Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD21 , REA940 , 50 , 130-115-609 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD21 , REA940 , 50 , 130-115-609 , FITC , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: Genes

Article Title: Establishment and Expression of Cytokines in a Theileria annulata -Infected Bovine B Cell Line

doi: 10.3390/genes10050329

Figure Lengend Snippet: Percentage of CD21 + cells in peripheral blood mononuclear cells (PBMCs) and analysis of their purity after magnetic separation. Here, ( A ) is the background control, ( B ) is the percentage of CD21 + cells in PBMCs, and ( C ) is the purity of magnetically isolated cells.

Article Snippet: The mouse anti-bovine CD21 (MCA1424PE), mouse anti-bovine IgM (AAI19F), mouse anti-bovine WC4 (MCA1648G), and negative control mouse IgG1 (MCA928F) antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Techniques: Control, Isolation

Journal: Genes

Article Title: Establishment and Expression of Cytokines in a Theileria annulata -Infected Bovine B Cell Line

doi: 10.3390/genes10050329

Figure Lengend Snippet: Percentage of CD21 in transformed B cells at the 3rd passage.

Article Snippet: The mouse anti-bovine CD21 (MCA1424PE), mouse anti-bovine IgM (AAI19F), mouse anti-bovine WC4 (MCA1648G), and negative control mouse IgG1 (MCA928F) antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Techniques: Transformation Assay

Journal: Genes

Article Title: Establishment and Expression of Cytokines in a Theileria annulata -Infected Bovine B Cell Line

doi: 10.3390/genes10050329

Figure Lengend Snippet: Percentages of B cell-specific surface markers (CD21, IgM, and WC4) present in transformed cells (6th and 10th generations), single-cell line clones (approximately 15th generation), and normal purified cells. The percentages of CD21 (98.8, 99.7, 98.0, 98.5, and 97%; mean ± SD of 98.4 ± 1.00), IgM (3.51, 0.64, 1.42, 0.99, and 2.83%; mean ± SD of 1.88 ± 1.23) and WC4 (0.51, 1.96, 0.71, 0.80, and 0.97%; mean ± SD of 0.99 ± 0.57) in five clones were determined. CD21 was present in mono- and polyclonal cell lines, while negligible percentage of IgM and WC4 were present.

Article Snippet: The mouse anti-bovine CD21 (MCA1424PE), mouse anti-bovine IgM (AAI19F), mouse anti-bovine WC4 (MCA1648G), and negative control mouse IgG1 (MCA928F) antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Techniques: Transformation Assay, Clone Assay, Purification

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Clinicopathological features and prognostic significance of tertiary lymphoid structures (TLS) (A) The distribution of clinical features and immature density for the 27 patients in cohort 1. (B) Kaplan-Meier survival curves for progression-free survival (PFS) of 27 patients with no treatment before surgery stratified based on the median density of TLSs (B) , density of intra-/peri- TLSs (C) , maturation of TLSs (D) and the existence of SFL-TLSs (In SFL-TLS, CD21+ follicular dendritic cells form dense clusters within the core germinal center) (E) . B–E: Differentiate Kaplan-Meier curves with distinct colors for better visual clarity. 3D & 3E: Clarify the difference between “mature TLS” and “SFL-TLS.”.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques:

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Characteristics of tertiary lymphoid structures (TLS) in treatment-naive group and neoadjuvant hormone therapy (NHT) group in cohort 2. (A) mIHC of immature and mature TLS in NHT group and treatment-naive group in cohort 2; (B) Comparison of the ratio of CD4 + cells, CD8+ cells, CD20+cells and CD21+cells in tumor microenvironment (TME) in the NHT group and treatment-naive group. (C) Proportion of different TLSs maturation stage in the NHT group and treatment-naive group. (D) Proportion of different density of TLS in the NHT group and treatment-naive group. (E) Proportion of different location of TLS in the NHT group and treatment-naive group. P values are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques: Comparison

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Characteristics and changes of immune cells in pre- and post- neoadjuvant hormone therapy (NHT) groups in GSE111177 . (A) Supervised clustering of PCa specimens by NHT (n = 24), displaying ssGSEA scores ( GSE111177 ). (B) The biological process of DEGs identified in pre-/post- NHT groups ( GSE111177 ). (C) Comparison of the expression of CD4, CD8A and CD8B, CD20, FOXP3, CD21, GZMB, PRF1 in PCa patients before and after NHT using GSE111177 cohort (n = 24). (D) Comparison of GSVA scores utilizing four TLS signatures in 24 paired pre-NHT and post-NHT samples from GSE111177 .

