14-4777-80

antibody from Invitrogen Antibodies

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

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Antibody Data
Antigen structure
References [132]
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Immunohistochemistry [1]
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Product number: 14-4777-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: FOXP3 Monoclonal Antibody (236A/E7), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The 236A/E7 antibody reacts with human Foxp3 protein also known as FORKHEAD BOX P3, SCURFIN, and JM2. Foxp3, a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in 'scurfy' (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.
Antibody clone number: 236A/E7
Concentration: 0.5 mg/mL

FOXP3 protein structure - 14-4777-80 shown in red.

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Experimental details: Figure 1 T FR expansion in human lymph nodes (LNs) during HIV infection. ( a ) Representative images of immunofluorescently stained LN sections from a subject with chronic, untreated HIV infection ( n =17) and an uninfected control subject ( n =8). LNs were stained with fluorescently labelled antibodies to CD20 (white), IgD (blue), Foxp3 (green) and CD4 (red). Follicle (F) was defined as CD20 + (white line) and germinal centre (GC) was defined as CD20 + IgD - (yellow line). Images were scanned at x 60 magnification and scale bars equal 20 mum. ( b ) Foxp3 + CD4 + cells were quantified in different regions of immunofluorescently stained LN shown in a from uninfected ( n =8) and HIV-infected ( n =17) subjects using visual inspection and quantitative image analysis to determine areas. ( c ) The average areas of total (LN), follicular and GC regions per LN cross-section were determined by quantitative image analysis. ( d ) The average number of CD4 + Foxp3 + cells per LN, F and GC cross-section was calculated by multiplying the frequency of CD4 + Foxp3 + cells per mm 2 ( b ) by the average area of each region ( c ) for each subject. The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Mann-Whitney (Wilcoxon) tests to compare unpaired, nonparametric values and significance is denoted by asterisks where * P

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Experimental details: Figure 2. Expression of CD25 and FOXP3 in peripheral blood lymphocytes and tumor-associated lymphocytes from neuroblastoma patients. ( A ) Flow cytometry data demonstrate the expression of CD25 on both peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) on the day of tumor excision in different CD3 + T-cell subsets. Gray bars represent PBLs and black bars represent TALs. One representative pattern of cell-surface CD25 expression in PBLs and TALs from patient 6 as detected by flow cytometry on the day of tumor excision is shown. Numbers indicate the percent of cells expressing or not expressing CD25 among CD4 + cells. ( B ) Expression of FOXP3 in CD4 + PBLs (gray bars) and CD4 + TALs (black bars) on the day of tumor excision, as analyzed by flow cytometry. One representative pattern of FOXP3 expression in PBLs and TALs of patient 6 is shown. Number indicates the percentage of positive cells. * = not performed.

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Experimental details: Figure 3. Tetramer staining of conventional and regulatory T cells from breast cancer patients. ( A-G ) Peripheral blood mononuclear cells (PBMCs) of a breast cancer patient were analyzed, upon gating on living CD3 + CD4 + T cells. The patient sample was stained with tetramers presenting either mam 34-48 ( B ) or the CLIP peptide ( C ). Numbers indicate the percentage of cells in the respective gate, referring to lymphocytes ( A ) or CD3 + CD4 + T cells ( B ) and ( C ). Within CD3 + CD4 + T cells, regulatory T cells (Tregs) were identified as CD25 high CD127 low ( D ) and ( G ) and CD25 high CD127 low FOXP3 + ( E ), ( F ) and ( H ). ( G ) reports the percentage of Tregs within tetramer-positive cells in a representative patient. ( I ) Frequency of mam 34-48 - and CLIP-presenting tetramer-positive cells among CD3 + CD4 + T cells of breast cancer patients and healthy donors (HD) (p values as per Mann-Whitney U tests are indicated). ( J and K ) Frequency of mam 34-48 -specific Tcon ( J ) cells and Tregs ( K ) of breast cancer patients and HDs.

