Nasal vaccination of β7 integrin-deficient mice retains elevated IgA immunity

Abstract

Understanding the migration of lymphocytes to nonintestinal mucosal sites is fundamental to developing mucosal vaccination strategies. Studies have shown that nasal and oral immunization with cholera toxin (CT) stimulates, in addition to α4β7⁺, the induction of αE (CD103)β7⁺ B cells. To determine the extent to which αE-associated β7 contributes to antigen (Ag)-specific immunoglobulin (Ig)A responses in the upper respiratory tract, nasal CT vaccination was performed in wild-type (wt) and β7^−/− mice. At 16 days postprimary immunization, upper respiratory tract IgA responses were greater in β7^−/− mice than in wt mice. IgA induction by distal β7^−/− Peyer’s patches, mesenteric lymph nodes and small intestinal lamina propria was minimal, in contrast to elevated gut IgA responses in wt mice. By 42 days postprimary immunization, β7^−/− gut IgA responses were restored, and upper respiratory tract Ag-specific IgA responses were equivalent to those of wt mice. Examination of homing receptor expression and cell-sorting experiments revealed that β7^−/− mice have increased usage of βi and αE integrins by upper respiratory tract B cells, suggesting that alternative integrins can facilitate lymphocyte migration to the upper respiratory tract, especially in the absence of β7.

INTRODUCTION

The mucosal immune system comprises inductive tissues in which antigens (Ags) are processed by Ag-presenting cells for presentation to naïve lymphocytes, thus initiating the stimulation of immune and memory B and T cells. Peyer’s patches (PPs) and the nasopharyngeal-associated lymphoid tissue (NALT) or adenoids are inductive sites for the gastrointestinal lymphoreticular tissue¹ and upper respiratory tract,² respectively. The PPs and NALT have three distinct areas: (1) the dome with a unique lymphoepithelium; (2) the B-cell follicles, which usually contain one or more germinal centers and (3) the perifollicular or T-cell-dependent area. The dome contains a specialized type of epithelial cell, referred to as a microfold cell that facilitates luminal Ag sampling to underlying Ag-presenting cells and lymphocytes. Subsequent to their activation, B and T cells migrate to, and ultimately seed, mucosal effector tissues in the epithelia of the gastrointestinal lymphoreticular tissue, upper respiratory tract and lungs.^3,4

Nasal immunization offers a productive means of inducing mucosal immunity both locally and distally.^1,3,4 The NALT and the various head and neck lymph nodes (HNLNs) support induction of respiratory immunity.^2,5,6 Notably, nasal immunization elicits pronounced humoral and cell-mediated immunity in distal mucosal tissues.^7–9 While the majority of these immune responses occur proximal to the site of nasal or oral vaccination, determining how to enhance the distal mucosal immune responses would expand the application of mucosal vaccines. In this context, elucidating the mechanisms by which Ag-specific lymphocytes migrate to mucosal tissues could result in new adjuvants that could directly modulate lymphocyte homing receptors or perhaps alter tissue addressin patterns. For migration to the intestinal compartment, lymphocytes with high expression of α4β7¹⁰ interact with their cognitive receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), on high endothelial venules.^9,11–13 However, the α4β7–MAdCAM-1 interaction emerges as being secondary to L-selectin and association with its receptor, peripheral node addressin (PNAd), in nonintestinal mucosal tissues.^5,6,14–16 The elevated presence of PNAd in the NALT⁵ and HNLNs,⁶ albeit variably associated with MAdCAM-1, is evidence for this. Moreover, immunoglobulin (Ig)A responses are delayed subsequent to nasal immunization with cholera toxin (CT) in L-selectin^−/− mice.^14,15 Interestingly, small intestinal lamina propria (iLP) IgA responses in L-selectin^−/− mice remained pronounced because of the involvement of a β7^high subset not being associated with α4, but rather with α_E.¹⁴ α4β7 is expressed on T and B lymphocytes,^17,18 and is largely responsible for lymphocyte homing in the intestine.¹⁰ Studies show that α4β7 is strongly expressed on B lymphocytes and on blasts with a memory phenotype.¹⁹ Retinoic acid derived from gut dendritic cells has been shown to promote gut homing of T and B cells by enhancing their α4β7 expression.^20–22

An integrin responsible for retention of lymphocytes in the epithelium is αE(CD103)β7.^10,23,24 Aside from the loss of intraepithelial lymphocytes and reduced iLP T cells in αE^−/− mice,²⁵ β7^−/− mice also showed reduced intraepithelial lymphocytes.²⁶ The αEβ7 integrin mediates lymphocyte adhesion to the epithelium via binding to E-cadherin, a member of the cadherin family of adhesion molecules expressed selectively on epithelial cells.^23,24,27,28 Alternative ligands for αEβ7 exist, as αEβ7⁺ cells bind to both E-cadherin intestinal microvascular endothelial cells²⁹ and oral and skin keratinocytes.³⁰ Few studies have investigated αEβ7 on B cells, and we showed that nasal CT immunization stimulated a novel gut B-lymphocyte subset expressing αEβ7.^14,15 This unique subset of IgA⁺ B cells, likely memory cells, resided in the HNLNs 4 weeks after the last nasal CT immunization.¹⁵ When examined for its applicability to oral vaccinations, 4 weeks after the last CT dose, αEβ7⁺ IgA-producing B cells were notably upregulated in HNLNs, PPs and iLPs of L-selectin^−/− mice.¹⁶

Collectively, past studies have shown that the absence of L-selectin accentuated the induction of αEβ7⁺ B cells distal to the vaccination site.^14–16 We queried whether regional and distal B cells in nonintestinal lymphoid tissues would be altered in β7^−/− mice after nasal CT immunization. In contrast to the reduced IgA responses in the small intestine, elevated IgA-producing B cells were observed in the NALT and nasal passages shortly after CT vaccination. These IgA responses did not differ from wild-type (wt) mice 6 weeks postprimary vaccination, further suggesting that β7 integrin is secondary to L-selectin in governing head and neck immunity.

