Fig 2: Influence of Extracellular ATP, AMP, and the Adenosine Analogs NECA, CGS 21680, and CADO on T Cell Proliferation(A and B) Human T cells were labeled with a proliferation dye, activated with anti-CD2/CD3/CD28 tetrameric antibody complexes, and treated with ATP, AMP, or the adenosine analogs NECA, CGS 21680, or CADO at the indicated concentrations on day 3 (A) or day 0 (B). As mock control, cells were not treated with ATP, AMP, NECA, CGS 21680, or CADO. Proliferation of T cells was analyzed by flow cytometry on day 5, and proliferation indices of CD8+ and CD4+ T cells were calculated. The mean + SD of three technical replicates is depicted. Representative results from two independent experiments are shown.

Fig 3: MAVS minor genotype is associated with sustained CD4+ T cell counts and a decreased level of cell-associated proviral DNA load during human immunodeficiency virus 1 (HIV-1) infection. (a) CD4+ T cell counts were assessed at 3, 5, and 7 years post seroconversion (p.SC) in untreated HIV-1-infected individuals carrying a MAVS minor or MAVS major genotype. (b) CD8+ T cell counts were assessed at 3, 5, and 7 years p.SC in individuals carrying a MAVS minor or MAVS major genotype. (c) CD4+ T cell counts and (d) the number of HIV-1 DNA copies per 5000 CD4+ T cells using qPCR were obtained from untreated HIV-1-infected individuals carrying the MAVS minor or MAVS major genotype 3.5–8.5 years p.SC. Each square or dot represents a different study participant (median ± IQR). All significant differences are indicated: * p < 0.05, ** p < 0.01, paired Wilcoxon test (a) and unpaired Mann–Whitney test (d).

Fig 4: Nectin-4-expressing tumor cells and CD137-expressing immune cells co-localize in human cancers. (A) Transcript co-expression across tumor types in TCGA. (B) MultiOmyx imaging allows for simultaneous interrogation of immune infiltrate and spatial proteomic profiling within human tumors. A single ROI (Region Of Interest) from a representative HNSCC (Head and Neck Squamous Cell Carcinoma) sample is shown. T cells (CD3+, red), macrophages (CD68+, blue), NK cells (CD56+, green), and tumor cells (PanCK+, cyan) detected throughout tumor (top left). Examples of CD137+CD4+ and CD137+CD8 T cells are shown and represented by white and gray arrows respectively (top right). Co-expression of Nectin-4 (red) and PanCK (blue) on tumor cells (bottom left). Tumor and stroma regions were identified using a PanCK and DAPI mask, respectively (bottom right, in red and blue, respectively). (C) Tumor Nectin-4 expression where total Nectin-4+PanCK+ cells are normalized to total cells (left) and CD137+ immune infiltrate where total CD137+ cells detected are normalized to total cells (right). Within each box, the horizontal line represents the mean of five samples shown. (D) Subset analysis of CD137+ immune infiltrate within stroma (left) and tumor (right) regions across samples are shown and included T cells (CD3+CD4+ and CD3+CD8+), macrophages (CD68+), NK cells (CD56+), and B cells (CD19+). Data are total cells per phenotype normalized to total CD137+ cells detected across samples within each indication. Within each box, the horizontal line represents the mean of five samples shown. In (A): ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; COADREAD, colorectal adenocarcinoma; DLBC, diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; GBMLGG, glioma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIPAN, Pan-kidney cohort (KICH+KIRC+KIRP); KIRC, renal clear cell carcinoma; KIRP, renal papillary cell carcinoma; LGG, brain lower grade glioma; LIHC, hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; NK, natural killer; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; STES, esophagus-stomach cancers; TCGA, The Cancer Genome Atlas; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Fig 5: Granule-Permissive-Sized Clearances Persist following Degranulation and Are a Feature of NK and Cytotoxic T Cells(A) Representative images of NK92 activated on anti-CD18- and anti-NKp30-coated glass for the times indicated, stained for F-actin, and imaged by time-gated STED microscopy. Scale bar, 5 µm.(B) Quantification of the clearances/µm2 measured in activated NK92. Each data point represents one cell from N = 58, 59, 59, and 59 cells from 4 pooled independent repeats. The p value was calculated by ordinary one-way ANOVA with Tukey’s post hoc comparison.(C) Quantification of mean hole area measured in fixed activated NK92 cells acquired by SIM at the indicated time points. N = 20 cells per condition; the experiment is representative of 3 independent repeats.(D) Representative images of NK92.LifeAct-mTurquoise cells showing F-actin (gray), lytic granules (red), and CD107a (green). Scale bar, 5 µm. The kymograph (below) produced from the region highlighted by the red square shown for and showing F-actin (gray) and CD107a (green). Images are representative of 20 cells from 2 independent experiments.(E) Primary human T cells were isolated from peripheral blood of healthy donors and activated on anti-CD3, anti-CD28, or anti-CD3/-CD28 for the times indicated prior to fixation and staining with phalloidin Alexa Fluor 488. Cells were imaged by time-gated STED microscopy. The regions inside the red boxes are magnified on the right of each panel. Scale bars, 5 µm and 1 µm (insets). Mean hole area was measured and is shown in the graph (right) of each condition. N = 10, 13, 24, 37, 41, 50, 49, 50, and 61 cells, respectively, pooled from 3 independent repeats. The p value was calculated by one-way ANOVA Kruskal-Wallis test (Dunn’s). Similar analysis was performed in primary T CD8+ and NK cells in Figure S2.