phosphate buffered saline pbs tablets primescripttm rt reagent kit foxp3 transcription factor staining buffer set matrigel matrix  (Thermo Fisher)

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

Journal: Journal of Neuroinflammation

Article Title: Macrophage expression of P2X7 controls autoimmune uveitis

doi: 10.1186/s12974-025-03529-w

Figure Lengend Snippet: P2X7 deficiency decreases the severity of EAU P2X7 +/+ and P2X7 −/− mice were immunized, or not (naïve), with 200 µg IRBP 651−670 peptide and injected with 1 µg Pertussis Toxin (PTX). A Representative fundus images and clinical scores of P2X7 +/+ and P2X7 −/− mice 12 days after immunization. Each dot represents one mouse ( n = 9 for P2X7 +/+ and n = 7 for P2X7 −/− , ** p = 0.005). The data shown are representative of four independent experiments. B Bars represent the percentages of immune cells in the retina: CD11b + CD45 med (Microglia), CD11b + CD45 high (monocyte-derived macrophages (MdMs)), CD3 + CD4 + Foxp3 neg (CD4 + T cells) and CD3 + CD4 + Foxp3 + (Tregs), 13 days after immunization. Each dot represents one retina ( n = 5 for P2X7 +/+ and n = 6 for P2X7 −/− , CD11b + CD45 high * p = 0.030, CD3 + CD4 + Foxp3 neg * p = 0.035). C Bars represent the percentages of immune cells in the cervical lymph nodes 13 days after immunization. Each dot represents one mouse ( n = 10 for P2X7 +/+ and n = 11 for P2X7 −/− ). Data were analyzed by Mann–Whitney test and are expressed as mean ± SEM. D Cells from the retinas of P2X7 +/+ and P2X7 −/− naïve mice were analyzed by flow cytometry. Live cells were gated on CD11b + CD45 med (Microglia) (left panel) and a representative plot of P2X7 expression is shown (right panel). E After 13 days, cells from the retinas of P2X7 +/+ and P2X7 −/− immunized mice were analyzed by flow cytometry. Live cells were gated on CD11b + CD45 med (Microglia), CD11b + CD45 high (MdMs), CD3 + CD4 + Foxp3 neg (CD4 + T cells), and CD3 + CD4 + Foxp3 + (Tregs) to evaluate P2X7 expression on each subset. Representative plots of gating and P2X7 expression in the retina of EAU mice are shown (left panel). The percentage of P2X7 + cells for each cell subset is shown in a bar graph (right panel), each dot represents one retina ( n = 16 for CD11b + cells and n = 11 for CD4 + cells)

Article Snippet: For the FOXP3 detection, a FOXP3 staining kit and anti-Foxp3 antibody (REA788) (Miltenyi Biotec) were used according to the manufacturer’s instructions.

Techniques: Injection, Derivative Assay, MANN-WHITNEY, Flow Cytometry, Expressing