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques: Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Characterization of tumor immune microenvironment (TME) and tertiary lymphoid structures (TLS) before and after neoadjuvant hormone therapy (NHT). (A) Comparison of the ratio of CD4 + cells, CD8+ cells, CD20+cells and CD21+cells in TME before and after NHT. (B) Comparison of the TLS presence in TME before and after NHT. (C) Comparison of the intra-tumor TLS presence in TME before and after NHT. (D) Comparison of the TLS maturation stage in TME before and after NHT. (E) Evaluation of TLS maturation stage using mIHC staining of CD4, CD8 CD20, CD21 before and after NHT. P values are denoted as follows: **p < 0.01, ***p < 0.001, NS p>0.05.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques: Comparison, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Hemophagocytic lymphohistiocytosis presenting with annular erythema multiforme-like eruptions in a patient with angioimmunoblastic T cell lymphoma: A case report

doi: 10.3892/etm.2018.6420

Figure Lengend Snippet: Histological and immunohistochemical staining findings of the patient. A biopsy sample taken from the left cervical lymph node revealed that normal structure had disappeared, and small to medium-sized atypical lymphoid cells had infiltrated the lymph node region. (A) H&E staining; original magnification, ×10. (B) H&E staining; original magnification, ×40. (C-G) On immunohistochemical staining, the lymphoid cells were diffusely positive for CD2, CD3, CD5, CD7 and CD10 (H&E staining; original magnification, ×40). (H) CD20, (I) paired-box domain 5 and (J) telomerase B staining were weak positive (H&E staining; original magnification, ×40). (K) CD21 staining suggested follicular dendritic cells net damage (H&E staining; original magnification, ×40). (L) Proliferative index with Ki-67 stain was high (H&E staining; original magnification, ×40). H&E, hematoxylin and eosin; CD, cluster of differentiation.

Article Snippet: The antibodies utilized included: with CD2 (cat. no. UMAB86), CD3 (cat. no. UM570048), CD5 (cat. no. UM570009), CD7 (cat. no. TA506337), CD10 (cat. no. TA590055), CD20 (cat. no. UM800065), CD21 (cat. no. TA327627) and Ki-67 (TA801577) all diluted at 1:100. paired-box domain 5 (cat. no. TA801884) and telomerase B (cat. no. TA301588) were diluted at 1:50 (all antibodies were obtained from OriGene Technologies, Inc.) and hematoxylin staining as previously described ( 17 ).

Techniques: Immunohistochemical staining, Staining

Journal: Cell reports

Article Title: B Cells Produce the Tissue-Protective Protein RELMα during Helminth Infection, which Inhibits IL-17 Expression and Limits Emphysema.

