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: Cytotherapy

Article Title: Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in CAR T cell products

doi: 10.1016/j.jcyt.2022.09.004

Figure Lengend Snippet: Assay #2 was designed at the site between the two distinct CARs within the ORF of the vector, spanning the junction between the linker domains and the 5’-end of the FMC63 scFv of CD19. This primer-probe set was designed to be specific for the bicistronic CD19x22 CAR T cell product. A representative example of droplet readout is pictured here demonstrating that ddPCR confirms specificity of this assay to detect only the bicistronic CAR product as demonstrated with CAR positive droplets (FAM) in blue in the last column. Albumin control (VIC), depicted in green, appropriately demonstrates signal in all samples containing T cells. No CAR positive droplets (in blue) are detected in untransduced T cells or T cells transduced with either CD19 only CARs or CD22 only CARs. NTC = No template control, negative control.

Article Snippet: Following staining and subsequent wash, cells were stained with Recombinant Human CD19-Fc Chimera Atto 647N Protein (R&D Biotechne, ATM9269-020, 1ug/test), Recombinant Human Singlec2/CD22-Fc Chimera Atto 488 (R&D Biotechne, ATJ1968-050, 0.5ug/test), and lyophilized T cell cocktail 6-color Kit (Biolegend, San Diego, CA) containing CD45-PE/Dazzle 594 (clone HI30), CD3 BV510 (clone UCHT1), CD4 Alexa Fluor 700 (clone SK3), CD8 Pacific Blue (clone SK1), CD45RA PE (clone KI100), and CD196 (CCR7) PE/Cy7 (clone G043H7) for 15 minutes at room temperature.

Techniques: Plasmid Preparation, Transduction, Negative Control

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

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: Representative donor samples (n=2) were tested to determine an optimal CFSE concentration for PBMC and MMC. Panels a-g show results from one representative donor (a) PBMC stim (No CFSE), (b) PBMC stim (CFSE 2.5 μM), (c) PBMC unstim (CFSE 2.5 μM), (d) MMC stim (No CFSE), (e) MMC stim (CFSE 2.5 μM), (f) MMC stim (CFSE 5.0 μM) and (g) MMC unstim (CFSE 2.5 μM) compartments. All culture stimulations were performed using 5 μl anti-CD2/3/28 microbeads.

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Concentration Assay

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: Staining 2–3×10 6 gut-derived T-cells (MMC) with no CFSE, 2.5 μM CFSE, and 5.0 μM CFSE indicates no toxicity as shown by D5 T Cell Fold Change (D5 number of live CD3 T cells/initial number of live CD3 T cells) with CFSE doses range 0–5.0 μM (n=2). All samples were stimulated with 5 μl anti-CD2/3/28 microbeads.

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Staining

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: A. CFSE histogram of live CD3 T-cells following a representative 5 day culture of (i) PBMC unstim, (ii) PBMC stim, (iii) MMC unstim, (iv) MMC stim cultures. B. Representative bivariate plots from blood and gut derived cultures (stimulated culture and unstimulated control) of CFSE-FITC (x-axis) versus BrdU-APC (Y-axis) gating used to enumerate replicating (CFSEloBrdU+) verus non-replicating (CFSEhiBrdU−) CD3+ populations, and illustrating (CFSEloBrdU−) non-replicating populations. All cultures include 0.5×106 irradiated autologous PBMC feeders. C. Mean % of live blood- and gut-derived CD3+8+ cells that are (CFSEloBrdU−) following bead stimulation and 5 day culture (n=29); ***, p<0.005 (two-side, two-tailed Student’s paired t test). All culture stimulations were performed using 5 μl anti-CD2/3/28 microbeads.

