Fig 2: Mechanism associated with the inhibitory function of SLAMF6.(A) RNA expression of Sh2d1a transcript (SAP) in WT and SLAMF6 -/- splenocytes. (B) Immunoblot analysis of expression of SLAMF6, SAP and SHP-1 in WT and SLAMF6 -/- splenocytes. (C) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were activated with gp10025-33 peptide for the indicated time points. At the end of the activation, cells were fixed and stained for phosphorylated S6. (D) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were either activated with gp10025-33 peptide in the presence of IL-2 (30 IU/ml) for 18 hr or kept only with IL-2 for 18 hr (non-activated). After 18 hr, the cells were lysed, RNA was extracted, and quantitative RT-PCR for transcription factors expression was performed. Data was normalized to Hprt expression for each mouse strain. Values for each condition were normalized to Pmel-1 non-activated values for each gene. (E) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml) for 7 days. After the expansion phase, the cells were kept for an additional 5 days without supplements. Expression of exhaustion markers was measured in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes. (F) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml) and 10 µg/ml anti-LAG-3 or isotype control for 7 days, and then incubated overnight with B16-F10/mhgp100 melanoma cells at a 1:1 effector-to-target ratio. IFN-? secretion was measured by ELISA. (G–I) B16-F10/mhgp100 mouse melanoma cells were injected s.c. into the back of C57BL/6 mice. Pmel-1 x SLAMF6 -/- mouse splenocytes were expanded with gp10025-33 peptide and IL-2 (30 IU/ml) in the presence of either Anti-Lag3 or Isotype control. On day 7, Isotype or Anti-Lag3 activated cells were adoptively transferred i.v. into the irradiated tumor-bearing mice. Anti-Lag3 or Isotype control were injected i.p. five times in the 2 weeks post-transfer. N = 5 mice per group. Tumor size was measured three times a week. (G) Scheme of the experimental layout. (H) Tumor volume (Mean ± SEM) until day 30 post-tumor inoculation. (I) Tumor volume on day 16 post-tumor inoculation. *, p<0.05, **, p<0.01.

Fig 3: SLAMF6 is constitutively expressed on T cells and increases upon activation.(A–C) SLAMF6 expression in human TIL412 cells, activated for five days. (A) Flow cytometry at the indicated time points. (B) Median fluorescence intensity (MFI) of SLAMF6, days 1–5. (C) Quantitative RT-PCR for SLAMF6. RNA was extracted at the indicated time points. Data normalized to HPRT expression at each time point and to the basal expression level on day 0. One-way ANOVA. **, p<0.01, ***, p<0.001. (D) SLAMF6 expression by flow cytometry in human TIL412 cells activated for 5 days with anti-CD3 or with anti-CD3 plus IL-2, at the indicated time points. (E) SLAMF6 expression by flow cytometry in Pmel-1 mouse splenocytes activated for 6 days, at the indicated time points. (F) Row normalized expression of immune-related genes from RNAseq, clustered according to similar expression patterns. CD4+ T cells from two donors were stimulated with anti-CD3 plus anti-CD28 for 72 hr, RNA was extracted and sequenced. Numbers in the top panel indicate hours. (G) Magnification of cluster C. SLAMF6 is marked.Figure 1—source data 1.RNA sequencing of healthy donors CD4 T cells along activation.

Fig 4: Pmel-1 x SLAMF6 -/- T cells have a better functional capacity.(A–D) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated overnight with B16-F10/mhgp100 melanoma cells. (A) The cells were incubated at a 1:1 effector-to-target ratio. IFN-? secretion was measured by ELISA. Each point represents one mouse. (B) The cells were incubated at the indicated effector-to-target ratios. IFN-? secretion was measured by ELISA. (C) The cells were incubated at a 1:1 effector-to-target ratio. GM-CSF secretion was measured by ELISA. Each point represents one mouse. (D) Conditioned medium was collected and analyzed with Quantibody mouse cytokine array. (E, F) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated for 16 hr with B16-F10/mhgp100 melanoma cells. Granzyme-B expression was detected by flow cytometry. One representative experiment (E) and a summary of triplicates (F) are shown. (G–J) B16-F10/mhgp100 mouse melanoma cells were injected s.c. into the back of C57BL/6 mice. Pmel-1 or Pmel-1 x SLAMF6 -/- mouse splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml). On day 7, Pmel-1 cells or Pmel-1 x SLAMF6 -/- cells were adoptively transferred i.v. into the irradiated tumor-bearing mice. N = 8 mice per group. Tumor size was measured three times a week. (G) Scheme of the experimental layout. (H) Spider plots showing tumor volume [calculated as L (length) x W (width)2 x 0.5]. CR, complete response. (I) Normalized tumor volume (Mean ± SEM) until day 45, on which the first mouse had to be sacrificed. Tumor dimensions were normalized to the 1st measurement. (J) Kaplan Meier survival curve. (K) Percent T cells specific for gp10025-33 peptide in the spleen or tumor draining lymph nodes (DLN) of mice sacrificed 7 days post-ACT. Tet, tetramer. Student t test. *, p<0.05, **, p<0.01, ***, p<0.001.

Fig 5: SLAMF6 expressed in trans by a melanoma target inhibits anti-tumor T cell reactivity.(A) SLAMF6 expression on B16-F10/mhgp100 parental or transfected (SLAMF6 or empty) melanoma cells. (B) Pmel-1 splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml), and then incubated overnight with B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 melanoma cells at the indicated effector-to-target ratios. IFN-? secretion was measured by ELISA. (C) Pmel-1 splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml), and then incubated overnight with B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 melanoma cells. IFN-? production was detected by intracellular staining and flow cytometry (gated on CD8+). Three replicates. The gating strategy is illustrated in Figure 2—figure supplement 1. (D, E) Pmel-1 splenocytes were expanded with gp10025-33 peptide (1 µg/ml) and IL-2 (30 IU/ml) for 7 days. On day 7, cells were transferred i.v. into irradiated C57Bl/6 mice bearing palpable (1 week) B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 tumors. IL-2 (0.25 × 106 IU) was administered i.p. twice a day for 2 days. Tumor growth was measured twice a week. Mice were sacrificed when the tumor reached 15 mm in diameter. (D) Scheme showing experimental layout. (E) Spider plot showing tumor volume [calculated as L (length) x W (width)2 x 0.5]. One-way ANOVA. *, p<0.05, **, p<0.01, ***, p<0.001.