RESULTS

Fecal, but not nasal, CT-B-specific IgA titers are reduced in CT-immunized β7^−/− mice

To assess the impact of β7 integrin on respiratory antibody (Ab) responses to a soluble Ag, groups of β7^+/+ and β7^−/− mice were nasally immunized with CT on days 0, 7 and 14. Serum and mucosal secretions were collected at various intervals over the course of 42 days.^14–16 Ab measurements focused on the induced Abs to the B subunit of CT (CT-B) because >90% of the induced Abs are against CT-B. Serum CT-B-specific IgG titers were identical for both groups (Figure 1a). A significant difference in serum IgA endpoint titers in β7^+/+ mice was observed at day 21 postprimary immunization (Figure 1b). Kinetic analysis of fecal Ab responses revealed that CT-B-specific IgA responses in β7^−/− mice were significantly diminished at days 16, 21 and 28 postprimary immunization (Figure 1c). Nasal washes from β7^−/− mice showed a significant increase in CT-B-specific IgA titers on day 16 (Figure 1d), suggesting that local immune responses were not compromised in the absence of β7 integrin. Nasal wash IgG endpoint titers were not significantly different between β7^+/+ and β7^−/− mice (Figure 1e). Naïve β7^+/+ and β7^−/− mice showed no detectable serum and mucosal anti-CT-B Abs. These data show that the differences in regional and systemic CT-B-specific IgA responses between β7^+/+ mice and β7^−/− mice are seen early in their development.

Figure 1.

Fecal, but not nasal wash, IgA responses in β7^−/− mice are suppressed. (a) Serum IgG and (b) IgA, (c) fecal IgA, and nasal (d) IgA and (e) IgG cholera toxin B subunit (CT-B)-specific ELISA results are shown 16–42 days postnasal CT immunization. Naïve mice showed no detectable serum or mucosal Abs reactive to CT-B. Data depict the mean of 19 mice ± s.e.m. (four experiments), and statistical differences between β7^+/+ and β7^−/− mice were determined as follows: *P < 0.001; **P < 0.01; ***P < 0.05. Ab, antibody; Ig, immunoglobulin.

Regional effector antibody-forming cell responses in β7^−/− mice are enhanced in nasal passages and submandibular glands while compromised in iLPs 16 days postprimary nasal CT immunization

To account for the differences in mucosal and serum IgA responses, a CT-B-specific ELISPOT assay was performed on nasal passage, submandibular gland and iLP lymphocytes collected 16 days postprimary nasal CT immunization. The IgA anti-CT-B Ab-forming cell (AFC) responses in the nasal passages and submandibular glands were notably augmented in β7^−/− mice relative to β7^+/+ mice (Figure 2a, c); total IgA AFCs were also significantly increased in β7^−/− nasal passages and submandibular glands. By contrast, the β7^−/− iLP CT-B-specific and total IgA AFC responses were markedly reduced by 8.2- and 5.2-fold, respectively, when compared with β7^+/+ mice (Figure 2a). No significant differences in the CT-B-specific IgG responses were observed in the nasal passages, nor in the iLPs, although the total IgG AFCs were increased in the β7^−/− nasal passages, and the total iLP IgG AFCs were reduced in the β7^−/− mice (Figure 2b). The submandibular gland CT-B-specific and total IgG AFCs were elevated by 7.8- and 6.3-fold in the β7^−/− mice, respectively, relative to β7^+/+ mice (Figure 2d). Splenic CT-B-specific IgA and IgG AFCs were similar, but total IgA and IgG AFCs were increased in β7^−/− spleens (Figure 2c, d). Collectively, these results show that in the absence of β7 integrin, regional (respiratory and salivary) IgA AFC responses are augmented early postvaccination with CT, and that distal (iLP) responses are compromised.

Figure 2.

Intranasal (i.n.) immunization of β7^−/− mice with cholera toxin (CT) results in enhanced IgA and IgG antibody-forming cell (AFC) responses in nasal passages (NPs) and submandibular glands (SMGs), and reduced IgA (CT-B-specific and total) and IgG (total) responses in small intestinal lamina propria (iLP) at 16 days postprimary immunization. B-cell ELISPOT analysis was performed to determine CT-B-specific and total (a, c) IgA and (b, d) IgG AFC responses in (a, b) NPs and iLPs and (c, d) in SMGs and spleen (SPL). For each graph, the label is the same for both left and right y axes. Results depict the mean of three experiments ± s.e.m. per tissue (n = 15 mice/group), and statistical differences between β7^+/+ and β7^−/− mice were determined as follows: *P < 0.001; **P < 0.01; ***P < 0.05. Ig, immunoglobulin.

Enhanced effector B-cell responses in β7^−/− mice are supported by elevated NALT and HNLN AFCs 16 days postprimary nasal CT immunization

To examine AFC responses in regional and distal inductive lymphoid tissues from β7^+/+ and β7^−/− mice at 16 days postprimary nasal immunization, an AFC assay was conducted. β7^−/− CT-B-specific NALT IgA and IgG AFCs were augmented 3.2-fold and 8.7-fold, respectively, relative to similarly vaccinated β7^+/+ mice (Figure 3a, b). Total IgA and IgG AFCs were also elevated 2.9-fold and 5.2-fold, respectively (Figure 3a, b). Although the CT-B-specific IgA and IgG AFCs were strikingly elevated in the HNLNs, no significant difference was observed between β7^+/+ and β7^−/− mice (Figure 3c, d). Examination of distal mucosal inductive tissues revealed no significant differences in CT-B-specific IgA or IgG AFCs between β7^+/+ and β7^−/− PPs (Figure 3a, b); total β7^−/− PP IgA and IgG AFCs were reduced by 3- and 38-fold, respectively, when compared with β7^+/+ mice. β7^−/− CT-B-specific IgA and IgG AFCs were reduced in mesenteric LNs (MLNs) by 16.6- and 10.1-fold, respectively, when compared with β7^+/+ mice (Figure 3c, d). Although the total IgA AFCs did not differ between mouse strains, the reduction of β7^−/− CT-B-specific AFCs in MLNs was accompanied by an increase in the total IgG AFCs. These results show that local respiratory tract and salivary gland AFC responses were significantly augmented in β7^−/− mice at 16 days postprimary nasal immunization with CT relative to β7^+/+ mice, whereas the reduced distal β7^−/− iLP AFC responses were supported by reduced PP and MLN function.

