Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Viability and Surface Expression of CD19 and CD22 in treated B-cell leukemia cell lines and normal B cells. Left panel: Epigenetic modifiers/differentiation agents (Bryostatin, 5-Azacytidine, ATRA, Panobinostat or Vorinostat) were added at increasing concentrations (x-axis, as indicated) to the culture media of B cell lines (Raji-green circle, NALM6-magenta square, REH-blue triangle) for 48 hours (or 24 h for bryostatin). Following drug exposure, cell viability was calculated and plotted (column 1). Each agent adversely affected viability as concentration increased, except for bryostatin. Surface expression of CD19 (column 2) and CD22 (column 3) in leukemia cell lines was qualified by flow cytometry using Quanti-Brite PE beads. Average of triplicate wells is shown, values differing from untreated controls are indicated, * indicates p<0.05. Right panel: Expanded peripheral blood B cells from three donors, cultured on CD40L expressing feeder cells in media supplemented with (squares) or without (circles) B cell growth factors (IL-2, IL-4, IL-21, BAFF, see Materials and Methods ), were tested for changes in cell surface expression induced by bryostatin. The number of CD19 and CD22 molecules differed between donors to a degree, but was not significantly impacted by bryostatin, paired t-test p>0.05, grand median, solid bar, shown for reference.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing, Concentration Assay, Flow Cytometry, Cell Culture

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Total RNA and protein levels of CD19 and CD22 in bryostatin treated leukemia cell lines. Western blot analysis of (A) CD19 and (B) CD22 protein expression in Raji, NALM6 and REH cell lines with (+B) or without bryostatin treatment. (C) CD19 and (D) CD22 band intensity from three independent experiments was quantified and normalized to β-actin. For each line, treated and non-treated groups were compared. There was no significant difference (ns) between groups of at the protein level. RNA levels for (E) CD19 and (F) CD22 were quantified by ddRT-PCR. CD19 and CD22 copies per ng RNA were calculated and analyzed. Significant differences between treated and untreated groups were seen for CD22 in Raji cells ( p < 0.05 ). *p < 0.05. ns, not significant.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Surface expression of CD19 and CD22 upon co-culture with anti-CD22 CAR-T. Using Quanti-Brite analysis, the number of CD19 and CD22 molecules (y-axis) on the surface of Raji, NALM6, and REH cell lines was quantified, following co-culture with CD22 CAR-T, at the indicated effector to target ratios, x-axis. The leftmost pair of columns quantifies surface expression on untreated cell lines. Significant differences from control are shown *p < 0.05. The x-axis lists the cell line tested, exposure to bryostatin (B, magenta bars) or CD22 CAR-T alone (green).

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Surface expression of CD19 and CD22 following bryostatin wash-out and CAR-T removal. After overnight culture with anti-CD22 CAR-T, the number of cell surface proteins was quantified using Quanti-Brite analysis, average of triplicate wells and standard deviations are shown. 0 hr, x-axis, is after the overnight culture, and each time point represents cell surface proteins on the surface of untreated Raji, NALM6, or REH (Leukemia cell, green bars), treated with bryostatin alone (Leukemia + Bryostatin, magenta bars), treated with CAR-T alone (Leukemia+CART, blue bars), or treated with both CAR-T and bryostatin (Leukemia+CART+Bryostatin, red bars), at the time points listed, x-axis. Significant differences from leukemia alone are shown *p < 0.05.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Transfer of CD19 and CD22 to CD22 CAR-T following overnight culture with leukemia cell lines. The number of CD19 and CD22 molecules acquired by anti-CD22 CAR-T (trogocytosis) was quantified using Quanti-Brite analysis, as per Figure 3 , however in this case the T cells were analyzed. Average antigen expression and standard deviation are shown. Background signal is shown as a gray bar for each subgroup (CART). The x-axis lists the anti-CD22 CAR-T to leukemia cell ratio (E:T) used for each condition and indicates if the leukemia line had been treated with bryostatin (B, magenta bars).

