Journal: Cold Spring Harbor Molecular Case Studies

Article Title: Tumor-immune microenvironment revealed by Imaging Mass Cytometry in a metastatic sarcomatoid urothelial carcinoma with a prolonged response to pembrolizumab

doi: 10.1101/mcs.a006151

Figure Lengend Snippet: Full list of antibodies and their properties

Article Snippet: CD20 , Dy161 , L26 , 1:200 , 0.5 , Novus Biologicals , NBP2-80486.

Techniques: Concentration Assay

Journal: Materials Today Bio

Article Title: Sophora flavescens-derived extracellular vesicles loaded with oncolytic vaccinia virus/IR1061 for NIR-II photoacoustic imaging guided multimodal treatment of diffuse large B-cell lymphoma

doi: 10.1016/j.mtbio.2025.102177

Figure Lengend Snippet: SFOVV@IR1061 induces apoptosis and ICD in A20 lymphoma cells. (A) Flow cytometric analysis of apoptosis in A20 cells treated with PBS, SFNPs, OVV, IR1061 + laser, OVV@IR1061+laser, or SFOVV@IR1061 + laser (1064 nm, 5 min, 0.8 W/cm 2 ) for 12 h. Cells were stained with Annexin V-FITC and propidium iodide (PI). (B) Representative CLSM images of live/dead cell staining using Calcein-AM (green, live cells) and PI (red, dead cells) under the same treatment conditions. (C) Quantification of the dead/live cell ratio from CLSM images shown in (B). Data are presented as mean ± SD (n = 3). (D) Western blot analysis of apoptosis-related proteins including PARP, and Caspase-3 in A20 cells treated as indicated. β-actin was used as a loading control: 1. PBS; 2. SFNPs; 3. OVV; 4. IR1061+laser; 5. OVV@IR1061+laser; 6. SFOVV@IR1061+laser. (E) Western blot analysis of ICD-related markers CRT, HSP70, and HMGB1 following the same treatments: 1. PBS; 2. SFNPs; 3. OVV; 4. IR1061+laser; 5.OVV@IR1061 +laser; 6. SFOVV@IR1061+laser. (F) Flow cytometric analysis of macrophage polarization markers CD86 (left) and CD206 (right) in RAW264.7 cells. (G) Quantification of fluorescence intensity for CD86 and CD206. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To analyze M1/M2 macrophage polarization, cells were incubated with FITC Anti-Mouse CD86 Antibody (Elabscience, China) to detect M1 polarization and APC Anti-Mouse CD206 Antibody (Elabscience, China) to detect M2 polarization, according to the manufacturer's instructions.

Techniques: Staining, Western Blot, Control, Fluorescence

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: Cancer Immunology, Immunotherapy : CII

Article Title: Association of CCL21 + CAFs with B-cell recruitment and TLS maturation in penile squamous cell carcinoma

doi: 10.1007/s00262-026-04348-5

Figure Lengend Snippet: Evaluation of TLS maturation and prognostic analysis of PSCC patients. A UMAP plot of the cell types identified in PSCC through spatial transcriptomic sequencing, depicting 7 distinct cell populations. B Dot plot displaying the expression of cell type-specific markers. The dot size represents the proportion of cells expressing the marker, while the colour intensity indicates the mean expression level. C Regions with AUCell scores for mTLS genes ( BCL6 , AICDA , CD38 , ICOS , CXCR5 , CXCL13 , and CD21 ) above the median were defined as mTLSs, whereas those below the median were classified as iTLSs. D Spatial visualization of AUCell scores for mTLS genes, where warmer colours indicate higher scores. E Spatial distribution of iTLSs (green) and mTLSs (orange) across different regions of PSCC tissues. F Representative images H&E staining and immunofluorescence staining for CD20 (green) and CD21 (red) in iTLSs and mTLSs. G GO enrichment analysis of upregulated genes in mTLS. Darker colours denote more significant enrichment of the pathway, and larger dots represent a greater number of genes involved. H Kaplan–Meier survival curves showing that patients with mTLSs experienced significantly longer overall survival than those with iTLSs, as determined using ssGSEA ( P < 0.05)

Article Snippet: The following antibodies were used: CD20 (Proteintech, 60,271–1-Ig, 1:500), CD21 (Proteintech, 28,206–1-AP, 1:250), ACTA2/α-SMA (Sigma, SAB5700835, 1:300), and CCL21 (Abcam, ab9851, 1:300).

Techniques: Sequencing, Expressing, Marker, Staining, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Association of CCL21 + CAFs with B-cell recruitment and TLS maturation in penile squamous cell carcinoma

doi: 10.1007/s00262-026-04348-5

Figure Lengend Snippet: Characterization of CCL21 + CAF subtypes and their association with TLS maturation in PSCC. A UMAP plot of CAF subtypes in PSCC derived from spatial transcriptomics, including CCL21 + CAFs (green), COL16A1 + CAFs (blue), MYH11 + CAFs (orange), and XBP1 + CAFs (red). B Dot plot of key markers identified across CAF subtypes. The dot size represents the proportion of cells expressing the marker, and colour intensity indicates the mean expression level. C Radar plot showing the distribution of CCL21 + CAF proportions across the iTLS-core, mTLS-core, iTLS-edge, and mTLS-edge regions. D Bar chart illustrating the spatial distances between different CAF subtypes and mTLS structures; ***, P < 0.001. E In situ visualization of TLS regions: green areas indicate the iTLS-core, blue circles indicate the iTLS-edge, orange areas represent the mTLS-core, and yellow circles denote the mTLS-edge. F Spatial localization of CCL21 + CAFs in PSCC tissues showing their association with distinct TLS regions. G Heatmap overlay of the CCL21 + CAF spatial density and TLS distribution, with red indicating a higher CCL21 + CAF abundance. H Scatter plots of the correlation between the CCL21 + CAF proportion and B-cell (left panel) and naive T-cell (right panel) numbers in the iTLS/mTLS regions. I Representative images of mIHC of TLS regions in PSCC showing the spatial distribution and colocalization of CD20 (purple), ACTA2/α-SMA (green), and CCL21 (yellow). Scale bar = 150 μm. ( J ) Patients with high CCL21 + CAF signature scores (n = 29) experienced significantly longer overall survival than those with low scores (n = 26)

Article Snippet: The following antibodies were used: CD20 (Proteintech, 60,271–1-Ig, 1:500), CD21 (Proteintech, 28,206–1-AP, 1:250), ACTA2/α-SMA (Sigma, SAB5700835, 1:300), and CCL21 (Abcam, ab9851, 1:300).

Techniques: Derivative Assay, Spatial Transcriptomics, Expressing, Marker, In Situ

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