cd8 staining  (Servicebio Inc)

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) Heatmaps depict the enrichment of immune and non-immune cell types in the immediate neighborhood of CCR7 + DCs in NSCLC spatial transcriptomic data ( n = 4). (B) (Left) Representative FOV displaying CCR7 + DCs (HLA-DR + LAMP3 + ; yellow) located near BVs (CD31 + PDPN − ; magenta) and Tregs (CD4 + FOXP3 + ; white) in one HNSCC sample using high-plex whole-tissue imaging. Scale bar represents 20 μm. (Right) Box plots display the frequencies of BV-associated, LV-associated, and non-vessel-associated CCR7 + DCs close (<5 μm) to Tregs among all tumor CCR7 + DCs with nearby Tregs. Wilcoxon test, whiskers represent min to max; * p < 0.05. (C) Correlations between CCR7 + DCs and Tregs within CD45 + cells, as determined by scRNA-seq in multiple human cancer types. Spearman rank correlation; significant correlations are shown with a fitted red line. (D) (Left) Scheme outlining the analyses of CCR7 + DCs and Tregs in NSCLC samples. Patients with numerous (>5) CCR7 + DC clusters ( n = 12) were selected for downstream analyses. (Right) Frequency of CCR7 + DCs (CD11c + LAMP3 + ) with at least one nearby (<50 μm) Treg (CD4 + FOXP3 + ) in each individual patient. Numbers of FOVs analyzed per sample are as follows: NR01, n = 126; NR06, n = 455; NR09, n = 180; NR12, n = 79; NR26, n = 293; R11, n = 122; R15, n = 205; R35, n = 175; R37, n = 459; R45, n = 276. (E) (Left) Scheme outlining the analysis of tumor biopsies from HNSCC patients before immunotherapy (pre-IO). Patients were divided into non-responders (NR, n = 5) and responders (R, n = 5) based on the assessment of clinical response at 6 months. (Right) CCR7 + DC shortest distance to Tregs, T CONV , and CD8 + T cells in NR versus R tumors. Data are shown for all CCR7 + DCs compiled (NR tumors, n = 1,457 cells; R tumors, n = 1,324 cells). Unpaired t test, whiskers represent min to max; **** p < 0.0001. Numbers of FOVs analyzed per sample as in (D). (F) (Left) Scheme outlining the analyses of CCR7 + DC-CD8 + T cell niches. (Right) Frequencies of CCR7 + DC-CD8 + T cell niches with or without Tregs in their proximity (<100 μm). Two-way ANOVA with multiple comparisons, whiskers represent min to max; * p < 0.05. Numbers of FOVs analyzed per sample as in (D). (G) Representative FOV displaying CCR7 + DCs (FSCN1 + cells; FSCN1 in yellow) located near BVs (CD31 + LYVE-1 − cells; CD31 in magenta) and Tregs (FOXP3 + cells; FOXP3 in white) in untreated MC38 tumors. Scale bar represents 50 μm. (H) Correlations between the numbers of CCR7 + DCs and Tregs per mg of tumor tissue, as determined by fluorescence-activated cell sorting (FACS) analyses of MC38 and D4M3. A tumors. Spearman rank correlation; significant correlations are shown with a fitted red line. (I) Box plots show the frequencies of tumor CCR7 + DCs close (<5 μm) to Tregs that are associated to BVs or LVs in MC38 tumors ( n = 7). Whole-tumor sections were analyzed. Paired t test, whiskers represent min to max; **** p < 0.0001. See also and .

Article Snippet: AF550 mouse anti-human CD8 (Clone AKYP0028) , Akoya Biosciences , Cat#S6501001.

