Journal: Mucosal immunology

Article Title: Single cell and tissue-transcriptomic analysis of murine bladders reveals age- and TNFα–dependent but microbiota-independent tertiary lymphoid tissue formation

doi: 10.1038/s41385-020-0290-x

Figure Lengend Snippet: ( A ) Frequency of B cells, T cells, and T cell subsets among live CD45 + cells in young and aged bladders by flow cytometry. n=5–8 per group. ( B ) Representative H&E images of young and aged bladders. ( C ) Number of bTLT over the life course of mice n=5–10/group. ( C ) Representative image of B cells (B220 + , green) and T cells (CD3 + , red) in segregated zones within bTLT in aged mice. ( D ) Representative image of CD31 + (red) PNAd + (green) high endothelial venules (white arrowheads) within bTLT in aged mice. ( E ) Representative image of CD35 hi follicular dendritic cell network within bTLT in aged mice. All nuclei stained with DAPI (blue). All scale bars, 50 μm.

Article Snippet: Primary antibodies against B220 (13–0452, eBioscience), CD3 (14–0031-82, eBioscience) PNAd (120803, BioLegend), CD31 (ab28364, Abcam), CD138 (142511, BioLegend), CD35 (558768, BD Biosciences), GL7 (13–5902-81, eBioscience), IgA (1040–31, Southern Biotech), and IgD (1120–01, Southern Biotech) were incubated overnight at 4° and detected with streptavidin-conjugated and species-specific secondary antibodies followed by Hoechst dye.

Techniques: Flow Cytometry, Staining

Journal: Cancer cell

Article Title: RHOA G17V induces T follicular helper cell specification and promotes lymphomagenesis

doi: 10.1016/j.ccell.2018.01.001

Figure Lengend Snippet: (A) Analysis of CXCR5+ CD4+ T cells in peripheral blood of mice transplanted with bone marrow cells from Rhoaco-G17V/+;Tet2f/f;CD4CreERT2 mice and treated with vehicle (n=10) or tamoxifen (TMX) (n=10). (B) Kaplan-Meier survival curve of animals transplanted with bone marrow progenitor cells from Rhoaco-G17V/+;Tet2f/f;CD4CreERT2 mice treated with vehicle only or tamoxifen (n=10 mice per group). Tamoxifen administration and serial sheep red blood cells (SRBC) immunizations are indicated by arrows in the timeline (red: tamoxifen; black: SRBC). (C) Flow cytometry analysis of Tfh cell markers in spleen and bone marrow cells from diseased tamoxifen-treated mice: CXCR5, PD1 FACS plot (upper panel), BCL6 and ICOS histograms (lower panel). (D) Pie chart representation of the results of Tcr Vβ clonality analysis by flow cytometry on spleen samples from diseased tamoxifen-treated animals. Three representative tumors are depicted. (E) Histological micrographs of representative lymph node and spleen tissues obtained from diseased tamoxifen-treated mice (F) Immunohistochemical analysis of the expression of CD4, PD1, PAX5, B220, CD21 and CD31 in spleen sections from tamoxifen-treated tumor affected mice. p values were calculated with two-tailed Student’s t-test. ***p ≤ 0.001.

Article Snippet: The antibody against CD45R (B220) (RA3-6B2) is from Miltenyi and the anti-IFNG (XMG1.2) was purchased from BD Biosciences.

Techniques: Flow Cytometry, Immunohistochemical staining, Expressing, Two Tailed Test

Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/jir.s347777

Figure Lengend Snippet: Figure 1 CD66b+ neutrophils were predominantly located within the peritumoral stroma region in LSCC patients, which were associated with poorer prognosis. (A) Representative images were shown for CD45 and CD66b IHC staining of tumor tissues and normal tissues from LSCC patients. (B) Representative LSCC tumor tissues exhibited various density of CD66b+ neutrophils in peritumoral stroma and intratumoral regions. Bars = 50μm. (C) Infiltrating CD66b+ neutrophils in intratumoral and peritumoral region of tumor tissue, and epithelium and sub-epithelium region of normal tissues were analyzed. (D and E) LSCC patients with advanced T stage had more intratumoral and peritumoral infiltrated CD66b+ neutrophils. (F–I) Kaplan–Meier curves for overall survival and disease-free survival of 45 LSCC patients according to different density of CD66b+ neutrophils infiltrated in peritumoral stroma and intratumoral regions of tumor tissues. 50X, 200X, 400X: magnification of 50, 200, and 400 fold; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Immunohistochemistry (IHC) Staining of Tissue Microarray Formalin-fixed and paraffin-embedded tumor tissues and matched normal mucosa tissues from the first LSCC patient cohort mentioned above were prepared for tissue microarray (TMA) construction as previously described.14 The TMA slides were incubated overnight at 4°C in wet box, with primary mouse anti-human CD45 antibody (60,287, Clone 4E9B2, Proteintech, Rosemont, USA), mouse anti-human CD66b antibody (392,902, Clone 6/40c, Biolegend, San Diego, USA) or rabbit polyclonal anti-human GM-CSF antibody (ab9741, Abcam, Cambridge, USA), followed with the incubation of biotin-conjugated secondary goat anti-mouse/rabbit IgG antibody at room temperature for one hour.

Techniques: Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/jir.s347777

Figure Lengend Snippet: Figure 2 Immune cell infiltration profiles in LSCC. (A) Representative polychromatic dot plots displayed CD45+ leukocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells γδT cells (γδTCR+) and neutrophils (CD66b+) in peripheral blood, tumor tissues and normal tissues from LSCC patients or healthy controls. (B) Calculative distribution of lymphocytes, neutrophils, NLR, CD3+T cells, CD4+T cells, CD8+T cells, γδT cells, and CD4+T cells /CD8 + T cells ratio in peripheral blood, tumor tissues and normal tissues. (C) Representative PD-1 expression on CD4+T/CD8+T/γδT cells and PDL-1 expression on CD66b+ neutrophils from the peripheral blood, tumor tissues and normal tissues. (D) Statistics analysis of PD-1+ CD4+T, PD-1+ CD8+T, PD-1+ γδT cells and PDL-1+ CD66b+ neutrophils. (E) Association analysis of TANs, NLR, CD3+ T cells or CD8+ T cells and tumor staging. (F) Linear correlation between TANs and CD3+ T cells, CD4+T cells, CD8+ T cells, or γδT cells. (G) Linear correlation between PD-L1+ TANs and PD-1+ CD4+ T cells, PD-1+ CD8+ T cells, or PD-1+ γδT cells. Each dot represents an independent data point as determined by flow cytometry. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Immunohistochemistry (IHC) Staining of Tissue Microarray Formalin-fixed and paraffin-embedded tumor tissues and matched normal mucosa tissues from the first LSCC patient cohort mentioned above were prepared for tissue microarray (TMA) construction as previously described.14 The TMA slides were incubated overnight at 4°C in wet box, with primary mouse anti-human CD45 antibody (60,287, Clone 4E9B2, Proteintech, Rosemont, USA), mouse anti-human CD66b antibody (392,902, Clone 6/40c, Biolegend, San Diego, USA) or rabbit polyclonal anti-human GM-CSF antibody (ab9741, Abcam, Cambridge, USA), followed with the incubation of biotin-conjugated secondary goat anti-mouse/rabbit IgG antibody at room temperature for one hour.

Techniques: Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag -dependent immunodeficiency

