Journal: Frontiers in Immunology

Article Title: Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations

doi: 10.3389/fimmu.2021.739675

Figure Lengend Snippet: DNA damage response to ionizing radiation differs among T, B, and NK lymphocytes. Peripheral blood mononuclear cells (PBMCs) from 26 healthy donors were irradiated with 2Gy and fixed at indicated time points. Surface markers of lymphocyte subsets and intranuclear DDR biomarkers were analyzed by mass cytometry. Induction of γH2AX (A) , p-ATM (B) , p-CHK2 (C) , and p53 (D) were calculated in CD45 + lymphocytes, CD45 + CD3 + T cells, CD45 + CD56 dim CD16 + NK cells and CD45 + CD19 + CD20 + B cells based on mean fluorescence intensities normalized on unirradiated samples. Bars represent mean values; error bars represent standard deviations. Significance is shown for NK and B lymphocytes in comparison to T cells (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Article Snippet: The following surface antibodies were used at concentrations of 1μl/100μl staining buffer: Mouse anti-human CD45 (HI30)-154Sm (Fluidigm), anti-human CD3 (UCHT1)-170Er (Fluidigm), anti-human CD4 (SK3)-174Yb (Fluidigm), anti-Human CD8 (Sk1)-168Er (Fluidigm), anti-human CD45RA (HI100) 169Tm (Fluidigm), anti-human CD45RO (UCHL1) 164-Dy (Fluidigm), anti-human CD197/CCR7 (G053H7) 159Tb (Fluidigm), anti-human CD69 (FN50) 144Nd (Fluidigm), anti-human CD56 (HCD56) 176Yb (Fluidigm), anti-Human CD16 (3G8)-165Ho/Fluidigm, anti-human CD57 (HCD57) 163Dy (Fluidigm), anti-human CD19 (HIB19) 142Nd (Fluidigm), anti-human CD20 (2H7) 171Yb (Fluidigm), anti-human IgD (IA6-2) 146Nd (Fluidigm), anti-human IgM (MHM-88) 172Yb (Fluidigm), anti-human IgG kappa (MHK-49) 160Gd (Fluidigm), anti-human IgG lambda (MHL-38) 151Eu (Fluidigm), anti-human CD27 (L128) 158Gd (Fluidigm), anti-human CD38 (HIT2) 167Er (Fluidigm), anti-human CD21 (BL-13) 152Sm (Fluidigm).

