Intrinsic Defects in B Cell Development and Differentiation, T Cell Exhaustion and Altered Unconventional T Cell Generation Characterize Human Adenosine Deaminase Type 2 Deficiency

Abstract

Purpose: Deficiency of adenosine deaminase type 2 (ADA2) (DADA2) is a rare inborn error of immunity caused by deleterious biallelic mutations in ADA2. Clinical manifestations are diverse, ranging from severe vasculopathy with lacunar strokes to immunodeficiency with viral infections, hypogammaglobulinemia and bone marrow failure. Limited data are available on the phenotype and function of leukocytes from DADA2 patients. The aim of this study was to perform in-depth immunophenotyping and functional analysis of the impact of DADA2 on human lymphocytes.

Methods: In-depth immunophenotyping and functional analyses were performed on ten patients with confirmed DADA2 and compared to heterozygous carriers of pathogenic ADA2 mutations and normal healthy controls.

Results: The median age of the patients was 10 years (mean 20.7 years, range 1-44 years). Four out of ten patients were on treatment with steroids and/or etanercept or other immunosuppressives. We confirmed a defect in terminal B cell differentiation in DADA2 and reveal a block in B cell development in the bone marrow at the pro-B to pre-B cell stage. We also show impaired differentiation of CD4⁺ and CD8⁺ memory T cells, accelerated exhaustion/senescence, and impaired survival and granzyme production by ADA2 deficient CD8⁺ T cells. Unconventional T cells (i.e. iNKT, MAIT, Vδ2⁺ γδT) were diminished whereas pro-inflammatory monocytes and CD56^bright immature NK cells were increased. Expression of the IFN-induced lectin SIGLEC1 was increased on all monocyte subsets in DADA2 patients compared to healthy donors. Interestingly, the phenotype and function of lymphocytes from healthy heterozygous carriers were often intermediate to that of healthy donors and ADA2-deficient patients.

Conclusion: Extended immunophenotyping in DADA2 patients shows a complex immunophenotype. Our findings provide insight into the cellular mechanisms underlying some of the complex and heterogenous clinical features of DADA2. More research is needed to design targeted therapy to prevent viral infections in these patients with excessive inflammation as the overarching phenotype.

Keywords: ADA2 deficiency; DADA2; Humoral immunodeficiency; Monocytes; SIGLEC-1; T cell exhaustion; Type I IFN signature.

Fig. 1

Impairment in peripheral B cell development and differentiation in DADA2 patients. Immunophenotyping was determined by flow cytometry on PBMCs from healthy controls (HC), heterozygous carriers (Crs) and DADA2 patients (Pts). Flow cytometric plots and graphs showing the frequencies of A CD20⁺ B cells within the lymphocyte population; B transitional (CD10⁺CD27⁻), naïve (CD10⁻CD27⁻) and memory (CD10⁻CD27⁺) subsets within the CD20⁺ B cell population; C, D IgA⁺ and IgG⁺ cells within the (C) CD27⁻ and (D) CD27⁺ memory B cell populations; E Representative histogram plots (top panels) show expression of CD38, CD21, CD23, and IgM on transitional (red), naïve (blue) and memory (orange) B cells of healthy donors and DADA2 patients, and summarized in graphs (bottom panels) depicting geometric mean fluorescent intensity (gMFI) normalized to the transitional B cell population of healthy controls. F Sort-purified naïve B cells from healthy controls (HC), heterozygous carriers (Crs) and DADA2 patients (Pts) were stimulated in vitro with CD40L/IL-21 for 7 days. IgM, IgG and IgA secretion was determined by ELISA. Graphs represent the mean ± S.E.M.; each symbol represents an individual. Significant differences were determined by multiple t-tests with P < 0.05 indicating statistical significance or by Mann–Whitney t-tests with *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

ADA2 mutations impede B cell development in the bone marrow at the preB to immature B cell stage. A, B BM aspirates from healthy donors, or a DADA2 patient were labeled with mAbs against CD34, CD19, CD20, CD10, IgM and IgD. A Proportions of B-lineage cells (CD19⁺), pro-B (CD19⁺CD34⁺CD10^hi), pre-BI (CD19⁺CD34⁻CD10^hi), pre-BII (CD19⁺CD34⁻CD10^hi), immature (CD19⁺CD34⁻CD10^int) and recirculating mature (CD19⁺CD34⁻CD10⁻CD20⁺) B cells. B Each BM B cell subset in healthy donors (black histogram) and DADA2 patients (red histogram) was assessed for expression of IgM (upper) and IgD (lower panel). Values in B correspond to the gMFI of IgM and IgD expression on BM B cell subsets from in healthy donors (black) and the DADA2 patient (red)

Fig. 3

Aberrant CD4⁺ and CD8⁺ T cell differentiation in DADA2 patients. Immunophenotyping by flow cytometry on PBMCs from healthy controls (HC), heterozygous carriers (Crs) and DADA2 patients (Pts) was performed. Flow cytometric plots (left) and graphs (right) show the frequencies of A CD4⁺ and CD8⁺ T cells within the CD3⁺ lymphocyte population; B CD25^hiCD127^loTreg within CD4⁺ T cells; C, D naïve (CD45RA⁺CCR7⁺), central memory (CM, CD45RA⁻CCR7⁺), effector memory (EM, CD45RA⁻CCR7⁻) and terminally differentiated effector memory (EMRA, CD45RA⁺CCR7⁻) subsets within C CD4⁺ T cells and D CD8⁺ T cells; E naïve (CD45RA⁻CXCR5⁻CCR7⁺), memory (CD45RA⁻CXCR5⁻) and cTfh (CD45RA⁻CXCR5⁺) within CD4⁺CD25^loCD127^lo/hI T cells. F Representative histogram plots (left) showing PD-1 expression on naïve (red), memory (blue) and cTfh (orange) CD4⁺ T cells from healthy controls and DADA2 patients, and summarized in graphs (right) as gMFI normalized to naïve CD4⁺ T cells of healthy controls. A–F Graphs represent the mean ± S.E.M.; each symbol represents an individual. Significant differences were determined by multiple t-tests with P < 0.05 indicating statistical significance or by Mann–Whitney U-tests with *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

