Journal:

Article Title: Protection against Experimental Autoimmune Myocarditis Is Mediated by Interleukin-10-Producing T Cells that Are Controlled by Dendritic Cells

doi:

Figure Lengend Snippet: Polarized CD4+ T-cell response after culture with DCs of S2-16 and adjuvant-treated rats: S2-16:IFA DCs promoted CD4+ T cells to produce more IL-10, whereas S2-16:CFA DCs promoted IFN-γ production from CD4+ T cells. Lewis rats were treated with S2-16:IFA or S2-16:CFA. Fourteen days later, CD4+ T cells and DCs were isolated from spleens of treated rats or untreated naïve rats, and co-cultured together with S2-16 peptide. Proliferation of cells was measured by 3H-thymidine incorporation after 96 hours. Cytokine production in cell culture supernatants was measured by ELISA. A: Proliferative response, IL-10, and IFN-γ production by CD4+ T cells from S2-16:CFA-treated rats after co-culture (5 × 105 CD4+ T cells) with DCs from three groups of rats at various concentrations. B: Proliferative response and IL-10 and IFN-γ production of CD4+ T cells from S2-16:IFA-treated rats after co-culture (5 × 105 CD4+ T cells) with DCs at various concentrations. C: Proliferative response and IFN-γ production of CD4+ T cells from naive rats after co-culture (5 × 105 CD4+ T cells) with DCs at various concentrations. Error bars represent SEMs. Mann-Whitney test was used to determine the difference between S2-16:IFA DC or S2-16:CFA DC versus naïve DC groups (proliferation and IFN-γ), and S2-16:IFA DC versus S2-16:CFA DC groups (IL-10). *P ≤ 0.05, **P ≤ 0.005. D: Intracellular staining suggested that S2-16:IFA DCs induced higher level IL-10 production from CD4+ T cells than S2-16:CFA DCs. DCs were purified from spleens of S2-16:CFA- or S2-16:IFA-treated rats, and cultured with purified CD4+ T cells from these groups of rats, together with S2-16 peptide. Cells were removed from co-culture on day 4 and stained for CD4+ cells and intracellular IL-10-positive cells. Numbers denote percentage of cells in each quadrant. The data are representative of two to three independent experiments with similar results.

Article Snippet: For in vitro stimulation assay of CD4 + T cells, splenocytes were incubated with a saturating concentration of magnetic anti-rat CD4 (OX-38) microbeads (Miltenyi Biotec, Auburn, CA) at 4°C for 20 minutes.

Techniques: Adjuvant, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, MANN-WHITNEY, Staining, Purification

Journal:

Article Title: CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response

doi:

Figure Lengend Snippet: Blockade of CTLA-4 at the time of induction of an immune response to cryptococcal antigens significantly augments anticryptococcal DTH. Mice were treated i.p. with either hamster IgG, as a control, or anti-CTLA-4 Fab to block CTLA-4 1 day before, the day of, and days 1 through 5 after immunization with cryptococcal culture filtrate antigen in CFA (CneF-CFA) or HKC or treatment with saline or saline-CFA on day 0. On day 7 after immunization, the mouse footpads were challenged with CneF or saline, and footpad swelling was measured on day 8. Five mice were used per group, and the experiment was repeated four times with similar results. Error bars show standard errors of the means.

Article Snippet: The splenocytes were stained with anti-CD4 (Cy-Chrome; rat immunoglobulin G2a (IgG2a); Pharmingen, San Diego, Calif.) and anti-Vβ8 TCR (fluorescein isothiocyanate, mouse IgG2a, Pharmingen) Abs.

