Differentiating the roles of STAT5B and STAT5A in human CD4+ T cells

Abstract

STAT5A and STAT5B are highly homologous proteins whose distinctive roles in human immunity remain unclear. However, STAT5A sufficiency cannot compensate for STAT5B defects, and human STAT5B deficiency, a rare autosomal recessive primary immunodeficiency, is characterized by chronic lung disease, growth failure and autoimmunity associated with regulatory T cell (Treg) reduction. We therefore hypothesized that STAT5A and STAT5B play unique roles in CD4(+) T cells. Upon knocking down STAT5A or STAT5B in human primary T cells, we found differentially regulated expression of FOXP3 and IL-2R in STAT5B knockdown T cells and down-regulated Bcl-X only in STAT5A knockdown T cells. Functional ex vivo studies in homozygous STAT5B-deficient patients showed reduced FOXP3 expression with impaired regulatory function of STAT5B-null Treg cells, also of increased memory phenotype. These results indicate that STAT5B and STAT5A act partly as non-redundant transcription factors and that STAT5B is more critical for Treg maintenance and function in humans.

Keywords: FOXP3; Regulatory T cells (Treg); STAT5; T cell development; T cell receptor excision circles; TREC; Treg; forkhead box P3; regulatory T cell.

Figure 1

Efficient knockdown of STAT5A and STAT5B in human primary CD4⁺ T cells. STAT5A and STAT5B were silenced in vitro using siRNA. Purified CD4⁺ T cells from healthy subjects (n = 3) were transfected with STAT5A siRNA (gray bars), STAT5B siRNA (black bars), or control siRNA (white bar). Relative mRNA expression of (A) STAT5A or (B) STAT5B was assessed by QT-PCR at 24 and 72 h post transfection and calculated on fold-change values over those of β-glucuronidase. ***P < 0.001, data represent mean ± SEM.

Figure 2

STAT5A and STAT5B differentially regulate T cell mRNA. (A–D) Human primary CD4⁺ T cells from healthy subjects were transfected in triplicate and assessed by QT-PCR at 24 or 72 h post transfection with STAT5A siRNA (gray bars), STAT5B siRNA (black bars), or control siRNA (white bars) for mRNA expression of (A) IL-2R, (B) BCL-X, (C) IGF1 and (D) FOXP3. Relative mRNA expression was calculated on fold-change values over those of β-glucuronidase. Data are expressed as mean ± SEM, *P < 0.001. (E) FOXP3 protein expression in CD4⁺ T cells was assessed by flow cytometry at 72 h post transfection with control siRNA (n = 4), STAT5A siRNA (n = 2) or STAT5B siRNA (n = 6) and graphed as mean fluorescence intensity (MFI). Levels of FOXP3 from non-electroporated T cells are shown as a control. **P < 0.005.

Figure 3

Reduced Treg FOXP3 expression in STAT5B-deficient patients correlates to reduced Treg function. Treg were purified from 4 subjects with STAT5B mutations, 1 heterozygote patients (A630Pwt/−) and 6 healthy controls. CD4⁺CD25^hi Treg were assessed for FOXP3 expression via flow cytometry and suppressive capability was assayed by [³H]-thymidine incorporation, in which cells were cultured in a 1:1 ratio of CD4⁺ effector T cells to Treg. Data were recorded as mean cpm, and representative data are reported as fold differences compared with unstimulated healthy control. Linear regression analysis was preformed comparing % of Treg expressing FOXP3 and % Treg suppressive function (r² = 0.87).

Figure 4

Memory Treg with low sjTREC account for FOXP3⁺ Treg in STAT5B-deficient patients. A) Purified CD4⁺CD25^hi Treg from age-matched healthy control (n = 3) and STAT5B^−/− patients (n = 4, patients #1–4) were assessed for sjTREC. B) Linear regression analysis was preformed comparing the number of TRECs/10⁵ cells and FOXP3 expression (as assessed by flow cytometry) in CD4⁺CD25^hi Treg from 4 STAT5B^−/− patients. C) Phenotypes of CD4⁺CD25^hi Treg from PBMCs from age-matched healthy controls (n = 6) or STAT5B^−/− patients were identified by flow cytometry for CD45RA⁺CD31⁺ Ki67neg (white bars), CD45RA⁺CD31⁺ Ki67⁺ (gray bars), CD45RO⁺Ki67neg (black) and CD45RO⁺Ki67⁺ (dashed) Treg. D) Suppressive function of naïve or memory Treg from STAT5B-deficient patients or healthy control subjects age-matched (n = 6) was assessed. CD3-depleted irradiated antigen presenting cells were incubated with purified Treg (CD4⁺CD25^hiCD45RO⁺, black bars) or CD4⁺CD25^hiCD45RA⁺, white bars) and were incubated with autologous CD4⁺CD25^low effector T cells. Proliferation was measured by [³H]-thymidine incorporation during the last 18 h of a 5-day culture. Cell proliferation is expressed as mean cpm ± SEM for triplicate wells from two separate experiments. Data is shown as mean ±SEM, *P < 0.05, n.s.; not significant.