61-4776-42

antibody from Invitrogen Antibodies

Targeting: FOXP3 AIID, DIETER, IPEX, JM2, PIDX, SCURFIN, XPID

Provider product page for 61-4776-42

Flow cytometry

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Antibody data

Antibody Data
Antigen structure
References [44]
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Validations
Flow cytometry [1]
Other assay [42]

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Product number: 61-4776-42 - Provider product page
Provider: Invitrogen Antibodies
Product name: FOXP3 Monoclonal Antibody (PCH101), PE-eFluor™ 610, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: eBioscience offers a panel of monoclonal antibodies to different epitopes of human Foxp3, providing useful tools for investigating the complete expression pattern of Foxp3 at the protein level, and discerning the precise subsets of Foxp3^+ cells.
Antibody clone number: PCH101
Concentration: 5 µL/Test

FOXP3 protein structure - 61-4776-42 shown in red.

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Supportive validation

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Experimental details: Figure 4 Intra-tumoral Treg Cell Depletion Is Required for the Anti-tumor Activity of Anti-CTLA-4 Mice were treated with 200 mug of anti-CTLA-4 on days 6 and 9 after s.c. inoculation of MCA205 tumor cells (n = 9-21). TILs, LNs, and PBMCs were processed on day 11 and stained for flow cytometry analysis. (A) Percentage of FoxP3 + CD4 + Treg cells from total CD4 + T cells. (B) CD8 + /Treg cell ratio in the indicated sites. Horizontal bars represent the mean. (C) Percentage of Ki67-expressing CD4 + FoxP3 - and CD8 + T cells. (D) Percentage of CD4 + FoxP3 - and CD8 + T cells expressing IFNgamma following re-stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin; cumulative data of three separate experiments. Error bars show +-SEM. (E and F) hFcgammaR mice were treated with anti-CTLA-4 on days 6, 9, and 12 after s.c. inoculation of MCA205 (50 mug/dose), MC38 (100 mug/dose) or B16 (200 mug/dose) tumor cells. (E) MCA205 tumor growth in individual hFcgammaR mice in each treatment group. Inset numbers show the fraction of mice with complete long-term response. (F) Kaplan-Meier curves demonstrating survival of hFcgammaR mice for each tumor model. The total number of mice in each treatment group is shown at the right. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. See also Figure S4 .

Supportive validation

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Experimental details: Figure 1. Flow cytometric mapping of the CD4 + CD25 + Foxp3 + cells in peripheral blood withdrawn from the syphilitic patients with sero-resistance. Foxp3, forkhead box P3.

Supportive validation

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Experimental details: Fig. 4 OVA DNT treatment selectively inhibited Tfh cells and CD11b + DCs. OVA-sensitized mice were treated with an intravenous transfer of OVA DNTs after the first OVA challenge. The mice were challenged daily for the next two days and sacrificed 48 h after the last aerosol challenge. a The lung and BALF Tfh cell (CD4 + B220 - CXCR5 + PD-1 + ), b CD4 + T cell (CD4 + B220 - ) and Treg cell (CD4 + B220 - Foxp3 + ) proportions were measured by flow cytometry. c The lung and mLN CD11b + DC (CD11c + MHC-II + CD11b + ), d DC (CD11c + MHC-9II + ) and CD103 + DC (CD11c + MHC-II + CD103 + CD11b - ) proportions were measured by flow cytometry. e The proportions of B cells (B220 + CD4 - ) in the mLN (mediastinum lymph node), BALF and lungs were measured by flow cytometry. OVA-stimulated bone marrow cells were cocultured with OVA DNTs and stimulated with GM-CSF (20 ng/ml) for 3 days to test the direct effect of OVA DNTs on OVA DC proliferation and differentiation. f The proportions of bone marrow-derived DCs and CD11b+ DCs were measured by flow cytometry. The direct effects of OVA DNTs on g costimulatory molecule expression and h apoptosis in DCs were measured by flow cytometry. i OVA tetramer + and tetramer - DNT cells were sorted by flow cytometry from OVA-primed DNT cells. Tetramer + or tetramer - DNT cells were cocultured with lung DCs from allergic asthma mice for 3 days. The MFIs of CD86 and MHC-II in DCs were measured by flow cytometry. j Lung DCs (2.5 x 10 4 ) from OVA DNT cell-t

