Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: The intestinal microbial metabolite acetyl l-carnitine improves gut inflammation and immune homeostasis via CADM2.

doi: 10.1016/j.bbadis.2024.167089

Figure Lengend Snippet: Fig. 6. (A) Immunoprecipitation was used to detect the interaction between CADM2 and MyD88. (B) Colocalization of CADM2 and MyD88 was detected by immunofluorescence confocal microscopy. (C) Immunohistochemical detection of CADM2 expression in the intestinal tissues of CD patients and healthy people. (D) Immunofluorescence detection of Th17 cells in DSS group and DSS + ALC group and the ratio of Th17 cells in DSS group and DSS + ALC group was analyzed by ImageJ. (E) Immunofluorescence detection of Treg cells in DSS group and DSS + ALC group and the ratio of Treg cells in DSS group and DSS + ALC group was analyzed by ImageJ. (F) Immunofluorescence detection of Macrophages in DSS group and DSS + ALC group and the ratio of macrophages in DSS group and DSS + ALC group was analyzed by ImageJ. (G) Expression profiles of immune cell-related in MCEC cells under different treatments.

Article Snippet: To isolate Treg cells, we collected cells from mouse spleens and sorted out CD4+ and CD25+ Treg cells using the Miltenyi Mouse Treg Cells Isolation Kit (Miltenyi, #130–091-041).

Techniques: Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Immunohistochemical staining, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: The intestinal microbial metabolite acetyl l-carnitine improves gut inflammation and immune homeostasis via CADM2.

doi: 10.1016/j.bbadis.2024.167089

Figure Lengend Snippet: Fig. 7. (A–D) Colonic length, body weight and DAI score in DSS colitis mice. (E) HE staining of mice colonic tissue. (F, G) Inflammatory factors expression in HCT116 and NCM460 cells between TNF-α group and TNF-α + Treg group. (H) Flow cytometry detection of Treg cells ratio changes.

Article Snippet: To isolate Treg cells, we collected cells from mouse spleens and sorted out CD4+ and CD25+ Treg cells using the Miltenyi Mouse Treg Cells Isolation Kit (Miltenyi, #130–091-041).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Epinephrine Production in Th17 Cells and Experimental Autoimmune Encephalitis

doi: 10.3389/fimmu.2021.616583

Figure Lengend Snippet: PNMT expression in T H 17 cells. (A) Diagram of de novo epinephrine synthesis and converting enzymes. (B) PNMT expression in T H 17 cells. Naïve CD4+ T cells from spleens were isolated and T H 0 or T H 17 cells were differentiated. Lysates of each cell subset (20 μg each) and adrenal gland tissue lysates (1 μg) were used for western blotting. Expression of actin was detected as a control protein. One representative image from 3 independent experiments. Graph is a quantitation and normalization of PNMT to ACTIN. (C) Total RNA was prepared and levels of Th , Ddc , Dbh , and Pnmt mRNA was measured by qPCR. RNA samples from the mouse brain (control mice) were used as positive controls. Relative expression was normalized to the level of Actin. Each dot represents an individual sample and the bar represent mean ± SEM (n = 5).

Article Snippet: To isolated CD4+ Treg cells and T H 17 cells, PBMCs were stained with antibodies and live CD127-Treg cells (FVD-CD4+ CD127loCD49d-CD25+) , CD25+ Treg cells (FVD-CD4+CD25+), and TH17 cells (FVD-CD4+CD25-CCR6+CXCR3-) were isolated by FACSAria (BD Biosciences).

Techniques: Expressing, Isolation, Western Blot, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Epinephrine Production in Th17 Cells and Experimental Autoimmune Encephalitis

doi: 10.3389/fimmu.2021.616583

Figure Lengend Snippet: Pnmt expression in T H 17 cells in dLNs. CD4+ T cells were isolated from LNs from 6-8 weeks old C57BL/6 mice. (A) IL-17+ T H 17 cells and IL-17- non-T H 17 cells were enriched and isolated by cell sorter. Purity of IL-17+ and IL-17- cells after sorting was confirmed and ~75% of cells are enriched in the positive fraction and less than 5% of cells are contaminated in the negative fraction. (B) Total RNA was prepared from the sorted cells and the levels of Il17A and Pnmt mRNA was measured by qPCR. RNA from whole brain tissue was used as a positive control. Relative expression was calculated based on the level of Polr2a . Each dot represents an individual mouse and the bar represents mean ± SEM (n = 5).

