Regulatory B cells and T follicular helper cells are reduced in allergic rhinitis

To the Editor

Although the role of human regulatory B cells (B_REGS) in auto-immunity has been extensively studied, there are limited studies exploring the potential importance of B_REGS in allergy.^1–7 A subset of peripheral blood B_REGS that produces IL-10 (ie, CD19⁺CD73⁻CD25⁺CD71⁺ B_REGS) has recently been demonstrated to inhibit T_H2 responses.^1–3 B_REGS express the cell surface markers CD19 (a B cell marker), CD25 (the alpha chain of the IL-2 receptor also expressed by regulatory T cells or T_REGS), and CD71 (a known activation marker of B cells), but express low amounts of CD73 (an ectonucleotidase thought to play a role in T cell suppression).^1,2 Murine in vivo studies have demonstrated that antigen specificity is required to develop B_REGS capable of secreting IL-10.⁸ Human B_REGS also exhibit allergen specificity as demonstrated in studies of bee venom allergic subjects in whom lower circulating phospholipase A₂-specific B_REGS were found in venom allergic patients compared to venom tolerant beekeepers and venom immunotherapy patients.¹ Importantly, T follicular helper (T_FH) cell production of IL-21 is critical for the maturation of B_REGS and their production of IL-10.⁸

In this study we investigated whether subjects with allergic rhinitis had reduced numbers of B_REGS, as well as reduced numbers of B_REGS expressing CD25^hi, as prior studies of T_REGS have identified the CD25^hi population as the T_REG subset most effective in inhibiting T cell responses.⁹ In addition to examining levels of B_REGS and T_FH cells in peripheral blood, we ascertained whether B_REGS and T_FH cells could be detected in human lymph nodes, a potential site of interaction between B_REGS, T_FH cells, and T_H2 cells not previously studied in humans.

To compare circulating levels of B_REGS and T_FH cells, PBMCs were isolated from 10 allergic rhinitis individuals (mean age: 33.8; gender: 3 males, 7 females) and 7 non-allergic individuals (mean age: 37.9; gender: 2 males, 5 females) in a protocol approved by the University of California San Diego Human Subjects Protection Committee. Allergy status was confirmed by ImmunoCAP specific IgE levels or skin prick testing to cat, dog, cockroach, dust mite, grasses, trees, weeds, and molds (see Table E1 in the Online Repository). Using a previously described gating strategy¹, B_REGS (CD19⁺CD73⁻CD25⁺CD71⁺) were identified in isolated PBMCs by FACS using fluorescently labelled CD19, CD73, CD25, and CD71 antibodies as well as their corresponding isotype controls (eBioscience, San Diego, CA)(Fig 1, A). We also examined the percentage of B_REGS expressing CD25^hi, as prior studies of T_REGS have identified the CD25^hi population as the T_REG subset most effective in inhibiting T cell responses (Fig 1, B).⁹ T_FH-like cells (previously defined in peripheral blood as CD4⁺PD-1⁺CXCR5⁺ T cells)^E1,E2 were also detected in PBMCs by FACS using fluorescently labeled CD4, CXCR5, PD-1 antibodies and their corresponding isotype controls (eBioscience)(Fig 1, A). Human lung lymph nodes were obtained from human lungs post-mortem as previously described at the Arkansas Children’s Hospital Research Institute in an IRB exempted protocol.^E3 A single cell suspension was prepared by mechanical disruption of lymph nodes for FACS analysis in order to identify B_REGS and T_FH cells (as described above for PBMCs). Flow cytometry was performed using the BD Accuri C6 (BD Biosciences, San Jose, CA) and Novocyte Flow Cytometer (ACEA Biosciences, Inc., San Diego, CA) and was analyzed using FlowJo software (version 10.0.7, Tree Star, Inc., Ashland, OR). Statistical analyses were performed using one-tailed t tests and results reported as mean ± SEM.

FIG 1. FACS Gating strategy to detect B_REGS, CD25^hi B_REGS and T_FH cells in peripheral blood.

A, Circulating B_REGS and T_FH-like cells were identified from the circulating lymphocyte population as CD19⁺CD73⁻CD25⁺CD71⁺ and CD4⁺PD-1⁺CXCR5⁺ respectively using the gating strategy shown. B, Example of FACS of circulating B_REGS from a non-allergic individual (left) and an allergic individual (right) including isotype controls were used to quantitate their total CD25⁺ and CD25^hi subsets.