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Anti-CD22 CAR-T mediated cellular cytotoxicity (CTL) of bryostatin-treated leukemia. (A–D) Average lysis from triplicate wells for four cell lines (Raji, NALM6, REH, and K562) by anti-CD22 CART (CD22 CART, open circle) or un-transduced T cells from the same donor (UTD, open triangle), treated with bryostatin (+B, closed shape) or untreated (open shape), a the E:T ratios listed on the x-axis. (E–H) Assay tested in parallel including cold-target inhibition (addition of K562 at a 30:1 E:T ratio). Representative results for T cells from 3 donors are shown, each data point showing the average and standard deviation from three replicate wells.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Lysis, Inhibition, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Bryostatin treatment reveals multiple pathways that CAR-T cells use to eliminate leukemia. In the center of the diagram, pre-B ALL cells are illustrated, displaying the CAR-T target antigen, CD22, innate immune receptor ligands induced by bryostatin that are recognized by activated T cells (Bryostatin-induced NK ligands) and ligands recognized by T cells that have been: a) sensitized by CAR-T production, b) bryostatin-induced, and c) not blocked by cold-target inhibition (CTAK, CAR-T cell non-antigen-specific killing). Also shown are a non-overlapping set of alternative innate immune receptor ligands that are recognized by NK92 upon bryostatin-treatment (right-most effector cell). Cold-target inhibition does not affect NK92 or CD22-specific CAR-T killing. Cold-target does decrease killing evidenced by activated T cells (UTD), but incompletely for CTAK-mediated killing. Green arrows indicate successful cytolysis and blunt red arrow indicates killing impacted by K562-mediated cold target inhibition (classic NK killing).

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Inhibition

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Impact of EBV lytic cycle inhibition on CD22 CAR-T mediated killing of bryostatin-treated Raji leukemia cells. Raji cells were cultured for 2 weeks in the presence or absence of 15 uM ganciclovir (+Ganciclovir in legend), and for the final day of culture bryostatin was added where indicated (+Bryo in legend). Treated cells were then used as targets in CTL assays using anti-CD22 CAR-T (CART) or untransduced T cells (UTD) as effector cells. Average cytolysis of 3 replicate wells is plotted for each condition. Results are representative of three independent experiments.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Inhibition, Cell Culture

Journal: Frontiers in Immunology

Article Title: Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line

doi: 10.3389/fimmu.2022.825364

Figure Lengend Snippet: Blocking of innate immunoreceptor ligands during CAR-T mediated cytolysis. The NALM6 pre-B ALL cell line was exposed to anti-CD22 CAR-T or control UTD cells at an E:T ratio of 10:1, y-axis. Results are grouped in each panel by cytolysis seen with untreated target (gray, control), bryostatin treatment (black), or treated with blocking agent (pink) or blocking agent and bryostatin (purple), using (A) recombinant DNAM-1, (B) NKG2D, (C) NKp30, or (D) anti-ICAM1 antibody, for 30 minutes prior to addition of effector cells. Average of 3 replicate wells are shown, with statistical difference between groups plotted, ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. (E–H) REH leukemia cells were similarly analyzed.

Article Snippet: T cells were phenotyped with: anti-CD3 (BioLegend, clone HIT3a, PB), CD4 (BioLegend, clone SK3, FITC), CD8 (BD Biosciences, clone RPAT8, BUV395), biotinylated CD22 protein (Sino Biological, for CAR detection) and SA-PE (BioLegend).

