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: International journal of molecular sciences

Article Title: ULBP2 Promotes Tumor Progression by Suppressing NKG2D-Mediated Anti-Tumor Immunity.

doi: 10.3390/ijms26072950

Figure Lengend Snippet: Figure 4. ULBP2 inhibits anti-tumor immunity mediated by NK cells. (A) Tumor growth in C57BL/6 mice subcutaneously transplanted with B16F10-mock cells (1 × 106). Anti-NKG2D antibody (clone HMG2D), anti-mouse CD4 antibody (clone YTS191), anti-mouse CD8α antibody (clone 2.43), or anti-mouse NK1.1 antibody (clone PK136) was administered intraperitoneally at 300 µg/mouse on day 0 post-transplantation, followed by 200 µg/mouse on days 3, 7, and 13. PBS (−) was administered as a control on the same schedule. Arrows indicate treatment days. Tumor sizes were measured three times per week using an electronic caliper (n = 5). Due to the euthanization of one mouse because of tumor ulceration, the data point for day 17 post-transplantation in the anti-NK1.1 group was unavailable. (B) Photos of tumors harvested on day 17, post-transplantation, from the experiment shown in (A). (C) Tumor weights of all tumors harvested on day 17, post-transplantation, from (A). The anti-NK1.1 group was excluded from statistical comparisons due to data loss. NA indicates exclusion from statistical comparisons. (D) Tumor growth in C57BL/6 mice subcutaneously transplanted with B16F10-ULBP2 cells (1 × 106) and treated as described in (A), except that no antibody was administered on day 13. Tumor growth was monitored as described above (n = 5). (E) Photos of tumors harvested on day 13 post-transplantation from the experiment shown in (D). (F) Tumor weights of all tumors harvested on day 13 post-transplantation from (D). (G) Schematic representation of the proposed mechanisms. A question mark and a dotted line indicate a potential mechanism suggested by our observations, but not directly demonstrated in this study. Illustration was created with BioRender.com. In (A,D), data are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; ns: Not significant (Mann–Whitney U test: control vs. anti-NK1.1 group). In (C,F), individual values are shown with the mean ± SEM. * p < 0.05; ns: not significant (Mann–Whitney U test: control vs. each treatment group).

Article Snippet: NKG2D blockade, CD4+ T cell depletion, CD8+ T cell depletion, and NK cell depletion were performed by intraperitoneally administering anti-mouse NKG2D (clone HMG2D, BE0111, Bio X Cell; Lebanon, NH, USA; RRID:AB_10950118), anti-mouse CD4 (clone YTS191, BE0119, Bio X Cell; RRID:AB_10950382), anti-mouse CD8α (clone 2.43, BE0061, Bio X Cell; RRID:AB_1125541), and anti-mouse NK1.1 antibodies (clone PK136, BE0036, Bio X Cell; RRID:AB_1107737).

Techniques: Transplantation Assay, Control, MANN-WHITNEY

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