Journal: iScience

Article Title: Atypical B cells consist of subsets with distinct functional profiles

doi: 10.1016/j.isci.2023.108496

Figure Lengend Snippet:

Article Snippet: One day prior to sorting B cells, wells of a 96-well plate were each seeded with 30,000 adherent, CD40L-expressing 3T3 cells (kind gift from Dr. Mark Connors, NIH) in 100 μl IMDM/Glutamax/ 10% FBS containing 2× MycoZap Plus-PR (Lonza #VZA-2021), 100 ng/ml human IL-2 (GoldBio #1110-02-50), and 100 ng/ml human IL-21 (GoldBio #1110-21-10) to promote expansion and differentiation of B cells into antibody-secreting cells.

Techniques: Staining, Recombinant, Cell Isolation, Multiplex Assay, Sequencing, Plasmid Preparation, Software

Journal: The Journal of Experimental Medicine

Article Title: Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection

doi: 10.1084/jem.20082387

Figure Lengend Snippet: IL-10 production by NK cells from MCMV-infected Prf1 −/− mice. (A) The top dot plots show the purity of NK cells (CD49b + ) and non–NK cells (CD49b − ) isolated from day 4 MCMV-infected wt and Prf1 −/− mice after CD49b + magnetic isolation. The numbers indicate percentages of cells in each box. Cytokine production of IFN-γ and IL-10 in the serum of infected wt and Prf1 −/− mice, or in 24 h conditioned media prepared with total splenic leukocytes and isolated subsets were determined. (B) The top dot plots show purity of NK cells (CD49b + Ly49H + ) and non–NK cells (CD49b − Ly49H − ) cells of day 4 MCMV-infected Prf1 −/− mice after flow sorting. The numbers indicate percentages of cells in each box. Cytokine production of IFN-γ and IL-10 in serum of infected Prf1 −/− mice, or in 24 h conditioned media from total splenic leukocytes and isolated subsets were determined. (C) Expression of IL-10 , IL-21 , and IL-15 in the total and sorted populations from day 4 MCMV-infected Prf1 −/− mice was analyzed by semiquantitative RT-PCR. Gapdh was used as an internal control. (D) Total splenic leukocytes, highly purified NK, and non–NK subsets from day 4 MCMV-infected Prf1 −/− mice were stimulated either on anti-Ly49H antibody or with cytokines. After 24 h of incubation, IL-10 production was determined in conditioned media. Results are representative of at least two independent experiments using a total of two to four individual mice. The studies in A were repeated twice for wt and four times for Prf1 −/− mice. Those in B were repeated three times, and those in C were repeated twice. The studies in D with anti-Ly49H antibody were repeated twice, and those with cytokines were repeated four times. The dashed white line indicates that intervening lanes have been spliced out. φ, not detected.

Article Snippet: For further stimulation, 10 5 highly purified cells were incubated either on control antibody (mouse IgG1)– and anti-Ly49H antibody–coated plates or with 100 ng/ml of mouse recombinant IL-21 (>97% purity; R&D Systems) in the presence or absence of 50 ng/ml IL-15 (>95% purity; R&D Systems).

Techniques: Infection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Incubation

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Increased interleukin-21 (IL-21) in sera and CD4+ T cells of patients with systemic lupus erythematosus (SLE). (a) The concentrations of IL-21 in sera isolated from 22 SLE patients and 16 healthy controls were analysed by ELISA. (b) Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells were isolated from heparinized peripheral blood obtained from three patients and three healthy controls. The mRNA expression levels of IL-21 and IL-21 receptor were determined by RT-PCR. Results are expressed as means ± standard deviation (SD). (**P < 0·001).

Article Snippet: Mouse IL-21 monoclonal antibody (R&D Systems, Minneapolis, MN) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Oestrogen treatment increased interleukin-21 (IL-21) expression in CD4+ T cells of systemic lupus erythematosus (SLE) patients in a dose- and time-dependent manner. (a, b) Isolated CD4+ T cells were treated with 10, 100 and 1000 nm 17β-oestradiol for 48 hr. The mRNA expression of IL-21 was measured by RT-PCR (a). The concentration of IL-21 in the supernatant was determined by ELISA (b). (c) CD4+ T cells were cultured with 1000 nm 17β-oestradiol for 24, 48 and 72 hr. The concentration of IL-21 in the supernatant was determined by ELISA. Data are expressed as means ± SD of three independent experiments using cells obtained from three patients and three healthy controls. (*P < 0·05, **P < 0·01, versus untreated cells of SLE patients).