Journal: Journal of Neuroinflammation

Article Title: Macrophage expression of P2X7 controls autoimmune uveitis

doi: 10.1186/s12974-025-03529-w

Figure Lengend Snippet: P2X7 deficiency does not affect dendritic cell (DC) and CD4 + T cell activation P2X7 +/+ and P2X7 −/− mice were immunized with 200 µg IRBP 651−670 peptide. Inguinal lymph nodes were collected 10 days post-immunization. A DCs were defined as MHCII + CD11c + (left panel). Representative plots of P2X7 and CD11b expression for MHCII + CD11c + cells are shown (middle panel). Representative plots of P2X7 expression for each subset MHCII + CD11c + CD11b +/− are shown (right panel). Representative plots of CD80 B and CD86 C expression for each subset are shown (left panel). Quantification of the expression of co-stimulatory molecules CD80 (B) and CD86 (CD) for each subset is shown (right panel), each dot represents pooled cells from 3 mice ( n = 3 for P2X7 +/+ and P2X7 −/− ). D Representative plots of P2X7 expression by CD3 + CD4 + Foxp3 neg (CD4 + T cells) and CD3 + CD4 + Foxp3 + (Regulatory T cells) in the lymph nodes (left panel). The percentage of P2X7 + cells for each subset is shown (right panel), each dot represents one mouse ( n = 10). (E-F) Gating of P2X7 + cells for subsets of T cells (right panels). Bars represent the percentages of CD3 + CD4 + T cells E and CD4 + Foxp3 + T cells F in the lymph nodes, each dot represents one mouse ( n = 5 for P2X7 +/+ and n = 4 for P2X7 −/− ). Expression of cell activation markers by CD4 + T cells was evaluated by flow cytometry. CD4 + T cells were gated on CD44 − CD62l + or CD44 − CD45RB + (naïve T cells), CD44 + CD62l + or CD44 + CD45RB + (central memory T cells), and CD44 + CD62l − or CD44 + CD45RB − (effector T cells) ( G and H ). Each dot represents represent pool cells from 2–3 mice ( n = 3–4 for P2X7 +/+ and n = 5 for P2X7 −/− mice). Data were analyzed by multiple Mann–Whitney tests and are expressed as mean ± SEM

Article Snippet: For the FOXP3 detection, a FOXP3 staining kit and anti-Foxp3 antibody (REA788) (Miltenyi Biotec) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Macrophage expression of P2X7 controls autoimmune uveitis

doi: 10.1186/s12974-025-03529-w

Figure Lengend Snippet: Lack of P2X7 expression in CD4 + T cells does not alter their functions during EAU ( A ) EAU was induced in WT mice by adoptive transfer of IRBP-specific T lymphocytes from spleens and lymph nodes of P2X7 +/+ and P2X7 −/− mice. B Representative fundus images and clinical scores of WT recipient mice 4 days after adoptive transfer of P2X7 +/+ and P2X7 −/− T cells. Each dot represents one mouse ( n = 10 for P2X7 +/+ and n = 9 for P2X7 −/− ). The data shown are representative of three independent experiments. C Representative plots of P2X7 expression by CD3 + CD4 + Foxp3 neg (CD4 + T cells) and CD3 + CD4 + Foxp3 + (Regulatory T cells) in the retinas of WT mice after adoptive transfer of P2X7 +/+ or P2X7 −/− T cells. D Bars represent the percentages of immune cells in the retina 6 days after adoptive transfer. Each dot represents one retina ( n = 10 for P2X7 +/+ and n = 8 for P2X7 −/− ). E Bars represent the percentages of immune cells in the cervical lymph nodes, 6 days after adoptive transfer. Each dot represents one mouse ( n = 5 for P2X7 +/+ and n = 4 for P2X7 −/− ) ( F ) P2X7 lox and Foxp3 creER P2X7 lox mice were immunized with 200 µg IRBP 651−670 peptide and injected with 1 µg PTX, one week after the end of tamoxifen treatment. G Representative fundus images and clinical scores of P2X7 lox and Foxp3 creER P2X7 lox 12 days after immunization. Each dot represents one mouse ( n = 13 for P2X7 lox and n = 8 for Foxp3 creER P2X7 ox ). The data shown are representative of four independent experiments. H Representative plots of P2X7 expression by CD3 + CD4 + Foxp3 neg (CD4 + T cells) and CD3 + CD4 + Foxp3 + (Regulatory T cells) in the retinas of P2X7 lox and Foxp3 creER P2X7 lox mice 13 days after immunization. I Bars represent the percentages of immune cells in the retina 13 days after immunization. Each dot represents one retina ( n = 26 for P2X7 lox and n = 16 for Foxp3 creER P2X7 lox ). J Bars represent the percentages of immune cells in the cervical lymph nodes, 13 days after immunization. Each dot represents one mouse ( n = 13 for P2X7 lox and n = 8 for Foxp3 creER P2X7 lox ). Data were analyzed by Mann–Whitney test and are expressed as mean ± SEM

Article Snippet: For the FOXP3 detection, a FOXP3 staining kit and anti-Foxp3 antibody (REA788) (Miltenyi Biotec) were used according to the manufacturer’s instructions.