doi: 10.1016/j.celrep.2018.11.038

Figure Lengend Snippet: Figure 6. RELMa Expression by B Cells Is Required for Limiting Nb-Induced Emphysematous Pathology (A) B cell numbers in the lung at different time points after N. brasiliensis inoculation were assessed by flow cytometry. (B) At day 5 after Nb inoculation, sort-purified B cells were analyzed for mRNA expression of candidate regulatory factors. (C) Sort-purified B cells from Il4ra/ and WT BALB/c mice were analyzed for Retnla mRNA expression at day 5 after Nb inoculation. (D–F) Sort-purified B cells (D), alveolar macrophages (E), or non-alveolar macrophages (F) at different time points after Nb infection were analyzed for Retnla mRNA expression. (G and H) RELMa mean fluorescence intensity (MFI) of lung B cells (CD19+) (G) and alveolar macrophages (H) was determined by cytoplasmic staining and FACS at day 5 after N. brasiliensis inoculation. (I and J) Sort-purified B cells from either untreated Retnla/ or WT mice were transferred to recipient Jh/ mice at days 3, 0, and +1 after N. brasiliensis inoculation. Lungs were collected for analysis 7 days after inoculation, emphysematous pathology was digitally imaged as described in Figure 1 (I), and lung tissues were analyzed for the expression of Il17a by qPCR (J). (K) Retnla/ and WT mice were inoculated with Nb, and 2 days later, gd T cells were assessed for IL-17A production by intracellular staining and flow cytometric analyses. (L and M) FACS analysis of lung B cell subsets at day 5 after N. brasiliensis inoculation, showing expression of IgM and IgD (L) and CD21, CD23, and CD24 (M) on gated CD19+ lymphocytes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (A and D–I), or data shown are the mean and SEM from five individual mice per group (B–F and J) or a pool of five mice per group (K and L). Data shown are representative of at least two independent experiments (**p < 0.01). See also Figures S4 and S5.

Article Snippet: Cells were blocked with Fc Block (BD Biosciences, San Jose, CA), directly stained with fluorochrome-conjugated Abs against Ly6G (1A8), MHCII, CD11c, Siglec-F, F4/80, CD19, B220 (BD Biosciences), CD21, CD23, CD24, CD5, Cd1, CD138 (Biolegend), RELMa (Biorbyt), and analyzed by flow cytometry.

Techniques: Expressing, Cytometry, Infection, Staining

Journal: European journal of immunology

Article Title: Essential role of antigen-presenting cell-derived BAFF for antibody responses.

doi: 10.1002/eji.200636791

Figure Lengend Snippet: Figure 1. Phenotypic characterisation and purity of sorted PBMC. (A) Expression of SWC1 on APC. CD4/CD172a dot plot defining four subsets of APC and their SWC1 expression (histograms). (B) SWC1 expression on T and NK cells. (C) SWC1/ sIg expression on PBMC, and SWC1/CD21 expression on sIg+

Article Snippet: Phenotypic analysis For phenotypic analysis, cells were stained using murine mAb against SWC1, CD172a, CD6, CD3 (mAb 8E6; VMRD), CD8 (mAb 11-295-33; kindly provided by Prof. Dr. A. Saalm ller, University of Veterinary Medicine Vienna), CD21 (mAb BB6-11C9.6; SouthernBiotech), TCR1 d chain (VMRD), goat mAb against human BAFF (R&D Systems) and polyclonal affinity-purified goat anti-swine antibody (Jackson Immuno Research).

Techniques: Expressing

Journal: Oncotarget

Article Title: Inflammaging increases susceptibility to cigarette smoke-induced COPD

doi: 10.18632/oncotarget.4027

Figure Lengend Snippet: A. Representative micrographs of HE-stained lung tissue sections from young and old FA and CS-exposed mice showing the presence of iBALT in aged animals after CS exposure; scale bar 100 μm. B. Total volume of iBALT per basal membrane was determined via quantitative morphological assessment. C. Lung tissue mRNA expression of CXCL13 was measured by qPCR. D. Representative micrographs of immunohistochemical stainings for CD3 (T lymphocytes), B220 (B lymphocytes) and CD21 (FDCs) in lung sections from CS-exposed young and old mice; scale bar 100 μm. E. Flow cytometry analysis of whole lung for CD4 + T lymphocyte infiltration reveals increases in Th2 and Th17 cells after CS exposure in aged compared to young mice; Th1 cells were defined by expression of CD4 and IFNγ, Th2 cells by expression of CD4 and IL-4, Th17 cells by expression of CD4 and IL-17A, and regulatory T cells by expression of CD4, CD25 and Foxp3. F. Lung tissue mRNA expression of Th signature cytokines IFNγ and IL-13 and transcription factors Gata3, Rorγt and Foxp3 was measured by qPCR. Data were combined from 2 independent experiments with n = 8 and are given as mean values ± SD; one-way ANOVA following Bonferroni post test with * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After overnight incubation with primary antibodies against MMP12 (Abcam, Cambridge, UK), CD45R/B220 (BD Pharmingen), CD21 (Novus Biologicals, Littleton, CO), CD3 (Sigma Aldrich), p16 (Santa Cruz, CA) or SIRT1 (Millipore, Schwalbach, Germany) tissue slides were incubated with an alkaline phosphatase-labeled secondary antibody (Biocare Medical).

