Journal: The Journal of Experimental Medicine

Article Title: Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

doi: 10.1084/jem.20051060

Figure Lengend Snippet: In vivo dose-response titration of mAb JJ316. Adult Lewis rats were injected i.v. with the three indicated amounts of mAb JJ316. 3 d after injection, peripheral LNCs and splenocytes were analyzed for the prevalence of CD4 + CD25 + and CD4 + CD25 − cells by FACS analysis. (A) Representative dot plots of CD4 and CD25 expression in lymph nodes and spleens of control or JJ316-treated animals; the figure indicates the percentages of cells in the respective quadrant. (B) The proportion of CD25 + among CD4 + cells is summarized for all animals analyzed. Absolute cell numbers were obtained by multiplying total cell numbers with the relative cell numbers obtained by FACS analysis (C). The plots in (B) and (C) show pooled data obtained on eight different occasions. Each circle represents one animal (i.e., 2 to 12 animals per group). Horizontal bars indicate medians. Mann-Whitney rank sum tests were performed between groups as indicated by the brackets, and the respective P values are given.

Article Snippet: The following monoclonal antibodies were used: anti–rat CD4-CyChrome (clone OX35, BD Biosciences); anti–rat CD25-FITC or -biotin (clone Ox39); anti–rat CTLA-4–biotin (clone WKH203, all Serotec), and anti–mouse FoxP3 (clone mFOXY, eBioscience).

Techniques: In Vivo, Titration, Injection, Expressing, Control, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

doi: 10.1084/jem.20051060

Figure Lengend Snippet: Direct monitoring of T reg and T conv cell proliferation upon superagonistic anti-CD28 stimulation in vivo. T reg and T conv cells were purified from pooled spleens and lymph nodes, labeled with CFSE, and adoptively transferred into syngeneic recipients. 0.1 mg of mAb JJ316 or control mAb MOPC-31C was injected on the following day. CFSE dilution was analyzed 2 d after antibody injection (A). Percentages represent the proportion of undivided cells among transferred cells. The result of one of at least three experiments is shown. (B) CD25 expression on transferred T reg and T conv cells isolated from MOPC-31C–treated or JJ316-treated animals was determined by counter-staining with anti-CD25 mAb. (C) CFSE dye dilution among, and CD25 expression on, T conv cells were assessed after stimulation with 1 mg of JJ316.

Article Snippet: The following monoclonal antibodies were used: anti–rat CD4-CyChrome (clone OX35, BD Biosciences); anti–rat CD25-FITC or -biotin (clone Ox39); anti–rat CTLA-4–biotin (clone WKH203, all Serotec), and anti–mouse FoxP3 (clone mFOXY, eBioscience).

Techniques: In Vivo, Purification, Labeling, Control, Injection, Expressing, Isolation, Staining

Journal: The Journal of Experimental Medicine

Article Title: Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

doi: 10.1084/jem.20051060

Figure Lengend Snippet: Phenotypic analysis of T reg and T conv cells from treated animals. 3 d after administration of 1 mg JJ316, lymph node and spleen cells were stained at the cell surface with mAbs against CD4 and CD25, followed by intracellular staining for FoxP3 and CTLA-4. Control animals were injected with PBS only. (A) Representative expression of CD25 and FoxP3 on gated CD4 + cells. The figure indicates the percentages of cells in the respective quadrant. (B) Detection of FoxP3 expression by Western blot analysis. CD4 + CD25 + and CD4 + CD25 − T cells were purified from untreated or JJ316-treated animals (1 mg), and protein lysates from whole cells were generated. FoxP3 expression was detected with a polyclonal rabbit-anti–mouse FoxP3 IgG. Lysates from cells purified after JJ316 treatment also were diluted serially and protein loading was assessed with polyclonal anti–rat ERK-2 Ig. The experiment was repeated with a similar result. (C) Representative anti–CTLA-4 staining profiles of CD4 + CD25 + FoxP3 + cells are depicted (filled line graphs). Staining specificity was controlled by preincubation with unconjugated anti–CTLA-4 mAb (gray profile). A repeat experiment rendered similar results.

Article Snippet: The following monoclonal antibodies were used: anti–rat CD4-CyChrome (clone OX35, BD Biosciences); anti–rat CD25-FITC or -biotin (clone Ox39); anti–rat CTLA-4–biotin (clone WKH203, all Serotec), and anti–mouse FoxP3 (clone mFOXY, eBioscience).

Techniques: Staining, Control, Injection, Expressing, Western Blot, Purification, Generated

Journal: The Journal of Experimental Medicine

Article Title: Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

doi: 10.1084/jem.20051060

Figure Lengend Snippet: Functional analysis of T reg and T conv cells from JJ316-treated animals ex vivo. (A) Purified T reg and T conv cells from the lymph nodes of animals treated as indicated 3 d before isolation were cocultured with CFSE-labeled CD4 + CD25 − T cells from the PBS-treated animal (indicator T cells), and stimulated with costimulatory beads for 5 d. Line graphs show proliferations of indicator T cells in the absence (gray shadow) or the presence of T reg or T conv cells at a 1:1 ratio (black line). (B) T reg and T conv cell/indicator T cell ratios of 1:1, 1:5, and 1:10 were determined by translating CFSE dilution profiles into the average number of cell divisions of the indicator T cells after 5 d of culture. The symbols indicate proliferation of indicator T cells in the absence of T reg/T conv cells (open diamonds) or in the presence of T reg/T conv cells from the animal treated with PBS (filled circles), 0.1 mg JJ316 (open squares), or 1 mg JJ316 (gray diamonds). The result is representative of two experiments performed.

