Primary Immunodeficiencies Unravel the Role of IL-2/CD25/STAT5b in Human Natural Killer Cell Maturation

Abstract

Natural killer (NK) cells play a pivotal role during immunity against viruses and circumstantial evidence also indicates that they can protect the host against developing tumors. Peripheral blood NK cells comprise CD56^brightCD16^lo/- cells that constitutively express CD25 (IL-2Rα) and CD56^dimCD16^hi cells that express CD25 upon activation. Using NK cells from two patients, one with a primary immunodeficiency characterized by a homozygous mutation in CD25 (born in year 2007 and studied since she was 3 years old) and one with a homozygous mutation in STAT5b (born in year 1992 and studied since she was 10 years old), we observed that the absence of IL-2 signaling through CD25 promotes the accumulation of CD56^brightCD16^high NK cells, and that CD56^brightCD16^lo, CD56^brightCD16^high, and CD56^dimCD16^high NK cells of this patient exhibited higher content of perforin and granzyme B, and proliferation capacity, compared to healthy donors. Also, CD56^bright and CD56^dim NK cells of this patient exhibited a reduced IFN-γ production in response to cytokine stimulation and increased degranulation against K562 cells. Also, the CD25-deficient patient presented a lower frequency of terminally differentiated NK cells in the CD56^dimCD16^hi NK subpopulation compared to the HD (assessed by CD57 and CD94 expression). Remarkably, CD56^dimCD16^high NK cells from both patients exhibited notoriously higher expression of CD62L compared to HD, suggesting that in the absence of IL-2 signaling through CD25 and STAT5b, NK cells fail to properly downregulate CD62L during their transition from CD56^brightCD16^lo/- to CD56^dimCD16^hi cells. Thus, we provide the first demonstration about the in vivo requirement of the integrity of the IL-2/CD25/STAT5b axis for proper human NK cell maturation.

Keywords: CD25; IL-2; STAT5b; natural killer cells; primary immunodeficiencies.

Figure 1

CD25 deficiency leads to an increased frequency of CD56^brightCD16^hi natural killer (NK) cells and a concomitant decreased frequency of CD56^dimCD16^hi NK cells in peripheral blood. (A) Absolute NK cell numbers in blood from the CD25-deficient patient (left graph) and the STAT5b-deficient patient (right graph) along time (indicated as month/year). Horizontal dotted lines delineate a gray zone that corresponds to the reference values for age-matched healthy donors (HDs). (B) Percentage of NK cells (CD3⁻CD56⁺ cells) in peripheral blood from one HD, the CD25-deficient patient (Pt1), and the STAT5b-deficient patient (Pt2). (C) Percentage of NK cell subpopulations according to the expression of CD56 and CD16, in peripheral blood from one HD, the CD25-deficient patient (Pt1) and the STAT5b-deficient patient (Pt2). (D) Percentage of CD56^brightCD16^lo/−, CD56^brightCD16^hi, and CD56^dimCD16^hi cells in blood from different HD, and in different blood samples from the CD25-deficient patient collected along three years (2015, 2016, and 2017). (E) Analysis of the expression of CD25 in CD56^brightCD16^lo/−, CD56^brightCD16^hi, and CD56^dimCD16^hi cells from one HD (dashed histograms), in the CD25-deficient patient (continuous black histograms) and in the STAT5b-deficient patient (dotted histograms). Gray histograms: FMO. Numbers in the zebra plots from (B,C) correspond to the percentages of each gated cell population; numbers in the histograms from (E) correspond to MFI. Results shown in (B,C,E) representative of three independent blood samples collected over the years. Pt1 began to be studied when she was 3 years old (year 2010). Pt2 began to be studied when she was 10 years old (year 2010). Data shown in (B,C,E) correspond to blood samples obtained in year 2017 (when Pt1 was 10 years old and Pt2 was 25 years old).

Figure 2

CD25 deficiency leads to higher amounts of perforin and granzyme B expression in natural killer cell subpopulations. Analysis of the expression of perforin (pfp), (A) and granzyme B (GzmB) (B), in CD56^brightCD16^lo/−, CD56^brightCD16^hi, and CD56^dimCD16^hi cells from one HD (dashed histograms) and in the CD25-deficient patient (continuous black histograms). Gray histograms: FMO. Numbers in the histograms correspond to MFI. Results are representative of three independent blood samples collected over the years.

