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: Frontiers in Immunology

Article Title: Double negative T cells (CD4 - /CD8 - ) are associated with Trypanosoma cruzi persistence in the mouse colon during chronic Chagas disease

doi: 10.3389/fimmu.2026.1761769

Figure Lengend Snippet: Multiparameter spectral flow cytometry analysis of immune cells of the colonic lamina propria during acute and chronic T. cruzi infection. C57BL/6 mice were infected with 10 4 T. cruzi (TcCol-Nluc). Colonic lamina propria cells were isolated from uninfected (Control), acutely infected (Acute, 30 dpi) and chronically infected (Chronic, 90 dpi) mice and analysed by flow cytometry using the gating strategy shown in Supplementary Figure 1 . (A) Automated T-distributed stochastic neighbour embedding (t-SNE) 2D map of the flow cytometry data acquired from control and infected mice colon. (B) Upper panel; tSNE 2D map showing scaled expression of CD11b for myeloid cells, B220 for B cells and CD3 for T cells. Lower panel; tSNE 2D map showing the location of CD11b + myeloid cells, B220 + B cells and CD3 + T cells. (C) Heat maps for (left) cell surface markers expression (CD45, CD11b, B220, CD3, CD4 and CD8) and (right) groups (control, acute and chronic colons) for 22 cell clusters identified. (D) Percentage of each cluster for each group. Panel (D) shows auto-scaled cluster frequencies optimized for visualization within each group. For standardized quantitative comparison across groups, refer to the heat map in panel (C) . Arrows in (C) and (D) indicate immune cell clusters of myeloid cells (blue), B cells (green) or T cells (red).

Article Snippet: Antibodies were polyclonal goat IgG anti-mouse CD4 antibody (R&D Systems, MN), polyclonal rabbit IgG anti-mouse CD8 antibody (Novus Biologicals, CO), monoclonal Rat IgG2b Alexa Fluor ® 647 anti-mouse CD3 Antibody (BioLegend, CA), Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 (Thermo Fisher Scientific, CA), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 568 (Thermo Fisher Scientific, CA) ( ).

Techniques: Flow Cytometry, Infection, Isolation, Control, Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: Double negative T cells (CD4 - /CD8 - ) are associated with Trypanosoma cruzi persistence in the mouse colon during chronic Chagas disease

doi: 10.3389/fimmu.2026.1761769

Figure Lengend Snippet: Phenotypic analysis of T cells via multiparameter spectral flow cytometry in the colonic lamina propria during acute and chronic T. cruzi infection. C57BL/6 mice were infected with 10 4 T. cruzi (TcCol-Nluc). Colonic lamina propria cells were isolated from uninfected (Control), acutely infected (Acute, 30 dpi) and chronically infected (Chronic, 90 dpi) mice and analysed by flow cytometry using the gating strategy shown in Supplementary Figure 1 . (A) T cells composition by flow cytometry, (B) tSNE 2D map showing scaled expression of CD3, CD4, and CD8 cell makers (C) Heat map of T cells (CD45 + , CD3 + ) (highlighted clusters 1, 7, 9, 16, 20, 22). Arrows indicate immune cell clusters of inflammatory (purple) and regulatory (pink) double-negative (DN) T cells. (D) tSNE 2D map of DN T cells and percentage of T cells cluster for each group. Arrows indicate DN T cell clusters of inflammatory (purple) and regulatory (pink) cells. (E) Percentage of DN T cells with inflammatory/regulatory phenotypes in total immune cells (CD45 + cells). (F) Relative percentage of DN T cells with inflammatory/regulatory phenotypes in total DN T cells. Panel (D) shows auto-scaled cluster frequencies optimized for visualization within each group. For standardized quantitative comparison across groups, refer to the heat map in panel (C) . Bars in A represent mean ± SD, and individual symbols denote values from single mice (n = 15; 5 per set, 3 individual sets). Statistical comparisons were made by an unpaired t test: *p < 0.05, ****p < 0.0001.

Article Snippet: Antibodies were polyclonal goat IgG anti-mouse CD4 antibody (R&D Systems, MN), polyclonal rabbit IgG anti-mouse CD8 antibody (Novus Biologicals, CO), monoclonal Rat IgG2b Alexa Fluor ® 647 anti-mouse CD3 Antibody (BioLegend, CA), Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 (Thermo Fisher Scientific, CA), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 568 (Thermo Fisher Scientific, CA) ( ).

