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
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