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Experimental details: Fig. 1 PegINFalpha increases Treg and highly suppressive Treg cells in peripheral blood of PV and ET patients. PBMC were collected from patients with PV or ET treated with PegINFalpha or HU for at least 70 days (range 70-616, median 112 for PegINFalpha and range 113-2422, median 175 for HU treated patients). Lymphocytes were analyzed by flow cytometry using surface markers, CD3, CD4, CD25, CD39, HLA-DR and intracellular markers Foxp3, Ki-67 and Helios. a represents CD4 + CD25 + Foxp3 + Treg cells. In b the frequency of Treg was analyzed at different time points after initiation of PegINFalpha treatment. c-e show frequency of Ki-67 + Treg, Helios + Treg, and CD39 + /HLA-DR + ) Treg

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Experimental details: Fig. 1 Ipilimumab downregulates cell CTLA-4. a CHO cells stably expressing human CTLA-4 were treated with either control human IgG or Ipilimumab (Ipi) for 4 h at 37 degC, then the CTLA-4 protein levels were analyzed by western blot. b Cell surface CTLA-4 on CHO cells used in ( a ) was measured by flow cytometry. To avoid the caveat associated with CTLA-4 masking by residual antibodies from the treatment phase, the cells were incubated with Ipilimumab (10 mug/mL) for 30 min at 4 degC before the staining with the non-competing anti-CTLA-4 antibody, clone BNI3. c Plasma membrane and cytosolic proteins purified from ( b ) were detected for CTLA-4 by immunoblot. Na + -K + ATPase and Tubulin were used as controls for purity of cellular fractionation. d Ten-days old Ctla4 h/h mice (body weight: 4.5-5.3 g; n = 6) were treated with anti-mouse PD-1 antibody intraperitoneally (i.p.) (100 mug/mouse). 24 h later, mice were further treated (i.p.) with 100 mug of control hIgG Fc or Ipilimumab for 4 h. e , f Cell surface ( e ) and total CTLA-4 ( f ) in Tregs isolated from spleen or lung were evaluated by flow cytometry. g , h Human PBMCs from healthy donors were stimulated with anti-CD3/anti-CD28 for 2 days and then treated with either control hIgGFc or Ipilimumab for 4 h at 37 degC. Cell surface CTLA-4 on CD4 + CD25 + FOXP3 + Tregs, CD4 + CD25 + FOXP3 - non-Tregs and CD8 + CD25 + cells were measured by flow cytometry. Again, for all the flow cytometry data of CTLA-4-positive cells treated w

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Experimental details: Figure 2 Representative micrographs of CD3, CD8, CD45RO, CD68 IHC (x10) as well as FoxP3 and PD-L1 IHC (x20) in pediatric and adult tonsils, respectively, from the bridging study.

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Experimental details: Figure 3 DIA results from IHC. Representative IHC stained images and their corresponding DIA segmentation overlays of CD3, CD8, CD45RO, CD68, FoxP3, and PD-L1 IHC (x20) from ganglioneuroblastoma, the indication showing the highest PD-L1 expression, far left, and from nephroblastoma, the indication showing the lowest PD-L1 expression, far right. Same cases and similar regions from serial sections were selected. Classification colors indicate blue for negative nuclei, brown for CD3, CD8, CD45RO, CD68, and FoxP3 positive cells; and yellow, orange, and red for PD-L1 positive cells of increasing stain intensity. The boxplots in the middle panel show distribution profiles of investigated biomarkers across investigated indications. Indications are sorted by descending order of mean PD-L1 positive cell densities. Horizontal lines mark indication pairs where the differences in distribution are statistically significant and corresponding adjusted P values are indicated above.