We found that percentages of CD25⁺ B_REGS (2.45 ± 0.41 vs 5.05 ± 1.42)(P < 0.05) and CD25hi B_REGS (0.25 ± 0.05 vs 0.49 ± 0.06)(P < 0.01) were lower in allergic rhinitis individuals compared to non-allergic controls (Fig 2, A). The lower levels of B_REGS in allergic rhinitis subjects tended to cluster in a similar range, while non-allergic individuals exhibited a wider distribution. Our studies are consistent with reports showing that B_REGS are reduced in allergic individuals compared to controls.^1,2,4–5,7

FIG 2. FACS Quantitation of B_REGS, CD25^hi B_REGS, and T_FH-like cells in allergic rhinitis subjects and controls.

A, Peripheral blood total CD25⁺ (left) and CD25^hi (right) B_REGS from allergic rhinitis (n = 10) and non-allergic individuals (n = 7) were quantitated as percentages of CD19⁺ cells. B, Circulating CD4⁺PD-1⁺CXCR5⁺ T_FH-like cells from allergic rhinitis (n = 7) and non-allergic individuals (n = 5) were quantitated as percentages of CD4⁺ T cells. *P < .05, **P < .01.

Levels of T_FH-like cells (CD4⁺PD-1⁺CXCR5⁺) were significantly lower in allergic rhinitis individuals (1.40 ± 0.26) compared to non-allergic individuals (2.81 ± 0.51)(P < 0.01)(Fig 2, B). As B_REGS have been recently demonstrated to undergo expansion under the direction of IL-21 produced by CD4⁺CXCR5⁺PD-1⁺ T_FH cells in autoimmunity^E4, the reduced numbers of T_FH-like cells in allergic rhinitis may contribute to the reduced numbers of B_REGS observed. We further determined that B_REGS and T_FH cells are detectable in human lung lymph nodes as has been demonstrated in secondary lymphoid organs in autoimmunity.^E1,E2 Thus, B_REGS and T_FH cells are present in human lung lymph nodes and may regulate adaptive T_H2 responses in allergic inflammation in asthma (see Fig E1 and E2 in the Online Repository).

To determine whether the B_REG and T_FH cells we quantitated by FACS expressed prototypic cytokines characteristic of B_REGS (i.e. IL-10) and T_FH cells (i.e. IL-21), we used cell sorting to obtain purified populations of B_REGS and T_FH cells (>99% pure populations) from one non-allergic blood donor who we had previously noted had increased numbers of these cells (Figures 1 and 2). Using these cell sorted populations, we demonstrated in vitro increased IL-10 protein production by B_REGs stimulated with CpG (see Figure E3, A in the Online Repository), as well as increased IL-21 mRNA expression by T_FH cells stimulated with anti-CD3 and anti-CD28 antibodies (see Figure E3, B in the Online Repository). A previous study⁴ in allergic rhinitis and asthma has also noted a reduced % of B_REGS and T_FH cells which were not phenotyped as in this study to determine whether they expressed prototypic cytokines (IL-10, and IL-21). Another difference between the two studies is the use of different cell surface markers to phenotype B_REGS. In this study we used cell surface markers (CD19⁺CD73⁻CD25^hiCD71⁺) previously used to characterize B_REGS expressing IL-10 in allergic disease¹, whereas the B_REGS investigated in another study of allergic disease⁴ used B_REG cell surface markers (CD3⁻CD19⁺CD24^hiCD27⁺) previously characterized in auto-immune diseases.^E5 B_REGS identified in auto-immune disease may or may not function the same as B_REGS identified in allergic disease and further studies are required to investigate the functional similarities and differences in these B_REG subsets. Interestingly, CD38 has recently been reported to identify IL-10 producing B_REGS in allergic disease.² In this study, we did not examine antigen-specific IL-10 production by B_REGS. However, previous studies have demonstrated that B_REGS (using the same surface markers we used) had antigen-specificity to Phospholipase A2 in bee tolerant beekeepers.¹

In summary, our findings show that B_REGS (total and CD25^hi) are reduced in subjects with allergic rhinitis. We have demonstrated that these B_REGS produce IL-10 and as such, could suppress T_H2 responses and play an important role in tolerance induction. We also demonstrated a significant reduction in levels of T_FH-like cells and their corresponding IL-21 production in allergic subjects, and made the novel observation that these cells are present in human lung lymph nodes. At present, the relative contribution of B_REGS compared to the T_REGS in inducing tolerance to allergens is unclear. Interestingly, B_REGS are able to induce T_REGS suggesting that B_REGS can have both a direct IL-10 effect on inducing tolerance, as well as an indirect effect through the induction of T_REGS.³ Future studies of B_REGS and T_FH cells in allergic disease may identify their relative importance as compared to T_REGS in natural and acquired tolerance induction by allergen immunotherapy.

Abbreviations

B_REG

Regulatory B cell

T_REG

Regulatory T cell

T_FH

T follicular helper cell