Supportive validation

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Experimental details: Extended Data 9. Fetal Micrococcus isolate promotes distinct APC and T cell phenotypes. a. Proportion of live cells after treatment with media (n=9) or Micrococcus (Micro36 n=6, MicroRef1 n=9, MicroRef2 n=3) strains, where n represents biologically independent fetal specimens for the indicated treatment. ANOVA test for significance. b. HLA-DR + CD45 + lin - cells pre- (left) and post- (right) fluorescence activated cell sorting (FACS). c. Proportion of naive (CD45RA + CCR7 + ), central memory (TCM, CD45RA - CCR7 + ), and effector memory T cells (TEM, CD45RA - CCR7 - ) among live, TCRbeta + , CD4 + cells (left panel) and PLZF and CD161 expression among memory subsets, numbers indicate proportion in TEM (right panel). d. Pre- (left) and post- (right) FACS of effector memory T cells. e. Proportion of PLZF + T cells or f. left, proportion of CD25 hi FoxP3 + regulatory T cells (T regs ) and right, representative flow plots of FoxP3 and CD25 expression among intestinal live, TCRbeta + , CD4 + , Valpha7.2 - , cells after exposure to splenic APCs pretreated with media or Micrococcus (Micro36, MicroRef1) strains for n=5 biologically independent fetal specimens. Concentration of g. IL-17A, h. IL-17F, i. GM-CSF, j. IL-4, k. IL-10, l. IL-13, m. TNFalpha in culture supernatants of lamina propria T cell co-cultures with splenic antigen presenting cells pre-exposed to media (n=7) or Micrococcus (Micro36 n=6, MicroRef1 n=7, MicroRef2 n=7) strains, where n represents biologically independent

Supportive validation

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Experimental details: Figure 1. Patients with Parkinson's disease (PD) and mice with experimental PD exhibit decreased regulatory T (Treg) and increased T helper 1 (Th1) cell numbers in the blood. Treg cells were defined as CD3+CD4+CD25+FoxP3+ cells, whereas Th1 cells were identified as CD3+CD4+T-bet+ cells. (a) Representative plots of Treg and Th1 cells in patients with PD and healthy volunteers (HVs). (b) Representative plots of Treg and Th1 cells in control mice (Saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. (c) Patients with PD exhibited lower Treg cell levels and greater Th1 cell levels in the blood than HVs. n = 20. ****, p < 0.0001 by a two-tailed Student's t tests. (d) MPTP-induced experimental PD mice had fewer Treg and more Th1 cells in the circulation than saline-treated mice. n = 9/group in each experiment performed in triplicate. ***, p < 0.001; ****, p < 0.0001, according to Student's t test.

Supportive validation

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Experimental details: Figure 1. The percentage of T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMCs) and the Th17/Treg ratio varies in patients with a successful pregnancy after immunotherapy. The percentage of Th17 and Treg cells in PBMCs were detected by flow cytometry in patients with unexplained recurrent spontaneous abortion (URSA) before and after therapy. Representative CD3 + CD8 + IL-17A + flow cytometry plots from patients with URSA are shown (A) before and (B) after therapy. Representative CD4 + CD25 + Foxp3 + flow cytometry plots from patients with URSA are shown (C) before and (D) after therapy. (E) The percentage of Th17 cells in PBMCs significantly decreased after therapy. The percentage of Treg cells in PBMCs significantly increased after therapy (*P

Supportive validation

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Experimental details: Figure 1 Elevation of FABP4 is accompanied with Treg/Th17 imbalance in PE (A) The serum level of FABP4 was determined by ELISA (normal pregnant women, n = 10; PE, n = 20). (B) The immunoreactivities of FABP4, IL-17A, and FOXP3 in placental tissues were monitored by IHC analysis. (C and D) The percentages of Treg (C) and Th17 cells (D) in peripheral blood were assessed by FACS (normal pregnant women, n = 10; PE, n = 20). (E) The serum level of IL-17A was determined by ELISA (normal pregnant women, n = 10; PE, n = 20). (F) Pearson's correlation analysis between IL-17A and FABP4 expression. Data were representative images. *p < 0.05.

