Fig 1: Galectin 7 binds to PD-1 by recognizing N-glycosylation on residues N74 and N116 of PD-1. (a) Cell lysates from 293 T cells overexpressing human-Fc-tagged PD-1 were incubated with His-tagged human galectin 7 (1 μM) and sugars (50 mM). After pull-down using protein A beads, purified proteins were subjected to Western blot with anti-human-Fc HRP (for PD-1) or anti-His-HRP antibodies (for galectin 7). The anti-His-HRP signal (galectin 7) was normalized against the anti-Fc-HRP signal (PD-1). (t-test, *** p = 6 × 10−8), (n = 4) (b) PD-1-Fc overexpressed 293 T cell lysates were treated with or without peptide-N-glycosidase F for 16 h. Subsequently, the lysate was incubated with recombinant His-tagged human galectin 7 (1 μM) for 2 h. After the pull-down, purified proteins were subjected to Western blot. The signal intensity of the PNGase F treatment group was normalized against the control. (t-test, *** p = 2 × 10−4), (n = 3) (c) Cell lysate from 293 T cells overexpressing human-Fc-tagged PD-1 wild type and PD-1 glycosylation site mutants were incubated with His-tagged human galectin 7 (1 uM). After pull-down using protein A beads, purified proteins were subjected to Western blot. (t-test, * p = 0.039, *** p = 6 × 10−4), (n = 3). Error bars show mean ± SD.
Fig 2: Galectin 7 binding to PD-1 leads to SHP-2 recruitment and suppression of NFAT. (a) Jurkat cells transduced with lentivirus encoding Fc tagged human PD1 were treated with BSA or Galectin-7 (1 μM), and the interaction of PD1 and shp2 was analyzed with co-IP. (b) Jurkat NFAT reporter cells were transfected with control siRNA or siRNA against PD1. Another set of Jurkat NFAT reporter cells was transduced to overexpress WT or dominant negative shp2 mutant (SHP2CS). Cells were cultured in a plate coated with anti-CD3 in the presence of BSA or Galectin-7 (1 μM). The activation of NFAT signaling was analyzed by quantifying the luciferase activity using a plate reader. (t-test, ** p = 0.0023, *** p = 0.00078), (n = 3). Error bars show mean ± SD.
Fig 3: Galectin-7 shows an inverse correlation with intratumoral CD4 cells in human esophageal cancer tissue. Representative Immunostaining images show galectin 7, CD4, and CD8 staining. Galectin 7 high and low areas have been selected for visualization. The number of CD4+ and CD8+ cells within areas with high or low galectin 7 expression have been counted from twelve image sets, and the percentages are represented in the graph. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars show mean ± SEM.
Fig 4: Galectin 7 treatment leads to a relative reduction in the percentage of CD4+ T cells. (a) Human PBMC were cultured in galectin 7 or BSA control for 7 days. The percentage of CD4+ , CD8+ T cells and regulatory T cells (CD4+ FoxP3 +) was quantified by flow cytometry (n = 3). (b) NSG mice were transplanted with human PBMC. From day 14 of the PBMC injection, the mice were treated with PBS or Galectin-7 (I.V. injection, 1.5 mg/kg) every 4 days. The human T cells in the peripheral blood (PB, depleted red blood cells) were detected by FACS with anti-hCD4 and anti-hCD8. (n = 4). (c) Activated human primary CD4+ and CD8+ T cells were treated with coated galectin 7 (20 μg/ml) or BSA control. Cells were stained with Annexin V FITC and propidium iodide to quantify apoptosis using flow cytometry. (n = 3). Error bars show mean ± SD.
Fig 5: Galectin 7 reduced the percentage of CD4+ T cells and suppressed tumor growth. (a) WT C57BL/6 or PD-1 KO mice were injected with mouse colon cancer cell line MC38 at a dose of 1 × 105. The mice received intravenous injections of mouse Galectin-7 (1.5 mg/kg) or PBS every 4 days from day 15 of tumor transplantation. On day 35, mice were sacrificed, and the percentages of CD4+ , CD8+ , and regulatory T cells were analyzed by flow cytometry. RBC depleted peripheral blood, spleen, and tumor cells were stained with anti-CD4, anti-CD8 and anti-CD25 antibodies for flow cytometry analysis. (CD4 group, t-test, p = 0.002, p = 0.017, p = 0.002 for WT), (Regulatory T cell group, t-test, p = 0.004, p = 0.049, p = 0.017 for WT), (n = 4 for WT, n = 3 for PD-1 KO). (b) Growth of MC38 cancer cells injected into WT or PD-1 KO mice. (one way ANOVA with Tukey HSD, p = 0.0289 for PBS vs. mGalectin 7, p = 5.96 × 10−5 for PBS vs. PD-1 KO, p = 1.03 × 104 for PBS vs. PD-1 KO with mGalectin 7, and p = 0.9657 for PD-1 KO vs. PD-1 KO with mGalectin 7 injection group), (n = 4 for WT, n = 3 for PD-1 KO). (c) MC38 cancer cells were injected into WT mice with an isotype control or anti-mPD-1 antibody. (one way ANOVA with Tukey HSD, p = 0.00018 for PBS vs. mGalectin 7, p = 1.1 × 10−5 for PBS vs. anti-mPD-1, p = 7.6 × 10−6 for PBS vs. mGalectin 7 and anti-mPD-1, p = 0.9889 for mGal7 vs. mGalectin 7 and anti-mPD-1), (n = 4). Error bars show mean ± SD.
Supplier Page from Abcam for Recombinant Mouse Galectin 7 protein (His tag N-Terminus)