16-1278-82

antibody from Invitrogen Antibodies

Targeting: IL7R CD127, IL7RA

Provider product page for 16-1278-82

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Antibody data

Antibody Data
Antigen structure
References [19]
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Validations
Flow cytometry [1]
Other assay [24]

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Product number: 16-1278-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD127 Monoclonal Antibody (eBioRDR5), Functional Grade, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The eBioRDR5 monoclonal antibody reacts with human CD127 (Interleukin-7 Receptor alpha). CD127 complexes with CD132, also known as the common gamma chain (gamma c), to form the multi-functional IL-7 receptor (IL-7R). CD127 is a type I glycoprotein with a molecular weight of 75-80 kDa and is expressed by immature B cells through the early pre-B stage, by thymocytes during several stages of their development, and on most mature T cells, with transient down-regulation upon activation. Binding of IL-7 results in signal transduction which occurs through several tyrosine kinase pathways including the Jak/STAT pathway. IL-7 is indispensible for the development of lymphocytes, and the control of homeostatic proliferation of T-cells in the periphery. In addition, IL-7R signaling is know to be involved in the regulation of T cell receptor (TCR) locus rearrangement in gamma delta T cells. Interestingly, recently it has been demonstrated that CD127 expression is down-regulated on CD4+CD25+ regulatory T cells (T regs). While the co-expression of CD4 and CD25 has become widely used as an indicator of T regs, this method of identification may also include cells without suppressive activity. It has clearly been shown that CD4+CD25+ cells that have down-regulated the expression of CD127 are significantly more highly-enriched for the regulatory T population, as defined by expression of the T reg-specific transcription factor Foxp3 and the suppressive activity of these cells, in vitro. Binding of the eBioRDR5 monoclonal antibody to PBMCs is blocked by pre-incubation of the cells with recombinant human IL-7 (Product # 14-1079-80). Applications Reported: This eBioRDR5 antibody has been reported for use in flow cytometric analysis. Applications Tested: This eBioRDR5 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at less than or equal to 0.06 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Storage and handling: Use in a sterile environment. Filtration: 0.2 µm post-manufacturing filtered. Purity: Greater than 90%, as determined by SDS-PAGE. Endotoxin Level: Less than 0.001 ng/µg antibody, as determined by LAL assay. Aggregation: Less than 10%, as determined by HPLC.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: eBioRDR5
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: 4° C

IL7R protein structure - 16-1278-82 shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Example staining and gating. Cryopreserved PBMCs from healthy human donors were thawed, rested over night, and stained as outlined in the online protocol. The gating strategy for the identification of T cell subsets is shown.

Supportive validation

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Experimental details: Figure 2. Expression of FoxP3 on different CD4 + CD127 +/- human T cell subsets. (a) PBMCs were harvested from human peripheral blood and stained with CD4, CD25, CD127 as well as intracellularly with FoxP3-specific mAbs, then analyzed on a Becton-Dickinson FACSCalibur. (b) Human PBMCs were stained for cell surface expression of CD4 and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD127 and FoxP3 expression. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. Data are representative of >20 independent individuals and >10 experiments.

Supportive validation

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Experimental details: Figure 3. Expression of FoxP3 on different CD4 + CD127 +/- mouse T cell subsets. Mouse spleen and lymph node cells were stained for cell surface expression of CD4 and CD127. For analysis, the spleen cells from FoxP3-GFP mice were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD127 and FoxP3 (GFP) expression. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. The letters A and B in the quadrants represent the gray and thick solid lines (bottom). (b) Spleen and lymph node cells isolated from FoxP3 transgenic mice were stained for cell surface expression of CD4, CD25, and CD127. For analysis, the spleen cells were gated on CD4 + lymphocytes (based on forward and side light scatter) and analyzed for CD127 and FoxP3 expression. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. The letters A, B, and C in the quadrants represent the dotted, thin solid, and thick solid lines (bottom).

Supportive validation

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Experimental details: Figure 4. Expression of FoxP3 on different CD4 + T cell subsets. (a) Human PBMCs were stained for cell surface expression of CD4, CD25, and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on CD4 + lymphocytes (based on forward and side light scatter and CD4 staining) and analyzed for CD127 and FoxP3 expression. The boxes represent arbitrary designations of CD25 + versus CD25 - cells. The numbers in the histograms indicate the percentage of gated cells expressing the relevant marker. (b) Human PBMCs were stained for cell surface expression of CD4, CD25, and CD127. The stained cells were fixed and stained intracellularly for FoxP3. For analysis, the PBMCs were gated on lymphocytes (based on forward and side light scatter) and analyzed for CD4, CD25, CD127, and FoxP3 expression. The boxes represent arbitrary designations of CD127 + versus CD127 lo/- cells. The numbers in the dot plot indicate the percentage of gated cells expressing the relevant marker. (c) Similar staining and analysis was performed on whole blood obtained from 10 healthy individuals. Each symbol represents an individual person and the narrow bar represents the mean percentage of FoxP3 + T cells on either CD4 + T cells gated based on CD25 and/or CD127 expression.

