61-0049-42

antibody from Invitrogen Antibodies

Targeting: CD4

Provider product page for 61-0049-42

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Product number: 61-0049-42 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD4 Monoclonal Antibody (RPA-T4), PE-eFluor™ 610, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The RPA-T4 monoclonal antibody reacts with human CD4, a 59 kDa cell surface receptor expressed by a majority of thymocytes, subpopulation of mature T cells (T-helper cells) and in low levels on monocytes. CD4 is a receptor for the human immunodeficiency virus (HIV). RPA-T4 blocks HIV binding and mixed lymphocyte reaction. The RPA-T4 antibody recognizes a different epitope than the OKT4 monoclonal antibody, and these antibodies do not cross-block binding to each other's respective epitopes. Applications Reported: This RPA-T4 antibody has been reported for use in flow cytometric analysis. Applications Tested: This RPA-T4 antibody has been pre-titrated and tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.125 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. PE-eFluor® 610 can be excited with laser lines from 488-561 nm and emits at 607 nm. We recommend using a 610/20 band pass filter (equivalent to PE-Texas Red®). Please make sure that your instrument is capable of detecting this fluorochome. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 607 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: RPA-T4
Vial size: 100 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

CD4 protein structure - 61-0049-42 shown in red.

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Experimental details: Figure 2 PD-1 and PD-L1 were upregulated on T cells and monocytes in septic shock patients . Blood samples were obtained from 19 septic shock patients and 22 healthy controls and were stained for programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) gated on CD4 + T cells, CD8 + T cells, and CD14 + monocytes. (a) to (c) Percentage of PD-1 expression on (a) CD4 + T cells and (b) CD8 + T cells, and (c) percentage of PD-L1 expression on CD14 + monocytes. Each dot represents one individual. Data are mean +- standard error of the mean (SEM) of three independent experiments. ** P < 0.01 compared with healthy controls. (d) to (f) Mean fluorescence intensity (relative fluorescence units) of PD-1 expression on (d) CD4 + T cells, (e) PD-1 expression on CD8 + T cells, and (f) PD-L1 expression on CD14 + monocytes Each dot represents one individual. Data are mean +- SEM of three independent experiments. * P < 0.05 compared with healthy controls. (g) Representative PD-1 expression levels on CD4 + T cells and CD8 + T cells, and PD-L1 expression on CD14 + monocytes. Values in the upper-right quadrant indicate the percentage of cells that express PD-1 or PD-L1.

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Experimental details: Fig 7 Measurement of ROS in CD14 + cells, CD4 + T-cells, and CD8 + T-cells by cellROX stain mean intensity in T2DM patients compared to healthy. CD14 + cells were stained with cellROX green reagent, a marker of ROS, and a CD14 cell marker, CD14-PE. CD14 + -ROX + cells' mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD14 + cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7A). CD4 + cells were also stained with cellROX green reagent and a CD4 cell marker, CD4-Cy5. CD4 + -ROX + cells' mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD4 + T-cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7B). CD8 + cells were stained with cellROX green reagent and a CD8 cell marker, CD8-Cy5. CD8 + -ROX + cells' mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD8 + T-cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7C). Data represents mean +-SE from 5 healthy individuals and 5 individuals with T2DM.

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Experimental details: FIG. 2. Baseline comparison of the interleukin-6 (IL-6) and reactive oxygen species (ROS) markers between healthy volunteers and HIV-positive individuals. We observed a significant increase in the levels of the proinflammatory cytokine, IL-6 in plasma samples collected from individuals with HIV infection compared to healthy individuals (A) . Data represent mean+-SE from comparing baseline levels of 10 healthy volunteers and 15 HIV-positive individuals, **** P

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Experimental details: FIG. 6. Difference in plasma IL-6 levels and ROS markers pre- and post-GSH supplementation. Sandwich ELISA was performed to compare the cytokine levels between pre-supplementation (V1) and post-supplementation (V3). Assay of cytokines showed a significant decrease in the levels of IL-6 in plasma samples collected from the lGSH-treatment group. There was no significant difference between the levels of IL-6 from the placebo group when comparing visit 1 and visit 3 (A) . Data represent mean+-SE, * P

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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.

