Cells in Human Stat5b Deficiency T High

Cutting Edge: Decreased Accumulation and Regulatory Function of CD4 +CD25high T Cells in Human STAT5b Deficiency

This information is current as Aileen C. Cohen, Kari C. Nadeau, Wenwei Tu, Vivian Hwa, of September 26, 2021. Kira Dionis, Liliana Bezrodnik, Alejandro Teper, Maria Gaillard, Juan Heinrich, Alan M. Krensky, Ron G. Rosenfeld and David B. Lewis J Immunol 2006; 177:2770-2774; ;

doi: 10.4049/jimmunol.177.5.2770 Downloaded from http://www.jimmunol.org/content/177/5/2770

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JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: Decreased Accumulation and Regulatory Function of CD4؉CD25high T Cells in Human STAT5b Deficiency1 Aileen C. Cohen,* Kari C. Nadeau,* Wenwei Tu,* Vivian Hwa,† Kira Dionis,* Liliana Bezrodnik,‡ Alejandro Teper,§ Maria Gaillard,‡ Juan Heinrich,‡ Alan M. Krensky,* Ron G. Rosenfeld,*† and David B. Lewis2*

ϩ We show that STAT5b is important for the in vivo accu- CD4 CD25high regulatory T cell (Treg)3 immunity in mice or ؉ mulation of CD4 CD25high T cells with regulatory cell man is unknown.

function. A patient homozygous for a missense A630P The roles of IL-2 and IL-2-dependent STAT5 activation in Downloaded from STAT5b mutation displayed immune dysregulation and regulating human immune responses in vivo remain poorly un- ؉ decreased numbers of CD4 CD25high T cells. derstood. The high-affinity IL-2R consists of CD25, which is -A630P/A630P ؉ high unique to IL-2R, the IL-2R␤1 chain (CD122), and the com STAT5b CD4 CD25 T cells had low ex- ␥ ␥ pression of forkhead box P3 and an impaired ability to mon -chain ( c), which is also a component of IL-4R, IL-7R, ؉ ؊ suppress the proliferation of or to kill CD4 CD25 T IL-9R, IL-15R, and IL-21R (9). Human CD25 deficiency, which specifically ablates high-affinity signaling by IL-2, results http://www.jimmunol.org/ cells. Expression of CD25, a component of the high-affin- in an abnormal accumulation of lymphocytes in extralymphoid ity IL-2R, was also reduced in response to IL-2 or after in tissues, suggesting perturbed homeostasis, and in increased sus- vitro propagation. The impact of the STAT5b mutation ceptibility to opportunistic infections (10), which indicates a was selective in that IL-2-mediated up-regulation of the ␥ role for IL-2 in human T cell effector function. This latter role common -chain cytokine receptor and perforin, and ac- may be species dependent, because blockade of IL-2/IL-2R sig- ␥ tivation-induced expressions of CD154 and IFN- were naling in mice impairs Treg development and promotes lym- normal. These results indicate that STAT5b propagates phoid hyperplasia and autoimmunity but does not appear to

an important IL-2-mediated signal for the in vivo accu- substantially decrease T cell effector function (11–13). by guest on September 26, 2021 mulation of functional regulatory T cells. The Journal of We examined the immunologic consequence of human Immunology, 2006, 177: 2770–2774. STAT5b deficiency focusing on IL-2 and its signal transduction pathway in influencing effector T cell and Treg immunity. TAT5a and STAT5b are highly homologous proteins whose role in human immunity remains unclear (1). Murine studies have revealed both overlapping and Materials and Methods S Cells nonredundant immunological effects in vivo for these proteins Ϫ Ϫ Ϫ Ϫ (2–4). Both STAT5a / and STAT5b / mice have decreased PBMCs were isolated (14) from a 20-year-old STAT5b-deficient (STAT5bA630P/A630P genotype) patient (15), her STAT5bwt/A630P (where wt is numbers, proliferation of mononuclear leukocytes, and de- wild type) genotype parents, and from age-matched controls who were either creased up-regulation of CD25 in response to IL-2. In contrast, healthy or immunosuppressed (control (IS)) similarly as the patient, who was Ϫ Ϫ only STAT5b / mice have a profound defect in NK cell num- on chronic immunosuppressive glucocorticoid therapy (0.5–1 mg/kg/day pred- nisone or equivalent for Ͼ1 year). Peripheral T cells were either used directly or ber, cytolytic activity, and IL-2-mediated perforin up-regula- ␮ Ϫ/Ϫ Ϫ/Ϫ primed into blasts by incubation with 10 g/ml PHA (Pharmacia) and 100 tion (3, 4). STAT5a STAT5b double-knockout mice U/ml recombinant human IL-2 (Chiron). Complete RPMI 1640 medium (14) was used for in vitro incubation. CD4ϩCD25high (Treg-enriched) and have markedly decreased IL-2-dependent T cell proliferation ϩ Ϫ (5) and decreased fetal thymopoiesis (6), as well as an autoim- CD4 CD25 (Treg-depleted) cell populations were isolated from PBMCs us- ϩ high ing magnetic beads (Miltenyi Biotec) with a final cell purity of 87–95%. T cell mune diathesis and a reduced number of CD4 CD25 T blasts were generated as described above from CD4ϩCD25high or cells (7, 8). However, the importance of STAT5a vs STAT5b in CD4ϩCD25Ϫ T cells.