Techniques: Irradiation, Mass Cytometry, Fluorescence, Comparison

Journal: Frontiers in Immunology

Article Title: Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations

doi: 10.3389/fimmu.2021.739675

Figure Lengend Snippet: DDR in naïve lymphocyte subsets is differential to mature memory populations. PBMCs obtained from 26 healthy donors were irradiated with 2Gy and fixed at indicated time points. Surface markers of lymphocyte subsets and intranuclear DDR biomarkers were assessed by mass cytometry. Fold inductions of γH2AX, p-ATM, p-CHK2, and p53 were calculated in CD45 + lymphocyte subsets, based on mean fluorescence intensities normalized on unirradiated samples. T cell subsets were characterized as CD3 + , CD3 + CD4 + , CD3 + CD8 + , CD45RA + CCR7 + (naïve CD4/CD8), CD45RO + CCR7 +/- (central and effector memory CD4/CD8) (A) . NK lymphocyte subsets were defined as CD3 - CD56 bright CD16 - , CD3 - CD56 bright CD16 + , CD3 - CD56 dim CD16 + (B) , which were further stratified to CD57 expression on CD56 bright CD16 + and CD56 dim CD16 + subsets. CD3 - B lymphocytes were characterized as CD19 + CD20 + , CD27 - IgD + (naïve B), CD27 + (memory B), CD27 + IgM + (unswitched memory B) CD27 + IgM - IgGκ + (class switched memory B IgGκ), CD27 + IgM - IgGΛ + (class switched memory B IgGΛ), CD27 + IgM + IgD + (Marginal Zone (MZ)-like B), IgM ++ CD38 ++ (transitional B), CD27 + IgM + IgD - (IgM only B), CD27 - IgM - IgD - (atypical memory B), CD19 + CD20 - CD27 + CD38 + IgM + (unswitched plasmablasts), CD19 + CD20 - CD27 + CD38 + IgM - (class switched plasmablasts), and CD21 low CD38 low B cells. (C) Bars represent mean values of fold induction; error bars represent standard deviations. Significance is shown for naïve T and B cell subsets compared to memory subsets, and immature CD56 bright CD16 - to mature CD56 bright CD16 + and CD56 dim CD16 + NK lymphocytes (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Article Snippet: The following surface antibodies were used at concentrations of 1μl/100μl staining buffer: Mouse anti-human CD45 (HI30)-154Sm (Fluidigm), anti-human CD3 (UCHT1)-170Er (Fluidigm), anti-human CD4 (SK3)-174Yb (Fluidigm), anti-Human CD8 (Sk1)-168Er (Fluidigm), anti-human CD45RA (HI100) 169Tm (Fluidigm), anti-human CD45RO (UCHL1) 164-Dy (Fluidigm), anti-human CD197/CCR7 (G053H7) 159Tb (Fluidigm), anti-human CD69 (FN50) 144Nd (Fluidigm), anti-human CD56 (HCD56) 176Yb (Fluidigm), anti-Human CD16 (3G8)-165Ho/Fluidigm, anti-human CD57 (HCD57) 163Dy (Fluidigm), anti-human CD19 (HIB19) 142Nd (Fluidigm), anti-human CD20 (2H7) 171Yb (Fluidigm), anti-human IgD (IA6-2) 146Nd (Fluidigm), anti-human IgM (MHM-88) 172Yb (Fluidigm), anti-human IgG kappa (MHK-49) 160Gd (Fluidigm), anti-human IgG lambda (MHL-38) 151Eu (Fluidigm), anti-human CD27 (L128) 158Gd (Fluidigm), anti-human CD38 (HIT2) 167Er (Fluidigm), anti-human CD21 (BL-13) 152Sm (Fluidigm).

Techniques: Irradiation, Mass Cytometry, Fluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations

doi: 10.3389/fimmu.2021.739675

Figure Lengend Snippet: DDR is diminished in all lymphocyte subsets of patients with ataxia telangiectasia. PBMCs obtained from healthy donors and 3 patients with ataxia telangiectasia (AT) were treated with 2Gy ionizing radiation and fixed after 1h, 4h, 8h and 24h. Fold inductions of γH2AX (A) , p-ATM (B) , p-CHK2 (C) , and p53 (D) were calculated in CD45 + , CD45 + CD3 + , CD45 + CD3 - CD56 dim CD16 - , and CD45 + CD3 - CD19 + CD20 + lymphocyte subsets, based on mean fluorescence intensities normalized on unirradiated samples. Box plots indicate distribution of fold inductions obtained from 26 healthy donors and 3 AT patients; error bars represent standard deviations. Significance is shown for differences between controls and patients (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001).

Article Snippet: The following surface antibodies were used at concentrations of 1μl/100μl staining buffer: Mouse anti-human CD45 (HI30)-154Sm (Fluidigm), anti-human CD3 (UCHT1)-170Er (Fluidigm), anti-human CD4 (SK3)-174Yb (Fluidigm), anti-Human CD8 (Sk1)-168Er (Fluidigm), anti-human CD45RA (HI100) 169Tm (Fluidigm), anti-human CD45RO (UCHL1) 164-Dy (Fluidigm), anti-human CD197/CCR7 (G053H7) 159Tb (Fluidigm), anti-human CD69 (FN50) 144Nd (Fluidigm), anti-human CD56 (HCD56) 176Yb (Fluidigm), anti-Human CD16 (3G8)-165Ho/Fluidigm, anti-human CD57 (HCD57) 163Dy (Fluidigm), anti-human CD19 (HIB19) 142Nd (Fluidigm), anti-human CD20 (2H7) 171Yb (Fluidigm), anti-human IgD (IA6-2) 146Nd (Fluidigm), anti-human IgM (MHM-88) 172Yb (Fluidigm), anti-human IgG kappa (MHK-49) 160Gd (Fluidigm), anti-human IgG lambda (MHL-38) 151Eu (Fluidigm), anti-human CD27 (L128) 158Gd (Fluidigm), anti-human CD38 (HIT2) 167Er (Fluidigm), anti-human CD21 (BL-13) 152Sm (Fluidigm).

