PMC8438314_01
Timeline of the clinical events and treatment strategies. AIHA, autoimmune hemolytic anemia; IVIG, intravenous immunoglobulin.
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The clinical picture was further worsened by multiple relapses of Evans syndrome, which required full-dose corticosteroid (prednisone, 2 mg/kg) and two hospitalizations at age 18 months and 3 years (Figure 1).
Genetic and functional investigations (A) Apoptosis assay: The IPEX patient exhibits a significantly altered apoptosis. Fas-induced apoptosis in EBV-lymphoblastoid cell lines (EBV-LCL) or PHA-activated peripheral blood lymphocytes of the patient and a healthy control were stimulated for 24 h with or without APO-1-3 in the presence of protein A. Subsequently apoptosis was measured by flow cytometric detection of Annexin V-PE and 7AAD. For apoptosis in EBV-LCL, data represent two separate experiments and the T bars indicate the standard deviation. The dotted red line represents 50% of the cell death observed in the control. (B) Genomic DNA sequence analysis of FOXP3 gene showing a missens hemizygous mutation p.Pro75Leu in the patient, the mother was heterozygous for the mutation. (C) Multiple protein alignment showing the position of the conserved proline 75 residue in different species using the NCBI Multiple Sequence Alignment Viewer, Version 1.19.1. (D) FOXP3 expression in PBMCs after staining with anti-CD4/APC, anti-CD25/BB-515 and anti-Foxp3/PE as compared to healthy control. Treg cells were absent in patient's unstimulated cells and induced after activation with anti-CD3 (aCD3)/anti-CD28 (aCD28) mAb during 72 h.
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Apoptosis functional assay was performed on the patient's EBV-transformed B-cells and showed severe resistance to Fas-mediated cell death in comparison to a healthy control, as confirmed in two separate experiments (Figure 2A). It has also been performed on the patient's PHA-activated peripheral blood lymphocytes and confirmed the resistance to apoptosis as compared with healthy control (Figure 2A). The patient's mother was heterozygous for the same mutation (Figure 2B). The affected residue is conserved among vertebrates (Figure 2C), and the mutation is predicted to be deleterious in multiple in silico prediction tools (probably damaging in Polyphen2, damaging in SIFT and CADD score of 24). FOXP3 mutation, FOXP3 expression was performed in patient's PBMCs as compared to healthy control, after staining with anti-CD4/APC, anti-CD25/BB-515 and anti-Foxp3/PE with or without activation for 72 h using anti-CD3/anti-CD28 monoclonal antibodies. FOXP3+ CD25+ cells were absent in patient's unstimulated cells (0.1% of CD4+) but were induced after activation (27.8%) (Figure 2D).
PMC8438314_02
Investigation of T cell exhaustion. (A) High percentage of TEMRA cells (Q3) was found in patient after whole blood staining with anti-CD3/PerCP, anti-CD4/FITC, anti-CD45RA/PE-Cy7 and anti-CCR7/PE as compared to one representative healthy control. (B) Increased surface expression of PD1 on the patient's CD4+ lymphocytes as compared to one representative control..
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We found increased percentage of CD4+ TEMRA cells in the patient reaching 24% (normal range for the age, 4-15%) (Table 1 and Figure 3A). Moreover, the patient showed high PD1 expression by 10.9% on CD4+T cells, one of the hallmarks of T cell exhaustion, in comparison to healthy controls (Figure 3B).