*Department of Pediatrics, Stanford University School of Medicine, Stanford, California Jeffrey Modell Center for Primary Immunodeficiency (to D.B.L), and a Berry Fellowship 94305; †Department of Pediatrics, Oregon Health and Sciences University, Portland, Or- in Children’s Health (to K.C.N.). egon 97239; and ‡Department of Endocrinology and §Department of Pulmonology, Hos- 2 Address correspondence and reprint requests to Dr. David B. Lewis, Division of Immu- pital General de Nin˜os Ricardo Gutie´rrez, Buenos Aires, Argentina nology and Transplantation Biology, Center for Clinical Sciences Research Building, Received for publication March 7, 2006. Accepted for publication June 29, 2006. Room 2115b, Stanford University School of Medicine, 269 Campus Drive, Stanford, CA 94305-5164. E-mail address: [email protected] The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. 3 Abbreviations used in this paper: Treg, CD4ϩCD25high regulatory T cell; AC, allophy- Section 1734 solely to indicate this fact. cocyanin; Foxp3, forkhead box P3; ␥c, common ␥ chain; control (IS), control treated with immunosuppressive glucocorticoid therapy; SEB, Staphylococcus aureus enterotoxin B; wt, 1 This work was supported by National Institutes of Health Grant K08 AI057961-01 (to wild type. A.C.C), an American Society of Hematology Fellow Basic Science Award (to A.C.C), the