Article Snippet: To isolated CD4+ Treg cells and T H 17 cells, PBMCs were stained with antibodies and live CD127-Treg cells (FVD-CD4+ CD127loCD49d-CD25+) , CD25+ Treg cells (FVD-CD4+CD25+), and TH17 cells (FVD-CD4+CD25-CCR6+CXCR3-) were isolated by FACSAria (BD Biosciences).

Techniques: Expressing, Isolation, Positive Control

Journal: Frontiers in Immunology

Article Title: Epinephrine Production in Th17 Cells and Experimental Autoimmune Encephalitis

doi: 10.3389/fimmu.2021.616583

Figure Lengend Snippet: Th and PNMT expression in human and mouse helper T cell subsets. (A) Th and PNMT expression in Treg cells and Th17 cells. Human CD25+ Treg cells (hTreg1 in figure), CD127lo Treg cells (CD49d-CD127 lo CD25+) (hTreg2 in figure) and T H 17 cells (CD25-CXCR3-CCR6+) was isolated from PBMCs and murine FOXP3+ Treg and T H 17 cells were isolated from spleen of 6-8 weeks of Foxp3 -GFP C57BL/6 mice. Total RNA was isolated and Th and PNMT expression was measured by qPCR. One representative image from 2 independent experiments (n = 6). (B) Mouse CD4+ T H cells were differentiated from naïve CD4+ T cells and protein level of PNMT was measured by western blot. Differentiation was confirmed by cytokine production of IFNγ (T H 1), IL-4 (T H 2), and IL-17 (T H 17) at the end of culture. Levels of actin was measured as a protein loading control. One representative image of 3 independent experiments. Intensity of protein expression was quantified by densitometry and normalized to the level of ACTIN.

Article Snippet: To isolated CD4+ Treg cells and T H 17 cells, PBMCs were stained with antibodies and live CD127-Treg cells (FVD-CD4+ CD127loCD49d-CD25+) , CD25+ Treg cells (FVD-CD4+CD25+), and TH17 cells (FVD-CD4+CD25-CCR6+CXCR3-) were isolated by FACSAria (BD Biosciences).

Techniques: Expressing, Isolation, Western Blot

Journal: Frontiers in Immunology

Article Title: Epinephrine Production in Th17 Cells and Experimental Autoimmune Encephalitis

doi: 10.3389/fimmu.2021.616583

Figure Lengend Snippet: Epinephrine expression and lymphocytes in Th-CKO mice. (A) T H 17 cells were isolated from SCs from control and Th-CKO EAE mice and Th expression was measured by qPCR. The relative expression was calculated to the level of Porl2a . Each dot represents an individual animal. 3 independent experiments (n = 6). (B) Naïve CD4+ T cells were isolated from spleens of 6-8 weeks old control and Th-CKO mice and differentiated into T H 0 or T H 17 cells. Total RNA was prepared and Th expression was measured by qPCR. The relative expression was calculated to the level of Porl2a . Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25 th to 75 th percentile, and the whiskers indicate 10 th and 90th percentile. Three independent experiments (n = 6). Cells were washed with PBS and epinephrine was measured by mass-spectrometry. Each dot represents and individual sample and the bar represents the mean ± SEM (n = 3). T lymphocytes in spleen and MLN of control and Th-CKO mice were characterized by flow. (C) Total TCRβ+ T cells, CD4+ T cells, CD8+ T cells, and FOXP3+ Treg cells were measured by flow cytometry. (D) T H 17 cells were differentiated from naïve CD4+ T cells of control and Th-CKO mice and IL-17a-positive CD4+ T cells were quantified. Each dot representative of individual mouse and the bar represents the mean ± SEM (n = 2).

Article Snippet: To isolated CD4+ Treg cells and T H 17 cells, PBMCs were stained with antibodies and live CD127-Treg cells (FVD-CD4+ CD127loCD49d-CD25+) , CD25+ Treg cells (FVD-CD4+CD25+), and TH17 cells (FVD-CD4+CD25-CCR6+CXCR3-) were isolated by FACSAria (BD Biosciences).

