Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: pH-responsive blocking and restoration of IL-2 receptor binding by UPS 5.3 (A) Schematic of the experimental design to assess the intrinsic interaction between UPS 5.3 micelles and proteins. (B) FPLC chromatograms showing individual proteins or UPS 5.3 /protein mixtures. (C) Molecular docking model of the DBA dimer (blue) with IL-2, highlighting predicted interaction sites. (D–F) Biolayer interferometry analysis of IL-2-Fc binding to IL-2Rα, IL-2Rβ, and IL-2Rγ, with and without UPS 5.3 NP encapsulation. (G) Schematic illustrating ex vivo assessment of IL-2-Fc binding to immune cells from various organs. (H) Quantification of IL-2-Fc binding to lymphocytes isolated from liver, lung, spleen, and tumor ( n = 5). Data are presented as mean ± SEM. See also and .

Article Snippet: InVivoMAb anti-mouse CD122 (IL-2Rβ) , BioXcell , Cat# BE0298 RRID: AB_2687820.

Techniques: Blocking Assay, Binding Assay, Encapsulation, Ex Vivo, Isolation

Journal: Frontiers in Immunology

Article Title: In Vivo Priming of Peritoneal Tumor-Reactive Lymphocytes With a Potent Oncolytic Virus for Adoptive Cell Therapy

doi: 10.3389/fimmu.2021.610042

Figure Lengend Snippet: Vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) treatment leads to enrichment of memory CD8 + T cells with an activated phenotype. Immunocompetent B6 mice were inoculated IP with 5.0x10 5 MC38-luc cells (day −9), randomized and treated with intraperitoneal (IP) injection of PBS, vvDD or vvDD-IL15/Rα at a dose of 5.0x107 pfu/mouse (day 0). Ten days following i.p. oncolytic virotherapy, peritoneal CD8 + T cells were isolated via peritoneal lavage and characterized via flow cytometry (in addition to day 3, 6, 8, and 14 for figure A). (A) Regional delivery of vvDD-IL15/Rα greatly enhanced and prolonged peritoneal CD8 + T cell infiltration on day 6 to 14 following virotherapy. (B) The amount of peritoneal central memory (Tcm, CD44 + CD62L + ) and effector memory (Tem, CD44 + CD62L - ) T cells is increased 10 days following vvDD-IL15/Rα injection. (C) Representative flow cytometry charts of memory CD8 + T cell subsets. vvDD-IL15Rα treatment resulted in increased Ki67 (D) , CXCR3 (E) , CD122 (F) , Tim3 - PD1 int (G) , CD103 (H) , and CD44 + PD-1 + (I) staining compared to vvDD or PBS 10 days following injection. PD-1 high (J) , TIGIT + (K) , and Tim-3 + (L) staining were not increased after vvDD-IL15/Rα treatment compared to vvDD or PBS treatment. Representative flow cytometry charts of PD-1 expression by treatment group are depicted in . N=8–12. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. Data are combined from two independent experiments.

Article Snippet: FITC conjugated anti-human CD122 (clone REA167) was purchased from Miltenyi Biotec.

Techniques: Virus, Expressing, Injection, Isolation, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: In Vivo Priming of Peritoneal Tumor-Reactive Lymphocytes With a Potent Oncolytic Virus for Adoptive Cell Therapy

doi: 10.3389/fimmu.2021.610042

Figure Lengend Snippet: Characterization of human peritoneal CD8 + and CD4 + T cells derived from peritoneal metastasized tumors. Peritoneal fluid was collected from 14 patients undergoing peritoneal surgery, and peritoneal lymphocytes were extracted and characterized. (A) Flowchart of flow cytometry gating to identify peritoneal CD4 and CD8 T cells. Single cells gating, dead cells exclusion via Zombie aqua staining. Subsequently CD3 + cells were separated into CD4 + or CD8 + cells. (B) Distribution of naïve T cells (Tn, CD45RO - CCR7 + ), central memory T cells (Tcm, CD45RO + CCR7 + ), effector memory T cells (Tem, CD45RO + CCR7 - ) and highly differentiated effector T cells (Temra, CD45RO - CCR7 - ) gated from CD8 + T cells. (C) Representative flow cytometry chart of peritoneal memory CD8 + T cell subsets. (D) Levels of 4-1BB and Ox40 on CD8 + T cells were analyzed. (E–H) Analysis of PD-1 and Tim-3 demonstrated the majority of CD8 + cells were PD-1 high and an average of 20% expressed Tim-3. Representative flow charts of PD-1 and Tim-3 expression are shown in (F, H) respectively. (I, J) (representative flow chart): The shared beta-subunit of the IL-2- and IL15-receptor (CD122) showed a mean expression in peritoneal CD8 + T cells of 29.1%. (K–M) Peritoneal CD8 + T cells from 7 patients were tested for secretion of cytotoxic IFN-γ by ELISA (K) and 4-1BB surface expression by flow (L, M) (representative images) after overnight coculture with correlating tumor digest. Monocytes were used as negative controls, CD3/CD28 dynabeads as a positive control. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Article Snippet: FITC conjugated anti-human CD122 (clone REA167) was purchased from Miltenyi Biotec.

