Journal: HemaSphere

Article Title: Functional differences between CLL‐ and ALL‐derived CAR T cells in a 3D tumor microenvironment highlight CXCR4 and IL‐10 as potential modulatory targets

doi: 10.1002/hem3.70279

Figure Lengend Snippet: Characterization of chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) patient‐derived T cells prior to and after transduction. (A) Cellular composition of peripheral blood mononuclear cells (PBMCs) derived from CLL ( n = 8) and ALL ( n = 8) patients before T‐cell enrichment. The percentages of viable PBMCs are shown, subdivided into CD3 + T cells, CD19 + B cells, and other PBMCs. (B) Percentages of CD4 + and CD8 + T cells, enriched from CLL‐derived ( n = 7) and ALL‐derived ( n = 8) PBMCs. (C) Percentages of exhausted TIM‐3 + and LAG‐3 + (left panel) and PD‐1 + (right panel) CD4 + and CD8 + T cells, after T‐cell enrichment from CLL‐derived ( n = 6) and ALL‐derived ( n = 4) PBMCs. Data show individual values with indication of the mean. (D) In vitro expansion per well of transduced CD19 chimeric antigen receptor (CAR) T cells derived from CLL ( n = 8) and ALL ( n = 8) patients on Day 14 after transduction. (E) Transduction efficiency of CLL and ALL patient‐derived CAR T cells ( n = 8) assessed on Day 14 after transduction. Data show individual values with indication of the mean. (F) Percentages of CD4 + and CD8 + T cells of CLL and ALL patients ( n = 8) within the CD3 + mock‐transduced (gray scale) and CD3 + CAR T‐cell population (colored) on Day 14 after transduction. Data are presented as mean ± SEM. (G) Percentages of exhausted TIM‐3 + and LAG‐3 + (left panel), PD‐1 + (middle panel), and intracellular CTLA‐4 + (right panel) CD4 + and CD8 + T cells within the mock‐transduced T‐cell and CAR T‐cell population of CLL and ALL patients on Day 14 post‐transduction. Data show individual values with indication of the mean. Data were analyzed by Šidák's multiple comparisons test. Significance is indicated by *P = 0.05.

Article Snippet: CD19 + Nalm‐6 (ACC 128, DSMZ, Braunschweig, Germany), CD19 + Mec‐1 (ACC 497, DSMZ), and CD19 – U937 cells (ACC 5, DSMZ) were used as control cell lines for 2D functionality analysis and cultivated in medium for primary CLL cells (Mec‐1) or RPMI supplemented with 10% FCS and 0.4% penicillin/streptomycin (Nalm‐6, U937).

Techniques: Derivative Assay, Transduction, In Vitro

Journal: HemaSphere

Article Title: Functional differences between CLL‐ and ALL‐derived CAR T cells in a 3D tumor microenvironment highlight CXCR4 and IL‐10 as potential modulatory targets

doi: 10.1002/hem3.70279

Figure Lengend Snippet: Cellular distribution of immune cells and bone marrow‐derived stromal cells (BMSCs) in the autologous chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) three‐dimensional (3D) co‐culture system. (A) Percentages of CD19 + malignant B cells, viable CD4 + and CD8 + T cells, and viable CD3 + CAR + T cells in the peripheral (P) and the core (C) regions of the 3D co‐cultures with primary CLL cells (upper panels, n = 8) and ALL cells (lower panels, n = 7) after co‐culture with mock‐transduced autologous T cells or chimeric antigen receptor (CAR) T cells for 18 h. Data were analyzed by Tukey's multiple comparisons test and paired t ‐test for comparisons between periphery and core. A gating strategy is shown in Figure . (B) Representative 3D reconstructions of co‐cultures of CLL (upper panel) and ALL (lower panel) cells with autologous T cells (left panel) or CAR T cells (right panel) for 18 h. The visualizations show the distribution of different cell types within the scaffold‐based 3D co‐culture model. z ‐Stacks of the stained 3D co‐cultures including BMSCs (CD90, red), B cells (CD19, green), and T cells (CD3, blue) were analyzed using Fiji software (upper panel: number of stacks from left to right: 114, 114, and 207; size of stacks: 1.04, 1.04, and 0.4 µm; lower panel: number of stacks from left to right: 119, 158, and 162; size of stacks: 1.04, 1.04, and 0.34 µm). The higher magnification on the right highlights the co‐localization of CAR T cells (CAR + , magenta) with malignant B cells (magenta and green overlap, white arrows), which strongly co‐localize with mesenchymal stromal cells (green and red overlap). Corresponding 3D reconstructions showing only CD3⁺/CAR⁺ T cells and CD19⁺ B cells are provided in Figure . (C) Quantitative image analysis of CLL ( n = 3) and ALL ( n = 4) patient‐derived 3D scaffolds demonstrates the fraction of CD19 + B cells co‐localized with CD3 + T cells (CD19 + /CD3 + cells). Data points represent means of analyzed images per patient, with indication of the mean, and statistical comparisons performed by Tukey's multiple comparisons test. The number of analyzed z ‐stacks and regions for each patient is provided in Figure . (D) Quantitative image analysis of cellular composition within CLL and ALL patient‐derived 3D scaffolds across the individual patient measurements. Data are presented as mean ± SEM. Significance is indicated by *P = 0.05, **P = 0.01, and ***P = 0.001.

Article Snippet: CD19 + Nalm‐6 (ACC 128, DSMZ, Braunschweig, Germany), CD19 + Mec‐1 (ACC 497, DSMZ), and CD19 – U937 cells (ACC 5, DSMZ) were used as control cell lines for 2D functionality analysis and cultivated in medium for primary CLL cells (Mec‐1) or RPMI supplemented with 10% FCS and 0.4% penicillin/streptomycin (Nalm‐6, U937).

Techniques: Derivative Assay, Co-Culture Assay, Staining, Software