Immunoglobulin E (IgE), a key mediator in allergic diseases, is spontaneously elevated in mice with disrupted commensal microbiota such as germ-free (GF) and antibiotics-treated mice. However, the underlying mechanisms for aberrant IgE elevation are still unclear. Here, we demonstrate that food antigens drive spontaneous IgE elevation in GF and antibiotics-treated mice by generating T helper 2 (T H 2)–skewed T follicular helper (T FH ) cells in gut-associated lymphoid tissues (GALTs). In these mice, depriving contact with food antigens results in defective IgE elevation as well as impaired generation of T FH cells and IgE-producing cells in GALT. Food antigen–driven T FH cells in GF mice are mostly generated in early life, especially during the weaning period. We also reveal that food antigen–driven T FH cells in GF mice are actively depleted by colonization with commensal microbiota. Thus, our findings provide a possible explanation for why the perturbation of commensal microbiota in early life increases the occurrence of allergic diseases.

In this study, we demonstrate that IgE elevation in GF and ABX-treated mice is not a direct result of the absence of intestinal microbiota but is induced by aberrant immune responses to ingested food Ags. We show that GF mice deprived of contact with food Ags have basal IgE levels. In GF mice, food Ag–specific T FH cells are found predominantly in mesenteric lymph nodes (MLNs) and Peyer’s patches (PPs) and are major producers of IL-4. T FH cells are required for food Ag–driven IgE elevation in the absence of commensal microbiota. Food Ag–driven T FH cells in GF mice are mostly generated in early life, especially during the weaning period, and actively depleted by colonization with commensal microbiota. Furthermore, we reveal that, although food Ag–driven T FH cell development is crucial for initial IgE production, prolonged maintenance of IgE is largely due to generation of long-lived IgE-producing plasma cells.

T follicular helper (T FH ) cells are a specialized CD4 + T cell subset for helping B cell immunity ( 6 ). After engaging with Ag-bearing dendritic cells (DCs), CD4 + T cells commit to differentiation into T FH cells by expressing BCL-6, a key transcription factor orchestrating T FH cell differentiation ( 7 , 8 ). T FH -committed T cells migrate into the T-B border and B cell follicles, and fully differentiate into PD-1 hi CXCR5 + germinal center (GC) T FH cells ( 9 ). In mice infected with helminths and sensitized with allergens, interleukin-4 (IL-4)–producing T FH cells were effectively generated; these T FH cells, rather than conventional T helper (T H 2) cells, are mainly required for IgE production ( 10 , 11 ). In contrast, IgE can be elevated in a T FH cell–independent manner, as shown in BCL-6–deficient mice ( 12 ). Although IgE elevation in GF mice is dependent on CD4 + T cells and IL-4 ( 5 ), it is unclear whether spontaneous IgE elevation observed in GF mice is mediated by T FH cells or caused by T FH cell–independent mechanisms.

Commensal microbiota profoundly influence host physiology. Perturbation of commensal microbiota is widely believed to be one of the major causative factors in allergic diseases ( 4 ). Experimental evidence has shown that the commensal microbiota plays a critical role in suppressing aberrant IgE production. Serum IgE levels are abnormally elevated in germ-free (GF) mice or conventional mice treated with broad-spectrum antibiotics (ABX) ( 2 , 5 ). However, the underlying mechanisms for spontaneous IgE elevation in GF mice, including the responsible Ags and immune subsets involved, remain to be elucidated.

Immunoglobulin E (IgE) is a key mediator for allergic reactions to innocuous foreign antigens (Ags), despite its beneficial role in protection against parasite infection ( 1 ). In healthy individuals, serum IgE is rare relative to other isotypes and is tightly regulated to avoid excessive allergic responses. IgE elevation is generally observed in allergic patients, contributing to the pathophysiology of allergic disease. Degranulation of mast cells and basophils by allergen-mediated IgE/FceR cross-linking causes fatal allergic symptoms ( 1 ). Furthermore, circulating IgE promotes hematopoiesis of basophils and survival of mast cells, the effector cells mediating allergic symptoms ( 2 , 3 ).