Supportive validation

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Experimental details: Figure 3. Dex treatment increases the proliferation and activation state of Tregs. (A) Representative flow cytometry data, demonstrating the gating strategy used for Treg identification and analysis. Forward scatter (FSC) area vs. FSC-height was used for doublet discrimination, and lymphocytes subsequently selected by FSC vs. side scatter. A ""dump"" channel was used to gate out dead cells (LIVE/DEAD fixable aqua viability stain), CD14 + , CD56 + , and CD19 + cells. CD4 + T cells were subsequently selected on the basis of CD4 vs. CD3 staining, followed by the identification of Tregs as CD25 hi , CD127 lo , and Foxp3 + . Tregs, or non-Treg CD4 + T cells, were further gated for expression of Ki67 and ICOS. (B-F) Analysis of Tregs in patient PBMC samples collected before (pre-Dex) and after (post-Dex) administration of Dex. (B) Percentage of Tregs (CD25 + CD127 lo Foxp3 + ) as a proportion of total CD4 + T cells in PBMC samples. (C-D) Proportion of Tregs (C) and non-Treg CD4 + lymphocytes (D) expressing the proliferation marker Ki67. (E-F) Changes in proportional expression of the activation marker ICOS, in Treg (E) and non-Treg CD4 + lymphocytes (F). Each dot represents an individual patient; significant difference between pre-Dex and post-Dex values: *** p < 0.0001, paired students t-test.

Supportive validation

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Experimental details: Example gating strategy for major immune cell subsets on stained PBMCs from healthy donors. [Color figure can be viewed at wileyonlinelibrary.com ]

Supportive validation

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Experimental details: FIGURE 1 Identification of Tscm CD4 + CD45RA + CD45RO - CD62L + CCR7 + CD127 + CD27 + CD28 + CD95 + CD122 + T (Tscm) cell in human blood and lymph nodes. PBMCs were isolated from the blood of non-small cell lung cancer (NSCLC) patients (n=15) (NSCLC-PBMC) and healthy donors (n=11) (HD-PBMC); lymphocytes were isolated from the tumor-infiltrated lymph node of NSCLC patients who were collected blood at same time (n=7) (NSCLC-Ly); lymphocytes were isolated from the healthy lymph node of non lung cancer patients (n=7) (Normal-Ly), analyzed by flow cytometry. A, Representative flow cytometric analyses of CD4 + CD45RA + CD45RO - CD62L + CCR7 + CD127 + CD27 + CD28 + CD95 + CD122 + T cells, indicating Tscm cells. B, The frequency of the CD4 + Tscm cells in the HD-PBMC, NSCLC-PBMC, Normal-Ly, NSCLC-Ly. The events of CD4 + Tscm cells in the blood and lymph node from NSCLC patients and healthy donors, expressed as the mean+-SEM. C, The frequency of the CD8 + Tscm cells in the HD-PBMC, NSCLC-PBMC, Normal-Ly, NSCLC-Ly, expressed as the mean+-SEM. D, The events of Tscm of CD4 + and CD8 + cells in the blood and the lymph node from NSCLC patients and healthy donors. HD indicates healthy donors; IFN, interferon; PBMC, peripheral blood mononuclear cells; Tscm cell, stem cell-like memory T cell. * P

Supportive validation

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Experimental details: FIGURE 3 CD4 + CD45RA + CD45RO - CD95 - CD122 + CD127 + T cell population displays different phenotypes in human blood and lymph nodes. A, Flow cytometry plots showing IFN-gamma expression in the CD4 + CD45RA + /CD45RO - CD95 - CD122 + CD127 + T cells from the blood and lymph node of the non-small cell lung cancer (NSCLC) patients and healthy donors. B, The mean frequency (+-SEM) of the events of CD4 + CD45RA + /CD45RO - CD95 - CD122 + CD127 + T cells. C, The mean IFN-gamma production (+-SEM) of the CD4 + CD45RA + /CD45RO - CD95 - CD122 + CD127 + T cells. HD indicates healthy donors; IFN, interferon; PBMC, peripheral blood mononuclear cells; PMA, phorbol 12-myristate13-acetate. * P

Supportive validation

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Experimental details: Fig. 6 a Representative flow cytometry data, demonstrating the gating strategy used on PBMC for Treg identification and analysis. FSC-area vs. FSC-height was used for doublet discrimination. A ""dump"" channel was used to gate out dead cells (LIVE/DEAD fixable viability stain), CD14 + monocytes and CD19 + B cells, and lymphocytes were subsequently selected by FSC vs. SSC. CD4 + T cells were gated on the basis of CD4 vs. CD3 staining, then examined for expression of Ki67 and ICOS. Tregs were identified within the CD4 + T cell population as CD25 hi CD127 lo and Foxp3 + . b Longitudinal empirical data, linear mixed models and estimated means (left, centre and right-hand panels respectively) for Ki67+ and ICOS+ expression on CD4+ T cells, and the Treg proportion of CD4 cells ( P -values: *