Techniques: Blocking Assay

Journal:

Article Title: CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response

doi:

Figure Lengend Snippet: Blockade of CTLA-4 Fab with either anti-CTLA-4 IgG or anti-CTLA-4 Fab augments the anticryptococcal DTH response if the blocking reagent is given during the induction period (days −1, 0, and +1 through +3) but not if the blocking reagent is given after the sensitized T cells have been induced (days 6 and 7)

Article Snippet: The splenocytes were stained with anti-CD4 (Cy-Chrome; rat immunoglobulin G2a (IgG2a); Pharmingen, San Diego, Calif.) and anti-Vβ8 TCR (fluorescein isothiocyanate, mouse IgG2a, Pharmingen) Abs.

Techniques: Blocking Assay

Journal:

Article Title: CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response

doi:

Figure Lengend Snippet: Blockade of CTLA-4 during induction of the immune response results in increased early clearance of C. neoformans from lungs, spleens, and brains. Mice were treated with hamster IgG or anti-CTLA-4 Fab and immunized as described in the Fig. ​Fig.11 legend, but instead of footpad challenge on day 7 after immunization, the mice were infected i.v. with 105 C. neoformans 184A cells. Seven days after infection, the numbers of cryptococcal CFU were determined in the designated tissues. Five mice were used per group, and the experiment was repeated twice with similar results. Error bars show standard errors of the means.

Article Snippet: The splenocytes were stained with anti-CD4 (Cy-Chrome; rat immunoglobulin G2a (IgG2a); Pharmingen, San Diego, Calif.) and anti-Vβ8 TCR (fluorescein isothiocyanate, mouse IgG2a, Pharmingen) Abs.

Techniques: Infection

Journal:

Article Title: CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response

doi:

Figure Lengend Snippet: Blockade of CTLA-4 results in mice developing anticryptococcal DTH reactivity as early as 7 days after infection. Mice were treated with hamster IgG or anti-CTLA-4 Fab and injected with saline or saline-CFA as indicated in the Fig. ​Fig.11 legend. On day 7 after treatment with saline or saline-CFA, the mice were infected i.v. with 105 C. neoformans 184A cells. Seven days after infection, the mouse footpads were challenged and the footpad swelling was measured the following day. Five mice were used per experimental group, and the experiment was repeated twice with similar results. Error bars show standard errors of the means.

Article Snippet: The splenocytes were stained with anti-CD4 (Cy-Chrome; rat immunoglobulin G2a (IgG2a); Pharmingen, San Diego, Calif.) and anti-Vβ8 TCR (fluorescein isothiocyanate, mouse IgG2a, Pharmingen) Abs.

Techniques: Infection, Injection

Journal:

Article Title: CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response

doi:

Figure Lengend Snippet: Increased protection against C. neoformans as measured by increased survival times is seen only in mice in which CTLA-4 is blocked during immunization with the protective immunogen, CneF-CFA. Mice were immunized and treated with hamster IgG or anti-CTLA-4 Fab as described in the Fig. ​Fig.11 legend. The animals were infected i.v. with 105 C. neoformans 184A cells on day 7 after immunization or treatment with control reagents. Survival of the mice was monitored for 80 days before the experiment was terminated. Ten mice were used per experimental group, and the experiment was repeated twice with similar results.

Article Snippet: The splenocytes were stained with anti-CD4 (Cy-Chrome; rat immunoglobulin G2a (IgG2a); Pharmingen, San Diego, Calif.) and anti-Vβ8 TCR (fluorescein isothiocyanate, mouse IgG2a, Pharmingen) Abs.

Techniques: Infection

Journal:

Article Title: CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response

doi:

Figure Lengend Snippet: Blockade of CTLA-4 during a C. neoformans infection with a highly virulent isolate results in significantly increased survival times for the mice. Mice were infected i.t. with 105 C. neoformans cells of the highly virulent isolate NU-2 on day 0. The animals were injected i.p. with 100 μg of hamster IgG or anti-CTLA-4 IgG on day 0 and day 3 and then twice weekly for the duration of the experiment. Ten mice were used per group.

