Supplementary Material for: Dynamic observation: Immune-privileged microenvironment limited the effectiveness of immunotherapy in an intraocular metastasis mouse model

Introduction: Intraocular metastasis (IM) occurred in approximately 8-10% of patients with metastatic malignancy, for whom oncological immunotherapies showed poor visual potential. However, the mechanism for that inefficiency remains unclear and requires further exploration. Methods: we established a novel mouse model of intraocular metastasis by intracarotid injection of cutaneous melanoma cells. We investigated disease progression using ophthalmic and histological examinations. We used combined anti-PD-1 and anti-CTLA4 antibodies for immunotherapy and evaluate the therapeutic effects in mice model. In addition, we characterized the immune microenvironment of tumor infiltrating CD8+ T by fluorescence staining and assessed their cytotoxicity by flow cytometry. Results: All mice presented IM in the left eye, while the right eye was healthy. Uveal tissues with rich vascularity (e.g., the iris, ciliary body, and choroid) initiated intraocular metastasis at an early stage, and intraocular metastasis development resulted in several secondary changes, including corneal swelling, retinal detachment, and intratumoral haemorrhage. Immunotherapy could inhibit intraocular metastasis, prolong the time to eye rupture but did not prevent rupture ending. This inefficiency might be attributed to ocular tissues specificities that inhibited CD8+ T cells infiltration via PD-L1 expression. PD-L1low corneal tissue resisted tumor invasion with high levels of CD8+ T cells infiltration, whereas CD8+ T cells were deficient in PD-L1high uveal metastasis. Furthermore, we found a significantly increased PD-1+/- CD4+ and PD-1+/- CD8+ T cells infiltrating the intratumoral haemorrhage area. Although these CD8+ T cells in the IM were not exhausted and had a higher capacity of cytotoxicity (higher Interferon-γ ratio) than CD8+ T cells in the blood, FasL+ PD-L1+ ocular tissue can strongly inhibit these IM infiltrating T cells. Conclusions: Immunotherapy can inhibit the disease progression of intraocular metastasis. Enhancing the effects of tumor-infiltrating CD8+ T cells should be one of the highest potentials to improve the visual potential.