Journal: Frontiers in Immunology

Article Title: IL-12-armed oncolytic HSV-2 enhances CAR T cell efficacy against pancreatic cancer in xenografted models

doi: 10.3389/fimmu.2025.1664289

Figure Lengend Snippet: Mesothelin chimeric antigen receptor (CAR)-T cells SS1-ICOSBBZ-CAR-T showed specific and modest anti-tumor capacity in vitro and in vivo . (A) Schematic diagram of the CAR structure. We constructed a third-generation MSLN-targeted CAR-T. This CAR incorporates the single-chain variable fragment (scFv) derived from the SS1 antibody for MSLN recognition, an ICOS-derived transmembrane domain, the intracellular domains contained two co-stimulatory molecules ICOS and 4-1BB, and the T cell receptor ζ (TCR-ζ) signaling domain. (B) Percentage of CAR positive T cells at day 12 (left) and CD4+ and CD8+ subsets (right) composition of the final CAR-T cell product. The CAR was stained by human mesothelin protein conjugated to PE. (C) Specific cytolytic activity of SS1-ICOSBBZ-CAR-T cells in vitro using luciferase assay. CAR-T cells were incubated with tumor cell lines for 30 hours at different E:T ratio. Panc-1-luc (mesothelin negative, left), Capan-2-luc (mesothelin positive, middle), Panc-1-luc-MSLN (mesothelin positive, right). (D) Schematic timeline of the antitumor efficacy experiment design. 2 × 10 6 Capan-2-luc cells were subcutaneously inoculated (s.c.) into the right flank of six-week-old B-NDG mice, tumors were established for 10 days prior to treatment initiation. Then, SS1-ICOSBBz-CAR-T cells or PBS (200 μL control) were administered via tail vein injection, (n = 5). (E) Bioluminescence imaging of mice at indicated days after treatment. (F) Quantification of tumor burden dynamics following CAR-T therapy. Tumor volume was monitored weekly during 35 days, and tumor volume changes were calculated relative to baseline (Day 0 set as 0% change). The tumor size at day 7 served as the reference for weekly measurements. Statistical significance of volume changes was assessed using t-test (n = 5 per group). (G) Body weight of mice treated with SS1-ICOSBBZ-CAR-T cells were measured. All data are presented as means ± standard deviation. *P < 0.05, **P < 0.01.

Article Snippet: After 24 h recovery, SS1-ICOSBBZ-CAR-T cells were co-cultured with Capan-2 target cells at E:T = 2:1 for 48 h. CD4 + and CD8 + subsets were separated using the human CD8 MicroBeads (Miltenyi), stained with AF647-conjugated antibodies (IsoPlexis,STAIN-1002–1 and STAIN-1003-1), loaded onto IsoLight chips (IsoPlexis), and analyzed using IsoSpeak v3.0.1.

Techniques: In Vitro, In Vivo, Construct, Derivative Assay, Staining, Activity Assay, Luciferase, Incubation, Control, Injection, Imaging, Standard Deviation

Journal: Therapeutic Advances in Medical Oncology

Article Title: Mismatch repair protein imbalance in head and neck squamous cell carcinoma: associations with clinical features and survival

doi: 10.1177/17588359251408596

Figure Lengend Snippet: Correlation between MMR protein expression and immune cell subsets in HNSCC. (a–f) Correlation of MSH2, MSH6, PMS2, MLH1, MSH2/MSH6, and PMS2/MLH1 ratios with immune cell subsets. (g) Diagram of correlation patterns among MMR proteins. (h) Correlation between MSH2 expression and CD8+ T cells in nontumor tissues. (i) Correlation between MSH6 expression and CD4+ T cells in tumor tissues. (j) Correlation between PMS2/MLH1 ratio expression and CD4+ T-cell in tumor tissues. HNSCC, head and neck squamous cell carcinoma; MMR, mismatch repair.

Article Snippet: Briefly, MSH2 (ZSGB-BIO ® ; ZA-0622, 1:2000), MSH6 (Roche Diagnostics GmbH ® ; SP93, ready-to-use), PMS2 (Dako ® ; EP51, ready-to-use), MLH1 (Dako ® ; ES05, ready-to-use), CD4 (Abcam; ab133616, 1:2000), CD8 (SinoBiological; 10980-R081, 1:500), and Foxp3 (Abcam; ab191416, 1:1000) antibodies were used to immunohistochemically stain the TMA slides, with brown staining considered as positive immunoreaction and blue staining considered as negative immunoreaction (nuclear, cytoplasm, or membrane, based on cells of interest).

Techniques: Expressing

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