Article Snippet: The splenocytes were stained with anti-CD4 (Cy-Chrome; rat immunoglobulin G2a (IgG2a); Pharmingen, San Diego, Calif.) and anti-Vβ8 TCR (fluorescein isothiocyanate, mouse IgG2a, Pharmingen) Abs.

Techniques: Infection, Injection

Journal:

Article Title: Neuroprotective autoimmunity: Naturally occurring CD4 + CD25 + regulatory T cells suppress the ability to withstand injury to the central nervous system

doi: 10.1073/pnas.232565399

Figure Lengend Snippet: Depletion of naturally occurring regulatory CD4+CD25+ T cells in BALB/c mice improves neuronal survival after optic nerve crush injury. (A) BALB/c mice were thymectomized (ThyX) 3 days after birth to deplete their regulatory T cells and were subjected as adults to severe unilateral crush injury inflicted on the intraorbital portion of the optic nerve. Surviving neurons were labeled by the application, 3 days before injury, of the neurotracer dye FluoroGold. Significantly more neurons survived in the thymectomized mice than in control (nonthymectomized) age-matched mice (n = 7–8 in each group; P < 0.001; Student's t test). (B) Neuronal survival after optic nerve crush injury in BALB/c nu/nu mice (devoid of T cells) was worse than in wild-type mice of matched background. Endogenous neuroprotection in the nude mice was restored by injection of 5 × 107 wild-type splenocytes. Injection of splenocytes depleted of regulatory CD4+CD25+ T cells increased neuronal survival in these mice beyond even that seen in the wild type (n = 5–6 in each group; P values between different groups, obtained by two-tailed Student's t test, are indicated by asterisks above the graph bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Mouse anti-rat CD4 antibodies conjugated to phycoerythrin (PE) and mouse anti-rat CD25 antibodies conjugated to fluorescein isothiocyanate were purchased from Serotec.

Techniques: Labeling, Injection, Two Tailed Test

Journal:

Article Title: Neuroprotective autoimmunity: Naturally occurring CD4 + CD25 + regulatory T cells suppress the ability to withstand injury to the central nervous system

doi: 10.1073/pnas.232565399

Figure Lengend Snippet: Naturally occurring regulatory CD4+CD25+ T cells diminish spontaneous neuroprotection. BALB/c mice, endowed with the spontaneous ability to evoke a protective beneficial autoimmune response, were injected with 2 × 106 purified activated regulatory CD4+CD25+ T cells. Control mice were injected with 2 × 106 purified activated effector cells (CD4+CD25−) or with PBS. Injection of regulatory T cells had an adverse effect on neuronal survival (more neurons underwent secondary degeneration). The results shown are of one representative experiment of three independent experiments; n = 5–6 mice in each group (P < 0.01 and 0.05; respectively).

Article Snippet: Mouse anti-rat CD4 antibodies conjugated to phycoerythrin (PE) and mouse anti-rat CD25 antibodies conjugated to fluorescein isothiocyanate were purchased from Serotec.

Techniques: Injection, Purification

Journal:

Article Title: Mink Cell Focus-Forming Murine Leukemia Virus Infection Induces Apoptosis of Thymic Lymphocytes

doi:

Figure Lengend Snippet: Number of cells in each thymic subpopulation a

Article Snippet: After being washed twice with PBS, the cells were resuspended in 100 μl of PBS containing hamster anti-CD3 antibody conjugated to phycoerythrin, rat anti-CD4 antibody conjugated to Cy-Chrome, rat anti-CD8 antibody conjugated to biotin (PharMingen), and NeutraLite avidin conjugated to Cascade blue (Molecular Probes, Eugene, Oreg.) at dilutions previously determined by titration assays.