Article Snippet: Mouse IL-21 monoclonal antibody (R&D Systems, Minneapolis, MN) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Synergistic effect of T-cell receptor (TCR) stimulation on interleukin-21 (IL-21) production by oestrogen-pretreated CD4+ T cells in systemic lupus erythematosus (SLE) patients. Isolated CD4+ T cells from SLE patients were cultured with 10, 100 and 1000 nm 17β-oestradiol for 48 hr in the presence of 0·5 μg/ml CD3 antibody. The concentration of IL-21 in the supernatant was determined by ELISA. Data are expressed as means ± SD of three independent experiments using cells obtained from three different patients. (*P < 0·05, versus cells treated with 1000 nm 17β-oestradiol only).

Article Snippet: Mouse IL-21 monoclonal antibody (R&D Systems, Minneapolis, MN) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°.

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Signalling pathways involved in oestrogen-induced interleukin 21 (IL-21) production. (a) Isolated CD4+ T cells from systemic lupus erythematosus (SLE) patients were pre-treated with 20 μm PD98509, 10 μm SB203850, 1 μm SP600125, 20 μm LY294002, 50 μm AG490, 50 μm BAY 11, or 10 μm curcumin for 2 hr, and then cultured with 1000 nm of 17β-oestradiol for 48 hr. The concentration of IL-21 in the supernatant was measured by ELISA. Data are expressed as means ± SD of three independent experiments using cells obtained from three different patients. (b) CD4+ T cells from SLE patients were treated with 1000 nm 17β-oestradiol for 20 min. Phosphorylated forms of mitogen-activated protein kinases were measured by Western blot analysis. The representative Western blots of three independent experiments using cells from three different patients are shown. (*P < 0·05, **P < 0·01).

Article Snippet: Mouse IL-21 monoclonal antibody (R&D Systems, Minneapolis, MN) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°.

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Increased antibody secretion by B cells co-cultured with oestrogen-stimulated CD4+ T cells. (a, b) CD4+ T cells from systemic lupus erythematosus (SLE) patients were treated with 1000 nm 17β-oestradiol for 48 hr. CD4+ T cells and culture supernatants were then separated by centrifugation. B cells isolated from the peripheral blood of healthy controls were cultured for 96 hr with oestrogen-pretreated T cells (a) and the culture supernatant (b), respectively, in the absence or presence of interleukin-21 (IL-21) blocking antibody. The concentrations of secreted IgG, IgG1 and IgG2a by B cells were measured by ELISA. Data are expressed as means ± SD of three independent experiments using cells from three patients and three healthy controls. (*P < 0·05, **P < 0·01, versus untreated cells, #P < 0·05, ##P < 0·01, versus 1000 nm 17β-oestradiol-treated cells).

Article Snippet: Mouse IL-21 monoclonal antibody (R&D Systems, Minneapolis, MN) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°.

Techniques: Cell Culture, Centrifugation, Isolation, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: IL-2 and IL-21 confer opposing differentiation programs to CD8 + T cells for adoptive immunotherapy

doi: 10.1182/blood-2007-09-113050

Figure Lengend Snippet: Antigen-induced acquisition of effector CD8+ T-cell phenotype is promoted by IL-2 and IL-15 but suppressed by IL-21. Naive pmel-1 Thy1.1 CD8+ T cells were primed with cognate antigen and the indicated cytokine for 3 days. (A-C) Flow cytometric determination of granzyme B, CD44, and IL-2Rα expression by Thy1.1 lymphocytes. (D) Real-time RT-PCR determination of Eomes expression 3 days after priming in 10 ng/mL of the indicated cytokine. Error bars indicate the standard error of the mean. (E) CFSE dilution 4 days after antigen priming of naive pmel-1 Thy1.1 CD8+ T cells with the indicated cytokine. Histograms are gated on Thy1.1 cells. The open histogram overlay indicates CFSE labeling prior to stimulation.

