Multiparametric Flow Cytometric Analysis of Signal Transducer and Activator of Transcription 5 Phosphorylation in Immune Cell SubsetsIn vitroand following Interleukin-2 Immunotherapy
Authors: Varker Kimberly A. , Kondadasula Sri Vidya , Go Michael R. , Lesinski Gregory B. , Ghosh-Berkebile Rupa , Lehman Amy , Monk J. Paul , Olencki Thomas , Kendra Kari , Carson William E. 
Published: October 2006

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.