Circulating Human Neonatal Naïve B Cells are Deficient in CD73 Impairing Purine Salvage

Abstract

Background: Extracellular purines, in particular adenosine (Ado) and adenosine-triphosphate, are critical immunoregulatory molecules. Expression and activity of purine ecto-enzymes on B cells in neonatal and adult blood may influence their function and has been incompletely characterized.

Methods: Mononuclear cells were isolated from human neonatal (cord blood) or adult (peripheral blood) subjects and evaluated directly by flow cytometry for expression of purine ecto-enzymes. Additionally, B cell subsets were isolated from mononuclear cell fractions by fluorescence-activated cell sorting and gene transcription of purine ecto-enzymes (CD39 and CD73), Ado deaminase (ADA1), purine nucleoside phosphorylase, and select purine receptors (A2a) were evaluated by reverse transcription followed by qRT-PCR. Immuno-magnetic-bead isolated naïve B cells were evaluated for enzymatic activity by incubation with radio-labeled purines followed by thin-layer chromatography, and subsequent B cell Ado acquisition was evaluated by liquid scintillation quantitation of radio-labeled Ado uptake.

Results: Relative to their adult counterparts, neonatal circulating naïve B cells were markedly and selectively deficient in CD73 as observed by gene transcription, surface protein expression, and enzyme activity. Neonatal naïve B cell deficiency of CD73 expression significantly impaired their capacity to acquire extracellular purines for purine salvage.

Conclusion: Human neonatal circulating naïve B cells are selectively deficient in CD73, impairing extracellular purine acquisition and potentially contributing to impaired B cell responses in early life.

Keywords: B cells; CD73; adenosine; neonatal; purine; purinergic; salvage.

Figure 1

Gating strategy for B cell flow cytometry. Briefly, CD19⁺ B cells were analyzed for differential expression CD24/CD38 (transitional naïve B cells CD38⁺⁺CD24⁺⁺) or IgD/CD27 (naïve B cells IgD⁺CD27⁻, IgD⁺ memory B cells IgD⁺CD27⁺, switched memory B cells and plasma cells are both IgD⁻CD27⁺). CD19⁺CD27⁺IgD⁻ cells were further analyzed by CD38 expression (switched memory B cells CD38^±, plasmablasts CD38⁺⁺). A similar gating strategy was used for fluorescence-activated cell sorting (FACS) with the exception that each population was a more conservative independent gate inside of each quadrant for the CD27 vs. IgD parameters Transitional B cells were not flow sorted.

Figure 2

Neonatal naïve B cells have a significant deficiency of CD73 surface expression. Flow cytometry analysis reveals that CD39 surface expression was similar on naïve B cells from neonatal cord blood or adult peripheral blood (A,C), but CD73 is strikingly deficient on neonatal naïve B cells (B,D). Data shown in (A,B) are from one independent experiment, representative of N = 5–9 subjects for each target, respectively. (C,D) present mean fluorescent intensity (MFI) for N = 5 newborn subjects and 9 adult subjects, bars represent population means and error lines are the standard error mean (SEM), **p < 0.01, two-tailed Student’s t-test.

Figure 3

Human neonatal naïve B cells are selectively deficient in CD73 mRNA compared to adult counterparts. Adult and newborn circulating naïve B cells were isolated by FACS and evaluated for mRNA expression of purine enzymes CD39, CD73, ADA1, PNP, and the Ado receptor A2a. N = 8 (neonatal) or 10 (adult), horizontal lines represent the mean, **p < 0.01, two-tailed Student’s t-test.

Figure 4

CD39 and CD73 expression varies on different subsets of adult B cells. Surface protein was evaluated by flow cytometry [CD39 in (A,C), CD73 in (B,D)] and mRNA from FACS-isolated B cell subsets was evaluated by reverse transcription and qPCR (E,F). (A,B) are representative of, and (C,D) quantitative analysis of, N = 9 adult subjects, statistical analysis repeated measures ANOVA with Holm–Sidak multiple comparison correction. (E,F) N = 5–9 (nine adults evaluated, insufficient numbers of plasmablasts for analysis of some samples). Statistical analysis by paired Student’s t-test, bars represent population means and error lines are standard error means (SEM), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Neonatal naïve B cells demonstrate lower levels of CD73-mediated Ado generation, and subsequently lower levels of Ado uptake. (A) Isolated neonatal and adult naïve B cells were incubated with radio-labeled adenosine mono-phosphate (C¹⁴-AMP) to observe the rate of CD73-mediated adenosine generation. Purine composition of the extracellular supernatant was evaluated by thin-layer chromatography, and cellular uptake of radio-labeled purine nucleosides was measured in cell fractions using liquid scintillation. Positions for AMP, Ado, and Inosine (Ino) were determined using radiolabeled controls. (A) is 1 representative of four to six thin-layer chromatographs of extracellular fluid, (B) is quantitative analysis of chromatographs for N = 6 newborn and N = 4 adult subjects. (C) is quantitative analysis of C¹⁴ purine uptake into cell fractions for N = 6 newborn and N = 4 adult subjects. Impaired neonatal AMP de-phosphorylation and purine acquisition can be overcome by the addition of exogenous recombinant CD73 protein. Statistical analysis by Student’s t-test, unpaired for newborn vs. adult, paired for newborn control vs. newborn with rCD73, bars represent population means and error lines are standard error means (SEM), **p < 0.01, ***p < 0.001.

Figure 6

Model comparing neonatal and adult naïve B cell purine metabolism. Charged purine nucleotides are not acquired via nucleoside transporters (equilibrative nucleoside transporters, ENTs) or concentrative nucleoside transporters (CNTs) and require de-phosphorylation prior to uptake of nucleosides. Newborn (A) and Adult (B) naïve B cells express similar levels of CD39 and dephosphorylate ADP/ATP to generate AMP, but newborn naïve B cells have low expression of CD73 and, therefore, a reduced capacity to dephosphorylate AMP to generate the nucleoside Ado which can be internalized.