Techniques: Blocking Assay, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Binding of IgG1-Fc variants to glycan receptors. A , mutants lacking the Asn-297 glycan are severely restricted in their capacity to bind DC-SIGN by ELISA. The addition of an N -linked sugar at position 221 results in proteins with a reduced capacity to bind DC-SIGN compared with their equivalent variants in which Asn-221 is absent. B , the hypersialylated D221N mutants bind Siglec-1. No binding was observed with the N297A/N563A glycan-deficient mutant ( error bars represent standard deviations around the mean value, n = 2 independent experiments).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Model showing the contribution of different N -linked glycan and cysteine residues on Fc stoichiometry. The presence of Cys-575 allows optimal disulfide bonding between tail pieces of monomeric-Fcs. The tail piece glycan Asn-563 controls the number of monomeric tails that fit into the central corona (five to six in the case of hexa-Fc) while still allowing Cys-309 interdisulfide bridge formation. Cys-575 allows disulfide bonding between tail pieces of different monomers, but the absence of the Asn-563 glycan (the N563A mutant) allows many more tail pieces (up to twelve in the case of dodecamers) to fit into the central corona while still allowing disulfide bond formation through Cys-309 and/or Cys-575. The absence of Cys-575 prevents disulfide bonding between tail pieces, thereby generating sialylated monomers at Asn-563. The additional Asn-563 tail piece glycan in these monomers must explain the increased binding seen to Siglec-1 ( , A and B , and inset in this figure). The bulkier Asn-563 glycan with its predicted overall negative charge may lead to repulsion between two monomers, thus preventing disulfide bond formation between two Cys-309 residues in each monomeric Fc. The loss of both Asn-563 and Cys-575 (the N563A/C575A mutant) means that the observed laddered multimers must arise through Cys-309–mediated disulfide bonding in the Cγ2 domain. The presence of monomers, dimers, trimers, tetramers, pentamers, hexamers, and other intermediates in this mutant ( C ) suggests that these structures arise through a different mechanism, most likely via the sequential addition of 25-kDa half-mer Fc units at Cys-309. The lack of observable ladders with the L448STOP mutant implies that other amino acids in the tail piece are involved in bringing about monomer interactions that then facilitate disulfide bonding through either Cys-309 and/or Cys-575. Monomers with glycans located at both the N- and C-terminal ends of the Fc (Asn-221 and Asn-563) may allow for binding to receptors in cis as shown ( inset ).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Binding of monomeric IgG1-Fc glycan variants to sialic acid-binding immunoglobulin-type lectins (Siglecs) with specificity for α2,3-linked sialic acid. A , the C575A monomer binds Siglec-1. B , the D221N/C575A monomer binds Siglec-1 and Siglec-4. ELISA as described under “Experimental procedures” with receptors coated down at 2 μg/ml and Fc-fragments at 20 μg/ml in TMS buffer ( error bars represent standard deviations around the mean value, n = 2 independent experiments).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Cochrane Database of Systematic Reviews

Article Title: Interventions for cutaneous disease in systemic lupus erythematosus

doi: 10.1002/14651858.CD007478.pub2

Figure Lengend Snippet: Summary of treatments for cutaneous disease in SLE

Article Snippet: Tabalumab (LY‐2127399) is a fully human immunoglobulin G subclass 4 (IgG4) monoclonal antibody that binds and neutralises both soluble and membrane‐bound BAFF ( Merrill 2016 ). . CD molecules Epratuzumab is a "humanized anti‐CD22 monoclonal antibody that modulates B‐cell signalling without total B‐cell depletion" ( Clowse 2015 ).

Techniques: Filtration, Binding Assay, Activation Assay, Recombinant, Ligand Binding Assay, Activity Assay, Injection, Purification, Cell Function Assay, DNA Synthesis, Inhibition, Selection, Cell Differentiation

Journal: Journal of Neuroinflammation

Article Title: CD22 modulation alleviates amyloid β-induced neuroinflammation

doi: 10.1186/s12974-025-03361-2

Figure Lengend Snippet: CD22 promotes neuroinflammation via microglia. A Schematic diagram of sCD22 i.c.v. injection into wildtype C57BL/6 mice. B Volcano plot of sCD22-treated mouse cortex after 3 days treatment. N = 3. C-D Gene ontology analysis of sCD22-treated mice (3 days treatment) in KEGG pathway ( C ) and Biological function ( D ). E Volcano plot of sCD22-treated mouse cortex after 7 days treatment. N = 4. F Gene ontology analysis of sCD22-treated mice (7 days treatment) in Biological function. G GSEA showing enrichment of IL6/JAK/STAT3, TNFα Signaling via NFκB, and Cholesterol Homeostasis of mouse cortex after sCD22 7 days treatment relative to PBS group. H Representative image and quantitation showing the effect of sCD22 on IbaI and GFAP expression in mouse cortex. IbaI: Student t -test, ** P < 0.01; GFAP: ns: not significant. N = 6. I Schematic diagram of MDMi differentiation and sCD22 treatment. J Principal component analysis showing sCD22 treated MDMi versus control MDMi. N = 4. K Volcano plot of sCD22-treated MDMi. L Gene ontology analysis of sCD22-treated MDMi in Biological function. M Gene ontology analysis of sCD22-treated MDMi in KEGG