CD4⁺ and CD8⁺ T cells in DADA2 patients display an exacerbated senescent/exhausted phenotype. Expression of CD95, CD28, CD57 and CX3CR1 on naïve (red), CM (blue), EM (orange), and EMRA (green) subsets of A CD4⁺ and B CD8⁺ T cells from healthy donors, heterozygous carriers and DADA2 patients. Data are depicted as histogram plots for healthy controls and DADA2 patients (top panels) and summarized in graphs (bottom panels) which as gMFI normalized to the naive CD4⁺ (A) or CD8⁺ (B) T cells from healthy controls. Graphs represent the mean ± S.E.M.; each symbol represents an individual. Significant differences were determined by multiple t-tests with *P < 0.05; **P < 0.01; ***P < 0.001, indicating statistical significance

Fig. 5

Reduced viability and impaired cytokine secretion by ADA2-deficient CD8⁺ T_EM/EMRA cells. Sort-purified combined T_EM/EMRA CD8⁺ T cells from PBMCs of healthy controls, heterozygous carriers and DADA2 patients were stimulated in vitro with anti-CD2/CD3/CD28 mAbs beads ± IL-2 for 4 days and were analyzed for viability and secretion of cytokines and cytotoxic granules. A Representative flow cytometric plot (left) showing viable cells. The values on the contour plots represent the percentage of ZombieAqua⁻ cells, and thus viable cells, and are summarized in graphs (right). B Secretion of IFN-γ, TNF, granzyme A and granzyme B by T_EM/EMRA CD8⁺ T cells. Graphs represent the mean ± S.E.M.; each symbol represents an individual. Significant differences were determined by multiple t-tests with *P < 0.05; **P < 0.01; ***P < 0.001, indicating statistical significance

Fig. 6

Diminished unconventional T cells and NK cell subsets in DADA2 patients. Proportions of A γδ⁺ T cells (top) and Vδ2⁺ within γδ⁺ T cells (bottom), B Vα7.2⁺CD161⁺ MAIT cells, and C Vβ11⁺Vα24⁺ NKT were analyzed in healthy donors, heterozygous carriers and DADA2 patients, presented as flow cytometry plots (A-C left) and summarized in graphs (A–C right). D representative flow cytometry plots showing total CD56⁺CD3⁻ NK cells as a proportion of lymphocytes (top), and proportions of immature CD56^bright, mature CD56^dimCD57⁻ and terminally differentiated CD57^dimCD57⁺ NK cell subsets within the total NK cell population (bottom). Graphs represent the mean ± S.E.M.; each symbol represents an individual. Significant differences were determined by Mann–Whitney t-tests with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. A–D or multiple t-tests with P < 0.05 indicating statistical significance (D bottom)

Fig. 7

Dendritic cell subsets in ADA2 deficiency. A Gating strategy for DC subsets. A broad gate containing lymphocyte and monocyte populations was first gated against SSC-A vs FSC-A, followed by single cells gated as FSC-H vs FSC-A. DCs were defined as negative for lineage markers (CD3, CD19, CD20, CD56, CD14, CD235a), and HLA-DR⁺. DCs were gated as CD123 and CD11c to distinguish CD123⁺CD11c⁻ plasmacytoid DCs (pDC) and CD123⁻CD11c⁺ myeloid (conventional) DCs. mDCs were gated as CD1c and CD16 to distinguish CD1c^hi, CD1c^lo/− and CD16⁺CD1c^lo/− mDCs and for CD1c and CD141 to identify CD141⁺ mDCs. B–D Representative flow cytometry plots and summary graphs showing proportions of B Lin⁻HLA-DR⁺ DCs, C pDCs and mDCs within the DC population and D CD1c^hi, CD1c^lo/−, CD16 + CD1c^lo/− and CD141⁺ mDC subsets. Graphs represent mean ± S.E.M.; each symbol represents an individual. Significant differences were determined by Mann–Whitney (B) or multiple (C, D) t-tests (**P < 0.01)

Fig. 8

Classical monocyte are reduced and IFN-signature detected in ADA2 deficiency. A Gating strategy for monocyte subsets. A broad monocyte gate was set according to SSC-A vs FSC-A, then single cells defined by FSC-H by FSC-A. Lineage markers CD3, CD19/CD20 and CD56 were used to exclude T, B and NK cells. CD14⁻CD16⁻ cells were also excluded. HLA-DR^int/+ cells that were CD16⁻ or CD16⁺ were defined as “true” monocytes. Finally, three subsets were defined: CD14⁺CD16⁻ classical, CD14⁺CD16⁺ intermediate and CD14⁻CD16⁺ non-classical monocytes (23). B Flow cytometry plots (left) showing the frequencies of classical (CD14⁺CD16⁻), intermediate (CD14⁺CD16⁺) and non-classical (CD14⁻CD16⁺) monocytes and summarized in graphs (right). C Histograms showing CD169 expression on classical, intermediate and non-classical monocytes, and gMFI of CD169 summarized in graphs (right). Significant differences were determined by multiple t-tests (*P < 0.05; **P < 0.01)

Fig. 9

Graphical overview of major immunophenotypic findings in ADA2 deficiency. Arrows depict a hypothetical link of the immunophenotype with a clinical presentation (drafted in BioRender)