Techniques: Expressing, Isolation, Whisker Assay, Mass Spectrometry, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: New Paradigm in the Role of Regulatory T Cells During Pregnancy

doi: 10.3389/fimmu.2019.00573

Figure Lengend Snippet: Immunological balance at the feto-maternal interface during early pregnancy. EVTs did not express polymorphic HLA-A, B whereas HLA-C and non-polymorphic HLA-E, G, and F were expressed. Maternal CD8 + T cells and NK cells can directly recognize paternal HLA-C and CD4 + T cells can indirectly recognize it. HLA- E and G protect EVTs from NK-cell mediated cytotoxicity. Treg cells can recognize fetal antigens via maternal antigen presenting cells (APCs) and induce tolerance in an antigen-specific manner. EVT, Extravillous trophoblast; NK, natural killer cell; Treg; regulatory T cell; APC, antigen-presenting cell.

Article Snippet: Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4 + CD25 + FoxP3 + regulatory T (Treg) cells is established during pregnancy.

Techniques:

Journal: Frontiers in Immunology

Article Title: New Paradigm in the Role of Regulatory T Cells During Pregnancy

doi: 10.3389/fimmu.2019.00573

Figure Lengend Snippet: Paternal antigen-specific Treg cells in mouse models.

Article Snippet: Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4 + CD25 + FoxP3 + regulatory T (Treg) cells is established during pregnancy.

Techniques: Derivative Assay

Journal: Frontiers in Immunology

Article Title: New Paradigm in the Role of Regulatory T Cells During Pregnancy

doi: 10.3389/fimmu.2019.00573

Figure Lengend Snippet: Single-cell based TCR repertoire analysis method. To study the clonality of effector Treg cells, a single-cell based T cell receptor (TCR) repertoire analysis method was used. Paired samples of maternal peripheral blood mononuclear cells and decidual lymphocytes were obtained. CD4+CD25+CD45RA-CD127low/- effector Treg cells were single-cell sorted. The cDNAs of complementarity determining lesion 3 (CDR3) in TCRβ chain and FoxP3 were amplified by RT-PCR. The nucleotides and amino acid sequences of CDR3 were analyzed.

Article Snippet: Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4 + CD25 + FoxP3 + regulatory T (Treg) cells is established during pregnancy.

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Immunity

Article Title: The polyamine-regulating enzyme SSAT1 impairs tissue regulatory T cell function in chronic cutaneous inflammation.

doi: 10.1016/j.immuni.2025.02.011

Figure Lengend Snippet: Figure 1. Treg cells are reduced in chronic inflammation and show a tissue-specific inflammation signature (A and B) Uniform manifold approximation and projection (UMAP) projection of FoxP3 expression and differential abundance analysis of (A) skin T cells, including healthy, psoriasis, and sarcoidosis samples, and (B) healthy colon and UC samples.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant human 4-1BBL Peprotech Cat #310-11 Recombinant human B7-1 Fc Peprotech Cat #310-31 Accell Non-targeting Control Pool HorizonDiscovery, Dharmacon Cat #D-001910-10-05 Accell siRNA SAT1 (SMARTPool) Horizon Discovery, Dharmacon Cat #E-010382-00-0005 Ficoll-Paque GE Healthcare Cat #17-1440-03 ImmunoCult CD3/CD28 Human T cell activator StemCell Cat #10971 Dynabeads Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation Thermo Fisher Scientific Cat #11456D Collagen G Sigma Cat # L7213 ZombieUV viability dye BioLegend Cat #423107 ZombieNIR viability dye BioLegend Cat #423105 eFluor780 viability dye Thermo Fisher Scientific Cat #65-0865-14 CellTrace Violet Thermo Fisher Scientific Cat #C34557 Cell Activation Cocktail with Brefeldin A BioLegend Cat #423303 DAPI Sigma Cat #D9542-1MG OCT Compound Cryostat Embedding medium Scigen Cat #4586 Diminazene aceturate Merck Cat #D7770 Acetyl-Coenzym A Trilithiumsalt Merck Cat #A2181 Spermine Merck Cat #S4264 Spermidine Merck Cat #S0266 TWEEN 20 Sigma Cat #P1379 PermaFluor Epredia Lab Vision aqueous mounting medium ePredia Cat #TA-030-FM RPMI-1640 Gibco Cat #52400-025 ImmunoCult -XF T Cell Expansion Medium StemCell Cat #10981 KGM-2 Promocell Cat #C-20111 OptiMEM Gibco Cat #31985062 5X siRNA Buffer HorizonDiscovery/Dharmacon Cat #B-002000-UB-100 Bovine Serum Albumin Sigma Cat #A2153 Fetal Bovine Serum Gibco Cat #10500064 Penicillin-Streptomycin Gibco Cat #15140122 Trypsin/EDTA Lonza Cat #LONCC-5012 Trypsin neutralizing solution Lonza Cat #LONCC-5002 EDTA ThermoFisher Cat #15575020 DMSO Sigma Cat # 317275-500ML Critical Commercial Assays CD4+CD25+CD127dim/- human Treg Isolation Kit II Miltenyi Biotec Cat #130-094-775 CD4+CD25+ Regulatory T Cell Isolation Kit, mouse Miltenyi Biotec Cat #130-091-041 Multi Tissue Dissociation Kit I Miltenyi Biotec Cat #130-110-201 Amaxa Human T cell Nucleofector Kit Lonza Cat #VPA-1002 Fixation Buffer BioLegend Cat #420801 Intracellular Staining Permeabilization Wash Buffer (10X) BioLegend Cat #421002 eBioscience FoxP3/Transcription Factor Staining Buffer Set Invitrogen Cat #00-5523-00 RNeasy Mini spin kit Qiagen Cat #74106 RNeasy Mico kit Qiagen Cat #74004 Biozym cDNA synthesis kit Biozym Cat #331470X/L PowerUP SYBR Green Master Mix ThermoFisher Cat #A25741 (Continued on next page) Immunity 58, 1–16.e1–e12, March 11, 2025 e3