RESULTS

Ingested food Ags are responsible for spontaneous IgE elevation in GF mice To investigate the role of ingested food Ags in spontaneous IgE elevation in GF mice, we examined serum IgE levels in “Ag-free (AF)” mice, i.e., F1 offspring of GF breeders raised on a diet devoid of macromolecules such as proteins and polysaccharides [AF diet (AFD)] (13). These F1 mice were raised on AFD. As reported previously (5), notable elevation of serum IgE levels in GF mice with age was detected after weaning mice onto normal chow diet (NCD), whereas serum IgE levels remained at basal level (<100 ng/ml) in specific pathogen–free (SPF) mice (Fig. 1A). In contrast to GF mice, AF mice had basal serum IgE levels (<100 ng/ml) comparable to those in SPF mice (Fig. 1A). IgE elevation was not observed in GF mice weaned onto AFD; conversely, in AF mice weaned onto sterile NCD, serum IgE levels were as high as in age-matched normal GF mice (Fig. 1B). Fig. 1 Ingested food Ags are responsible for spontaneous IgE elevation in GF mice. (A) Serum IgE levels of SPF, GF, and AF mice with ages were measured by enzyme-linked immunosorbent assay (ELISA) (n = 4). Statistically significant difference between GF and AF mice at indicated age was shown. (B) AF and GF pups were weaned onto NCD (AF weaned on NCD) and AF diet (GF weaned on AFD), respectively. After 7 weeks of feeding, serum IgE levels were measured by ELISA. Age-matched AF and GF mice were used as control mice (n = 6). (C) GF mice were weaned onto NCD or AAD. After 6 weeks, serum IgE levels were measured by ELISA (n = 7). (D) Levels of IgE specific to water-soluble fraction of chow diet (diet extract) in sera from 12-week-old AF and GF mice were measured by direct ELISA. OVA at an equivalent amount was used as an irrelevant control (n = 4 for AF sera and n = 18 for GF sera). (E) GF mice were weaned onto AAD mixed with indicated proteins (W.Glu: wheat gluten and EW). After 6 weeks of feeding, serum IgE levels were measured by ELISA (n = 4). Each symbol represents an individual mouse. (F) Levels of serum IgE specifically bound to wheat gluten (n = 4 for AF sera and n = 9 for GF sera). Data in (A) and (E) are representative data of two or three independent experiments. Data are pooled from two or three independent experiments (B, C, D, and F). Statistical differences were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (A, B, and D to F) or by unpaired two-tailed Student’s t test (C). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM. To exclude the possibility that the absence of IgE elevation in AF mice and GF mice weaned onto AFD was caused by an artifact of the AFD, GF mice were weaned onto a commercially available sterile amino acid diet (AAD). AAD is devoid of protein Ags as the result of replacing protein components with a mixture of amino acids. GF mice fed with AAD for 6 weeks after weaning failed to display the elevation of serum IgE (Fig. 1C), confirming that aberrant IgE elevation in GF conditions is caused by ingested food Ags. In accordance with these findings, in GF mice, serum IgE specifically bound to diet extracts was significantly higher relative to IgE bound to an irrelevant Ag, ovalbumin (OVA), and that of AF mice (Fig. 1D). Furthermore, in GF mice raised on AAD alone or mixed with individual food proteins such as wheat gluten, casein, egg white (EW), and peanut, IgE elevation was observed only in wheat gluten–fed mice (Fig. 1E). Wheat gluten is a component of NCD. Hence, serum IgE specifically bound to wheat gluten in GF mice was higher than IgE bound to OVA (Fig. 1F). These results suggest that not all food Ags are capable of inducing IgE elevation and that distinct immunogenic properties of food Ags determine IgE elevation in GF mice. Collectively, our data indicate that spontaneous IgE elevation in GF mice is driven by specific immune responses to individual food Ags, rather than a nonspecific consequence of GF conditions as proposed previously (5, 14).

