Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-α (LTα). However, the structure and function of NALT are impaired in Ltα−/− mice, suggesting a link between LTα and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltα−/− mice. We also show that the NALT of Cxcl13−/− and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltα−/− mice. Like the NALT of Ltα−/− mice, the NALT in Cxcl13−/− mice lacks follicular dendritic cells, BP3+ stromal cells, and ERTR7+ lymphoreticular cells. However, unlike the NALT of Ltα−/− mice, the NALT of Cxcl13−/− mice has peripheral node addressin+ high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3+ stromal cells, ERTR7+ lymphoreticular cells, and peripheral node addressin+ HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltα−/− and Cxcl13−/− mice. In contrast, CD8 T cell responses to influenza are impaired in Ltα−/− mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltα−/− mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltα−/− mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTα.

Like other mucosal lymphoid tissues, nasal-associated lymphoid tissue (NALT),3 is specialized to provide immunity to Ags that contact the mucosal surface (1). Thus, unlike lymph nodes, NALT is not encapsulated and is not supplied by afferent lymphatics (1). Instead, NALT has a specialized dome epithelium, with membranous cells (M cells) that transport Ag from the lumenal surface of the epithelium to the APCs directly underneath (2). Membranous cells also allow the transport of bacteria or viruses into NALT (2, 3), where immune responses to these pathogens are initiated (4). In response to stimulation by pathogens or Ags, NALT facilitates B cell differentiation and isotype switching to IgA (5), which is efficiently transported across the epithelium to the mucosal surface (6). CD4 responses to group A streptococcus are induced in NALT (4) as are CD8 responses to respiratory viruses, such as influenza (7). Thus, the NALT of mice serves as an important mucosal inductive site, similar in function to the palatine tonsils in humans.

Despite the structural and functional differences between encapsulated lymph nodes and NALT, all secondary lymphoid organs share a related architecture, with separated B and T cell areas and specialized populations of stromal cells (8). The architecture of lymphoid organs is maintained by homeostatic chemokines, which are constitutively expressed and direct the recruitment and placement of lymphocytes and dendritic cells (DCs) (9). CXCL13 (B lymphocyte chemoattractant) is expressed in B cell follicles and attracts CXCR5+ B cells (10) and activated CXCR5+ T cells (11). CXCL13 is also expressed by some high endothelial venules (HEVs) and helps to recruit CXCR5-expressing cells into lymphoid organs (12). In contrast, CCL21 (secondary lymphoid organ chemokine) and CCL19 (EB1 ligand chemokine) are expressed in the T cell areas of secondary lymphoid organs and are primarily responsible for the recruitment of naive CCR7+ T cells and activated CCR7+ APCs to these areas (13, 14). CCL21 is also strongly expressed on HEVs and is instrumental for the recruitment of T cells from blood into lymph nodes (15). Together, the homeostatic chemokines organize the B and T cell areas of secondary lymphoid organs and help to orchestrate the recruitment and interaction of lymphocytes and APCs.

The constitutive expression of homeostatic chemokines in spleen is dependent on the lymphotoxin-α (LTα) signaling pathway (16, 17). As a result, the production of CXCL13, CCL21, and CCL19 is reduced in the spleens of Ltα−/− mice (16), which prevents the formation of B cell follicles, germinal centers, marginal zones, and follicular DCs (FDCs) and disrupts the proper segregation of B and T cell areas (18, 19). In addition, Ltα−/− mice lack lymph nodes and Peyer’s patches due to an inability of lymphoid tissue inducer cells to trigger the differentiation of mesenchymal cells into mature stromal cells during embryogenesis (20, 21). Homeostatic chemokines are also required to recruit lymphoid tissue inducer cells to sites of lymph node development (22, 23). Thus, the interplay between LT signaling and the expression of homeostatic chemokines controls the development of secondary lymphoid organs as well as their organization and function.