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Experimental details: FIGURE 5 Flow cytometry revealed central memory T cell, Th1, Th17, Th22 and Treg proportions increased in PBMCs derived from CP/CPPS patients than healthy controls. PBMCs were incubated with various fluorescein-labelled antigens for surface staining. PerCP/Cyanine5.5-conjugated CD3 and FITC-conjugated CD4 were used for the central memory T cell, Th1, Th17 and Th22 cell staining, and PE-conjugated CD25 and FITC-conjugated CD4 were incubated for staining Treg cells. After fixing and permeabilizing with cell fixation/permeabilization kit, (A) for samples staining central memory T cells were incubated with PE-conjugated CD45RA and APC-conjugated CD62L; (C) for samples staining, Th1 cells were incubated with PE-conjugated IFN-gamma; (E) for samples staining, Treg cells were incubated with eFluor 660-conjugated FoxP3; (G) for samples staining, Th17 cells were incubated with PE-conjugated IL-17A; and (I) for samples staining, Th22 cells were incubated with PE-conjugated IL-17A and APC-conjugated IL-22. The quantification data were presented in B, D, F, H and J. * P < 0.05; PBMC, peripheral blood mononuclear cell

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Experimental details: Figure 5. Immune and stromal cells survive in slice culture. IHC for (A) T cell markers (CD3, CD8 + , and FOXP3) (N = 3) and (B) macrophage markers (CD68, HLA-DR and CD163) was performed on days 1 and 6 of PDA slice cultures (all Bar = 50 mum). (N = 3) (C) Quantitation of cells staining for each marker demonstrates stability of immune cell populations. (N = 3) Error bars represent STDEV. (D) Day 1 and 6 slices were stained with alpha smooth muscle actin (alphaSMA) (Bar = 100 mum). (N = 4) The right panels are the expanded views of the region of interest indicated by the black boxes in the left panels.

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Experimental details: Figure 1 Immunohistochemistry (IHC) of CD3, CD8, and FoxP3 T lymphocytes in CRC. Representative IHC showing low and high CD3 + , CD8 + , and FoxP3 + T lymphocytes densities. Magnification: 200x.

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Experimental details: Figure 3 The MPNST immune landscape is determined by genetic background. ( A ) Levels of CD8+ T cells in terminally-harvested MPNSTs are similar across all host strains. ( B ) Infiltration of CD4+ T cells are significantly lower in tumors from C57BL/6 mice compared to MPNSTs in mice from 129X1, BALB/c, and 129/SvJae backgrounds. ( C ) Foxp3+ Tregs are detected at higher levels in tumors from 129X1 mice compared to C57BL/6 and BALB/c mice. ( D ) MPNSTs from BALB/c mice have significantly higher levels of infiltrating F4/80+ macrophages compared to C57BL/6 mice. ( E ) Mast cell infiltration is higher in tumors from BALB/c mice compared to 129/SvJae, C57BL/6, and 129X1 mice. Mast cell levels are lowest in MPNSTs from C57BL/6 mice. 129X1 (red circles), C57BL/6 (black triangles), BABL/c (white squares), and 129/SvJae (blue triangles). Analyzed by one-way ANOVA with Tukey's multiple comparison test. A p -value of less than 0.05 is considered statistically significant and is denoted by ""*"" ( n = 5 tumors per strain).

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Experimental details: FIGURE 9 Results of positive correlation analysis. In this study, we analyze two pairs of markers: CD3+, CD8+ and CD4+, FoxP3+. Each marker within the correlation pair is defined as a ""channel"". (A) DoC scores distributions for Cases 1A (left island) and 1B (right island). (B) Histograms of CD3+ and CD8+ DoC scores for Case 1A (left) and 1B (right). Red line is the predetermined threshold to select potential correlated cells. (C) Selected cells for both channels are mapped for clustering analysis (left) using HDBSCAN algorithm. We then define those clusters that both channels are involved as correlation hotspots (right, each cluster is represented by a colored outline). Green: normal tissue (N); Red: invasive front (IF); Yellow: central tumor (CT). (D) Statistical analysis. Top row: proportions of correlated CD3+ and CD8+ cells and density of correlated hotspots in different regions; bottom row: same metrics for CD4+ and FoxP3+.

