case_images: PMC6759462_01
case_images: PMC6759462_02
case_images: (A) The results of hematological testing and infectious diseases during the course of the disease. (B) The results of immunological testing during the course of the disease. BM, bone marrow; CMV, cytomegalovirus; IFNg, interferon gamma; SCT, hematopoietic stem cell transplantation.
case_images: fimmu-10-02194-g0001.jpg
case_images: PMC6759462_04_fimmu-10-02194-g0001.jpg
case_images: F1
case_images: Figure 1B. Flow cytometry (FC) determination (first performed at the age of 5 months) of major lymphocyte subsets did not provide conclusive results.
case_images: PMC6759462_03
case_images: PMC6759462_04
case_images: (A) The results of hematological testing and infectious diseases during the course of the disease. (B) The results of immunological testing during the course of the disease. BM, bone marrow; CMV, cytomegalovirus; IFNg, interferon gamma; SCT, hematopoietic stem cell transplantation.
case_images: fimmu-10-02194-g0001.jpg
case_images: PMC6759462_04_fimmu-10-02194-g0001.jpg
case_images: F1
case_images: Figure 1B. Flow cytometry (FC) determination (first performed at the age of 5 months) of major lymphocyte subsets did not provide conclusive results.
case_images: (A) T-cell culture viability determined by flow cytometry. Live cells were gated based on propidium iodide and forward scatter signals. (B) Western blot analysis of Bcl-2 and Bcl-xL expression in T cells after 2, 3, and 5 weeks of culture. Ctrl, control, Pt, patient. (C) Flow cytometry analysis of T-cell calcium response to activation with 1 and 100 mug/ml anti-CD3epsilon antibody. The arrow indicates the time point when antibody was added to the sample.
case_images: fimmu-10-02194-g0002.jpg
case_images: PMC6759462_04_fimmu-10-02194-g0002.jpg
case_images: F2
case_images: T-cell cultures were established by stimulating PBMCs with immobilized anti-CD3epsilon antibody followed by propagation in the presence of IL-2. These cultures displayed reduced viability, accompanied by the lack of anti-apoptotic Bcl-2 protein expression (Figures 2A,B). Upon CD3 re-stimulation, they showed slightly decreased calcium flux (Figure 2C), while no difference in overall tyrosine phosphorylation after TCR stimulation was observed (data not shown).
case_images: Analysis of proliferation (A), cell death (B) of HEK293T cells transfected with wt SAMD9-GFP, mutant SAMD9-GFP or empty GFP-positive control expression vector. Representative data are shown with results depicted as a fraction of GFP+ cells. Statistical significance: (*P <= 0.05; **P <= 0.001; ***P <= 0.0001).
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case_images: PMC6759462_04_fimmu-10-02194-g0003.jpg
case_images: F3
case_images: The impact of mutant SAMD9 was even more profound resulting in a dramatic drop in the number of proliferating cells (Figure 3A). Interestingly, mutant SAMD9 induced primary necrosis of the cultured cells further demonstrating the gain-of-function impact of p.R824Q on SAMD9 (Figure 3B).