Food Ags induce T H 2-skewed immune responses in MLN and PP in GF conditions In accordance with a previous study (5), CD4+ T cells were found to be crucial for IgE elevation in GF mice, as serum IgE levels were decreased by depleting CD4+ T cells (fig. S1A). We found that relative to SPF and GF mice, CD44hi activated CD4+ T cells in AF mice were profoundly reduced in gut-associated lymphoid tissues (GALTs) such as MLN and PP (fig. S1, B and C). In GF mice, relative to SPF mice, CD4+ T cells in MLN and PP were more polarized into IL-4–producing cells, which are essential for IgE production (15), but IL-4–producing CD4+ T cells were significantly reduced in AF mice (fig. S1D). Reduction of IL-4–producing CD4+ T cells was not caused by the reciprocal increase of Foxp3+ regulatory CD4+ T (T reg ) cells because the number of T reg cells was also significantly reduced in AF mice relative to GF and SPF mice (fig. S1E). These results suggest that food Ags are responsible for the generation of IL-4–producing CD4+ T cells in MLN and PP in GF mice.

Food Ag–driven IL-4–producing T FH cells in MLN and PP are key mediators of IgE elevation in GF mice The T FH cell subset is a key mediator for antibody production and is also involved in IgE production (10, 11). To elucidate the role of T FH cells in spontaneous food Ag–driven IgE production in GF mice, we examined T FH cell populations in MLN and PP, where CD4+ T cells are activated in response to food Ags. In GF mice, PD-1hi CXCR5+ T FH cell populations were abundant among CD44hi activated CD4+ T cells in MLN and PP relative to SPF mice (Fig. 2A). Despite the higher frequency of T FH cells in both MLN and PP of GF mice, the number of T FH cells in PP in GF mice was comparable with that in SPF mice, presumably due to reduced size and cellularity of PP in GF mice (Fig. 2B). The emergence of T FH cells in GALT of GF mice was observed upon weaning of mice onto NCD (fig. S2A). In contrast to the high levels of T FH cells in GF mice, both the percentage and number of T FH cells were profoundly reduced in MLN and PP of AF mice, indicating that food Ags are responsible for the generation of T FH cells in GALT in GF conditions (Fig. 2, A and B). Consistent with the defective T FH cell generation in AF mice, GC B cell generation, which is mainly mediated by T FH cells, was profoundly reduced in MLN and PP of AF mice (fig. S2B). Fig. 2 Food Ag–driven T FH cells generated in GALT mediate IgE elevation in GF conditions. (A to C) Single-cell suspension was prepared from MLN and PP from 10-week-old SPF, GF, and AF mice. (A) Representative fluorescence-activated cell sorting (FACS) plot of CXCR5 and PD-1 (left) and frequency of PD-1hi CXCR5+ T FH cells (right) gated on CD4+ TCRβ+ Foxp3− CD44hi cells (n = 5). (B) Number of PD-1hi CXCR5+ T FH cells at indicated tissues. (C) Representative FACS plot showing PD-1hi CXCR5+ T FH cells among IL-4+ CD4+ T cells (left) and frequency of PD-1hi CXCR5+ T FH cells gated on IL-4–producing CD4+ T cells in indicated tissues from 10-week-old GF mice (n = 4) (right). (D) Number of IL-4–producing T FH cells in indicated tissues from SPF, GF, and AF mice. (E and F) GF mice (4.5 weeks old) were treated with isotype (Control) or anti-ICOSL antibody (T FH -depleted) every 3 days for 3 weeks to deplete T FH cells. Representative FACS plot showing PD-1hi CXCR5+ T FH cells in MLN and PP (E) and serum IgE levels (n = 4) (F). (G) FACS-sorted T FH cells and non-T FH cells in MLN and T FH cells in SPL from 9- to 12-week-old GF mice were cotransferred into GF Rag1−/− mice with naïve B cells isolated from SPF mice. Serum IgE levels at 1 and 2 weeks after transfer were shown (n = 3). Each symbol represents an individual mouse. Data are pooled from two independent experiments (A and B). Data in (C), (D), (F), and (G) are representative of two or three independent experiments. Statistical differences were determined by one-way (A to D) or two-way (G) ANOVA with Tukey’s multiple comparisons test or by unpaired two-tailed Student’s t test (F). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM. In T H 2-skewed conditions, T FH cells are known to produce a T H 2 signature cytokine, IL-4 (16, 17). In accordance with these studies, about 40 and 70% of IL-4–producing CD4+ T cells in MLN and PP, respectively, were PD-1hi CXCR5+ T FH cells, whereas only 20% were T FH cells in spleen (SPL) (Fig. 2C). IL-4–producing T FH cells were profoundly increased in GF mice relative to SPF mice but markedly reduced in AF mice, suggesting that IL-4–producing T FH cells are generated in response to food Ags in GF conditions (Fig. 2D). Furthermore, IgE-producing plasma cells were detected only in MLN and PP of GF mice by means of enzyme-linked immunospot (ELISPOT) assay, but not in AF mice (fig. S2, C and D). To further elucidate the critical role of T FH cells in IgE elevation in GF mice, young GF mice were treated with anti-ICOSL antibody to prevent the generation of T FH cells (8, 18). By blocking ICOS-ICOSL (inducible T cell co-stimulator – ICOS ligand) interaction, food Ag–induced T FH cell generation was greatly reduced and IgE elevation was consequently suppressed (Fig. 2, E and F). Furthermore, transfer of T FH cells from MLN of GF mice, but not of non-T FH cells from MLN or splenic T FH cells, led to the efficient increase of serum IgE levels in GF Rag1−/− mice cotransferred with naïve B cells (Fig. 2G). IgE specifically bound to wheat gluten was also increased in GF Rag1−/− mice reconstituted with naïve B cells by the transfer of MLN T FH cells but not by the transfer of SPL T FH cells or MLN non-T FH cells (fig. S3). Collectively, these results indicate that IL-4–producing T FH cells generated in response to food Ags, especially in MLN, are critical for IgE elevation in GF mice.