Article Snippet: All other materials included RPMI-1640 (Gibco, A10491-01), Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995-040), FBS (Gibco, A5256701), Pen/strep (Gibco, 15140-122), IL-34 (Sino Biological, 10948-H08S), GM-CSF (Sino Biological, 10015-HNAH), Geltrex (Gibco, A14133-02), human CD22 ECD (CD22 FL) (Sino Biological, 11958-H08H), human CD22 a.a. 176–687 (CD22 δ1) (Sino Biological, 11958-H08H1), mouse CD22 ECD (Sino Biological, 51177-M08H), Tanzisertib (Selleckchem, S8490), Perifosine (Selleckchem, S1037), Ravoxertinib (Selleckchem, S7554), SB856553 (Selleckchem, S7215).

Techniques: Injection, Quantitation Assay, Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: CD22 modulation alleviates amyloid β-induced neuroinflammation

doi: 10.1186/s12974-025-03361-2

Figure Lengend Snippet: sCD22 promotes microglial neuroinflammation via MAPK-signaling pathway and in a sialic acid-dependent manner. A Immunostaining with anti-IbaI antibody to examine microglia activation after sCD22 treatment in MDMi. Student t -test, **** P < 0.0001, ns: not significant. N = 26–27, from 3 independent experiments. B-D Effect of sCD22 on viability of MDMi ( B ), HMC-3 ( C ) and BV-2 cells ( D ). E Representative and quantitation of western blot examining ERK1/2 and p38 phosphorylation in sCD22-treated MDMi. p38: One-way ANOVA, F = 16.01, P = 0.001, Tukey post hoc test ** P < 0.01; ERK1/2: One-way ANOVA, F = 7.38, P = 0.0108, Tukey post hoc test * P < 0.05. F Effect of ERK1/2 inhibitor (Ravoxertinib) and p38 inhibitor (SB856553) on sCD22-mediated TNFα, IL-6 & CCL3 release. TNFα: One-way ANOVA, F = 16.09, Tukey post hoc test **** P < 0.0001; IL-6: One-way ANOVA, F = 4.917, Tukey post hoc test * P < 0.05, ** P < 0.01; CCL3: One-way ANOVA, F = 6.672, Tukey post hoc test ** P < 0.01. N = 4–5. G Effect of pan JNK inhibitor (Tanzisertib) and Akt inhibitor (Perifosine) on sCD22-mediated CCL3 release. H Schematic diagram of sCD22 with complete extracellular domain (CD22-FL) and with D1-truncated (CD22-δ1). I Full length and D1-truncated sCD22 effect on TNFα, IL-6 & CCL3 release in MDMi. TNFα: One-way ANOVA, F = 7.847, Tukey post hoc test * P < 0.05, ** P < 0.01; IL-6: One-way ANOVA, F = 4.375, Tukey post hoc test * P < 0.05; CCL3: One-way ANOVA, F = 3.669, Tukey post hoc test * P < 0.05. ns: not significant. N = 5–6. J Effect of CHO-derived sCD22 on CCL3 release in MDMi. Student t -test, P = 0.83. N = 2. K Effect of HEK293-derived sCD22 and CHO-derived sCD22 on CCL3 release in THP-1. Two-way ANOVA, source of sCD22: F (1,4) = 124, P = 0.0007; CCL3 release: F (1,4) = 87.57, P = 0.0007. Tukey post hoc test, i < 0.001. ns: not significant. N = 2. All data are presented as mean ± SEM

Article Snippet: All other materials included RPMI-1640 (Gibco, A10491-01), Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995-040), FBS (Gibco, A5256701), Pen/strep (Gibco, 15140-122), IL-34 (Sino Biological, 10948-H08S), GM-CSF (Sino Biological, 10015-HNAH), Geltrex (Gibco, A14133-02), human CD22 ECD (CD22 FL) (Sino Biological, 11958-H08H), human CD22 a.a. 176–687 (CD22 δ1) (Sino Biological, 11958-H08H1), mouse CD22 ECD (Sino Biological, 51177-M08H), Tanzisertib (Selleckchem, S8490), Perifosine (Selleckchem, S1037), Ravoxertinib (Selleckchem, S7554), SB856553 (Selleckchem, S7215).

