Fig 2: DNAM-1, TIGIT and CD96 are dose-dependently upregulated by rhIL-15 and DNAM-1 is downregulated by OC tumor cells. (a) DNAM-1 (n = 8), TIGIT (n = 6) and CD96 (n = 5) expression on NK cells of healthy donors co-cultured with SKOV-3, IGROV-1 or OVCAR-3 spheroids and increasing rhIL-15 concentrations for 7 days. Data is shown as mean+SEM. One-Way ANOVA with Bonferroni correction was used for statistical analysis, * p < .05, ** p < .01 and *** p < .001. (b) DNAM-1, TIGIT and CD96 expression on healthy donor NK cells co-cultured with a patient-derived primary tumor cell line and increasing rhIL-15 concentrations for 7 days cultured in duplicate. (c) DNAM-1, TIGIT and CD96 expression on healthy donor NK cells co-cultured with patient-derived tumor cells and increasing rhIL-15 concentrations for 7 days cultured in duplicate

Fig 3: SKOV-3 tumor-bearing mice have significantly reduced DNAM-1 expression in CD56dim and CD56bright NK cells. (a) Schematic overview of the mouse experiment. (b) Bioluminescence imaging (BLI) signal of the SKOV-3 tumor-bearing mice over time (n = 5). (c) Expression of DNAM-1, TIGIT and CD96 on CD56dim (left) and CD56bright (right) NK cells by flow cytometric measurement on day 0. The top graphs depict percentage positive cells and the bottom graphs depict ?MFI. (d) Expression of DNAM-1, TIGIT and CD96 on intraperitoneal NK cells harvested 14 days after adoptive transfer in SKOV-3 tumor-bearing NSG mice and control mice. Day 0 data is represented by dotted lines as a reference. The top graphs depict percentage positive cells and the bottom graphs depict ?MFI. Cumulative data are shown (lines indicate mean, n = 5 per group). A One-Way ANOVA with Bonferroni correction was used for statistical analysis, * p < .05 and *** p < .001. (e) Representative plots are shown of CD107a and IFN? expression by CD56dim NK cells after 4 h co-culture with SKOV-3 target cells, low dose rhIL-15 and TIGIT blockade or matching isotype control. (f) The percentage of CD107a and IFN? expressing CD56dim NK cells are shown on day 0 and day 14. Day 14 results are pooled NK cells from 5 different mice from either tumor-bearing or control mice

Fig 4: CD96 provides a negative signal in autoimmune vasculitisCD4+ T cells from patients with GCA were transfected with CD96-specific or control siRNA and adoptively transferred into chimeric NSG mice that had been engrafted with human arteries. Human artery grafts were explanted after 2 weeks and processed for RNA extraction and immunohistochemical analysis.(A) Tissue sections of explanted arteries were stained with hematoxylin and eosin to assess the invasion of the arterial media by inflammatory cells.(B) Density of the infiltration was quantified based on nuclear counts in H&E-stained images (n = 7).(C and D) Tissues were stained for CD3 (red) to estimate the density of tissue infiltrating T cells. Cell numbers were enumerated.(E and F) Immunostaining of microvessels in the arterial wall. Vascular lumina visualized as vWF+/aSMA+ structures. Enumeration of microvessels shown as bar graphs.(G) Tissue transcriptomics for T cell receptor (TCR) and cytokines in grafts from mice injected with control or CD96 siRNA-transfected CD4+ T cells. RT-PCR data shown as heatmap.Scale bars: 100 µm (A) and 20 µm (C and E). Mean ± SEM with individual values shown. (B and D) Two-tailed paired t test. (F) Two-tailed unpaired t test. (G) Two-tailed paired t test or Wilcoxon matched-pairs signed-rank test. *p < 0.05, **p < 0.01, n.s., not significant.

Fig 5: Enrichment of CD96hi CD4+ T cells in giant cell arteritis (GCA)(A–D) Freshly harvested peripheral blood mononuclear cells (PBMCs) from patients with GCA and age-matched healthy individuals were immunophenotyped by multiparametric flow cytometry for the expression of 6 immunoinhibitory receptors. CD4+ memory T cell clusters were classified based on CD45RA and CCR7 expression.(A) Uniform manifold approximation and projection (UMAP) clustering of memory CD4+ T cells, delineating 10 clusters among central memory cells (CD45RA- CCR7+), effector memory cells (CD45RA- CCR7-), and terminally differentiated effector memory cells (TEMRAs; CD45RA+ CCR7-). Data are a mix of samples from 8 patients and 8 controls. Plot of 10 identified PhenoGraph clusters is overlaid on the UMAP projection.(B) Heatmap demonstrating expression of the 6 checkpoint receptors in the 10 clusters based on mean fluorescence intensity (MFI). z scores are shown.(C–E) UMAP clustering of memory CD4+ T cells from 8 healthy individuals (C) and 8 patients with GCA (D). Quantified frequencies (%) of each cluster are presented as pie charts. T cell subsets that are enriched or reduced in patients with GCA are indicated (E).(F) CD96 mRNA transcripts were quantified by RT-PCR. Data from 10 patients and 10 controls.(G) CD96 expression quantified by flow cytometry in PBMCs from a confirmation cohort of 16 patients and 16 age-matched controls. MFIs are shown, and each dot represents one patient or control.(H) CD4+ T cells isolated from controls and patients with GCA were stimulated for 0, 3, or 6 days. Kinetics of CD96 expression measured by flow cytometry (n = 4 patients, n = 4 controls).(I and J) Temporal arteries were collected from diagnostic biopsies of patients with GCA and sinus biopsies from patients with granulomatosis with polyangiitis (GPA).(I) Targeted tissue transcriptomic analysis of GCA-affected temporal arteries compared with non-inflamed arteries. CD96 transcripts evaluated by RT-PCR. Each lane represents one tissue sample.(J) Temporal artery (left) and sinal tissue (right) sections were stained for CD3 (red) and CD96 (green). Nuclei marked by DAPI. Representative images from 4 patients. Frequencies of CD3+ CD96high T cells quantified in 50 randomly selected high-power fields. Scale bar: 20 µm. Mean ± SEM with individual values shown.(F) Mann-Whitney test. (G) Two-tailed unpaired t test. (H) Mann-Whitney test and two-tailed unpaired t test. *p < 0.05, **p < 0.01.