NBAS Variants Are Associated with Quantitative and Qualitative NK and B Cell Deficiency

The general clinical parameters included country of origin, sex, and age at onset of clinical symptoms. For clinical phenotyping, human phenotype ontology terms were used (see Supplemental Material). For immunological phenotyping, blood from all individuals was drawn outside episodes of inflammation. If more than one measurement per patient was performed, the arithmetic mean was calculated. For flow cytometry and gene expression analysis, gender-matched and age-matched healthy donors were included. For NK cell degranulation assays additional blood samples from six patients were obtained and gender-matched healthy donors were recruited. Both recruitments took place at the University Hospital Heidelberg. Database was closed on December 1, 2019.

Heparinized whole blood of 0.5 ml was incubated with 0.1 ml RPMI1640 or stimulated in RPMI1640 with 100 ng/ml PMA and 5 μg/ml ionomycin (Sigma) for 3 h at 37 °C and 5% CO₂. Red cells were lysed twice with ACK buffer. Afterward, the leukocytes were lysed with 400 μl of MagNA-Pure lysis buffer (Roche) containing 1% DTT (Roche, cat. no. 10708 984001), and the samples were frozen at − 70 °C. After thawing, the lysates were thoroughly mixed and transferred into the MagNA-Pure sample cartridge and total RNA was isolated with the MagNA-Pure-LC device using the RNA standard protocol for cells. The elution volume was set to 50 μl. An aliquot of 8.2 μl mRNA was reversely transcribed using AMV-RT and oligo-(dT) as primers (First Strand cDNA synthesis kit, Roche) in a thermocycler according to the manufacture’s protocol. After termination of the cDNA synthesis, the reaction mixture was diluted to a final volume of 500 μl and stored at − 20 °C until PCR analysis.

Primer sets optimized for the LightCycler® (RAS) were developed and purchased from SEARCH-LC GmbH (www.​Search-LC.​com). The PCR was performed with the LightCycler® FastStart DNA Sybr Green kit (RAS) according to the protocol provided in the parameter-specific kits. To control for specificity of the amplification products, a melting curve analysis was performed. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value.

To correct for differences in the content of mRNA, the calculated transcript numbers were normalized according to the expression of the housekeeping gene peptidylprolyl isomerase B (PPIB). Values were thus given as transcripts per 1,000 transcripts of PPIB.

Cytokine measurement was performed by Myriad RBM (TX, USA) as follows: Using automated pipetting, an aliquot of each sample was added to individual microsphere multiplexes of the selected multi-analyte profile (MAP) and blocker. This mixture was thoroughly mixed and incubated at room temperature for 1 h. Multiplexed cocktails of biotinylated reporter antibodies were added robotically and after thorough mixing, incubated for an additional hour at room temperature. Multiplexes were labeled using an excess of streptavidin–phycoerythrin solution, thoroughly mixed, and incubated for 1 h at room temperature. The volume of each multiplexed reaction was reduced by vacuum filtration and washed 3 times. After the final wash, the volume was increased by addition of buffer for analysis using a Luminex instrument, and the resulting data was interpreted using proprietary software developed by Myriad RBM. For each multiplex, both calibrators and controls were included on each microtiter plate. Eight-point calibrators to form a standard curve were run in the first and last column of each plate and controls at 3 concentration levels were run in duplicate. Study sample values for each of the analytes were determined using 4 and 5 parameter logistics, with weighted and non-weighted curve fitting algorithms included in the data analysis package.

Peripheral blood mononuclear cells (PBMCs) were isolated from unfractionated peripheral blood by Ficoll-Hypaque density gradient (density 1.077 g/ml, Biochrom, VWR) centrifugation. For functional and phenotypic analyses, PBMCs were resuspended in SCGM medium (CellGenix) containing 10% human serum (pooled human AB serum, # P041702, Pan-Biotech) and cultured for 2 days without or with 400 U/ml of IL-2 (kindly provided by the NIH, USA).

CD107a upregulation on NK cells of NBAS-deficient patients or healthy controls was measured after co-culture of NK cells without or with the erythroleukemic cell line K562 (ATCC) at 1:1 ratio (each 5 × 10⁴ cells) in duplicate for 4 h in the presence of anti-CD107a FITC (clone A4A3, Biolegend) or anti-CD107a PE (clone A4A3, Biolegend, and GolgiPlug, 1/100 v/v, BD Bioscience) in 96-well plates in RPMI 1640 (Invitrogen) supplemented with heat-inactivated 10% FCS (Invitrogen) and 1% penicillin/streptomycin (Sigma-Aldrich).

