Journal: Nature medicine

Article Title: Antigen-independent activation enhances the efficacy of 41BB co-stimulated CD22 CAR T cells

doi: 10.1038/s41591-021-01326-5

Figure Lengend Snippet: a, Pipeline for development and evaluation of new CD22-f2-short CAR. b, Affinity and size of purified CD22-f2-long and short scFvs. c, Expression of CD22 CARs on primary T cells. d, Measurement of secreted IFNγ by CD22-engineered T cells after 24h exposure to CD22+ target cells. e, Progression of Nalm6 disease burden in xenograft mice treated with CD22-f2-short and long T cells (Representative of 4 replicate experiments, n=4–7 mice per condition; see Supplementary Figure 5 for individual animal responses and Supplementary Figure 6 for experimental replicates). f, Survival of Nalm6-bearing xenograft mice after treatment with m971 or CD22-f2 CAR T cells. Data are presented as mean values +/− standard error of the mean (S.E.M.) Statistics reflect differences between CAR22-short and long T cells.

Article Snippet: 40 All animal studies were approved and supervised by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). scFv design and optimization: To identify novel binders to human CD22 extracellular domain, three rounds of panning were done against recombinant human CD22 (R&D systems, Cat. # 1968-SL-050) using a fully human derived scFv phage library derived internally.

Techniques: Purification, Expressing

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) In-silico analysis of CAR-T cell-treated patients (n=4,219) revealed a high relapse rate, with 42.11% (n=216 of n=513 overall relapse patients) experiencing CD19-negative recurrence after monospecific CAR-Therapy (n=2,916). (B) Schematic overview of the CAR design strategy showing mono, bi, and trispecific constructs targeting CD19, CD20, and CD22. (C) Experimental workflow illustrating CAR screening: 1,452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). (D) Categorization of CARs into low (L), medium (M3), and high (H) levels based on fluorescence intensity cutoffs determined by monospecific CD19 CARs. (E) Distribution of 1,452 screened CARs across L-, M-, and H-CARMSeD categories using the CAR-Mediated Self-Destruction (CARMSeD) scoring system. (F) AI model development pipeline for CAR dysfunction risk prediction, based on 1,452 CAR constructs with an 80:20 split for training and testing. (G–J) Performance metrics of AI model predicting CAR-Mediated Self-Destruction (CAR-MSED) scores using 1,452 CAR constructs (G) Model accuracy over 50 epochs, achieving a training accuracy of 0.98 and validation accuracy of 0.95. (H) Scatter plot comparing measured versus predicted CAR-MSED scores for training (R 2 = 0.87) and validation (R 2 = 0.83) sets. (I) Predicted versus measured CAR-MSED scores on the validation set, categorized into low (L-CARMSED, blue), medium (M-CARMSED, orange), and high (H-CARMSED, green) groups. (J) Box plot of predicted signal scores for 9,372 unknown sequences, classified as L-CARMSED (2,749 sequences), M-CARMSED (1,468 sequences), and H-CARMSED (5,155 sequences). (K) Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing CAR-CAR interaction. Structural conformations at 0 ns, 50 ns and 200 ns for different CAR scFv arrangements highlighting CDR regions (surface transparency 30%), Root Mean Square Deviation (RMSD) plots over 200 ns for the both constructs, respectively, indicating structural stability and conformational changes. (L) In vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs (n=6).

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: In Silico, Construct, Activation Assay, Fluorescence, Biomarker Discovery, In Vitro, Binding Assay

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (yellow), and CD22 (red) antigens. (B) Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. (B) Cytotoxicity assays showing potent and antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR-T cells (n=5). (C) Comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR-T cells. Trispecific constructs showed reduced proliferation, consistent with increased structural rigidity predicted by CARMSeD scoring. (D) Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (yellow), and CD22 (red) antigens, edited using CRISPR-Cas9 to generate knockout variants. (E) Cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR-T cells in eliminating antigen-negative Raji variants, compared to ineffective m19 CARs (n=5). (F) Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, while purple circles indicate the timing of the Raji CD19-/- rechallenge. (G) Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis, and the number of CAR-T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge (n=5). (H) Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR-T cells. Mice were xenografted with Raji WT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR-T cells on day 5 and subsequent Raji CD19-/- TR on day 12, 19 and 26. (I–J) Bioluminescent imaging and tumor burden quantification show effective tumor control by b20/19 CAR-T cells versus m19 CARs. (K) CAR-T cell persistence over time. (L) Kaplan-Meier survival curves showing survival outcomes over 70 days (n=5). (M) Analysis of residual tumor CD19 or CD20 tumor cells over time. (N, O) Bar plot showing Granzyme B and IFN-γ secretion from CD8 + CAR-T cells isolated post-treatment with m19 and b20/19 confirm functional cytotoxicity of b20/19 against CD19⁻ targets (n=5). (P–Q) Repeated TR induced upregulation of exhaustion markers PD-1 and LAG-3 (n=5). (R) Immunophenotyping of CAR-T cells post-TR shows loss of central memory (T cm ) populations and increased PD-1 expression, consistent with functional exhaustion and limited persistence (n=5). Data represents mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Lysis, In Vivo, Imaging, Control, Isolation, Functional Assay

