Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity

Abstract

Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8(+) T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8(+)STAT5(+) T cells with lytic potential infiltrated the skin, even though FOXP3(+) Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.

Figure. 1

A c.497G>A mutation in the IL2RA gene inhibits surface and soluble CD25 expression. A, Sequence trace of genomic DNA of the patient (left panel) showing a G to A substitution at position 497 (down arrow) in exon 4 leading to the amino acid substitution S166N. Sequence trace of paternal genomic DNA shows a single copy of the G497A mutation (right panel). B, Representative dot plots from 3 independent experiments of CD25 expression on CD4⁺ T cells from PBMCs of the CD25 deficient patient, the patient's father, mother and sister and a healthy control (HC) (n = 6) as determined by flow cytometry. C, Representative histograms of surface (left column) and cytoplasmic (right column) expression of CD25 on T cell lines were determined for the patient (bottom histograms) and healthy controls (upper histograms) after stimulation with anti-CD3 and anti-CD28 alone (solid line) or in combination with IL-2 and IL-15 (dotted line) or left untreated (grayed area). Embedded histograms of CD69 expression for the patient and healthy controls are shown. D, Soluble CD25 (sCD25) was measured from the plasma at 8 different time points from the patient and from 9 healthy controls. E, Representative histograms showing the expression of CD122 (upper left) and CD132 (lower left) were determined on PBMCs of the patient (large histograms) and healthy controls (embedded histograms). The MFI of CD122 and CD132 on the CD4⁺ and CD8⁺ T cells of the patient (each symbol indicates a unique time point) and healthy controls (n = 6) was determined using FACS analysis on PBMCs.

Figure. 2

CD25 deficiency promotes a T memory cell phenotype. A, Representative FACS plots depicting the average percentage of FOXP3⁺ CD4⁺ T cells (upper panels), CD127^low cells expressing FOXP3 (middle upper panels with embedded histograms), CD127^lowCD49d^low cells expressing FOXP3 (middle bottom panels with embedded histograms), FOXP3⁺ and HELIOS⁺ staining were determined in the CD25 deficient patient (average of 2–4 different time points is shown) and healthy controls (HC; average percentage of 2–6 different donors is shown). B, The average percentage of naïve (CD45RA⁺CD62L⁺), central memory (TCM, CD45RA⁻CD62L⁺), effector memory (TEM; CD45RA⁻CD62L⁻) for CD4⁺ (upper row) and CD8⁺ (lower row) T cells for the patient (left column) and healthy controls (right columns). The percentages shown are the average of 4 different time points for the patient and the average of six healthy donors. C, The overall percentage of memory cells (CD45RA⁻) from the patient and healthy controls. D, The percentage of the effector populations within the memory pool (CD45A⁻) of the patient and healthy controls.

Figure. 3

Reduced IL-2 response of T cells from the CD25 deficient patient but FOXP3⁺ T cells remain first responders. A, The percentage of CD4⁺ (upper row) and CD8⁺ (lower row) T cells from PBMCs of the patient (left column) or healthy controls (right column; n = 5) responding to IL-2lo (10 U/ml), IL-2med (100 U/ml), IL-2hi (1000 U/ml), or IL-15 (10 ng/ml) at 0, 10 or 30 min. pSTAT5 was evaluated by flow cytometry from barcoded cells. B, Representative dot plots of the IL-2 responsiveness of CD4⁺FOXP3⁺ and CD4⁺FOXP3⁻ T cells from the patient and healthy control were evaluated for using IL-2lo (10 U/ml), IL-2med (100 U/ml), IL-2hi (1000 U/ml) conditions at the indicated time points determined by pSTAT5. C, The hierarchy of IL-2 signaling was determined from PBMCs of the patient (left column; closed shapes) or healthy control (right column; open shapes (n = 3)) for CD4⁺FOXP3⁺, CD4⁺FOXP3⁻ and CD8⁺ T cells in response to different concentrations of IL-2 as used in B.

Figure. 4

Abundant innate and adaptive cytokines in vivo correlate with predominate CD8⁺ activation and proliferation. Sera from the CD25 deficient patient (unique symbol for each time point) and healthy donors (each symbol represents a unique patient) were measured for innate (A) and adaptive (B) associated cytokines. C, Representative FACS plots of pSTAT5 (upper row; bold line) and pSTAT3 (lower row; bold line) in CD4⁺ and CD8⁺ T cells of the CD25 deficient patient (larger histogram) and healthy controls (embedded histogram) using isotype control antibody staining (grayed area) as a reference. D, Proliferating CD4⁺ and CD8⁺ T cells from fresh PBMCs were determined by staining for Ki-67⁺ (representative FACS plot shown). The average proliferative rate of CD4⁺ and CD8⁺ T cells from the CD25 deficient patient and healthy controls (E) was taken at different time points and averaged (p value; students t test). F, Representative histograms of HLA-DR and FAS-L expression on proliferating (Ki-67⁺; solid line) and non proliferating (Ki-67⁻; dashed line) CD8⁺ T cells from the CD25 deficient patient at two different time points or total CD8⁺ T cells from healthy controls (dotted lines). Gray line is the staining control on the CD8⁺ T cells.

Figure. 5

Epidermal hyperplasia due to CD8⁺ T cell infiltration, proliferation and granzyme B production. A, A skin biopsy of the patient showed hyperplasia with hyper-orthokeratosis. (B), Lymphocytic infiltrate was observed mainly in upper dermis, (C) displaying mostly a T cell phenotype (CD3⁺). Skin infiltrating cells were mostly CD8⁺ T cells (D–F) and many were proliferating as determined by double immunofluorescence staining with CD8 and Ki-67 (E). F, A high frequency of CD8⁺ T cells was also granzyme B⁺.

Figure. 6

Defective T cell responses to polyclonal mitogens and viral Ags. A, PBMC from the patient and healthy controls were stimulated with anti-CD3 and anti-CD28 in the presence or absence of IL-2 (+ 10 U/ml, ++ 100 U/ml, and +++ 1000 U/ml) and/or IL-15 (10 ng/ml) and/or IL-15 (10 ng/ml), or PHA and proliferation was determined by ³H-thymidine after 3 days. B, T cell responses to C. albicans, TT, were measured after 4 days and (C) different viruses (CMV, VZV, and HSV) were measured after 3 days of stimulation in the presence or absence of IL-2 and IL-15. D, PBMC ELIspot assays from the patient and healthy control were stimulated with CMV Ags alone or in the presence of IL-2 or IL-15, or with IL-2, IL-15, or TPA/Ionomyocin alone.