Techniques:

Journal:

Article Title: Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia

doi: 10.1128/JVI.75.7.3152-3163.2001

Figure Lengend Snippet: Changes in the numbers of TER-119+ erythroid cells (a), F-MuLV gp70-expressing cells detected with MAb 720 (b), and CD4+ (c) and CD8+ (d) cells in the spleens of mice inoculated with FV. CB6F1 mice were either immunized with peptide i (○) or given CFA without a peptide (●). Four weeks later, they were inoculated with 150 SFFU of FV. A group of three or four animals were killed at each indicated point, and their spleen cells were subjected to flow-cytometric analyses. Data presented here are means ± SEM. The dashed line in panel b indicates the limit of detection by the flow-cytometric analysis.

Article Snippet: Antibodies and their final concentrations used in the present study were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (rat immunoglobulin G2b [IgG2b]; Seikagaku Corporation, Tokyo, Japan) at 0.5 μg/10 6 cells, phycoerythrin (R-PE)-conjugated anti-mouse CD8 (rat IgG2a; Caltag Laboratories, Burlingame, Calif.) at 1 μg/10 6 cells, FITC-conjugated anti-mouse CD69 (hamster IgG; PharMingen, San Diego, Calif.) at 1 μg/10 6 cells, R-PE-conjugated anti-mouse B220 (rat IgG2a; Coulter Immunology, Hialeah, Fla.) at 0.5 μg/10 6 cells, FITC-conjugated anti-NK1.1 (mouse IgG2a; PharMingen) at 2 μg/10 6 cells, biotin-conjugated anti-mouse Pan-NK (DX5, rat IgM; PharMingen) at 1 μg/10 6 cells, and allophycocyanin-conjugated anti-mouse TER-119 (PharMingen) at 0.2 μg/10 6 cells.

Techniques: Expressing

Journal:

Article Title: Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia

doi: 10.1128/JVI.75.7.3152-3163.2001

Figure Lengend Snippet: Detection of cytotoxic effector cells in FV-infected CB6F1 mice. Mice were either immunized with 10 μg of peptide i/mouse or given CFA emulsion without a peptide. B220− spleen cells were separated into CD8+, CD4+, and CD4− CD8− populations, and their cytotoxic activities against FBL-3 (○), Y57-2C (□), and EL-4 (●) cells were tested by incubating the effector and labeled target cells for 12 h. Representative data obtained from a set of experiments performed at PID 9 are shown here, and the results obtained from the six repeated experiments were consistent with these charts.

Article Snippet: Antibodies and their final concentrations used in the present study were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (rat immunoglobulin G2b [IgG2b]; Seikagaku Corporation, Tokyo, Japan) at 0.5 μg/10 6 cells, phycoerythrin (R-PE)-conjugated anti-mouse CD8 (rat IgG2a; Caltag Laboratories, Burlingame, Calif.) at 1 μg/10 6 cells, FITC-conjugated anti-mouse CD69 (hamster IgG; PharMingen, San Diego, Calif.) at 1 μg/10 6 cells, R-PE-conjugated anti-mouse B220 (rat IgG2a; Coulter Immunology, Hialeah, Fla.) at 0.5 μg/10 6 cells, FITC-conjugated anti-NK1.1 (mouse IgG2a; PharMingen) at 2 μg/10 6 cells, biotin-conjugated anti-mouse Pan-NK (DX5, rat IgM; PharMingen) at 1 μg/10 6 cells, and allophycocyanin-conjugated anti-mouse TER-119 (PharMingen) at 0.2 μg/10 6 cells.

Techniques: Infection, Labeling

Journal:

