Dysfunctional mitochondria, disrupted levels of reactive oxygen species, and autophagy in B cells from common variable immunodeficiency patients

Abstract

Introduction: Common Variable Immunodeficiency (CVID) patients are characterized by hypogammaglobulinemia and poor response to vaccination due to deficient generation of memory and antibody-secreting B cells. B lymphocytes are essential for the development of humoral immune responses, and mitochondrial function, hreactive oxygen species (ROS) production and autophagy are crucial for determining B-cell fate. However, the role of those basic cell functions in the differentiation of human B cells remains poorly investigated.

Methods: We used flow cytometry to evaluate mitochondrial function, ROS production and autophagy processes in human naïve and memory B-cell subpopulations in unstimulated and stimulated PBMCs cultures. We aimed to determine whether any alterations in these processes could impact B-cell fate and contribute to the lack of B-cell differentiation observed in CVID patients.

Results: We described that naïve CD19⁺CD27^- and memory CD19⁺CD27⁺ B cells subpopulations from healthy controls differ in terms of their dependence on these processes for their homeostasis, and demonstrated that different stimuli exert a preferential cell type dependent effect. The evaluation of mitochondrial function, ROS production and autophagy in naïve and memory B cells from CVID patients disclosed subpopulation specific alterations. Dysfunctional mitochondria and autophagy were more prominent in unstimulated CVID CD19⁺CD27^- and CD19⁺CD27⁺ B cells than in their healthy counterparts. Although naïve CD19⁺CD27^- B cells from CVID patients had higher basal ROS levels than controls, their ROS increase after stimulation was lower, suggesting a disruption in ROS homeostasis. On the other hand, memory CD19⁺CD27⁺ B cells from CVID patients had both lower ROS basal levels and a diminished ROS production after stimulation with anti-B cell receptor (BCR) and IL-21.

Conclusion: The failure in ROS cell signalling could impair CVID naïve B cell activation and differentiation to memory B cells. Decreased levels of ROS in CVID memory CD19⁺CD27⁺ B cells, which negatively correlate with their in vitro cell death and autophagy, could be detrimental and lead to their previously demonstrated premature death. The final consequence would be the failure to generate a functional B cell compartment in CVID patients.

Keywords: B cells; CVID; ROS; autophagy; mitochondria.

Figure 1

B-cell subpopulations with dysfunctional mitochondria in healthy controls and common variable immunodeficiency (CVID) patients. (A) Dot plots of B-cell subpopulations with dysfunctional mitochondria from a representative control and CVID patient. (i) Gating strategy: viable B cells were selected based on forward and side scatter, non-expression of Live/Dead marker, and CD19⁺ expression. B-cell subpopulations were identified as naïve CD19⁺CD27⁻ (green color) and memory CD19⁺CD27⁺ (purple color). (ii) Cells with dysfunctional mitochondria were selected as MitoTracker Deep Red^low and MitoTracker Green⁺ from healthy control (left panels) and CVID patients (right panels). (B) Comparison between the percentages of naïve CD19⁺CD27⁻ (green dots) and memory CD19⁺CD27⁺ (purple dots) B cells with dysfunctional mitochondria in (i) healthy controls and (ii) CVID patients after 24 h of culture without or with stimulation with anti-BCR, anti-CD40, CpG-ODN, or the combinations anti-CD40+IL-21 and anti-BCR+CpG-ODN. (C) Percentages of unstimulated naïve CD19⁺CD27⁻ (green dots) and memory CD19⁺CD27⁺ (purple dots) B cells with dysfunctional mitochondria from healthy controls (empty dots) and CVID patients (filled dots). (B, C) Each dot represents an individual; black horizontal lines illustrate the median of the group. Mann–Whitney test p-values: **p < 0.01.

