The impact of hypoxia on B cells in COVID-19

Abstract

Background: Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This presentation resembles the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19.

Methods: Detailed B cell phenotyping was undertaken by flow-cytometry on longitudinal samples from patients with COVID-19 across a range of severities (NIHR Cambridge BioResource). The impact of hypoxia on the transcriptome was assessed by single-cell and whole blood RNA sequencing analysis. The direct effect of hypoxia on B cells was determined through immunisation studies in genetically modified and hypoxia-exposed mice.

Findings: We demonstrate the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes also observed in B cell VHL-deficient mice. These findings were associated with hypoxia-related transcriptional changes in COVID-19 patient B cells, and similar B cell abnormalities were seen in mice kept in hypoxic conditions.

Interpretation: Hypoxia may contribute to the pronounced and persistent B cell pathology observed in acute COVID-19 pneumonia. Assessment of the impact of early oxygen therapy on these immune defects should be considered, as their correction could contribute to improved outcomes.

Funding: Evelyn Trust, Addenbrooke's Charitable Trust, UKRI/NIHR, Wellcome Trust.

Keywords: B cells; COVID-19; Hypoxia; Lymphopenia.

Figure 1

B cells in COVID-19 and VHL-deficient mice. a, Cohort details. Time post positive swab (group A) or symptom onset (groups B-E). b, Median absolute cell counts (left) or proportions relative to total B cells (right) (log2 fold change relative to healthy controls). (Wilcoxon test FDR adjusted p-value (q)): *<0.05, **<0.005, ***<0.0005. c, Serum IgG and IgM (g/L) at enrolment. Grey band: 5-95^th centiles of healthy reference range (see methods). Significant P values listed. d, Somatic hypermutation frequency in IgA and IgG within 0-12 days post symptom onset, calculated over the CDR1/CDR2 regions using BCR sequencing of whole blood. (Wilcoxon test). c,d, Circles represent individual donors. e, Phenotype comparison: COVID-19 patients versus mice with Vhl-deficient B cells.

Figure 2

Hypoxia-related transcription signatures in COVID-19. a, Eigenvalues of Hallmark Hypoxia geneset grouped by severity at 0-12 days post symptom onset, (unpaired, two-sided Student's t-test). Circles represent individual donors. b, B cell subpopulations identified using CITEseq with Gene set enrichment analysis (GSEA) of Hallmark hypoxia geneset assessed on a single cell level comparing HC to COVID-19, grouped by severity (A/B n=8, C/D/E n =20), within 24 days of symptom onset (B-E)/positive swab(A). c, GSEA of Hallmark genesets in COVID-19 versus HC grouped by severity and time. Outlined circles: nominal P value <0.05 and FDR adjusted P <0.2. Mean hsCRP represented. d, Correlation between Hallmark hypoxia geneset eigengenes and parameters shown at 0-12 days post symptom onset in COVID-19 patients. Boxes coloured by strength of correlation, Pearson correlation.

Figure 3

The response of mouse B cells to hypoxia in vivo. a, WT mice exposed to 21% or 10% O₂, were immunized with NP-KLH at day 1, then absolute spleen B cells enumerated at day 21. (Unpaired, two-sided Student's t-test). FO, (Unpaired, two-sided Mann-Whitney U-test). Gating in methods, mean ± s.e.m, circles represent individual mice (n=8 per group), data pooled from two experiments, results confirmed in a third. b, Serum NP-specific antibodies after NP-KLH immunization, (unpaired, two-sided Student's t-test). mean ± s.e.m, circles represent individual mice, (n=8 per group), data pooled from two experiments, confirmed in a third. *P < 0.05,**P < 0.01,***P < 0.001,**** P < 0.0001.

Figure 4

The response of mouse B cells to re-oxygenation in vivo. a, Spleen confocal images from immunised mice (Figure 3a), MZ B cells (magenta, B220⁺CD23⁻), FO B cells (yellow, B220⁺CD23⁺) and MZ metallophilic macrophages (blue, CD169⁺). B cell area, circles represent individual follicles from one spleen per condition, mean ± s.e.m. b, Experiment outline and absolute spleen B cells, (two-way ANOVA with Tukey's multiple comparisons test). Gating in methods. Mean ± s.e.m, data pooled from two experiments. *P < 0.05,**P < 0.01,***P < 0.001,**** P < 0.0001.