RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect

Abstract

Background: Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG₄ . IgG₄ competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy.

Methods: Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort.

Results: Four months of SLIT increased RGP-specific IgE and IgG₄ in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG₄ ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1⁺ Bmem expressing surface IgG₄ , CD23, and CD29 after SLIT.

Conclusions: A clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

Keywords: allergen immunotherapy; biomarkers; grass pollen allergy; memory B cells; transcriptomics.

FIGURE 1

Four months of SLIT increases Lol p 1‐specific IgE and IgG₄ in serum. A, Patient recruitment in May and June 2019 and timeline of 4 months pre‐seasonal SLIT for grass pollen allergy. B, Total and RGP‐specific IgE in serum at timepoint 0. Red bars, median values. Statistics, Mann–Whitney U‐test; ns, not significant. C, Lol p 1‐specific IgE, IgG₂ and IgG₄ in serum at t = 0 and 4 months. B, C, Data from the four subjects that were included for transcriptomic analysis of Lol p 1‐specific Bmem are highlighted in blue. Statistics, Wilcoxon signed rank test; **p < 0.01; ****p < 0.0001

FIGURE 2

Identification of Lol p 1‐specific B cells for single‐cell transcriptomic analysis. Single‐cell RNA sequencing of PBMC sorted for Lol p 1‐specific Bmem by FACS from four patients before and after SLIT was conducted using the BD Rhapsody Whole Transcriptome Analysis pathway. A, Exemplary plot of Lol p 1‐specific Bmem sorted for single‐cell transcriptomics. Number indicates proportion of Bmem. B, Selection of genes used for the identification of B cells (square gate), excluding those with low expression (insufficient sequencing) or high and ubiquitous expression (housekeeping genes). C, The top 200 genes with highest dispersion in expression among all cells were used to generate D, PCA plot with two distinct clusters. E, Heatmaps of expression patterns for canonical B‐cell gene MS4A1 and myeloid gene CD14 identified separate clusters. Monocytes (lower cluster; 233 cells) were excluded from further analyses

FIGURE 3

SLIT increases proportions of IGHE and IGHG4 transcripts in Lol p 1‐specific Bmem. A, Schematic of the constant genes in the human IGH locus depicting their position relative to the variable domain of the VDJ exon. B, Proportion of transcripts for IGH or IGK and IGL genes in single Lol p 1‐specific Bmem before (n = 189) and after SLIT (n = 323) isolated from four allergic patients used for single‐cell transcriptomic analysis. Cells arranged left‐to‐right based on maximal expression of IGH transcripts in the order of IGH genes in the human IGH locus (IGHM to IGHA2). C, Proportion of total IGH or IGHG gene transcripts in these Lol p 1‐specific Bmem before and after SLIT. Statistics for the proportional distribution of IGH and IGHG in (C), χ ² test; ****p < 0.0001

FIGURE 4

SLIT increases the proportion of IGHE, IGHG1 and IGHG4‐expressing Lol p 1‐specific Bmem and promotes differentiation toward two transcriptionally distinct clusters A, DEG in Lol p 1‐specific Bmem before and after SLIT. Genes with >1.5‐fold change and q < 0.05 identified by red arrows. Genes with >1.5‐fold change and q < 0.05 identified by red arrows. B, Pseudotime clustering based on the 268 genes (Q < 0.05) identified in 4A. C, B‐cell clusters identified by pseudotime analysis before or after SLIT. D, Heat map of IGHM, IGHE and E, IGHG1‐4 gene expression in B cells clustered by pseudotime analysis. F, IGH gene expression in Bmem from the four distinct pseudotime clusters. Statistics: Mann–Whitney U‐test;*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant

FIGURE 5

Lol p 1‐specific Bmem generated by SLIT show increased expression of ITGB1 (CD29), FCER2 (CD23), and IL13RA1 (CD213A1). A, Pseudotime clustering of all Lol p 1‐specific Bmem from four patients before and after SLIT using genes identified in Figure 3A. Numbers indicate proportion of clusters. B, Volcano plots of DEG comparing pseudotime clusters 1 to 4, 1 to 3, and 3 to 4. Significant genes (p > 0.05) with largest fold change or known immunological interest indicated by red arrows. C, Heatmap of gene expression for ITGB1, FCER2, and IL13RA1 on pseudotime plot from A

FIGURE 6

Increased numbers of circulating IgG₄ ⁺ Lol p 1‐specific Bmem and surface expression of CD29 and CD23 after SLIT. A, Number of circulating Lol p 1‐specific Bmem and B, number of IgM⁺, IgG⁺, IgG₄ ⁺, and IgA⁺ Lol p 1‐specific Bmem from PBMC of RGP‐allergic patients before and after SLIT (red lines; SLIT n = 13) or from RGP‐allergic patients that did not receive SLIT (blue lines; no SLIT n = 14). The only patient to report no clinical benefit of SLIT is indicated by red dots and black lines. C, Representative histogram of CD29 expression on Lol p 1‐specific Bmem from one RGP‐allergic patient before (orange) and after (red) SLIT. Fluorescence minus one (FMO) control from after SLIT in gray. D, Number of CD29hi Lol p 1‐specific Bmem and MFI of E, CD29, F, CD23 and G, CD213A1 on Lol p 1‐specific Bmem. Statistics, Wilcoxon signed rank test; *p < 0.05; **p < 0.01; ns, not significant

FIGURE 7

Positive predictive values of biomarkers for allergen immunotherapy. A, Receiver‐operator curves of ratios before/after 4 months for patients treated with SLIT vs untreated RGP allergic patients. Shown are Lol p 1⁺ Bmem numbers, CD29^hi Lol p 1⁺ Bmem numbers and MFI of CD29, CD23, and CD213A1 on Lol p 1⁺ Bmem. B, Receiver‐operator curves of data obtained at the 4‐month timepoint for patients treated with SLIT vs untreated RGP‐allergic patients. Shown are Lol p 1⁺ Bmem numbers, CD29^hi Lol p 1⁺ Bmem numbers and MFI of CD29, CD23, and CD213A1 on Lol p 1⁺ Bmem