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OriGene full length cd33
Full Length Cd33, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/full+length+cd33/CD33+(NM_001772)+Human+Untagged+Clone/pmc06391081-555-10-12
Average 90 stars, based on 1 article reviews

full length cd33 - by Bioz Stars, 2026-10

90/100 stars

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Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen
Article Snippet: .. Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912). .. Electroporation of the targeting construct into PRX ES cells (C57BL/6N background), blastocyst injections, and chimera breeding with C57BL/6J albino mice (The Jackson Laboratory, stock no. 000058) were performed according to standard protocols ( 66 ).

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen
Article Snippet: .. Rosa26-Stopfl/flCD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank BC028152.1) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912). .. Electroporation of the targeting construct into PRX ES cells (C57BL/6N background), blastocyst injections, and chimera breeding with C57BL/6J albino mice (The Jackson Laboratory, stock no. 000058) were performed according to standard protocols (66).

Subcloning:

Sequencing:

Plasmid Preparation:

Image Search Results

Identification of VH binders targeting human CD33 antigen. (A) Schema illustrating the workflow of VH binders screening using a phage display library. (B) Non-reducing and reducing SDS-PAGE of the selected VH binders. Molecular masses of standards are shown on the left. (C) ELISA binding to recombinant human CD33. (D) FACS binding of the selected binders to CD33-negative CHO and acute lymphoblastic leukemia cell line RS4;11, and CD33-positive acute myeloid leukemia cell line MV4-11.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Identification of VH binders targeting human CD33 antigen. (A) Schema illustrating the workflow of VH binders screening using a phage display library. (B) Non-reducing and reducing SDS-PAGE of the selected VH binders. Molecular masses of standards are shown on the left. (C) ELISA binding to recombinant human CD33. (D) FACS binding of the selected binders to CD33-negative CHO and acute lymphoblastic leukemia cell line RS4;11, and CD33-positive acute myeloid leukemia cell line MV4-11.

Article Snippet: Detection of A431 cells expressing CD33 full length isoform variant 1, or the V domain -truncated isoform variant 2 was facilitated by flow cytometry using domain-specific antibodies (Clone WM53 reactive with the V domain, and clone HIM3-4, detecting the C2 domain, common to both isoforms (BioLegend), or clone AC104.3E3 detecting the full-length CD33 isoform (Miltenyi Biotec).

Techniques: SDS Page, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant

Structure and surface expression of anti-CD33 CAR constructs in primary human T cells. CAR33VH and the positive control My96CAR , are represented (A) Each binder sequence was connected in frame to CD8 linker (H), CD8 transmembrane domain (TM), CD137/4-1BB and CD3 zeta signaling domains. Lentiviral vectors encoding each of the constructs under the control of EF1a promoter were generated and used to transduce primary human T cells at 10%v/v. (B) Flow cytometric analysis of CAR T expression. Transduced live singlet cells were stained with primary CD33-Fc followed by anti Fc–APC F(ab)′2 (light blue). Non-transduced cells (pink) served as negative controls. Results are representative of five transduction experiments from five separate donors. (C) Summary of CAR surface expression as a function of MOI. The expression of CAR33VH or My96CAR for five separate donors transduced at MOI of 20 or 60 was measured by flow cytometry. Average CAR expression ±SEM for each determination is shown.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Structure and surface expression of anti-CD33 CAR constructs in primary human T cells. CAR33VH and the positive control My96CAR , are represented (A) Each binder sequence was connected in frame to CD8 linker (H), CD8 transmembrane domain (TM), CD137/4-1BB and CD3 zeta signaling domains. Lentiviral vectors encoding each of the constructs under the control of EF1a promoter were generated and used to transduce primary human T cells at 10%v/v. (B) Flow cytometric analysis of CAR T expression. Transduced live singlet cells were stained with primary CD33-Fc followed by anti Fc–APC F(ab)′2 (light blue). Non-transduced cells (pink) served as negative controls. Results are representative of five transduction experiments from five separate donors. (C) Summary of CAR surface expression as a function of MOI. The expression of CAR33VH or My96CAR for five separate donors transduced at MOI of 20 or 60 was measured by flow cytometry. Average CAR expression ±SEM for each determination is shown.