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Experimental details: Figure 1. Serum sFGL2 levels and circulating Tregs frequencies are decreased in patients with IHF. (A) Serum sFGL2 levels were analyzed by ELISA. (B) The frequency of Tregs was compared in each group. (C) Representative FSC/SSC pseudo-color density image shows the gated CD4 + T cells and representative fluorescence-activated cell sorting plots of CD4 + CD25 + Foxp3 + Tregs from a single patient in each group. ***P

Supportive validation

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Experimental details: Figure 2. Serum sFGL2 levels and circulating Tregs frequencies are decreased with the elevation of NYHA classification. (A) sFGL2 levels were analyzed by ELISA. (B) The proportion of Tregs was compared in each subgroup. (C) Representative fluorescence-activated cell sorting plots of CD4 + CD25 + Foxp3 + Tregs from a single person in each subgroup. **P

Supportive validation

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Experimental details: Figure 3. Serum sFGL2 levels and circulating Tregs frequencies are decreased with the reduction of LVEF. (A) sFGL2 levels were analyzed by ELISA. (B) The proportion of Tregs was compared in each subgroup. (C) Representative fluorescence-activated cell sorting plots of CD4 + CD25 + Foxp3 + Tregs from a single person in each subgroup. *P

Supportive validation

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Experimental details: Fig. 3 Investigation into PD Treg populations shows reduced numbers and impaired function. The number of Tregs in PD patients are decreased via a flow cytometric analysis of CD4 + CD25 + FOXP3 + cells as a percent of total CD4 + population and b counting of viable CD4 + CD25 + immune cells following bead/column-based isolation methods from peripheral blood (C n = 28, PD n = 34). Treg health and function markers were deceased in PD patients compared to controls when examining c CD25 protein MFI and d FOXP3 protein MFI from CD4 + CD25 + FOXP3 + cells during flow cytometry (C n = 28, PD n = 34). e Treg suppression of Tresp proliferation is impaired in PD patients compared to controls at ratios (Tresp:Treg) of 1:1, 1:1/2, and 1:1/4 (C n = 25, PD n = 30). f The suppressive capacity of PD Tregs on Tresp proliferation decreases with increasing PD disease burden using the H&Y disease scale (C n = 25, H&Y1 n = 4, H&Y2 n = 17, H&Y3 n = 5, H&Y4 n = 4). Numbers shown as averages +- SEM with Student's t test or one-way ANOVA with appropriate post hoc testing for multiple comparisons as appropriate. P -values are * p < 0.05, ** p < 0.01, and *** p < 0.001.

Supportive validation

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Experimental details: Figure 5 Kinetics of regulatory T cells is not affected significantly in mild, moderate and convalescent patients. Foxp3 + expression on CD19 - CD3 + CD4 + CD45RA - T cells to identify the regulatory T cells in HC, outpatient, outpatient and convalescent (upper FACS panel). There was a statistically significant difference among HC, mild, moderate and convalescent (upper FACS panel). Figure 5

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: Figure 2 Proportions of CD4+CD25+FoxP3+ Treg and CD4+IL-17+ Th17 cells in gingivitis patients and healthy controls. (A) Dot plot int the upperright quadrant represents CD4+ CD25 + T Cells. (B) Isotype control staining of FoxP3. (C) Dot plot represents Treg cells from a healthy control subject (D) Treg cells from a representative patient with gingivitis. (E) CD4+CD25+Foxp3+Treg percentages. (F) Non stimulation of Leukocyte Activation Cocktail of the Th17 cells in a healthy control . (G) Dot plot in the upper right quadrant represents Th17 cells from a healthy control subject. (H) Th17 cells from a representative patient with gingivitis. (I) CD4+IL-17+Th17 percentages. (J) the ratio of Th17/Tre. Data presented are means +- SD. ** P < 0.01, *** P < 0.001 versus healthy control.

Supportive validation

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Experimental details: Figure 2 Phenotypic analysis of circulating regulatory T cells (Treg). A) Isolated PBMC were analyzed directly ex-vivo by flow cytometry. In the lymphogate, CD3 + CD4 + cells were assessed for the proportions of CD25 + FoxP3 + and CD25 + CD127 - Treg cells. Expression of CD45RA was analyzed to phenotype naive (CD45RA + ) and memory (CD45RA - ) Treg. B-D) The proportions of circulating CD25 + FoxP3 + (B) and CD25 + CD127 - (C)Treg, and of naive Treg (D) before and after vitamin D 3 supplementation (week 0 and 12). Significance was assessed with the Wilcoxon signed ranks comparison test.