Techniques: Fluorescence

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: A) This diagram depicts the dual‐targeting strategy of biHSNPs in facilitating tumor cell elimination. Each biHSNP features two functional arms: one specifically binds to a tumor‐associated antigen on lymphoma cells (CD19/CD20), while the other engages and activates immune effector cells (CD3/CD16). By bridging tumor cells and immune cells, this approach enhances immune‐mediated tumor cell lysis, leveraging the immune system's inherent ability to identify and eliminate abnormal cells for precise and efficient cancer cell killing. B) Schematic illustration of the T cell activation and tumor cell death mechanism mediated by biHSNPs.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Functional Assay, Lysis, Activation Assay

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Schematic illustration of the chemical synthesis and characterization of antibody‐conjugated hollow silica nanoparticles. A) Synthetic pathway of PEGylated hollow silica nanoparticles. B) Fourier transform‐infrared spectroscopy (FT‐IR) characterization of HSNP@PEG (2). C) Proton nuclear magnetic resonance ( 1 H NMR) characterization of HSNP@PEG. D) DLS and TEM images of HSNP@PEG. E) DLS and TEM images of HSNP@PEG@NH 2 (3). F) Fluorescence spectrum of HSNP@PEG@NH 2 @FITC (9.62 µ m ), confirming successful FITC conjugation. G) DLS and TEM images of HSNP@PEG@NH 2 @FITC (4). H–K) DLS and TEM results for monospecific nanoparticles (HSNP αCD3 , HSNP αCD16 , HSNP αCD19 , HSNP αCD20 ). L–O) DLS and TEM results for bispecific nanoparticles (HSNP αCD3 + αCD19 , HSNP αCD3 + αCD20 , HSNP αCD16 + αCD19 , HSNP αCD16 + αCD20 ). All scale bars in the TEM images represent 100 nm. HSNP αCD3 , HSNP αCD16 , HSNP αCD19 and HSNP αCD20 : Monospecific HSNPs conjugated with a single type of antibody (anti‐CD3, anti‐CD16, anti‐CD19, or anti‐CD20, respectively). HSNP αCD3 + αCD19 , HSNP αCD3 + αCD20 , HSNP αCD16 + αCD19 and HSNP αCD16 + αCD20 : Bispecific HSNPs conjugated with two different types of antibodies (e.g., anti‐CD3 and anti‐CD19). Antibody types are indicated using lowercase Greek letters (e.g., αCD3).

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Fourier Transform Infrared Spectroscopy, Spectroscopy, Nuclear Magnetic Resonance, Fluorescence, Conjugation Assay

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Binding specificity and cell–cell linkage induced by biHSNPs. Target cells were incubated with FITC‐labeled, antibody‐coated HSNPs at 4 °C for 30 min, followed by flow cytometry analysis to confirm binding specificity. A–C) Depict cell–cell linkage induced by biHSNPs: A) Schematic illustration of cell‐cell complex formation mediated by biHSNPs. B) Confocal microscopy images showing stable junctional complexes between immune effector cells and tumor cells facilitated by HSNP αCD3 + αCD19 and HSNP αCD3 + αCD20 . C) Flow cytometry analysis of cell–cell complexes between pre‐stained Jurkat (green, CellTracker Green CMFDA) and Raji cells (red, CellTracker Red CMTPX), demonstrating effective linkage by biHSNPs. D) Verification of biHSNPs binding to CD3 + cells using PBMCs as the CD3 + cell line. E) Verification of biHSNPs binding to CD19 + /CD20 + cells using Raji cells as the target. F) Verification of biHSNPs binding to CD16 + cells using PBMCs as the CD16 + cell line. G) Verification of biHSNPs binding to CD19 + /CD20 + cells using Raji cells as the target.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry, Confocal Microscopy, Staining