Techniques: Immunostaining, Activation Assay, Quantitation Assay, Western Blot, Derivative Assay

Journal: Journal of Neuroinflammation

Article Title: CD22 modulation alleviates amyloid β-induced neuroinflammation

doi: 10.1186/s12974-025-03361-2

Figure Lengend Snippet: CD22 modulation by suciraslimab alleviates Aβ-induced neuroinflammation in human CD22 transgenic mice. A Schematic diagram of Aβ-induced neuroinflammation model in human CD22 transgenic mice. B Effect of suciraslimab on Aβ-injected model mice in Y-maze test. Alternation: One-way ANOVA, F = 4.724, P = 0.0196. Tukey post hoc test, * P < 0.05. Number of arm entry: One-way ANOVA, F = 0.07, P = 0.93. N = 8–9. C Volcano plot of suciraslimab-treated mouse cortex. N = 3. D Gene ontology analysis of suciraslimab-treated mouse cortex in Biological function. E Gene ontology analysis of suciraslimab-treated mouse cortex in molecular function. F Effect of suciraslimab on chemokine release in mouse brain of model mice. Student t -test, P value as stated in the figure. N = 3. All data are presented as mean ± SEM

Article Snippet: All other materials included RPMI-1640 (Gibco, A10491-01), Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995-040), FBS (Gibco, A5256701), Pen/strep (Gibco, 15140-122), IL-34 (Sino Biological, 10948-H08S), GM-CSF (Sino Biological, 10015-HNAH), Geltrex (Gibco, A14133-02), human CD22 ECD (CD22 FL) (Sino Biological, 11958-H08H), human CD22 a.a. 176–687 (CD22 δ1) (Sino Biological, 11958-H08H1), mouse CD22 ECD (Sino Biological, 51177-M08H), Tanzisertib (Selleckchem, S8490), Perifosine (Selleckchem, S1037), Ravoxertinib (Selleckchem, S7554), SB856553 (Selleckchem, S7215).

Techniques: Transgenic Assay, Injection

Journal: Journal of Neuroinflammation

Article Title: CD22 modulation alleviates amyloid β-induced neuroinflammation

doi: 10.1186/s12974-025-03361-2

Figure Lengend Snippet: Suciraslimab suppresses Aβ-induced inflammation in microglia and human PBMC. A Effect of CD22 overexpression on Aβ-induced NFκB signaling in HEK293. Two-way ANOVA: CD22 expression, F (1,20) = 62.97, i < 0.0001; Aβ treatment, F (4,20) = 16.83, P < 0.0001. Tukey post hoc test, **** P < 0.0001. N = 3. B Effect of suciraslimab on Aβ-induced IL-1β release in MDMi. One-way ANOVA, F = 7.767, P = 0.004. Tuley post hoc test, * P < 0.05, ** P < 0.01. N = 6–7. C Immunofluorescent staining and quantitation of NLRP3 and ASC after Aβ and suciraslimab treatment in MDMi. NLRP3: One-way ANOVA, F = 10.09, P < 0.0001, Tukey post hoc test * P < 0.05, *** P < 0.001; ASC, One-way ANOVA, F = 19.10, P < 0.0001, Tukey post hoc test **** P < 0.0001. N = 6–15. D Effect of suciraslimab on Aβ-induced IL-1β release in human PBMC. One-way ANOVA, F = 6.833, P = 0.0052. Tukey post hoc test, ** P < 0.01, ns = not significant. N = 8. E Effect of suciraslimab on IFNγ + LPS-induced IL-23 and IL-12 release in human PBMC. IL-23: One-way ANOVA, F = 26.93, P = 0.0002. Tukey post hoc test, ** P < 0.01, *** P < 0.001; IL-12: One-way ANOVA, F = 10.21, P = 0.0008. Tukey post hoc test, * P < 0.05, *** P < 0.001. N = 4–8. F Effect of suciraslimab on TLR4 surface expression on monocyte upon IFNγ and LPS activation. Two-tailed paired Student’s t test, P = 0.0293, t = 3.322, df = 4. N = 5 G Effect of suciraslimab on α4 integrin surface expression on T cell of human PBMC. One-way ANOVA, F = 0.7059, P = 0.5131. N = 5. H Effect of suciraslimab on α4 integrin surface expression on B cell of human PBMC. One-way ANOVA, F = 66.02, P < 0.0001. Tukey’s post hoc test, * P < 0.05, **** P < 0.0001. N = 4–5. I Effect of suciraslimab on α4 integrin surface expression on T cell-depleted human PBMC. One-way ANOVA, F = 16.91, P = 0.0009. Tukey’s post hoc test, ** P < 0.01. N = 4. J Effect of suciraslimab on α4 integrin surface expression on monocyte-depleted human PBMC. One-way ANOVA, F = 8.565, P = 0.0083. Tukey’s post hoc test, IgG1 vs. αCD22 Ab, P = 0.1748. N = 4. All data are presented as mean ± SEM