Techniques: Staining, Membrane, Expressing, Immunohistochemical staining, Flow Cytometry

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Synergistic activation by MB‐CD40L, BCR, and TLR‐9 signaling for improved B‐cell expansion and differentiation. (a) Representative flow cytometry plot of CD27 versus CD38 for days 0–13 cells (gated on IgD‐ B‐cells from day‐4 onwards) for the activation of naïve B‐cells by MB‐CD40L with or without the B‐cell receptor signal by anti‐human IgM/G/A (H + L) antibody plus co‐receptor signal by anti‐human CD21 antibody (referred to as BCR) and/or toll‐like receptor signal by the TLR‐9 agonist CpG ODN 2006 in the presence of BAFF and the interleukins Il‐2, ‐4, ‐10 and ‐21. Bar graphs of (b) fold expansion and the yield of (c) memory B‐cells and (d) ASCs on day‐13 for each group. Data were acquired each day as duplicates for each group and represented as mean + standard deviation. Statistical analysis was conducted by applying ordinary one‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns p > 0.05).

Article Snippet: For the activation of the B‐cell receptor, a combination of goat anti‐human IgA + IgG + IgM (H + L) antibody (109006064; Jackson Immuno Research Laboratories, USA) was added to the culture at 6 μg/mL concentration, together with a monoclonal mouse anti‐human CD21 antibody (MAB4909; R&D Systems, USA) at 1 μg/mL.

Techniques: Activation Assay, Flow Cytometry, Standard Deviation

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Switching interleukins and CD40L dosage for the terminal differentiation of naïve B‐cells for effective generation of functional ASCs. (a) Representative flow cytometry plot of CD27 versus CD38 gated on IgD‐ B‐cells on day‐10 and day‐13. High‐purity naïve B‐cells were activated with MB‐CD40L, BAFF, anti‐BCR/CD21 antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for initial 10 days. On day‐10, CD40L dosage was either maintained (MB‐CD40L) or reduced by 10‐fold (MB‐CD40L low ). For each CD40L dosage, three different conditions were tested up to day‐13: All factors or all factors except IL‐21 or all factors except anti‐BCR antibodies and IL‐4. (b), (c) Bar graphs comparing the yield percentage of memory B‐cells (IgD‐CD38‐/lo + CD27) and ASCs (IgD‐CD38++CD27+) on day‐13 from indicated conditions. Data were acquired as duplicates and presented as mean + standard deviation. (d) Number of ASCs generated by day‐13 from the culture of 10,000 naïve B‐cells (day‐0). Either MB‐CD40L or Sol Multimer CD40L was employed for the entirety of the culture period. Other indicated culture conditions were varied between day‐10 and day‐13. Data were acquired as triplicates and presented as mean + standard deviation. (e), (f) The concentrations of antibodies of different isotypes, IgM and IgG, secreted by ASCs generated by activation of high‐purity naïve B‐cells using the indicated method of CD40L presentation in the presence of BAFF, anti‐BCR antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for the initial 10 days. On day‐10, cells were reseeded at 10,000 cells per well, CD40L dosage was reduced by 10‐fold, and all soluble factors except anti‐BCR antibodies and IL‐4 were employed. ELISA was performed using supernatant samples harvested on day‐12. Data were acquired as triplicates and presented as mean + standard deviation. (b)−(f) Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001; ns p > 0.05).

Article Snippet: For the activation of the B‐cell receptor, a combination of goat anti‐human IgA + IgG + IgM (H + L) antibody (109006064; Jackson Immuno Research Laboratories, USA) was added to the culture at 6 μg/mL concentration, together with a monoclonal mouse anti‐human CD21 antibody (MAB4909; R&D Systems, USA) at 1 μg/mL.

Techniques: Functional Assay, Flow Cytometry, Standard Deviation, Generated, Activation Assay, Enzyme-linked Immunosorbent Assay