Article Title: Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia

doi: 10.1128/JVI.75.7.3152-3163.2001

Figure Lengend Snippet: In vivo depletion of NK cell activity by injection of anti-asialo-GM1 Ab. (a and b) CB6F1 mice immunized with peptide i were injected either with 60 μg of anti-asialo-GM1 Ab each (b) or with normal rabbit serum (a) and were infected with FV. Spleen cells were obtained at PID 9, and the NK cell activity of the B220− population was tested by using YAC-1 (▵) and EL-4 (●) target cells. Data from two separate experiments are shown together here. Injection of higher doses of anti-asialo-GM1 Ab gave the same results when B200− cells were similarly tested for their YAC-1-killing activities. (c and d) Flow cytometric analyses for the expression of the NK cell markers on spleen cells obtained from mice injected with normal rabbit serum (c) or anti-asialo-GM1 Ab (d). Experiments were performed twice and gave essentially the same results as those shown here. (e through j) Cytotoxicity assays using different cell populations isolated from spleen B220− cells of peptide-immunized, FV-infected mice. CD8+, CD4+, and CD4− CD8− populations were purified as described for the experiments shown in Fig. ​Fig.33 from CB6F1 mice injected with anti-asialo-GM1 Ab (f, h, and j) or from those injected with control rabbit serum (e, g, and i). The experiments were performed twice at PID 7 and 9, and the results from the repeated experiments were consistent with the representative data shown here. Target cells used were YAC-1 (Δ), FBL-3 (○), and EL-4 (●).

Article Snippet: Antibodies and their final concentrations used in the present study were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (rat immunoglobulin G2b [IgG2b]; Seikagaku Corporation, Tokyo, Japan) at 0.5 μg/10 6 cells, phycoerythrin (R-PE)-conjugated anti-mouse CD8 (rat IgG2a; Caltag Laboratories, Burlingame, Calif.) at 1 μg/10 6 cells, FITC-conjugated anti-mouse CD69 (hamster IgG; PharMingen, San Diego, Calif.) at 1 μg/10 6 cells, R-PE-conjugated anti-mouse B220 (rat IgG2a; Coulter Immunology, Hialeah, Fla.) at 0.5 μg/10 6 cells, FITC-conjugated anti-NK1.1 (mouse IgG2a; PharMingen) at 2 μg/10 6 cells, biotin-conjugated anti-mouse Pan-NK (DX5, rat IgM; PharMingen) at 1 μg/10 6 cells, and allophycocyanin-conjugated anti-mouse TER-119 (PharMingen) at 0.2 μg/10 6 cells.

Techniques: In Vivo, Activity Assay, Injection, Infection, Expressing, Isolation, Purification

Journal:

Article Title: Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia

doi: 10.1128/JVI.75.7.3152-3163.2001

Figure Lengend Snippet: Cytotoxic activity of a CD4+ T-cell clone, SB14-31, specific for an F-MuLV env-encoded epitope. (a) SB14-31 cells were incubated with various target cells with or without preincubation with peptide fn. 51Cr release during 4 h of incubation at an E:T ratio of 20 was measured. (b) LB 27.4 target cells were either incubated with the indicated Ab-binding peptides after 51Cr labeling or infected with the indicated recombinant vaccinia virus for 16 h at a multiplicity of infection of 10 and then labeled. Pretreated LB 27.4 cells were then incubated with SB14-31 cells for 4 h at an E:T ratio of 20:1. Experiments were performed at least twice at various E:T ratios, and the results were consistent with the representative data shown here.

Article Snippet: Antibodies and their final concentrations used in the present study were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (rat immunoglobulin G2b [IgG2b]; Seikagaku Corporation, Tokyo, Japan) at 0.5 μg/10 6 cells, phycoerythrin (R-PE)-conjugated anti-mouse CD8 (rat IgG2a; Caltag Laboratories, Burlingame, Calif.) at 1 μg/10 6 cells, FITC-conjugated anti-mouse CD69 (hamster IgG; PharMingen, San Diego, Calif.) at 1 μg/10 6 cells, R-PE-conjugated anti-mouse B220 (rat IgG2a; Coulter Immunology, Hialeah, Fla.) at 0.5 μg/10 6 cells, FITC-conjugated anti-NK1.1 (mouse IgG2a; PharMingen) at 2 μg/10 6 cells, biotin-conjugated anti-mouse Pan-NK (DX5, rat IgM; PharMingen) at 1 μg/10 6 cells, and allophycocyanin-conjugated anti-mouse TER-119 (PharMingen) at 0.2 μg/10 6 cells.