Techniques: Expressing, Construct, Positive Control, Sequencing, Generated, Transduction, Staining, Flow Cytometry

Heavy chain only anti-CD33 CAR demonstrates tumor-specific cytokine responses in vitro . Cytokine analysis was performed on supernatants from 5 × 10 4 CAR T cells cultured with CD33 high cell lines MOLM-14, or HL60, or CD33 low cell line KG-1a at E:T ratio of 1:1 in triplicate. CAR alone group was included to control for spontaneous CAR T cytokine release. Levels of cytokines in culture supernatants were measured by ELISA. Mean values + SEM for CAR33VH, My96CAR and untransduced control (UTD) from three independent donors are shown. Groups were compared by Two Way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns-non significant. UTD, untransduced T cells.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only anti-CD33 CAR demonstrates tumor-specific cytokine responses in vitro . Cytokine analysis was performed on supernatants from 5 × 10 4 CAR T cells cultured with CD33 high cell lines MOLM-14, or HL60, or CD33 low cell line KG-1a at E:T ratio of 1:1 in triplicate. CAR alone group was included to control for spontaneous CAR T cytokine release. Levels of cytokines in culture supernatants were measured by ELISA. Mean values + SEM for CAR33VH, My96CAR and untransduced control (UTD) from three independent donors are shown. Groups were compared by Two Way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns-non significant. UTD, untransduced T cells.

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Heavy chain only anti-CD33 CAR demonstrates antigen-specific cytotoxicity in vitro . (A–E) CD33-targeting CAR constructs CAR33VH, My96CAR or untransduced T cells (UTD) were incubated with tumor lines (listed above each plot) for 18 h at effector to target ratios of 5, 10, or 20, x-axis, in triplicate. CAR cytotoxic effect was measured by luminometry. Mean values ± SEM from four independent donors are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only anti-CD33 CAR demonstrates antigen-specific cytotoxicity in vitro . (A–E) CD33-targeting CAR constructs CAR33VH, My96CAR or untransduced T cells (UTD) were incubated with tumor lines (listed above each plot) for 18 h at effector to target ratios of 5, 10, or 20, x-axis, in triplicate. CAR cytotoxic effect was measured by luminometry. Mean values ± SEM from four independent donors are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Techniques: In Vitro, Construct, Incubation

Heavy chain only anti-CD33 CAR T persist and eliminate tumors in a long-term co-incubation assay. VH33CAR persistence and long-term cytotoxic activity were evaluated by co-incubating CAR cells with CD33 hi HL-60 leukemia cells at low E:T ratios (5:1-0.04:1) for 11 days. My96CAR served as a positive control, and untransduced cells (UTD) as negative controls. On days 5 and 11, co-cultures were stained with anti-CD3, and anti-CD33 antibody, and signal from 7-AAD (–) cells acquired by flow cytometry to determine the percentage of live effector and target cells in each culture. (A) Representative plots from one of three donors at E:T ratio of 5:1 on day 5 is shown. HL-60 leukemia cells are gated in the upper left box of each plot, HL60 targets bound to T cells (HL60+T cells) are gated at the upper right box of each plot, activated T cells are gated at the bottom left box, and resting CD3 + effector T cells are gated in the lower right box of each plot, and percentage of gated cells for each population is noted next to corresponding box. (B) Mean %Live HL-60 +SEM from three independent co-incubation experiments performed with CAR T cells from three separate donors. **** p < 0.0001, ** p < 0.01, NS-non-significant, two way ANOVA followed by Dunnett's multiple comparisons test.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only anti-CD33 CAR T persist and eliminate tumors in a long-term co-incubation assay. VH33CAR persistence and long-term cytotoxic activity were evaluated by co-incubating CAR cells with CD33 hi HL-60 leukemia cells at low E:T ratios (5:1-0.04:1) for 11 days. My96CAR served as a positive control, and untransduced cells (UTD) as negative controls. On days 5 and 11, co-cultures were stained with anti-CD3, and anti-CD33 antibody, and signal from 7-AAD (–) cells acquired by flow cytometry to determine the percentage of live effector and target cells in each culture. (A) Representative plots from one of three donors at E:T ratio of 5:1 on day 5 is shown. HL-60 leukemia cells are gated in the upper left box of each plot, HL60 targets bound to T cells (HL60+T cells) are gated at the upper right box of each plot, activated T cells are gated at the bottom left box, and resting CD3 + effector T cells are gated in the lower right box of each plot, and percentage of gated cells for each population is noted next to corresponding box. (B) Mean %Live HL-60 +SEM from three independent co-incubation experiments performed with CAR T cells from three separate donors. **** p < 0.0001, ** p < 0.01, NS-non-significant, two way ANOVA followed by Dunnett's multiple comparisons test.

