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: 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

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Compound Kushen Injection inhibits epithelial-mesenchymal transition of gastric carcinoma by regulating VCAM1 induced by the TNF signaling pathway.

doi: 10.1016/j.phymed.2023.154984

Figure Lengend Snippet: Fig. 3. CKI improves immunity in the tumor xenograft model in cooperation with DDP. (A) Mean thymus and spleen indices of different groups. (B) Representative flow cytometry images of CD3+ T cells, CD4+ T cells, CD8+ T cells in tumor tissues. (C) Representative flow cytometry images of CD3+ T cells, CD4+ T cells, CD8+ T cells in spleen tissues. (D) H & E staining of tumor sections. Data are presented as mean ± SD of 6 mice per group. *p < 0.05, **p < 0.01 vs. model group. #p < 0.05, ##p < 0.01 vs. DDP group. CKIMD, CKI middle dose; CKIHD, CKI high dose.

Article Snippet: BB515 Rat Anti-Mouse CD45 (BD, USA), APC/FireTM 750 antimouse CD3, APC anti-mouse CD4, and PE/Cyanine7 anti-mouse CD8a (BioLegend, USA) were used to label CD3+, CD4+, and CD8+ T cells, and measurement was performed by flow cytometry (Cytek, USA).

Techniques: Flow Cytometry, Staining

Journal: iScience

Article Title: Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

doi: 10.1016/j.isci.2025.114572

Figure Lengend Snippet: Tregs traffic from the tumor to the TdLN at higher proportions and are equally susceptible to radiation as non-Treg CD4 T cells (A) Kaede mice were injected with either MC38, MOC1, or MOC2 cancer cells subcutaneously. Tumors were photoconverted on day 14 via ultraviolet light. TdLNs were harvested on day 3 post-photoconversion. All cell populations were pre-gated on live singlets. (B) Photoconverted CD8 T cells were defined as CD3 + CD4 − CD8 + Red + . Photoconverted Tregs were defined as CD3 + CD8 − CD4 + CD25 + Red + . Photoconverted non-Treg CD4 T cells were defined as CD3 + CD8 − CD4 + CD25 − Red + . (C) Frequencies of photoconverted CD8 T cells, Tregs, and non-Treg CD4 T cells were quantified via flow cytometry. (D) Mice were injected with MC38 cancer cells subcutaneously. Tumors were treated with 12 Gy RT on day 14 and harvested for analysis via flow cytometry on day 1–3 post-RT. Non-Treg CD4 T cells and Tregs frequencies as a percentage of live cells were quantified. (E) MC38-bearing Kaede mouse tumors were photoconverted on day 14 followed by immediate treatment with 12 Gy RT. (F) TdLNs were harvested on day 1–3 and percentages of photoconverted non-Treg CD4 T cells and Tregs were analyzed via flow cytometry. A total of 5–6 mice were analyzed per group. Data are represented as mean ± SD. Statistics were performed using an unpaired Student’s t test between groups. ns = not significant, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Samples were enriched magnetically using CD4/CD8 TIL MicroBeads (Miltenyi Biotec, 130-116-480).

Techniques: Injection, Flow Cytometry

Journal: Biomedicines

Article Title: CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

doi: 10.3390/biomedicines10030704

Figure Lengend Snippet: Figure 6. Removal of CD5 functionally increased glycolysis and mitochondrial respiration in un- stimulated Th cells. (A,E) Using a standard Seahorse protocol, approximately 200,000 CD4+ T cells were seeded into a poly-d-lysine coated 8-well XFp plate and analyzed for metabolic function using stepwise injections of oligomycin, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin A. Three wells were plated for each mouse, and the average was taken for

Article Snippet: Unstimulated CD4+ T cells were selected using positive selection CD4+ (L3T4) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany; kit #130-117-043) according to manufacturer instructions.

Techniques:

Journal: Biomedicines

Article Title: CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

doi: 10.3390/biomedicines10030704

Figure Lengend Snippet: Figure 7. Unstimulated CD5KO CD4+ T cells were phenotypically different from CD5WT CD4+ T cells. (A) CD4+ T cells were stained with CD44, CD62L, and CD25 to determine activation state and subset polarization. (B) Gating strategy to determine naïve and memory populations between CD5WT

Article Snippet: Unstimulated CD4+ T cells were selected using positive selection CD4+ (L3T4) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany; kit #130-117-043) according to manufacturer instructions.

Techniques: Staining, Activation Assay