Techniques: Imaging, Fluorescence, FACS

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) (Left) Scheme outlining the experimental setup for bulk RNA-seq analyses of tumor-derived CCR7 + DCs. (Right) GO pathway enrichment analyses performed on differentially expressed genes (DEGs) in CCR7 + DCs in MC38 tumors ( n = 4) from Treg-depleted ( FoxP3 -DTR) compared with Treg-sufficient (WT) mice. Bar plot indicates the −log 10 raw binomial p -values of the top 10 most enriched pathways in CCR7 + DCs. (B) (Left) Experimental setup for ex vivo stimulation of OT-I CD8 + T cells with tumor CCR7 + DCs. (Right) Percentage of OT-I CD8 + T cells that proliferated after 5-day culture with OVA 257–264 peptides-loaded CCR7 + DCs isolated from WT or Treg-depleted tumors. As a control, CCR7 + DCs without OVA 257–264 peptides were used. Two-way ANOVA with multiple comparisons, whiskers represent min to max; ** p < 0.01. (C) (Left) Relative gene expression levels analyzed by bulk RNA-seq. Each dot represents one mouse ( n = 4), whiskers represent mean to max. Unpaired t test with multiple comparisons; * p < 0.05. (Right) Representative histogram of CD40 protein expression and relative mean fluorescence intensity (MFI) measured by FACS and expressed both as normalized values and absolute MFI. Each dot represents one mouse ( n = 18), whiskers represent min to max. Unpaired t test; ** p < 0.01. (D) Analyses of cDCs in tumor-draining lymph nodes. Absolute cell counts (left, n = 10) and MFI of CD40 expression (right, n = 18) measured by FACS in migratory cDCs (CCR7 + CD8α − ) from WT or Treg-depleted mice. Whiskers represent mean to max. (E) (Left) Experimental setup for ex vivo analyses of tumor CCR7 + DCs isolated from anti-PD-1-treated mice that received or not αCD25 NIB mAbs. (Right) CD40 protein expression measured by FACS and expressed both as normalized values and absolute MFI. Each dot represents one mouse ( n = 4 WT and n = 6 FoxP3-DTR), whiskers represent min to max. Unpaired t test; ** p < 0.01. (F) (Left) Overall survival analyses of MC38 tumor-bearing mice treated, or not treated, with αPD-1 and αCD25 NIB mAbs, and in which CD4 + or CD8 + cells were depleted or not ( n = 8 or 9 mice/group). Log-rank Mantel-Cox test; * p < 0.05, *** p < 0.001, and *** p < 0.0001. (Right) Percentage of tumor-free mice on day 60 in the indicated treatment groups. (G) (Left) Experimental setup for ex vivo stimulation of OT-I CD8 + T cells with tumor CCR7 + DCs as in (B). The DCs were obtained from mice receiving anti-PD-1 immunotherapy and that were treated or not with αCD25 NIB mAbs. (Right) Percentage of OT-I CD8 + T cells that proliferated after 5-day culture with OVA 257–264 peptide-loaded CCR7 + DCs. Each dot represents one mouse ( n = 8 and n = 7), whiskers represent min to max. Two-way ANOVA with multiple comparisons; * p < 0.05. (H) (Left) Scheme outlining bone marrow chimeras with inducible Cd40 -deficiency in cDCs and the treatment schedule. (Right) Growth curves of MC38 tumors inoculated in zDC iDTR : Cd40 WT and zDC iDTR : Cd40 KO bone marrow chimeras treated with αPD-1, αCD25 NIB , or αPD-1 + αCD25NIB combination ( n = 8–10 mice/group). Mean with SEM. Two-way ANOVA with multiple comparisons; * p < 0.05 and **** p < 0.0001. See also and .

Article Snippet: AF550 mouse anti-human CD8 (Clone AKYP0028) , Akoya Biosciences , Cat#S6501001.

