Multiparametric flow cytometric analysis of signal transducer and activator of transcription 5 phosphorylation in immune cell subsets in vitro and following interleukin-2 immunotherapy.
OBJECTIVE
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.


METHODS
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.
Varker
Kimberly A
KA
Department of Surgery, Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA.
Kondadasula
Sri Vidya
SV
Go
Michael R
MR
Lesinski
Gregory B
GB
Ghosh-Berkebile
Rupa
R
Lehman
Amy
A
Monk
J Paul
JP
Olencki
Thomas
T
Kendra
Kari
K
Carson
William E
WE
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000378:metabolism / Q000556:secondary; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000627:therapeutic use*; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor / Q000097:blood* / Q000187:drug effects; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000556:secondary; D013601:T-Lymphocytes / Q000378:metabolism
D000970:Antineoplastic Agents; D007376:Interleukin-2; D050799:STAT5 Transcription Factor