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Derivative Assay, Irradiation, Two Tailed Test

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: MMC were stained with 5 μM CFSE and cultured for 5 days following CD3 stimulation via three different methods, at the recommended concentrations; (1) Miltenyi anti-CD2, 3, 28 Ab beads (5μl/ml) (n=4), (2) OKT3 (1μg/ml) + CD28 Ab (20μg/ml) (n=1), (3) Bispecific CD3/CD4 (blocking) Ab (1μl/ml) (n=2). At day 5, cells were stained with CD3-PE and %CD3+ cells that had proliferated (CFSElo) was determined. (A) Comparison of % proliferating (CFSElo) cells using Miltenyi beads (n=4), OKT3 (n=1) and Bispecific Ab (n=2). (B) Representative 1D flow histogram demonstrating proliferating (CFSElo) versus non-proliferating (CFSEhi) CD3+ cell populations of gut derived cells for each stimulation method (bead method 72% proliferating, OKT3 17% proliferating, bispecific ab 54% proliferating).

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Staining, Cell Culture, Blocking Assay, Comparison, Derivative Assay

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: Representative donor samples (n=2) were tested to determine an optimal CFSE concentration for PBMC and MMC. Panels a-g show results from one representative donor (a) PBMC stim (No CFSE), (b) PBMC stim (CFSE 2.5 μM), (c) PBMC unstim (CFSE 2.5 μM), (d) MMC stim (No CFSE), (e) MMC stim (CFSE 2.5 μM), (f) MMC stim (CFSE 5.0 μM) and (g) MMC unstim (CFSE 2.5 μM) compartments. All culture stimulations were performed using 5 μl anti-CD2/3/28 microbeads.

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Concentration Assay

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: Staining 2–3×10 6 gut-derived T-cells (MMC) with no CFSE, 2.5 μM CFSE, and 5.0 μM CFSE indicates no toxicity as shown by D5 T Cell Fold Change (D5 number of live CD3 T cells/initial number of live CD3 T cells) with CFSE doses range 0–5.0 μM (n=2). All samples were stimulated with 5 μl anti-CD2/3/28 microbeads.

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Staining

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: A. CFSE histogram of live CD3 T-cells following a representative 5 day culture of (i) PBMC unstim, (ii) PBMC stim, (iii) MMC unstim, (iv) MMC stim cultures. B. Representative bivariate plots from blood and gut derived cultures (stimulated culture and unstimulated control) of CFSE-FITC (x-axis) versus BrdU-APC (Y-axis) gating used to enumerate replicating (CFSEloBrdU+) verus non-replicating (CFSEhiBrdU−) CD3+ populations, and illustrating (CFSEloBrdU−) non-replicating populations. All cultures include 0.5×106 irradiated autologous PBMC feeders. C. Mean % of live blood- and gut-derived CD3+8+ cells that are (CFSEloBrdU−) following bead stimulation and 5 day culture (n=29); ***, p<0.005 (two-side, two-tailed Student’s paired t test). All culture stimulations were performed using 5 μl anti-CD2/3/28 microbeads.

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Derivative Assay, Irradiation, Two Tailed Test

Journal: Journal of immunological methods

Article Title: Human Immune Compartment Comparisons: Optimization of Proliferative Assays for Blood and Gut T Lymphocytes

doi: 10.1016/j.jim.2017.03.014

Figure Lengend Snippet: MMC were stained with 5 μM CFSE and cultured for 5 days following CD3 stimulation via three different methods, at the recommended concentrations; (1) Miltenyi anti-CD2, 3, 28 Ab beads (5μl/ml) (n=4), (2) OKT3 (1μg/ml) + CD28 Ab (20μg/ml) (n=1), (3) Bispecific CD3/CD4 (blocking) Ab (1μl/ml) (n=2). At day 5, cells were stained with CD3-PE and %CD3+ cells that had proliferated (CFSElo) was determined. (A) Comparison of % proliferating (CFSElo) cells using Miltenyi beads (n=4), OKT3 (n=1) and Bispecific Ab (n=2). (B) Representative 1D flow histogram demonstrating proliferating (CFSElo) versus non-proliferating (CFSEhi) CD3+ cell populations of gut derived cells for each stimulation method (bead method 72% proliferating, OKT3 17% proliferating, bispecific ab 54% proliferating).