Techniques: RNA Sequencing, Derivative Assay, Ex Vivo, Isolation, Control, Gene Expression, Expressing, Fluorescence

Journal: Cell Reports Medicine

Article Title: ENPP1 inhibitor with ultralong drug-target residence time as an innate immune checkpoint blockade cancer therapy

doi: 10.1016/j.xcrm.2025.102336

Figure Lengend Snippet: STF-1623 boosts intratumoral cGAMP levels to suppress murine breast cancer growth and metastasis (A–C) cGAMP levels (A), IFN-γ levels (B), and relative Ifn-γ mRNA expression (C) in subcutaneous EMT6 (top) or EMT6 shEnpp1 (bottom) tumors at time points indicated after STF-1623 (50 mg/kg) injection. For cGAMP measurement, background measured in vehicle controls was subtracted from the measurements. Mean ± SEM is plotted, n = 3 for EMT6 tumors; n = 4–5 for EMT6 shEnpp1 tumors. (D) BALB/c female mice with established subcutaneous EMT6 tumors received treatment as indicated. Mean ± SEM is plotted. (E) Percent lung metastatic burden determined by hematoxylin and eosin staining of mice in (D) on day 29 of the study. Mean ± SD is plotted, n = 9–10 mice. (F) Immunohistochemistry of CD8 + T cells in randomly selected tumors of mice in (D) on day 29 of the study. Mean ± SD is plotted, n = 5 mice. (G) BALB/c female mice with established orthotopic 4T1 tumors received treatment as indicated. Mean ± SEM of tumor volume is plotted. (H) BALB/c female mice with established orthotopic 4T1 tumors received treatment as indicated. Mice were sacrificed when exhibiting symptoms related to metastasis. The p value for the Kaplan-Meier analysis was determined by log rank test. p values were determined by two-sided unpaired t test unless otherwise mentioned. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; p value is shown if between 0.05 and 0.1. See also .

Article Snippet: To characterize tumor infiltrating lymphocytes, CD8 primary antibody staining (CST, #98941; 1:400) followed by anti-rabbit poly-HRP-IgG (Leica, #DS9800) of tumor samples were performed.

Techniques: Expressing, Injection, Staining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: ENPP1 inhibitor with ultralong drug-target residence time as an innate immune checkpoint blockade cancer therapy

doi: 10.1016/j.xcrm.2025.102336

Figure Lengend Snippet: STF-1623 delays murine colorectal tumor growth (A) C57BL/6 female mice with established subcutaneous MC38 tumors received treatment as indicated. Mean ± SEM of tumor volume is plotted. (B) Percent weight change of mice in (A) on day 14 compared to day 1 of the study. Mean ± SD is plotted, n = 10 mice per group. (C) ENPP1 activity of WT, ENPP1 WT-OE , and ENPP1 T238A-OE MC38 cell lysates assessed by [ 32 P] cGAMP hydrolysis by thin-layer chromatography. (D) MC38 ENPP1 WT-OE and MC38 ENPP1 T238A-OE cells were subcutaneously injected in WT female BALB/c mice. n = 9 for ENPP1 WT-OE and n = 10 for ENPP1 T238A-OE . Mean ± SEM of tumor volume is plotted. (E) C57BL/6 female mice with established subcutaneous MC38 ENPP1 WT-OE tumors received treatment as indicated. Mean ± SEM of tumor volume is plotted, n = 8–9 mice. (F) Percent weight change of mice in (E) on day 14 of study compared to day 1 of the study. Mean ± SD is plotted, n = 8 or 9 mice per group. (G) BALB/c mice with established CT26 tumors received treatment as indicated. Mean ± SEM is plotted, n = 10 mice. (H) Percent weight change of mice in (G) on day 13 compared to day 1 of the study. Mean ± SD is plotted, n = 10 mice. (I) BALB/c mice with established subcutaneous CT26 tumors received 5 or 50 mg/kg of STF-1632 on days 9–11, 16–18 post inoculation ( pi ), 10 mg/kg anti-PD-1 intraperitoneally on day 9, 12, 16, and 19 pi , or a combination of the two. On day 23 of the study, tumors were isolated and processed for flow cytometry. The number of CD3 + T cells, CD4 + CD3 + T cells, and CD8 + CD3 + T cells per gram of tumors is shown. Mean ± SEM is plotted, n = 10 mice. p values were calculated by two-sided unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: To characterize tumor infiltrating lymphocytes, CD8 primary antibody staining (CST, #98941; 1:400) followed by anti-rabbit poly-HRP-IgG (Leica, #DS9800) of tumor samples were performed.

