Journal: Cancer Immunology Research

Article Title: Phenotypic Characterization and Prognostic Impact of CD103 + Tissue-Resident Memory T Cells in Diffuse Large B-cell Lymphoma

doi: 10.1158/2326-6066.CIR-25-0445

Figure Lengend Snippet: Characterization of the T RM population in DLBCL by spectral flow cytometry. A, Representative flow plots of CD103 + CD69 + T cells, highlighted in red. B, % CD103 + CD69 + of T cells per COO ( n = 2–4). C, UMAP reduction of T cells collected from patients with DLBCL ( n = 8) and rLN ( n = 2). Colors represent clusters determined by PhenoGraph clustering ( k = 45). D, Overlay of COO classification of DLBCL samples ( n = 2 for each). E, Overlay of markers of interest is highlighted in red. F, Heatmap showing the percent positivity of all T-cell phenotype markers in spectral panel. G, Boxplots showing the percent of CD103 + (red) or CD103 − (gray) T cells (CD45 + CD3 + CD5 + CD2 + cells) expressing each marker for samples from patients with DLBCL ( n = 10). P values displayed on plot were calculated using paired t tests for each marker. H, Intracellular IFNγ and TNFα expression by CD103 + or CD103 − T cells measured by flow cytometry after bulk single-cell lymphoma biopsy samples ( n = 3) or healthy donor peripheral blood mononuclear cells ( n = 1) were cultured alone, with PMA/ionomycin, or with HLA class I and class II CEF peptide pools for 24 hours. I, Enumeration of remaining live B cells by flow cytometry after 24-hour coculture of sorted CD103 + or CD103 − T cells (sorted as CD8 + or CD4 + cells) with autologous sorted B cells from single-cell biopsy samples of patients with lymphoma ( n = 4). Samples containing CD103 + and CD103 − cells from the same donor are connected with a line. P value generated using a ratio paired t test. The bar graph represents the mean, with error bars representing SEM. For box plots, the center line represents the median, the box represents the IQR, and the whiskers represent 1.5× the IQR. ICS, intracellular staining; UNCLASS, unclassified.

Article Snippet: Bulk-cell suspensions (1 × 10 6 ) were cultured alone, with MHC class I and class II cytomegalovirus, Epstein–Barr virus, and influenza virus (CEF) peptides (Miltenyi Biotech, cat. #130-098-426 and Cedarlane, cat. #PT-PA-CEFT-001-1), or with 1× cell activation cocktail [phorbol 12-myristate 13-acetate (PMA) and ionomycin; BioLegend, cat. #423301] for 24 hours in RPMI-1640 supplemented with 1% (vol/vol) GlutaMAX (Gibco, cat. #35050061), 1% (vol/vol) penicillin–streptomycin (Thermo Fisher Scientific, cat. #15140122), 50 μmol/L β-mercaptoethanol (Sigma-Aldrich, cat. #M3148), 5% (vol/vol) male AB-plasma human serum (Sigma-Aldrich, cat. #H4522), and 5 U/mL IL2 (PeproTech, cat. #200-02) in 96-well flat-bottom plates.

Techniques: Flow Cytometry, Expressing, Marker, Cell Culture, Generated, Staining

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: OVA 257-264 H-2Kb-restricted OVA MHC class I epitope , InvivoGen , Cat#vac-sin.

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