Figure 3.

Intranasal (i.n.) immunization of β7^−/− mice with cholera toxin (CT) results in enhanced CT-B-specific and total IgA and IgG antibody-forming cell (AFC) responses in nasopharyngeal-associated lymphoid tissue (NALT) 16 days postprimary immunization, whereas total IgA and IgG AFC responses in Peyer’s patches (PPs) as well as CT-B-specific IgA and IgG AFC responses in mesenteric lymph nodes (MLNs) are reduced. B-cell ELISPOT determined (a, c) IgA and (b, d) IgG CT-B-specific and total responses in the (a, b) NALT and PPs and (c, d) in head and neck lymph nodes (HNLNs) and MLNs. For each graph, the label is the same for both left and right y axes. Results depict the mean of three experiments ± s.e.m. per tissue (n = 15 mice/group), and statistical differences between β7^+/+ and β7^−/− mice were determined as follows: *P < 0.001; **P < 0.01; ***P < 0.05. Ig, immunoglobulin.

Upper respiratory tract β7^+/+ and β7^−/− IgA AFCs are elevated, but equivalent by 6 weeks postprimary immunization

Both β7^−/− and β7^+/+ CT-B-specific IgA and IgG AFC responses in the nasal passages were greatly augmented by 6 weeks postprimary immunization (Figure 4a, b) when compared with results obtained at day 16 (Figure 2a, b). Nonetheless, both strains of mice showed a similar magnitude of Ag-specific AFCs in the nasal passages, and in fact, the total IgA and IgG AFCs from β7^+/+ mice exceeded those in the β7^−/− mice (Figure 4a, b). In contrast to results obtained at 16 days postprimary immunization, CT-B-specific IgA AFCs in the iLP were not significantly different between β7^−/− and β7^+/+ mice (Figure 4a). Albeit low, the IgA AFC responses in the β7^−/− iLP were restored, relative to β7^+/+ mice, despite the total IgA AFCs in the β7^−/− iLPs remaining diminished. The CT-B-specific IgG AFC responses were nonexistent in both groups of mice at 16 days postprimary immunization (Figure 2b), but were present by week 6, with no significant difference between β7^−/− and β7^+/+ iLPs being observed (Figure 4b). β7^−/− and β7^+/+ IgA and IgG CT-B-specific AFC responses in the submandibular glands revealed no significant difference (Figure 4c, d), although the IgA responses were relatively enhanced compared with day 16 IgA responses. Total IgA and total IgG AFC responses in the β7^−/− submandibular glands remained elevated (Figure 4c, d). Furthermore, total, but not CT-B-specific, IgA and IgG AFCs remained elevated in β7^−/− spleens at 6 weeks. These data indicate that a lack of β7 integrin does not permanently affect the effector function of the iLP subsequent to nasal immunization with CT. No significant difference was observed for splenic Ag-specific IgA and IgG AFCs between β7^−/− and β7^+/+ mice (Figure 4c, d), although total IgA and total IgG AFCs were significantly elevated in β7^−/− mice (Figure 4c, d). These data show that CT-B-specific IgA AFC responses were restored in the β7^−/− iLPs. Although no significant differences in CT-B-specific IgA AFC responses between β7^−/− and β7^+/+ nasal passages were observed, the overall magnitude of these responses was increased relative to day 16 responses.

Figure 4.

Partial restoration of antibody-forming cell (AFC) responses in β7^−/− intestinal lamina propria (iLP) is accompanied by a decrease of immune responses in β7^−/− nasal passage (NP) at 42 days postprimary cholera toxin (CT) intranasal (i.n.) immunization. CT-B-specific and total (a, c) IgA and (b, d) IgG responses (a, b) in the NPs and iLPs and (c, d) in submandibular glands (SMGs) and spleen (SPL) were determined by B-cell ELISPOT. For each graph, the label is the same for both left and right y axes. Results depict the mean of three experiments ± s.e.m. per tissue (n = 15 mice/group), and statistical differences between β7^+/+ and β7^−/− mice were determined as follows: *P < 0.001; **P < 0.01; ***P < 0.05. Ig, immunoglobulin.

CT-B-specific IgA and IgG AFC responses remain elevated in β7^−/− NALT and HNLNs with weak distal AFC responses in the PPs and MLNs

IgA and IgG AFCs in the respiratory/salivary and gut inductive tissues were also assessed. At week 6 postprimary immunization (Figure 5a, b), CT-B-specific IgA and IgG AFC responses in β7^−/− NALT remained elevated, and equivalent increases were observed in β7^+/+ mice. Total IgA and IgG AFCs in β7^−/− NALT remained increased, relative to β7^+/+ NALT (Figure 5a, b). Comparison of day 16 (Figure 3c, d) and day 42 HNLN AFCs (Figure 5c, d) revealed that the magnitude of CT-B-specific IgA and IgG AFC responses lessened with time for both β7^−/− and β7^+/+ mice. At this later time point, no significant differences in Ag-specific IgA or IgG AFCs were evident, although total IgA and IgG AFC responses were significantly increased in β7^−/− HNLNs.

Figure 5.