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: A) Cytotoxicity and cytokine release assays of biHSNPs in luciferase‐transfected Raji cells. B) In vitro cytotoxicity of HSNP@PEG, HSNP αCD3 , HSNP αCD19 , and bispecific HSNP αCD3 + αCD19 . C) In vitro cytotoxicity of HSNP@PEG, HSNP αCD3 , HSNP αCD20 , and bispecific HSNP αCD3 + αCD20 . D,E) Cytokine secretion levels (IFN‐γ, IL‐2, and TNF‐α) for HSNP αCD3 , HSNP αCD19 , and HSNP αCD3 + αCD19 ; and HSNP αCD20 and HSNP αCD3 + αCD20 . F) In vitro cytotoxicity of HSNP@PEG, HSNP αCD16 , HSNP αCD19 , and bispecific HSNP αCD16 + αCD19 . G) In vitro cytotoxicity of HSNP@PEG, HSNP αCD16 , HSNP αCD20 , and bispecific HSNP αCD16 + αCD20 . H,I) Cytokine secretion levels (IFN‐γ, IL‐2, and TNF‐α) for HSNP αCD16 , HSNP αCD19 , HSNP αCD16 + αCD19 , HSNP αCD20 , and HSNP αCD16 + αCD20 . J) Cytotoxicity of combined bispecific nanoparticle treatments: HSNP αCD3 + αCD19 with HSNP αCD16 + αCD19 , and HSNP αCD3 + αCD20 with HSNP αCD16 + αCD20 . K) In vitro cytotoxicity of HSNP αCD3 + αCD19 combined with HSNP αCD16 + αCD19 , and HSNP αCD3 + αCD20 combined with HSNP αCD16 + αCD20 . Data are presented as mean ± SD ( n ≥ 3), Statistical differences in B–I) were analyzed by One‐way ANOVA and K) were analyzed by Student's t‐test and the statistical significance is indicated as ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Luciferase, Transfection, In Vitro

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: Rituximab conjugation confers γδ2 T cells with CD20 binding capacity. ( a ) A representative histogram of rituximab conjugation is illustrated. DNA linker-1 and linker-2 were conjugated with γδ2 T cells and rituximab, respectively. Linker-1-conjugated γδ2 T cells and linker-2-conjugated rituximab were mixed and ACE1831, rituximab-linked γδ2 T cells, were generated through DNA hybridization. Un-conjugated γδ2 T cells and ACE1831 were stained with R-phycoerythrin-coupled anti-F(ab’)2 antibody to determine the rituximab conjugation efficiency through flow cytometry. Un-conjugated γδ2 T cells (light blue line) represent negative staining, and efficient rituximab conjugation on ACE1831 (dark blue line) is shown. Percent of Max is the highest point of each peak of the overlaid histogram derived from ACE1831 and γδ2 T cells. ( b ) CD20 binding capacity of ACE1831 and γδ2 T cells was determined through flow cytometry analysis. The cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of human CD20-His recombinant protein, and the CD20-bound cell population was identified through staining with Fluorescein-coupled anti-6X His tag antibody. The study was performed in triplicate in five different experiments, and the representative results are shown. Statistical analysis was performed using the t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Conjugation Assay, Binding Assay, Generated, DNA Hybridization, Staining, Flow Cytometry, Negative Staining, Derivative Assay, Incubation, Recombinant

