Journal: Frontiers in Immunology

Article Title: Lymphoid Aggregates in the CNS of Progressive Multiple Sclerosis Patients Lack Regulatory T Cells

doi: 10.3389/fimmu.2019.03090

Figure Lengend Snippet: Ectopic lymphoid structures in progressive MS are characterized by infiltration of lymphocytes, FDCs and plasma cells. (A) Parenchyma of FFPE sections of brain and spinal cord of progressive MS patients were screened for infiltrated regions by H&E staining. (B) IF staining for CD3 + T cells and CD20 + B cells on serial sections were used to determine the infiltration score. Score 0, no or <5 lymphocytes; score 1, at least five but <30 lymphocytes; score 2, 31 to 60 lymphocytes; score 3, more than 60 lymphocytes. (C) Meninges and sulci of FFPE sections of brain and spinal cord of progressive MS patients were screened for infiltrated regions by H&E staining. (D) IF staining for CD3 + T cells and CD20 + B cells on serial sections were used to determine the infiltration score. Score 0, no or <5 lymphocytes; score 1, at least five, but <30 lymphocytes; score 2, 31 to 60 lymphocytes; score 3, more than 60 lymphocytes. (E) Whole slides were screened for infiltration on H&E, representative infiltration area depicted in the box (F) , and serial sections were stained, depicted in the box (G–O) . eLFs are characterized by (G) CD3 + T cells and CD20 + B cells, (H) CD4 + T cells and CD138 + plasma cells, (I) CD3 + T and CD3 + CD4 + T helper cells (J) Ki67 + proliferating cells, (K) CD35 + and, (L) CD21 + FDCs, (M) CD68 + macrophages as well as (N) BCL-6 + and (O) CXCR5 + GC-like lymphocytes. (P) CD3 + CD8 + cytotoxic T cells as well as some CD3 + CD27 + memory T cells were also present in eLFs. Scale bars (A–D) , (F) indicate 100 μm; (E) indicates 2,000 μm; (G–P) indicate 50 μm.

Article Snippet: Sections were incubated with the primary antibodies CD20 (1:200, Dako, #M0755), CD3 (1:100, Dako, #A0452), CD3 (1:50, abcam, #11089), CD4 (1:200, R&D Systems #AF-379-NA), CD8 (1:100, Dako #M7103), CD27 (1:50, Sigma-Aldrich, #HPA038936), CD138 (1:200, BioLegend, # 356502), CD69 (1:100, ThermoFisher, #PA5-84010), CXCR5 (1:200, abcam, #ab225575), FOXP3 (1:100, ThermoFisher, #14-4776-82), NFATc1 (1:100, BD Pharmigen, #556602), and/or PD-1 (1:100, abcam, #ab52587) in Antibody Diluent for 1 h. Secondary antibodies (1:400, all from ThermoFisher)—donkey-anti-goat Alexa Fluor 546 (#A-11056), donkey-anti-mouse Alexa Fluor 647 (#A-31571), donkey-anti-rabbit Alexa Fluor 488 (#A-21206), donkey-anti-rabbit Alexa Fluor 555 (#A-31572), donkey-anti-rat Alexa Fluor 488 (#A-21208), donkey-anti-rat DyLight 550 (#SA5-10027)—were applied in PBS containing 0.05% Tween20 and Hoechst (1:5.000, Sigma, #B2261) for 1 h at RT.

Techniques: Clinical Proteomics, Staining

Journal: Frontiers in Immunology

Article Title: Lymphoid Aggregates in the CNS of Progressive Multiple Sclerosis Patients Lack Regulatory T Cells

doi: 10.3389/fimmu.2019.03090

Figure Lengend Snippet: Follicle-like structures of SPMS brains exhibit CD3 + CD4 + T cells, which neither express PD-1 nor FOXP3. (A–E) IF staining of CD3, CD4 and PD-1 reveal CD3 + CD4 + PD-1 − T-helper cells in progressive MS. Inserts in the upper right corners show magnification of the white box. (F–I) IF staining of CD3 and FOXP3 on serial sections of a representative meningeal follicle-like structure in SPMS (same region as ). CD3 + T cells, but no FOXP3 + cells were detected. Inserts show magnification of the white box. Scale bars indicate 100 μm, inserts 10 μm.