Techniques: Activity Assay, Thin Layer Chromatography, Injection, Isolation, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: ENPP1 inhibitor with ultralong drug-target residence time as an innate immune checkpoint blockade cancer therapy

doi: 10.1016/j.xcrm.2025.102336

Figure Lengend Snippet: STF-1623 controls Panc02 pancreatic tumor growth by activating anti-cancer immunity (A and B) BALB/c female mice with established subcutaneous Panc02 tumors received treatment indicated. On day 12 of the study, tumors were processed for flow cytometry: the percentage of IA-IE + CD206 low M1 macrophage (F4/80 + GR-1 − ) over IA-IE − CD206 high M2 macrophage, the number of CD335 + CD3 − NK cells, CD335 + CD3 + NKT cells, CD4 + CD3 + T cells, and CD8 + CD3 + T cells per gram of tumors, and the ratio of CD8 + T cells over Foxp3 + CD4 + Treg cells (A), CD69 + CD4 + T cells, PD-1 + CD4 + T cells, Ki67 + CD4 + T cells, CD69 + CD8 + T cells, PD-1 + CD8 + T cells, and Ki67 + CD8 + T cells (B). Mean ± SD is plotted, n = 6 mice for vehicle and STF-1623 groups, and n = 3 for all other combination therapy groups. (C) BALB/c female mice with established Panc02 tumors received the treatment as indicated. Mean ± SEM of tumor volume is plotted, n = 9–12 mice. Spider plots of individual tumor growth are shown. (D) Percent weight change of mice in (C) on day 45 compared to day 1 of the study. Mean ± SD is plotted, n = 9–12 mice. NK, natural killer; NKT, natural killer T; Treg, regulatory T cells; IR. ionizing radiation. p values were calculated by two-sided unpaired t test unless otherwise noted. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; p value is shown if between 0.05 and 0.1.

Article Snippet: To characterize tumor infiltrating lymphocytes, CD8 primary antibody staining (CST, #98941; 1:400) followed by anti-rabbit poly-HRP-IgG (Leica, #DS9800) of tumor samples were performed.

Techniques: Flow Cytometry

Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 5. Cytotoxic T cells (CTLs) exhibit close proximity to tumors with a suppressive impact on tumor proliferation (A) Density gradient in the background indicates distance to tumor islands, with brighter yellow representing greater distance from tumor cells. Left panel only has the gradient whereas the middle, zoomed panel additionally depicts yellow masks of the CTLs and the right panel red dots of helper T cells (TH), respectively. (B) Representative images showcasing markers for T cell phenotyping. (C) T cell gating based on the expression of CD3, CD8, CD45RA, CD45RO, and PD1 to analyze the corresponding T cell subpopulations. (D) Representative images showing CD3- and CD8-positive CTLs in tumor parenchyma (TIL-CTLs) with absent CD45RA and CD45RO expression. (E) Distance heatmap depicting proximity and interactions between tumor cells and T and B lymphocyte subtypes, with lower values indicating higher proximity as in Figure 4E. (F) Histogram of Ki67/Ki67+ tumor cell number as a function of distance from RARO CTLs. Data represent three technical replicates. Statistical significance was determined using one-way ANOVA. ***p < 0.001, **p < 0.01. (G) Representative image displaying masks of tumor cells at varying proximities to RARO TIL-CTLs.

Article Snippet: For CD8 + HIF1a + GSTP1/TRAP1 staining, sections were incubated overnight at 4 Cwith primary antibodies (anti-GSTP1, 1:100, Proteintech 15902-1-AP; or anti-TRAP1, 1:100, Proteintech 10325-1-AP) in a humidified chamber.

Techniques: Expressing