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Experimental details: Figure 2. Flow cytometric mapping of the proportion of CD4 + IL-17 + cells in peripheral blood withdrawn from the syphilitic patients with sero-resistance. IL, interleukin.

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Experimental details: Figure 4. Flow cytometric mapping of the expression of forkhead box P3 and retinoic acid-related orphan receptor gammat (ROR-gammat) molecules in CD4 + cells of peripheral blood.

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Experimental details: Figure 4 Murlentamab opsonization of SKOV3-R2 + activates an effective anti-tumor T cell immune response. SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). After 3 days of co-culture, activated T cells coming from the same healthy donor were added in the culture well for 4 more days. ( A ) The CD4 + Th1/Th2 polarization profile, ( B ) the proportion of CD3 + CD4 + CD25 + regulatory T cells and ( C ) the activation of T CD8 + cells were determined by flow cytometry after four days of co-culture. Data shown (boxplots) are the results from two different experiments (performed with two different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001. p values were determined using one-way ANOVA analysis followed by Tukey''s multiple comparisons test.

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Experimental details: Figure 5 Murlentamab/pembrolizumab combination accentuates the anti-tumoral effect of murlentamab monotherapy through the enhancement of T cell activation. ( A - C ) SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors stimulated with M-CSF and IL-10 (TAMs). After 3 days of co-culture, activated T cells coming from the same healthy donor were added in the culture well for 4 more days. Pembrolizumab was added into co-culture wells everyday from day 3 to day 10. ( A ) Opsonized-SKOV3-R2 + cell number was determined by flow cytometry after one and two days of co-culture with TAMs. Data shown (mean +- SEM) are the results from three different experiments (performed with one healthy donors). ** p < 0.01 compared 3C23K-FcKO vs. Murlentamab. # p < 0.05; ## p < 0.01 compared 3C23K-FcKO + anti-PD-1 vs. Murlentamab + anti-PD-1 as determined using one-way ANOVA analysis followed by Dunnett''s multiple comparisons test. ( B , C ) The CD4 + Th1/Th2 polarization profile and the activation of T CD8 + cells were determined by flow cytometry after four days of co-culture. Data shown (mean +- SEM) are the results from three different experiments (performed with one healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey''s multiple compa

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Experimental details: Figure 2. The frequency of CD4 + T cell subsets in the tumor tissues ( n = 44) with regard to human papillomavirus (HPV)-status. To evaluate the subtypes of tumor-infiltrating CD4 + T cells, tumor-derived single cell suspensions were stimulated with PMA and ionomycin in the presence of Brefeldin A and analyzed by flow cytometry. ( A, B, D, F, and H ) The plots represent the numbers of Th1 cells, Th17 cells, Tregs or naive T cells within 10 6 isolated tumor-derived cells; the lines in the box represent the median. ( C, E, G, and I ) The data are expressed as the proportion of Th1 cells, Th17 cells, Tregs and naive T cells, respectively, among the tumor-infiltrating CD4 + cells. The boundaries of the box indicate the SEM, and the lines in the box represent the mean. Whiskers indicate the SD * p < 0.05 (General Linear Models; age was added as a covariate).

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Experimental details: Figure 5. The levels of cyclooxygenase 2 ( Cox-2 ), programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1) and T cell immunoglobulin and mucin domain containing protein-3 (Tim-3) in control and metastatic lymph nodes and tumor tissue from head and neck squamous cell carcinoma (HNSCC) patients. ( A ) The white columns represent the relative mRNA expression of Cox-2 , PD-1 , PD-L1 and Tim-3 in control lymph nodes (LN; n = 14); the gray columns represent the relative mRNA expression of genes in metastatic lymph nodes ( n = 14); the black columns represent the relative mRNA expression of these genes in tumor tissues ( n = 14). To assess the expression levels of mRNA, cDNA was synthesized from total tumor and lymph node RNA and amplified by quantitative real time PCR. As an internal reference, beta-actin housekeeping gene was used. ( B ) The columns represent the mean proportion of IFNgamma + cells among Tim-3-PD-1- (white column), Tim-3-PD-1+ (gray column) and Tim-3+PD-1+ (black column) cells in the tumor tissue ( n = 6). To analyze the IFNgamma production, tumor-derived single cell suspensions were stimulated with PMA and ionomycin in the presence of Brefeldin A and analyzed by flow cytometry. ( C ) Dot plots show the expression of CD3 + on PD-1 + or Tim-3 + tumor-infiltrating cells from a representative patient. ( D ) Dot plots are gated on CD3 + CD8 + cells (upper line) and CD3 + CD4 + cells (lower line) and show the expression of Tim-3 and PD-1 in the peripher