Figure 2

Reactive oxygen species (ROS) in B-cell subpopulations from healthy controls and common variable immunodeficiency (CVID) patients. (A) Dot plots with histograms representing ROS production in B-cell subpopulations from a representative control and CVID patient. (i) Gating strategy: viable B cells were selected based on forward and side scatter, non-expression of SYTOX marker, and CD19⁺ expression. B-cell subpopulations were identified as naïve CD19⁺CD27⁻ (green color) and memory CD19⁺CD27⁺ (purple color). (ii) ROS production was identified as the percentage of cells positive for CellROX Deep Red probe (% CellROX⁺ cells). Histograms show the percentage (upper right corner) of ROS-producing cells from healthy control (left histograms) and CVID patients (right histograms). (B) Comparison between the percentages of CellROX⁺ naïve CD19⁺CD27⁻ (green dots) and memory CD19⁺CD27⁺ (purple dots) B cells from (i) healthy controls and (i) CVID patients after 24 h of culture without or with stimulation with anti-BCR, anti-CD40, CpG-ODN, anti-BCR+IL-21, anti-CD40+IL-21, anti-BCR+CpG-ODN, or anti-BCR+anti-CD40. (C) Percentages of unstimulated CellROX⁺ naïve CD19⁺CD27⁻ (green dots) and memory CD19⁺CD27⁺ (purple dots) from healthy controls (empty dots) and CVID patients (filled dots). (B, C) Each dot represents an individual; black horizontal lines illustrate the median of the group. Mann–Whitney test p-values: ****p < 0.0001.

Figure 3

Autophagy in B-cell subpopulations from healthy controls and common variable immunodeficiency (CVID) patients. (A) Dot plot and histograms of autophagy levels in B-cell subpopulations from a representative control and CVID patient. (i) Gating strategy: viable B cells were selected based on forward and side scatter, non-expression of Live/Dead marker, and CD19⁺ expression. B-cell subpopulations were identified as naïve CD19⁺CD27⁻ (green color) and memory CD19⁺CD27⁺ (purple color). (ii) Anti-LC3-II mean fluorescence intensity (MFI) was used to evaluate autophagy levels. Histograms represent Anti-LC3-II MFI from a healthy control (left histograms) and a CVID patient (right histograms). Within individual plots, upper histogram refers to basal autophagy, and lower histogram refers to autophagic flux. Numbers in the histogram refer to Anti-LC3-II MFI. (iii) Representative histograms of autophagic flux without or with stimulation with anti-BCR, anti-CD40, or CpG-ODN in naïve CD19⁺CD27⁻ (green color) and memory CD19⁺CD27⁺ (purple color) from a healthy control (left histograms) and a CVID patient (right histograms). Numbers in the histogram refer to Anti-LC3-II MFI. (B) Comparison of autophagic flux between naïve CD19⁺CD27⁻ (green dots) and memory CD19⁺CD27⁺ (purple dots) B cells from (i) healthy controls and (ii) CVID patients after 24 h of culture without or with stimulation with anti-BCR, anti-CD40, CpG-ODN, anti-BCR+IL-21, anti-CD40+IL-21, anti-BCR+CpG-ODN, or anti-BCR+anti-CD40. (C) Basal autophagy in unstimulated naïve CD19⁺CD27⁻ (green color) and memory CD19⁺CD27⁺ (purple color) from healthy controls (empty dots) and CVID patients (filled dots). (D) Autophagic flux in unstimulated naïve CD19⁺CD27⁻ (green color) and memory CD19⁺CD27⁺ (purple color) from healthy controls (empty dots) and CVID patients (filled dots). (B–D) Each dot represents an individual; black horizontal lines illustrate the median of the group; Mann–Whitney test p-values: *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 4

B-cell subpopulations with dysfunctional mitochondria in healthy controls and common variable immunodeficiency (CVID) patients after stimulation. (A) Percentages of naïve CD19⁺CD27⁻ (i) and (ii) and memory CD19⁺CD27⁺ (iii) and (iv) B cells with dysfunctional mitochondria in healthy controls (i) and (iii) and CVID patients (ii) and (iv) after 24 h of culture without or with stimulation with anti-BCR, anti-CD40, CpG-ODN, anti-CD40+IL-21, or anti-BCR+CpG-ODN. (B) Fold increase in the percentage of B-cell subpopulations with dysfunctional mitochondria induced by each single stimulus or combination, related to unstimulated sample in (i) naïve CD19⁺CD27⁻ and (ii) memory CD19⁺CD27⁺ B cell from healthy controls and CVID patients. Each dot represents an individual. Green dots (naïve CD19⁺CD27⁻) and purple dots (memory CD19⁺CD27⁺); empty dots (healthy controls) and filled dots (CVID patients). Black horizontal lines illustrate the median of the group. (A) Wilcoxon test p-values: *p < 0.05; **p < 0.01; ***p < 0.001; ^#p < 0.05 (“*” refers to p-value of samples stimulated with a single stimulus compared to unstimulated sample; “#” refers to p-value of stimulus combinations compared to single stimulus). (B) Mann–Whitney test p-values: *p < 0.05, **p < 0.01.