At 42 days postprimary cholera toxin (CT) intranasal (i.n.) immunization, CT-B-specific, but not total, Ab responses are recovered to the normal/control state in β7^−/− nasopharyngeal-associated lymphoid tissue (NALT), whereas total antibody-forming cell (AFC) responses are still compromised in β7^−/− Peyer’s patches (PPs). B-cell ELISPOT analysis was performed to determine CT-B-specific and total (a, c) IgA and (b, d) IgG AFC responses (a, b) in the NALT and PPs and (c, d) in head and neck lymph nodes (HNLNs) and mesenteric lymph nodes (MLNs). For each graph, the label is the same for both left and right y axes. Results depict the mean of three experiments ± s.e.m. per tissue (n = 15 mice/group), and statistical differences between β7^+/+ and β7^−/− mice were determined as follows: *P < 0.001; **P < 0.01; ***P < 0.05. Ab, antibody; Ig, immunoglobulin.

Examination of gut inductive tissues revealed that CT-B-specific responses in the PPs remained weak for the duration of the experiment. Although the IgA responses did not significantly differ between β7^−/− and β7^+/+ mice, the IgG responses were significantly less (Figure 5a, b). Total IgA and IgG AFC responses at week 6 remained reduced in β7^−/− PPs relative to those in β7^+/+ PPs (Figure 5a, b). The β7^−/− and β7^+/+ MLN CT-B-specific IgA and IgG AFC responses remained weak with no significant difference in IgA, and a reduced IgG (Figure 5c, d). By week 6 postprimary immunization, CT-B-specific IgA AFC responses were restored in β7^−/− MLNs. β7^−/− MLNs showed an increase in total IgG AFCs although total IgA AFCs did not significantly differ (Figure 5c, d). Thus, these data show that NALT and HNLN CT-B-specific IgA and IgG AFC responses are longer lived than PP and MLN AFC responses.

L-selectin and αE expression and alternative β-integrin usage by β7^−/− upper respiratory tract B cells following intranasal CT immunization

Despite their deficiency, β7^−/− mice still retained their capacity to elicit sustained IgA and IgG Ab responses to CT in the upper respiratory tract. To account for possible compensation for the β7 deficiency, other homing receptors and integrins necessary to enable B-cell migration in the upper respiratory tract lymphoid tissues were examined. Using flow cytometry, L-selectin levels were measured to determine whether these were impacted before and after CT immunization in wt and β7^−/− mice (Figure 6). Examination of 16-day responses revealed significant increases in CT-immunized β7^−/− mice for nasal passage, submandibular gland LNs (SMLNs; or superficial cervical LN), parotid gland LNs (posterior to the parotid gland), along with splenic L-selectin^high and SMLN, parotid gland LN and splenic L-selectin^low B cells (Figure 6a–h). By 42 days, only the β7^−/− NALT showed a significant increase in L-selectin^high B cells (Figure 6j) and the β7^−/− MLNs showed significant reductions in L-selectin^high and L-selectin^low B cells (Figure 6l, o). On day 16 postprimary immunization, αE, α4β1 and β2 integrin expression levels were measured on B cells from β7^+/+ and β7^−/− mice (Figure 7). Data showed a similar magnitude of α4β1⁺, α2^bright and αE⁺ B cells in the β7^−/− nasal passages (Figure 7a, b, e, g), and the α4β1⁺ and the β2^bright B cells from β7^−/− mice were significantly greater than those present in wt nasal passages. The wt and β7^−/− NALT also showed equivalent levels of α4β1⁺, α4β2^bright and aE⁺ B cells; the β7^−/− NALT amount did not significantly differ from that of β7^+/+ NALT (Figure 7e–g). The number of α4β1⁺ and αE⁺ B cells increased in β7^−/− compared with that of the wt SMLNs; the number of α4β1⁺, β2^bright and αE⁺ B cells significantly decreased in the β7^−/− deep cervical LNs relative to those in the β7^+/+ cervical LNs (Figure 7c, d, h–j). For the β7^−/− parotid gland LNs, only the β2^bright B cells were significantly reduced compared with the wt parotid gland LNs; no difference was observed between numbers of wt and β7^−/− parotid gland LN α4β1⁺ and αE⁺ B cells. These integrin⁺ B-cell subsets were each independent of the other and did not coexpress the various integrins.

Figure 6.

Increased L-selectin⁺ B cells are evident in β7^−/− nasal passage (NP) and head and neck lymph nodes (HNLNs) at 16 and in nasopharyngeal-associated lymphoid tissue (NALT) at 42 days postprimary immunization. Groups of B6 and β7^−/− mice were nasally vaccinated with cholera toxin (CT) as described in text. Flow cytometry analysis was performed on lymphocytes isolated from individual naïve mice or from immunized mice at (a–h) 16 days and (i–o) 42 days postprimary immunization. (a, b, i) Gated on lymphocytes isolated from the submandibular gland lymph nodes (SMLNs) and NPs, cells were stained for CD19 and L-selectin expression with the latter staining positive as L-selectin^hi or L-selectin^lo. The total number of L-selectin^hi (c–e, j–l) and L-selectin^lo B cells (f–h, m–o) was determined for (c, f, j, m) NPs, NALT, (d, g, k, n) spleen (Spl), SMLNs, (e, h, l, o) mesenteric lymph nodes (MLNs), cervical lymph nodes (CLNs) and parotid gland lymph nodes (PRLNs). Results depict the mean of two experiments ± s.e.m. per tissue (n = 5–10 mice/group), and statistical differences between B6 (β7^+/+) and β7^−/− mice were determined as follows: *P < 0.001; **P < 0.01; ***P < 0.05.

Figure 7.