A notable exception to this model is the development of NALT (7, 24). Surprisingly, NALT develops in the absence of the cytokines, LTα, LTβ, and TNF-α; the receptors TNFR1 and LTβR; and the signaling molecule NF-κB-inducing kinase (7, 24), demonstrating that neither TNF nor LT signaling pathways are required for NALT development. Despite the fact that NALT development is initiated in the absence of LT signaling, the structure of NALT is severely compromised in the absence of LTα (7), suggesting that the loss of this signaling pathway leads to impaired chemokine expression in NALT (25). The function of NALT is also impaired in Ltα−/− mice, because it is unable to support germinal centers or promote the priming and expansion of influenza-specific CD8 T cells (7). However, it is unclear whether these functional defects are related to the loss of LTα-induced chemokine expression or to the loss of LTα itself.

In this study we tested whether the structural and functional defects in the NALT of Ltα−/− mice are related to impaired chemokine expression or to the loss of LTα. We found that the expression of CXCL13 and CCL19, but not that of CCL21, is highly impaired in the NALT of Ltα−/− mice. We also found that stromal cell defects in the NALT of Ltα−/− mice are related to the lack of LTα-induced CXCL13 expression, whereas defects in the development of peripheral node addressin (PNAd)-expressing HEVs are related primarily to the loss of LTα. Furthermore, we found that the NALT of Cxcl13−/− and Ltα−/− mice does not support germinal center formation or promote isotype switching to IgA, even though IgA+ B cells accumulate in the nasal mucosa of these mice. In contrast, although CD8 T cell responses are impaired in the NALT of Ltα−/− and plt/plt mice, they are normal in the NALT of Cxcl13−/− mice. Thus, many of the structural and functional defects in the NALT of Ltα−/− mice are due to the impaired expression of homeostatic chemokines, whereas impaired HEV development is directly due to the loss of LTα.

C57BL/6 and Ltα−/− mice were obtained from The Jackson Laboratory. Cxcl13−/− and plt/plt mice were obtained from Dr. J. Cyster (University of California, San Francisco, CA). All gene-targeted mice were on the C57BL/6 genetic background and were bred at the animal breeding facility of Trudeau Institute. All procedures using animals were approved by the Trudeau Institute Institutional Animal Care and Use Committee and were conducted according to the principles outlined by the National Research Council.

For histochemical staining of NALT, heads of euthanized mice were fixed in neutral buffered formalin for 24 h. The skull was decalcified in 7% EDTA in PBS for several days, then embedded in paraffin, sectioned, and stained with H&E. For immunofluorescent analysis of NALT, the heads of euthanized animals remained unfixed and were decalcified in several changes of 150 mM EDTA in PBS for 5–7 days at 4°C on a rocking platform, embedded in OCT medium (Sakura Finetek), and frozen above liquid nitrogen before sectioning. For analysis by flow cytometry, NALT was removed from the nasal cavity by cutting along the inside edges of the upper molars with a scalpel and then peeling the tissue away from the roof of the mouth. Cells from the NALT were scraped from the dorsal side of the soft palette, and single-cell suspensions were used for flow cytometric analysis. Cells from the nasal mucosa were prepared for ELISPOT analysis by first removing the skin, lower jaw, and brain from the head. The nose was removed from the incisors forward, as was any muscle. Finally the region from behind the incisors to the posterior end of the soft palette, including a large area of nasal mucosa and NALT, was mechanically disrupted through a metal screen. Viable cells were isolated by density centrifugation.

