PMC7611501_01
Clinical phenotype of the patient. (A) MRI scan of the brain at 3.5 years (upper panel), showing atrophy of the telencephalon (1), enlarged external and internal cerebrospinal fluid spaces (4), arachnoidal cyst (2) in the posterior fossa and insufficient myelination (3) indicated by arrow heads. The lower panel shows an MRI scan of a healthy 3.5 year old child. (B) X-ray of the pelvis showing flat, dyplastic acetabulae. (C) X-rays of the chest showing chronic interstitial pneumonia. (D) Neutrophil counts over time. Dashed line at 1000 neutrophiles/mul represent limit for neutropenia. (E) Bone marrow smear showing hypersegmented neutrophils.
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Furthermore, the myelination was insufficient for age corresponding rather to a 15 months old child: while cerebellar white matter was hypointense indicating myelin, only projection fibres and central parts of the supratentorial white matter were myelinated involving some subcortical domains, but not U-fibres (Fig. 1A). A hip radiograph showed flat acetabulae (Fig. 1B). Chest X-rays revealed chronic interstitial pneumonia (Fig. 1C). He had persistent hepatosplenomegaly and febrile episodes that were frequently associated with severe neutropenia (Fig. 1D), but did not develop thrombocytopenia or anemia. Bone marrow showed moderate dysplasias (Fig. 1E) with full maturation of all lineages.
Impaired degranulation and cytotoxicity of patient NK cells. (A) Ex vivo degranulation of NK cells (CD3-CD56+) from the patient (Pat) and a healthy donor (Ctr) after incubation with medium (left panel) or with K562 cells (middle panel) as assessed by flow cytometric analysis of CD107a surface expression. The right panel shows the difference in CD107a expression between unstimulated and stimulated cells of the patient (closed symbol) and a day control (open symbol) relative to healthy controls (gray area). The dashed line represents the level below which NK cell degranulation has the best positive and negative predictive value for a mutation in a gene relevant for cytotoxicity in an unfiltered cohort of patients with HLH. (B) Degranulation of NK cells prestimulated with IL-2 and PHA for 48 hours. (C) Cytotoxicity of patient (circle) and control (squares) PBMC on K562 target cells without (black symbols) or after overnight pre-incubation with IL-2 (grey symbols). (D) T cell degranulation. Cultured T cell of the patient and a healthy control were incubated in medium or stimulated with 3mug/ml of plate-bound anti-CD3. The increase in CD107a expression upon stimulation is shown as an overlay of histograms of unstimulated (dotted line) and stimulated (solid line) cells.
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NK cells (Fig. 2B) after incubation with the NK sensitive target cell K562. NK cell degranulation was clearly abnormal. These results were confirmed in a 51Cr release assay (Fig. 2C). T cell degranulation was impaired, but not absent (Fig 2D).
Genetic analysis and model of the AP-3 complex. (A) Electropherograms of the section of exon 32 harboring the homozygous deletion in the patient. The deletion leads to a frameshift at codon 1189 and a termination codon after seven residues. The encoded wild-type peptide sequence is shown at the top, the codons of the mutant sequence are shown below the DNA sequences. The parents are heterozygous for the deletion as expected. For technical reasons the reverse strand was sequenced in the DNA sample of the father but the complementary sequence is depicted here, i.e. it is shown in the same direction as the other samples. (B) Model of the AP-3 protein complex in the ubiquitous and the neuronal form (adapted from). AP-3delta is an essential part of both complexes, while AP-3beta3A, mutated in HPS-2, is substituted by AP-3beta3B in neuronal cells.
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The deletion of GT at positions c.3565-3566 in exon 32 of AP3D1 (c.3565_3566delGT; NM_001261826; Fig. 3A) leads to a frameshift and a termination codon after seven residues (p.Val1189Leufs*8) (Fig. 3A). AP3D1: In isoform 2, which is the shorter splicing variant lacking exons 23 and 24, the mutation is found in exon 30 (c.3379_3380delGT; NM_003938). AP3D1 is not only expressed in the ubiquitous AP-3 complex but also in the neuronal form (Fig. 3B).
Reduced expression of AP-3delta affects the stability of the AP-3 complex. Western blot analysis of lysates from Macs-purified CD8 T cell of two healthy controls, an AP-3beta3A deficient patient and the index patient (lane 1-4). Two separate blots are using rabbit antibodies against AP-3delta and AP-3sigma (left) and AP-3beta and AP-3mu (right). beta-actin was used as a loading control. AP-3delta protein was analysed using two different antibodies in three independent experiments each, each giving the same results. Lanes 5 and 6 show blots from patient PHA/IL-2 blasts retrovirally transduced with an AP3D1 expressing or empty vector analysed in the same experiments. These cells were FACS sorted for GFP positive cells to enrich for successfully transduced cells.
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Consistent with an instable heterotetramer formation, protein levels of other subunits (AP-3beta3A, AP-3sigma and AP-3mu) of the AP-3 complex were clearly reduced (Fig. 4). Consistent with published data, the control cells from the HPS-2 patient only showed reduced levels of AP-3beta3A and AP-3mu, whereas AP-3delta and AP-3sigma were present at normal levels. T cells revealed that retroviral transfection of T cells with the AP3D1 wildtype gene restored the presence of AP-3delta, AP-3beta3A, AP-3sigma and AP-3mu to normal levels (Fig.4). These data confirmed that the patient mutation AP3D1 was responsible for the destabilization of the AP-3 complex.
Genetic reconstitution with wild-type AP3D1 showing expression and function of the AP-3 complex. (A) Immunofluorescence analysis of patient PHA/IL-2 blasts reconstituted with either empty vector or wild-type AP3D1 using mouse anti-AP-3delta (SA4). Cells are shown in one 3D stack picture of 28 slices. Cells successfully transduced with the vector are GFP+ (white in the single stains, green in the merged picture), the nucleus is stained with Hoechst (blue) and AP-3delta is stained with SA4 provided by A. Peden (white in the single stains, red in the merged picture). The variable GFP intensity of reconstituted cells is explained by the use of an IRES-GFP construct where AP3D1 is placed before and GFP is placed after the IRES sequence. (B) Transduced T cells were rested in medium free of IL-2 for 24 hours and then incubated in medium or stimulated with 3mug/ml pb CD3 for 3 hours. Transduction efficiencies varied between 1 and 12%. (C) CD107a expression after incubation of transduced cells with medium or plate-bound anti-CD3. Plots are gated on successfully transduced GFP+ CD8+ cells. The solid line serves indicates peak CD107a expression in patient cells transduced with the empty vector. (D) Overlay of CD107a expression of transduced T cells kept in medium (dotted line) and stimulated stimulated with anti-CD3 (solid line) (E) The difference in mean fluorescence intensity (delta MFI) of CD107a expression between cells incubated in medium versus stimulated with pb anti-CD3 is shown for patient (pat) and ctonrol (ctr) GFP+CD8+ T cells transduced with the indicated vector. Results are shown from two independent experiments.
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Successful reconstitution of GFP positive cells with AP3D1 could be demonstrated by immunofluorescence (Fig.5A), showing the expression of AP-3delta as red dots in GFP positive cells only. Figure 5B shows the flow cytometric analysis of retrovirally transduced cells. For degranulation, we incubated the transduced T cell cultures either with plate-bound anti-CD3 or with medium and measured the CD107a expression in successfully transduced GFP+ CD8+ T cells (Fig. 5C). Importantly, transduction with the AP3D1 containing vector fully restored the degranulation response of patient cells to levels of wild-type cells in two independent experiments (Fig.5D+E), while transduction with the empty control vector had no effect.