PMC6549489_01
Imaging, bone marrow biopsy findings and characterization and location of the novel FAS heterozygous variant in a patient with CD4 lymphopenia and ALPS. (A) Computed tomography of abdomen and pelvis. (1) Coronal section: Splenomegaly. (2) Splenic volume estimation by 3D reconstruction: 1,034 milliliter (mL) (normal 200 +- 50 mL). (3) Axial section: Paraortic and mesenteric lymphadenopathy. (B) Bone marrow biopsy showing scattered lymphocytic aggregates with prevalence of CD8+ cells. (1) Lymphocytic aggregate in hematoxylin and eosin staining at 200x magnification. (2) CD3 Immunohistochemistry (IHC) staining. (3) CD8 IHC staining. (C) Diagram of the FAS gene identifying intron/exon structure and protein domains. Mutations are indicated at their approximate location and identified by symbols corresponding to mutation type. The new mutation identified in the index patient indicated by the red arrow. Figure adapted by Hsu et al.. (D) FAS-induced apoptosis was evaluated in terminal effector memory cells (TEM) and TCRalphabeta+ DN T cells of index patient and healthy subjects using an ex vivo flow cytometric assay based on FAS crosslinking. (1) Average fold increase +- standard error of mean (SEM) of Annexin-V expression in TEM (CD45RA-CCR7-CD27-) from 4 independent experiments with index patient (red) and 8 different healthy controls (blue) with different concentrations of anti-FAS crosslinking antibody. (2) Average fold increase +- SEM of Annexin-V expression in DNT cells (TCRalphabeta+CD4-CD8-) from 4 independent experiments with index patient (green) and 8 different healthy controls (purple) with different concentrations of anti-FAS crosslinking antibody.
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Idiopathic CD4 lymphopenia (ICL) was formulated, and she was referred to the NIH Clinical Center for further evaluation. Imaging confirmed splenomegaly (17 cm cranio-caudal length) and revealed modest mesenteric and retroperitoneal adenopathy (Figure 1A). Total IgG levels were moderately elevated (1,716 mg/dL), IgM were within normal range (54 mg/dL), while IgA undetectable. Bone marrow biopsy showed scattered lymphocytic aggregates with prevalence of CD8+ cells, consistent with previously reported findings in bone marrow biopsy of patients with ALPS (Figure 1B). Targeted NGS with custom target enrichment revealed a novel heterozygous missense variant in the death domain (NM_000043 c.719T>A p.M240K) of the FAS gene and was confirmed by direct Sanger sequencing (Figure 1D). Annexin-V expression was found with 1,000 ng/mL of crosslinking FAS antibody. At the same concentration of crosslinking FAS antibody (1,000 ng/mL), the Annexin-V expression remained unchanged in the index patient (1.06 +- 0.1-fold, n = 4, p = 0.01) (Figure 1C).
FAS-induced apoptosis and activation-induced-cell death (AICD) in T cell blasts from the index patient and healthy subjects. *Comparisons with statistically significant differences. (A) Average fold increase +- SEM of Annexin-V expression in CD4 T cell blasts from index patient (red) and 2 different healthy controls (blue) with different concentrations of anti-FAS crosslinking antibody. (B) Average fold increase +- SEM of Annexin-V expression in CD8 T cell blasts from index patient (green) and 2 different healthy controls (purple) with different concentrations of anti-FAS crosslinking antibody. (C) Average fold increase +- SEM in Propidium Iodide (PI) positive CD4 T cell blasts from index patient (red) and 2 different healthy controls (blue) with 2 different concentrations of PHA. (D) Average fold increase +- SEM in PI positive CD8 T cells blasts from index patient (green) and 2 different healthy controls (purple) with 2 different concentrations of PHA.
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A significant reduction of FAS-induced apoptosis was found in T cell blasts from the index patient at all concentration of crosslinking FAS antibody: at the highest concentration (1,000 mg/mL), Annexin-V expression increased in CD4 (3.06 +- 0.2-fold, n = 6) and CD8 (2.8 +- 0.3-fold, n = 6) blasts from healthy individuals but remained substantially unchanged in CD4 (1.2 +- 0.05-fold, n = 3, p = 0.02) and CD8 (1.2 +- 0.1-fold, n = 3, p = 0.02) blasts from the index patient (Figures 2A,B). Similarly, a significant reduction in AICD was found in T cell blasts of the index patient: the proportion of dead cells after stimulation with the highest concentration of PHA (50 mug/mL) increased in CD4 (6.8 +- 0.3-fold, n = 6) and CD8 (13.7 +- 2-fold, n = 6) blasts from healthy individuals but to a lower extent in CD4 (3.3 +- 0.25-fold, n = 3, p = 0.02) and CD8 (8 +- 1.2-fold, n = 3, p = 0.09) blasts from the index patient (Figures 2C,D).