1. Multiparametric flow cytometric analysis of signal transducer and activator of transcription 5 phosphorylation in immune cell subsets in vitro and following interleukin-2 immunotherapy.
- Author
-
Varker KA, Kondadasula SV, Go MR, Lesinski GB, Ghosh-Berkebile R, Lehman A, Monk JP, Olencki T, Kendra K, and Carson WE 3rd
- Subjects
- Carcinoma, Renal Cell drug therapy, Carcinoma, Renal Cell metabolism, Carcinoma, Renal Cell secondary, Gene Expression Regulation, Neoplastic drug effects, Humans, Immunoblotting, Kidney Neoplasms drug therapy, Kidney Neoplasms immunology, Killer Cells, Natural metabolism, Melanoma drug therapy, Melanoma immunology, Monocytes metabolism, Phosphorylation drug effects, Reverse Transcriptase Polymerase Chain Reaction, STAT5 Transcription Factor drug effects, Signal Transduction, Skin Neoplasms drug therapy, Skin Neoplasms metabolism, Skin Neoplasms secondary, T-Lymphocytes metabolism, Antineoplastic Agents therapeutic use, Flow Cytometry, Interleukin-2 therapeutic use, Kidney Neoplasms metabolism, Melanoma metabolism, STAT5 Transcription Factor blood
- Abstract
Purpose: Treatment with interleukin (IL)-2 (Proleukin) yields a 10% to 20% response rate in patients with metastatic melanoma or metastatic renal cell carcinoma. IL-2 is known to activate distinct signals within lymphocytes, including the Janus-activated kinase-signal transducer and activator of transcription (STAT) pathway. We examined the phosphorylation of STAT5 (P-STAT5) in IL-2-stimulated immune cells of normal subjects and in patients receiving IL-2 therapy using a novel flow cytometric assay to characterize the pattern and level of activation within immune subsets., Experimental Design: Normal peripheral blood mononuclear cells (PBMC) were treated in vitro with IL-2 and analyzed for P-STAT5 using an intracellular flow cytometric assay. PBMC were simultaneously evaluated for the induction of STAT5-regulated genes at the transcript level. PBMC were also obtained from patients immediately before and 1 hour after treatment with high-dose IL-2 and analyzed for the presence of P-STAT5 within immune cell subsets by dual-variable intracellular flow cytometry., Results: In vitro IL-2 treatment produced a rapid and dose-dependent increase in P-STAT5 within normal PBMC that correlated with the induction of transcript for the IL-2-responsive genes CIS, Pim-1, and SOCS1 (correlation coefficients 0.8628, 0.6667, and 0.7828, respectively). Dose-dependent induction of P-STAT5 was detected in PBMC for up to 18 hours following in vitro pulse stimulation with IL-2. P-STAT5 was detected within a subset of normal donor CD4(+) T cells (52.2 +/- 15.0%), CD8(+) T cells (57.6 +/- 25.8%), and CD56(+) natural killer (NK) cells (54.2 +/- 27.2%), but not CD14(+) monocytes or CD21(+) B cells, following in vitro IL-2 treatment. The generation of P-STAT5 within immune cell subsets after the therapeutic administration of IL-2 varied significantly between individuals. NK cells were noticeably absent in the posttreatment sample, a finding that was consistent for all patients examined. Surprisingly, activated STAT5 persisted within CD4(+) and CD8(+) T lymphocytes, as well as CD56(+) NK cells, for up to 3 weeks post-IL-2 treatment in three patients who exhibited a clinical response to therapy and in a fourth who exhibited a significant inflammatory response after 11 doses of therapy (first cycle)., Conclusions: The flow cytometric assay described herein is a highly efficient and reliable method by which to assess the cellular response to IL-2 within PBMC and specific immune effector subsets, both in vitro and in the clinical setting. Assessment of P-STAT5 in patient PBMC in response to therapeutic IL-2 administration reveals disparate responses between immune cell subsets as well as interpatient variation. Persistent activation of STAT5 within NK and T cells was an unexpected observation and requires further investigation.
- Published
- 2006
- Full Text
- View/download PDF