Changhe Li's research works | Tsinghua University and other places

Figure 1. Silencing of ICAM-1 confers resistance to CTL and NK cell-mediated killing (A) The mRNA expression level of MHC and co-stimulatory molecules among 1,086 human cancer cell lines from the CCLE database. (B) Pan-cancer analysis of ICAM-1 expression level on different cancer types from TCGA database. ICAM-1 median expression value is show in red line. (C and D) FACS analysis of surface ICAM-1 level on indicated human (C) and murine (D) cancer cell lines. Murine cells were either untreated or treated with IFN-g (50 ng/mL) for 24 hours. (E) In vitro competition assay of tumor and CTL co-culture. Control (sgControl) SW480 cells were either mixed with tdTomato-labeled control (sgControl) cells or ICAM-1 KO cells. These mixture cells were then co-cultured with NY-ESO-1-specific T cells or control T cells without the expression of TCR against NY-ESO-1. Log 2 fold changes of the percentage of mixture SW480 cells upon co-culture with NY-ESO-1-specific CTLs as compared with that co-cultured with control T cells were shown (n = 3).

Figure 2. Tumor-intrinsic ICAM-1 is critical for immune evasion for both MHC-I-sufficient and deficient tumors (A and B) Vector-transduced or Icam1 OE B16F10 and 4T1 tumors were inoculated in wild-type mice (A) and NSG mice (B), respectively. Tumor growth curves were recorded and shown. n = 5-6 mice per group. (C) Control (sgControl) and Icam1 KO (sgIcam1) 4T1 tumors were inoculated in the wild-type and NSG mice, respectively. Tumor growth curves were recorded and shown. n = 4-5 mice per group. (D) Summary of FACS analysis comparing the number of indicated tumor-infiltrating immune cells between control and Icam1 KO 4T1 tumors on day 16 after tumor inoculation (n = 5-6). (E) B2m/Icam1 double KO (sgB2m + sgIcam1) or B2m single KO (sgB2m + sgControl) 4T1 tumors were inoculated in the wild-type and NSG mice, respectively. Tumor growth curves were recorded and shown. n = 4-6 mice per group. (F) Summary of FACS analysis comparing the number of indicated tumor-infiltrating immune cells between B2m/Icam1 double KO and B2m single KO 4T1 tumors on day 15 after tumor inoculation (n = 6). Data are presented as means ± SEM (A-F). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA (A-C and E) and one-way ANOVA (D and F). ns, not significant. Data are representative of at least two independent experiments (A-F).

Figure 3. ICAM-1 is co-expressed with a wide range of pro-inflammatory genes and is epigenetically regulated in tumor cells (A) Workflow of CRISPR screen to identify regulators of ICAM-1 expression. Cas9-expressing A549 cells were transduced with a genome-wide sgRNA library. CRISPR-edited A549 cells were then sorted into ICAM-1 high and ICAM-1 low fractions, followed by genomic DNA extraction and sequencing to determine the sgRNA abundance. (B) Volcano plot showing the log 2 fold change and p values of ICAM-1 regulators identified from CRISPR screen. The left graph shows the depleted hits (KO of the gene reduced ICAM-1 expression) and the right graph shows the enriched hits (KO of the gene enhanced ICAM-1 expression). Annotated genes represent the NFkB pathway (blue) and epigenetic regulators (red). (C) Log 2 fold change of sgRNAs against indicated genes in ICAM-1 high A549 cells as compared with control. Depleted sgRNA (KO leads to reduced ICAM-1) and enriched sgRNAs (KO leads to enhanced ICAM-1) are labeled in blue and red bars, respectively. The control sgRNAs are indicated by gray bars. (D) Gene ontology (GO) analysis in top 100 enriched hits from ICAM-1 high A549 cells of CRISPR screen. (E) FACS analysis of ICAM-1 level on A549-Cas9 cells expressing control sgRNA or sgRNAs targeting UHRF1, DNMT1, EED, BPTF, and STAG2. The same control sample was used for all comparisons shown in the panel. Data are representative of two independent experiments (E).