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Experimental details: Figure 5. Flow cytometry analysis of HDAC9 expression by different cell subsets from patients with AITD. Peripheral blood mononuclear cells from patients with AITD and healthy controls were immunostained for CD3 + (T lymphocytes), CD4 + CD25 + Foxp3 + (T regulatory cells), CD19 + (B lymphocytes), CD14 + (monocytes) and HLA-DR + CD11c + BDCA1 + (myeloid dendritic cells), and HDAC9, and the cells were analyzed by flow cytometry, as stated in ""Methods."" Analysis strategy is shown in the dot plot left panel. A significant increase in the percent of HDAC9 + Treg (CD4 + CD25 + Foxp3 + ) cells was detected in patients with AITD compared with healthy controls. (right panel). * P < 0.05; **** P < 0.001.

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Experimental details: Figure 4 Single vs. triple stainings, immunohistochemistry on tonsil sections. ( A - C ) Single stainings: ( A ) Bcl6 red after 15 min TRIS pH 9, ( B ) FoxP3 brown after 20 min TRIS pH 6.1, ( C ) PAX5 blue after 20 min TRIS pH 6.1. ( D ) Human tonsil with triple staining IHC, according to the FOLGAS protocol: Bcl6 red, FoxP3 brown, PAX5 blue; antigen retrievals equivalent to the corresponding single staining. Magnifications indicated by bar. ( E ) Sum of H-scores of the respective stainings under single and triple staining conditions, from two independent observers.

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Experimental details: Fig. 4 CP DCL cells inhibit the allogeneic immune rejection of the same hESC line-derived cardiomyocytes in Hu-mice. (a) Schematic description of the experiments. DCL cells derived from WT and CP hESCs were delivered via the tail vein into Hu-mice. The experiments were performed using 2 different hESCs lines, H1 and HUES3. Seven days after intravenous injection of DCLs, the same hESC line-derived cardiomyocytes were transplanted into the skeletal muscle of Hu-mice and NSG control mice. (b) Cardiomyocyte survival and T cell infiltration into the cardiomyocyte graft 2 weeks after transplantation ( n = 5, H1 n = 3, HUES3 n = 2). (c) Cardiomyocyte survival and T cell infiltration into the cardiomyocyte graft 4 weeks after transplantation ( n = 4, H1 n = 2, HUES3 n = 2). Sections were stained with anti-CD3 (shown in green) and anti-CTnI (shown in red) antibodies. For each section, the numbers of CD3 + T cells and CTnI cells were counted, 3 sections per graft were analyzed. The graft survival rate was calculated in comparison to the graft survival in NSG mice. The T cell infiltration rate was calculated in comparison to that of grafts in Hu-mice without any DCL cell injection. Data are presented as mean values +- SEM. Data from different groups were compared using one-way ANOVA test. * P

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Experimental details: Fig. 5 CP DCL cells inhibit the allogeneic immune rejection of the same hESC line-derived smooth muscle cells in Hu-mice. (a) Characterization of hESC-derived smooth muscle cells (SMCs). The expression of SM22 was analyzed by Flow cytometry and immunofluorescence microscopy, scale bar 50 mum. (b) SMC survival and T cell infiltration in the grafts 2 weeks after transplantation. Seven days after intravenous injection of DCLs, the same hESC line-derived SMCs were transplanted into the skeletal muscle of Hu-mice and NSG control mice ( n = 5). Data are presented as mean values +- SEM. Data from different groups were compared using one-way ANOVA test. ** p

