Fig 2: Artemisinin (ART) reduces T cell infiltration in cardiac allografts. Lymphocytes (CD45+), T cells (CD3+), CD4+ T cells (CD3+CD4+), and CD8+ T cells (CD3+CD8+) in cardiac allografts were detected by flow cytometry. Representative histograms and quantitative analysis of frequencies and counts of lymphocyte cells (A–C), T cells (D–F), CD4+ T cells and CD8+ T cells in allografts (G–K) (n = 5/group). (L–O) Representative immunohistochemistry staining images and results of quantitative analysis of cell numbers/view based on CD3, CD4, CD8, and Foxp3 in the Control and ART groups (n = 5/group). Magnification: 200× and 400×. *P < 0.05; ** P < 0.01; *** P < 0.001. NS, no significance.

Fig 3: 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 4: Intratumoral distribution of CD4+ and CD8+ TILs correlates strongly with the intratumoral penetration of HCT-mono-mIL12. (A–C) Treatment scheme of CT26-HER2/neu tumor-bearing mice initiated at a tumor volume of ~300 mm3 with i.p. injection of HCT-mono-mIL12 at 1.6 μg per dose (an equimolar amount of 0.5 μg rmIL12 per dose) three times on day 14, 18, and 21 after tumor inoculation (A) to determine the intratumoral distribution of total CD4+ and CD8+ TILs in relation to the blood vessels (B) and IFNγ-producing cells among CD4+ and CD8+ TILs (C) at 2 days after the third dosing determined by IF staining. In (A), the arrows indicate each time point for treatment or assay. In (B, C), tumor tissues were excised and stained for CD4 or CD8 (Alexa Fluor 488, green) with CD31 (TRITC, red) (B) and/or IFNγ (TRITC, red) (C). Blue represents nuclei staining. Image magnification, ×200; scale bar, 50 μm. The bar graphs depict the number of the indicated cells per mm2 in a tumor section. Data represent mean ± SEM of four fields per tumor (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups; ns, not significant.

Fig 5: AAI induces robust renal infiltration of cytotoxic T cells.(A) The UMAP visualization shows unsupervised scRNA-Seq clustering, revealing 9 distinct subtypes of T lymphocyte and NK cells. CD4+Tn, CD4+ T naive; CD4+Te, CD4+ T effector; CD4+Tem, CD4+ T memory; CD8+ Tn, CD8+ Tnaive; CD8+CTL, CD8+ cytotoxic T cell; CD8+Tem, CD8+ T memory; T Pro, T proliferation; NK, NK cell. (B) The heatmap depicts the cell markers expression of each cell subtype in the T lymphocyte and NK cells subpopulation. (C) The pie chart revealed the relative proportion of each cell subtype of T lymphocyte and NK cells in the Con (upper panel) and the AAN groups (lower panel). (D) Cumulative distribution function shows the distribution of naive, cytokine, cytotoxic, and regulatory state scores across T lymphocyte and NK cell subpopulation. (E) The UpSet plot depicts the concordance of upregulated differentially expressed gene (DEG) numbers of each cell subtype in T lymphocyte and NK cell subpopulations. The Venn plot shows the overlap genes number between subgroup union DEGs and whole T lymph/NK DEGs. (F) The bubble plot shows the GO enrichment BP items of AAN versus Con upregulated DEGs in the whole T lymph/NK subgroup. (G) The scatter plot shows the relative gene expression level of 12 cytokines (upper), cytotoxic (middle), and regulatory (lower) genes in pseudotime, colored according to group types. (H) Representative immunofluorescence staining of CD4 (green) and CD8 (red) (n = 3 per group). Scale bar: 100 μm.