Article Snippet: All other materials included RPMI-1640 (Gibco, A10491-01), Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995-040), FBS (Gibco, A5256701), Pen/strep (Gibco, 15140-122), IL-34 (Sino Biological, 10948-H08S), GM-CSF (Sino Biological, 10015-HNAH), Geltrex (Gibco, A14133-02), human CD22 ECD (CD22 FL) (Sino Biological, 11958-H08H), human CD22 a.a. 176–687 (CD22 δ1) (Sino Biological, 11958-H08H1), mouse CD22 ECD (Sino Biological, 51177-M08H), Tanzisertib (Selleckchem, S8490), Perifosine (Selleckchem, S1037), Ravoxertinib (Selleckchem, S7554), SB856553 (Selleckchem, S7215).

Techniques: Over Expression, Expressing, Staining, Quantitation Assay, Activation Assay, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: CD22 modulation alleviates amyloid β-induced neuroinflammation

doi: 10.1186/s12974-025-03361-2

Figure Lengend Snippet: Suciraslimab promotes Aβ phagocytosis. A BLI analysis of mouse CD22-Aβ interaction. Association: 600s; Dissociation: 600s. B BLI analysis of human CD22-Aβ interaction. Association: 600s; Dissociation: 600s. C Immunofluorescent staining and quantitation of FITC-Aβ on HEK293 and HEK293-hCD22 cells. Student t -test, ** P < 0.01. N = 20–21, from 3 independent experiments. D Representative image and quantitation of Proximity-ligation assay of CD22-Aβ complex in HMC-3. Student’s t -test, **** P < 0.0001. N = 41, from 3 independent experiments. E Structural alignment of mouse CD22 and human CD22. The structures of both mouse and human CD22 extracellular domain were generated with Alphafold2. Pairwise structural alignment score (TM-score) higher than 0.5 assumes generally proteins aligned of the same fold. F Surface CD22 expression in HMC-3 after suciraslimab treatment. One-way ANOVA, F = 2.892, P = 0.0139. Tukey post hoc test, * P < 0.05. N = 76–83. G Surface suciraslimab binding on HMC-3. One-way ANOVA, F = 125, P < 0.0001. Tukey post hoc test, ** P = 0.002. N = 3. H Effect of suciraslimab on FITC-Aβ phagocytosis in HMC-3. One-way ANOVA, F = 43.92, P < 0.0001. Tukey post hoc test, * P = 0.046, ** P = 0.0018, **** P < 0.0001. N = 3. I Effect of suciraslimab on FITC-Aβ phagocytosis in PMA-differentiated MO3.13. Two-tailed Student’s t test, P = 0.0477, t = 2.482, df = 6

Article Snippet: All other materials included RPMI-1640 (Gibco, A10491-01), Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11995-040), FBS (Gibco, A5256701), Pen/strep (Gibco, 15140-122), IL-34 (Sino Biological, 10948-H08S), GM-CSF (Sino Biological, 10015-HNAH), Geltrex (Gibco, A14133-02), human CD22 ECD (CD22 FL) (Sino Biological, 11958-H08H), human CD22 a.a. 176–687 (CD22 δ1) (Sino Biological, 11958-H08H1), mouse CD22 ECD (Sino Biological, 51177-M08H), Tanzisertib (Selleckchem, S8490), Perifosine (Selleckchem, S1037), Ravoxertinib (Selleckchem, S7554), SB856553 (Selleckchem, S7215).