Article Snippet: Based on these experimentally defined parameters over 7 day observation periods, subsequent method development retained the manufacturer’s recommendation of a 1:2 bead:cell ratio for both PBMC and MMC. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 4 caption a7 caption a8 Stimulation with Miltenyi anti-CD2,3,28 Ab microbeads was tested with bead:cell ratios of 1:4, 1:2 and 1:1 on MMC from a single donor (n=1) On Day 5 post-stimulation the percentage of non-dividing (CFSE hi BrdU − ) gut derived CD3 T cells was 4%, 3.5% and 3.2%, respectively; the percentage of dividing (CFSE lo BrdU + ) gut-derived CD3 T cells was 75.6%, 77.4% and 79.3%, respectively, indicating no major quantitative difference in proliferative dynamics.

Techniques: Staining, Cell Culture, Blocking Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model

doi: 10.3390/ijms23031866

Figure Lengend Snippet: CerS4 deficiency impaired CD8+ T-cell development and enhanced cytokine secretion. ( A ) Isolated thymocytes of CerS4 WT and LCK/Cre mice were differentiated into cytotoxic T-cells. T-cells were stained with various surface markers and detected by FACS. The enrichment of CD8+ T-cells was higher in WT than in CerS4 LCK/Cre mice over the time of analysis. ( B ) For cytokine secretion, the primary T-cells were activated with IL-2 and CD2/3/28 beads for 4 h and 8 h. Cytokine concentration in the supernatant was determined with a cytometric bead array flex set (mouse IL-6, IL-10, IL-17A, IFNγ, and TNFα; BD Biosciences). Data are mean ± SEM or median ± min to max of n = 3–4. Statistically significant differences were determined by a two-way ANOVA with a Tukey’s multiple comparison test. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For T-cell activation, the Jurkat cells were stimulated with 200 units/mL IL-2 (PeproTech GmbH, Hamburg, Germany) and anti-CD2/3/28 activation beads (Miltenyi Biotec, Bergisch Gladbach, Germany) in a 1:1 bead-to-cell ratio for either a long (0–48 h) or a short term (0–30 min).

Techniques: Isolation, Staining, Concentration Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model

doi: 10.3390/ijms23031866

Figure Lengend Snippet: Downregulation of CerS4 in human Jurkat cells affected CerS expression and T-cell signaling. ( A ) The stimulation of transduced Jurkat cells (shNC/shCerS4) with IL-2 (200 units/mL) and anti-CD2/3/28 activation beads (1:1 bead-to-cell ratio) for 24 or 48 h changed the CerS mRNA expression profile compared with that of Jurkat shNC 0 h (set to 1). ( B ) CerS4 downregulation in Jurkat cells reduced the mRNA expression of IFNγ and IL-17a. ( C ) T-cell signaling after short- and long-term activation analyzed by Western blot. NF-κB signaling was detected by anti-phospho-p65 and anti-p65 at 0, 24, and 48 h after activation. The protein expression was normalized to β-actin. All timepoints were related to shNC Jurkat cells at 0 h (set to 1). The T-cell receptor activation was detected with anti-phospho-Zap70, anti-Zap70, phospho-LAT, and anti-LAT at short-term timepoints. Protein expression levels were normalized to p84 and compared with those of shNC Jurkat cells at 0 h (set to 1). Data are median ± min to max of n = 5, 3, and 2. Statistically significant differences were calculated by a two-way ANOVA with a Tukey’s multiple comparison posttest or unpaired t -test. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For T-cell activation, the Jurkat cells were stimulated with 200 units/mL IL-2 (PeproTech GmbH, Hamburg, Germany) and anti-CD2/3/28 activation beads (Miltenyi Biotec, Bergisch Gladbach, Germany) in a 1:1 bead-to-cell ratio for either a long (0–48 h) or a short term (0–30 min).