DCs initiate food Ag–driven T FH cell differentiation in GF mice by elevating ICOS expression on activated T cells via CD40-CD40L interaction Next, to define the underlying mechanisms for the generation of food Ag–driven T FH cells in GF mice, we first examined ICOS expression on CD4+ T cells because ICOS expressed on CD4+ T cells is critical for T FH cell differentiation (18, 19). In GF mice, ICOS expression levels on CD44hi activated CD4+ T cells were considerably higher in MLN and PP relative to those in SPL (Fig. 3A). Moreover, in AF mice, ICOS expression on activated CD4+ T cells in MLN and PP was significantly lower relative to GF mice regardless of the presence of T FH cells (Fig. 3A and fig. S4A). These results show that ICOS is up-regulated in CD4+ T cells activated by food Ags in MLN and PP of GF mice. Fig. 3 Food Ag–induced CD40 elevation on DCs promotes ICOS up-regulation on activated T cells. (A) Representative histograms (left) and mean fluorescence intensity (MFI) of ICOS expression (right) gated on CD44hi or CD44lo CD4+ T cells at indicated tissues from 9- to 10-week-old GF and AF mice (n = 3). (B) MFI of CD40 expression gated on DCs (Lin− CD11c+ MHCII+) or B cells at indicated tissues from 9- to 10-week-old GF and AF mice (n = 4 to 5). (C and D) GF mice (4.5 weeks old) were treated with isotype or anti-CD40L antibody every 3 days for 2.5 weeks (n = 3). (C) Representative histograms (left) and MFI of ICOS expression (right) gated on CD44hi or CD44lo CD4+ T cells at indicated tissues. (D) Representative FACS plot of CXCR5 and PD-1 (left) and frequency of PD-1hi CXCR5+ T FH cells (right) gated on CD4+ TCRβ+ Foxp3− CD44hi cells. (E) Number of T FH cells in indicated tissues from GF mice treated with isotype and anti-CD40L antibody. (F) Total serum IgE levels in GF mice treated with isotype and anti-CD40L antibody. Each symbol represents an individual mouse. Data in (A) and (C) to (F) are representative of three independent experiments. Data were pooled from two independent experiments (B). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM. ICOS expression on CD4+ T cells is known to be up-regulated through stimulation by DCs (7). Examination of costimulatory molecules on DCs in GALT of GF and AF mice revealed that the expression of CD40, but not other costimulatory molecules, was significantly reduced in DCs from MLN and PP of AF mice relative to those in GF mice (Fig. 3B and fig. S5). In contrast, the expression levels of CD40 on B cells in MLN and PP were comparable between GF and AF mice (Fig. 3B). Blocking CD40-CD40L interaction substantially reduced ICOS expression on activated CD4+ T cells in MLN and PP of GF mice (Fig. 3C) and consequently suppressed food Ag–driven T FH cell generation (Fig. 3, D and E). As a result of defective generation of T FH cells, IgE levels in GF mice treated with anti-CD40L antibody were profoundly reduced relative to GF mice treated with isotype antibody (Fig. 3F). These results suggest that CD40 up-regulated on DCs by exposure to NCD is a key molecule for ICOS up-regulation on CD4+ T cells activated by food Ags. In contrast, B cell–derived signals are not required for ICOS up-regulation in CD4+ T cells. Although B cells constitutively express CD40 (20), ICOS expression levels on activated CD4+ T cells from MLN and PP of GF B cell–deficient Jh−/− mice were comparable to those in GF wild-type (WT) mice (fig. S4B). These results collectively indicate that CD40 expressed on DCs, but not on B cells, in MLN and PP plays a critical role in ICOS up-regulation on food Ag–reactive T cells and consequent differentiation of T FH cells in GF conditions.