Supportive validation

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Experimental details: Figure 3 Naive CD4 + T cells are converted to functional Tregs by tumor-infiltrating DCs and tumor conditioned medium (CM). (A-C) Naive CD4 + T cells from peripheral blood of patients with invasive breast carcinoma were co-cultured with or without autologous pDCs isolated from tumor (TI) or peripheral blood (PB) for 9 days in the presence or absence of 30% CM from autologous tumor slices or adjacent normal tissue slices. (A , B) Non-adherent cells from co-cultures were stained for CD3, CD4, CD25 and intracellular Foxp3, and analyzed by flow cytometry. Representative plots of gated CD3 + CD4 + cells (A) and quantification of percentage of Foxp3 + CD25 + cells among CD3 + CD4 + cells (B) are shown (mean +- SEM, n = 19; * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t -test). (C) Expression of Treg-associated genes, assessed by qRT-PCR normalized to GAPDH , in sorted CD4 + T cells, relative to expression in cultures without DCs or CM (mean +- SEM, n = 19; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with naive CD4 + T cells cultured alone by Student's t -test). (D-G) Effect of naive CD4 + T cell-derived Tregs, obtained by co-culture with TI pDCs and tumor CM as above, on function of autologous tumor-specific CD8 + T cells. Tumor-specific CD8 + T cells were generated for each subject by stimulating autologous PB CD8 + T cells with autologous tumor lysate-pulsed autologous DCs. Tregs were recovered from co-cultures by magnetic sorting. (D) CFSE-labeled CD8 + T ce

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 3 T-lymphocyte subset distribution in glaucoma. Mononuclear cells isolated from peripheral blood samples from glaucoma (n = 32) and control (n = 21) groups were analyzed by multicolor flow cytometry for T-cell subset markers. (A) Similar to CD4+ Tregs, the percentage of CD8+/CD25+/FoxP3+ Tregs (CD8-Tregs) was also significantly lower in glaucomatous samples than nonglaucomatous controls (ANOVA, P < 0.001). (B) Shown are representative flow cytometry images after CD8/CD25/FoxP3 immunostaining for Tregs.

Supportive validation

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Experimental details: Figure 3 CG-745 increases helper T cells, cytotoxic T cells and natural killer T cells, and decreases Treg: (A) hPBMCs were incubated with CG (CG-745) for 36 hours and a subset of hPBMCs was analyzed using the antibodies indicated in the text; (B) hPBMCs were co-cultured with Huh7, Hep3B, HepG2 or PLC/PRF/5 cells for 36 hours with or without CG, and a subset of hPBMCs was analyzed by Attune Nxt (Invitrogen, USA).

Supportive validation

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Experimental details: Figure 2 Identification of a CD4 + Foxp3 + Subset in Tumor-Infiltrating MAIT Cells (A) UMAP plots showing expression of selected markers on MAIT cells; intensities are red (high), yellow/green (intermediate), blue (low). (B) Tet-MR1 staining plotted against Foxp3 on total T cells from two CRC tumor samples. (C) Representative staining of CD4 + Foxp3 + MAIT cells from PBMC, adjacent tissue, and tumors of two patients, gated on total MAIT cells. Shown are frequencies of Foxp3 expression among total MAIT cells (PBMC = 13, colon = 10, tumor = 19). Data are mean with SD from at least 7 experiments. Mann-Whitney U test. (D) Expression intensities of Treg-related markers and CD161 on different T cells compared with the CD4 + Foxp3 + MAIT subset; one representative tumor sample. (E) Correlation of Foxp3 expression on MAIT cells with CD4 + MAIT cell frequency; n = 20, two-tailed paired t test, Pearson's correlation. (F) Co-expression of Foxp3 and TNF-alpha gated on total MAIT cells (left) and cytokine production (IFNgamma, TNF-alpha, and IL-17) by CD4 + Foxp3 + tumor-infiltrating MAIT cells compared with Tregs upon 4 h of PMA/ionomycin stimulation (right); n = 6. Data are mean with SD from 2 experiments; two-tailed paired t test. See also Figure S2 .