Techniques: Activity Assay, Incubation, Binding Assay, Labeling, Infection, Recombinant

Journal:

Article Title: Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia

doi: 10.1128/JVI.75.7.3152-3163.2001

Figure Lengend Snippet: Cytotoxic activities of four different CD4+ T-cell clones specific for peptide i. T-cell clones F5-5 (a), FP3-10 (b), FP8-7 (c), and FP10-16 (d) were tested for their ability to lyse LB 27.4 target cells by incubation at the indicated E:T ratios for 3 h. LB 27.4 cells were incubated either with peptide i (□, ○, ▵) or with the control peptide of the same length, ie (●). Killing assays were performed in the absence (○, ●) or presence of anti-CD4 (□) or anti-CD8 (▵) MAb. Assays were performed at least twice, and the results were consistent with the representative data shown here.

Article Snippet: Antibodies and their final concentrations used in the present study were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (rat immunoglobulin G2b [IgG2b]; Seikagaku Corporation, Tokyo, Japan) at 0.5 μg/10 6 cells, phycoerythrin (R-PE)-conjugated anti-mouse CD8 (rat IgG2a; Caltag Laboratories, Burlingame, Calif.) at 1 μg/10 6 cells, FITC-conjugated anti-mouse CD69 (hamster IgG; PharMingen, San Diego, Calif.) at 1 μg/10 6 cells, R-PE-conjugated anti-mouse B220 (rat IgG2a; Coulter Immunology, Hialeah, Fla.) at 0.5 μg/10 6 cells, FITC-conjugated anti-NK1.1 (mouse IgG2a; PharMingen) at 2 μg/10 6 cells, biotin-conjugated anti-mouse Pan-NK (DX5, rat IgM; PharMingen) at 1 μg/10 6 cells, and allophycocyanin-conjugated anti-mouse TER-119 (PharMingen) at 0.2 μg/10 6 cells.

Techniques: Clone Assay, Incubation

Journal:

Article Title: Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia

doi: 10.1128/JVI.75.7.3152-3163.2001

Figure Lengend Snippet: In vivo depletion of asialo-GM1+ cells and its effect on T cells and protective immunity against FV infection induced by peptide immunization. (a through d) Mice used for the experiments whose results are shown in Fig. ​Fig.77 were also analyzed for the presence of CD4+ and CD8+ T cells in the spleen and their ability to mount viral-antigen-specific CD4+ T-cell responses. Flow-cytometric analyses for the expression of CD4 and CD8 were performed by using pooled whole spleen cells obtained from the mice injected with anti-asialo-GM1 Ab (b) or normal rabbit serum (a). Experiments were performed twice at PID 7 and 9, and results obtained from the repeated experiments were consistent with the representative data shown here. Numbers indicate percentages of CD4+ and CD8+ cells among live nucleated spleen cells. B220− CD8− CD4+ T cells purified for the experiments whose results are shown in Fig. ​Fig.7g7g and h were also tested for their proliferative activities in response to stimulation with peptide i. CD4+ T cells purified from the mice injected with anti-asialo-GM1 Ab (d) and those purified from control mice given normal rabbit serum (c) were incubated with X-irradiated syngeneic spleen cells and the indicated amount of peptide i (○). As controls, the CD4+ T cells purified from the anti-asialo-GM1 Ab-injected mice were also stimulated with an endogenous retroviral env-derived peptide ie (●) and the influenza virus nucleoprotein-derived peptide NP366–374 (▵). Experiments were performed twice, and results obtained from the repeated experiments were consistent with the representative data shown here. (e) Development of FV-induced leukemia in CB6F1 mice immunized with peptide i. Mice were either immunized with 10 μg of peptide i each (○, ▵, □) or given CFA alone (●). Two groups of the immunized mice were then injected with anti-asialo-GM1 Ab (▵) or control rabbit serum (□), while the remaining group (○) was not injected with any Ab. All mice were inoculated with 150 SFFU of FV.