B cells are also indispensable for the generation of food Ag–driven T FH cells in GF mice Previous studies showed that B cells are involved in the generation of T FH cells. Thus, the lack of Ag presentation by B cells to cognate T cells causes defective T FH cell generation (21). In addition, ICOSL expressed on B cells is required for T FH cell generation (22). Despite the dispensable role of B cells in ICOS up-regulation on activated CD4+ T cells, the generation of food Ag–driven T FH cells in GF mice was profoundly defective in GF Jh−/− mice (fig. S6, A and B). To further address the relative contribution of Ag presentation and ICOSL provided by B cells to the development of food Ag–driven T FH cells in GF mice, we generated mixed bone marrow chimeras (BMCs) to make major histocompatibility complex (MHC) II and ICOSL expression defective only on B cells, but not on the other Ag-presenting cells such as CD11c+ DCs (fig. S7A). T FH cell populations in MLN and PP were deficient in mixed BMCs with B cell–specific ablation of both MHCII and ICOSL (fig. S7, B and C). Consequently, GC B cell generation and IgE elevation were defective in both mixed BMCs (fig. S7, D and E). However, whereas CXCR5+ cell generation was defective in ICOSL−/− BMC, CXCR5+ population was significantly higher in MHCII−/− BMC than in ICOSL−/− BMC (fig. S7F). These results indicate that B cell–derived ICOSL signaling is required for the generation of CXCR5+ T cells at an early stage of food Ag–driven T FH cell differentiation and the presentation of cognate Ags by B cells is required for further differentiation of CXCR5+ T cells into GC T FH cells. Together, our results indicate that DCs and B cells exert different roles in stepwise differentiation of food Ag–driven T FH cells in GF mice.