Article Snippet: The following monoclonal antibodies were used: anti–rat CD4-CyChrome (clone OX35, BD Biosciences); anti–rat CD25-FITC or -biotin (clone Ox39); anti–rat CTLA-4–biotin (clone WKH203, all Serotec), and anti–mouse FoxP3 (clone mFOXY, eBioscience).

Techniques: Functional Assay, Ex Vivo, Purification, Isolation, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

doi: 10.1084/jem.20051060

Figure Lengend Snippet: mAb JJ316-primed T reg cells suppress the proliferation of gpMBP-specific T cells in vitro . T reg and T conv cells were isolated from the draining lymph nodes of animals that had been immunized with gpMBP in CFA 9 d before functional analysis. mAb JJ316 (0.1 mg or 1 mg per animal) or PBS was administered 3 d before the in vitro suppression assay. Conventional CD4 + CD25 − T cells from the PBS-treated animal (gray bars) served as indicator T cells, and were cocultured with T reg or T conv cells from the animal treated with PBS, 0.1 mg JJ316, or 1 mg of JJ316. T cells were stimulated with gpMBP in the presence of irradiated splenic APCs. A repeat experiment rendered similar results.

Article Snippet: The following monoclonal antibodies were used: anti–rat CD4-CyChrome (clone OX35, BD Biosciences); anti–rat CD25-FITC or -biotin (clone Ox39); anti–rat CTLA-4–biotin (clone WKH203, all Serotec), and anti–mouse FoxP3 (clone mFOXY, eBioscience).

Techniques: In Vitro, Isolation, Functional Assay, Suppression Assay, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

doi: 10.1084/jem.20051060

Figure Lengend Snippet: CD4 + CD25 + T reg cells mediate reduced disease severity. Donor animals were immunized with gpMBP in CFA and received 1 mg of mAb JJ316 i.v. 3 d later, draining LNCs were prepared and separated, in part, into CD4 + CD25 − and CD4 + CD25 + cells. Recipient animals received PBS only, 4 (4.7) × 10 7 total draining LNCs, 4 (4.7) × 10 6 purified CD4 + CD25 + T cells, or 3.6 (4.2) × 10 7 CD4 + CD25 − T cells; numbers in parentheses denote the amount of cells transferred in AT-EAE (see B). EAE was induced in recipient animals by active immunization with gpMBP in CFA (A) or by adoptive transfer of encephalitogenic T cell line cells (B). These experiments were reproduced twice with similar results. Generation of protective CD4 + CD25 + T reg cells is related to the application of superagonistic CD28-specific mAb and is antigen priming-independent (C). On the day of immunization with gpMBP in CFA, animals received 6 × 10 7 JJ316-primed total draining LNCs (open squares) or 6 × 10 6 separated CD4 + CD25 + T cells (open circles). Control treatment consisted of the transfer of 5.4 × 10 7 CD28 superagonist-activated CD4 + CD25 − T cells (gray circles) or in the administration of PBS only (black circles).

Article Snippet: The following monoclonal antibodies were used: anti–rat CD4-CyChrome (clone OX35, BD Biosciences); anti–rat CD25-FITC or -biotin (clone Ox39); anti–rat CTLA-4–biotin (clone WKH203, all Serotec), and anti–mouse FoxP3 (clone mFOXY, eBioscience).

Techniques: Purification, Adoptive Transfer Assay, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Serine protease inhibitor from the muscle larval Trichinella spiralis ameliorates non-alcoholic fatty liver disease in mice via anti-inflammatory properties and gut-liver crosstalk.

doi: 10.1016/j.biopha.2024.116223

Figure Lengend Snippet: Fig. 4. rTs-SPI attenuated macrophage infiltration and promoted CD4þCD25þFoxp3þ Tregs population in NAFLD mice. (A) Macrophage infiltration and (B) CD4+CD25+Foxp3+ Tregs in spleen using Flow cytometry analysis; Quantitative analysis of (C) F4/80+ macrophages and (D) CD4+CD25+Foxp3+ Tregs in spleen; (E) Microphotographs show total macrophages staining with F4/80 (red) and DAPI (blue) in liver (600x); (F) Quantitative analysis of F4/80+ macrophages in liver; The hepatic mRNA expression levels of (G) F4/80 and (H) Foxp3. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For Treg analysis, cells were stained with Percp/cy5.5 anti-mouse CD4 and APC anti-mouse CD25 antibodies (Elabscience, China), followed by fixing and permeabilizing using a Foxp3/transcription factor staining kit (Elabscience, China) and staining with PE anti-mouse Foxp3 antibody (Elabscience, China).

Techniques: Flow Cytometry, Staining, Expressing