PMC7674281_01
Flow cytometric analysis of BTK expression in a severe XLA patient (left plot) and the index patient (right plot). For each plot, histograms representing BTK expression measured as Mean Fluorescence Intensity (MFI) are presented for the following cell subsets: B lymphocytes of a healthy control (green line), patient's B lymphocytes (red line), T lymphocytes of a healthy control (blue line), patient's T lymphocytes (purple line).
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Flow cytometry analysis displayed the presence of a residual BTK protein expression of both patient's B lymphocytes ( Figure 1 ) and myeloid lineage cells (monocytes and granulocytes). To note, BTK was identified as a single peak for each population, with a Mean Fluorescence Intensity (MFI) corresponding respectively to approximately 40% ( Figure 1 ) and 70% (data not shown) of the values measured on the healthy control cells. Conversely, BTK was absent in circulating B lymphocytes of a patient diagnosed with a severe form of XLA, whose MFI was comparable to one of those lymphocyte subsets which intrinsically lack BTK expression, such as T cells ( Figure 1 ).
Analysis of peripheral B cell subset distribution a healthy control (left column), the index patient (central column), and a severe XLA patient (right column). For each subject the following B cell subsets are presented: recent bone marrow emigrants (RBE) (CD20+CD38++CD10+), immunoglobulin-secreting cells (ISC) (CD38+++CD27++CD20-) and mature cells (CD20+CD38+/-CD10-) which are further classified in naive cells (IgD+IgM+CD27-) and both switched memory (IgD-IgM-CD27+) and unswitched memory cells (IgD+IgM+CD27+).
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Percentages of cells having the phenotypical characteristics of recent bone marrow emigrants (RBE) (CD20+CD38++CD10+) and naive cells (IgD+IgM+CD27-), as well as switched and unswitched memory cells (IgD-IgM-CD27+ and IgD+IgM+CD27+ respectively), did not significantly differ from values observed in healthy age-matched individuals, while only immunoglobulin-secreting cells (ISC) (CD38+++CD27++CD20-) were below the normal range ( Figure 2 , central column). On the opposite, peripheral B cells of XLA patients with severe clinical manifestations and undetectable BTK expression did not display any ability to differentiate into mature cells ( Figure 2 , right column).