αE and alternative β-integrin expression is selectively enhanced on B cells from the nasal passages (NPs) and submandibular gland lymph nodes (SMLNs), and reduced on cervical lymph node (CLN) B cells relative to similarly immunized wild-type (wt) (β7^+/+) mice. Groups of wt and β7^−/− mice (6–12 mice/group) were intranasally (i.n.) vaccinated three times with cholera toxin (CT) as described in Figure 1. On day 16 following primary i.n. CT immunization, dot plots of total B cells from (a, c) β7^+/+ and (b, d) β7^−/− (a, b) NPs and (c, d) SMLNs are depicted. Gating on CD19⁺ B cells, they were evaluated for α4, αEβ7 and β2 integrin expression, and α4⁺ cells were measured for coexpression with β1 integrin. (e–j) The total number of wt and β7^−/− B cells that are (e, h) α4β1⁺, (f, i) β2^bright, (g, j) αE⁺ and present in the (e–g) nasopharyngeal-associated lymphoid tissue (NALT) and NPs and in the (h–j) CLNs, parotid gland lymph nodes (PRLNs) and SMLNs is shown. Differences in integrin expression on B cells taken from the same tissue between β7^+/+ and β7^−/− mice are depicted as *P ≤ 0.001, **P ≤ 0.010, ***P < 0.05. TCR, T-cell receptor.

HNLN, nasal passage and splenic αE⁺ B cells from cell-sorted β7^−/− mice contain more CT-B-specific IgA-forming cells than those in β7^+/+ mice at 16 days postprimary vaccination

Following nasal vaccination with CT, IgA⁺ upper respiratory tract B cells increased in β7^−/− mice compared with β7^+/+ mice. To examine how these B cells partition relative to their αE expression, cell-sorting experiments were performed to purify αE⁺, L-selectin, β1⁺ and β2⁺ B-cell subsets present in the nasal passages, HNLNs and spleens from vaccinated wt and β7^−/− mice. IgA anti-CT-B-specific activity was measured by B-cell ELISPOT (Table 1). In β7^−/− mice, CT-B-specific and total αE⁺ IgA B cells in nasal passages were elevated fivefold in relative to those present in B6 mice (Table 1). CT-B-specific and total β7^−/− αE⁺ splenic IgA AFCs significantly exceeded those in wt spleens. Only total, not CT-B-specific, β7^−/− αE⁺ HNLN IgA B cells exceeded by nine-fold B6 αE⁺ HNLN B cells. A significant increase in β7^−/− nasal passage CT-B-specific β1⁺ IgA B cells relative to those present in B6 nasal passages was observed. Likewise, this significant increase was observed in β7^−/− splenic CT-B-specific and total β1⁺ IgA B cells when compared with wt spleens. No significant difference between β7^−/− and B6 nasal passages and spleens for CT-B-specific and total L-selectin⁺ IgA B cells was observed (Table 1). CT-B-specific and total L-selectin⁺ IgA B cells were significantly elevated for B6 HNLN relative to β7^−/− HNLNs. The number of B6 nasal passage and splenic CT-B-specific β2⁺ IgA B cells was significantly elevated when compared with β7^−/− tissues. Hence, these studies show the induction of alternative integrins expressed by B cells in the absence of β7 integrin.

Table 1.

CT-B-specific and total IgA antibody-forming cell responses by sorted B cells.

Mousea	Tissue	Integrin	IgA+ CT-B-specific B cells ± s.e.m./1 × 106 Integrin+ B cells	P-value	Total IgA+ B cells ± s.e.m./1 × 106 Integrin+ B cells	P-value
B6	NP	αE	182 ± 27	<0.001	517 ± 120	<0.001
β7−/−	”	αE	950 ± 97		2733 ± 349
B6	”	L-selectin	544 ± 53	NS	1549 ± 165	<0.001
β7−/−	”	L-selectin	489 ± 43		1128 ± 61
B6	”	β1	234 ± 78	<0.001	677 ± 521	NS
β7−/−	”	β1	639 ± 7.9		976 ± 212
B6	”	β2	1053 ± 132	0.021	1842 ± 402	NS
β7−/−	”	β2	614 ± 7.3		1067 ± 35
B6	HNLNs	αE	826 ± 264	NS	7132 ± 1242	0.006
β7−/−	”	αE	12710 ± 7011		63853 ± 37054
B6	”	L-selectin	69 ± 13	0.011	590 ± 92	0.003
β7−/−	”	L-selectin	13 ± 1.2		19 ± 8.7
B6	”	β1	ND	—	ND	—
β7−/−	”	β1
B6	”	β2	ND	—	ND	—
β7−/−	”	β2
B6	Spleen	αE	45 ± 8.6	<0.001	87 ± 9.7	0.008
β7−/−	”	αE	153 ± 6.6		248 ± 31.1
B6	”	L-selectin	20 ± 7.4	NS	20 ± 7.4	0.003
β7−/−	”	L-selectin	27 ± 3.8		84 ± 6.4
B6	”	β1	23 ± 0.9	0.003	56 ± 5.3	<0.001
β7−/−	”	β1	172 ± 22.6		209 ± 13.3
B6	”	β2	101 ± 6.0	<0.001	124 ± 13	NS
β7−/−	”	β2	38 ± 2.9		110 ± 14

CT, cholera toxin; HNLNs, head and neck lymph nodes; Ig, immunoglobulin; ND, not done; NP, nasal passages; NS, not significant.

^aMouse strain: B6, C57BL/6; β7^−/−, β7-deficient B6 mice.