Article Snippet: Recombinant human IL-2 (Novartis, Emeryville, CA), recombinant human IL-15 (Peprotech, Rocky Hill, NJ), or recombinant murine IL-21 (R&D Systems, Minneapolis, MN) were added to the culture conditions as indicated.

Techniques: Expressing, Quantitative RT-PCR, Labeling

Journal:

Article Title: IL-2 and IL-21 confer opposing differentiation programs to CD8 + T cells for adoptive immunotherapy

doi: 10.1182/blood-2007-09-113050

Figure Lengend Snippet: In contrast to IL-2 and IL-15, IL-21 does not promote acquisition of effector CD8+ T-cell function. (A,B) Pmel-1 CD8+ T cells were antigen-primed with 10 ng/mL of the indicated cytokine for 4 days, washed, and cocultured overnight with irradiated splenocytes pulsed with hgp10025-33. IFN-γ and IL-2 concentrations in the supernatants were determined by ELISA. (C) Chromium release cytolysis assay showing specific target killing by pmel-1 CD8+ T cells antigen-primed with 10 ng/mL of the indicated cytokine.

Article Snippet: Recombinant human IL-2 (Novartis, Emeryville, CA), recombinant human IL-15 (Peprotech, Rocky Hill, NJ), or recombinant murine IL-21 (R&D Systems, Minneapolis, MN) were added to the culture conditions as indicated.

Techniques: Cell Function Assay, Irradiation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: IL-2 and IL-21 confer opposing differentiation programs to CD8 + T cells for adoptive immunotherapy

doi: 10.1182/blood-2007-09-113050

Figure Lengend Snippet: The antitumor efficacy of CD8+ T cells for adoptive transfer is impaired by IL-2 and IL-15, but enhanced by IL-21. Pmel-1 CD8+ T cells were primed with cognate antigen and 10 ng/mL of the specified cytokine for 4 days then adoptively transferred into tumor-bearing hosts. Vaccination and exogenous IL-2 were administered to mice in all but the “No treatment” group. (A) Tumor response to adoptive transfer of 2.5 × 105 cells. The cytokine present during priming is indicated. Error bars reflect the standard error of the mean. (B) Pmel-1 Thy1.1 CD8+ T cells were antigen-primed with 10 ng/mL of the indicated cytokine. At 4 days after priming, 5 × 105 cells per mouse were infused, and vaccine and IL-2 were administered. The number of Thy1.1 splenocytes was determined 12 days after transfer. The scatter plots indicate individual mice. The horizontal lines represent the means. *P < .05 compared with “No cytokine.”

Article Snippet: Recombinant human IL-2 (Novartis, Emeryville, CA), recombinant human IL-15 (Peprotech, Rocky Hill, NJ), or recombinant murine IL-21 (R&D Systems, Minneapolis, MN) were added to the culture conditions as indicated.

Techniques: Adoptive Transfer Assay

Journal:

Article Title: IL-2 and IL-21 confer opposing differentiation programs to CD8 + T cells for adoptive immunotherapy

doi: 10.1182/blood-2007-09-113050

Figure Lengend Snippet: IL-21 suppresses IL-2–induced effector CD8+ T-cell differentiation and substantially prevents IL-2–mediated impairment of CD8+ T cells for adoptive transfer. Naive pmel-1 CD8+ T cells were antigen-primed with 10 ng/mL IL-2, IL-21, or 10 ng/mL IL-2 combined with 10 ng/mL IL-21. (A) Eomes expression was determined by RT-PCR 3 days following priming. (B) Granzyme B expression was assessed by flow cytometry 3 days after priming. The mean fluorescence intensity is indicated. (C) Pmel-1 CD8+ T cells were primed with cognate antigen and 10 ng/mL of the indicated cytokine(s). After 4 days, 5 × 105 cells per mouse were adoptively transferred into tumor-bearing recipients. Vaccine and IL-2 were administered to all but the “No treatment” group. Tumor responses were assessed with serial measurements. Error bars represent the standard error of the mean.