Techniques: Expressing, Activation Assay, Western Blot, Comparison

Journal: EMBO Reports

Article Title: Mitochondrial translocation of TFEB regulates complex I and inflammation

doi: 10.1038/s44319-024-00058-0

Figure Lengend Snippet: ( A ) TFEB, LONP1, NDUFA8, NDUFB10, and GAPDH expression in HeLa cells transfected with control siRNA (siCTRL), siRNA against LONP1 (siLONP1), TFEB specific siRNA (siTFEB) and siRNA against both LONP1 and TFEB. Western blot is representative of three independent experiments showing similar results. ( B ) Mean densitometric analysis of the immunoblot from ( A ). Shown are mean ± SEM, n = 3 biological replicates. Unpaired t -test was done to determine statistical significance and represented as * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. ( C ) LONP1, TFEB, NDUFA8, NDUFB10, NDUFS5, NDUFA9, and GAPDH protein expression in shCTRL and shTFEB HeLa cells transfected with siCTRL and siLONP1. Western blot is representative of three independent experiments showing similar results. ( D ) Relative mRNA expression of LONP1 and TFEB (relative to RPL13A) in HeLa cells transfected with siCTRL and siLONP1. Shown are mean ± SEM, n = 6 biological replicates. Unpaired t -test was done to determine statistical significance and represented as *** p < 0.005. ( E ) OXPHOS profile of shCTRL and shTFEB cells transfected with siCTRL and siLONP1. Shown are mean ± SEM, n = 3 biological replicates. ( F ) ATP-linked respiration and Maximal respiration rates normalized to Basal respiration in shCTRL and shTFEB HeLa cells transfected with siCTRL or siLONP1. Shown are mean ± SEM, n = 3 biological replicates.. One-way ANOVA test followed by Tukey’s multiple comparison was done to determine statistical significance and represented as ** p < 0.01; *** p < 0.005. ( G ) Endogenous TFEB was immunoprecipitated and immunoblotted for LONP1 and TFEB in HeLa cells. Western blot is representative of two independent experiments showing similar results. IgG was used as negative control. ( H ) TFEB-Flag was pulled down from cells transfected with Empty vector (EV), LONP1-HA, TFEB-FLAG and TFEB-FLAG + LONP1-HA and immunoblotted for LONP1-HA and TFEB-FLAG to confirm TFEB-LONP1 interaction. EV was used as a negative control. Quantifications of the immunoblot are also shown. The experiment was performed in biological triplicates ( n = 3). Unpaired t-test was done to determine statistical significance and represented as * p < 0.05; ** p < 0.01. ( I ) LONP1 was purified and incubated with control beads as negative control and with TFEB- FLAG-tagged beads. The experiment was performed in duplicate showing similar results. .

Article Snippet: For the ectopic immunoprecipitations, 10 7 cells overexpressing TFEB- FLAG and LONP1 were lysed in cell lysis buffer (98035S, Cell Signaling Technology) containing protease inhibitors (4693159001, Roche) and incubated for 15 min on ice.

Techniques: Expressing, Transfection, Control, Western Blot, Comparison, Immunoprecipitation, Negative Control, Plasmid Preparation, Purification, Incubation