Biotinylated anti-B220, anti-CD3, anti-CD11c, and anti-CD21/CD35 Abs were purchased from BD Pharmingen. Biotinylated Abs were detected with strepavidin conjugated to either Alexa 488 or Alexa 594. Purified anti-PNAd was a gift from Dr. R. Mebius (Vrije Universiteit Medical Center, Amsterdam, The Netherlands). Anti-PNAd was detected with rabbit anti-rat IgM. Polyclonal anti-CXCL13, anti-CCL21, and anti-CCL20 Abs (R&D Systems) were detected using Alexa 594-conjugated donkey anti-goat IgG. Anti-BP3 (Research Diagnostics) was biotinylated using NHS-LC-biotin (Pierce). Anti-ERTR7 was obtained from Acris Antibodies. The ERTR7 Ab was detected using Alexa 488-conjugated donkey anti-rat IgG. All slides were viewed with a Zeiss Axioplan 2 microscope. Images were recorded with a Zeiss AxioCam digital camera. Images were cropped and rotated in Adobe Photoshop 7.0 and saved as TIFF files.

Mice were killed at 8 wk of age, and NALT was dissected with the help of a stereoscopic microscope. Total RNA was extracted using the RNeasy kit (Qiagen). DNase-treated RNA was reverse transcribed with oligo(dT) and SuperScript II (Invitrogen Life Technologies). Quantitative PCR was performed using TaqMan Universal PCR Master Mix, following the Applied Biosystems protocol. Primers and probes for GAPDH, CXCL12, CXCL13, CCL19, CCL20, LTβ, and activation-induced cytidine deaminase (AID) were obtained from Applied Biosystems. Primers for CCL21 (5′-AGACTCAGGAGCCCAAAGCA-3′ and 5′GTTGAAGCAGGGCAAGGGT-3′) were synthesized by IDT, and the probe for CCL21 (5′FAM-CCACCTCATGCTGGCCTCCGT-BHQ-3′) was synthesized by Biosearch Technology. Quantitative PCRs were performed using a PRISM 7700 instrument from Applied Biosystems available through the Molecular Biology Core Facility at Trudeau Institute. Standard PCRs were performed with 50 ng of cDNA using the primers 5′-CTCTGGCCCTGCTTATTGTTG-3′ and 5′-GAGCTGGTGGGAGTGTCAGTG-3′ to amplify Iμ-Cα transcripts and the primers 5′-GGCGGAAACCCAGAGGCATTGACA-3′ and 5′-GTAGCCTGGCGTTGGGATTGGTGACTC-3′ to amplify RPL32. Conditions for the amplification included a denaturing step of 95°C for 5 min, followed by 35 PCR cycles (94°C for 30 s, 58°C for 1 min, 72°C for 1 min). At the end, an extension step of 5 min at 72°C was added to the amplification reaction. PCR products were resolved on 1% agarose gels.

Mice were infected intranasally with 100 egg infectious units of A/PR8/34 influenza and were killed 10 days after infection. Cells from the NALT were isolated and analyzed by flow cytometry. Viral titers in the nasal mucosa were determined by homogenizing the excised tissue in 2.5 ml of PBS and inoculating embryonated chicken eggs with 100 μl of 10-fold serial dilutions of the homogenate. Allantoic fluid was harvested from inoculated eggs 4 days later, and infected eggs were scored by hemagglutination of chicken RBC. The viral end-point titer was defined as the highest dilution in which two or more eggs scored positively in the hemagglutination assay.

Cells from NALT were incubated in 3% FCS in PBS containing 10 μg/ml 2.4G2 to block FcR binding, followed by the addition of fluorochrome-conjugated Abs or MHC class I tetramers. Fluorochrome-conjugated Abs were obtained from BD Biosciences. Peanut agglutinin (PNA) was obtained from Vector Laboratories. The H-2Db class I tetramers containing either the nucleoprotein366–374 peptide (NP) or the acidic polymerase224–236 peptide (PA) were generated by the Trudeau Institute Molecular Biology Core Facility. Flow cytometry was performed on a dual-laser FACSCalibur available through the Flow Cytometry Core Facility at the Trudeau Institute.

