Low dose antigen administration in TLS-enriched, but not TLS-depleted, zone induces specific IgE production.
To reconstitute typical clinical situation in which allergens enter over a long time period in the organism, mice were repeatedly immunized with low (100 ng) and high (10 µg) antigen doses in the regions which are relatively enriched in TLSs (withers) or lack them (foot pad). Intraperitoneal administration was carried out for comparison. For each experiment female 6–8 weeks old (18–25 g weight) naïve BALB/c mice were used. Mice were kept in SPF conditions. The results (Fig. 1A-F) show that specific IgE production was induced only after chronic antigen administration in withers region and mostly in low dose group. Specific IgE production was still significant after 7th immunization, and at this time point was comparable both in high and low dose groups. However, after 14th immunization it was markedly enhanced in low dose group and only slightly in high dose group. In contrast to this observation specific IgG1 production (Fig. 1G-L) was significant almost in all immunized mice groups. It is not surprising that high antigen doses induce more pronounced IgG1 production. This production was comparable between high dose groups, which probably indicate the same efficacy of antigen delivery to SLOs from all these regions. Nevertheless, chronically administrated antigen doses induced significant though lower IgG1 production as well, and between low doses groups, it was more pronounced upon antigen administration in withers and less pronounced in i.p. and f.p. administrated groups, according to titer values. Specific IgG2a production was minimal in all groups, and increased significant after prolonged (14th ) immunization with high antigen doses in withers region (Fig. 2) and was triggered only after prolonged, but not after short-time (7th ) immunizations.
The degree of mice sensitization by low antigen doses is associated mostly with specific IgE production.
It is well appreciated that in humans type I hypersensitivity reactions mediated by mast cell and basophil mediators is linked exclusively with allergen-specific IgE antibodies. However, in mice IgG1- and even IgG2a- mediated type I hypersensitivity becomes possible because of basophils degranulation in response to IgG immune complexes . In addition, PAF-mediated macrophage-dependent anaphylactic response which closely resembles histamine and leukotriene mediated reactions becomes possible as well . As mentioned above, in most currently used allergic models, IgE production is triggered together with high levels of specific IgG1 production [4–9] which makes clinical situation in these models slightly different from that in human individuals. We presume that in our low-dose allergic model situation may be substantially different. To clarify this, we first measured systematic (Fig. 3A) and local (Fig. 3B) anaphylaxis intensity in mice after long-time administration of high and low antigen doses. Indeed, mice in low dose groups had more pronounced symptoms of local and systemic anaphylaxis. In the second step we estimated whether systematic anaphylaxis intensity (-dT, which could be easily measured) was associated with specific IgE or IgG1 production. Results (Figs. 3C and 3D) show that the degree of sensitization measurement by systematic anaphylaxis intensity is linked with both IgE and IgG1 production, but mainly associated with elevated IgE levels. During the course of allergic sensitization process not only specific but also total IgE levels rise significantly. But the impact of such IgE antibodies with undefined specificity on antigen-induced mast cell activation may be negative especially in the case when specific IgE production is low . Indeed, systematic anaphylaxis intensity did not correlate with total IgE levels (Fig. 3E) but there was correlation with ratio between specific and total IgE levels (Fig. 3F). It is also need to be mentioned that despite the common mechanisms of induction, specific IgE are produced independently of IgG1 (Fig. 3G) and, surprisingly, the same is observed in case of total and specific IgE (Fig. 3H).
B-cell activation by low antigen doses occurs exclusively in tissue in the site of antigen administration.
To confirm our hypothesis about predominant local (in the site of antigen administration) B-cell activation, we estimated expression of genes corresponding to B-cells in general (cd19), their activated state during germinal center formation (Bcl6)  or extrafollicular foci formation (Ebi2) , as well as to the class switch DNA recombination in general (Aicda)  or to the IgE and IgG1 switching (germline ε and germline γ1 transcripts respectively) . As shown in Fig. 4A B-cell immunoglobulin class switching induced by low antigen doses occurs exclusively in withers tissue. In low dose mouse groups, expression of Aicda, germline ε and germline γ1 were triggered exclusively in withers tissue but not in regional lymph nodes. It is interesting that even high antigen doses did not trigger germline transcripts expression in lymph nodes though these doses trigger lymph nodes’ Aicda expression. It should be mentioned that high but not low antigen doses induce relative B-cell accumulation in withers tissue. In contrast, low antigen doses induce B-cell depletion from withers tissue as evident from relative drop of cd19 expression. Both doses of antigen induced comparable levels of extrafollicular B-cell activation marker (Ebi2) but low doses induce more pronounced germinal center formation in withers characterized by Bcl6 upregulation. However, it is not surprising that high antigen doses induce significant accumulation of both types of activated B-cells in lymph nodes. It should be noted, that predominant expression of transcripts corresponding to B-cell activation in low dose group was observed not only when expression was normalized to Gapdh but also when it was normalized to Cd19 (data not shown). B-cell IgE class switching depends on IL-4 and less on IL-13 which could be produced by either T helper 2 or ILC2 cells. Both cell types express transcription factor GATA3 [3, 35], but among all immune cells only ILC2 express NMUR1 . We have measured expression of these genes in withers and lymph nodes. Low antigen doses induced substantial increase in Gata3 and Nmur1 genes expression in withers tissue. Only high antigen doses triggered expression of Gata3 in lymph nodes (Fig. 4B). Long time antigen administration via needle induced damage of adipose tissue and, therefore, stimulated the expression of tissue cytokines. Expression of Il25 (only in low dose group) and Il33 (both groups) but not TSLP was induced (Fig. 4C). It should be noted that due to normalization to respective control samples taken from the same organ the intensity of expression between W and LN could not be directly compared. The comparison is performed only between the samples from the same tissue.
