case_images: PMC7333364_01
case_images: IFNgammaR1 cell-surface expression and activity in cells of patient heterozygous for 818del4 IFNGR1 mutation. (A) The expression of IFNgammaR1 molecules (measured as Mean Fluorescence Intensity, MFI) at the surface of CD14-positive mononuclear blood cells, in the patient (P1) and a healthy donor (HD), by flow cytometry assay with a mouse antibody specific for human IFNgammaR1, and an isotype control. (B) The p-STAT1 level expression (measured as MFI), to analyze IFNgammaR1-mediated signaling, is detected by flow cytometry assay with a mouse antibody specific for human p-STAT1, and an isotype control, after incubation with IFN-gamma (1,000 UI/ml) for 30 min, or medium alone, in CD14-positive mononuclear blood cells in the patient (P1) and a healthy donor (HD).
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case_images: F1
case_images: The flow cytometry analysis of IFNgammaR1 expression on patient's monocytes showed that the receptor was present at a higher level on cell's surface (Figures 1A,B), while STAT1 phosphorylation (pY-701) following stimulation with IFNgamma was markedly reduced, as compared to the healthy control (Figures 1C,D).
case_images: Functional analysis of 818del4 mutated IFNgammaR1 and study of patient's DCs. (A) Percentage of plasmacytoid dendritic cells (%pDCs) and myeloid dendritic cells (%mDCs) in P1 in timing (months) of therapy and follow-up (the dotted lines indicate the normal values, gray-colored for mDCs and black-colored for pDCs). IFN-alpha (B) and CXCL10 production (C) are measured in triplicate, by freshly isolated PBMCs after stimulation for 24 h with 6 mg/ml CpG, HSV-1 (copies/200 ml), or medium alone, in the patient (P1) and a healthy donor (HD). Statistical analysis by non-parametric test shows a significant difference (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Plasmacytoid dendritic cells (%pDCs) and myeloid dendritic cells (%mDCs) are analyzed in other patients with mycobacterial infection: P2 has AD partial IFNgammaR1 deficiency and had acute mycobacterial infection during the evaluation of flow cytometry, while P3 has AD partial IFNgammaR1 deficiency but blood was collected when he was free of infection; P4, P5, and P6 presented a mycobacterial infection without any identified predisposing genetic cause (the dotted lines indicate the normal values, gray-colored for mDCs and black-colored for pDCs).
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case_images: F2
case_images: Monitoring of DCs subsets during the infection treatment showed that the percentage of DCs progressively increased, reverting to the normal levels by the end of the treatment (18 months) and remaining normal in the following months (Figure 2A). In addition, ELISA measurements of IFNalpha and CXCL10 in 24-h supernatants of PBMCs incubated with CpG or with increasing copies of human Herpes Simplex Virus Type 1 (HSV1) demonstrated lower levels of these cytokines in P1 as compared to the healthy control (Figures 2B,C), which is in accord with the blood depletion of pDCs. We detected a low number of both pDCs and mDCs subsets in P2, while DCs counts were normal in all the other investigated patients (Figure 2D).
case_images: Study of 818del4 mutated DCs maturation pattern. DCs are cultured for 24 h with LPS for both the patient (P1) and a healthy donor (HD) and the expression of DCs maturation markers (HLA-DR, CD80, CD83, CD86) are analyzed by flow cytometry, together with an isotype control (PE- or PerCP- IgG): a similar pattern of maturation is present in both the patient and the healthy subject.
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case_images: F3
case_images: Finally, in P1, we studied DCs maturation by flow cytometry: the analysis of HLA-DR, CD80, CD83, and CD86 markers revealed that, after 24 h of in vitro culture with LPS, DCs exhibited a normal pattern of maturation (Figure 3).
case_images: Abdominal lymph node histology. Sections are from lymph nodes of P1 (A-D) and two age-matched control (E-L) with mycobacterial infection, stained as labeled. On H&E, necrotizing granulomas are surrounded by lymphoid cells represented by CD20+ B-cells and CD3+ T-cells. CD303/BDCA2+ pDC are severely reduced as compared to matched controls. Original magnification: 40x (A-C,E-G,I-K; scale bar 500 mum), 100x (D,H,L; scale bar 200 mum).
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case_images: F4