Cell-sorted β7^−/− HNLN and splenic αE⁺ and L-selectin⁺ B cells contain more CT-B-specific IgG-forming cells than that seen in B cells from β7^+/+ mice

AFC analysis for IgG⁺ B cells was also conducted for β7^−/− and β7^+/+ mice at 16 days postnasal vaccination with CT using sorted integrin⁺ B cells present in the nasal passages, HNLNs and spleens (Table 2). The β7^−/− HNLNs and spleens were significantly increased by 1.7-fold and three-fold, respectively, in CT-B-specific αE⁺ IgG B cells relative to those present in B6 mice (Table 2). No significant difference was observed between β7^−/− and wt CT-B-specific αE⁺ nasal passage IgG B cells, but the total B6 mice nasal passage αE⁺ IgG B cells were significantly increased. Total β7^−/− splenic, not HNLN, αE⁺ IgG B cells were elevated 2.9-fold relative to B6 mice. β7^−/− HNLN and splenic CT-B-specific L-selectin⁺ IgG B cells were significantly elevated compared with B6 mice; no significant difference between β7^−/− and wt total HNLN and splenic IgG B cells was observed. In addition, no significant difference between β7^−/− and B6 nasal passage CT-B-specific and total β1⁺ IgG B cells was found. For the spleen, B6 CT-B-specific IgG β1⁺ B cells were significantly increased over β7^−/− IgG B cells, and no significant difference was evidenced for total splenic β2⁺ IgG B cells. The number of nasal passage CT-B-specific β2⁺ IgG B cells in β7^−/− mice was significantly increased when compared with that in B6 mice. Hence, these studies demonstrate that alternative integrins are expressed by B cells in the absence of β7 integrin.

Table 2.

CT-B-specific and total IgG antibody-forming cell responses by sorted B cells.

Mousea	Tissue	Integrin	IgG+ CT-B-specific B cells ± s.e.m./1 × 106 Integrin+ B cells	P-value	Total IgG+ B cells ± s.e.m./1 × 106 Integrin+ B cells	P-value
B6	NP	αE	4270 ± 653	NS	17 350 ± 1382	0.002
β7−/−	”	αE	4005 ± 109		5903 ± 854
B6	”	L-selectin	1464 ± 204	NS	15 536 ± 964	<0.001
β7−/−	”	L-selectin	2320 ± 233		3367 ± 349
B6	”	β1	2391 ± 95	NS	4994 ± 889	NS
β7−/−	”	β1	2554 ± 171		3183 ± 71
B6	”	β2	2978 ± 169	0.038	8967 ± 269	NS
β7−/−	”	β2	4114 ± 330		8967 ± 841
B6	HNLNs	αE	445 ± 40	0.016	9810 ± 924	NS
β7−/−	”	αE	746 ± 110		9625 ± 1455
B6	”	L-selectin	69 ± 12	0.002	3471 ± 667	NS
β7−/−	”	L-selectin	208 ± 15		3932 ± 327
B6	”	β1	ND	—	ND	—
β7−/−	”	β1
B6	”	β2	ND	—	ND	—
β7−/−	”	β2
B6	Spleen	αE	1184 ± 21	<0.001	2684 ± 404	0.006
β7−/−	”	αE	3510 ± 131		7790 ± 867
B6	”	L-selectin	229 ± 7.8	<0.001	843 ± 292	NS
β7−/−	”	L-selectin	381 ± 3.2		1188 ± 111
B6	”	β1	479 + 51	0.044	872 ± 113	NS
β7−/−	”	β1	265 ± 53		938 ± 111
B6	”	β2	943 ± 68	NS	3023 ± 335	0.014
β7−/−	”	β2	1216 ± 140		1381 ± 200

CT, cholera toxin; HNLNs, head and neck lymph nodes; Ig, immunoglobulin; ND, not done; NP, nasal passages; NS, not significant.

^aMouse strain: B6, C57BL/6; β7^−/−, β7-deficient B6 mice.

DISCUSSION

Past studies have demonstrated the importance of β7 integrin in intestinal B-cell responses subsequent to rotavirus infection^31,32 as well as after nasal immunization with rotavirus virus-like particles.³³ However, in nonintestinal mucosal tissues, the role of α4β7 and αEβ7 in IgA B-cell responses is unknown. We have reported that naïve HNLN lymphocyte homing is dependent on L-selectin interacting with PNAd, and that anti-β7 monoclonal Abs (mAbs) treatment had minimal impact on lymphocyte homing and retention in the various HNLNs.⁵ Yet, anti-β7 mAb treatment did inhibit lymphocyte binding to HNLN/high endothelial venules.⁵ Interestingly, the amount of PNAd and MAdCAM-1 varied among the NALT and different HNLNs,^5,6 as did the amount of PNAd decorating MAdCAM-1.^5,6 This evidence points to a role for α4β7 in mediating lymphocyte trafficking in the NALT and HNLNs. Nasal CT immunization of L-selectin^−/− mice showed delayed IgA responses in the reproductive and nasal tracts, but not in the intestine and serum.¹⁴ Ultimately, the B cells could reach the reproductive and nasal tracts with heightened potency, particularly in the nasal passages, as a result of elevated levels of immune B cells in the HNLNs.¹⁵ The importance of L-selectin and the reduced relevance of α4β7 present on circulating lymphocytes were also shown in other studies.^34,35 While L-selectin was found to be important for B-lymphocyte trafficking in the NALT and HNLNs,^5,6,14,15 the reason why increased numbers of αEβ7⁺ B cells are seen in the iLPs following nasal CT immunization of L-selectin^−/− mice remains unclear.^24,25 Similar distal sequestration of αEβ7⁺ B cells has been observed in oral CT-immunized L-selectin^−/− mice, but these B cells resided in the NALT and HNLNs, not in the iLPs.¹⁶

Given these observations, the studies embarked upon here examine the role of β7 in mice nasally immunized with CT, in order to learn how local and distal B-cell responses develop in the absence of β7 integrin. The results show that β7 integrin deficiency severely inhibits the effector function of the iLP. This reduction in B lymphocytes is consistent with previous observations in β7^−/− mice, and supports the notion that lymphocyte migration to intestinal tissues is α4β7 dependent.^26,32 Clearly, the absence of β7 integrin reduced the recruitment of circulating, likely nasally induced, IgA-producing B cells to intestinal effector sites. Based on the analysis of CT-B-specific IgA AFC and fecal IgA responses, the conclusion can be drawn that in β7^−/− mice the intestinal IgA responses are delayed, but inducible.