Figure 3

CD25 deficiency leads to impaired IFN-γ production but does not negatively affect degranulation by CD56^bright and CD56^dim natural killer (NK) cells. (A) Percentage of IFN-γ-producing CD56^bright (left histograms) and CD56^dim NK cells (right histograms) from one HD (dashed histograms) and from the CD25-deficient patient (continuous black histograms) in response to stimulation with IL-12, IL-15, and IL-18. (B) Percentage of T-bet-expressing CD56^bright (left histograms) and CD56^dim NK cells (right histograms) from one HD (dashed histograms) and from the CD25-deficient patient (continuous black histograms). (C) Degranulation of CD56^bright (left histograms) and CD56^dim NK cells (right histograms) from one HD (dashed histograms) and from the CD25-deficient patient (continuous black histograms) in response to stimulation with K562 cells. In all panels, gray histograms correspond to unstimulated cells. Numbers in the histograms from (A,C) correspond to the percentage of positive cells; numbers in the histograms from (B) correspond to MFI. Results are representative of three independent blood samples collected over the years.

Figure 4

CD25 deficiency leads to increased proliferative response of natural killer (NK) cells. (A) Percentage of CFSE^low NK cells from one HD (dashed histograms) and in the CD25-deficient patient (continuous black histograms) in response to stimulation with IL-2 plus IL-15 in CD16^lo/− NK cells (left histograms) and in CD16^hi NK cells (right histograms). (B) Percentage of CFSE^low NK cells from one HD (dashed histograms) and in the CD25-deficient patient (continuous black histograms) in response to stimulation with IL-2 plus IL-15 in CD56^brightCD16^lo/− NK cells (left histograms), in CD56^brightCD16^hi NK cells (middle histograms) and in CD56^dimCD16^hi NK cells (right histograms). Numbers in the histograms correspond to the percentage of CFSE^low NK cells. Gray histograms: CFSE-labeled NK cells cultured in the absence of IL-2 and IL-15. Results are representative of three independent blood samples collected over the years.

Figure 5

CD25 and STAT5b deficiencies leads to accumulation of CD27⁺CD11b⁺ natural killer (NK) cells with different consequences on the frequency of CD57⁺CD56^dimCD16^hi NK cells. (A) Analysis of maturation stages according to the expression of CD27 and CD11b in CD56^brightCD16^lo/−, CD56^brightCD16^hi, and CD56^dimCD16^hi NK cells from peripheral blood from one healthy donor (HD), the CD25-deficient patient (Pt1), and the STAT5b-deficient patient (Pt2). (B) Analysis of the expression of CD57 in CD56^dimCD16^hi NK cells from peripheral blood from one HD, the CD25-deficient patient (Pt1) and the STAT5b-deficient patient (Pt2). Numbers in the zebra plots correspond to the percentages of each cell population. (C) Analysis of the expression of CD94 in CD56^brightCD16^lo, CD56^brightCD16^hi, and CD56^dimCD16^hi NK cells from peripheral blood from one HD (dashed histograms) and the CD25-deficient patient (continuous black histograms). Gray histograms: FMO. Numbers in the histograms correspond to MFI.

Figure 6

CD25 and STAT5b deficiencies do not affect CCR7 expression, but negatively affect downregulation of CD62L on CD56^brightCD16^hi and CD56^dimCD16^hi natural killer cell subpopulations. Analysis of the expression of CCR7 (A) and CD62L (B) on CD56^brightCD16^lo/− cells (gray histograms), CD56^brightCD16^hi cells (continuous thick line), and CD56^dimCD16^hi cells (continuous thin lines) from one HD, in the CD25-deficient patient (Pt1) and in the STAT5b-deficient patient (Pt2). The appended table summarizes the percentage and MFI for CCR7 and CD62L on each cell population from each sample.

Figure 7

Schematic representation of the role of IL-2 during human natural killer (NK) cell maturation. (A) In healthy individuals, IL-2 promotes the maturation of CD56^brightCD16^lo NK cells into CD56^brightCD16^hi NK cells and then, into CD56^dimCD16^hi NK cells. The first process occurs probably in the paracortical areas of the lymph nodes while the second process occurs once CD56^brightCD16^hi NK cells left the lymph nodes and circulate in periphery, thanks to the imprint provided by IL-2. The transition from CD56^brightCD16^lo NK cells into CD56^brightCD16^hi NK cells and then, into CD56^dimCD16^hi NK cells is accompanied by the downregulation of CCR7 and CD62L, the calibration of the normal content of pfp and granzyme B, and the acquisition of optimal effector functions (IFN-γ production and cytotoxic response). (B) In the absence of IL-2 signaling through the high affinity receptor, the transition from CD56^brightCD16^lo NK cells into CD56^brightCD16^hi NK cells and then, into CD56^dimCD16^hi NK cells cannot occur normally and, consequently, NK cells cannot normally progress from CD56^brightCD16^hi NK cells to CD56^dimCD16^hi NK cells, resulting in the accumulation of CD56^brightCD16^hi NK cells in peripheral blood. Moreover, in the absence of adequate IL-2 signaling, NK cells display increased content of pfp and granzyme B, proliferation, and expression of CD62L, and impaired IFN-γ production, suggesting that they achieve an incomplete maturation program.