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Experimental details: Fig. 5 The immunomodulatory role of anti-RANKL in BC. a Change from baseline in serum levels of free-sRANKL ( n = 23, p = 2.384e-07) and CTX ( n = 17, p = 1.526e-05) (significance assessed by the two-tailed sign test), the percentage of Ki-67-positive cells ( p = 0.485) and the staining of activated caspase-3 ( p = 0.391) (H-score) ( n = 24) (significance assessed by two-tailed paired t -tests). Boxplots display median line, IQR boxes, 1.5 x IQR whiskers, and data points. b Each bar plot shows the change from baseline (Delta; post- minus pretreatment values) of the immune parameters assessed using HE (TILs) and IHC (CD3, CD20, CD8, FOXP3, proliferative TILs (TILs Ki67+ ), CD68, and CD163). Each bar represents one patient, which are ranked by their increase in stromal TIL levels. Geometric mean changes, 95% CIs, and p -values are shown below each bar plot. For each measured parameter, the corresponding boxplot is displayed on the right-hand side. Boxplots display median line, IQR boxes, 1.5 x IQR whiskers, and data points. Tumor characteristics and tumor RANK metagene expression at baseline are shown above. p; p -values derived from two-tailed paired t -tests (* p < 0.05) # . c Representative micrographs of multiplex IHC of pre- and posttreatment tumor sections from the four patients with the highest immunomodulatory response. White scale bar, 100 mum. d Top 20 significantly enriched pathways after denosumab treatment, identified by GAGE. e Comparison of baseline serum levels

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Experimental details: Figure 3 NPC tumor-released exosomes inhibited Th1 and Th17 cell induction but promoted Tregs by altering STAT protein phosphorylation A. Differentiation of naive CD4 + T cells into Th1 and Th17 cells and Tregs after stimulation with tumor cells with or without EXOs. Purified naive CD4 + T cells from healthy donors were co-cultured with the irradiated NPC cell line TW03 (EBV + ) and then treated with EXOs or left or untreated in the presence of OKT3 in IL-2 medium for 5 days. INFgamma, IL-17, and Foxp3 staining following by FACS analysis was performed after stimulation with PMA/ionomycin. Representative data of five experiments are shown. B. Numerous data showing the mean percentage +- S.E.M. of positive cell subsets in three independent experiments. * means P < 0.05. C. Western blot analysis for the expression of P-STAT1, P-STAT3, and P-STAT5 proteins. Stimulation with EXOs decreased the expression of P-STAT1 and P-STAT3 but increased the expression of P-STAT5 in OKT3 and NPC cells stimulated by CD4 + T cells; the GAPDH gene was included as a control. Representative data of three experiments are shown.

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Experimental details: Figure 3 HIV entry and replication promote T FR expansion. Disaggregated tonsil cells were spinoculated with X4 or R5 HIV and T FR populations were analysed by flow cytometry ( n =15). ( a ) A representative example of tonsil cell flow gating. From viable CD3 + CD8 - cells, T FR are defined as CXCR5 + and CD25 hi CD127 - . T FR cells contain Foxp3 + cells, whereas remaining T FH (CXCR5 + CD25 lo/- ) cells are Foxp3 - . ( b ) Percentages of T FR determined by gating strategies in a are shown. Experimental conditions include mock-spinoculated cells cultured with PMA (50 ng ml -1 ) and ionomycin (1 mug ml -1 ) or exogenous TGF-beta (100 ng ml -1 ) for 24 h and cells pretreated to block CXCR4 (AMD, 200 muM) and CCR5 (MVC, 2 muM). ( c ) Using flow cytometry counting beads, the number of cells per mul were determined for total (CD3 + CD8 - ), T FH (CXCR5 + CD25 lo/- ) and T FR (CXCR5 + CD25 hi CD127 - ) subsets in mock- and X4-spinoculated samples ( n =3). ( d ) Bcl-6 expression is shown in CXCR5- (grey), T FH (blue) and T FR (red) populations after mock-, X4- or R5-spinoculation ( n =5). ( e ) Blimp-1 expression was also determined as in d . The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Friedman nonparametric tests ( b , d , e ) and significance is denoted by asterisks where * P

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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

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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