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: Rituximab conjugation confers γδ2 T cells with superior cytotoxicity against CD20-expressing cancer cells. ( a – c ) ACE1831 and γδ2 T cells were co-incubated with CD20-expressing cancer cells. ( a ) Daudi, ( b ) Raji and ( c ) rituximab-resistant Raji cells at effector to target (E:T) ratios of 1:1, 2:1, 5:1 and 10:1, analyzed via CellTiter-Glo ® luminescent cell viability assay after 4 h of co-incubation. ( d ) CD107a, ( e ) granzyme B and ( f ) IFNγ of ACE1831 in the absence and presence of Raji cells at an E:T ratio of 2:1 after 2 h of incubation were analyzed via flow cytometry. Each group was performed in triplicate from two donor lots, and the representative results are shown. Statistical analysis was performed using the t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ( g , h ) ACE1831 and γδ2 T cells were co-incubated with ( g ) CD20-negative K562 and ( h ) donor PBMC cells at effector to target (E:T) ratios of 2:1, 5:1 and 10:1 and analyzed using a CellTiter-Glo ® luminescent cell viability assay after 4 h of co-incubation.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Conjugation Assay, Expressing, Incubation, Cell Viability Assay, Flow Cytometry

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: ACE1831 shows superior potency against CD20-expressing cancer cells in vivo. Four doses of intravenously delivered ACE1831 effectively suppressed tumor growth. Tumor-bearing SCID–Beige mice were treated with ACE1831, γδ2 T cells and a Vehicle (serum-free medium) twice per week for two weeks. ( a ) Tumor burden of mice (n = 5 per group) was determined through bioluminescence imaging. ( b ) The bioluminescence intensity of the tumor burden is presented as mean values ± SD. The difference in mean tumor burden between groups was examined using a two-way ANOVA. ***, p < 0.001. ( c ) The survival rate of mice with different treatments was analyzed using the Kaplan–Meier method. **, p < 0.01. ( d ) The body weight of each group of mice is presented as mean value ± SD.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Expressing, In Vivo, Imaging

Journal: Cancers

Article Title: A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma

doi: 10.3390/cancers15194844

Figure Lengend Snippet: T cell activation and cytotoxicity mediated by the antigen recognition of ACC-linked antibody. ( a ) Jurkat-NFAT-Luc cells were conjugated with different amounts of rituximab using ACC technology and were stained with anti-F(ab’)2 antibody to examine the levels of rituximab conjugated on Jurkat-NFAT-Luc cells. Un-conjugated Jurkat-NFAT-Luc cells (grey line) represent negative staining, and Jurkat-NFAT-Luc cells with low (light blue) and high (dark blue) rituximab conjugation are shown. Percent of Max is the highest point of each peak of the overlaid histogram. ( b ) Jurkat-NFAT-Luc cells conjugated with different amounts of rituximab were co-incubated with different Raji cell numbers (+, 5 × 10 4 ; ++, 2 × 10 5 ; +++, 5 × 10 5 ), and NFAT signaling activation was determined based on NFAT-regulated luciferase activity. Each condition was applied in triplicate in two different experiments, and the representative results are shown. Mean ± SD. Statistical analysis was performed using a t test. **, p < 0.01; ****, p < 0.0001. ( c ) The effector cells were preincubated with or without 1 μg/mL of TCRγδ blocking antibody for 1 h at 37 °C. After 4 h of co-incubation with Raji cells, cytotoxicity against Raji cells was analyzed using a CellTiter-Glo ® luminescent cell viability assay. Each condition was applied in triplicate in two different experiments, and the representative results are shown. Mean ± SD, *, p < 0.05, ***, p < 0.001. ( d ) Illustration delineating the activation of ACE1831 upon encountering CD20-expressing cancer cells.

Article Snippet: To examine their CD20 binding capacity, the cells were incubated with 0.001, 0.01, 0.1, 1, 10 and 100 μg/mL of recombinant human CD20-His protein (ACROBiosystems, Newark, DE).

Techniques: Activation Assay, Staining, Negative Staining, Conjugation Assay, Incubation, Luciferase, Activity Assay, Blocking Assay, Cell Viability Assay, Expressing