Journal: The EMBO Journal

Article Title: Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens

doi: 10.15252/embj.201694177

Figure Lengend Snippet: A–E B1‐8 B cells, 3046SM cells, 3046M cells, 3046SD cells or 3046D cells were stimulated with 1NIP‐pep (80 nM), or anti‐CD19 antibody (62 nM), or co‐stimulated with 1NIP‐pep and anti‐CD19 antibody. The calcium fluxes were measured by FACScan. F–J B1‐8 B cells, 3046SM cells, 3046M cells, 3046SD cells or 3046D cells were stimulated with 1NIP‐pep (80 nM), or anti‐CD20 antibody (62 nM), or co‐stimulated with 1NIP‐pep and anti‐CD20 antibody. The calcium fluxes were measured by FACScan. Data information: Addition of reagents to the cells is indicated by arrows. Data are representative of three independent experiments.

Article Snippet: For stimulating cells and measuring Ca 2+ response, the following antibodies were used: anti‐IgD (goat anti‐mouse, polyclonal antiserum, eBioscience), anti‐IgM (goat anti‐mouse, polyclonal antiserum, Southern Biotech), anti‐λ (goat anti‐mouse, polyclonal antiserum, Southern Biotech), anti‐CD19 (eBio1D3, eBioscience), anti‐CD20 (AISB12, eBioscience), anti‐MHCII (NIMR‐4, eBioscience), anti‐CD21 (7E9, BioLegend), anti‐CD22 (OX‐97, BioLegend), anti‐CD81 (JS‐81, BD Pharmingen), Ac146 and Ac38 (both prepared from in‐house hybridoma).

Techniques:

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 2 SPPL3KO suppresses CD19 CAR T cell activation. a Schematic of T cell activation studies. b–d, Expression of b CD69, c PD1, and d Tim3 on T cells after overnight co-culture with WT or SPPL3KO Nalm6 cells or alone (control). e expression of CD107a on T cells after a 4-h co-culture with WT or SPPL3KO

Article Snippet: Proteins were labeled with SPPL3 (EMD Millipore #MABS1910), CD19 (#3574), CD22 (#67434), Actin (#4970) or GAPDH (#2118, all from Cell Signaling, all diluted 1:1000), followed by secondary antibody staining using either anti-rabbit (Cell Signaling #7074, diluted 1:5000), or anti-mouse (Cell Signaling #7076, diluted 1:5000) HRP-linked antibodies followed by visualization.

Techniques: Activation Assay, Expressing, Co-Culture Assay, Control

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 3 Hyperglycosylation impairs detection of CD19. a Western blot of lysates from WT or SPPL3KO Nalm6 and OCI-Ly10 cells probed for CD19. Protein electrophoresis was performed on a 6% polyacrylamide gel. Representative of n = 4 individual experiments. b Western blot of lysates from WT or SPPL3KO

Article Snippet: Proteins were labeled with SPPL3 (EMD Millipore #MABS1910), CD19 (#3574), CD22 (#67434), Actin (#4970) or GAPDH (#2118, all from Cell Signaling, all diluted 1:1000), followed by secondary antibody staining using either anti-rabbit (Cell Signaling #7074, diluted 1:5000), or anti-mouse (Cell Signaling #7076, diluted 1:5000) HRP-linked antibodies followed by visualization.

Techniques: Western Blot, Protein Electrophoresis

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 5 Overexpression of SPPL3 results in loss of CD19 surface expression. a Sequential flow cytometric evaluation of FMC63-APC binding on SPPL3KO + Nalm6 after overexpression of SPPL3. b Western blot of fractionated cellular lysates from WT, SPPL3KO or SPPL3KO with overexpression of SPPL3 (SPPL3KO+) Nalm6 cells probed for CD19 performed 20 days after engineering. Representative of n = 2 individual experiments. c Survival of WT, SPPL3KO, or SPPL3KO + Nalm6 over time when established on 20 days after re-introduction of SPPL3 (E:T 0.25:1). Representative data from n = 2 experiments with independent T cell donors. Error bars reflect mean ± standard error of the mean (s.e.m.). ****P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Statistical annotations reflect differences between WT and SPPL3KO (upper) or WT and SPPL3KO + Nalm6 (lower). Source Data are provided as a Source Data file.

Article Snippet: Proteins were labeled with SPPL3 (EMD Millipore #MABS1910), CD19 (#3574), CD22 (#67434), Actin (#4970) or GAPDH (#2118, all from Cell Signaling, all diluted 1:1000), followed by secondary antibody staining using either anti-rabbit (Cell Signaling #7074, diluted 1:5000), or anti-mouse (Cell Signaling #7076, diluted 1:5000) HRP-linked antibodies followed by visualization.

Techniques: Over Expression, Expressing, Binding Assay, Western Blot

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 6 Proposed model for the mechanism of glycosylation-mediated antigen escape. (left) Normal SPPL3 function results in standard glycosylation of CD19. (Middle) Loss of SPPL3 results in increased and altered glycosylation, disrupting the CAR binding epitope. (Right) Increased SPPL3 results initially in decreased glycosylation followed by degradation of CD19, preventing surface presenation.

Article Snippet: Proteins were labeled with SPPL3 (EMD Millipore #MABS1910), CD19 (#3574), CD22 (#67434), Actin (#4970) or GAPDH (#2118, all from Cell Signaling, all diluted 1:1000), followed by secondary antibody staining using either anti-rabbit (Cell Signaling #7074, diluted 1:5000), or anti-mouse (Cell Signaling #7076, diluted 1:5000) HRP-linked antibodies followed by visualization.

Techniques: Glycoproteomics, Binding Assay