Journal: EMBO Reports

Article Title: Mitochondrial translocation of TFEB regulates complex I and inflammation

doi: 10.1038/s44319-024-00058-0

Figure Lengend Snippet: ( A ) Endogenous TFEB subcellular localization upon 24 h of S . Typhimurium infection (MOI 100) in HeLa cells. LAMINB, TOMM20, and GAPDH served as controls for nucleus, mitochondria, and cytosol, respectively. Western blot is representative of three independent experiments showing similar results. ( B ) Densitometric quantitation of immunoblots depicting the subcellular endogenous TFEB localization in uninfected (Ul) and S . Typhimurium-infected (MOI = 100) cells from ( A ). Shown are mean ± SEM, n = 3 biological replicates. Unpaired t -test was done to determine statistical significance and represented as **** p < 0.001. ( C , D ) ( C ) IL-6 and ( D ) IL-8 in the supernatants of 24 h S . Typhimurium-infected shCTRL and shTFEB HeLa cells. Shown are mean ± SEM, n = 6 biological replicates for IL-6 ( C ) and 3 biological replicates for IL-8 ( D ). Shown are mean ± SEM, n = 3 biological replicates. Unpaired t-test was done to determine statistical significance and represented as ** p < 0.01; *** p < 0.005. ( E ) IL-6 in supernatants of shCTRL and shTFEB cells transfected with FLAG, WT-TFEB-FLAG or MLS-TFEB-FLAG plasmids and infected for 24 h with S . Typhimurium (MOI 100). Shown are mean ± SEM, n = 3 biological replicates. Shown are mean ± SEM, n = 3 biological replicates. One-way ANOVA followed by Tukey’s multiple comparison analysis was performed to determine statistical significance and represented as * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. ( F ) Flow cytometric analysis of Mitochondrial ROS (mtROS) production was estimated on MitoSOX-stained shCTRL and shTFEB HeLa cells transfected with empty FLAG and WT-TFEB-FLAG and the Mean Fluorescence Intensity (MFI) was plotted; mean ± SEM, n = 3 biological replicates. Unpaired t -test was done to determine statistical significance and represented as ** p < 0.01; *** p < 0.005; **** p < 0.001. ( G ) IL-6 secretion in supernatants of shCTRL and shTFEB cells infected with S . Typhimurium (MOI 100) for 24 h and treated with MitoTempo 2 h prior infection and during infection. Shown are mean ± SEM, n = 3 biological replicates with n = 3 technical replicates each. One-way ANOVA followed by Tukey’s multiple comparison analysis was performed to determine statistical significance and represented as * p < 0.05; **** p < 0.001. ( H ) IL-6 expression in supernatants of shCTRL and shTFEB HeLa cells transfected with siCTRL and siLONP1 and infected with S . Typhimurium (MOI 100) for 24 h. Shown are mean ± SEM, n = 3 technical replicates. The experiment was repeated twice with similar results. One-way ANOVA followed by Tukey’s multiple comparison analysis was performed to determine statistical significance and represented as * p < 0.05; **** p < 0.001. ( I ) MitoSOX-based flow cytometric detection of mitochondrial ROS production in shCTRL and shTFEB HeLa cells transfected with siCTRL and siLONP1. Shown is the Mean Fluorescence Intensity (MFI). Shown are mean ± SEM, n = 3 biological replicates. One-way ANOVA followed by Tukey’s multiple comparison analysis was performed to determine statistical significance and represented as * p < 0.05; **** p < 0.001. ( J ) A model showing that S . Typhimurium prevents mTOR-dependent mitochondrial translocation where it interacts with LONP1 to co-regulate complex I assembly and function. .

Article Snippet: For the ectopic immunoprecipitations, 10 7 cells overexpressing TFEB- FLAG and LONP1 were lysed in cell lysis buffer (98035S, Cell Signaling Technology) containing protease inhibitors (4693159001, Roche) and incubated for 15 min on ice.