Our work revealed a dramatic level of acceleration/enhancement of the IgA AFC responses in β7^−/− nonintestinal mucosal effector sites at 16 days postprimary immunization. The β7^−/− nasal passage IgA AFC responses were increased at 16 days, and by 6 weeks postprimary immunization, the levels were similar to those in wt mice, indicating that even in the absence of β7 integrin, the IgA responses remained enhanced. Furthermore, CT-B-specific IgA endpoint titers were elevated at day 16 in the nasal washes relative to levels present at 6 weeks postprimary immunization, and this trend also manifested for β7^−/− submandibular gland AFC responses relative to wt levels.

To determine the mechanisms supporting these AFC responses in both β7^−/− nasal passage and submandibular gland, subsequent analysis of the regional inductive immune responses was performed. The greatest impact noted in β7^−/− mice was the augmentation of upper respiratory tract immunity relative to that observed for wt mice, which occurred shortly after vaccination as opposed to the 6-week time point. The absence of β7 integrin augments lymphocyte migration to this site. At 16 days postprimary immunization, CT-B-specific and total IgA and IgG AFC levels were significantly enhanced in β7^−/− NALT. In the remaining upper respiratory tract inductive sites, the β7^−/− HNLNs contained 13% (day 16) or 15% (day 42) more lymphocytes compared with wt mice (data not shown). This finding is similar to that reported by Steeber et al.,³⁶ who showed that β7^−/− mice exhibited a 10% increase in peripheral LN cellularity. Furthermore, we observed β7^−/− mice parotid gland LNs and SMLNs to have significantly more B cells than wt mice, resulting in a higher count of B cells in the β7^−/− HNLNs (data not shown).

β7^−/− mice have markedly hypocellular PPs, whereas the CT-specific IgA AFC responses were equivalent to those of wt mice at 16 days postprimary immunization. MLNs in β7^−/− mice were also affected, with nearly no IgA and IgG CT-B-specific responses at 16 days postprimary immunization. This lack of a response persisted for 6 weeks postprimary immunization, which may be attributable to the hypocellularity of the MLNs at this time point when the total MLN lymphocyte number was reduced by half (data not shown). Previous studies, which focused on naïve MLNs, showed no difference in lymphocyte numbers when compared with wt mice.^26,36 These diametric results may reflect how differences in activation status can affect the response to vaccination. A study by Lefrançois et al.³⁷ demonstrated a dichotomy in β7 integrin requirements for entry into the MLNs, caused by the need for α4β7 becoming activated, but not naïve CD8⁺ T cells in order to migrate. Whether this is applicable to B cells remains to be determined. Past studies have shown that β7^−/− lymphocyte migration to the MLNs was reduced by 49%,²⁶ and that lymphocytes deficient in both L-selectin and β7 were not retained in wt MLNs.^36,38 Although lymphocyte migration to PPs is dependent on β7,^26,36 migration to MLNs may be mediated by homing receptors other than β7.

Considering that these nasal immunizations were conducted with β7^−/− mice which eliminates the possibility for α4β7 expression, the expectation would be that L-selectin and alternate β-integrins are induced. Indeed, an increase in B-cell subsets expressing L-selectin^high, L-selectin^low, α4β1⁺ or β2^bright in β7^−/− nasal passages relative to wt mice was detected shortly after nasal CT immunization. No significant differences were noted between wt and β7^−/− NALT. Increased α4β1⁺, not β2^bright, B cells were found in the β7^−/− SMLNs. The αE⁺ SMLN B cells were also increased relative to wt SMLNs, suggesting that these integrins collectively contribute to the compensation for the β7 integrin deficiency. Cellsorting studies also substantiated the relevance of αE⁺ and β1⁺ B cells in the β7^−/− nasal passages and spleens being increased. The total β7^−/− HNLN αE⁻ B cells also exceeded those found in B6 mice. Of note, both α4β1⁺ and β2^bright B cells were significantly reduced in β7^−/− cervical LNs and parotid gland LNs, but the β7^−/− α4β1⁺ B cells were significantly enhanced in the SMLNs compared with wt mice, further manifesting the diversity of HNLNs.⁶

Such a compensation was previously observed in how β1 and β7 integrins compete for association with α4, noted by the increased α4β7 in the absence of β1 integrin.³⁹ By contrast, no increased β1 expression was observed in β7^−/− mice, and the α4β7 expression did not reduce until the overexpression of β1 integrin.³⁹ Another possibility to account for increased α4β1⁺ B cells is through the increased interaction of α4β1 with its receptor, VCAM-1.⁴⁰ Previous studies showed that NALT and parotid gland LNs express VCAM-1,^5,6 as do tonsils,^41,42 implicating VCAM-1’s contribution to lymphocyte homing in the upper respiratory tract. VCAM-1 expression is increased in mammary gland blood vessels, resulting in enhanced α4β1⁺ cytoplasmic IgA⁺ B-cell retention in these glands.⁴³ CT is known for promoting α4β7 expression via dendritic cell-derived retinoic acid.⁴⁴ In addition, CT stimulates transforming growth factor-β1,⁴⁵ resulting in increased αE expression.^14–16,46 Results with wt mice are consistent with these findings; however, the observation that β7^−/− mice had enhanced induction of CT-specific B-cell immunity within the upper respiratory tract raises the question of whether this response may be in part a result of the absence of β7 integrin’s competition for a4 integrin. While such findings can explain the observed increases in β1 integrin, how the increase in β2 integrin expression occurs is unclear. Others have found plasma cell emergence from LNs⁴⁷ to be β2 integrin dependent. The augmentation of αE in β7^−/− upper respiratory tract, along with the ability of β7^−/− αE⁺ B cells to successfully migrate to nasal passages, suggests that αE can function independently of β7.