doi: 10.1084/jem.20091927

Figure Lengend Snippet: Rag1 S723C mut/mut mice have a severe but incomplete block in B cell development. (A) Distribution of B lymphocyte subsets in the bone marrow. Left: representative FACS analysis of bone marrow cells from indicated 8-wk-old mice labeled with B220 and IgM antibodies, and histograms of CD43 expression gated on B220 + IgM − subset. Right: Percentage (mean and SD) of indicated bone marrow B cell subsets in Rag1 S723C ( mut/mut ) and wild type (+/+) mice. Imm, immature B cells; Mat, mature recirculating B cells. P-values (**, P < 0.01; ****, P < 0.001) were determined by the Mann-Whitney U test. Seven mice per group were analyzed in three experiments. (B) Distribution of B lymphocyte subsets in the spleen. Top: strategy for gating on transitional (T1-3), MZ precursor (MZP), Fo type I and II (Fo1-2), MZ B cells, CD93 hi B220 int CD138 − pPB, and CD93 hi B220 int CD138 + PC subpopulations of splenic B cells after excluding DAPI + , Dump + (CD11c, CD11b, and Gr-1), and B220 − cells. One representative example is shown for both wild-type (+/+) and mut/mut mice out of three wild-type (+/+) and seven mut/mut 8-wk-old mice that were analyzed in two experiments. Bottom: distribution (percentage of B220 + splenocytes) and absolute numbers (mean ± SD) of splenic B cell subsets, defined as in A, in 8-wk-old wild-type (+/+, n = 3) and mut/mut ( n = 7) mice. sIgG1/sIgG2a, lymphocytes expressing surface IgG1 or surface IgG2a. P-values (**, P < 0.01; ****, P < 0.001) were determined by the Mann-Whitney U test.

Article Snippet: The following primary antibodies were used: rat anti-B220 (clone RA3-6B2, 1:100; Invitrogen), rabbit anti-CD3 (1:100; Dako), and rabbit anti–Iba-1 (1:500; Wako Chemicals USA, Inc.).

Techniques: Blocking Assay, Labeling, Expressing, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag -dependent immunodeficiency

doi: 10.1084/jem.20091927

Figure Lengend Snippet: Impaired receptor editing and increased serum BAFF levels in mut/mut mice. (A) Levels of Vκ-RS DNA rearrangement products were measured by quantitative PCR in sorted bone marrow B220 + CD43 − AA4.1 + IgM − (Fraction D) cells from wild-type (+/+, n = 4) and mut/mut ( n = 5) mice. Results were expressed as relative levels of Vκ-RS DNA rearrangement products as compared with a reference value of 1 attributed to DNA from sorted Fraction D bone marrow cells from one selected wild-type (+/+) mouse. Genomic DNA extracted from the tail of wild-type mice was run in parallel. For each of two experiments performed, quantitative PCR was run in triplicates. Horizontal bars identify mean values. P-values (****, P < 0.001) were determined by a two-tailed Student’s t test for unpaired data. (B) Serum BAFF levels in 8-wk-old, 16-wk-old, and 9–10-mo-old wild-type (+/+, n = 6–8) and mut/mut ( n = 8–20) mice. Horizontal bars represent mean values. For each serum sample, three replicates were run and three experiments were performed. P-values (*, P < 0.05; **, P < 0.01; ****, P < 0.001) were determined by a two-tailed Student’s t test for unpaired data.

Article Snippet: The following primary antibodies were used: rat anti-B220 (clone RA3-6B2, 1:100; Invitrogen), rabbit anti-CD3 (1:100; Dako), and rabbit anti–Iba-1 (1:500; Wako Chemicals USA, Inc.).

Techniques: Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: PLoS ONE

Article Title: Diverse Hematological Malignancies Including Hodgkin-Like Lymphomas Develop in Chimeric MHC Class II Transgenic Mice

doi: 10.1371/journal.pone.0008539

Figure Lengend Snippet: (A–D). Histology and immunohistochemistry of LTCL (all bars equal 50 µm). (A) LTCL consists of uniform cells with scant cytoplasm showing high mitotic and apoptotic rates and the typical starry sky pattern (hematoxylin & eosin, H&E). (B) CD3+ infiltrates in the liver. (C). Tumor cells are positively stained for Tdt (terminal deoxynucleotidyl transferase) and (D) CD3. (E–F) Flow cytometry analysis of T cell receptor Vβ-repertoire on tumor-bearing spleen samples. (E) Monoclonal single positive (SP) CD4+ LTCL. Dot plots - top: gated on CD3+B220− cells: CD4 versus CD8, middle: gated on SP CD4+ T cells showing the Vβ8.2+ tumor cell population and an irrelevant Vβ-chain (Vβ6); bottom: gated on DP CD4/Vβ8.2 as shown in histogram all other Vβ-families were negative. (F) Monoclonal precursor LTCL gated on CD3+ B220− cells: top: CD4 versus CD8, middle: monoclonal DP CD4+CD8+ tumor cells expressing two Vβ-chains (Vβ7&Vβ10), bottom: gated on DP Vβ7/Vβ10 tumor cells, as shown in histogram all other Vβ-families were not expressed. (G) Lymphocytes were stained as for FACS-analyses; top: Vβ7-PE in red, middle: Vβ10-FITC in green, bottom: merged image showing co-localization of Vβ7-PE and Vβ10-FITC as dual T cell receptors on splenic tumor cells with deconvolution microscopy. All bars equal 5 µm.