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Experimental details: Figure 2 Protein delivery efficiency of dNP2 in primary mouse and human immune cells. ( a , b ) Mouse primary splenocytes were isolated from 6-week-old female C57BL/6 mice and the cells were incubated with 5 muM EGFP, TAT- and dNP2-EGFP for 2 h. Intracellular fluorescence was analysed by flow cytometry and the data are represented as dot plots or mean fluorescence intensity (MFI) of the cells. ( c , d ) Human PBMCs were isolated from healthy donor blood and the cells were incubated with 5 muM EGFP, TAT-, dNP2-EGFP for 2 h. The data were analysed as described above. ( e ) Total splenocytes were incubated with 1 muM EGFP, TAT-, and dNP2-EGFP for 2 h. Cells were gated using markers specific for CD4 T cells (CD4 + ), B cells (CD19 + ), macrophages (CD11c lo CD11b hi F480 + ) and DCs (CD11c hi MHCII hi ). The EGFP signal in each cell population was then analysed by flow cytometric analysis. The relative MFI value was normalization to PBS treated cells. The red line indicates relative MFI of PBS-treated cells. ( f ) Total PBMCs were incubated with 1 muM EGFP, TAT-, and dNP2-EGFP for 2 h. Cells were gated with markers specific for CD4 T cells (CD4 + ), B cells (CD19 + ), macrophages (CD11b + ) and DCs (CD11c + ) and the data were then analysed as described above. ( g ) Time-lapse images of mouse CD4 T cells incubated with 1 muM EGFP, TAT- and dNP2-EGFP were acquired for 2 h (Scale bar, 15 mum) and ( h ) the average fluorescence intensities of 10 cells from each sample were calculate

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Experimental details: Figure 3 Intracellular delivery mechanisms of dNP2. ( a ) Splenocytes from 6-week-old female C57BL/6 mice were incubated with 5 muM TAT- or dNP2-EGFP at various temperatures (4, 25 or 37 degC) for 2 h. The intracellular EGFP signal of gated CD4 T cells was analysed by flow cytometric analysis and the data are represented as mean fluorescence intensity (MFI). ( b ) The splenocytes were pretreated with 0, 10, 20 or 50 mug ml -1 heparin at 37 degC for 30 min and the cells were then further incubated with 5 muM TAT- or dNP2-EGFP at 37 degC for 2 h. The intracellular EGFP protein signal of gated CD4 T cells was analysed and the data are represented as described above. ( c - h ) The splenocytes were pre-treated with the indicated concentrations of chlorpromazine (CPZ), amiloride (Am) or methyl-beta cyclodextrin (MbetaCD) at 37 degC for 30 min and cells were further incubated with ( c - e ) 5 muM TAT- or dNP2-dTomato or ( f - h ) 5 muM TAMRA-labeled TAT or dNP2 peptide at 37 degC for 1 h. The intracellular dTomato protein signal or TAMRA signal in the CD4 T cells were analysed by flow cytometry. ( i ) HeLa cells were pre-treated with 1-5 mM MbetaCD or PBS on ice for 10 min and the cells were further incubated with 20 muM dTomato or dNP2-dTomato at 37 degC for 1 h. The intracellular localization of the dTomato protein was visualized by fluorescent microscopy ( x 400, Scale bar, 75 mum). Values are mean+-s.e.m. and * P

