Fig 2: Porins-induced tumor-infiltrating TCF-1+ Trm CD8+ T cell correlates with melanoma protection. Vaccination scheme as in Figure 6A , data is presented 21 days after the melanoma challenge. (A) CD8+ (green) T cells expressing TCF-1 (cyan) from tumors (representative immunofluorescence micrographs from a zoomed area) (Scale bar = 20μm). (B) TCF-1 medium fluorescent intensity (MFI) from CD8+ and PD-1+ CD8+ TILs. (C) Flow cytometry of PD-1+ expressing CD8+ Trm T cells. Frequencies and absolute numbers of CD8+ Trm (D) and PD-1+ CD8+ Trm (E). (F) Flow cytometry of CD8+ Trm T cells expressing PD-1+ and TCF-1+. (G) Frequencies and absolute numbers of TCF-1+ PD-1+ CD8+ Trm T cells. (H) TCF-1 MFI from CD8+ Trm and PD-1+ CD8+ Trm. Statistical data were pooled from three independent experiments with 3–4 mice per experimental condition. *p < 0.05, **p < 0.01.

Fig 3: Model of vaccination strategy protecting against melanoma using Salmonella Typhi Porins. Depicted are the circulating from skin-draining lymph nodes (SDLN), skin and tumor-infiltrating Trm T cell response induced by Porin immunization. Porins as an adjuvant induce a circulating memory T cell response. In addition, the immunization induces a Trm response in the skin and a formation of functional Trm T cells in the tumor. A Porins immunization is capable to induce tumor-infiltrating TIM-3+ PD-1+ CD8+ Trm T cells (potential terminally exhausted Trm) and importantly TCF-1+ PD-1+ CD8+ Trm T cells (progenitor exhausted Trm), being the latter associated with tumor growth control and anti-PD-1 immunotherapy cooperation. Created with BioRender.com.

Fig 4: TCF-7+ progenitor exhausted Trm CD8+ TILs correlates with disease control in melanoma patients. Merged images of TCF-7+ (green) PD-1+ (yellow) CD8+ (red) (A) and TCF-1+ (green) CD103+ (yellow) CD8+ (red) (B) TILs from healthy control skin or from tumor stroma derived from metastatic or disease-free melanoma patients (representative immunofluorescence micrographs from a zoomed area) (Scale bar = 20μm). (C) Percentages of TCF-7+ CD103+ CD8+ T cells are shown. Statistical data were pooled from a comparison of healthy skin (N = 7), and metastatic (N = 5) and disease-free melanoma patients (N = 7). ***p < 0.001, ****p < 0.0001.

Fig 5: Bemcentinib induced type I interferon secretion is required for TCF1+PD-1+ CD8 cell expansion(A) BMDCs were co-cultured with irradiated KPL cells (40 Gy) and treated with BGB324 (40 nM) for 24 h. Conditioned medium was collected for IFN-ß ELISA. **p < 0.01.(B) C57BL/6J mice (n = 5) were injected with 1 × 106 KPL tumor cells and treated with a control IgG (IgG) or interferon alpha receptor blocking antibody (anti-IFN-aR, see STAR Methods). Mice were treated with BGB324 (50 mg/kg, twice daily) and anti-PD-1 (10 mg/kg, day 7, 10, 14) (combined), or corresponding IgG and vehicle (Ctrl) starting on day 7 post tumor cell injection. Tumor volume was measured every 3 days. ****p < 0.001.(C) Abundance of TCF1+PD-1+ cells among gated CD8+ T cells (per mm3 of tumor) at day 7 post therapy initiation. Experimental schema is same as (B). ****p < 0.001.(D) Mean fluorescent intensity (MFI) for TCF1 in CD8+ OT-1 cells. BMDCs were co-cultured with isolated CD8+ T cells stimulated with ovalbumin (see STAR Methods). Each dot represents one biological replicate. ****p < 0.001.(E) MFI of TCF1 in CD8+ OT-1 cells. Wild-type BMDCs or Axl-/- BMDCs were co-cultured with isolated CD8+ T cells stimulated with ovalbumin for 48h, respectively. IFN-aR blocking strategy is as in (B). **p < 0.01.(F) Rag1-/- mice (n = 5) were transferred with CD8+ T cells isolated from wild-type or Ifnar1-/- mice. After 7 days of being transferred, mice were injected with 1 × 106 KPL tumor cells and treated with BGB324 (50 mg/kg, twice daily) and anti-PD-1 (10 mg/kg, day 7, 10, 14) (combined), or corresponding IgG and vehicle (Ctrl). Tumor volume was measured every 3 days. ****p < 0.001.