Evaluating STAT5 Phosphorylation as a Mean to Assess T Cell Proliferation

Abstract

Here we present a simple and sensitive flow cytometric—based assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to evaluate phosphorylation of STAT5A as an indicator of T cell proliferation. We determined pSTAT5A in T cell treated with either CD3/CD28 or PHA. After stimulation, T cells from adult healthy donors displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 h. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3% (CD3/CD28) and to 48.4 ± 9.7% (PHA). Treatment with specific JAK3 and STAT5 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation. Compared with currently available methods, STAT5A phosphorylation is well-suited to predict T cell proliferation. Moreover, the method presented here is not very time consuming (several hours) and delivers functional information from which conclusions about T cell proliferation can be drawn.

The IL-2RG R328X nonsense mutation allows partial STAT-5 phosphorylation and defines a critical region involved in the leaky-SCID phenotype

“…STAT-5 phosphorylation was examined in residue Y694 to evaluate the functionality of the receptor. Phosphorylation of residue Y694 is the most widely studied in T cell function experiments in patients with suspected severe combined immunodeficiency and shows a strong correlation with lymphocyte proliferation results, as demonstrated by Bitar et al [37]. Our results, using three different concentrations of IL-2, showed an average decrease of 30% in STAT-5 phosphorylation compared to that of a healthy control (Fig.…”

Role of the JAK/STAT Pathway in Cervical Cancer: Its Relationship with HPV E6/E7 Oncoproteins

“…The STAT5a/b target genes are involved in proliferation, survival, differentiation, and apoptosis. STAT5 is necessary for the activation of immune system cells because it regulates the transcription of target genes such as the IL-2 α receptor ( IL-2RA ) (CD25), necessary for the formation of the high-affinity receptor (IL-2Rαβγ), and IL-2 signaling, essential for G1 phase progression and for mounting an effective immune response [ 118 ]. STAT5 is aberrantly active in various cancers, such as melanoma, glioblastoma multiforme, leukemia, and prostate and colorectal cancers, as well as in myeloid and lymphoproliferative diseases, among others.…”

Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells

“…Ingenuity upstream regulator analysis (Qiagen) comparing polyfunctional and monofunctional cells suggests that STAT5A may be critical regulator of polyfunctional T cell activity. Evidence suggests that STAT5 is required for effective CD8+ memory and effector responses and subsequent T cell proliferation ( 69 – 75 ). In polyfunctional cells, there is significant upregulation of STAT5A/B as well as several downstream transcriptional targets of STAT5 activity including myc, Bcl-2, Bcl-xL, Mcl1, PIM2 , and Cyclin D1 ( 72 ).…”

“…In this study, we demonstrated that STAT5 activity is required for antigen-induced polyfunctionality and maximal proliferation in healthy subjects, and that a strong association between STAT5 signaling with polyfunctional T cells is maintained in immunosuppressed patients. STAT5 has been shown to play a critical role in antigen-specific effector and memory cell survival and proliferation, although its role in polyfunctional T cell activity has not previously been explored ( 69 – 75 , 83 , 85 , 86 ). STAT5 activity downstream of TCR and/or IL-2 has also been shown to function as a stabilizer of gene regulation initiated following antigen re-exposure ( 84 ).…”

“…Additionally, transfection with constitutively-active STAT5 has been associated with superior anti-tumor activity in mouse models of malignancy, and CAR-T cells engineered toward increased STAT5 activity demonstrate improved proliferation and polyfunctionality while preventing terminal differentiation ( 71 , 72 , 74 , 75 , 86 ). Several studies have also demonstrated that ex-vivo expansion of antigen-specific T cells in the presence of IL-7, an upstream regulator of STAT5 activity, is associated with a STAT5-dependent increase in polyfunctional cells ( 69 – 74 , 85 , 87 – 89 ). This is of particular interest given that polyfunctional T cells have increased surface expression of IL-7R, which regulates IL-7 activity.…”