Article Snippet: Automated immunohistochemical staining (Ventana Medical Systems) was performed as published using the following primary antibodies: CD45R/B220 (BD), CD3, CD79a, Tdt (Dako), CD30 (Chemicon), CD49b (eBioscience), MPO (NeoMarkers).

Techniques: Immunohistochemistry, Staining, Flow Cytometry, Expressing, Microscopy

Journal: PLoS ONE

Article Title: Diverse Hematological Malignancies Including Hodgkin-Like Lymphomas Develop in Chimeric MHC Class II Transgenic Mice

doi: 10.1371/journal.pone.0008539

Figure Lengend Snippet: (A–K) Histology (H&E) and immunohistochemistry (all bars equal 50 µm). (A) Lymphoblastic B cell lymphoma (LBCL) shows blastic nuclei with prominent nucleoli and numerous mitotic figures. (B) Staining for CD79a+. (C) Diffuse large B cell lymphoma (DLBCL) consists of immunoblastic and centroblastic cells and is (D) CD79a+. (E) The histiocyte-associated variant (DLBCL-HA) shows participation of many histiocytes and granulocytes. (F) B220+ DLBCL-HA. (G–I) The splenic marginal zone lymphoma (SMZL) is usually (H) CD79a+ but (I) B220−. (J) H&E of follicular B cell lymphoma. (K–L) Well-differentiated plasmacytomas display (L) CD79+ plasma cells. (M–O) The cellular phenotype of the B natural killer cell lymphoma (BNKL) is confirmed by (N) positively stained CD79a B cells and (O) DP B220/NK1.1 cells in FACS.

Article Snippet: Automated immunohistochemical staining (Ventana Medical Systems) was performed as published using the following primary antibodies: CD45R/B220 (BD), CD3, CD79a, Tdt (Dako), CD30 (Chemicon), CD49b (eBioscience), MPO (NeoMarkers).

Techniques: Immunohistochemistry, Staining, Variant Assay

Journal: PLoS ONE

Article Title: Diverse Hematological Malignancies Including Hodgkin-Like Lymphomas Develop in Chimeric MHC Class II Transgenic Mice

doi: 10.1371/journal.pone.0008539

Figure Lengend Snippet: (A-P+R-T) Histology and immunohistochemistry of Hodgkin-like lymphoma samples in composite compartments, surrounded by activated B and T cells as well as histiocytes. (A–H). H&E staining of (A–D) lymph node and (E–H) spleen sections containing Hodgkin/Reed-Sternberg (H/RS)-like cells (in white boxes). Magnification of characteristic (D+H) giant mononucleated Hodgkin-like and (B+F) multinucleated RS-like cells. (K+L & O+P) CD30+ Hodgkin-like and (I+J & M+N) RS-like cells in (I–L) lymph node and (M–P) spleen sections. (Q) CDR3-region of IgH-transcripts of microdissected H/RS-like cells from five different mice. (R–T) Immunohistochemistry of either B220 positive or negative (R) Hodgkin-like and (S) RS-like cells surrounded by (T) rosettes of CD3+ T cells. All bars equal 50 µm.

Article Snippet: Automated immunohistochemical staining (Ventana Medical Systems) was performed as published using the following primary antibodies: CD45R/B220 (BD), CD3, CD79a, Tdt (Dako), CD30 (Chemicon), CD49b (eBioscience), MPO (NeoMarkers).

Techniques: Immunohistochemistry, Staining