Figure 4. UHRF1-DNMT1-mediated methylation is a major ICAM-1 silencing mechanism in cancer cells (A) Illustration of functional domains in UHRF1. The indicated point mutations abolish the corresponding functions of the domains. (B) Western blot analysis of UHRF1 protein level in control and UHRF1 KO A549 cells expressing indicated UHRF1 mutants. (C) Mean fluorescence intensity (MFI) of surface ICAM-1 level determined by flow cytometry in cells expressing indicated UHRF1 mutants (n = 3). (D) RNA-seq and WGBS profiles of ICAM1 in UHRF1 KO and control A549 cells. CpG region is shaded in blue. One of representative biological replicates is shown for each sample. (E) Bisulfite sequencing of the ICAM1 CpG region in control (left) and UHRF1 KO (right) A549 cells. Each line represents a single clone (n = 20). Methylated CpG sites are shown in black circles and unmethylated sites in blank circles. The percentages of overall methylated CpGs are indicated. (F) Pearson's correlation of tumor ICAM-1 expression and ICAM1 promoter methylation score from the CCLE database. (G and H) Control or UHRF1 KO A549 cells co-cultured either with NY-ESO-1-specific CTLs (G) or NK-92MI cells (H) in the presence of isotype (mouse IgG1 kappa antibodies) or anti-ICAM-1-blocking antibodies (5 mg/mL). Specific lysis percentage was determined by FACS, counting the number of alive cells after co-culture with NY-ESO-1-specific CTLs or NK-92MI cells, as compared with control group (n = 3). Data are presented as means ± SEM (C and G and H). *p < 0.05 and ****p < 0.0001 by one-way ANOVA (C) and two-way ANOVA (G and H). ns, not significant. Data are representative of at least two independent experiments (B, C, G, and H).

Figure 5. Reconstitution of ICAM-1/LFA-1 signaling through fusion protein Cet3ICAM1-D1 (A) Schematic structure of Cet3ICAM1-D1 fusion protein (left) and working hypothesis (right). Cet3ICAM1-D1 is composed of Fab fragment of cetuximab and murine natural D1 domain of ICAM-1, fused to a ''LALA-PG'' human Fc fragment. Working hypothesis: in the absence of ICAM-1, the fusion protein could interact and activate with LFA-1 signaling through the ICAM-1 D1 domain. (B) Binding affinity of cetuximab and Cet3ICAM1-D1 to EGFR in MC38 cells (n = 3). (C and D) OT-1 T cells were co-cultured with MC38 (C) and B16F10 (D) tumor cells with serial dilutions of Cet3ICAM1-D1 or cetuximab. FACS analysis showing the percentage of intracellular IFN-g-producing OT-I T cells (n = 3). (E) OT-1 cells were co-cultured with SIINFEKL-pulsed or unpulsed MC38 tumor cells in the presence of 10 nM Cet3ICAM1-D1. FACS analysis showing the percentage of intracellular IFN-g-producing OT-I T cells (n = 3).

Potentiating anti-tumor immunity by re-engaging immune synapse molecules

February 2025

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Cell Reports Medicine

[...]

The formation of immune synapses (ISs) between cytotoxic T cells and tumor cells is crucial for effective tumor elimination. However, the role of ISs in immune evasion and resistance to immune checkpoint blockades (ICBs) remains unclear. We demonstrate that ICAM-1, a key IS molecule activating LFA-1 signaling in T and natural killer (NK) cells, is often expressed at low levels in cancers. The absence of ICAM-1 leads to significant resistance to T and NK cell-mediated anti-tumor immunity. Using a CRISPR screen, we show that ICAM-1 is epigenetically regulated by the DNA methylation pathway involving UHRF1 and DNMT1. Furthermore, we engineer an antibody-based therapeutic agent, “LFA-1 engager,” to enhance T cell-mediated anti-tumor immunity by reconstituting LFA-1 signaling. Treatment with LFA-1 engagers substantially enhances immune-mediated cytotoxicity, potentiates anti-tumor immunity, and synergizes with ICB in mouse models of ICAM-1-deficient tumors. Our data provide promising therapeutic strategies for re-engaging immune stimulatory signals in cancer immunotherapy.

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Changhe Li’s research while affiliated with Tsinghua University and other places

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