determined by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) (n = 3). (J) The expression of LTBR in TAMs treated with Wnt3a or DMSO was tested by qRT‐PCR (n = 3). (K) The binding of β‐catenin to the promoter of LTBR gene was analyzed via ChIP array (n = 3). (L) TAMs were treated as (I) and then co‐cultured with granulocytic myeloid‐derived suppressor cell (G‐MDSC) in a transwell system. The migration of G‐MDSC was analyzed by microscopy (n = 3). (M) TAMs were treated as (I) and then co‐cultured with CD8⁺ T cells. The proliferation of CD8⁺ T cells was analyzed by flow cytometry (n = 3). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by paired Student's t test (D, G, H, J, and K) or one‐way analysis of variance with Tukey's multiple comparison test (I, L, and M). AGR2, arginase 2; COX2, cyclooxygenase 2; IgG, immunoglobulin G; IL, interleukin; PDL1, programmed cell death 1 ligand 1.

Figure 5

Knockout of lymphotoxin β receptor (LTBR) in tumor‐associated macrophages (TAMs) impedes tumor growth via disrupting TAM immunosuppressive activities and M2 phenotype. (A) Orthotopic lung cancer model was established by intratracheally instillation of luciferase‐carried Lewis lung carcinoma (LLC) cells in macrophage‐specific LTBR knockout (LTBR ^cKO) mice and the control (Ctrl) mice. (B) Three weeks after LLC inoculation as (A), the growth of orthotopic lung cancer was monitored by an in vivo imaging system (n = 6). (C) The quantification of average radiance in each group from (B) (n = 6). (D) The representative images of tumors from (B) were displayed. (E) The tumor weight of mice from (D) was measured and compared (n = 6). (F–H) The mean fluorescence intensity of LTΒR (F), mannose receptor (G), and programmed cell death 1 ligand 1 (PDL1) (H) in TAMs isolated from (D) was measured by a fluorescence‐activated cell sorter (n = 6). (I) In the sorted TAMs from (D), the expression of the indicated genes was tested by quantitative reverse transcription polymerase chain reaction (n = 3). (J, K) The infiltration of T cells, myeloid‐derived suppressor cells (MDSC), and M2‐like TAMs was measured by flow cytometry (n = 6). (L) The survival curves of LLC‐bearing LTBR ^cKO and Ctrl mice were compared by log‐rank test, **p < 0.01. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 using paired Student's t test (C, E–K). AGR2, arginase 2; COX2, cyclooxygenase 2; G‐MDSC, granulocytic myeloid‐derived suppressor cell; siLTBR, LTBR siRNA.

Figure 6

Tumor‐associated macrophage (TAM)‐targeted delivery of lymphotoxin β receptor (LTBR) small interfering RNA disrupts TAM immunosuppressive ability and improves immunotherapy response. (A) The distribution of Cy5‐labled LTBR siRNA (siLTBR) in different organs of tumor‐bearing mice was observed by IVIS Lumina system at 6 h after tail vein administration of naked Cy5‐siLTBR, and vector (V)＆Cy5‐siLTBR. (B) Tumor sections from tumor‐bearing mice described in (A) were stained with fluorescein isothiocyanate (FITC)‐F4/80 antibody and Hoechst, followed by images acquisition with confocal microscopy. (C, D) The orthotopic lung cancer model was established by intratracheally instillation of Lewis lung carcinoma (LLC) cells. One week after instillation, tumor‐bearing mice were intravenously injected with V&siCtrl, siLTBR, and V&siLTBR every 3 days for five times. Three days after the last treatment, the tumors were collected (n = 6). The representative images and quantitative comparison of tumor weights are shown in (C) and (D), respectively. (E, F) The mean fluorescence intensity of LTΒR in TAMs from mice treated as (C) was measured and compared by a fluorescence‐activated cell sorter (FACS) (n = 6). (G) TAMs from mice treated as (C) were sorted. The expression of the indicated genes in these sorted TAMs was measured by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) (n = 3). (H) The infiltration of T cells, myeloid‐derived suppressor cells (MDSC), and M2‐like TAMs was measured by FACS. (I) Serum from tumor‐bearing mice treated as (C) was collected, and the concentration of the indicated cytokines was determined by enzyme‐linked immunosorbent assays (n = 3). (J) After V&siCtrl, siLTBR, or V&siLTBR treatment, the survival curves of tumor‐bearing mice were analyzed by log‐rank test. Versus V&siCtrl: **p < 0.01; ***p < 0.001; versus siLTBR: ^# p < 0.05 by log‐rank test. (K) After the establishment of the orthotopic lung cancer model, tumor‐bearing mice were treated with saline, V&siLTBR, programmed cell death 1 ligand 1 (PDL1) antibody (aPDL1), and V&siLTBR + aPDL1 every 3 days for five times. After that, the tumors were dissected and photographed. (L) The tumor weight of different treatment as (K) were compared (n = 6). (M) In sorted TAMs from (K), the expression of genes involved in chemotaxis, T cell exhaustion, and immune suppression was measured by qRT‐PCR (n = 3). (N, O) After different treatments as (K), the proportion of MDSC (N) and T cells (O) was measured by FACS (n = 6). (P) The survival curves of tumor‐bearing mice were observed after treatment with different drugs as shown in (K); versus saline: **p < 0.01; ***p < 0.001; versus V&siLTBR: ^### p < 0.001; versus aPDL1: ${}^{@@@}p$ < 0.001 by log‐rank test. (Q) The schematic diagram shows that integrative analysis of immune multiomics data and single cell RNA‐seq data (iMOS) reveals LTΒR as the immune checkpoint of TAMs to exhaust cytotoxic CD8⁺ T cells and recruit G‐MDSC by noncanonical nuclear factor kappa B signaling and Wnt/β‐catenin signaling. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 by paired Student's t test (I and M) or one‐way analysis of variance with Tukey's multiple comparison test (D, F, G, L, N, and O).