Tertiary lymphoid structure related B-cell IgE isotype switching and secondary lymphoid organ linked IgE production in mouse allergy model

Abstract

Background: Numerous data obtained by different research laboratories indicate that specific IgE production is triggered independently of specific IgG or IgA ones and so it is not linked to fully matured germinal centers formation in the secondary lymphoid organs. The aim of this study was to clarify whether specific IgE production is triggered by low antigen doses administrated in tertiary tissues enriched by lymphoid structures.

Methods: Ovalbumin (OVA) in different doses (100 ng to 10 μg) was administrated three times a week for 4-5 weeks intraperitoneally (i.p.) or subcutaneously (s.c.) to female BALB/c mice in the wither region which is enriched in fat-associated lymphoid clusters or in the foot pad region not containing them.

Results: OVA-specific IgE was predominantly induced by low but not high antigen doses and only after immunization into the withers. IgE isotype switching was triggered exclusively in the withers adipose tissue but not in the regional lymph nodes while mature IgE expressing cells were observed both in the withers and lymph nodes. Anti-proliferative genotoxic stress inducing drugs shifted the balance from IgG1 towards IgE production.

Conclusions: Tertiary lymphoid structures possess unique environment where B-cell antibody isotype switching to IgE predominantly occurs. This phenomenon is partially explained by hampered proliferation of B-cells in these structures.

Keywords: Anti-proliferative drugs; IgE-mediated allergy; Local IgE class switching; Low allergen doses; Tertiary lymphoid structures.

Fig. 1

Specific IgE, but not IgG₁ production, is induced mostly by low antigen doses, and depends on the immunization site in BALB/c mice. BALB/c mice (n = 45) were immunized by OVA in different doses (10, 100, 1000, and 10,000 ng/injection) intraperitoneally (i.p.), subcutaneously in withers region (s.c.w) or in the foot pad (f.p.). Antigen administration was performed three times a week for 5 weeks. a-f: OVA specific IgE (a,c,e) and IgG₁ (b, d, f) production after 14th immunizations compared to preimmune (intact) or saline immunized mice. g-h: Time-dependent OVA specific IgE (g) and IgG1 (h) response in saline (white circles), 100 ng (grey circles) or 10,000 ng (black circles) immunized mice after 7th or 14th immunizations. Data represent mean values ±SD from three independent experiments, (n = 5 mice per group). Mann-Whitney test was used for p value estimation.*p < 0.05; **p < 0.01 from preimmune and saline immunized mice

Fig. 2

Specific IgG2a production is not induced during long-term low dose immunization regardless administration route. IgG2a production after immunization i.p (a), s.c.w. (b), or f.p. (c) with different doses of OVA (see the details in the legend to Fig. 1)

Fig. 3

Systematic and local anaphylactic response in BALB/c mice is associated mostly with specific IgE production. a: BALB/c mice (n = 6–7 per group) intact (0) or immunized with 100 or 10,000 ng/injection OVA in the withers 3 times a week for 4 weeks. A decrease in the temperature (−dT C^o) was estimated after systematic allergen challenge 3 days after the last OVA immunization by injecting 200 ul of 0,25% OVA i.p. Significance *p < 0.05; **p < 0.01 between indicated groups. b: Representative images of Evans Blue local anaphylaxis assay in different immunization groups. Doses in μg of OVA injected are shown. Arrows show the sites of reactions. c-e: Correlation between specific IgE (c), IgG1 (d), total IgE (e) production and temperature decrease after systematic allergen challenge in low dose (100 ng) immunized mice (n = 14). f: Correlations between OVA-specific to total IgE ratio and temperature decrease. g: Correlations between specific IgG1 and specific IgE production. h: Correlations between specific and total IgE

Fig. 4

B-cell activation in low-dose immunization protocol occurs in the withers adipose tissue, but not in the lymph nodes. a: Expression of B-cell specific gene cd19 and the genes associated with B-cell antibody class switching Aicda, germline ε and γ1 in the withers (W) or axillary lymph nodes (LN) after 14th s.c.w. with saline (0), 100 or 10,000 ng/injection of OVA. All data were normalized to intact control. b: expression of genes linked with immune cell activation bcl6, ebi2, gata3, and nmur1. c: expression of innate-immunity linked pro-allergic cytokines genes IL-25, 33, and TSLP in the same samples. Data are representative from 3 independent experiments (n = 5 mice per group, total number of mice 15). Error bars show SD. Statistical significance between immunized and intact mouse groups is shown with asterisks: * - p < 0.05; ** - p < 0.01; *** - p < 0.001; statistical significance between low and high dose groups is shown in each figure in upper right pannel

Fig. 5

Upon allergen challenge, a portion of antigen-activated B-cells migrates to SLOs where specific IgE production primarily occurs. Expression of postswitch ε (a-b) or postswitch γ1 (c-d) transcripts normalized to GAPDH (a, c) or CD19 (b, d) in the withers (W) and regional lymph nodes (LN) of intact (0), low-dose (100) and high dose (10000) immunized mice. Data are representative from 3 independent experiments (n = 5 mice per group, total number of mice 15). Error bars show SD. Statistics significance between immunized and intact mouse groups: * - p < 0.05; ** - p < 0.01; *** - p < 0.001; statistical significance between low and high dose groups is shown in each figure in upper right pannel

Fig. 6

Water soluble and lipophilic anti-proliferative drugs stabilize and enhance specific IgE rather than IgG₁ production. BALB/c mice were immunized by low (100 ng) dose of OVA alone or with low doses (2 and 20 μg) of anti-proliferative drugs Doxorubicin (D 2 and D 20) and Etoposide (E 2 and E 20) in withers tissue. Three days after the 7th immunization (2 weeks after the start, 2 w) and 3 days after the last immunization (4 weeks after the start, 4 w) blood was taken for the measurement of antigen-specific humoral response. a: Specific IgE antibody production. b: Specific IgG1 antibody production. Antibody production was compared between saline and immune mice as well as between control (antigen alone immunized) mice and mice immunized together with anti-proliferative drugs. Total number of mice 23, 6 mice in saline and 7 in antigen control group and 5 mice in each test group. Mann-Whitney test was used for p value estimation.* - p < 0.05; ** - p < 0.01 from p.i. and saline immunized mice, § - p < 0.05 between bar indicated groups