Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Naive CD4+ and CD8+ T-cells, were separately isolated by negative selection from PBMCs, and rested overnight prior to compound exposure. (A) Cells were treated with saturating concentrations of IL-7 (10 nM), MDK1472 (1 μM), and MDK-703 (1 μM) for up to 2 hours and scored for pSTAT5 accumulation by ELISA. (B) The dose response for each compound was measured in human naive CD4+ and CD8+ cells, utilizing an exposure time of 15 min (T max ).

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Frozen PBMCs from 5 healthy donors were rested overnight and treated with 100 nM MDK-703 or 1nM IL-7, or no added compound, and cultured for 30 days in the presence of compounds. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry for (A) Ki-67 expression and (B) absolute numbers of CD8 + , CD4 + , Treg, and NK cells. Data are shown as mean ± SEM. Flow cytometry gating data shown in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: NSG mice (n = 10 per treatment) engrafted with human CD34+ cells from two donors (5 mice/donor) were dosed once intravenously with 1 mg/kg Fc (gray bars) or MDK-703 (blue bars), and peripheral blood (B-D) and spleen (E-G) were collected and analyzed at the indicated times by flow cytometry. (A) Diagram of the experimental plan. (B) Frequencies of Ki-67+ in CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 7. (C) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 12. (D) CD8+ T memory subpopulations in peripheral blood on day 12. (E) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in the spleen on day 12. (F) CD8+ T memory subpopulations in the spleen on day 12. (G) TCF1 expression in CD8+ T memory subpopulations in the spleen on day 12. Population gates were drawn based on FMO controls. Statistical analysis was done using Student’s T-Test. *p<0.05, **p<0.005, and ***p<0.0005. Detailed gating information is provided in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: (A) PK of MDK-703: Animals (n = 3) were administered a single dose of 1 mg/kg MDK-703 via IV, SC, or IM. The serum concentration of MDK-703 at the indicated time points was determined by sandwich ELISA. (B and C) PD effect of MDK-703: Three animals were dosed once subcutaneously with 0.3 mg/kg, and blood samples were collected at the indicated time points for absolute lymphocyte counts (B) and immune profiling of CD8, CD4, Treg, and NK cells by flow cytometry (C). Data show mean ±SEM.

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Concentration Assay, Sandwich ELISA, Flow Cytometry