Techniques: Infection, Western Blot, Quantitation Assay, Transfection, Comparison, Staining, Fluorescence, Expressing, Translocation Assay

Journal: Frontiers in Genome Editing

Article Title: CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

doi: 10.3389/fgeed.2025.1649993

Figure Lengend Snippet: Genomic deletion of target antigens in Raji-Luc lines does not alter proliferation or viability. (A) Schematic representation of CRISPR-Cas9–mediated strategy to generate single, double, and triple knockout (KO) Raji-Luc cell lines. (B–D) Amplicon sequencing data showing genomic deletions at the CD19 (B) , CD20 (C) , and CD22 (D) loci in the respective KO lines, confirming targeted gene disruption. (E) Proliferation assay comparing all the Raji-Luc KO lines and control Raji-Luc-WT over 96 h. Data plotted as fold change normalized to Raji WT. (F) Viability analysis after 2 h incubation with AlamarBlue reveals no significant differences between Raji WT, Raji-Luc WT, and KO lines. Statistical analysis was performed using ordinary one-way ANOVA followed by the Dunnett’s multiple comparisons test. *, p < 0.05; ns = not significant.

Article Snippet: For CD19 and CD22 staining, cells were incubated with CD19-PE (Miltenyi Biotec) and CD22-APC (Miltenyi Biotec) antibodies for 30 min at 4 °C in the dark.

Techniques: CRISPR, Triple Knockout, Amplification, Sequencing, Disruption, Proliferation Assay, Control, Incubation

Journal: Frontiers in Genome Editing

Article Title: CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

doi: 10.3389/fgeed.2025.1649993

Figure Lengend Snippet: Raji knockout lines generated via CRISPR-Cas9 exhibit complete loss of CD19, CD20, and CD22 transcript and protein expression. (A–C) Transcript level analysis via qRT-PCR revealed significant reduction in the mRNA levels of (A) CD19, (B) CD20 and (C) CD22. K562 was used as CD-marker negative control. Gene expression was normalized to Raji WT and GAPDH was used as an internal control. Data plotted as fold change (mean ± SD, n = 3). (D) Immunofluorescence staining for CD19 (yellow), CD20 (green), CD22 (red), and Hoechst (blue) in all Raji cell lines and K562. Loss of surface antigen staining is evident in all corresponding knockout lines. Images are representative of three independent experiments. Scale bar = 50 μm. (E–G) Flow cytometry analysis of surface CD19 (E) , CD20 (F) , and CD22 (G) expression in nonpermeabilized Raji-derived knockout cells and K562 control. Data represent mean ± SD of fluorescence intensity (MFI) from three independent experiments, confirming complete loss of surface expression of the respective target antigen in each knockout line. Statistical analysis was performed using ordinary one-way ANOVA followed by the Dunnett’s multiple comparisons test. ****, p < 0.0001; ***, p < 0.001; ns = not significant.

Article Snippet: For CD19 and CD22 staining, cells were incubated with CD19-PE (Miltenyi Biotec) and CD22-APC (Miltenyi Biotec) antibodies for 30 min at 4 °C in the dark.

Techniques: Knock-Out, Generated, CRISPR, Expressing, Quantitative RT-PCR, Marker, Negative Control, Gene Expression, Control, Immunofluorescence, Staining, Flow Cytometry, Derivative Assay, Fluorescence

Journal: Frontiers in Genome Editing

Article Title: CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

doi: 10.3389/fgeed.2025.1649993

Figure Lengend Snippet: Raji KO lines model antigen loss and resist CAR-T cell–mediated cytotoxicity. (A) Schematic representation of CAR constructs targeting CD19, CD20, and CD22. (B) Flow cytometric analysis of isolated T cells confirmed the expression of T cell–specific markers and negligible expression of non–T cell markers. (C) Expression of T-cell activation markers on isolated T cells post 48 h of activation with CD3/CD28 beads. (D) Flow cytometry of GFP expression in T cells 48 h post-transduction. Histogram shown from one of three independent transductions using T cells from three different donors. (E–G) Viability of Raji KO lines assessed by bioluminescence following 16 h co-culture with CAR T cells expressing CAR19 (E) , CAR20 (F) or CAR22 (G) at indicated effector-to-target (E:T) ratios. Percent live cells was calculated based on luminescence signal relative to target-only control and normalized to untransduced controls. CAR-T cells selectively eliminated WT Raji cells expressing the corresponding target antigen, while respective single, double, and triple KO lines remained resistant. K562 cells served as antigen negative control. Data are presented as mean ± SD for three independent biological replicates. Statistical analysis was performed using multiple unpaired t-tests (one per row) with Holm–Šidák correction for multiple comparisons (C) or by comparing area under the curve for each cell line group in an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (E–G) . ***, p < 0.001; ****, p < 0.0001; ns = not significant.