Techniques: Expressing

Journal: Immunity

Article Title: The polyamine-regulating enzyme SSAT1 impairs tissue regulatory T cell function in chronic cutaneous inflammation.

doi: 10.1016/j.immuni.2025.02.011

Figure Lengend Snippet: Figure 2. SAT1 expression defines an inflammation-specific Treg cell population in the skin (A and B) UMAP projections of skin Treg cells showing (A) distribution of SAT1hi and SAT1lo cells and (B) SAT1 expression. (C) Differential abundance testing of skin Treg cells in inflamed vs. healthy skin.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant human 4-1BBL Peprotech Cat #310-11 Recombinant human B7-1 Fc Peprotech Cat #310-31 Accell Non-targeting Control Pool HorizonDiscovery, Dharmacon Cat #D-001910-10-05 Accell siRNA SAT1 (SMARTPool) Horizon Discovery, Dharmacon Cat #E-010382-00-0005 Ficoll-Paque GE Healthcare Cat #17-1440-03 ImmunoCult CD3/CD28 Human T cell activator StemCell Cat #10971 Dynabeads Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation Thermo Fisher Scientific Cat #11456D Collagen G Sigma Cat # L7213 ZombieUV viability dye BioLegend Cat #423107 ZombieNIR viability dye BioLegend Cat #423105 eFluor780 viability dye Thermo Fisher Scientific Cat #65-0865-14 CellTrace Violet Thermo Fisher Scientific Cat #C34557 Cell Activation Cocktail with Brefeldin A BioLegend Cat #423303 DAPI Sigma Cat #D9542-1MG OCT Compound Cryostat Embedding medium Scigen Cat #4586 Diminazene aceturate Merck Cat #D7770 Acetyl-Coenzym A Trilithiumsalt Merck Cat #A2181 Spermine Merck Cat #S4264 Spermidine Merck Cat #S0266 TWEEN 20 Sigma Cat #P1379 PermaFluor Epredia Lab Vision aqueous mounting medium ePredia Cat #TA-030-FM RPMI-1640 Gibco Cat #52400-025 ImmunoCult -XF T Cell Expansion Medium StemCell Cat #10981 KGM-2 Promocell Cat #C-20111 OptiMEM Gibco Cat #31985062 5X siRNA Buffer HorizonDiscovery/Dharmacon Cat #B-002000-UB-100 Bovine Serum Albumin Sigma Cat #A2153 Fetal Bovine Serum Gibco Cat #10500064 Penicillin-Streptomycin Gibco Cat #15140122 Trypsin/EDTA Lonza Cat #LONCC-5012 Trypsin neutralizing solution Lonza Cat #LONCC-5002 EDTA ThermoFisher Cat #15575020 DMSO Sigma Cat # 317275-500ML Critical Commercial Assays CD4+CD25+CD127dim/- human Treg Isolation Kit II Miltenyi Biotec Cat #130-094-775 CD4+CD25+ Regulatory T Cell Isolation Kit, mouse Miltenyi Biotec Cat #130-091-041 Multi Tissue Dissociation Kit I Miltenyi Biotec Cat #130-110-201 Amaxa Human T cell Nucleofector Kit Lonza Cat #VPA-1002 Fixation Buffer BioLegend Cat #420801 Intracellular Staining Permeabilization Wash Buffer (10X) BioLegend Cat #421002 eBioscience FoxP3/Transcription Factor Staining Buffer Set Invitrogen Cat #00-5523-00 RNeasy Mini spin kit Qiagen Cat #74106 RNeasy Mico kit Qiagen Cat #74004 Biozym cDNA synthesis kit Biozym Cat #331470X/L PowerUP SYBR Green Master Mix ThermoFisher Cat #A25741 (Continued on next page) Immunity 58, 1–16.e1–e12, March 11, 2025 e3