Specific IgE production predominantly occurs in regional lymph nodes after migration of activated IgE-switched B-cells from TLSs.
In order to develop new allergen-specific immunotherapy methods based on elimination of IgE-producing B-cells and their precursors, one should entirely know not only the site where IgE antibody isotype switching occurs but also the site of IgE antibody production. Because B-cells from TLSs can recirculate between these structures and SLOs [37, 38] one could not exclude the possibility that the final differentiation of IgE+ B-cells into antibody producing cells takes place not only in TLSs but also in SLOs. Indeed, data from Fig. 6 indicate that expression of postswitch ε and postswitch γ1 transcripts was induced in regional lymph nodes. It is not surprising that in case of regional lymph nodes postswitch ε and postswitch γ1 transcripts were mainly induced by low and high antigen doses, respectively, regardless of reference gene, i.e. Gapdh or Cd19, the expression of genes of interest was normalized to (Fig. 5B and 5D). However, in adipose tissue the effects of low vs high doses on genes expression were different. When normalized to Gapdh, the predominant expression of postswitch ε, rather than postswitch γ1, in high, rather than low, dose groups was evident (Fig. 5A and 5C). When normalized to Cd19, the expression of postswitch ε transcripts was comparable in both groups (Fig. 5В and 5D). Despite inefficient IgE class switch in adipose tissue observed in high dose group, and considering absolute number of B cells rather than percentage of total B cell population, the accumulation of IgE expressing B cells in tissue was more prominent in high vs. low dose group.. Together with the data that indicates B-cell depletion from adipose tissue in low but not high dose group (Fig. 4A), our results suggest that activated B-cells migrate from TLSs to SLOs.
Dampening of cell proliferation by anti-proliferative drugs enhances specific IgE but not IgG1 production.
One of the most interesting observations from data described above is that expression of cd19 marker in adipose tissue is downregulated in low dose group compared to control and high dose group, despite the fact that B-cell activation in withers occurs mostly in low dose group. In contrast, relative CD19 accumulation in withers of high dose group was accompanied by relatively weak intensity of B-cell activation and IgE class switching. Because of the harmless nature of OVA diluted in saline the administration of low antigen dose is unlikely to induce necrotic or apoptotic death of B-cells. It is known that activated B-cells tend to migrate between TLSs and SLOs and the data shown in Fig. 4 support this point of view . Upon antigen-based activation, B-cells tend to proliferate as well but the number of B-cells’ niches in TLSs must be more limited than in SLOs. So, it is reasonable to suppose that restriction of proliferation caused by limited number of niches (or limited proliferation supporting factors) in TLSs in contrast to SLOs shifts the balance in former from IgG1 to IgE production by yet unknown molecular mechanism. To verify this, we used two commercially available anti-proliferative drugs doxorubicin and etoposide. The water-soluble doxorubicin can be rapidly distributed in internal milieu. Etoposid is more hydrophobic and its specific action would be probably more restricted to adipose tissue. Drugs were administrated with antigen for long time period. Data from Fig. 6A-D indicate that both drugs induced significant (p < 0.05) increase in OVA specific IgE production after prolonged (14th immunizations) treatment, though only high (20 µg/injection) dose had effect. After short time treatment (7th immunizations) only etoposide’s effect was significant. In contrast, specific IgG1 production at short time period was not affected by these drugs but was slightly, though significantly (p < 0.05), dampened after long time treatment (Fig. 6E-H). Although the drug-induced effects on IgE and IgG1 specific response were not very pronounced in numerical expression (about 2–3 times) they were significant and differed in sign between antibody classes and could not be accidental. This observation supports our hypothesis that dampened B-cell proliferation could shift the balance towards specific IgE rather than IgG1 production.