immpress excel polymer staining kit  (Vector Laboratories)

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: 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: AF647 mouse anti-human FOXP3 (Clone AKYP0102) , Akoya Biosciences , Cat#S6501007.

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: AF647 mouse anti-human FOXP3 (Clone AKYP0102) , Akoya Biosciences , Cat#S6501007.

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

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) Outline of in vivo two-photon time-lapse microscopy in MC38-H2b-Cerulean tumors. (Middle) Representative FOVs displaying the migratory behavior of Tregs ( FoxP3 -mRFP, magenta) in untreated (top) or anti-PD-1-treated (bottom) mice in perivascular regions containing Il12 -eYFP + DCs (yellow). Scale bar represents 20 μm. Treg migratory tracks are shown in white. (Right) Observed contact duration between DCs and Tregs in each experimental group ( n = 4). Each dot represents one DC, whiskers represent mean to max. Unpaired t test; *** p < 0.001. (B) As in (A), but in Il12-p40 -eYFP; FoxP3 -mRFP; Ccl22 −/− mice (n = 5). (C) Change in contact duration between DCs and Tregs after anti-PD-1 treatment, comparing Ccl22 WT and Ccl22 −/− mice. Each dot represents one mouse, bar represents mean with SEM. Unpaired t test; * p < 0.05. (D) Quantification of the cumulative interactions of CCR7 + DCs with Tregs over time of acquisition, comparing Ccl22 WT and Ccl22 −/− mice. Shaded areas indicate confidence intervals. (E) Change in tumor volume in WT and Ccl22 -deficient mice left untreated (left), or upon anti-PD-1 therapy (right). Unpaired t test, whiskers represent min to max; * p < 0.05. See also .

Article Snippet: AF647 mouse anti-human FOXP3 (Clone AKYP0102) , Akoya Biosciences , Cat#S6501007.

Techniques: In Vivo, Time-lapse Microscopy