Techniques: Staining, Quantitation Assay, Proximity Ligation Assay, Generated, Expressing, Binding Assay, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

doi: 10.1136/jitc-2025-011759

Figure Lengend Snippet: NCtx-dual induces robust and durable in vivo CAR-T generation, tumor control and extended survival in CD34+ HSC-engrafted NCG mice. ( a ) Schematic representation of study design: NCG mice engrafted with CD34+ HSC (NCG-His) were injected intravenously with 5×10 5 luciferase-expressing Nalm6 tumor cells, followed by IP injection of 200 ng IL-7. Mice were treated intravenously with NCtx-dual or a NCtx vehicle control encapsulating eGFP mcDNA and SB100x mRNA (vehicle control) at a total nucleic acid dose of 50 µg/kg. ( b ) CD19/CD22 dual CAR mcDNA expression was assessed by flow cytometry in circulating T cells for 40 days post-NCtx administration. n=12, data are presented as mean with individual values. ( c ) Nalm6 tumor burden was monitored by BLI. ( d ) Kaplan-Meier survival analysis. n=6 (vehicle control) or n=12 (NCtx-dual). ( e ) Expression of the exhaustion marker PD-1 in CAR+ and CAR− T cell populations over time in NCtx-dual-treated mice, analyzed by flow cytometry. n=12, data represent mean±individual values. ( f ) T cell phenotype characterization (Tnaive/Tscm, Tcm, Tem, and Teff) based on CD45RA and CD62L expression in CAR+ and CAR− T cells after NCtx-dual administration. n=12, data represent mean±SD. P values were calculated using log-rank Mantel-Cox test ( b ) or two-way ANOVA, mixed effect model ( d, e ). Significance is plotted with ns for p>0.0332 and *p<0.0332. ANOVA, analysis of variance; BLI, bioluminescent imaging; CAR, chimeric antigen receptor; HSC, hematopoietic stem cell; IL-7, interleukin 7; IP, intraperitoneal; mcDNA, minicircle DNA; mRNA, messenger RNA; ns, not significant; PD-1, programmed cell death protein-1; Tcm, central memory T cell; Teff, effector T cell; Tem, effector memory T cell; Tnaive, naïve T cell; Tscm, stem cell memory T cell.

Article Snippet: CD19/CD22 dual CAR expression was detected using CD22 CAR detection reagent (Miltenyi Biotec, REA130-126-727, 1:1000) and streptavidin-APC-Cy7 (BD, REA746, 1:200).

Techniques: In Vivo, Control, Injection, Luciferase, Expressing, Flow Cytometry, Marker, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients

doi: 10.1172/JCI130059

Figure Lengend Snippet: (A) BM samples obtained on day 28 after CAR T cell infusion. H&E-stain shows decreased trilineage hematopoiesis with increased macrophages. CD3 immunohistochemical (IHC) stain highlights extensive T cell infiltration with flow cytometric confirmation of anti-CD22 CAR positivity in 59% of T cells. CD68 IHC stain highlights hemophagocytic macrophages. Giemsa stain of BM aspirate also shows hemophagocytosis. Original magnification, 50× (H&E, CD3, CD68 stains) and 100× (Giemsa stain). (B) Representative chronological changes in serum cytokine levels from patient 52 who had CRS without subsequent HLH. (C) Representative chronological changes in serum cytokine levels from patient 37 who had CRS and subsequent HLH. (D) The percentages of circulating T cells (CD3+) that stained positive for surface CAR expression were assessed by flow cytometry at the indicated time points. (E–G) Peak levels (during the first 28 days) of (E) IFN-γ, (F) IL-1β, and (G) IL-18 in serum/plasma. Data shown in D–G include all patients who were diagnosed with CRS according to previously published criteria (9), and patients who had never been diagnosed with CRS are not included. Data were stratified according to the presence or absence of HLH diagnosis (in addition to CRS) after CAR T cell infusion. Data are reported as the mean ± SD (D–G). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Mann-Whitney U test (D–G).

Article Snippet: Human anti-CD22 CAR detection was performed using a CD22-Fc chimera protein (R&D Systems) as previously described ( 1 ).

Techniques: Staining, Immunohistochemical staining, Giemsa Stain, Expressing, Flow Cytometry, MANN-WHITNEY