METHODS

Mice and immunizations

β7^−/− B6 mice (Jackson Laboratory, Bar Harbor, ME, USA) were bred locally. β7^−/− and wt C57BL/6N (β7^+/+; National Cancer Institute, Frederick, MD, USA) females were kept under pathogen-free conditions in individually ventilated cages under HEPA-filtered barrier conditions, and fed sterile food and water ad libitum. All animal care and procedures were in accordance with institutional policies for animal health and well-being and approved by Montana State University and University of Florida Institutional Animal Care and Use Committees. Mice (5–8 weeks of age) were vaccinated via nasal drip on day 0 with 5 μg CT (List Biological Laboratories, Campbell, CA, USA) in 10 μL sterile phosphate-buffered saline, and boosted with 2.5 μg CT on days 7 and 14. Saphenous blood and fresh fecal pellets were collected from individual mice and processed.¹⁵

Isolation of mucosal inductive and effector tissue lymphocytes

Parotid gland LNs, SMLNs (also known as superficial cervical LNs)^6,15 and cervical LNs^6,15 comprise the HNLNs. Isolated MLNs, PPs, spleens and NALTs from immunized β7^+/+ and β7^−/− mice were dounce homogenized and subjected to Lympholyte-M (Accurate Chemical and Scientific Corp., Westbury, NY, USA) density gradient centrifugation.^5,6,14–16 Nitex (Fairview Fabrics, Hercules, CA, USA) filtered single-cell suspensions were washed and resuspended in a complete medium [Roswell Park Memorial Institute medium-1640 + 10% fetal bovine serum (HyClone, Logan, UT, USA), and supplements (Thermo-Fisher Scientific, Waltham, MA, USA) 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate and 10 U/mL penicillin/streptomycin], or in a fluorescence-activated cell-sorting buffer.

Nasal passages devoid of NALT and small intestines devoid of PPs and isolated lymphoid follicles were isolated and prepared as previously described.^5,6,14–16 Submandibular glands were removed, minced into 1–2 cm pieces and then digested with 200 U/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) solution containing 0.08 U/mL DNase (Sigma-Aldrich) under vigorous magnetic stirring for 45 min at 37°C. Nitex-filtered single-cell suspensions were washed in complete medium, and subjected to Ficoll gradient centrifugation.¹⁵ Lymphocytes were removed from the interface layer, washed and resuspended in complete medium or in fluorescence-activated cell-sorting buffer.

For IgA and IgG B-cell ELISPOT, integrin⁺ B cells from CT-immunized B6 and β7^−/− mice were isolated by cell sorting similar to that previously described.¹⁶ B cells were negatively presorted by magnetic bead separation using a Pan B-Cell Isolation Kit (STEMCELL Technologies, Vancouver, BC, Canada). B cells were then stained for αE using the anti-CD103 mAb (clone 2E7), for L-selectin with clone MEL-14 mAb, for β1 with anti-CD29 mAb (clone HM β1-1) and for β2 with anti-CD18 mAb (clone M18/2); all mAbs from BioLegend (San Diego, CA USA). Each sorted B-cell subset achieved ≥95% purity.

Mucosal secretion collections

Fecal pellets (100 mg) from individual mice were freshly collected, and solubilized in 1.0 mL sterile phosphate-buffered saline with 50 μg/mL soybean trypsin inhibitor (Sigma-Aldrich) by continuous vortexing for 30 min at 4°C. Following microcentrifugation, supernatants were collected and then frozen until assayed.^14–16 Nasal washes were collected by intubation of the trachea to access the nasopharyngeal cavity. A 1-in. (2.54 cm) long Tygon tubing [internal diameter 0.010 in. (0.0254 cm), outside diameter 0.030 in. (0.0762 cm); Cole-Parmer; Vernon Hills, IL, USA] was attached to a 1-mL syringe, an approach used to avoid any blood contamination of the nasal washes. A total of 200 μL phosphate-buffered saline was administered via the trachea, and the exudate from the nares was collected into microfuge tubes. Following microcentrifugation, supernatants were collected, and then frozen until assayed.¹⁶

Anti-CT-B ELISA and B-cell ELISPOT

Sera were collected from individual mice as previously described,^14,15 and frozen until assayed. Individual serum and mucosal samples were assayed in duplicate using IgA or IgG (H-chain specific) CT-B-specific ELISA and B-cell ELISPOT similar to those previously described.^14–16 For CT-B ELISA and ELISPOT, CT-B (List Biological Laboratories) or unconjugated goat antimouse IgA and IgG Abs (Southern Biotechnology Associates, Birmingham, AL, USA) were used as coating Ags. Horseradish peroxidase-conjugated goat antimouse IgA and IgG Abs (Southern Biotechnology Associates) were used as secondary (detecting) Abs. For ELISAs, soluble 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (Moss, Pasadena, CA, USA) was used; for B-cell ELISPOT, precipitable 3-amino-9-ethylcarbazole (Moss) substrate was used.

Cell surface staining

To measure homing receptor levels, a multicolor analysis was performed using fluorochrome-conjugated mAbs to B cells and α- and β-integrins. Abs for staining B lymphocytes were obtained from BD Biosciences (San Diego, CA, USA). Rat antimouse B220 (RA3-6B2), rat antimouse CD19 (clone 1D3), rat antimouse L-selectin (MEL 14), anti-CD103 (α_E integrin; M290), rat anti-β₇ (FIB504), rat antimouse α₄ β7 (DATK 32), hamster antirat α1 (Ha2/5) and rat antimouse β2 (C71/16), and rat antimouse α₄ (R1-2) were from BioLegend. Flow cytometry analysis was performed using a FACSCanto (BD Biosciences), and 10 000 events/sample were collected.

Statistical analysis

One-way ANOVA was performed to analyze endpoint titers, B-cell ELISPOT and flow cytometry results. Data were considered statistically significant if the P-value was <0.05.