l1210 mouse cell lines  (ATCC)

Journal: EJHaem

Article Title: Innate Immune Cell Infiltration Induced by Polatuzumab Vedotin Contributes to the Antitumor Effect in Mouse Models

doi: 10.1002/jha2.70219

Figure Lengend Snippet: Pola‐induced infiltration of MΦs and NK cells contributes to the antitumor effect of Pola in the L1210‐hCD79b‐9 syngeneic mouse model. (A) Sensitivity of L1210 and L1210‐hCD79b‐9 cells to Pola at the indicated concentrations (3 replicates). The data represent the mean + SD. (B) Percentage of the area of CD68‐positive staining cells to the total area of tumor cells and (C) percentage of NCR1‐positive cells among tumor cells after 4 mg/kg ctrl IgG or 4 mg/kg Pola administration in the L1210‐hCD79b‐9 syngeneic mouse model ( n = 8). * p < 0.05 by Student's t ‐test. In dot plots, horizontal bars represent median values. (D) L1210‐hCD79b‐9 tumor growth curves from Day 0 to Day 9 after administration of 4 mg/kg ctrl IgG, 4 mg/kg Pola, or 4 mg/kg Pola plus clodronate liposomes plus anti‐asialo GM1 ( n = 6–7). (E) Numbers of mice presenting L1210‐hCD79b‐9 tumor regression on Day 9 following Pola administration. In vivo experiments (B–E) were conducted in DBA/2J mice. (F) Schematic diagram illustrating the relationship between the antitumor effect of Pola and innate immune cells.

Article Snippet: The DB (human) and L1210 (mouse) cell lines were purchased from the American Type Culture Collection.

Techniques: Staining, Liposomes, In Vivo

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: Following gentle washes to eliminate non-adherent cells, the adherent cells—which predominantly consist of monocytes—were differentiated into macrophages by incubating them in a medium supplemented with 10% human serum and 10 ng ml -1 CSF-1 (Cat# 300-25, Peprotech, Rocky Hill, NJ) for 7 d. The cell lines L1210 (CCL-219), P815 (TIB-64), EL-4 (TIB-39), L929 (CCL-1), HEK293T (CRL-3216), Phoenix-Eco (CRL-3214), and Raji (CCL-86) were sourced from the American Type Culture Collection (Rockville, MD).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Following gentle washes to eliminate non-adherent cells, the adherent cells—which predominantly consist of monocytes—were differentiated into macrophages by incubating them in a medium supplemented with 10% human serum and 10 ng ml -1 CSF-1 (Cat# 300-25, Peprotech, Rocky Hill, NJ) for 7 d. The cell lines L1210 (CCL-219), P815 (TIB-64), EL-4 (TIB-39), L929 (CCL-1), HEK293T (CRL-3216), Phoenix-Eco (CRL-3214), and Raji (CCL-86) were sourced from the American Type Culture Collection (Rockville, MD).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Following gentle washes to eliminate non-adherent cells, the adherent cells—which predominantly consist of monocytes—were differentiated into macrophages by incubating them in a medium supplemented with 10% human serum and 10 ng ml -1 CSF-1 (Cat# 300-25, Peprotech, Rocky Hill, NJ) for 7 d. The cell lines L1210 (CCL-219), P815 (TIB-64), EL-4 (TIB-39), L929 (CCL-1), HEK293T (CRL-3216), Phoenix-Eco (CRL-3214), and Raji (CCL-86) were sourced from the American Type Culture Collection (Rockville, MD).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis