Fig 2: Flow cytometry analysis of LAG-3 expression on T cells and T cell subsets within B16-F10 tumors treated by intratumoral CpG injection on day 8. (A) The ratio of CD8+ T cells to CD4+ T cells in tumors of CpG-treated (n=9) or control mice (n=5). (B, C) Expression of LAG-3 (cumulative %) on T cells (B) and T cell subsets (C) in tumors of CpG-treated (n=9) or control mice (n=5). (D) Expression of LAG-3 (MFI) on T cell subsets in tumors of CpG-treated (n=9) or control mice (n=5). **p<0.01, ***p<0.001.

Fig 3: HE staining and immunofluorescence staining of tumors of B16-F10 bearing mice after CpG therapy. (A) HE staining of tumor tissue sections of B16-F10. There was a large amount of necrosis in the tumor of the responders while tumors of the non-responders and the control mice mainly contained tumor cells. Scale bar=1 mm. (B) Immunofluorescence staining showed colocalization (yellow) of LAG-3 (red) and CD3 (green) in tumor tissue sections of B16-F10. T cells infiltration in both the responders and the non-responders was significantly higher than that in the control group, and the expression of LAG-3 in the non-responders was higher than that in the responders. DAPI (blue) was used to visualize cell nucleus. Scale bar=100 μm.

Fig 4: 68Ga-NOTA-XH05 clearly visualizes B16-F10 tumors and its uptake correlates with LAG-3 expression. (A) Representative PET/CT images of B16-F10 bearing mice injected with 68Ga-NOTA-XH05 after 0.5, 1, 1.5, 2 hours. Tumors are circled and marked with a ‘T’, kidneys are marked with a ‘K’, and gallbladders are indicated with a white arrow and marked with a ‘GB’. (B) Uptake of 68Ga-NOTA-XH05 in tumor (blue), blood (red), muscle (green) and liver (purple) at different time points after injection (n=4). (C) Tumor to blood ratio (T/B, red), tumor to muscle ratio (T/M, green) and tumor to liver ratio (T/L, purple) at different time points after injection (n=4). (D) Biodistribution analysis of 68Ga-NOTA-XH05 accumulation in selected organs. Mice were sacrificed 1 h post-injection (n=4). (E) Correlation plot of tumor uptake of 68Ga-NOTA-XH05 vs LAG-3 expression (MFI) on tumors from mice undergoing PET imaging, followed by subsequent ex vivo analysis by flow cytometry (n=10). PET, positron emission tomography.