Article Snippet: Recombinant human IL-2 (Novartis, Emeryville, CA), recombinant human IL-15 (Peprotech, Rocky Hill, NJ), or recombinant murine IL-21 (R&D Systems, Minneapolis, MN) were added to the culture conditions as indicated.

Techniques: Cell Differentiation, Adoptive Transfer Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Fluorescence

Journal:

Article Title: IL-2 and IL-21 confer opposing differentiation programs to CD8 + T cells for adoptive immunotherapy

doi: 10.1182/blood-2007-09-113050

Figure Lengend Snippet: Antigen priming with IL-21 imparts CD8+ T cells with a distinct gene expression program. Pmel-1 CD8+ T cells were antigen-primed with the cytokine indicated or without cytokine for 4 days, then restimulated for 4 hours without cytokine. Microarray gene expression analysis was then performed. Expression levels relative to the reference cells primed without cytokine are indicated. (A) Dendrogram showing the relatedness of gene expression patterns. (B) Microarray expression of selected genes associated with mature and immature effector CD8+ T cells. (C,D) Wild-type CD8+ T cells were stimulated with anti-CD3/anti-CD28 and 10 ng/mL of the indicated cytokine for 3 days and then restimulated in cytokine-neutral conditions for (C) 4 hours or (D) 3 days before real-time RT-PCR analysis. Expression of Tcf7 and Lef1 relative to β-actin is shown.

Article Snippet: Recombinant human IL-2 (Novartis, Emeryville, CA), recombinant human IL-15 (Peprotech, Rocky Hill, NJ), or recombinant murine IL-21 (R&D Systems, Minneapolis, MN) were added to the culture conditions as indicated.

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal:

Article Title: IL-2 and IL-21 confer opposing differentiation programs to CD8 + T cells for adoptive immunotherapy

doi: 10.1182/blood-2007-09-113050

Figure Lengend Snippet: Antigen priming with IL-21 directs CD8+ T cells to express L-selectin during secondary stimulation. (A) Wild-type CD8+ T cells were anti-CD3/anti-CD28 stimulated with 10 ng/mL of the indicated cytokine for 3 days. Sell expression relative to β-actin expression was determined 4 hours after restimulation in cytokine-neutral conditions. (B) Four days after antigen priming with the indicated cytokine, pmel-1 Thy1.1 CD8+ T cells were restimulated with cognate antigen and 10 ng/mL IL-2 (regardless of the initial priming cytokine). Flow cytometry was performed 3 days after initiating the secondary response and histograms of L-selectin expression are displayed. The indicated cytokines and concentrations refer to the initial priming conditions. (C) Dot plots showing CFSE dilution and L-selectin expression by pmel-1 Thy1.1 CD8+ T cells that were antigen-primed with 100 ng/mL of the indicated cytokine for 4 days, labeled with CFSE, and restimulated with antigen and 10 ng/mL IL-2 (regardless of the cytokine present during initial priming). (D) Graphs of the mean fluorescence intensity of CFSE and L-selectin during restimulation. (E) Secondary expansion of cells antigen-primed with 10 ng/mL of the indicated cytokine for 4 days then restimulated with antigen and 10 ng/mL IL-2. Error bars indicate the standard error of the mean. (F) Tumor regression in response to adoptive transfer of 106 pmel-1 CD8+ T cells primed in 10 ng/mL of the indicated cytokine for 4 days, restimulated with antigen and IL-2 for 6 days, and then adoptively transferred. Vaccine and IL-2 were administered to mice in all except the “No treatment” group. Error bars reflect the standard error of the mean.

Article Snippet: Recombinant human IL-2 (Novartis, Emeryville, CA), recombinant human IL-15 (Peprotech, Rocky Hill, NJ), or recombinant murine IL-21 (R&D Systems, Minneapolis, MN) were added to the culture conditions as indicated.

Techniques: Expressing, Flow Cytometry, Labeling, Fluorescence, Adoptive Transfer Assay