Article Snippet: Antibodies and their final concentrations used in the present study were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (rat immunoglobulin G2b [IgG2b]; Seikagaku Corporation, Tokyo, Japan) at 0.5 μg/10 6 cells, phycoerythrin (R-PE)-conjugated anti-mouse CD8 (rat IgG2a; Caltag Laboratories, Burlingame, Calif.) at 1 μg/10 6 cells, FITC-conjugated anti-mouse CD69 (hamster IgG; PharMingen, San Diego, Calif.) at 1 μg/10 6 cells, R-PE-conjugated anti-mouse B220 (rat IgG2a; Coulter Immunology, Hialeah, Fla.) at 0.5 μg/10 6 cells, FITC-conjugated anti-NK1.1 (mouse IgG2a; PharMingen) at 2 μg/10 6 cells, biotin-conjugated anti-mouse Pan-NK (DX5, rat IgM; PharMingen) at 1 μg/10 6 cells, and allophycocyanin-conjugated anti-mouse TER-119 (PharMingen) at 0.2 μg/10 6 cells.

Techniques: In Vivo, Infection, Expressing, Injection, Purification, Incubation, Irradiation, Derivative Assay

Journal: PLoS ONE

Article Title: A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

doi: 10.1371/journal.pone.0000353

Figure Lengend Snippet: (A) Lymph nodes cells from 6–8 week old Ag pos and Ag neg mice were stained with antibodies against CD4, CD8 and Vβ7 and analysed by flow cytometry. Numbers in quadrants are percentage of live-gated lymphocytes. (B) Lymph node cells were stained with antibodies against CD8, Vβ7 and the activation markers CD44, CD62L, CD69 and CD25. Histograms display gated viable CD8 + Vβ7 + cells. Numbers in histograms represent the specific MFI of the activation marker staining for the gated population. Data are representative of at least three independent experiments.

Article Snippet: The following mAbs were used for flow cytometric staining: rat-anti-mouse Vβ7 FITC (Serotec, UK), rat-anti-mouse CD4 FITC, rat-anti-mouse CD8α APC, rat-anti-mouse CD8α Cy-Chrome 5 (CY-5), rat-anti-mouse Vβ7 PE, hamster-anti-mouse CD69 PE, rat-anti-mouse CD25 PE, rat-anti-mouse CD62L-FITC, rat-anti-mouse CD44 PE, rat-anti-mouse CD43 activation-associated glycoform-PE (all BD Biosciences).

Techniques: Staining, Flow Cytometry, Activation Assay, Marker

Journal: The Journal of Experimental Medicine

Article Title: Anergy in Peripheral Memory Cd4 + T Cells Induced by Low Avidity Engagement of T Cell Receptor

doi:

Figure Lengend Snippet: Anergized cells are able to expand in the presence of rIL-2. Splenocytes and lymph node cells from anergized and nonanergized mice were cultured without peptide, with peptide (10 μM) alone or with rIL-2 (10 U/ml) for 8 d. Cells were stained with HA-DR1-SA-PE oligomers and anti-CD4–FITC.

Article Snippet: 9 d after the second peptide injections, 5 × 10 6 cells of HLA-DR1 Tg mice were cultured either with peptide (10 μM) alone or with rIL-2 (10 U/ml) for 8 d. Cells were stained with HA-DR1-SA-PE oligomers (provided by T. Cameron and L. Stern, MIT, Cambridge, MA) for 3.5 h at 37°C followed by anti–mouse CD4-FITC or anti–mouse CD4-Cyc and anti–mouse CTLA-4–FITC (Southern Biotechnology Associates, Inc.).

Techniques: Cell Culture, Staining