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. (B) Relative mRNA expression levels (normalized to beta actin) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR-T. (C1) Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR-T cells after TR with Raji CD19-/- cells (C2) Histogram analysis of the flow cytometry plots (n=10). (D) Bar graph shows the percentage of CD8 + CAR-T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions. (E) Persistence of CAR-T cells over 15 days under various conditions (n=4). (F) Violin plots show the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown significantly elevated mTOR activity (n=6). (G) Bar plots show the percentage of MFI of autophagy from autophagic flux assay (n=8). (H) ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from oxidative phosphorylation (OXPHOS) to glycolysis (n=12 data points). (I) Similarly, OCR with FOXO4 knockdown decreasing mitochondrial respiration. Individual data points are shown for each condition (n=12 data points). (J1) Percentage of expression (% expression) of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker. (J2) Bar graph displays the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. (K) Schematic illustration of K562 WT cells based on CD20 expression levels, resulting in three populations: CD20 L (low), CD20 M (medium), and CD20 H (high). (L) Violin plots show the percentage of CD20 expression (% CD20 expression) in the sorted K562 WT cell populations, confirming distinct expression levels (n=10). (M) Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show DAPI (blue, nuclear staining) and CD20 (red) in K562-C20 L (low), K562-C20 M (medium), and K562-C20 H (high) cell. Scale bar indicates 10 μm. (N) Survival of K562 cells expressing varying CD20 expression levels under CAR-T cell treatments. Panels N1 (K562-CD20 L ), N2 (K562-CD20 M ), and N3 (K562-CD20 H ) show the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) (N=4). (O) Persistence of CAR-T cells with varying CD20-targeting CAR constructs over 15 days (N=5). Data represents mean ± SEM. ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups. Scale bar indicates 10 μm.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Phospho-proteomics, Marker, Super-Resolution Microscopy, Staining, Construct

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic of the engineering strategy for trispecific CAR-T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). (B) Correlation of expression of nbCD3, nb22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin and data was obtained using intracellular flow cytometry. (C) Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. (D) Dose-dependent T cell activation (CD69 expression) in response to culture supernatants with nbCD3/22, using db coated with CD3 for validation. (E) HEK-293T synNotch reporter assay shows dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR-T cell supernatants, confirming BiTE functionality under two conditions. (F) Experimental timeline for in vivo CAR-T cell therapy study in Raji WT or NALM6 WT model followed by CAR-T cell administration and TR with Raji CD19/CD20-/- or NALM6 CD19/CD20-/- cells (G) Bioluminescence imaging of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 CAR-T cells, monitored from Day 7 to Day 84. (H) Quantified tumor radiance over time, showing sustained tumor control in Raji and NALM6 models with b20/19-AKT3 PROTAC+nbCD3/22 . (I1) Percentage of CAR-T cells in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days (I2) Bar graph of CAR-T cell populations in blood at various time points. (J) Levels of nbCD3/22 (pg/mL) in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days, showing sustained secretion. (K) Kaplan-Meier survival curves demonstrating improved survival with nbCD3/22-modified CAR-T cells. (L) Bar graph and pie charts compare b20/19-AKT3 PROTAC and b20/19-AKT3 PROTAC+nbCD3/22 , showing various memory T cell subsets over time (n=5) in all conditions. Data represents mean ± SEM. ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Briefly, CD19, CD22 CAR expression was evaluated using CD19 and CD20 CAR detection antibodies and CD22 CAR expression (Miltenyi Biotec) was evaluted using Protein L-APC (Cell signaling) followed by PE-conjugated anti-biotin secondary antibodies (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vivo, Imaging, Control, Modification