Techniques: Expressing

Journal: Immunity

Article Title: The polyamine-regulating enzyme SSAT1 impairs tissue regulatory T cell function in chronic cutaneous inflammation.

doi: 10.1016/j.immuni.2025.02.011

Figure Lengend Snippet: Figure 5. Inhibition of SSAT in a mouse model of psoriasis rescues Treg cell number and function in the skin (A) Quantification of immunofluorescence staining for Treg cells (CD3+CD4+CD25++) in skin of DKO*K15 mice as absolute numbers/mm2. (B) Quantification of MFI of SSAT activity in the skin of DKO*K15 mice.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant human 4-1BBL Peprotech Cat #310-11 Recombinant human B7-1 Fc Peprotech Cat #310-31 Accell Non-targeting Control Pool HorizonDiscovery, Dharmacon Cat #D-001910-10-05 Accell siRNA SAT1 (SMARTPool) Horizon Discovery, Dharmacon Cat #E-010382-00-0005 Ficoll-Paque GE Healthcare Cat #17-1440-03 ImmunoCult CD3/CD28 Human T cell activator StemCell Cat #10971 Dynabeads Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation Thermo Fisher Scientific Cat #11456D Collagen G Sigma Cat # L7213 ZombieUV viability dye BioLegend Cat #423107 ZombieNIR viability dye BioLegend Cat #423105 eFluor780 viability dye Thermo Fisher Scientific Cat #65-0865-14 CellTrace Violet Thermo Fisher Scientific Cat #C34557 Cell Activation Cocktail with Brefeldin A BioLegend Cat #423303 DAPI Sigma Cat #D9542-1MG OCT Compound Cryostat Embedding medium Scigen Cat #4586 Diminazene aceturate Merck Cat #D7770 Acetyl-Coenzym A Trilithiumsalt Merck Cat #A2181 Spermine Merck Cat #S4264 Spermidine Merck Cat #S0266 TWEEN 20 Sigma Cat #P1379 PermaFluor Epredia Lab Vision aqueous mounting medium ePredia Cat #TA-030-FM RPMI-1640 Gibco Cat #52400-025 ImmunoCult -XF T Cell Expansion Medium StemCell Cat #10981 KGM-2 Promocell Cat #C-20111 OptiMEM Gibco Cat #31985062 5X siRNA Buffer HorizonDiscovery/Dharmacon Cat #B-002000-UB-100 Bovine Serum Albumin Sigma Cat #A2153 Fetal Bovine Serum Gibco Cat #10500064 Penicillin-Streptomycin Gibco Cat #15140122 Trypsin/EDTA Lonza Cat #LONCC-5012 Trypsin neutralizing solution Lonza Cat #LONCC-5002 EDTA ThermoFisher Cat #15575020 DMSO Sigma Cat # 317275-500ML Critical Commercial Assays CD4+CD25+CD127dim/- human Treg Isolation Kit II Miltenyi Biotec Cat #130-094-775 CD4+CD25+ Regulatory T Cell Isolation Kit, mouse Miltenyi Biotec Cat #130-091-041 Multi Tissue Dissociation Kit I Miltenyi Biotec Cat #130-110-201 Amaxa Human T cell Nucleofector Kit Lonza Cat #VPA-1002 Fixation Buffer BioLegend Cat #420801 Intracellular Staining Permeabilization Wash Buffer (10X) BioLegend Cat #421002 eBioscience FoxP3/Transcription Factor Staining Buffer Set Invitrogen Cat #00-5523-00 RNeasy Mini spin kit Qiagen Cat #74106 RNeasy Mico kit Qiagen Cat #74004 Biozym cDNA synthesis kit Biozym Cat #331470X/L PowerUP SYBR Green Master Mix ThermoFisher Cat #A25741 (Continued on next page) Immunity 58, 1–16.e1–e12, March 11, 2025 e3

Techniques: Inhibition, Staining, Activity Assay