Techniques: Incubation, Activity Assay, Positive Control, Staining, Flow Cytometry

Heavy chain only CAR CD33VH targets V domain-containing the full length isoform of CD33. The CD33-targeting CAR constructs CAR33VH, My96CAR or negative controls were incubated with tumor lines (A) A431 (CD33-), (B) A431v1 (containing the full length CD33 isoform) or (C) A431v2 (containing the V domain-truncated CD33 isoform) for 18 h at effector to target ratios of 5, 10 or 20 in triplicate. All target lines stably expressed firefly luciferase, and CAR cytotoxic effect was measured by luminometry. Data from one representative experiment out of three experiments utilizing CART cells derived from different donors is shown. (D) Supernatants from co-cultures of CAR cells with tumor lines in (A) were harvested after 18 h co-incubation and analyzed for IFN-gamma by ELISA in triplicate. Mean values +SEM from four independent experiments performed in different donors are shown. UTD-untransduced T cells, GFP-transduced T cells. NS-non-significant.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only CAR CD33VH targets V domain-containing the full length isoform of CD33. The CD33-targeting CAR constructs CAR33VH, My96CAR or negative controls were incubated with tumor lines (A) A431 (CD33-), (B) A431v1 (containing the full length CD33 isoform) or (C) A431v2 (containing the V domain-truncated CD33 isoform) for 18 h at effector to target ratios of 5, 10 or 20 in triplicate. All target lines stably expressed firefly luciferase, and CAR cytotoxic effect was measured by luminometry. Data from one representative experiment out of three experiments utilizing CART cells derived from different donors is shown. (D) Supernatants from co-cultures of CAR cells with tumor lines in (A) were harvested after 18 h co-incubation and analyzed for IFN-gamma by ELISA in triplicate. Mean values +SEM from four independent experiments performed in different donors are shown. UTD-untransduced T cells, GFP-transduced T cells. NS-non-significant.

Techniques: Construct, Incubation, Stable Transfection, Luciferase, Derivative Assay, Enzyme-linked Immunosorbent Assay

Heavy chain only construct VH33CAR activity in vivo . (A) Study design schema: NOD- scid IL2Rg null (NSG) mice were injected with 0.5 × 10 6 luciferase-enabled MOLM-14 tumor cells i.v . on day 0. On day 6, mice were dosed i.v . with 5 × 10 6 CART + cells. Tumor burden was evaluated weekly by bioluminescence, between days 14-35. Blood was collected for analysis on Day 19. (B) Immunofluorescent Imaging of experimental groups was performed on study days 14, 21, 28, 35. TA- tumor alone, UTD-untransduced T cell control, CAR33VH–single chain only anti CD33 CAR, My96CAR–positive control CAR. Images were acquired on IVIS Lumina and analyzed by Living Image software (PerkinElmer). (C) Tumor burden was assessed by bioluminescent imaging on study days 14, 21, 28, 35. N = 6 mice per group, average radiance ± SEM was plotted for each time point. TA- tumor alone, UTD-untransduced T cell control. (D) Survival analysis of treated mice. While all animals survived to day 35 in CAR33VH and My96CAR groups, animals in the control groups survived only up to day 21. Retro-orbital bleeds were obtained from surviving mice on study day 19. (E) MOLM-14 tumor cells (CD45 + /Singlets/Live/GFP + ) and (F) . CAR T + cells (CD45 + /Singlets/Live/CD3 + /CAR + ) in blood samples were analyzed by flow cytometry. Total cell count was determined by volumetric flow cytometry, normalized using CountBright beads added during sample preparation. N = 6 for CAR groups, N = 4 for UTD; mean ± SEM. One way ANOVA with multiple comparisons analysis, **** p < 0.0001. Survival data were compared by log-rank Mantel-Cox test, *** p < 0.001.