Techniques: Expressing, Adoptive Transfer Assay, Injection, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Macrophage expression of P2X7 controls autoimmune uveitis

doi: 10.1186/s12974-025-03529-w

Figure Lengend Snippet: P2X7 deficiency in resident microglial cells and infiltrating MdMs reduces the severity of EAU ( A ) P2X7 lox and Cx3cr1 cre P2X7 lox mice were immunized with 200 µg IRBP651-670 peptide and injected with 1 µg Pertussis Toxin (PTX). B Representative fundus images and clinical scores of P2X7 lox and Cx3cr1 cre P2X7 lox 12 days after immunization. Each dot represents one mouse ( n = 16 for P2X7 lox and n = 12 for Cx3cr1 cre P2X7 lox mice, * p = 0.044). The data shown are representative of at least five independent experiments. C Representative plots of P2X7 expression by CD11b + CD45 med (Microglia) and CD11b + CD45 high (Infiltrating MdM) in the retinas of P2X7 lox and Cx3cr1 cre P2X7 lox mice 13 days after immunization. D Bars represent the percentages of immune cells in the retina 13 days after immunization. Each dot represents one retina (P2X7 lox ( n = 32) vs. Cx3cr1 cre P2X7 lox mice ( n = 24); CD11b + CD45 med ** p = 0.006, CD11b + CD45 high * p = 0.011, CD3 + CD4 + Foxp3 neg ** p = 0.003 and CD3 + CD4 + Foxp3 + ** p = 0.006). ( E ) Bars represent the percentages of immune cells in the cervical lymph nodes, 13 days after immunization. Each dot represents one mouse ( n = 16 for P2X7 lox and n = 12 for Cx3cr1 cre P2X7 lox mice). Data were analyzed by Mann–Whitney test and are expressed as mean ± SEM

Article Snippet: For the FOXP3 detection, a FOXP3 staining kit and anti-Foxp3 antibody (REA788) (Miltenyi Biotec) were used according to the manufacturer’s instructions.

Techniques: Injection, Expressing, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Macrophage expression of P2X7 controls autoimmune uveitis

doi: 10.1186/s12974-025-03529-w

Figure Lengend Snippet: P2X7 deficiency on resident microglia is sufficient to reduce the severity of EAU ( A ) P2X7 lox and Cx3cr1 creER P2X7 lox mice were immunized with 200 µg IRBP 651−670 peptide and injected with 1 µg Pertussis Toxin (PTX). B Representative fundus images and clinical scores of P2X7 lox and Cx3cr1 cre P2X7 lox 12 days after immunization. Each dot represents one mouse ( n = 14 for P2X7 lox and n = 13 for Cx3cr1 creER P2X7 lox mice, * p = 0.042). The data shown are representative of three independent experiments. C Representative plots of P2X7 expression by CD11b + CD45 med (Microglia) and CD11b + CD45 high (Infiltrating MdM) in the retinas of P2X7 lox and Cx3cr1 creER P2X7 lox mice 13 days after immunization. D Bars represent the percentages of immune cells in the retina 13 days after immunization. Each dot represents one retina (P2X7 lox ( n = 12) vs. Cx3cr1 creER P2X7 lox mice ( n = 16); CD11b + CD45 med ** p = 0.009, CD11b + CD45 high * p = 0.032, CD4 + Foxp3 neg ** p = 0.005 and CD4 + Foxp3 + * p = 0.019). E Bars represent the percentages of immune cells in the cervical lymph nodes, 13 days after immunization. Each dot represents one mouse ( n = 6 for P2X7 lox and n = 8 for Cx3cr1 creER P2X7 lox mice). Data were analyzed by Mann–Whitney test and are expressed as mean ± SEM

Article Snippet: For the FOXP3 detection, a FOXP3 staining kit and anti-Foxp3 antibody (REA788) (Miltenyi Biotec) were used according to the manufacturer’s instructions.

Techniques: Injection, Expressing, MANN-WHITNEY