Fig 2: CD8+ T cells express SLAMF7 dependent on their activation status.A, B WT (A) or OT-I (B) CD8+ T cells were activated with microspheres coated with increasing concentrations of αCD3 and αCD28 (A) or with T4- or N4-peptide pulsed MHC I complexes in combination with CD80 (B), respectively, or not (resting) and SLAMF7 abundances on CD8+ T cells were analyzed by flow cytometry after 24 h to 72 h. C WT (upper panel) or OT-I TCR transgenic CD8+ T cells (lower panel) were activated with microspheres coated with αCD3 and αCD28 or N4-peptide pulsed MHC I complexes, respectively, or not (resting). SLAMF7 surface expression and intracellular IFN-γ of CD8+ T cells was analyzed by flow cytometry after 24 h to 72 h. D Bar graph showing SLAMF7 expression on IFN-γ positive activated WT (C) CD8+ T cells (n = 4); an outlier was eliminated according to Grubbs test (α = 0.01). E Correlation of surface SLAMF7 and intracellular IFN-γ of activated WT (C) CD8+ T cells. Numbers indicate percentages of positive cells (A–C) or correlation coefficient (R). Data points represent biological replicates with mean and SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p-values were calculated using two-way ANOVA with Sidak’s multiple comparisons test (A, B, D) or linear regression analysis (E).

Fig 3: SLAMF7 facilitates initial population formation of activated CD8+ T cells.A OT-I CD8+ T cells were activated with microspheres coated with T4- or N4-peptide pulsed MHC I complexes in combination with CD80, or not (resting) and correlation of CD44 and SLAMF7 expression on CD8+ T cells was assessed by flow cytometry after 24 h. B, C WT (B) or OT-I (C) CD8+ T cells were activated with microspheres coated with αCD3 and αCD28 antibodies or as in A in combination with or without agonistic αSLAMF7 antibodies, respectively. Akt and Erk phosphorylation in non-activated WT CD8+ T cells or 90 and 300 min after activation was detected by Western Blot (B) and CD44 expression of T cells after 24 h by flow cytometry (C). D Inverted grayscale images of cell culture wells (left) and numbers (right) of CFSE-labelled population-forming WT CD8+ T cells activated by microspheres coated with increasing αCD3 and αCD28 antibody concentrations in combination with or without agonistic αSLAMF7 antibodies, respectively, or not (resting). Populations were counted by ImmunoSpot after 24 h, scale bar = 1 mm. E Size (left) and percentages of populations > 3000 µm2 (right) of WT CD8+ T cell populations (D). F T-cell population analysis as in D of OT-I CD8+ T cells activated by microspheres coated with CD80 and V4-, T4-, or N4-peptide pulsed MHC I complexes in combination with or without agonistic αSLAMF7 antibodies, respectively, or not (resting). Numbers indicate correlation coefficient (A), relative quantities of proteins (B), T-cell population counts (D, F), or percentages of positive cells (E). Data points represent biological replicates with mean and SD. ns - not significant, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p-values were calculated using two-way ANOVA with Sidak’s multiple comparisons test (C–F) or linear regression analysis (A).

Fig 4: SLAMF7 expression on CD8+ T cells is dependent on T-cell activation signals.A Multi-Epitope-Ligand-Cartography (MELC) analysis of CD3 (blue), CD8α (red), and SLAMF7 (green) distribution in mouse spleen sections. Arrows indicate overlapping areas (white) in the overlay. B WT or OT-I CD8+ T cells were activated with αCD3 and αCD28 coated microspheres or OVA257-264 (N4) peptide pulsed APCs, respectively, or not (resting) and SLAMF7 abundancies on CD8+ T cells were analyzed by flow cytometry after 24 h and 48 h. C, D WT CD8+ T cells were activated by microspheres coated with 0.25 or 0.75 µg/ml αCD3 (C) or αCD3 with or without αCD28 and/or IL-12 (D) and SLAMF7 expression on CD8+ T cells was analyzed as in B. E WT CD8+ T cells were activated with αCD3/αCD28-coated microspheres in the presence or absence (DMSO ctrl.) of STAT1 or STAT4 inhibitor or left untreated (resting). After 24 h and 48 h SLAMF7 mean fluorescent intensities (MFI) of CD44+ CD8+ T cells were analyzed by flow cytometry. Bar graph (right panel) shows SLAMF7 expression relative to controls. Numbers indicate percentages of positive cells (B) or MFI (E). Data points represent biological replicates with mean and SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p-values were calculated using two-way ANOVA with Sidak’s multiple comparisons test.

Fig 5: SLAMF7 signals enhance CD8+ T-cell responses towards low-affinity antigens.A WT CD8+ T cells were activated by microspheres coated with 0.25 µg/mL αCD3 and 0.5 µg/mL αCD28 antibodies in combination with or without increasing concentrations of agonistic αSLAMF7 antibodies, respectively, or not (resting). Proliferation of cells was monitored by CFSE dilution by flow cytometry after 24 h and 48 h. B Western Blot analysis of cell cycle regulators CDK6 and Cyclin D3 in WT CD8+ T cells at 24 h and 48 h after activation by microspheres coated with αCD3 and αCD28 antibodies in combination with or without agonistic αSLAMF7 antibodies, respectively, or not. C OT-I CD8+ T cells activated by microspheres coated with CD80 and V4-, T4-, or N4-peptide pulsed MHC I complexes in combination with or without agonistic αSLAMF7 antibodies, respectively, or not (resting). Proliferation of cells was analyzed by flow cytometry after 48 h and 72 h. D–F CFSE-labeled OT-I SLAMF7+/+ CD8+ T cells were activated by V4-peptide pulsed WT or SLAMF7−/− APCs and SLAMF7 expression of co-cultured cells was determined (D) and proliferation of CD8+ T cells (E, F) was analyzed by flow cytometry after 72 h and 96 h, respectively. G IFN-γ production of OT-I CD8+ T cells activated by microspheres coated with CD80 and V4- or T4-peptide pulsed MHC I complexes in combination with or without agonistic αSLAMF7 antibodies, respectively, or not (resting). H, I Cytotoxicity assay of OT-I CD8+ T cells 48 h pre-activated by microspheres coated with V4- (H), T4-, or N4-peptide (I) pulsed MHC I complexes in combination with or without CD80 and agonistic αSLAMF7 antibodies, respectively, or not (resting). Cytolytic capacity was assessed by decrease of N4-pulsed high fluorescent target cells in relation to non-pulsed low fluorescent control cells. Numbers indicate percentages of positive cells (A, C–H) or relative quantities of proteins (B). Data points represent biological replicates with mean and SD (A, C, G) or range, interquartile, and median (F, H, I). *p < 0.05; **p < 0.01; ****p < 0.0001; p-values were calculated using two-way ANOVA with Sidak’s multiple comparisons test.