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only construct VH33CAR activity in vivo . (A) Study design schema: NOD- scid IL2Rg null (NSG) mice were injected with 0.5 × 10 6 luciferase-enabled MOLM-14 tumor cells i.v . on day 0. On day 6, mice were dosed i.v . with 5 × 10 6 CART + cells. Tumor burden was evaluated weekly by bioluminescence, between days 14-35. Blood was collected for analysis on Day 19. (B) Immunofluorescent Imaging of experimental groups was performed on study days 14, 21, 28, 35. TA- tumor alone, UTD-untransduced T cell control, CAR33VH–single chain only anti CD33 CAR, My96CAR–positive control CAR. Images were acquired on IVIS Lumina and analyzed by Living Image software (PerkinElmer). (C) Tumor burden was assessed by bioluminescent imaging on study days 14, 21, 28, 35. N = 6 mice per group, average radiance ± SEM was plotted for each time point. TA- tumor alone, UTD-untransduced T cell control. (D) Survival analysis of treated mice. While all animals survived to day 35 in CAR33VH and My96CAR groups, animals in the control groups survived only up to day 21. Retro-orbital bleeds were obtained from surviving mice on study day 19. (E) MOLM-14 tumor cells (CD45 + /Singlets/Live/GFP + ) and (F) . CAR T + cells (CD45 + /Singlets/Live/CD3 + /CAR + ) in blood samples were analyzed by flow cytometry. Total cell count was determined by volumetric flow cytometry, normalized using CountBright beads added during sample preparation. N = 6 for CAR groups, N = 4 for UTD; mean ± SEM. One way ANOVA with multiple comparisons analysis, **** p < 0.0001. Survival data were compared by log-rank Mantel-Cox test, *** p < 0.001.

Techniques: Construct, Activity Assay, In Vivo, Injection, Luciferase, Imaging, Positive Control, Software, Flow Cytometry, Cell Counting, Sample Prep

(A) Schematic representation of an antigenic liposome (TNP-LP, left) or an antigenic liposome displaying human CD33 ligands (TNP-LP-CD33L, right). (B) Antibody staining of various Siglecs (Sig-) on LAD2 cells analyzed by flow cytometry. (C) Flow cytometric analysis of binding of fluorescent liposomes with or without CD33L (20 μM) to LAD2 cells pretreated with isotype control or anti-CD33 (clone WM53). (D) Calcium flux of LAD2 cells induced by addition (arrow) of TNP-LP or TNP-LP-CD33 (2.5 μM) or PBS (1 μl). Graph shows quantification of the AUC of calcium flux induced by 2.5 μM TNP-LP or TNP-LP-CD33L. Results were combined from 2 independent experiments. (E) Degranulation induced by TNP-LP or TNP-LP-CD33L as measured by the percentage of β-hex release (n = 3 per condition; values are plotted as the mean ± SD). (F) Degranulation induced by TNP-LP (30 μM), TNP-LP-CD33L (30 μM), or a mixture of TNP-LP and LP-CD33L (30 μM each). (G) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of LP-CD33L (10 μM). Control cells received buffer only. (H) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of isotype or anti-CD33 (clone WM53, 1 μg/ml). (I) Degranulation induced by Ah2-LP or Ah2-LP-CD33L (30 μM), with final Ah2 at 750 ng/ml using LAD2 cells sensitized with atopic plasma reactive to peanut (PlasmaLab). (J) Degranulation induced by OVA-LP or OVA-LP-CD33L (30 μM), with the final OVA dose at 1.5 μg/ml using LAD2 cells sensitized with human anti–OVA-IgE. Results in E–J are representative of 3 independent experiments. ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (D and E) and 1-way ANOVA followed by Tukey’s test (F–J). α, anti; Max, maximum.

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Schematic representation of an antigenic liposome (TNP-LP, left) or an antigenic liposome displaying human CD33 ligands (TNP-LP-CD33L, right). (B) Antibody staining of various Siglecs (Sig-) on LAD2 cells analyzed by flow cytometry. (C) Flow cytometric analysis of binding of fluorescent liposomes with or without CD33L (20 μM) to LAD2 cells pretreated with isotype control or anti-CD33 (clone WM53). (D) Calcium flux of LAD2 cells induced by addition (arrow) of TNP-LP or TNP-LP-CD33 (2.5 μM) or PBS (1 μl). Graph shows quantification of the AUC of calcium flux induced by 2.5 μM TNP-LP or TNP-LP-CD33L. Results were combined from 2 independent experiments. (E) Degranulation induced by TNP-LP or TNP-LP-CD33L as measured by the percentage of β-hex release (n = 3 per condition; values are plotted as the mean ± SD). (F) Degranulation induced by TNP-LP (30 μM), TNP-LP-CD33L (30 μM), or a mixture of TNP-LP and LP-CD33L (30 μM each). (G) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of LP-CD33L (10 μM). Control cells received buffer only. (H) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of isotype or anti-CD33 (clone WM53, 1 μg/ml). (I) Degranulation induced by Ah2-LP or Ah2-LP-CD33L (30 μM), with final Ah2 at 750 ng/ml using LAD2 cells sensitized with atopic plasma reactive to peanut (PlasmaLab). (J) Degranulation induced by OVA-LP or OVA-LP-CD33L (30 μM), with the final OVA dose at 1.5 μg/ml using LAD2 cells sensitized with human anti–OVA-IgE. Results in E–J are representative of 3 independent experiments. ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (D and E) and 1-way ANOVA followed by Tukey’s test (F–J). α, anti; Max, maximum.

Article Snippet: Full-length human CD33 was amplified from cDNA (OriGene, catalog SC122608) and subcloned into a pcDNA5/FRT/V5-His vector using the Nhe I and Age I site.

Techniques: Staining, Flow Cytometry, Binding Assay

(A) Flow cytometric analysis of GFP expression on representative peritoneal mast cells harvested from C57BL/6J, control-Tg (Mcpt5-Cre–Rosa26-Stopfl/fl-CD33+), and CD33-Tg (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+) mice. Mast cells were defined as PI–CD45+c-Kit+. Baseline GFP signal was determined by mast cells from C57BL/6J mice. (B) Quantification of the percentage of GFP+ peritoneal mast cells from mice of the 3 genotypes. Tg mice bearing 1 or 2 copies of CD33 were used. Both male and female mice 8 weeks or older were analyzed, with no difference observed. (C) Numbers of peritoneal mast cells from the same mice of the 3 genotypes as in B. (A–C) Results were compiled from 6 experiments. (D) Staining of peritoneal cells harvested from control-Tg or CD33-Tg mice with anti-CD33 (clone WM53) or isotype control, as analyzed by flow cytometry. (E) Binding of fluorescent liposome, with or without CD33L (20 μM), to peritoneal mast cells (c-Kit+FcεRI+CD45+). (F) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L. (G) Cytokine induction of CD33+ BMMCs following treatment with TNP-LP (40 μM), TNP-LP-CD33L (40 μM), LP-CD33L (40 μM), or a mixture of TNP-LP and LP-CD33L (40 μM each). Supernatant from the unstimulated cells was subtracted as a background. (H) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of anti-CD33 (2 μg/ml). (I and J) Cytokine production of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (40 μM) in the presence of anti-CD33 (10 μg/ml). Supernatant from untreated cells was subtracted as a background. Results shown are representative of 3 (D–G) or 2 (H–J) independent experiments. (F–J) Values are plotted as the mean ± SD (n = 3 per condition). ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (F) and 1-way ANOVA followed by Tukey’s test (C and G–J).

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Flow cytometric analysis of GFP expression on representative peritoneal mast cells harvested from C57BL/6J, control-Tg (Mcpt5-Cre–Rosa26-Stopfl/fl-CD33+), and CD33-Tg (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+) mice. Mast cells were defined as PI–CD45+c-Kit+. Baseline GFP signal was determined by mast cells from C57BL/6J mice. (B) Quantification of the percentage of GFP+ peritoneal mast cells from mice of the 3 genotypes. Tg mice bearing 1 or 2 copies of CD33 were used. Both male and female mice 8 weeks or older were analyzed, with no difference observed. (C) Numbers of peritoneal mast cells from the same mice of the 3 genotypes as in B. (A–C) Results were compiled from 6 experiments. (D) Staining of peritoneal cells harvested from control-Tg or CD33-Tg mice with anti-CD33 (clone WM53) or isotype control, as analyzed by flow cytometry. (E) Binding of fluorescent liposome, with or without CD33L (20 μM), to peritoneal mast cells (c-Kit+FcεRI+CD45+). (F) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L. (G) Cytokine induction of CD33+ BMMCs following treatment with TNP-LP (40 μM), TNP-LP-CD33L (40 μM), LP-CD33L (40 μM), or a mixture of TNP-LP and LP-CD33L (40 μM each). Supernatant from the unstimulated cells was subtracted as a background. (H) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of anti-CD33 (2 μg/ml). (I and J) Cytokine production of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (40 μM) in the presence of anti-CD33 (10 μg/ml). Supernatant from untreated cells was subtracted as a background. Results shown are representative of 3 (D–G) or 2 (H–J) independent experiments. (F–J) Values are plotted as the mean ± SD (n = 3 per condition). ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (F) and 1-way ANOVA followed by Tukey’s test (C and G–J).

Article Snippet: Full-length human CD33 was amplified from cDNA (OriGene, catalog SC122608) and subcloned into a pcDNA5/FRT/V5-His vector using the Nhe I and Age I site.