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Experimental details: Figure 5 Inhibition of effector T cell functions and amelioration of EAE by dNP2-ctCTLA-4. ( a ) DNA constructs and purified proteins. ( b ) Mouse splenocytes were incubated with 1 muM ctCTLA-4, Hph-1-, and dNP2-ctCTLA-4 for 1 h and the intracellular ctCTLA-4 proteins were stained with an anti-HA antibody and the signal was amplified with PE-conjugated anti-rabbit IgG antibody. Intracellular fluorescence was analysed by flow cytometry. ( c ) Splenocytes from 6-week-old female C57BL/6 mice were activated with anti-CD3/CD28 antibody or PMA/ionomycin in the presence of 1 muM PBS, dNP2-EGFP or dNP2-ctCTLA-4 for 24 h. The concentration of IL-2 was determined by ELISA assay. ( d ) The supernatants of anti-CD3/CD28 antibody stimulated cells were analysed for IFN-gamma and IL-17A by ELISA assay. ( e - i ) EAE was induced in 7-week-old female C57BL/6 mice as described in the Methods section. The mice were treated intraperitoneally with PBS or 25 mug dNP2-EGFP or dNP2-ctCTLA-4 on day 7 after MOG immunization and subsequently treated every other day (prevention scheme, n =15). ( e ) The clinical scores were monitored and ( f ) spinal cord tissues were harvested and observed after Luxol fast blue (LFB) and hematoxylin and eosin staining to determine demyelination and tissue inflammation levels (Scale bar, 100 mum). ( g ) The number of spinal cord tissue infiltrating cells was counted via Image J software. ( h ) The spinal cord cells were isolated and IL-17A and/or IFN-gamma expressing CD

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Experimental details: Figure 1 HIV-1 Nef is internalized by CD4 + T cells and activates Akt in PBLs which is mediated via PI3K in a dose and time dependent manners. ( A ), Dose-dependent (n = 3) and ( B ), Time-dependent activation of Akt (pAkt(Ser473)) in PBLs treated with rNef (n = 3). ( B ) Five million PBLs were either left untreated or treated with rNef (100 ng/ml) for various period of time (30 seconds to 30 minutes). As a positive control, PBLs were treated with anti-CD28 antibody. Expression of pAkt (Ser473, Thr308), total Akt and beta-actin were detected by standard western blotting method as described in materials and methods (n = 3). ( C , D ) A series of confocal images showing internalization and colocalization of HIV-1 Nef and Akt ( C ) at serum concentration of Nef (1 to 10 ng/ml) and a dose response of rNef treatment on Akt activation ( D ) in PBLs isolated from healthy donors. ( E ), Internalization of rNef by CD4+ T cells determined by flow cytometry. Five million CD4+ T cells were treated with rNef for 30 min at 37 degC and 4 degC with and without permeabilization. Expression of rNef was determined by confocal microscopy (n = 3).( F ), Activation of Akt in PBLs treated with rNef is mediated via PI3K. Western blot detection of activated pAkt (Ser473, Thr308) in the lysates derived from 5 x 10 6 PBLs treated with 100 ng/ml of Nef with or without Akt (Akt inhibitor VIII) and PI3K inhibitors (LY294002 and Wortmannin) (n = 3). ( G ), Akt activation in PBLs by wild-type HIV-1, but not

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Experimental details: Figure 3 Another PBMC-RPE MLR Assay with Allogeneic iPS-RPE Cells by Ki-67 Proliferation To evaluate the PBMC-RPE MLR assay with allogeneic HLA homozygote iPS-RPE cells (454E2, 453F2, and Ff-I01) and B cells as positive control cells, we used Ki-67 proliferation by FACS analysis using antibodies against CD4 + cells (helper T cells), CD8 + cells (cytotoxic T cells), CD11b + cells (macrophages/monocytes), CD19 + cells (B cells), and CD56 + (NK cells). (A) TLHD10 PBMCs versus both 454E2 and Ff-I01 iPS-RPE cells = HLA-A, -B, -DRB1 matched. (B) TLHD14 PBMCs versus TLHD1 iPS-RPE cells = HLA-A, -B, -DRB1 mismatched, and Ff-I01 iPS-RPE cells = HLA-A matched, and HLA-B and -DRB1 mismatched. Figure 3