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Experimental details: Figure 1 Multiplex IHC on tissue before and after sepsis. (A) Images showing T cell infiltration and macrophage infiltration in tissue before (omentum biopsy) and after (tumor resection) sepsis. CKAE1/AE3 was used to visualize tumor cells. Effector T cells were characterized as CD3 + CD8 + , regulatory T cells as CD3 + Foxp3 + , M1 macrophages as CD68 + , and M2 macrophages as CD163 + . (B) CD8 T cells, T Regs , M1 and M2 macrophages per mm 2 . Immune cells were quantified in control tissue, and in tissue obtained before and after sepsis. (C) CD8 effector T cell/T Reg ratio and M1/M2 ratio before and after sepsis. (D) Average staining intensity of immune cell markers before and after sepsis. Unstained tissue was used as control. Untreated primary tumor from stage III and IV ovarian cancer patient were used as control ( n = 16). Red line indicates mean.

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Experimental details: Figure 1. Spectrum of tumor-infiltrating lymphocytes characterized by MIBC PD-L1 staining. (A) Normalized histograms are shown with the TIL densities (TIL counts per 100 tumors cells) binned into the designated intervals on the x -axes and the sample proportions shown on the y -axes. In each case (overall, CD8, and FOXP3 + TILs) a significant (*Goodman-Kruskal p < 0.01) increase in the amount of TILs is observed with increased MIBC PD-L1 staining. (B) The percentage of cases with MIBC PD-L1 positivity (defined as > 0%) is shown on the x -axes and the per sample CD8/Treg ratio on the y -axes. No statistically significant (NS) association to MIBC PD-L1 expression status was observed.

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Experimental details: Figure 6 FoxP3 and Helios expression in CD4 + T cells PBMC from HD and PBC patients, NILs and TILs were stained for CD3 and CD4 antibodies followed by intracellular staining for FoxP3 and Helios. Live cells were first gated using Fixable Viability Dye 660. ( A ). Representative flow cytometric plots of FoxP3 staining from one cancer patient are shown. ( B ). Scatter plot showing the differences in frequencies of FoxP3 + Tregs between different samples. ( C ). IHC staining of FoxP3 + expression in one NT and TT samples. ( D ). Flow cytometric plots of FoxP3 and Helios co-expression in CD4 + T cells from different samples and whisker plots ( E ) showing differences in various FoxP3 and Helios-expressing Treg subsets. ( F ). Non-parametric Spearman's test showing correlations between FoxP3 and Helios expressions in NILs and TILs. ( G ). Pie charts show the relative percentages of different FoxP3 and Helios Treg subsets.

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Experimental details: Fig. 6 Major overlap between Treg and Tconv subsets in breast tumors. a Representative immunostaining of FFPE breast tumor tissue (TT) from n = 3 biologically independent replicates. CD4 (red), CD127 (purple), FOXP3 (green), DAPI (blue). TT Treg (CD4 + FOXP3 + CD127 - ), TT Tconv (CD4 + FOXP3 - CD127 + ), TT ActTconv (CD4 + FOXP3 + CD127 + ). Scale bar denotes 2.32 um. b Pie chart of all clonotypes (unique TCRbeta nucleotide sequences) detected among TT Treg (red), TT Tconv (green), and TT ActTconv (orange) in order of decreasing percentage (%). Gray slices correspond to the TCRbeta repertoire fraction that contains clonotypes with a frequency below 1%. Representative data from breast cancer patient with mammary gland adenocarcinoma MaCa 4554 from n = 3 biologically independent replicates. c Venn diagram of shared clonotypes (white) between TT Treg, TT Tconv, and TT ActTconv within each patient. d Frequency of common clonotypes between TT Treg, TT Tconv, and TT ActTconv in relation to the frequency distribution of all clonotypes per subset. N , total number of clonotypes recovered from TT Treg (red), TT Tconv (green), and TT ActTconv (orange). e Total frequency of Treg clonotypes overlapping with Tconv among Tconv (green), with Tconv and ActTconv among Treg (red) or with ActTconv among ActTconv (orange) either in peripheral blood (PB) or within TT in n = 3 MaCa patients and n = 3 healthy individuals (HD) (cumulative data, mean +- s.e.m.). n refers to biologically independent