Journal: Frontiers in Immunology

Article Title: Tailored strategies for improved control of CAR-T cells in multiple myeloma

doi: 10.3389/fimmu.2026.1740345

Figure Lengend Snippet: Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) BCMA expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.

Article Snippet: Antibodies used in this research report were specific for BCMA-Antibodies used in this research report were specific for BCMA (Miltenyi Biotec; 130-119-152), EGFR (Cetuximab, in-house labelled), CD8 (Miltenyi Biotec; 130-110-683) and CD4 (Miltenyi Biotec; 130-114-534).

Techniques: In Vitro, Incubation, Staining, Construct, Flow Cytometry, Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: B cell maturation antigen, the receptor for a proliferation-inducing ligand and B cell-activating factor of the TNF family, induces antigen presentation in B cells.

doi: 10.4049/jimmunol.175.5.2814

Figure Lengend Snippet: FIGURE 2. Expression of APRIL/BAFF receptors in B cells. A, Total RNA isolated from untransfected A20 B cells or those stably transfected with full-length BCMA, TACI, and the BCMA-C83 mutant were sub- jected to RT-PCR analysis. RNA isolated from purified splenic B cells was included in the analysis. No BCMA signal is detected in cells transfected with the BCMA-C83 mutant because the PCR primers used detect the intracellular domain of BCMA, which is absent in the mutant. B, Cyto- plasmic extracts from A20 cells transfected with the different receptors, which are HA-tagged, were resolved and probed with anti-HA mAb to indicate expression. In multiple transfections, only modest but equivalent expression of the full-length BCMA and TACI was achieved whereas the BCMA-C83 mutant is more highly expressed. This effect is most likely due to the fact that both BCMA and TACI can induce apoptosis when highly overexpressed. C, A20 B cells stably expressing the indicated re- ceptors were left unstimulated or stimulated for 20, 40, and 60 min with APRIL (100 ng/ml). Cytoplasmic extracts were resolved and probed with mAb specific for I-B. Membrane was stripped and reprobed with anti- -actin mAb to ensure equivalent loading. D, A20 B cells expressing the indicated receptors were stimulated for 0, 30, 60, and 90 min with APRIL (100 ng/ml). Nuclear extracts were prepared and gel shift analysis was performed with a radiolabeled NF-B probe.

Article Snippet: Abs used were from the following sources: TNFR2 (AB-226-PB) and BCMA (AF593 and MAB593; R&D Systems); I- B mAb (Imgenex); HA mAb (clone 3F10; Roche Applied Science); phospho-JNK, JNK, phospho-p38, p38, phospho-ERK, and ERK (Cell Signaling Technology).

Techniques: Expressing, Isolation, Stable Transfection, Transfection, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Membrane, Gel Shift

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: B cell maturation antigen, the receptor for a proliferation-inducing ligand and B cell-activating factor of the TNF family, induces antigen presentation in B cells.

doi: 10.4049/jimmunol.175.5.2814

Figure Lengend Snippet: FIGURE 6. BCMA-mediated Ag presentation is NF-B- and JNK-dependent. A, A20 cells expressing the indicated chimeric receptors were left unstimulated or stimulated for 20, 40, and 60 min with anti-TNFR2 (10 g/ml). Cytoplasmic extracts were probed with mAb specific for I-B. Membrane was subsequently stripped and reprobed with anti--actin mAb to ensure equivalent loading. The indicated cell lines were stimulated with anti-human TNFR2 for 0, 30, 60, and 90 min (bottom panel). Gel shift analysis with a radiolabeled NF-B probe was then performed. B, Cells were stimulated as in A, and the blot sequentially probed with phospho-JNK and JNK Abs (upper double panels). Cell lysates were also sequentially blotted with phospho-p38 and total p38 Abs (middle double panels), or with phospho-ERK and total ERK Abs (lower double panels). C, A20 cells expressing the indicated receptors were stimulated with APRIL (100 ng/ml) for varying lengths of time, and JNK phosphorylation was analyzed as in B. D, A20 B cells stably expressing BCMA were transduced with retroviruses encoding irrelevant control protein, I-BSR, or JNKK2/MKK7 (K149M) mutant upstream of GFP in a bicistronic manner. Sorted GFP-positive cells were left unstimulated or stimulated with APRIL (100 ng/ml) for 24 h. Surface molecule expression was analyzed using biotin-conjugated Abs followed by streptavidin-allophycocyanin. E, BCMA-expressing cells transfected with control, I-BSR, or JNKK2KM were stim- ulated with the indicated concentrations of APRIL for 24 h. B cells were then cocultured with DO11.10 T cells in the presence of 80 ng/ml OVA peptide. IL-2 production by T cells was measured as described in Fig. 3B.

Article Snippet: Abs used were from the following sources: TNFR2 (AB-226-PB) and BCMA (AF593 and MAB593; R&D Systems); I- B mAb (Imgenex); HA mAb (clone 3F10; Roche Applied Science); phospho-JNK, JNK, phospho-p38, p38, phospho-ERK, and ERK (Cell Signaling Technology).

Techniques: Expressing, Membrane, Gel Shift, Phospho-proteomics, Stable Transfection, Transduction, Control, Mutagenesis, Transfection