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: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . CD8+ T cells from 5 donors stimulated 3x with pools of 10 peptides, except pool 9 which had 6 peptides. C . 3D modeling of VP1 pentamer highlighting epitopes (Green: VP1 (100-108), Yellow: VP1 (251-259), (253-262), (274-283), Purple: Published epitopes, Blue: Predicted epitopes from IEDB. D . CD8+ T cells from 3 donors, 10 cell lines each, stimulated 3x with pools of peptides and then individual T2 IFN- γ response.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques:

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . Pooled CD8+ T cell lines from 3 healthy donors were split into three different fractions and stained with low, intermediate, or high tetramer concentration. From samples stained with low and intermediate tetramer concentration, live CD3+ CD8+ T cells were sorted from the 90th percentile tetramer(+) gates. Shown here is the cumulative clone count in the 90th percentile gates (y-axis) plotted against maximum enrichment score out of the 11 calculated for each clone (x-axis). Symbol size corresponds to clone count in tetramer-negative gate. Symbol color indicates clone abundance in the single cell TCR dataset (low tetramer concentration condition). High affinity TCRs have high cumulative clone count, high maximum enrichment score, and small symbol size. SumSort = total number in top 10% tetramer binder gate (i.e. 90th percentile) in the 1:2000 + 1:4000 tetramer staining condition. MaxFold = greatest value for fold enrichment (out of the calculated 11). C . Top 14 T cell receptors were virally transduced into human primary T cells in 2 donors. TCR-transduced cells were stimulated with different amounts of VP1(100-108) peptide and tested for intracellular INF- γ content as ratio of positive control (max). EC50 (M) scores populated in tabular format.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Staining, Concentration Assay, Positive Control

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . TCR 3 and TCR 8 tested against K562 presenting VP1(100-108) on HLA-A2 vs a control TCR (DMF5) with its corresponding peptide (MART1) after 48h incubation at unsorted E:T of 0.5 and 2. Error bars repres nt SEM for 3 technical replicates from one representative experiment. p<0.001 by unpaired T test. C . After 72 hours of co-culture with JCV-infected SVG cells and no T cells (no TCR), sham TCR, TCR 3, and TCR 8 looking at live CD8+ T cell CD137 positivity via flow cytometry. TCR 3 and 8 vs no TCR p value <0.001; TCR 3 vs sham TCR+JCV p=0.0032 and TCR 8 vs sham TCR+JCV p=0.029 D . Percent killing of SVG cells determined by flow cytometry based on gating of loss of live astrocytes. p value <0.001; TCR 3 vs sham TCR+JCV p<0.001 and TCR 8 vs sham TCR+JCV p=0.002 by unpaired T test. Done in duplicate at various unsorted E:Ts, mean results shown.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Control, Incubation, Co-Culture Assay, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: Flow cytometry data of live CD8+ T cells stained with HLA-A2:BK VP1 (100-108) and HLA-A2:JV VP1 (100-108) at 1:50 tetramer dilution with an HLA-A2 positive JCV antibody negative healthy donor on the left and an HLA-A2 confirmed PML survivor on the right.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Flow Cytometry, Staining