Supportive validation

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Experimental details: TIM-3 + Treg cells suppress Tresp-cell proliferation better than TIM-3 - Treg cells. CD4 + T cells were stained and sorted into Treg cells (CD4 + CD25 high CD127 low/- ) by flow cytometry and activated with anti-CD3/anti-CD28 for 3 days. Then, TIM-3 + and TIM-3 - Treg cells were resorted by flow cytometry and Tresp cells (CD4 + CD25 low/- CD127 + ) were sorted in parallel. CFSE-labeled Tresp cells were cocultured with TIM-3 + or TIM-3 - Treg cells at various Treg:Tresp ratios ranging from 1:2 to 1:8. At day 3, cells were collected, stained for viability and fixed. (A) Proliferation of unstimulated (upper left panel) or stimulated (upper right panel) Tresp cells alone and Tresp-cell cocultures with TIM-3 + or TIM-3 - Treg cells (lower panels left and right respectively) was determined by flow cytometry. One representative experiment of four independent experiments is shown. (B) Percentage of suppression was calculated from results obtained as in (A), per the following formula: % suppression = [1 - (proliferation of Tresp cells in coculture / Tresp cells alone)] x 100. Data are shown as mean +- SEM of eight samples pooled from four independent experiments. Statistical significance determined by a two-tailed standard t -test.

Supportive validation

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Experimental details: LBS effect on immune cells profile. Fluorescent-activated cell sorting (FACS) analysis was used to compare frequencies of (A-D) CD4 + IL-10 + T cells, CD39 + CD127 low CD25 high T cells, CD4 + LAP + T cells, and effector memory CD8 T cells in healthy controls (HC) and multiple sclerosis patients (MS) at the indicated time points determined by Wilcoxon signed-rank test. * p < 0.05; # p < 0.1. FACS analysis was used to compare (E,F) frequencies of intermediate monocytes, inflammatory monocytes, as well as CD80 mean fluorescence intensity (MFI) on classical monocytes and HLA-DR MFI on dendritic cells in HC and MS patients at the indicated time points determined by Wilcoxon signed-rank test, * p < 0.05; ** p < 0.01; # p < 0.1. The following markers were used for the lineage pool: CD3, CD14, CD16, CD19, CD20, and CD56. HLA-DR = human leukocyte antigen D related.

Supportive validation

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Experimental details: Figure 6 In vivo knockdown of PITPNM3 in CD4 + T cells reverses immunosuppression and inhibits tumor progression in humanized mice. (A) Humanized mice bearing palpable MDA-MB-231 orthotopic xenografts were intraperitoneally injected daily for 14 days with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 (sequence in A , AsiC-PI) to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Experimental schematic is provided in Supplementary information, Figure S9A . (B) Representative immunoblots showing selective knockdown of PITPNM3 protein in PB CD4 + T cells, but not tumor xenografts ( n = 3). (C) PITPNM3 knockdown did not affect the distribution of human CD45 + hematopoietic cells, CD4 + and CD8 + T cells, and CD14 + monocytes in the peripheral blood of humanized mice. Representative flow plots are shown ( n = 3). (D , E) Effect of PITPNM3 knockdown on TI naive CD4 + , Tregs and CD8 + T cell numbers, and apoptosis by TUNEL assay in xenografts. D shows representative immunofluorescence microscopy images. Top row indicates CD4 + naive T cells by arrows; the second row indicates CD4 + CD45RO + Foxp3 - CD4 + memory T cells (yellow arrows) and Foxp3 + Tregs (white arrows). Scale bar, 50 mum. E shows number of cells of each subtype/high power field in eight mice ( ** P < 0.01, *** P < 0.001 compared to PBS group by Student's t -test). (F) Flow cytometry analysis of gated human CD3 + CD4 + cells isolated from xenogra

Supportive validation

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Experimental details: Figure 4. miR-21 Promotes Effector Cell Differentiation through AP-1 Activation (A) Naive CD4 + T cells were transfected with either scrambled control or miR-21-blocking locked nucleic acid (LNA21). After 48 hr, PDCD4 and b-actin expression were assessed by western blot. Representative blots and mean normalized intensities from four experiments are shown (mean +- SEM, two-tailed paired t test). (B) Naive CD4 + T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control or anti-miR-21. The representative histogram shows phosphorylated JNK in GFP + cells on day 3. The filled gray histogram represents unstimulated naive CD4 + T cells. Results from 7 experiments are expressed relative to the geometric mean fluorescence intensity (MFI) of controls (mean +- SEM, two-tailed paired t test). (C) Naive CD4 + T cells were activated and transduced as described in (B) and co-transfected with the AP-1 luciferase reporter plasmid and the Renilla luciferase control construct. On day 3, the activity of AP-1 firefly luciferase was measured and normalized to that of Renilla luciferase (n = 4, mean +- SEM, two-tailed paired t test). (D-F) Naive CD4 + T cells isolated from 20- to 35-year-old and 65- to 85-year-old individuals were activated with beads coated with anti-CD3 and anti-CD28 antibodies. (D) PDCD4 expression was quantified by RT-PCR on day 3 and day 5. Results are normalized to ACTB and presented relative to those of cell