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Interleukin-2 (IL-2) activates extracellular signal-regulated protein kinase (ERK) within immune cells. To examine the profile of phosphorylated ERK (p-ERK) in IL-2 stimulated immune cells of normal donors and patients receiving IL-2 therapy, we developed a dual parameter flow-cytometric assay. An analysis of PBMCs stimulated with IL-2 indicated that IL-2 exposure induced p-ERK in CD56bright NK cells and CD14+ monocytes, but not in CD3+ T cells or CD21+ B cells. CD3+ T cells that were induced to express functional high-affinity IL-2R did not exhibit enhanced p-ERK following IL-2 treatment. Measurement of p-ERK within PBMCs from cancer patients 1 h following their first dose of IL-2 revealed a complete absence of circulating NK cells, consistent with earlier observations. However, the total number of circulating CD14+ monocytes increased in these samples and 97% of these cells exhibited ERK activation. p-ERK was not observed in T cells post-IL-2 therapy. Analysis of PBMCs obtained 3 weeks post-IL-2 therapy revealed high-p-ERK levels in CD56bright NK cells in a subset of patients, while levels of p-ERK returned to baseline in monocytes. These studies reveal an effective method to detect ERK activation in immune cells and demonstrate that IL-2 activates ERK in a subset of NK cells and monocytes but not T cells.

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.