Journal: bioRxiv

Article Title: MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

doi: 10.1101/2025.09.09.675083

Figure Lengend Snippet: (A) The K D values of CD22-miniCARbids were determined by titrations of soluble CD22-miniCARbids on NALM6 cells. (B) A representative example of titrations of miniCARbids 22_1611 and 22_1317 on NALM6 cells is shown. The binding intensity was assessed via anti-His-tag staining by flow cytometry. Data were fitted with a 1:1 binding model (solid lines) for the calculation of the respective K D values illustrated in (A) (average ± SD, n=3 or 4, biological replicates). (C) Thermostability of CD22-miniCARbids and their parental protein 5UMR was assessed using DSC (average ± SD of 3 independent measurements, technical replicates). (D) Aggregation properties of CD22-miniCARbids were assessed using SEC-HPLC. One representative analysis (n=3, technical replicates) of CD22-miniCARbids and their parental protein 5UMR is shown. (E) Binding specificity was assessed by incubating NALM6, Raji or Jurkat (CD22-negative) cells with 250 nM CD22-miniCARbid, followed by flow cytometric analysis (one of three biological replicates is shown).

Article Snippet: Selection campaigns started with magnetic bead selections using Dynabeads Biotin Binder (Thermo Fisher Scientific) as described previously., Yeast display selections for miniCARbids against CD22 were based on a soluble, biotinylated CD22 protein (AcroBiosystems, SI2-H82E3).

Techniques: Binding Assay, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

doi: 10.1101/2025.09.09.675083

Figure Lengend Snippet: (A) CAR architecture used for the in vitro assessment of CAR activity. (B) Expression of CARs based on ten CD22-specific miniCARbids and scFvs HA22, m971-1xG 4 S and m971-4xG 4 S as benchmarks in Jurkat Nur77 reporter cells was assessed via anti-MAP-tag staining by flow cytometry (average ± SD, n=3, biological replicates). (C) Activation of CD22-specific CARs in Jurkat Nur77 reporter cells in the presence or absence of a 2-fold excess of NALM6 target cells was assessed via the expression of mKO2 by flow cytometry (average ± SD, n=3, biological replicates). (D) Cytotoxicity of CD22-specific CAR T cells and mock T cells (no CAR) against Raji cells (E:T 2:1, average ± SD, n=4, biological replicates). (E and F) Release of IFN-γ (E) and IL-2 (F) analyzed via ELISA. The cytokines were analyzed in the supernatants of co-cultures with Raji cells (E:T 2:1, average ± SD, n=4, biological replicates). (G) Cytotoxicity of CD22-specific CAR T cells and mock T cells (no CAR) against NALM6 cells (E:T 2:1, average ± SD, n=4, biological replicates). (H and I) Release of IFN-γ (H) and IL-2 (I) analyzed via ELISA. The cytokines were analyzed in the supernatants of co-cultures with NALM6 cells (E:T 2:1, average ± SD, n=4, biological replicates). Statistical analysis was performed using a repeated measure One-Way ANOVA with a Tukey post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). The statistical analysis for the cytokine concentration was performed using log-transformed values. Parts of this figure were created with BioRender.com.

Article Snippet: Selection campaigns started with magnetic bead selections using Dynabeads Biotin Binder (Thermo Fisher Scientific) as described previously., Yeast display selections for miniCARbids against CD22 were based on a soluble, biotinylated CD22 protein (AcroBiosystems, SI2-H82E3).

Techniques: In Vitro, Activity Assay, Expressing, Staining, Flow Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transformation Assay

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail does not alter tyrosine phosphorylation of CD22 or ERK phosphorylation. Splenocytes from IgM and IgMG transgenic mice were stimulated with 50 μg/ml anti-IgM F(ab′) 2 . Total cellular proteins (A) or immunoprecipitated CD22 (B) were fractionated by SDS-PAGE and Western blotted with antiphosphotyrosine antibody (A and B, top) or anti-CD22 antibody (B, bottom). The ratios of phosphorylated CD22 to total CD22 are indicated. (C and D) Mean fluorescence intensity (MFI) of permeabilized B220 + CD21 medium CD23 + follicular B cells stained by flow cytometry for phosphorylated ERK either (C) at the indicated times after stimulation with 50 μg/ml anti-IgM F(ab′) 2 or (D) in unstimulated (−) versus stimulated (+) cells after 2 min in the presence of MEK inhibitors PD98059, U0126, or DMSO as diluent controls. Data are representative of two experiments.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Phospho-proteomics, Transgenic Assay, Immunoprecipitation, SDS Page, Western Blot, Fluorescence, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail evokes an augmented Ca 2+ response independent of CD22. RBC-depleted splenocytes from mice of the indicated genotypes were labeled with 1 μM Indo-1 for 30 min at 37°C. (A and B) The cells were counterstained with antibodies against B220 and CD21 for the final 10 min of Indo-1 loading. The stained cells were at first acquired for 30 s and then stimulated with the indicated concentrations of anti-IgM F(ab′) 2 antibody (A, 5 μg/ml; and B, 0.5 μg/ml). Lines show the mean Indo-1 ratio in B220 + CD21 medium cells. Data from one out of three independent experiments are shown. (C and D) Indo-1–loaded cells stained with antibody against B220 were simulated with the indicated concentrations of HEL conjugated to PE (HEL-PE; C, 5 μg/ml; and D, 0.5 μg/ml), and the Indo-1 ratio was measured on gated HEL + B220 + cells.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Labeling, Staining