Techniques: Expressing, Staining, Flow Cytometry, Binding Assay

(A) Phosphorylation of Syk, PLCγ1, MEK, and ERK in LAD2 cells after a 3-, 10-, or 30-minute stimulation using TNP-LP or TNP-LP-CD33L (2 μM), as evaluated by Western blotting. (B) Phosphorylation of Syk and ERK in LAD2 cells after a 10-minute stimulation using TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (C) Phosphorylation of Syk, PLCγ1, PLCγ2, JNK, AKT, and ERK in CD33+ BMMCs cells after a 10-minute stimulation with TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (A–C) Total Syk and ERK were used as loading controls. (D–F) Proposed mechanisms of IgE/FcεRI signaling induced by antigenic liposomes and recruitment of CD33 by CD33L. (D) TNP-LP stabilizes the anti–TNP-IgE–FcεRI complex in lipid rafts with Src kinases that initiate the FcεRI signaling cascade. We propose that CD33 has no basal impact on signaling, because it is not constitutively localized in the same microdomain with FcεRI. (E) TNP-LP-CD33L recruits CD33 to the anti–TNP-IgE–FcεRI immunological synapse. Our results suggest that the cytoplasmic ITIMs of CD33 were phosphorylated by Src kinases and then recruited tyrosine phosphatases such as Shp-1, which dephosphorylated Syk, and other kinases. (F) Proposed model showing that monoclonal anti-CD33 antibodies (or LP-CD33L) block recruitment of CD33 to the IgE-FcεRI complex and enable mast cell degranulation induced by TNP-LP-CD33L.

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Phosphorylation of Syk, PLCγ1, MEK, and ERK in LAD2 cells after a 3-, 10-, or 30-minute stimulation using TNP-LP or TNP-LP-CD33L (2 μM), as evaluated by Western blotting. (B) Phosphorylation of Syk and ERK in LAD2 cells after a 10-minute stimulation using TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (C) Phosphorylation of Syk, PLCγ1, PLCγ2, JNK, AKT, and ERK in CD33+ BMMCs cells after a 10-minute stimulation with TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (A–C) Total Syk and ERK were used as loading controls. (D–F) Proposed mechanisms of IgE/FcεRI signaling induced by antigenic liposomes and recruitment of CD33 by CD33L. (D) TNP-LP stabilizes the anti–TNP-IgE–FcεRI complex in lipid rafts with Src kinases that initiate the FcεRI signaling cascade. We propose that CD33 has no basal impact on signaling, because it is not constitutively localized in the same microdomain with FcεRI. (E) TNP-LP-CD33L recruits CD33 to the anti–TNP-IgE–FcεRI immunological synapse. Our results suggest that the cytoplasmic ITIMs of CD33 were phosphorylated by Src kinases and then recruited tyrosine phosphatases such as Shp-1, which dephosphorylated Syk, and other kinases. (F) Proposed model showing that monoclonal anti-CD33 antibodies (or LP-CD33L) block recruitment of CD33 to the IgE-FcεRI complex and enable mast cell degranulation induced by TNP-LP-CD33L.

Article Snippet: Full-length human CD33 was amplified from cDNA (OriGene, catalog SC122608) and subcloned into a pcDNA5/FRT/V5-His vector using the Nhe I and Age I site.

Techniques: Western Blot, Blocking Assay

Display of CD33L on antigenic liposomes suppresses PCA and PSA in CD33-Tg mice (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+), but not in control-Tg mice (Mcpt5-Cre– Rosa26-Stopfl/fl-CD33+). Mice bearing 1 or 2 copies of the CD33 transgene were used. In I, Mcpt5-Cre+/– mice expressing human CD33 (CD33-Tg) were crossed with Ptpn6fl/fl mice to yield mice with mast cells expressing CD33 and no Shp-1 (CD33-Tg/Shp-1–KO). (A) Injection scheme for the PCA model. The genotypes of the mice were determined by PCR after the experiments. (B) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in control-Tg mice. (C) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 14) or TNP-LP-CD33L (50 μg, n = 28) in control-Tg mice. (D) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in CD33-Tg mice. (E) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 21) or TNP-LP-CD33L (50 μg, n =27) in CD33-Tg mice. (F) Injection scheme for the PSA model. (G–I) Decrease in rectal temperature induced by TNP-LP or TNP-LP-CD33L (150 μg) in control-Tg mice (G), CD33-Tg mice (H), and CD33-Tg mice lacking Shp-1 (I) that were sensitized with 10 μg anti–TNP-IgE. (G–I) Values are plotted as the mean ± SEM at the indicated time points. Data are from 1 experiment (G and I) or were compiled from 3 (H) or 9 sets of experiments (C and E). ***P < 0.001 and ****P < 0.0001, by 1-way ANOVA followed by Tukey’s test (C and E), repeated-measures (RM) 2-way ANOVA (G and H), and RM 2-way ANOVA followed by Tukey’s test (I).