Article Snippet: Sections were incubated with the primary antibodies CD20 (1:200, Dako, #M0755), CD3 (1:100, Dako, #A0452), CD3 (1:50, abcam, #11089), CD4 (1:200, R&D Systems #AF-379-NA), CD8 (1:100, Dako #M7103), CD27 (1:50, Sigma-Aldrich, #HPA038936), CD138 (1:200, BioLegend, # 356502), CD69 (1:100, ThermoFisher, #PA5-84010), CXCR5 (1:200, abcam, #ab225575), FOXP3 (1:100, ThermoFisher, #14-4776-82), NFATc1 (1:100, BD Pharmigen, #556602), and/or PD-1 (1:100, abcam, #ab52587) in Antibody Diluent for 1 h. Secondary antibodies (1:400, all from ThermoFisher)—donkey-anti-goat Alexa Fluor 546 (#A-11056), donkey-anti-mouse Alexa Fluor 647 (#A-31571), donkey-anti-rabbit Alexa Fluor 488 (#A-21206), donkey-anti-rabbit Alexa Fluor 555 (#A-31572), donkey-anti-rat Alexa Fluor 488 (#A-21208), donkey-anti-rat DyLight 550 (#SA5-10027)—were applied in PBS containing 0.05% Tween20 and Hoechst (1:5.000, Sigma, #B2261) for 1 h at RT.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Lymphoid Aggregates in the CNS of Progressive Multiple Sclerosis Patients Lack Regulatory T Cells

doi: 10.3389/fimmu.2019.03090

Figure Lengend Snippet: CD4 + CXCR5 + T FH s mark positive for cytoplasmic NFATc1. (A–E) Consecutive IF staining of CD4, CXCR5 and NFATc1 on serial sections of follicle-like structures in SPMS (same region as , ). Inserts show magnification of the white box. (F) NFATc1 appears to be cytoplasmic in MS brains, compared to nuclear localization within tonsillar GCs (left insert) and cytoplasmic predominance in inter-follicular cells (right insert). Scale bars indicate 100 μm, inserts 10 μm.

Article Snippet: Sections were incubated with the primary antibodies CD20 (1:200, Dako, #M0755), CD3 (1:100, Dako, #A0452), CD3 (1:50, abcam, #11089), CD4 (1:200, R&D Systems #AF-379-NA), CD8 (1:100, Dako #M7103), CD27 (1:50, Sigma-Aldrich, #HPA038936), CD138 (1:200, BioLegend, # 356502), CD69 (1:100, ThermoFisher, #PA5-84010), CXCR5 (1:200, abcam, #ab225575), FOXP3 (1:100, ThermoFisher, #14-4776-82), NFATc1 (1:100, BD Pharmigen, #556602), and/or PD-1 (1:100, abcam, #ab52587) in Antibody Diluent for 1 h. Secondary antibodies (1:400, all from ThermoFisher)—donkey-anti-goat Alexa Fluor 546 (#A-11056), donkey-anti-mouse Alexa Fluor 647 (#A-31571), donkey-anti-rabbit Alexa Fluor 488 (#A-21206), donkey-anti-rabbit Alexa Fluor 555 (#A-31572), donkey-anti-rat Alexa Fluor 488 (#A-21208), donkey-anti-rat DyLight 550 (#SA5-10027)—were applied in PBS containing 0.05% Tween20 and Hoechst (1:5.000, Sigma, #B2261) for 1 h at RT.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Lymphoid Aggregates in the CNS of Progressive Multiple Sclerosis Patients Lack Regulatory T Cells

doi: 10.3389/fimmu.2019.03090

Figure Lengend Snippet: B cells enrich in lymphoid aggregates. (A) Absolute number of infiltrates that were positive for T FH in follicle-like structures (F+) and less defined infiltrates (F-). Fisher's exact test, N = 76, X 2 (1) = 4.55, p = 0.048, d = 0.505. (B) Mean percentage of T FH cells defined as CD4 + CXCR5 + cells of CD4 + cells in two serial FFPE sections of follicle-like structures (F+) and less defined infiltrates (F-) in SPMS brains and spinal cords. F-, M = 15.57, SD = 17.13, n = 39; F+, M = 17.04, SD = 15.85, n = 37. Mann Whitney test, U = 635.0, p = 0.369. (C) CD20/CD3 ratio in follicle-like structures (F+) and less defined infiltrates (F-) based on IF co-staining of CD3 and CD20. F-, M = 0.28, SD = 0.33, n = 39; F+, M = 0.38, SD = 0.34, n = 37; Mann Whitney test, U = 525.5, p = 0.042. * p < 0.05.

Article Snippet: Sections were incubated with the primary antibodies CD20 (1:200, Dako, #M0755), CD3 (1:100, Dako, #A0452), CD3 (1:50, abcam, #11089), CD4 (1:200, R&D Systems #AF-379-NA), CD8 (1:100, Dako #M7103), CD27 (1:50, Sigma-Aldrich, #HPA038936), CD138 (1:200, BioLegend, # 356502), CD69 (1:100, ThermoFisher, #PA5-84010), CXCR5 (1:200, abcam, #ab225575), FOXP3 (1:100, ThermoFisher, #14-4776-82), NFATc1 (1:100, BD Pharmigen, #556602), and/or PD-1 (1:100, abcam, #ab52587) in Antibody Diluent for 1 h. Secondary antibodies (1:400, all from ThermoFisher)—donkey-anti-goat Alexa Fluor 546 (#A-11056), donkey-anti-mouse Alexa Fluor 647 (#A-31571), donkey-anti-rabbit Alexa Fluor 488 (#A-21206), donkey-anti-rabbit Alexa Fluor 555 (#A-31572), donkey-anti-rat Alexa Fluor 488 (#A-21208), donkey-anti-rat DyLight 550 (#SA5-10027)—were applied in PBS containing 0.05% Tween20 and Hoechst (1:5.000, Sigma, #B2261) for 1 h at RT.