Ab staining and flow cytometric analysis Staining with mAbs (purchased from Caltag Laboratories or BD Biosciences, unless indicated otherwise) or appropriate mouse isotype controls (Caltag Lab- oratories) was performed (14) and analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences). Paraformaldehyde-fixed cells were used, except for annexin-V and propidium iodide staining. Western blotting Protein (15 ␮g/lane) from T cell blasts was electrophoresed, blotted, and probed with STAT5a (L20; rabbit polyclonal IgG) or STAT5b (G-2; mouse mAb IgG1) Abs (Santa Cruz Biotechnology) as described (15). IFN-␥ and CD69 expression PBMCs were stimulated with 10 ␮g/ml Staphylococcus aureus enterotoxin B (SEB) (Toxin Technologies) or CD3/CD28 mAb microbeads (Miltenyi Bio- tec) for 16 h, with 10 ␮g/ml brefeldin A (Sigma-Aldrich) added for the last 5 h. Cells were analyzed as described (14) after staining with PE-Cy5-CD4, allo- phycocyanin (AC)-CD8, PE-CD69, and FITC-IFN-␥ mAb. CD154 expression PBMCs (1 ϫ 106) were treated with CD3/CD28 mAb microbeads for6hand Downloaded from stained with CD154 mAb (clone 5C8) or an isotype control and PE-conjugated goat-anti-mouse IgG (Caltag Laboratories). CD25 and ␥c staining PBMCs (5 ϫ 105/well) were incubated in 96-well round-bottom microtiter ␮ plates coated with 3.5 g/ml purified CD3 mAb 64.1 (Bristol-Myers Squibb) A630P/A630P FIGURE 1. Decreased STAT5b and CD25 expression by STAT5b http://www.jimmunol.org/ with or without 10 U/ml recombinant human IL-2 for 24 h. T cell blasts or CD3 mAb-activated PBMCs were stained with PE-Cy5-CD4, AC-CD25, and T cell blasts is associated with chronic lymphopenia and hypergammaglobu- PE-␥c mAbs. linemia. A. Western blot showing STAT5a and STAT5b expression in T cell blasts. B. The numbers of CD4 and CD8 T cells, NK cells (CD56ϩ lympho- ϩ Perforin expression by CD8 T cells cytes), and B cells (CD19ϩ) were determined by flow cytometry and complete ϫ 3 blood counts with differentials. Total serum IgG and IgA levels that were de- PBMCs incubated with or without IL-2 (1.0 10 U/ml) for 3 days were Ϯ permeabilized/fixed (BD Biosciences) and stained with FITC-perforin and PE- termined in parallel by nephelometry are shown. Normal values (mean 1 SD) CD8 mAbs. for age are indicated. C, CD25 expression by CD4 T cell blasts. The CD25 mean fluorescence intensity values of CD4 T cell blasts of the Annexin-V staining and propidium iodide staining STAT5bA630P/A630P, STAT5bwt/A630P, and control (IS) subjects were 231, 721, and 1115, respectively. D, Annexin-V-FITC vs propidium iodide staining of by guest on September 26, 2021 T cell blasts were incubated with annexin V-FITC and propidium iodide (On- A630P/A630P cogene Sciences) and AC-CD4 mAb. control (IS) and STAT5b CD4 T cell blasts. Intracellular staining for Foxp3 Purified CD4ϩCD25high or CD4ϩCD25Ϫ T cells or their blasts were fixed/ years of age (Fig. 1B) revealed modest but consistently reduced permeabilized, blocked with 2% normal rat serum, and stained with FITC- circulating numbers of CD4 and CD8 T cells, low to normal Foxp3 mAb (eBioscience) or isotype control mAb for 30 min. levels of NK cells, and normal to elevated levels of B cells. In Treg-mediated suppression of CD4 T cell proliferation contrast, serum IgG and IgA concentrations were persistently elevated (Fig. 1B), suggesting immune dysregulation. T cell ϩ high ϫ 3 CD4 CD25 T cells (3.75 10 /well) were incubated in 96-well round- proliferation to mitogens, specific Ags, and CD3 mAb was nor- bottom plates with either a 1:1 or 1:4 ratio of autologous or allogeneic CD4ϩCD25Ϫ T cells, 3.75 ϫ 104 allogeneic irradiated (4000 rad) APCs (PB- mal at 10 and 16 years of age despite the T cell lymphopenia, MCs depleted of CD3ϩ T cells using magnetic beads from StemCell Technol- and specific Ab titers to several protein Ags were detectable, in- ogies), and 5.0 ␮g/ml purified HIT3a CD3 mAb (BD Biosciences). Control dicating that T cell proliferation and T cell-dependent Ab for- conditions lacked either CD4ϩCD25high or CD4ϩCD25Ϫ T cells. [3H]Thy- midine (1.0 ␮Ci/well) was added during the last 16 h of a 7-day culture, and mation were intact (data not shown). At 7 years of age she de- cellular incorporation was determined by liquid scintillation counting. veloped lymphocytic interstitial pneumonia, which is associated with autoimmunity, but only had two major infec- Treg cytotoxicity assay tious complications — severe varicella-zoster virus infection Autologous CD4ϩCD25Ϫ T cells were activated with 2 ␮g/ml PHA for 3 days, and Pneumocystis jiroveci pneumonia — that occurred after re- labeled with 51Cr, and added at 1 ϫ 103/well to 96-well round-bottom plates. ϩ ceiving potent immunosuppressive therapy for the lymphoid CD4 CD25high T cells or their blasts were added as effector cells, plates were incubated at 37°C for 4 h, and 100 ␮l of supernatant was counted for gamma pneumonitis. irradiation. Because STAT5 proteins have a well-defined role in enhanc- ing CD25 gene transcription in response to IL-2 (17), we com- Results and Discussion pared CD25 expression by STAT5b-deficient or control PHA- We investigated the immunological phenotype of a 20-year-old and IL-2-stimulated CD4 T cell blasts. STAT5bA630P/A630P woman with severe growth hormone insensitivity due to a ho- CD4 T cell blasts expressed ϳ20% of the amount of surface mozygous missense STAT5b mutation (A630P) (15). CD25 compared with control cells, based on the mean fluores- STAT5bA630P/A630P T cells (Fig. 1A) had undetectable levels of cence intensity measurements (Fig. 1C), a phenotype that STAT5b protein and normal levels of STAT5a, similar to those would be expected to reduce IL-2-mediated signaling. Interest- of STAT5bA630P/A630P fibroblasts or EBV-transformed B cell ingly, STAT5bwt/A630P T cell blasts also had a modest but con- lines (15, 16). Immunological evaluation between 3 and 19 sistently lower CD25 expression than did control cells (Fig. 2772 CUTTING EDGE: IMPORTANCE OF STAT5b IN ACCUMULATION OF FUNCTIONAL Tregs