Article Snippet: For CD19 and CD22 staining, cells were incubated with CD19-PE (Miltenyi Biotec) and CD22-APC (Miltenyi Biotec) antibodies for 30 min at 4 °C in the dark.

Techniques: Construct, Isolation, Expressing, Activation Assay, Flow Cytometry, Transduction, Co-Culture Assay, Control, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: The Notch1/CD22 signaling axis disrupts Treg function in SARS-CoV-2–associated multisystem inflammatory syndrome in children

doi: 10.1172/JCI163235

Figure Lengend Snippet: ( A ) Experimental scheme. Mice were injected i.p. with poly I:C daily for 12 days. ( B and C ) Weight indices of Foxp3 EGFPCre and Foxp3 EGFPCre R26 N1c/+ mice subjected to poly I:C treatment. ( D ) H&E-stained sections and inflammation score for liver, gut, and lung tissues isolated from mice in the indicated groups (original magnification, ×200). ( E ) Flow cytometric analysis and graphical representation of naive (CD4 + CD44 – CD62L + ) and activated (CD4 + CD44 + CD62L – ) Tconv cells. ( F and G ) Flow cytometric analysis and graphical representation of IFN-γ and IL-17 expression in Tconv cells ( F ) and Tregs ( G ) in the respective poly I:C–treated mouse groups. ( H ) Flow cytometric analysis and graphical representation of α4β7 expression in Tregs and Tconv cells from mice in the indicated groups. ( I ) Flow cytometric analysis and graphical representation of α4β7 expression in Tregs and Tconv cells from individuals in the indicated groups. ( J ) Relative expression of ITGB7 in the different clusters inferred from scRNA-Seq data. Max, maximum; Min, minimum. ( K ) Flow cytometric analysis and cell frequencies of α4β7 (ITGB7) expression on circulating CD4 + FOXP3 + Tregs in healthy controls and patients with MIS-C before and after treatment. ( L and M ) Frequencies of cells expressing Notch1 ( L ) and CD22 ( M ) on circulating CD4 + FOXP3 + Tregs from healthy controls and patients with MIS-C before and after treatment. Each symbol represents 1 mouse ( B – I ), 1 cell ( J ), or 1 human ( I and K – M ). Numbers in the flow plots indicate percentages. Error bars indicate the SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 2-way ANOVA with Šidák’s post hoc analysis ( B ), Student’s t test ( C and D ), and 1-way ANOVA with Dunnett’s post hoc analysis ( E – I , and K – M ).

Article Snippet: For blockading, CD22 mice were treated with an anti-CD22 antibody every 2 days for 12 consecutive days (InVivoMAb anti–mouse CD22; clone Cy34.1, Bio X Cell).

Techniques: Injection, Staining, Isolation, Expressing

Journal: The Journal of Clinical Investigation

Article Title: The Notch1/CD22 signaling axis disrupts Treg function in SARS-CoV-2–associated multisystem inflammatory syndrome in children