The immunophenotyping was performed in whole blood. Absolute cell count was measured using the BDMultitest™ 6-color TBNK reagent with BD Trucount™tubes (BD Biosciences, Heidelberg, Germany). The panels are modified recommendations of the “Human Immunophenotyping consortium” [11]. Briefly, Panel-1 consisted of 17 antibodies to characterize the major T cell populations, including naïve and various memory/effector populations (CD2; CD3; CD4; CD8; TCR ƴδ; CD45RA; CD197 (CCR7); CD28; CD57 and CD127). Th1, Th2; Th17, and Tfh cells were identified with the following antibodies: CD183 (CXCR3); CD185 (CXCR5); and CD196 (CCR6). Activation was monitored with antibodies against HLA-DR, CD38, CD278 (ICOS), and CD279 (PD1). Using CD3 as a backbone for absolute quantification, with this comprehensive panel more than 200 defined subpopulations have been quantified in absolute numbers as well as in various ratios. Panel-2 consists of 8 parameters (CD3; CD19; CD20; anti-IgD; CD27; CD10; CD24; and CD38), allowing the identification of transitional, naïve, and memory B cells as well as circulating plasmablasts both in absolute numbers (CD19 backbone) and in various ratios. Panel-3 identified various NK, monocyte and DC subsets and included antibodies with the following specificities: CD11c; CD123; CD14; CD16; CD19; CD2; CD20; CD25; CD3; CD45; CD56; CD57; CD8a; HLA-DR; and M-DC8. Using NK cells as backbone, absolute numbers were calculated for 50 different populations. Finally, the Treg subset was characterized using the following antibodies: CD127; CD194 (CCR4); CD25; CD3; CD4; CD45; CD45RO; and anti-HLA-DR. CD4 + T-lymphocytes served as backbone for the absolute quantification of the different subsets. The analysis was performed on an LSR Fortessa Analyzer (Special Order Research Product) (BD Biosciences). The detailed setup, staining, gating, and analysis procedures have been recently described [12]. Cell surface expression of CD107a and CD69 (marker for NK cell activation) and intracellular expression of perforin and granzyme B of CD3⁻ CD56⁺ NK cells in PBMCs was measured by flow cytometry (LSR Fortessa II, BD Bioscience) and analyzed with FlowJo 10 software (FlowJo LLC). Intracellular staining was performed using the “Cytofix/Cytoperm” kit (BD Bioscience). (For information on antibodies, see Supplemental Material.)

Statistical analyses were performed with GraphPad Prism. Mean values of two groups were compared by Mann–Whitney U-tests. Mean values of multiple groups were assessed by one-way ANOVA followed by multiple comparisons post hoc tests as indicated. Error bars represent the SEM and asterisks indicate the significance levels (*P < 0.05; **P < 0.01; ***P < 0.001). The correlation coefficient R² was calculated by linear regression.

Fifteen patients from 14 unrelated families carrying 19 different NBAS variants (Supplemental Fig. 1A) were included in this study. All three NBAS deficiency subgroups were represented (“β-propeller” n = 4; “Sec39” n = 5; “C-terminal n = 4”; not attributable to one of the subgroups n = 2) (Table 1). Clinical presentations of 14 of those patients have been reported previously [3, 4], whereas individual NBAS 88 has not been reported before. Thirteen patients originated from European countries, one patient from the USA, and one from Saudi Arabia. The median age at last assessment was 7 years (range: 0.4–43 years). First symptoms occurred in all patients within the first 2 years of life and consisted of acute liver failure or elevated liver transaminases in most cases (73%). Only individual NBAS 88 was diagnosed prenatally and showed immunological laboratory abnormalities and slightly elevated liver transaminases at 8 months of age when the database was closed.

All patients had immunological and hepatic features, while other organ affections were present to different extents according to their NBAS deficiency subgroup (Table 1 [3]). One prominent finding throughout all subgroups was the occurrence of recurrent infections (reported in 10/15 patients). These infections were mostly common with respect to localization and pathogens (Table 2), but intensity suggested a pathological susceptibility in some patients when assessed following the national guideline for clinical diagnostics of inborn errors of immunity [13].

To assess adaptive humoral abnormalities in NBAS-deficient patients, we examined serum immunoglobulin (Ig) levels. Ig levels of classes M, G, and A were reduced in 10 of the 15 patients, with consistently low IgG being most prevalent (n = 8; age at diagnosis: 2 months until 7 years, median: 7.5 months; Table 2). All patients from the “C-terminal” subgroup had low IgG. Six patients with consistently low IgG received Ig replacement therapy, resulting in a reduction of infections. Five patients showed insufficient responses to vaccination when tested sporadically, but also after scheduled vaccinations in 3 patients, pointing to an impaired adaptive humoral immune responsiveness (Table 2). Comparing the B cell phenotype of those patients with impaired Ig levels of all tree classes with those patients with normal levels of Ig, the relative number of transitional B cells seems to be increased (22.22 ± 10.44 vs. 6.73 ± 3.73; P = 0.034), pointing to delayed B cell maturation in the patients with humoral immunodeficiency. Differences in B cell subpopulations between patients with specific Ig isotype deficiencies could not be observed.