1C). A substantially greater percentage of the STAT5bA630P/A630P We next determined whether STAT5b deficiency had an im- T cell blasts was apoptotic as compared with control cells (Fig. pact on the coexpression by CD4 T cells of CD25 and the tran- 1D), and this likely accounted for the poor IL-2-mediated ex- scription factor forkhead box P3 (FoxP3), which identifies cells pansion of STAT5bA630P/A630P T cells from PBMCs compared highly enriched in Treg function (11, 18). STAT5bA630P/A630P ϩ with T cells from the STAT5bA630P/wt parents or from control CD4 CD25high T cells did not express detectable Foxp3 protein, (IS) donors (A. C. Cohen and D. B. Lewis, unpublished and this level of expression was also very low for freshly isolated ϩ observations). STAT5bwt/A630P CD4 CD25high T cells as compared with that for ϩ We next evaluated freshly isolated STAT5bA630P/A630P T cells controls (Fig. 3). The percentage of STAT5bA630P/A630P CD4 T for activation- and IL-2-dependent protein expression and ef- cells that were CD25high was also only ϳ10% and ϳ25% of fector function. CD154 (Fig. 2A), CD69, and IFN-␥ expres- that of control (IS) and STAT5bwt/A630P donors, respectively. sion in response to SEB (Fig. 2B) or CD3/CD28 mAb stimu- Foxp3 expression by T cell blasts generated in vitro from ϩ lation (data not shown) and perforin up-regulation in response STAT5bA630P/A630P CD4 CD25high T cells remained undetect- to IL-2 (Fig. 2C)bySTAT5bA630P/A630P T cells were similar to able, and CD25 expression was also lower relative to the basal level, ϩ those of T cells from healthy controls or control (IS) donors. As whereas control (IS) CD4 CD25high T cell blasts retained high ϩ Ϫ with T cell blasts, CD25 up-regulation by STAT5bA630P/A630P levels of both proteins. As expected, CD4 CD25 T cells or blasts CD4 T cells in response to IL-2 was decreased compared with from all subjects lacked detectable Foxp3 (data not shown), con- ϩ control T cells, whereas the up-regulation by IL-2 of ␥c was sistent with the CD4 Tregs being contained mainly in the high wt/A630P ϩ high normal (Fig. 2D). These results indicated that STAT5b defi- CD25 subset. Interestingly, STAT5b CD4 CD25 Downloaded from ciency selectively impaired the up-regulation of CD25 by IL-2 T cell blasts acquired Foxp3 levels similar to those of control (IS) in freshly isolated T cells but spared other IL-2- and activation- blasts, indicating that STAT5b haplo insufficiency did not impair dependent functions. responsiveness to activation- and IL-2-mediated signals for increased Foxp3 (19). Therefore, complete STAT5b deficiency im- ϩ paired the peripheral accumulation of CD4 CD25high Tregs and

their generation in vitro (18, 20). Whether this decreased accumu- http://www.jimmunol.org/ lation in vivo is the result of decreased intrathymic and/or peripheral Treg generation or, once generated, by impaired Treg survival or homeostatic proliferation, remains to be determined. ϩ The capacity of STAT5b-deficient vs control CD4 ϩ CD25high T cells to suppress autologous or allogeneic CD4 Ϫ CD25 T cells was examined using a standard assay (21). ϩ Ϫ CD4 CD25 T cells proliferated in response to allogeneic ϩ high

stimulation in the absence of CD4 CD25 T cells as ex- by guest on September 26, 2021 pected (21). Both control (IS) and STAT5bwt/A630P Treg-enriched cells suppressed proliferation of autologous and alloge- ϩ Ϫ neic CD4 CD25 T cells in a dose-dependent fashion, although the suppressive ability of the STAT5bwt/A630P ϩ CD4 CD25high T cells was consistently lower than that of control (IS) cells (Fig. 4, A and B). In contrast, STAT5bA630P/A630P

