Volume 123, Issue 2 , Pages 505-508, February 2009
Signal transducer and activator of transcription 5 tyrosine phosphorylation for the diagnosis and monitoring of patients with severe combined immunodeficiency
Article Outline
To the Editor:
T−B+ natural killer (NK)− severe combined immunodeficiency (SCID) is frequently caused by defects in the common γ chain (γc) or the tyrosine kinase Janus kinase (JAK) 3. However, not all γc or JAK3 SCIDs present with this typical phenotype because some might have low numbers of T cells, NK cells, or both. Other forms of SCID, such as IL-7 receptor alpha (IL-7Rα) SCID, might present in a similar manner.1 A rapid screen to distinguish among these patients is essential to direct genetic analysis and initiate appropriate therapy.2
Signal transducer and activator of transcription (STAT) 5 is a member of the STAT family of transcription factors. STATs are downstream of a number of cytokine and growth factor receptors, including γc receptors. In healthy individuals IL-2 binds a trimeric receptor consisting of the IL-2 α, β, and γ chains. This receptor complex then trimerizes, bringing together the tyrosine kinases JAK1 and JAK3. These cross-phosphorylate each other, resulting in their activation and enabling them to phosphorylate STAT5. Once phosphorylated, STAT5 dimerizes, translocates to the nucleus where it binds a defined DNA sequence, and activates transcription.3
Detection of tyrosine-phosphorylated STAT5 after IL-2 stimulation is therefore an indication of a functional IL-2/JAK3 signal transduction pathway, and abnormalities in STAT5 tyrosine phosphorylation might identify patients with defects in this pathway. Historically, STAT5 activation has been assessed by using the electrophoretic mobility shift assay (EMSA). EMSA specifically detects STAT5 binding to its consensus DNA sequence3; however, EMSA is time-consuming and requires large volumes of blood.
A functional STAT5 tyrosine phosphorylation (ptyr) assay was established to detect abnormalities in signaling through the γc/JAK3 pathway. Ethical approval and consent from parents/guardians was obtained for patients included in the study. Immunophenotyping by means of flow cytometry was performed with standard techniques to determine T, B, and NK cell numbers and γc expression,2 and phosphorylated STAT5 was detected by means of EMSA4 and flow cytometry (FACS). To detect STAT5 ptyr by means of FACS, 100 μL of blood was left unstimulated or stimulated with 104 units of IL-2, lysed, fixed, washed, and permeabilized before 5 μL of antibodies (STAT5 ptyr, CD19 phycoerythrin, and CD4 peridinin-chlorophyl-protein complex; BD Biosciences, San Jose, Calif) was added. Ten thousand lymphocyte events were acquired and analyzed.
STAT5 ptyr by means of FACS was compared with results obtained from staining for γc by means of flow cytometry, expression of JAK3 by means of immunoblotting, available genetic results, and detection of STAT5 by using EMSA. Fig 1 is a representative experiment and shows γc expression by FACS (Fig 1, A) in γc-deficient SCID (patient 8 in Table I) in comparison with that seen in a healthy control subject and demonstrates the correlation between a STAT5 EMSA (Fig 1, B) and STAT5 ptyr (Fig 1, C) by means of flow cytometry. By using STAT5 ptyr analysis, there is a significant shift in fluorescence in the normal blood control compared with the patient sample (Fig 1, C). By means of EMSA analysis (Fig 1, B), a large band could be detected in the control subjects where phosphorylated STAT5 had bound to the oligonucleotide; the specificity of this band was demonstrated by adding unlabeled oligonucleotide, which competed most of the protein away from the labeled oligonucleotide. This band was faint or undetectable when unlabeled oligonucleotide was added in the patient sample. The faint remaining band is likely to be due to activation of STAT5 by γc-independent signaling pathways, such as prolactin.
Fig 1.