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Experimental details: Figure 5 Expression of Th1-Associated Cytokines in Allogeneic iPS-RPE Cell-Exposed Helper T Cells In FACS analysis, purified CD4 + T cells exposed to allogeneic HLA homozygous 454E2 iPS-RPE cells were stained with anti-human CD4/IFN-gamma, IL-2, or TNF-alpha antibody for 48 hr. (A) TLHD1 donor (versus 454E2 MHC-mismatched). (B) TLHD21 donor (versus 454E2 MHC-matched). Figure 5

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Experimental details: Figure 7 Recognition of MHC-II Molecules on Allogeneic iPS-RPE Cells by CD4 + T Cells, but Not iPSCs (A) The graph depicts data for IFN-gamma ELISA by CD4 + T cells (TLHD1) exposed to 454E2 iPS-RPE cells. Anti-MHC-II (HLA-DR, -DP, -DQ) or isotype control antibody (mouse IgG) was used for the T cell RPE culture. Anti-human CD3 antibody was also used (right panel). Data represent the mean +- SEM of three independent experiments. (B) FACS data for IFN-gamma by CD4 + T cells (TLHD1) exposed to 454E2 iPS-RPE cells in the presence of anti-MHC-II blocking antibodies. (C) 454E2 human iPSCs were stained with anti-MHC-II antibody. Cells were also cultured with recombinant IFN-gamma for 48 hr. Red curve represents data for MHC-II molecules. Blue curve represents data for isotype controls. (D) Purified CD4 + T cells (TLHD1) were cultured with 454E2 iPSCs, 454E2 iPS-RPE cells, and 454E2 fibroblasts for 48 hr, and the supernatants were measured for IFN-gamma production by T cells. Data represent the mean +- SEM of four independent experiments. * p < 0.05, ** p < 0.005 compared with the positive control (open bars). n.s., not significant. Figure 7

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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 7 CD4-aptamer-siRNA targeting PITPNM3 reduces TI Tregs and inhibits tumor progression in humanized mice with circulating human Tregs. Humanized mice, implanted with MDA-MB-231 tumors and concurrently injected intravenously with autologous Tregs, were intraperitoneally injected daily for 14 days after tumors became palpable with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Tregs were administered every 10 days after the initial injection and mice were sacrificed 30 days after tumor cell inoculation. (A) Experimental schematic. (B , C) Peripheral blood cells of humanized mice were stained for human CD3, CD4 and Foxp3, and analyzed by flow cytometry. A representative flow plot (B) and the percentage (mean +- SEM) of PB CD4 + cells that are CFSE + Tregs in six mice per group (C) are shown. (D , E) Isolated cells from xenografts were stained for human CD3, CD4 and Foxp3. The percentage (mean +- SEM) of six mice per group (D) and representative flow plot (E) of FoxP3 + Tregs are shown. Most Tregs were CFSE - (i.e., did not come from infused Tregs) and the number of TI Tregs was reduced by knocking down PITPNM3 in CD4 + T cells ( *** P < 0.001 compared to the PBS group by Student's t -test). (F) Tumor size (mean +- SEM, n = 6 per group; *** P < 0.001 by two-way ANOVA with Bonferroni multiple comparison tests). (G) Lung metastases assessed by qRT-PCR

