PMC5326763_01
(A) The immunophenotyping of the T and B lymphocyte subsets of the patient was compared with the maturation of his 2-year-old brother who did not carry the mutation. (B) Standard screening of CD4+ regulatory T cells using CD127 and CD25 immunostaining (upper panel) was further supported by the assessment of a normal expression of intracellular forkhead box protein 3 (lower panel). (C) Function of the patient's helper cell differentiation was tested by measuring the cytokines for the Th1 (IFNgamma, TNFalpha), Th2 (IL-13), and Th17 (IL-17) in the supernatant of anti-CD3/anti-CD28-stimulated T cells as determined by Luminex (eBioscience, San Diego, CA, USA).
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F1
T cells (Table 1). The percentage of naive and switched memory B cells was within age-specific reference ranges. CD4+ and CD8+ T cell differentiation into memory/effector memory cells was normal (Figure 1A). The percentage of CD4+ regulatory T (Treg) cells determined by CD25 and CD127 was found to be unremarkable, and the intracellular FOXP3 expression was comparable to healthy controls (Figure 1B). Lymphocyte activation using T cell stimuli such as anti-CD3/anti-CD28 or IL-15 induced adequate proliferative responses of CD4+ and CD8+ T cells as assessed by carboxyfluorescein succinimidyl ester dilution (data not shown), although the cytokine release into the supernatant was considered high to normal, in particular for the Th2 signature cytokine IL-13 (Figure 1C).
(A) Mutation c.1010G>A; p.(Arg337Gln) in the forkhead box protein 3 (FOXP3) gene identified using whole exome sequencing (gene on reverse strand). The index patient is hemizygous, and the mother is a heterozygous carrier (left panel). Mutation c.1010G>A; p.(Arg337Gln) in the FOXP3 gene is not present in the grandmother, I:1 (right panel). (B) The cosegregation study in the family showing that the mutation had occurred as a de novo change in the mother of the patient. Arrow indicates index patient.
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F2
His mother was found heterozygous for the mutation (Figure 2A). Subsequent cosegregation showed that the mutation occurred de novo in the mother (Figure 2B).
(A) FACS panels showing the intracellular staining for forkhead box protein 3 (FOXP3) and Helios in the in vitro expanded regulatory T (Treg) cells from a healthy donor (in black) and the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome patient (in red). (B) FACS plots displaying the expression of CD25 and CTLA-4. (C) FACS plots of CD4 and CD8 staining. Gate encloses the CD4+CD8+ double-positive (DP) cells. (D) Immunophenotyping for CD3 and TCRalphabeta of the cells in the gate in panel (C). (E) Representative images of ImageStream analysis from Treg and conventional T cells. Nuclear localization was confirmed by colocalization of the FOXP3 staining (purple) with the nuclear DAPI signal (in blue). (F) Graphs representing the proliferation of responder CD4+ or CD8+ T cells gated from total allogeneic peripheral blood mononuclear cells normalized to the proliferation of the responders only condition (0:1).
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F3
Treg cells from the patient and a healthy donor were expanded upon stimulation with anti-CD3/anti-CD28. The cells were subsequently stained for the expression of the quintessential Treg cell transcription factors FOXP3 and Helios (Figure 3A) and the surface molecules CD25 and CTLA-4 (Figure 3B). Remarkably, the patient's Treg cells presented an elevated presence of CD4+CD8+ DP cells that were also CD3+TCRalphabeta+ (Figures 3C,D). Some FOXP3 mutations have been described to affect the nuclear localization of the protein; we therefore analyzed the nuclear localization of FOXP3 in the Treg cells of the patient by ImageStream (Figure 3E). Undoubtedly, the Treg cells were unable to fully suppress the proliferation of neither CD4+ nor CD8+ T cells, although the Treg cells still retained a limited capacity to suppress CD8+ T cell proliferation (Figure 3F).