Food Ag–induced T FH cells are generated more efficiently at early age than at adult age As shown previously, the generation of food Ag–driven T FH cells in GALT was effectively prevented by the treatment with anti-ICOSL antibody. After the cessation of anti-ICOSL antibody treatment, we kept tracking T FH cell populations (Fig. 4A). We found that the recovery of T FH cells was profoundly retarded and T FH cells were not fully recovered even at 4 weeks after cessation of anti-ICOSL treatment (Fig. 4, B and C). Consistent with the levels of T FH cells, serum IgE levels were not effectively increased relative to age-matched control (Fig. 4D). Because T FH cell differentiation occurred rapidly in MLN and PP of young GF mice weaned onto NCD (fig. S2A), these results suggest that food Ag–induced T FH cell generation is not efficient in adult mice relative to young mice, i.e., GF mice at weaning period. Fig. 4 T FH cell generation and IgE elevation in response to food Ags are developmentally regulated. (A to D) GF mice (6.5 weeks old) were treated with anti-ICOSL antibody for 2 weeks to deplete T FH cells. Mice were analyzed to examine the level of T FH cells at days 2 and 28 after cessation of anti-ICOSL antibody treatment (n = 3). (A) Schematic view of experimental design and time plan. (B) Representative FACS plots of PD-1 and CXCR5 gated on CD4+ TCRβ+ Foxp3− CD44hi cells. (C) Number of PD-1hi CXCR5+ T FH cells. (D) Serum IgE levels of control mice and mice with T FH depletion at days 0 and 28 were measured by ELISA. (E to G) Young and adult AF mice (4 and 8 weeks of age, respectively) were switched to NCD for 4 weeks (n = 4). Representative FACS plots showing PD-1hi CXCR5+ T FH cells gated on CD4+ TCRβ+ Foxp3− CD44hi cells (E) and number of PD-1hi CXCR5+ T FH cells (F) from MLN and PP of indicated mice. (G) Serum IgE levels in indicated mice by ELISA. Each symbol represents an individual mouse. All data are representative of two independent experiments. Statistical differences were determined by one-way (C, F, and G) or two-way (D) ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001. Error bars represent SEM. To further support these findings, we compared young AF mice with adult AF mice, both switched to NCD. T FH cells were efficiently generated in GALT of young AF mice fed with NCD (Fig. 4, E and F). Upon the exposure to NCD for the same period of 4 weeks, CD44hi activated CD4+ T cells were comparable between young and adult AF mice (fig. S8, A and B). Although adult AF mice switched to NCD showed increased levels of GATA3-expressing T H 2 cells in MLN and PP (fig. S8C), T FH cell generation was profoundly limited in adult AF mice exposed to NCD (Fig. 4, E and F). Paralleling T FH cell frequency in MLN and PP, serum IgE levels were lower in adult AF mice than in young AF mice exposed to NCD (Fig. 4G). These results support the idea that the host immune response to ingested food Ags in GF mice at an early age is highly favorable to T FH cell generation relative to adult mice and IL-4–producing T FH cells, rather than T H 2 cells, are more important in spontaneous IgE elevation driven by food Ags. Furthermore, these results also suggest that the abundant T FH cells in MLN and PP of adult GF mice are maintained by prolonged survival of T FH cells generated in early life rather than replenishment by newly generated T FH cells.