Techniques: Derivative Assay, Flow Cytometry, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: Analysis of IL-15R and JAK3 expression was performed by RT-PCR ( A ) and immunoblotting ( B ) on primary normal (RPTEC) and tumoral (RCC5, RCC7, RCC8) epithelial cells and the ACHN cell line. Data show that RPTEC express the three chains of the IL-15R (αβγ) and JAK3 whereas γc and JAK3 proteins were not detected in RCC. Specific primers or Abs against IL-15Rα (AF247), IL-2Rβ (sc-1046), IL-2Rγ (sc-670) and JAK3 (sc-513) were used. PBL, TF1β, MCF7 and IFNγ-activated U937 cells were used as controls. Housekeeping β-actin was used as loading control.

Article Snippet: Recombinant human non-glycosylated IL-15 (rhIL-15) and neutralizing anti-IL-2Rβ Mikβ1 mAb were obtained from ImmunoTools (Friesoythe, Germany) and Horseradish peroxidase (HRP)-conjugated and fluorescent-conjugated secondary Abs were from Jackson ImmunoResearch.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: Hematoxylin staining of biopsies from 2 normal samples reveals the presence of the normal kidney structures (glomerulus (Gl), distal (Dt) and proximal (Pt) tubules), while analysis of two different cancer specimens shows that the normal tissue architecture is totally lost and are replaced by tumor cells with clear cell morphology, characterized by optically clear cytoplasm and sharply outlined cell membrane. Whereas no difference on IL-2Rβ is observed between normal and tumoral tissue samples, the IL-2Rγ staining, localized to both proximal and distal tubuli in normal tissue samples, is not found in the tumor samples. A strong JAK3 staining is localized to both proximal and distal tubular cells of normal tissues, while a very faint JAK3 protein expression is detected in tumor samples. Two representative samples of a total of ten are shown for each staining. Negative control was subjected to all treatments omitting primary antibody. Scale bars, 50 µm. Staining was quantified using a morphometric TRIBVN software (Montrouge, France) and results are presented as histograms.

Article Snippet: Recombinant human non-glycosylated IL-15 (rhIL-15) and neutralizing anti-IL-2Rβ Mikβ1 mAb were obtained from ImmunoTools (Friesoythe, Germany) and Horseradish peroxidase (HRP)-conjugated and fluorescent-conjugated secondary Abs were from Jackson ImmunoResearch.

Techniques: Staining, Expressing, Negative Control, Software

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: A) Scatchard's plot analysis: Effects of anti-IL-15Rβ and γc mAbs on IL-15 binding to RCC. For the IL-15 binding experiments, RCC7 cells were incubated with increasing concentrations of radioiodinated rIL-15 in presence or not of the following neutralizing mAbs: anti-IL-2Rβ and IL-2Rγ. The nonspecific cell binding was determined in the presence of radioiodinated rhIL-15 and a 100-fold excess of unlabeled rhIL-15. Cell-bound (B) and unbound (free, F) fractions were measured, and the specific bound fraction was calculated by subtracting the nonspecific binding from the cell-bound fraction. On the ordinate is plotted the ratio of the specific bound fraction (expressed in sites per cell) over the total concentration (bound plus free) of radioiodinated rIL-15 (expressed in pM). On the abscissa bound fraction (expressed in sites per cell). The high affinity specific IL-15 binding (Kd = 375 pM, 413 IL-15 binding sites per cell), which was completely abrogated by neutralizing antibody against the IL-2Rβ (inset) but not the γc chain, suggested the presence on RCC of an IL-15Rα/IL-2Rβ complex. B ) Detection of IL-15Rαβ complex by immunoprecipitation (IP) with anti-IL-15Rα (M161) or mouse IgG protein G-Sepharose-conjugate on total lysate (TL) of RCC7. Immunoprecipitated complexes were blotted either with anti-IL-2Rβ (sc-1046) and anti-IL-15Rα (sc-9172). C ) Stimulation for 10 and 40 min with physiologic (10 pg/mL) and supra-physiologic (10 ng/mL) concentrations of rhIL-15 induces the phosphorylation of MAPK ERK1/2 and IκBα in RPTEC and RCC7, whereas STAT5 activation was only observed in RPTEC. Histograms represent densitometry comparison of each factor normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) and 3 RPTEC batches. * P<0.05 versus control, Mann-Whitney test. One experiment representative of a total of three is shown.

Article Snippet: Recombinant human non-glycosylated IL-15 (rhIL-15) and neutralizing anti-IL-2Rβ Mikβ1 mAb were obtained from ImmunoTools (Friesoythe, Germany) and Horseradish peroxidase (HRP)-conjugated and fluorescent-conjugated secondary Abs were from Jackson ImmunoResearch.

Techniques: Binding Assay, Incubation, Concentration Assay, Immunoprecipitation, Activation Assay, MANN-WHITNEY