Techniques: Flow Cytometry, Infection, Isolation, Control, Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: Double negative T cells (CD4 - /CD8 - ) are associated with Trypanosoma cruzi persistence in the mouse colon during chronic Chagas disease

doi: 10.3389/fimmu.2026.1761769

Figure Lengend Snippet: Double-negative T cells phenotypes in the colonic lamina propria of C57BL/6 mice during T. cruzi infection. C57BL/6 mice were infected with 10 4 T. cruzi (TcCol-Nluc). Mice were euthanized during the acute (30 dpi) and chronic (90 dpi) phases. Colonic lamina propria cells were isolated from uninfected (Control; blue), acutely infected (Acute; pink) and chronically infected (Chronic; green) mice. Cells were gated on single cells, live, CD45 + , CD3 + , CD4 - , CD8 - events and subsequently on (A) inflammatory immune cell markers including CCR5, CXCR3 or Granzyme B, and (B) regulatory immune cells markers including CCR4, IL10Rα or IL10. Bars represent mean ± SD, and individual symbols denote values from single mice (n = 8-12; 4 per set, 2–3 individual sets). Statistical comparisons were made by an unpaired t test: *p < 0.05, **p < 0.01, ***p < 0.001, **** p ≤ 0.0001.

Article Snippet: Antibodies were polyclonal goat IgG anti-mouse CD4 antibody (R&D Systems, MN), polyclonal rabbit IgG anti-mouse CD8 antibody (Novus Biologicals, CO), monoclonal Rat IgG2b Alexa Fluor ® 647 anti-mouse CD3 Antibody (BioLegend, CA), Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 (Thermo Fisher Scientific, CA), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 568 (Thermo Fisher Scientific, CA) ( ).

Techniques: Infection, Isolation, Control

Journal: Frontiers in Immunology

Article Title: Double negative T cells (CD4 - /CD8 - ) are associated with Trypanosoma cruzi persistence in the mouse colon during chronic Chagas disease

doi: 10.3389/fimmu.2026.1761769

Figure Lengend Snippet: Microscopy analysis of T. cruzi infected colons. C57BL/6 mice were infected with 10 4 T. cruzi (TcCol-Nluc-RFP). Colons were isolated from control and chronically infected mice (90 dpi), processed for microscopy and stained with CD3 (Blue), CD4 (Green) and CD8 (Red) specific antibodies ( Supplementary Table 1 , Antibody Panel 4) as described in the materials and methods. Immunofluorescence images of cross sections of colons obtained from non-infected mice (Control) and chronically infected mice (Chronic) are shown in panels (A, B) , respectively. In (A, B) , a low magnification image of the entire colon is shown in the upper left, a region of interest (ROI) 1 is shown at mid-magnification in the lower left, and a high magnification image of ROI 2 is shown in shown on the right. For ROI 2, an overlay image of the 3 channels CD3 (Blue), CD4 (Green) and CD8 (Red) is shown on the top right, and the individual channels are represented below as indicated. A representative result of 3 controls and 3 infected colons is shown in this figure. LP, Lamina Propria; solid white arrow heads, double-negative T cells (CD3 + , CD4 - , CD8 - ); open arrowheads, CD3 - , CD4 - , CD8 + cells; solid pink arrowheads, CD3 - , CD4 + , CD8 - cells; solid yellow arrowheads, CD3 - , CD4 + , CD8 + cells.

Article Snippet: Antibodies were polyclonal goat IgG anti-mouse CD4 antibody (R&D Systems, MN), polyclonal rabbit IgG anti-mouse CD8 antibody (Novus Biologicals, CO), monoclonal Rat IgG2b Alexa Fluor ® 647 anti-mouse CD3 Antibody (BioLegend, CA), Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 (Thermo Fisher Scientific, CA), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 568 (Thermo Fisher Scientific, CA) ( ).

Techniques: Microscopy, Infection, Isolation, Control, Staining, Immunofluorescence

Journal: The Tohoku Journal of Experimental Medicine

Article Title: Local Delivery of Nimustine Hydrochloride against Brain Tumors: Basic Characterization Study

doi: 10.1620/tjem.2023.j069

Figure Lengend Snippet: Fig. 4. Immune stimulation by local delivery of ACNU in a rodent 9L tumor model. (A) Immunofluorescence of rat CD4/CD8-positive T cells (green) in 9L tumors was performed using brains harvested 7 days after treatment. Nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. (B) and (C) The number of cells positively stained for CD4 and CD8 was counted under × 400 magnification. Bars indicate mean ± SD. **p < 0.01.

Article Snippet: Sections were incubated with a rabbit anti-rat CD4 IgG polyclonal antibody (1:200 dilution; NBP1-19371; NOVUS, Littleton, CO, USA) and mouse anti-rat CD8α IgG1 monoclonal antibody (1:500 dilution; MCA48R; Bio-Rad) overnight at 4°C and then washed three times with PBS, followed by incubation with the secondary antibodies, a goat anti-rabbit IgG Alexa Fluor 488 (1:1,000 dilution; A-11034; Thermo Fisher Scientific) and a goat anti-mouse IgG Alexa Fluor 488 (1:1,000 dilution; A-11029; Thermo Fisher Scientific).

Techniques: Immunofluorescence, Staining