Food Ag–driven T FH cells require cognate Ags and ICOS signaling for their maintenance To test whether continuous exposure to food Ags is required for maintaining T FH cells, we fed adult GF mice with AFD to remove contact with food Ags, because de novo food Ag–driven T FH cell generation is not efficient at the adult stage. As a consequence, T FH cell populations shrank to about 40% relative to those in GF mice (Fig. 5A), indicating partial dependence on contact with cognate Ags. A considerable proportion of T FH cells survived even after depriving exposure to food Ags, presumably representing the presence of long-lived memory T FH cells (Fig. 5A). These results demonstrate that food Ag–induced T FH cells are heterogeneous with regard to the requirement of contact with Ags for their maintenance. Fig. 5 Long-lived IgE-producing plasma cells contribute to sustained serum IgE levels regardless of the maintenance of T FH cells in adult mice. (A and D) GF mice (10 to 12 weeks old) were fed with NCD and AFD for 8 weeks (NCD versus AFD, n = 4 to 5). (A) Representative FACS plots showing PD-1hi CXCR5+ T FH cells gated on CD4+ TCRβ+ Foxp3− CD44hi cells in MLN and PP (left) and ratios of T FH cell frequency (right). (B and E) GF mice (12 weeks old) were untreated or treated with anti-ICOSL antibody every 3 days for 3 weeks (control versus anti-ICOSL, n = 4). (B) Representative FACS plots showing T FH cells in MLN and PP (left) and ratios of T FH cell frequency (right). (C) B cells in 10-week-old GF mice were gradually depleted by treating once with 0, 25, 100, and 250 μg of anti-CD20–depleting antibody (n = 2 per each dose). The correlation between the frequency of T FH cells and B220+ cells in MLN (left) and PP (right) was examined at 2 weeks after anti-CD20 antibody treatment. (D) Serum IgE levels in GF control (NCD, n = 5) and GF on AFD (AFD, n = 4) for indicated periods. (E) Serum IgE levels in GF untreated control (n = 3) and GF mice treated with anti-ICOSL antibody for indicated periods (n = 4). (F) GF mice (14 weeks old) were treated with isotype and anti-CD20 antibody (250 μg) every 3 days for 2 weeks. Serum IgE levels were shown. Each symbol represents an individual mouse. Data in (A) to (D) are pooled from two or three independent experiments. Data in (E) and (F) are representative of two independent experiments. Statistical differences were determined by two-way (A, B, D, and E) ANOVA with Tukey’s multiple comparisons test and by unpaired two-tailed Student’s t test (F). *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Error bars represent SEM. Maintenance of T FH cells generated after Ag immunization is known to depend on ICOS-ICOSL interaction (18). To examine this requirement for food Ag–driven T FH cells, we treated adult GF mice with anti-ICOSL antibody at 12 weeks of age. After preventing ICOS-ICOSL interaction, T FH cell frequencies quickly declined to the basal level seen in AF mice (Fig. 5B), indicating that ICOS-ICOSL interaction is essential for the maintenance of food Ag–induced T FH cells in GF mice. To examine whether B cells are also required for the maintenance of food Ag–driven T FH cells, we depleted B cells gradually in adult GF mice by injecting different doses of anti-CD20 antibody. At 2 weeks after B cell depletion, the frequency of T FH cells correlated closely with the percentage of B cells in GALT, indicating that B cells are required for the maintenance of T FH cells (Fig. 5C). Hence, we concluded that T cell receptor (TCR) stimulation by cognate Ags and ICOSL expressed on B cells collectively provide important survival signals to maintain T FH cells residing in B cell follicles.

Long-lived IgE-producing plasma cells support prolonged high levels of serum IgE in GF mice While T FH cells were reduced in adult GF mice fed with AFD (Fig. 5A), serum IgE levels remained at a high level, although constant IgE elevation ceased with AFD feeding (Fig. 5D). Depletion of T FH cells in adult GF mice by disrupting ICOS-ICOSL interaction for 3 weeks prevents IgE elevation (Fig. 5E), but IgE levels sustained higher than typical IgE levels in AF mice (Fig. 1A). Furthermore, serum IgE levels were not significantly reduced upon B cell depletion (Fig. 5F). As the half-life of mouse IgE is shorter than 48 hours (1), our observations suggest that long-lived IgE-producing plasma cells are responsible for sustaining high levels of serum IgE in T FH cell–depleted GF mice without the need for de novo generation of IgE-producing cells. Long-lived plasma cells are known to be radioresistant (23). In this respect, serum IgE levels could persist for 2 weeks even in adult GF mice lethally irradiated and reconstituted with BM cells from Rag1−/− mice (fig. S9, A and B). Also, irradiation-resistant IgE-producing cells could survive in MLN and BM of lethally irradiated GF mice (fig. S9, C and D). Together, our results suggest that T FH cells are essential for IgE elevation with age in GF mice but are not necessary for the maintenance of high level of serum IgE due to the generation of long-lived IgE-producing plasma cells.