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: Display of CD33L on antigenic liposomes suppresses PCA and PSA in CD33-Tg mice (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+), but not in control-Tg mice (Mcpt5-Cre– Rosa26-Stopfl/fl-CD33+). Mice bearing 1 or 2 copies of the CD33 transgene were used. In I, Mcpt5-Cre+/– mice expressing human CD33 (CD33-Tg) were crossed with Ptpn6fl/fl mice to yield mice with mast cells expressing CD33 and no Shp-1 (CD33-Tg/Shp-1–KO). (A) Injection scheme for the PCA model. The genotypes of the mice were determined by PCR after the experiments. (B) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in control-Tg mice. (C) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 14) or TNP-LP-CD33L (50 μg, n = 28) in control-Tg mice. (D) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in CD33-Tg mice. (E) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 21) or TNP-LP-CD33L (50 μg, n =27) in CD33-Tg mice. (F) Injection scheme for the PSA model. (G–I) Decrease in rectal temperature induced by TNP-LP or TNP-LP-CD33L (150 μg) in control-Tg mice (G), CD33-Tg mice (H), and CD33-Tg mice lacking Shp-1 (I) that were sensitized with 10 μg anti–TNP-IgE. (G–I) Values are plotted as the mean ± SEM at the indicated time points. Data are from 1 experiment (G and I) or were compiled from 3 (H) or 9 sets of experiments (C and E). ***P < 0.001 and ****P < 0.0001, by 1-way ANOVA followed by Tukey’s test (C and E), repeated-measures (RM) 2-way ANOVA (G and H), and RM 2-way ANOVA followed by Tukey’s test (I).

Article Snippet: Full-length human CD33 was amplified from cDNA (OriGene, catalog SC122608) and subcloned into a pcDNA5/FRT/V5-His vector using the Nhe I and Age I site.

Techniques: Expressing, Injection, Activation Assay

(A) Injection scheme for desensitization to TNP. CD33-Tg mice were used in the TNP-LP-CD33L–treated group (red). Both CD33-Tg and control-Tg mice were used in the 2 untreated groups (black, gray). (B) Changes in rectal temperature induced by treatment or the challenges indicated in A. (C) Injection scheme to determine antigen specificity of desensitization. CD33-Tg mice were used in the OVA-LP-CD33L–treated group (red circles and squares). Both CD33-Tg and control-Tg mice were used in the untreated group (gray circles and squares). (D) Rectal temperature induced by the treatment or challenge illustrated in C. (B and D) Values are plotted as the mean ± SEM. (E) Injection scheme used to evaluate the impact of TNP-LP-CD33L on mast cell frequency and anti–TNP-IgE on mast cells. Control mice received 200 μl PBS. (F) Frequencies of mast cells from peritoneal fluid from mice treated in E. Mast cell frequencies were determined by c-Kit+CD45+PI– cells. (G) In vitro binding of fluorescent TNP-LP (20 μM) to peritoneal mast cells harvested from mice treated as illustrated in C. (H) MFI of fluorescent TNP-LP binding to peritoneal mast cells quantified in G. The background was determined using untreated cells from a naive mouse. (I) Serum anti–TNP-IgE quantified prior to and 6 hours and 24 hours after treatment with TNP-LP-CD33L (450 μg) using CD33-Tg mice sensitized with 10 μg anti–TNP-IgE. Control mice received 200 μl PBS. Data in B were compiled from 2 experiments. Data are representative of 2 (F–H) or 3 (I) independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by RM 2-way ANOVA (B), RM 2-way ANOVA followed by Tukey’s test (D), and unpaired, 2-tailed Student’s t test (F–I).