Tyrosine-phosphorylated STAT5 is detected in a control subject but not in a γc-deficient patient. A, γc Staining. B, EMSA showing cells stimulated with IL-2. C, STAT5 ptyr staining on unstimulated (black) and IL-2–stimulated (gray) cells. C, Control; P, patient with γc SCID (patient 1); Co/Po, control/patient plus specific oligonucleotide; PMF, patient with maternal fetal engraftment (patient 14). D, Graph showing the mean and SD percentage change in STAT5 ptyr in control subjects and subjects with combined immunodeficiencies/SCIDs, γc/JAK3 SCIDs (samples with maternal-fetal engraftment not included), and γc SCIDs after therapy.
Table I. Patient data
| abs | ||||||||
|---|---|---|---|---|---|---|---|---|
| Patient no. | Diagnosis | Sex | T cells | B cells | NK cells | STAT5 | Change | Genetic mutation |
| 1 | γc SCID | Male | 0.00 | 0.56 | 0.02 | Absent† | 0.0% | c.670C>T (p.Arg224Trp) |
| 2 | γc SCID | Male | 0.02 | 1.65 | 0.09 | Absent | 0.6% | c.590G>A (Trp197X) |
| 3 | γc SCID | Male | 0.31 | 3.08 | 0.00 | Absent | 2.0% | c.317T>C (Leu106Pro) |
| 4 | γc SCID | Male | 0.07 | 0.61 | 0.00 | Absent | 1.3% | c.322T>C (Ser108Pro) |
| 5 | γc SCID | Male | 0.92 | 1.06 | 0.02 | Abnormal | 5.3% | c.272A>g(Tyr91Cys): a missense mutation with abnormal (not absent) α, β, and γ chains |
| 6 | γc SCID | Male | 0.05 | 0.21 | 0.00 | Absent | 3.3% | c.296G>A (trp90X) |
| 7 | γc SCID | Male | 0.00 | 1.16 | 0.00 | Absent | 0.8% | γc Gene deletion |
| 8 | γc SCID | Male | 0.00 | 4.08 | 0.01 | Absent† | 0.1% | c.545G>A (Cys182Tyr) |
| 9 | γc SCID | Male | 0.04 | 1.16 | 0.00 | Absent | 3.4% | c.3G>A (M1I) |
| 10 | γc SCID | Male | 4.53 | 1.39 | 0.01 | Absent | 5.9% | c.822-823delCAins/dup. 833-845 (Ile274MetfsX24) |
| 11 | γc SCID | Male | 0.04 | 0.93 | 0.21 | Absent | 2.6% | γc Gene deletion |
| 12 | γc SCID | Male | 0.19 | 0.01 | 0.00 | Absent∗ | 25.2% | c.676C>T (p.Arg226Cys) |
| 13 | JAK3 SCID | Male | 0.00 | 1.02 | 0.46 | Absent | 4.0% | c.184+22 C>T/wt, c.184+40G>A/wt, c.184+42G>A/wt, c.1701+9 A>G/wt, c.1915-30C>T/wt, c.2978+32T>C/ c.2978+32T>C |
| 14 | JAK3 SCID | Male | 1.82 | 1.38 | 0.04 | Absent∗ | 32.8% | JAK3 gene deletion |
| 15 | JAK3 SCID | Male | 0.26 | 1.17 | 0.00 | Absent | 0.2% | c.421-10G>A, c.1701+9A>G, c.2680+3G>C |
| 16 | JAK3 SCID | Male | 0.02 | 1.19 | 0.00 | Absent | 2.3% | c.2350+1G>T/c.2350+1G>T, c.2350+6C>T/ c.2350+6C>T |
| 17 | JAK3 SCID | Male | 0.00 | 0.14 | 0.01 | Absent | −3.6% | JAK3 sequence variants: IVS13-30c>t/wt, c.2625C>T (L875L)/wt, IVS 20+32T>C/wt (no mutation found in γc) |
| Posttreatment analysis | ||||||||
| 5 | γc SCID after HSCT | Male | 1.26 | 0.65 | 1.44 | Normal | 30.7% | |
| 6 | γc SCID after HSCT | Male | 1.01 | 0.32 | 0.09 | Normal | 31.6% | |
| 7 | γc SCID after GT | Male | 4.71 | 1.53 | 0.04 | Normal | 33.5% | |
| 8 | γc SCID after GT | Male | 3.12 | 1.98 | 0.02 | Normal | 20.5% | |
| 9 | γc SCID after GT | Male | 1.94 | 0.98 | 0.00 | Normal | 25.3% | |
| Control samples | ||||||||
| 18 | ADA SCID | Female | 0.12 | 0.2 | 0.05 | Normal | 29.1% | |