To analyze the composition of cellular immunity, peripheral blood leukocyte subsets were quantified. The majority of NBAS-deficient patients showed a consistent reduction in CD56⁺ NK cells, resulting in > 50% lower absolute NK cell numbers relative to healthy controls (Fig. 1A–B and Supplemental Fig. 1B). This reduction in CD56⁺ NK cells was maintained over time in 8 of 15 patients, who could be assessed at two to five time points over a period of 8 to up to 1100 days after the first visit/measurement (Supplemental Fig. 1C). Naïve CD19⁺ B cell numbers were considerably lower and correlated with the reduction in NK cells in most patients. Frequencies of other leukocyte subsets or B cell subsets were reduced to a lower extent or were heterogeneous (Fig. 1B, Supplemental Fig. 1B and D).

Next, the gene expression status of immune cell–secreted molecules in leukocytes (no subsets) was measured. The expression of key cytokines such as IL-2, IFN-γ, and TNF was not altered in steady state or after stimulation with PMA/ionomycin compared to healthy controls. Blood cytokine levels of IL-2, IFN-γ, and TNF did not differ between patients and controls (data not shown). Altogether, the quantification of leukocyte subsets revealed a significant reduction in NK cells and naïve B cells as the most consistent cellular biomarker of immune deficiency.

To extend the phenotypic characterization of NK cells in NBAS-deficient patients, we analyzed the percentage of NK cell subsets within PBMCs. The percentage of the CD56^dim CD16⁺ NK cell subset was significantly reduced in NBAS-deficient patients compared to healthy controls (Fig. 2A). The decrease of the percentage of CD56^dim CD16⁺ NK cells was paralleled by the reduction of the absolute cell number of the CD56^dim CD16⁺ NK cell subset, whereas the absolute cell number of the CD56^bright CD16⁻ NK cell subset was unchanged (Fig. 2B). Hence, the reduction in NK cells in PBMCs of NBAS-deficient patients was a consequence of the reduction in the CD56^dim CD16⁺ NK cell subset.

NBAS transcripts have been detected in numerous human tissues including blood [14], and NBAS protein was detected in primary NK cells and NK cell lines (Supplemental Fig. 1E); hence, NBAS deficiency could have an intrinsic effect on NK cell biology.

We investigated whether the remaining NK cells in NBAS-deficient patients retained responsiveness to the prototypical target cell line K562 by measuring CD107a upregulation on NK cells as a functional marker for cytolytic granule release (Fig. 3A–B). For this functional analysis, blood samples could be obtained from six patients that belonged to the “β-propeller” and the “Sec39” subgroup, but not from patients from the “C-terminal” subgroup. CD107a upregulation on NK cells was tested outside inflammatory episodes or liver decompensations. The mean percentage of CD107a⁺ NK cells of NBAS-deficient patients in response to K562 cells was considerably decreased, resulting in an approx. 50% reduction in CD107a upregulation as compared to NK cells from healthy controls (Fig. 3B). The reduction in CD107a upregulation was apparent in both the CD56^dim and the CD56^bright NK cell populations. Accordingly, the reduction in peripheral blood NK cell numbers was accompanied by a reduced responsiveness of the NK cells in NBAS-deficient patients. Intracellular perforin protein expression in NK cells was lower in NBAS-deficient patients but did not reach statistical significance, while granzyme B levels were unchanged (Fig. 3C). In conclusion, the reduced NK cell population in NBAS-deficient patients appeared to be less responsive to target cell stimulation in terms of degranulation.

Next, we assessed whether functionality of patients’ NK cells can be improved by the NK cell-activating cytokine IL-2 (Fig. 4A) in vitro. Exposure to IL-2 for 2 days induced upregulation of the early activation marker CD69 on the cell surface of NBAS-deficient NK cells, but still to a lower extend than observed in healthy control NK cells (Supplemental Fig. 2A). Importantly, IL-2-exposed NK cells of NBAS-deficient patients showed enhanced CD107a upregulation in response to K562, which was similar to that of IL-2-exposed NK cells from healthy controls (Fig. 4B). Activation by IL-2 substantially improved NK cell degranulation even of those patient cells which were very weak responders to K562 without IL-2 pre-activation (i.e., patients 2, 60, and 88). Congruent with the higher activation potential, patients exhibiting high CD107a upregulation also showed higher CD69 expression (Supplemental Fig. 2B). Moreover, intracellular perforin and granzyme B protein expression levels were increased and similar between IL-2-exposed NK cells of NBAS-deficient patients and healthy controls (Fig. 4C). Thus, IL-2 cytokine-induced activation can rescue the functionality of NBAS-deficient patient NK cells and their responsiveness to target cells.