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Injection scheme for desensitization to TNP. CD33-Tg mice were used in the TNP-LP-CD33L–treated group (red). Both CD33-Tg and control-Tg mice were used in the 2 untreated groups (black, gray). (B) Changes in rectal temperature induced by treatment or the challenges indicated in A. (C) Injection scheme to determine antigen specificity of desensitization. CD33-Tg mice were used in the OVA-LP-CD33L–treated group (red circles and squares). Both CD33-Tg and control-Tg mice were used in the untreated group (gray circles and squares). (D) Rectal temperature induced by the treatment or challenge illustrated in C. (B and D) Values are plotted as the mean ± SEM. (E) Injection scheme used to evaluate the impact of TNP-LP-CD33L on mast cell frequency and anti–TNP-IgE on mast cells. Control mice received 200 μl PBS. (F) Frequencies of mast cells from peritoneal fluid from mice treated in E. Mast cell frequencies were determined by c-Kit+CD45+PI– cells. (G) In vitro binding of fluorescent TNP-LP (20 μM) to peritoneal mast cells harvested from mice treated as illustrated in C. (H) MFI of fluorescent TNP-LP binding to peritoneal mast cells quantified in G. The background was determined using untreated cells from a naive mouse. (I) Serum anti–TNP-IgE quantified prior to and 6 hours and 24 hours after treatment with TNP-LP-CD33L (450 μg) using CD33-Tg mice sensitized with 10 μg anti–TNP-IgE. Control mice received 200 μl PBS. Data in B were compiled from 2 experiments. Data are representative of 2 (F–H) or 3 (I) independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by RM 2-way ANOVA (B), RM 2-way ANOVA followed by Tukey’s test (D), and unpaired, 2-tailed Student’s t test (F–I).

Article Snippet: Full-length human CD33 was amplified from cDNA (OriGene, catalog SC122608) and subcloned into a pcDNA5/FRT/V5-His vector using the Nhe I and Age I site.

Techniques: Injection, In Vitro, Binding Assay

(A) Flow cytometric analysis of mast cells isolated from discarded human skin (c-KithiFcεRI+ gated on PI–CD45+CD3–CD19–CD56– cells) (left) and overlay of isotype control and anti-CD33 staining of gated mast cells (right). (B) MFI of antibody staining of Siglecs on mast cells isolated from skin that was discarded following surgical procedures (n = 1 to 10 donors). (C) Time course of the percentage of bronchoconstriction of hPCLSs. Lung slices were sensitized with human IgE (4 mg/ml, gray) or anti–TNP-IgE (10 μg/ml, black and red) with recombinant human SCF (200 ng/ml, R&D Systems) overnight. Slices were challenged with anti–human IgE (20 μg/ml, gray), TNP-LP (50 μM, black), or TNP-LP-CD33L (50 μM, red) over a 10-minute period. The airway luminal area over time was compared with the baseline luminal area and expressed as the percentage of bronchoconstriction. Values represent the mean ± SEM. (D) AUC induced by the indicated treatments. AUC values below 0 are plotted as 0. (E) Percentage of bronchoconstriction induced by the indicated agents at 10 minutes. (F) Following stimulation with the indicated reagents, the percentage of bronchoconstriction induced by CCh (0.1 mM) was measured. *P < 0.05 and **P < 0.01, by 1-way ANOVA followed by Tukey’s test (D–F). Gating of skin mast cells in A is representative of the 10 donors. Data in C–F were compiled from 5 or 9 lung slices from 2 donors.

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Flow cytometric analysis of mast cells isolated from discarded human skin (c-KithiFcεRI+ gated on PI–CD45+CD3–CD19–CD56– cells) (left) and overlay of isotype control and anti-CD33 staining of gated mast cells (right). (B) MFI of antibody staining of Siglecs on mast cells isolated from skin that was discarded following surgical procedures (n = 1 to 10 donors). (C) Time course of the percentage of bronchoconstriction of hPCLSs. Lung slices were sensitized with human IgE (4 mg/ml, gray) or anti–TNP-IgE (10 μg/ml, black and red) with recombinant human SCF (200 ng/ml, R&D Systems) overnight. Slices were challenged with anti–human IgE (20 μg/ml, gray), TNP-LP (50 μM, black), or TNP-LP-CD33L (50 μM, red) over a 10-minute period. The airway luminal area over time was compared with the baseline luminal area and expressed as the percentage of bronchoconstriction. Values represent the mean ± SEM. (D) AUC induced by the indicated treatments. AUC values below 0 are plotted as 0. (E) Percentage of bronchoconstriction induced by the indicated agents at 10 minutes. (F) Following stimulation with the indicated reagents, the percentage of bronchoconstriction induced by CCh (0.1 mM) was measured. *P < 0.05 and **P < 0.01, by 1-way ANOVA followed by Tukey’s test (D–F). Gating of skin mast cells in A is representative of the 10 donors. Data in C–F were compiled from 5 or 9 lung slices from 2 donors.

Article Snippet: Full-length human CD33 was amplified from cDNA (OriGene, catalog SC122608) and subcloned into a pcDNA5/FRT/V5-His vector using the Nhe I and Age I site.

Techniques: Isolation, Staining, Recombinant

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