| 19 | ADA SCID | Female | 0.14 | 0.32 | 0.36 | Normal | 29.8% | |
| 20 | Undefined CID | Male | 0.58 | 0.63 | 0.20 | Normal | 11.7% | No mutation found in γc (subsequently given a diagnosis of ALL) |
| 21 | Undefined CID | Female | 2.78 | 0.23 | 0.10 | Normal | 76.0% | Polymorphisms in IL7RA |
| 22 | Undefined CID | Female | 0.23 | 0.00 | 0.06 | Normal | 60.2% | No RAG1 or RAG2 mutation |
| 23 | Undefined CID | Female | 1.12 | 0.24 | 0.04 | Normal | 35.4% | No RAG1 or RAG2 mutation |
| 24 | Undefined CID | Male | 0.35 | 0.01 | 0.04 | Normal | 48.2% | |
| 25 | Undefined CID | Male | 2.27 | 0.16 | 0.33 | Normal | 24.5% | No mutation found in RAG1 |
| 26 | Undefined CID | Male | 0.19 | 0.05 | 0.05 | Normal | 33.6% | |
| 27 | IL-7Rα SCID | Male | 0.00 | 2.22 | 0.08 | Normal | 8.2% | c.221+2T>G, c.221+2T>G in IL7RA |
| 28 | IL-7Rα SCID | Male | NA | NA | NA | Normal | 11.7% | Confirmed IL7RA mutation |
| 29 | MHC II SCID | Female | 2.04 | 1.35 | 0.02 | Normal | 36.3% | |
| 30 | RAG SCID | Male | 0.84 | 0.00 | 0.07 | Normal | 77.7% | c.1088G>A (p.Cys363Tyr)/C.1186C>T (p.Arg396Cys) in RAG1, no mutation found in RAG2 |
| 31 | STAT5 SCID | Female | 1.64 | 1.09 | 0.18 | Normal | 19.2% | Homozygous point mutation in exon 13 of STAT5 that results in expressed protein that binds DNA in response to IL-2 |
| 32 | ZAP 70 | Male | 0.52 | 1.66 | 0.47 | Normal | 31.4% | |
| Control | Mean of 40 control subjects | 45.6% | ||||||
∗Small second peak detected by means of FACS because of confirmed maternal engraftment. |
†Absent IL-2–induced STAT5, as measured by means of EMSA. |
Table I summarizes the data from 32 patients with SCID. Twelve γc and 4 JAK3 genetically defined SCIDs, including several patients who had atypical immunophenotypes (TlowB+NK+), had absent or highly abnormal STAT5 tyrosine phosphorylation in their lymphocytes. In 2 patients who had high levels of maternal engraftment (patients 12 and 14), a negative STAT5 ptyr peak was present, as well as a smaller positive STAT5 ptyr peak presumed to be due to maternal cells (Fig 1, C). A further patient with a missense mutation allowing partial cytokine binding (patient 5) showed abnormal but not absent STAT5 ptyr. In patient 17 there was absent IL-2–induced STAT5 ptyr, but a confirmed γc or JAK3 mutation has not been identified, although a number of sequence variants have been identified in his JAK3 gene. A second affected child carried these same variants, whereas an unaffected sibling did not. Forty control samples and 15 samples from other forms of SCID or undefined combined immunodeficiency, including 2 IL-7Rα SCIDs (patients 27 and 28) and 2 adenosine deaminase SCIDs (patients 18 and 19) had B-cell STAT5 tyrosine phosphorylation in response to IL-2. In 2 IL-7Rα and 1 undefined combined immunodeficiencies there was decreased STAT5 ptyr, but the STAT5 ptyr levels were significantly higher than for γc/JAK3 SCIDs. These data demonstrate the specificity of this FACS assay for detecting γc/JAK3 SCID defects.
