American Association for Cancer Research
ccr-22-3853_supplementary_material_1_suppms1.pdf (2.09 MB)

Supplementary Material 1 from Uncoupling CD4+ TIL-Mediated Tumor Killing from JAK-Signaling in Melanoma

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journal contribution
posted on 2023-05-12, 12:40 authored by Arianna Draghi, Mario Presti, Agnete W.P. Jensen, Christopher A. Chamberlain, Benedetta Albieri, Anne-Christine K. Rasmussen, Mads H. Andersen, Michael D. Crowther, Inge Marie Svane, Marco Donia

Supplementary Figures and Tables



Impaired MHCI-presentation and insensitivity to immune effector molecules are common features of immune checkpoint blockade (ICB)-resistant tumors and can be, respectively, associated with loss of β2 microglobulin (B2M) or impaired IFNγ signaling. Patients with ICB-resistant tumors can respond to alternative immunotherapies, such as infusion of autologous tumor-infiltrating lymphocytes (TIL). CD4+ T cells can exert cytotoxic functions against tumor cells; however, it is unclear whether CD4+ T-cell responses can be exploited to improve the clinical outcomes of patients affected by ICB-resistant tumors. Here, we exploited CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing to reproduce immune-resistant tumor phenotypes via gene knockout (KO). To determine the role of cytotoxic CD4+ TILs in ICB-resistant tumors, we investigated CD4+ TIL-mediated cytotoxicity in matched pairs of TILs and autologous melanoma cell lines, used as a model of patient-specific immune-tumor interaction. Around 40% of melanomas constitutively express MHC Class II molecules; hence, melanomas with or without natural constitutive MHC Class II expression (MHCIIconst+ or MHCIIconst−) were used. CD4+ TIL-mediated cytotoxicity was not affected by B2M loss but was dependent on the expression of CIITA. MHCIIconst+ melanomas were killed by tumor-specific CD4+ TILs even in the absence of IFNγ-mediated MHCII upregulation, whereas IFNγ was necessary for CD4+ TIL-mediated cytotoxicity against MHCIIconst- melanomas. Notably, although tumor-specific CD4+ TILs did not kill JAK1KO MHCIIconst- melanomas even after IFNγ stimulation, sensitivity to CD4+ TIL-mediated cytotoxicity was maintained by JAK1KO MHCIIconst+ melanomas. In conclusion, our data indicate that exploiting tumor-specific cytotoxic CD4+ TILs could help overcome resistance to ICB mediated by IFNγ-signaling loss in MHCIIconst+ melanomas.

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