FIGURE 2. Impairment of CD25 expression but not effector function by freshly isolated T cells in STAT5b deficiency. A, CD154 surface expression (dark gray trace) or control isotype staining (light gray trace) by lymphocytes; the upper right-hand corner indicates the percentage of positive cells. B, IFN-␥ and CD69 expression by CD4 and CD8 T cells after incubation with FIGURE 3. Decreased Foxp3 expression by CD4ϩCD25high T cells in or without SEB. C, IL-2 mediated perforin expression by CD8 T cells. STAT5b deficiency. CD25 and Foxp3 expression (open histograms) was ana- Shown is the percentage of cells expressing perforin. D, CD25 and ␥c sur- lyzed by flow cytometry for CD4ϩCD25high T cells directly ex vivo and after face expression by CD4 T cells after stimulation with CD3 mAb alone or in their propagation in vitro as T cell blasts. Filled histograms indicate isotype combination with IL-2. control mAb staining. The Journal of Immunology 2773

ϩ Ϫ or STAT5bwt/A630P CD4 CD25 T cells or their T cell blasts were used as effectors. Both control (IS) and STAT5bwt/A630P ϩ ϩ Ϫ CD4 CD25high T cells killed autologous CD4 CD25 T cell targets in a dose-dependent manner. In contrast, freshly isolated ϩ STAT5bA630P/A630P CD4 CD25high T cells failed to kill either when used directly or as T cell blasts (Fig. 4C). Cytotoxicity by ϩ STAT5bwt/A630P CD4 CD25high T cells and their blasts was also consistently lower than that by control (IS) effector cells. Our findings indicate an important and unexpected role in ϩ humans for STAT5b signaling in CD4 CD25high Treg immunity and, in contrast, only a modest impact on the size of the peripheral T cell compartment and its effector function. This Treg deficiency was associated with a role for STAT5b that was not redundant with that of STAT5a in the up-regulation of CD25 gene expression by IL-2. The major immunological phenotype of STAT5b haplo insufficiency was also on the up-regulation of CD25 by IL-2 and on Treg immunity. In contrast to

mice (12), the phenotype of human CD25 deficiency (10) and Downloaded from ability of CD25 mAb to block allograft rejection suggest that IL-2R signaling in humans may also be important for T cell effector function in vivo. Our results suggest that optimal human Treg immunity, but not most T cell effector functions, requires either higher levels of IL-2 signaling and/or a unique

transcriptional effect of STAT5b. If high levels of IL-2 signaling http://www.jimmunol.org/ are required for human Treg immunity, it is plausible that the

ϩ STAT5b-dependent up-regulation of CD25 by IL-2 may play FIGURE 4. Decreased Treg activity by CD4 CD25high (CD4ϩCD25hi)T cells in STAT5b deficiency. A,[3H]Thymidine incorporation by autologous an important role in achieving such signaling. CD4ϩCD25Ϫ (CD4ϩCD25neg) T cells or their T cell blasts alone (filled bars), CD4ϩCD25high T cells or their T cell blasts alone (light gray bars), and mix- Acknowledgments ϩ Ϫ ϩ tures of CD4 CD25 and CD4 CD25high T cells or their T cell blasts at We are grateful to Dr. Jennifer Frankovich for help in obtaining blood samples ratios of 1:1 (dark gray bars) and 4:1 (open bars), respectively. B, [3H]Thymi- from patients and their family members and to Dr. David Randolph for cri- ϩ Ϫ dine incorporation by CD4 CD25 T cells incubated with allogeneic tiquing the manuscript. CD4ϩCD25high T cells from a control or from the STAT5bA630P/A630P patient, by guest on September 26, 2021 as indicated. For each condition, the mean Ϯ SEM for eight replicates is shown. ϩ Ϫ Disclosures C, Percentage of cell lysis of 51Cr-labeled CD4 CD25 T cells after incubation The authors have no financial conflict of interest. with the indicated ratio of autologous CD4ϩCD25high and CD4ϩCD25Ϫ sub- sets or T cell blasts derived from these cell populations. Mean Ϯ SEM for three replicates are shown. References 1. Crispi, S., E. Sanzari, J. Monfregola, N. De Felice, G. Fimiani, R. Ambrosio, M. D’Urso, and M. V. Ursini. 2004. Characterization of the human STAT5A and STAT5B promoters: evidence of a positive and negative mechanism of transcriptional ϩ high regulation. FEBS Lett. 562: 27–34. CD4 CD25 T cells proliferated after allogeneic stimula- 2. Teglund, S., C. McKay, E. Schuetz, J. M. van Deursen, D. Stravopodis, D. Wang, tion. This proliferation after allogeneic stimulation is consistent M. Brown, S. Bodner, G. Grosveld, and J. N. Ihle. 1998. 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