Definitive treatment for SCID is either by means of hematopoietic stem cell transplantation (HSCT) or gene therapy (GT). The success of these therapies is usually monitored by engraftment of donor cells; immunophenotyping for the presence of naive, memory, and effector T cells; and proliferation assays to assess cell function.5, 6 Five patients were analyzed before and after HSCT (patients 5 and 6) or GT (patients 7, 8, and 9) for IL-2–induced STAT5 tyrosine phosphorylation. As Table I shows, all pretreatment samples demonstrated absent/abnormal STAT5 ptyr, whereas all posttreatment samples showed restoration of IL-2–induced STAT5 tyrosine phosphorylation. This suggests that this assay should be evaluated further for monitoring immune reconstitution in this group of patients. Because the STAT5 assay requires fewer numbers of cells, it can be performed earlier after therapy than proliferation assays and has a shorter analytic time, and where necessary, individual T-cell populations can be examined.
Analysis of IL-2–induced STAT5 tyrosine phosphorylation is a rapid assay that determines the functionality of the IL-2 signaling pathway. If STAT5 tyrosine phosphorylation is normal, this demonstrates the integrity of the upstream signaling molecules (γc and JAK3). If there is absent IL-2–induced STAT5 tyrosine phosphorylation, this implies a defect in this pathway, and further genetic investigations can focus on mutation detection in γc or JAK3. Examination of the restoration of IL-2–induced STAT5 tyrosine phosphorylation might be a specific method to evaluate immune reconstitution in γc and JAK3 SCIDs after HSCT or GT. This technique should prove useful for assessing the functionality of other STAT5 signaling pathways in the clinical setting.
We thank the Immunology Department at Great Ormond Street Hospital for immunophenotyping of the patients and the clinicians who provided samples for analysis.
References
- . Cytokines and immunodeficiency diseases: critical roles of the gamma (c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21 and their signaling pathways. Immunol Rev. 2004;202:67–83
- Rapid protein-based assays for the diagnosis of T-B+ severe combined immunodeficiency. Br J Haematol. 2001;112:671–676
- . The role of STAT5a and STAT5b in signalling by IL-2 family cytokines Oncogene. 2000;19:2566–2576
- . Interleukin-2 activates STAT5 (Mammary Gland Factor) and specific gene expression in T lymphocytes. Proc Natl Acad Sci U S A. 1995;92:10772–10776
- Bone marrow transplantation for severe combined immunodeficiency. JAMA. 2006;295:508–518
- Successful gene therapy of SCID-X1 using a pseudotyped gammaretroviral vector. Lancet. 2004;364:2181–2187
This work was undertaken at GOSH/UCL Institute of Child Health, which received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. A. Thrasher was funded by the Wellcome Trust. The STAT5 tyrosine antibody was a gift from BD Biosciences (San Jose, Calif).
Disclosure of potential conflict of interest: H. B. Gaspar has served as a consultant for Enzon, Inc, and has served as an expert witness for primary immunodeficiency. K. C. Gilmour has received research support from the Institute of Child Health, the Leukemia Research Foundation, and Becton, Dickinson, and Co and is a member of the European Society for Immunology. The rest of the authors have declared that they have no conflict of interest.
PII: S0091-6749(08)02356-7
doi:10.1016/j.jaci.2008.11.041
© 2009 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Volume 123, Issue 2 , Pages 505-508, February 2009