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail increases antibody production independent of CD22. 5 × 10 5 HEL-binding splenic B cells from IgMG or IgM transgenic donors of the indicated CD22 genotypes were adoptively transferred into nonirradiated C57BL/6 mice, and the recipient mice were immunized with HEL in CFA. The concentration of anti-HEL IgM a antibody 10 d after immunization was measured in the serum of individual recipient mice (circles) by ELISA. Data are representative of three separate experiments. Significant differences were determined by the Mann-Whitney test. *, P < 0.05; **, P < 0.01.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Binding Assay, Transgenic Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: CD22 deficiency suppresses IgM but not IgMG marginal zone B cell differentiation. Splenocytes were stained with HEL and combinations of antibodies against CD21/CD23/HyHEL9/B220 (A and B) or CD21/CD1d/HyHEL9/B220 (C and D). (A and C) The displayed profiles are gated on B220 + HEL-binding cells, and the percentage of B220 + HEL-binding cells in each window is shown. (B and D) Percentages of HEL-binding B cells in the marginal zone subset in individual mice (circles). Significant differences, as determined by the Student's t test, are indicated.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Cell Differentiation, Staining, Binding Assay

Journal: Arthritis Research & Therapy

Article Title: TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

doi: 10.1186/ar3294

Figure Lengend Snippet: Dual immunostaining for TWEAK and cell lineage markers and cells expressing Fn14 . Dual immunostaining for TWEAK (red) and CD68 (blue) in inflamed synovial tissue from a patient with active RA ( A ). Dual immunostaining for TWEAK (red) with CD38 (blue) with co-expression of TWEAK and CD38 (purple) indicated by arrow ( B ). C ) and D ) Dual immunostaining for TWEAK (red) with CD22 (blue). E ) TWEAK expression (red) in multinucleated cells (indicated by arrows), and F ) by plasma cells in tonsil tissue. Expression of Fn14 (brown) in multinucleated cells ( G ), and blood vessels of the synovial tissue ( H ), indicated by arrows. Sections shown in E, F, G, and H were counterstained with haematoxylin. Images shown in B and C were obtained with obj ×10; image shown in A obtained with obj ×20, D, F, G, H with obj ×40 and E with obj ×60.

Article Snippet: Anti-TWEAK antibody was combined with MAbs for human cell surface markers: CD68 (macrophage; clone KP-1, Dako), CD22 (B lymphocyte; MAB1968, R&D Systems, Minneapolis, MN, USA), Tryptase G3 (mast cell; Cell Marque, Rocklin, CA, USA) and CD38 (plasma cells, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Immunostaining, Expressing, Clinical Proteomics

Journal: Arthritis Research & Therapy

Article Title: TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

doi: 10.1186/ar3294

Figure Lengend Snippet: TWEAK expression by PBMC . PBMC from two healthy volunteers were sorted by FACS based on their expression of CD22, yielding CD22 + and CD22 - populations of greater than 94% purity based on post-sort analysis ( A ). Isolated cells were then analysed for TWEAK mRNA expression relative to that of GAPDH, by real-time RT-PCR ( B ). Data shown are means of triplicate reactions ± SD. Differences in relative expression of TWEAK mRNA between CD22 + and CD22 - populations were tested by Student's t -test (** P < 0.001).

Article Snippet: Anti-TWEAK antibody was combined with MAbs for human cell surface markers: CD68 (macrophage; clone KP-1, Dako), CD22 (B lymphocyte; MAB1968, R&D Systems, Minneapolis, MN, USA), Tryptase G3 (mast cell; Cell Marque, Rocklin, CA, USA) and CD38 (plasma cells, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR