human cd4 cd25 high t lymphocytes  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Loss of FOXP3 function causes expansion of two pools of autoreactive T cells in patients with IPEX syndrome

doi: 10.1101/2022.07.10.499494

Figure Lengend Snippet: (A) Representative image of gating strategy used to sort and analyze frequencies of Treg, Teff1 and Teff2 subpopulations. (B,C) Quantification of frequencies of Treg, Teff1, Teff2, and CD3 + CD4 + T cells from PBMC of 11 IPEX patients and 11 HD. (D) TSDR demethylation analyses from sorted Treg, Teff1, and Teff2 populations of 10 IPEX patients and 16 HD (sorted as shown in A ). (E) TSDR demethylation analyses of FOXP3-Teff1 and Teff2 populations and FOXP3+ Treg from 4 IPEX patients and 10 HD. ( Left panel ) Representative image of gating strategy for FOXP3, of Teff1, Teff2, and Treg cells gated as shown in A. ( Right panel ) Quantification of TSDR demethylation. Data points shown in grey (IPEX Teff2) represents samples with low cell number/genome copies, where the values may be partially imprecise. Significance was evaluated using Mann Whitney test.

Article Snippet: To isolate CD4 + CD25 high CD127 - Treg cells, we used PBMC from LRS chambers and enriched Treg cells using the CD4 + CD25 + CD127 dim/- Regulatory T Cell Isolation Kit II, human (Miltenyi Biotech) followed by FACS sorting of the CD4 + CD25 + CD127 dim/- cells to obtain a pure population.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Loss of FOXP3 function causes expansion of two pools of autoreactive T cells in patients with IPEX syndrome

doi: 10.1101/2022.07.10.499494

Figure Lengend Snippet: (A) FACS analysis of IPEX patient chimerism 2.5 years post transplantation show competitive advantage of healthy donor Treg cells over the patient’s Treg. Donor cells are HLA-A2 + and host cells are HLA-A2 - . (B) TSDR demethylation analyses of sorted host and donor Teff from patient before and 2.5 years post transplantation. In addition, HD Teff cells were sorted in parallel as a control (light blue). ( Left panel ) Gating strategy. ( Right panel ) Results from TSDR demethylation analyses. (C) TSDR demethylation analyses from whole blood at various time points pre- and post-transplantation. TSDR demethylation was normalized to % of CD4 + T cells also determined by epigenetic analyses. Mean and standard deviation of HD (internal data of Bacchetta lab) are depicted as full line and dashed lines, respectively. (D) Quantification of TSDR/CD4 ratio before, less than 1 year post transplantation, and more than one year post transplantation. Data were analyzed using Dunn’s multiple comparisons test.

Article Snippet: To isolate CD4 + CD25 high CD127 - Treg cells, we used PBMC from LRS chambers and enriched Treg cells using the CD4 + CD25 + CD127 dim/- Regulatory T Cell Isolation Kit II, human (Miltenyi Biotech) followed by FACS sorting of the CD4 + CD25 + CD127 dim/- cells to obtain a pure population.

Techniques: Transplantation Assay, Control, Standard Deviation

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: Activity of MEDI6383 using primary human T cells. Figure 3 shows the activity of MEDI6383 immobilized to tissue culture plastic or clustered by CD32A-expressing cells with OX40-expressing activated primary human T cells. (A) Schematic illustration of the bioactivity assay using plate immobilized MEDI6383. (B) IFNγ and (C) TNFα release into cell culture supernatants and (D) proliferation of cells plated under the conditions described next to each graph. Solution phase MEDI6383 indicates drug added to medium instead of captured on the plate surface, and no anti-CD3 indicates omission of anti-CD3 mAb clone OKT3 stimulation in the wells. (E) Bioactivity of MEDI6383 with OX40-expressing activated primary human CD4 T cells co-cultured with CD32A-expressing HEK293 cells and a sub-optimal amount of anti-CD3 mAb clone OKT3. No drug, no anti-CD3, and no HEK CD32A indicate bioassay conditions containing all components except MEDI6383, anti-CD3 mAb clone OKT3, or CD32A-expressing HEK293 cells, respectively. T cells only indicate the presence of CFSE-labeled CD4 T cells alone in the absence of drug, anti-CD3 mAb clone OKT3, and CD32A expressing HEK293 cells. Results for plate-immobilized or HEK CD32 clustered MEDI6383 are each representative of 3 independent experiments, with data points and error bars representing the mean and SEM of triplicate measurements, respectively. M, molarity.

Article Snippet: Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Expressing, Cell Culture, Bioassay, Labeling

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: MEDI6383 overcomes the suppressive activity of nTreg cells. Figure 4 demonstrates the ability of MEDI6383 to reverse the immunosuppressive activity of nTreg co-cultured with Teff. (A) Expression of cell surface OX40 by Treg cells at the time of isolation (Day 0) and after 5 days of Teff/Treg co-culture in the presence of anti-CD3 and anti-CD28 stimulation. (B) Examples of CFSE dilution of Teff cells or (C) Treg among untreated, MEDI6383-treated, or F180A OX40L FP control-treated Teff cell, Treg cell, or Teff/Treg co-cultures, as indicated. (D) Plot of CD4 Teff cell proliferation versus concentration of MEDI6383- or F180A OX40L FP control-treated Teff cell (Teff only) or Teff/Treg co-cultures (Teff + Treg), as indicated. (E) Plot of CD4 Treg cell proliferation versus concentration of MEDI6383- or F180A OX40L FP control-treated Treg cell (Treg only) or Teff/Treg co-cultures (Teff + Treg), as indicated. Data is representative of 3 independent experiments. Points and error bars represent mean of triplicate measures and standard error of the mean, respectively. † p<0.05 using two-tailed Student’s t-test comparing MEDI6383-treated versus F180A OX40L FP control-treated cells for points 0.15 nM and above for Teff + Treg and 0.62 nM and above for Teff only in (D) and for points 0.31 and above for Teff + Treg and 0.62 nM and above for Treg only in (E).

Article Snippet: Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Cell Culture, Expressing, Isolation, Co-Culture Assay, Control, Concentration Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: Anti-tumor activity of MEDI6383. Figure 5 demonstrates the T cell dependent activity of MEDI6383 in a human T cell/tumor cell admixed model in NOD/SCID mice. (A) Statistically significant tumor growth inhibition by MEDI6383 administered over a range of dose levels in mice engrafted with human A375 melanoma tumor cells admixed with allogeneic A375-reactive human CD4 and CD8 T cells. (B) Lack of MEDI6383 activity in mice engrafted with A375 tumor cells but in the absence of human T cells. (C) Activity of MEDI6383 when allogeneic A375-reactive human T cells were engrafted with A375 tumor cells. *, p<0.05 by Mann-Whitney rank sum test of mean tumor size at day 28 for MEDI6383 treated groups compared to isotype control group. Arrows indicate times after tumor injection when MEDI6383 was administered to mice. Statistically significant differences in mean tumor size between MEDI6383 and isotype control group was demonstrated in three independent experiments, with representative data shown.

Article Snippet: Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Inhibition, MANN-WHITNEY, Control, Injection

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: Pharmacodynamic effects of MEDI6383. Figure 6 shows the activity of MEDI6383 on peripheral T and B cells when administered to healthy rhesus macaques. (A) Schematic for test article dosing and blood draws for immunophenotyping by flow cytometry. (B) Percentage of Ki67+ CD4 (top) and CD8 (bottom) total memory, naïve, central and effector memory T cells, as indicated, prior to and after test article dosing on day 0. (C) Percentage of ICOS+ (left) and PD-1+ (right) total memory CD4 (top) and CD8 (bottom) T cells. (D) Ki67+ CD20 B cells. Error bars were withheld for (C) and (D) so that the comparisons between different treatments could be more easily visualized.

Article Snippet: Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Flow Cytometry

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: Activity of MEDI6383 using primary human T cells. Figure 3 shows the activity of MEDI6383 immobilized to tissue culture plastic or clustered by CD32A-expressing cells with OX40-expressing activated primary human T cells. (A) Schematic illustration of the bioactivity assay using plate immobilized MEDI6383. (B) IFNγ and (C) TNFα release into cell culture supernatants and (D) proliferation of cells plated under the conditions described next to each graph. Solution phase MEDI6383 indicates drug added to medium instead of captured on the plate surface, and no anti-CD3 indicates omission of anti-CD3 mAb clone OKT3 stimulation in the wells. (E) Bioactivity of MEDI6383 with OX40-expressing activated primary human CD4 T cells co-cultured with CD32A-expressing HEK293 cells and a sub-optimal amount of anti-CD3 mAb clone OKT3. No drug, no anti-CD3, and no HEK CD32A indicate bioassay conditions containing all components except MEDI6383, anti-CD3 mAb clone OKT3, or CD32A-expressing HEK293 cells, respectively. T cells only indicate the presence of CFSE-labeled CD4 T cells alone in the absence of drug, anti-CD3 mAb clone OKT3, and CD32A expressing HEK293 cells. Results for plate-immobilized or HEK CD32 clustered MEDI6383 are each representative of 3 independent experiments, with data points and error bars representing the mean and SEM of triplicate measurements, respectively. M, molarity.

Article Snippet: Natural Treg suppression assay Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Expressing, Cell Culture, Bioassay, Labeling

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: MEDI6383 overcomes the suppressive activity of nTreg cells. Figure 4 demonstrates the ability of MEDI6383 to reverse the immunosuppressive activity of nTreg co-cultured with Teff. (A) Expression of cell surface OX40 by Treg cells at the time of isolation (Day 0) and after 5 days of Teff/Treg co-culture in the presence of anti-CD3 and anti-CD28 stimulation. (B) Examples of CFSE dilution of Teff cells or (C) Treg among untreated, MEDI6383-treated, or F180A OX40L FP control-treated Teff cell, Treg cell, or Teff/Treg co-cultures, as indicated. (D) Plot of CD4 Teff cell proliferation versus concentration of MEDI6383- or F180A OX40L FP control-treated Teff cell (Teff only) or Teff/Treg co-cultures (Teff + Treg), as indicated. (E) Plot of CD4 Treg cell proliferation versus concentration of MEDI6383- or F180A OX40L FP control-treated Treg cell (Treg only) or Teff/Treg co-cultures (Teff + Treg), as indicated. Data is representative of 3 independent experiments. Points and error bars represent mean of triplicate measures and standard error of the mean, respectively. † p<0.05 using two-tailed Student’s t-test comparing MEDI6383-treated versus F180A OX40L FP control-treated cells for points 0.15 nM and above for Teff + Treg and 0.62 nM and above for Teff only in (D) and for points 0.31 and above for Teff + Treg and 0.62 nM and above for Treg only in (E).

Article Snippet: Natural Treg suppression assay Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Cell Culture, Expressing, Isolation, Co-Culture Assay, Control, Concentration Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: Anti-tumor activity of MEDI6383. Figure 5 demonstrates the T cell dependent activity of MEDI6383 in a human T cell/tumor cell admixed model in NOD/SCID mice. (A) Statistically significant tumor growth inhibition by MEDI6383 administered over a range of dose levels in mice engrafted with human A375 melanoma tumor cells admixed with allogeneic A375-reactive human CD4 and CD8 T cells. (B) Lack of MEDI6383 activity in mice engrafted with A375 tumor cells but in the absence of human T cells. (C) Activity of MEDI6383 when allogeneic A375-reactive human T cells were engrafted with A375 tumor cells. *, p<0.05 by Mann-Whitney rank sum test of mean tumor size at day 28 for MEDI6383 treated groups compared to isotype control group. Arrows indicate times after tumor injection when MEDI6383 was administered to mice. Statistically significant differences in mean tumor size between MEDI6383 and isotype control group was demonstrated in three independent experiments, with representative data shown.

Article Snippet: Natural Treg suppression assay Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Inhibition, MANN-WHITNEY, Control, Injection

Journal: Molecular cancer therapeutics

Article Title: Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein

doi: 10.1158/1535-7163.MCT-17-0200

Figure Lengend Snippet: Pharmacodynamic effects of MEDI6383. Figure 6 shows the activity of MEDI6383 on peripheral T and B cells when administered to healthy rhesus macaques. (A) Schematic for test article dosing and blood draws for immunophenotyping by flow cytometry. (B) Percentage of Ki67+ CD4 (top) and CD8 (bottom) total memory, naïve, central and effector memory T cells, as indicated, prior to and after test article dosing on day 0. (C) Percentage of ICOS+ (left) and PD-1+ (right) total memory CD4 (top) and CD8 (bottom) T cells. (D) Ki67+ CD20 B cells. Error bars were withheld for (C) and (D) so that the comparisons between different treatments could be more easily visualized.

Article Snippet: Natural Treg suppression assay Human CD4 effector T cells (Teff) and regulatory T cells (Treg: CD4 + CD25 high CD127 low ) were isolated from the same healthy donor Leukopacks (Hemacare, Los Angeles, CA) using an EasySep human CD4 + T cell isolation kit and a EasySep human CD4 + CD127 low CD25 high Regulatory T cell isolation kit (Stemcell Technologies), respectively (see Supplementary Figure 1 for CD25, CD127 and FoxP3 immunophenotypic profile).

Techniques: Activity Assay, Flow Cytometry