Figure 5

Reactive oxygen species (ROS) production in B cells from healthy controls and common variable immunodeficiency (CVID) patients after stimulation. (A) Percentages of CellROX⁺ naïve CD19⁺CD27⁻ (i) and (ii) and memory CD19⁺CD27⁺ (iii) and (iv) B cells in healthy controls (i) and (iii) and CVID patients (ii) and (iv) after 24 h of culture without or with stimulation with anti-BCR, anti-CD40, CpG-ODN, anti-BCR+IL-21, anti-CD40+IL-21, anti-BCR+CpG-ODN, or anti-BCR+anti-CD40. (B) Fold increase in the percentage of CellROX⁺ cells induced by each single stimulus or combinations, related to unstimulated sample in (i) naïve CD19⁺CD27⁻ and (ii) memory CD19⁺CD27⁺ B cells from healthy controls and CVID patients. Each dot represents an individual. Green dots (naïve CD19⁺CD27⁻) and purple dots (memory CD19⁺CD27⁺); empty dots (healthy controls) and filled dots (CVID patients). Black horizontal lines illustrate the median of the group. (A) Wilcoxon test p-values: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ^#p < 0.05; ^##p < 0.01; ^####p < 0.0001 (“*” refers to p-value of samples stimulated with a single stimulus compared to unstimulated sample; “#” refers to p-value stimulus combinations compared to single stimulus). (B) Mann–Whitney test p-values: *p < 0.05, **p < 0.01.

Figure 6

Autophagic flux in stimulated B-cell subpopulations from healthy controls and common variable immunodeficiency (CVID) patients. (A) Autophagic flux of naïve CD19⁺CD27⁻ (i) and (ii) and memory CD19⁺CD27⁺ (iii) and (iv) B cells in healthy controls (i) and (iii) and CVID patients (ii) and (iv) after 24 h of culture without or with stimulation with anti-BCR, anti-CD40, CpG-ODN, anti-BCR+IL-21, anti-CD40+IL-21, anti-BCR+CpG-ODN, or anti-BCR+anti-CD40. (B) Fold increase in autophagic flux induced by each single stimulus or combination, related to unstimulated sample in (i) naïve CD19⁺CD27⁻ and (ii) memory CD19⁺CD27⁺ B cells from healthy controls and CVID patients. Each dot represents an individual. Green dots (naïve CD19⁺CD27⁻) and purple color (memory CD19⁺CD27⁺); empty dots (healthy controls) and filled dots (CVID patients). Black horizontal lines illustrate the median of the group. (A) Wilcoxon test p-values: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ^#p < 0.05; ^##p < 0.01; ^####p < 0.0001 (“*” refers to p-value of samples stimulated with a single stimulus compared to unstimulated sample; “#” refers to p-value stimulus combinations compared to single stimulus)). (B) Mann–Whitney test p-values: *p < 0.05.

Figure 7

Principal component analysis (PCA) of experimental variables related to B-cell metabolism, “in vitro” B-cell death, and the degree of B-cell deficiency in our cohort of common variable immunodeficiency (CVID) patients. Loading plot of the first two principal components (PC1 and PC2) in naïve CD19⁺CD27⁻ (A) and memory CD19⁺CD27⁺ (B) B-cell subpopulations. M., R., and (A) refer to dysfunctional mitochondria (green), reactive oxygen species (ROS) production (red), and autophagy (blue) variables, respectively.