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Experimental details: Figure S6 Functional Analysis of CD56dim and CD56neg NK Subsets and Gating Strategy for Analysis of NK:Tfh:B Cell Co-culture, Related to Figures 4 and 5 (A) Spearman correlation of percentage of total NK cells undergoing degranulation (assessed by CD107a expression) on exposure to target cells with plasma HIV-1 neutralization breadth (PC1). (B and C) Summary boxplots for CD107a expression, IFN-gamma and TNF-alpha production from (B) CD56dim NK cells and (C) CD56neg NK cells following target cell stimulation in bnAb (red), control (blue) and HIV seronegative individuals (black). Each symbol represents data from an individual subject and the box-and-whisker plots show the median, quartiles and range. * p < 0.05, ** p < 0.01. P values corrected for MCA and viral load. (D) Class-switched memory B cells were identified as live CD3-CD4-CD56-CD19+IgD-IgM-CD20+CD38+/- cells and plasmablasts as live CD3-CD4-CD56-CD19+IgD-IgM-CD20-CD38+ cells. Tfh cells were identified as live CD3+CD4+CD19-CD56- cells. The division index was calculated using Flowjo software. CountBright absolute counting beads were used to calculate absolute numbers. The staining shown is from a representative subject (Tfh+B cell only condition).

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Experimental details: Figure 1. Purity of CD3 + CD4 + CD45RA + T cells, CD3 + CD8 + T cells and CD4 + CD25 + FoxP3 + Treg cells. CD3 + CD4 + CD45RA + T cells and CD3 + CD8 + T cells were purified by magnetic cell sorting, and their purity was determined by flow cytometry. (A) The purity of CD3 + CD4 + CD45RA + T cells was 99.45% (CD3 + T cells, 99.6%; CD4 + CD45RA + T cells, 99.85%). (B) The purity of CD3 + CD8 + T cells was 95.32%. (C) The purity of CD4 + CD25 + FoxP3 + Treg cells was 93.15% (CD4 + T cells, 99.5%; CD25 + FoxP3 + T cells, 93.62%). (D) miR-155 expression in T cells of the patients with vitiligo and healthy donor was detected by reverse transcription quantitative polymerase chain reaction. **P

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Experimental details: Fig. 5 Characterization of generated T cells against LDHC-derived peptides. Multi-parameter flow cytometry was conducted to assess the frequency of CD4 + and CD8 + central memory (T CM ), effector memory (T EM ), naive (T N ), and effector (T E ) T cells. a Frequency of immune cell subsets among T cell responses after in vitro stimulation with peptides or control (no peptides). Representative density plots show an increase in CD4 + and CD8 + T EM cells of donor D03 after stimulation with PP4 as compared to control (no peptides)-stimulated T cells. b Frequency of immune cell subsets among T cell responses after priming with autologous pulsed-DCs. Representative density plots show an increase in CD4 + and CD8 + T EM s of donor D09 after stimulation with P11- and P73-pulsed DCs as compared to the cell subpopulations of T cells primed by control (no peptides)-pulsed DCs

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Enhancer of zeste homolog 2 (Ezh2) was involved in the development of allergic rhinitis (AR) in human patients. (A) The mean Th1/Th2 cell ratio in the patients with AR and controls. (B) Identification of Th1 (CD4 + CD45RA - IFN-gamma + ) cells in peripheral blood mononuclear cells (PBMCs). (C) Identification of Th2 (CD4 + CD45RA - IL-4 + ) cells in PBMCs. (D) Expression of Ezh2 in Th1 cells in in the patients with AR and controls (two representatives in each group). (E) Expression of Ezh2 in Th2 cells in the patients and controls (two representatives in each group). (F) Relative Ezh2 expression of Th1 cells to naive CD4 + T cells. The mean fluorescence intensity (MFI) of Th1 cells divided by the MFI of naive CD4 + T cells in the same tube [(MFI of the Th1 cells/MFI of the naive CD4 + T cells) x 100%] ( n = 65 in the AR group and n = 49 in the control group). (G) Relative Ezh2 expression of Th2 cells to naive CD4 + T cells. The MFI of Th2 cells divided by the MFI of naive CD4 + T cells in the same tube [(MFI of Th2 cells/MFI of naive CD4 + T cells) x 100%] ( n = 65 in the AR group and n = 49 in the control group). (H) Correlation analysis between the relative Ezh2 MFI of Th1 and Th2 cells. (I) Total IgE levels in all subjects with lower relative Ezh2 MFI of Th1 cells (lower than the median relative Ezh2 MFI in the controls: 147.60%) compared with those with a lower relative Ezh2 MFI of Th1 cells. (J) Allergen-specific IgE to Dermatophag

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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).