Techniques: MANN-WHITNEY, Staining

Journal: Frontiers in Immunology

Article Title: Lymphoid Aggregates in the CNS of Progressive Multiple Sclerosis Patients Lack Regulatory T Cells

doi: 10.3389/fimmu.2019.03090

Figure Lengend Snippet: eLFs of brain and spinal cord exhibit more CD4 + CD69 + cells. (A–D) Consecutive IF co-staining of CD4 and CD69 in follicle-like structures of SPMS brains and spinal cords. Inserts show co-localization of CD4 + cells with CD69 suggesting tissue-resident T cells in a representative meningeal eLF of SPMS spinal cord (same region as , , ). Scale bar indicate 100 μm, scale bars of the inserts indicate 10 μm. (E) Percentage of tissue-resident cells defined as CD4 + CD69 + cells of CD4 + cells in follicle-like structures (F+) and less defined infiltrates (F-) in SPMS brains and spinal cords. F-, 5.70, SD = 10.67, n = 38; F+, M = 7.92, SD = 9.39, n = 32; Mann Whitney test, U = 434.0, p = 0.028.

Article Snippet: Sections were incubated with the primary antibodies CD20 (1:200, Dako, #M0755), CD3 (1:100, Dako, #A0452), CD3 (1:50, abcam, #11089), CD4 (1:200, R&D Systems #AF-379-NA), CD8 (1:100, Dako #M7103), CD27 (1:50, Sigma-Aldrich, #HPA038936), CD138 (1:200, BioLegend, # 356502), CD69 (1:100, ThermoFisher, #PA5-84010), CXCR5 (1:200, abcam, #ab225575), FOXP3 (1:100, ThermoFisher, #14-4776-82), NFATc1 (1:100, BD Pharmigen, #556602), and/or PD-1 (1:100, abcam, #ab52587) in Antibody Diluent for 1 h. Secondary antibodies (1:400, all from ThermoFisher)—donkey-anti-goat Alexa Fluor 546 (#A-11056), donkey-anti-mouse Alexa Fluor 647 (#A-31571), donkey-anti-rabbit Alexa Fluor 488 (#A-21206), donkey-anti-rabbit Alexa Fluor 555 (#A-31572), donkey-anti-rat Alexa Fluor 488 (#A-21208), donkey-anti-rat DyLight 550 (#SA5-10027)—were applied in PBS containing 0.05% Tween20 and Hoechst (1:5.000, Sigma, #B2261) for 1 h at RT.

Techniques: Staining, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

doi: 10.1371/journal.pone.0183398

Figure Lengend Snippet: Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Slides were stained with the following specific antibodies: anti-MHC-II, anti-CD163, anti-CD172a, anti-CD3, and anti-CD4 to detect and quantify positive cells in tissue sections or with toluidine blue to quantify mast cells (magnification x200). Representative photomicrographs of mucosal tissue sections are shown.

Article Snippet: The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam).

Techniques: Staining

Journal: PLoS ONE

Article Title: Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

doi: 10.1371/journal.pone.0183398

Figure Lengend Snippet: Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Cell counting was performed on slides labeled with Abs specific for APC (anti-MHC-II, anti-CD163, anti-CD172a) and T cell (anti-CD3 and anti-CD4) markers or stained with toluidine blue for mast cells to evaluate the mean number of positive cells per field using a light microscope (magnification x400). All areas (epithelium (Epith.), Lamina propria (LP) and muscle) were scored. Histograms represent the mean + SEM with n = 3.

Article Snippet: The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam).

Techniques: Cell Counting, Labeling, Staining, Light Microscopy

Journal: Vaccines

Article Title: Immunogenicity and Protection against Mycobacterium caprae Challenge in Goats Vaccinated with BCG and Revaccinated after One Year

doi: 10.3390/vaccines8040751

Figure Lengend Snippet: Gating strategy for the determination of frequency of the IFN-γ positive lymphocyte subsets. Peripheral blood mononuclear cells (PBMC) from all goats were cultured in the presence of M. bovis tuberculin (PPD-B). ( A , B ) Singlet lymphocytes were identified based on the degree of cellular differentiation determined by forward scatter (FSC) and side scatter (SCC). ( C ) Representative frequencies of the CD4/CD45RO cell populations. ( D ) Representative frequencies of intracellular IFN-γ staining of cells gated from prelabelled CD4 + CD45RO + .

Article Snippet: Cells were stained with mouse monoclonal antibodies (mAb) CC8 (IgG2A, conjugated to FITC), which recognizes bovine CD4, and mAb IL-A116 (IgG3, conjugated to RPE), which recognizes bovine CD45RO (both from Bio-Rad Laboratories Inc., Hercules, CA, USA).

Techniques: Cell Culture, Cell Differentiation, Staining