doi: 10.1172/JCI163235

Figure Lengend Snippet: ( A ) Flow cytometric analysis and graphical representation of CD22 expression on splenic Tregs and Tconv cells of poly I:C–treated Foxp3 EGFPCre and Foxp3 EGFPCre R26 N1c/+ mice. ( B ) Flow cytometric analysis and cell frequencies of CD22 expression on circulating CD4 + FOXP3 + Treg and CD4 + FOXP3 – Tconv cells from healthy controls and patients with either mild pediatric COVID or MIS-C. ( C ) Correlation analysis of CD22 expression on Tregs and Tconv cells of patients with MIS-C and controls as a function of Notch1 expression on these cells. ( D ) In vitro suppression of Tconv cell proliferation by Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs in the presence of increasing concentrations of anti-CD22 mAb. ( E ) In vitro suppression of human Tconv cell proliferation by Tregs isolated from healthy controls or patients with MIS-C in the absence of presence of anti-CD22 mAb. CTV, Cell Trace Violet. Each symbol represents 1 mouse ( A ) or 1 human ( B and C ). Numbers in flow plots indicate percentages. Error bars indicate the SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by Student’s t test ( A ), 1-way ANOVA with Dunnett’s post hoc analysis ( B ), 2-way ANOVA with Sidak’s post hoc analysis ( D and E ), and Pearson’s correlation analysis ( C ).

Article Snippet: For blockading, CD22 mice were treated with an anti-CD22 antibody every 2 days for 12 consecutive days (InVivoMAb anti–mouse CD22; clone Cy34.1, Bio X Cell).

Techniques: Expressing, In Vitro, Isolation

Journal: The Journal of Clinical Investigation

Article Title: The Notch1/CD22 signaling axis disrupts Treg function in SARS-CoV-2–associated multisystem inflammatory syndrome in children

doi: 10.1172/JCI163235

Figure Lengend Snippet: ( A ) Flow cytometric analysis and MFI of colonic markers of Tregs from poly I:C–treated Foxp3 EGFPCre and Foxp3 EGFPCre R26 N1c/+ mice cotreated with isotype a control mAb or an anti-CD22 mAb. ( B ) Flow cytometric analysis and MFI of p-Erk and p–PLC-γ expression induced by anti-CD3 mAb treatment of Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs. ( C ) Flow cytometric analysis and MFI of p-S6 and p-AKT (T308) expression induced by anti-CD3 mAb treatment of Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs. Numbers in the flow plots indicate percentages or MFI. Each symbol represents 1 mouse ( A – C ). Error bars indicate the SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA with Dunnett’s post hoc analysis ( A ) and 2-way ANOVA with Šidák’s post hoc analysis ( B and C ).

Article Snippet: For blockading, CD22 mice were treated with an anti-CD22 antibody every 2 days for 12 consecutive days (InVivoMAb anti–mouse CD22; clone Cy34.1, Bio X Cell).

Techniques: Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: The Notch1/CD22 signaling axis disrupts Treg function in SARS-CoV-2–associated multisystem inflammatory syndrome in children

doi: 10.1172/JCI163235

Figure Lengend Snippet: ( A and B ) Flow cytometric analysis ( A ) and MFI ( B ) of p-S6 expression induced by anti-CD3 mAb treatment of Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs that were treated or not with anti-CD22 mAb. ( C and D ) Foxp3 MFI in Tregs from in vitro suppression of Tconv cell proliferation by Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs in the presence of increasing concentrations of anti-CD22 mAb. ( E and F ) In vitro suppression of Tconv cell proliferation by Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs in the presence of increasing concentrations of rapamycin. ( G ) Foxp3 + MFI in Tregs from in vitro suppression of Tconv cell proliferation by Foxp3 EGFPCre and CD22 + Foxp3 EGFPCre R26 N1c/+ Tregs in the presence of increasing concentrations of rapamycin. Numbers in flow plots indicate percentages or MFI. Each symbol represents 1 mouse ( A – G ). Error bars indicate the SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 2-way ANOVA with Šidák’s post hoc analysis ( B , D , F , and G ).

Article Snippet: For blockading, CD22 mice were treated with an anti-CD22 antibody every 2 days for 12 consecutive days (InVivoMAb anti–mouse CD22; clone Cy34.1, Bio X Cell).

Techniques: Expressing, In Vitro