Multiscreen 96-well plates (Millipore) were coated overnight with 1 μg/ml protein from purified, disrupted influenza virus in 50 mM NaHCO3 pH 9.5. Plates were blocked with complete RPMI 1640 medium containing 10% FCS. Cells from NALT were cultured in 3-fold serial dilutions for 6 h. The plates were washed in PBS with 0.1% Tween 20 and incubated overnight with alkaline phosphatase-conjugated goat anti-mouse IgG or anti-IgA (Southern Biotechnology Associates). The plates were washed again and developed with 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich) and NBT (Sigma-Aldrich). Spots were counted under a dissecting microscope.

Because LTα controls the splenic expression of the chemokines, CXCL13, CCL19, and CCL21 (16), we hypothesized that the organizational and functional defects in the NALT of Ltα−/− mice were due to impaired expression of these molecules. To test this hypothesis, we first used histology to examine the structure of NALT in normal C57BL/6 mice as well as in mice lacking LTα (Ltα−/− mice), mice lacking CCL19 and CCL21 (plt/plt mice), and mice lacking CXCL13 (Cxcl13−/− mice). As shown in Fig. 1,A, the NALT of C57BL/6 mice formed large lymphoid structures on either side of the nasal passages. In contrast, the NALT of Ltα−/− mice was underdeveloped and lymphopenic (Fig. 1,B), consistent with previous reports (7, 24). The NALT of plt/plt mice was larger than that of Ltα−/− mice and had more lymphocytes (Fig. 1,C), whereas the NALT of Cxcl13−/− mice was similar to that of Ltα−/− mice (Fig. 1,D). We also found that the NALT of plt/plt × Cxcl13−/− mice was structurally similar to that of Ltα−/− mice (Fig. 1 E). Thus, the loss of CCL21/CCL19 and CXCL13 did not prevent NALT development, but did appear to impact the structure of NALT.

FIGURE 1.

LTα and the homeostatic chemokines control the structure of NALT. Paraffin sections of NALT from C57BL/6 (A), Ltα−/− (B), plt/plt (C), Cxcl13−/− (D), or plt/plt × Cxcl13−/− mice (E) were stained with H&E. Images were obtained using a ×2.5 objective. The data are representative of images obtained from at least three individual mice.

FIGURE 1.

LTα and the homeostatic chemokines control the structure of NALT. Paraffin sections of NALT from C57BL/6 (A), Ltα−/− (B), plt/plt (C), Cxcl13−/− (D), or plt/plt × Cxcl13−/− mice (E) were stained with H&E. Images were obtained using a ×2.5 objective. The data are representative of images obtained from at least three individual mice.

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We next used immunofluorescence to examine whether the loss of LTα impaired chemokine expression in NALT. As shown in Fig. 2, A–C, the NALT of C57BL/6 mice expressed CXCL13 in a reticular pattern in the center of the B cell follicle (Fig. 2,A), whereas CCL21 was expressed on reticular cells in the corners of NALT surrounding the follicle and was also expressed on vascular endothelial cells (Fig. 2,B). The chemokine CCL20 was expressed in the dome epithelium of NALT (Fig. 2,C, arrows), similar to its placement in the dome epithelium of Peyer’s patches (26). In contrast, the NALT of Ltα−/− mice (Fig. 2, D–F) did not express CXCL13 at levels detectable by immunofluorescence (Fig. 2,D) and CCL21 expression was reduced relative to that in normal mice, particularly on reticular cells around the edge of the follicle (Fig. 2,E). The expression of CCL20 in the dome epithelium was also reduced to near-background levels (Fig. 2 F). These data demonstrate that LTα is important for the proper expression of chemokines in NALT.

FIGURE 2.

Impaired chemokine expression in the NALT of Ltα−/− mice. Frozen sections of NALT from C57BL/6 mice (A–C), Ltα−/− mice (D–F), plt/plt mice (G–I), and Cxcl13−/− mice (J–L) were probed with Abs to CXCL13 (A, D, G, and J), CCL21 (B, E, H, and K), and CCL20 (C, F, I, and L). Sections were counterstained with 4′,6-diamido-2-phenylindole hydrochloride (DAPI). Arrows in A, I, and L indicate epithelial expression of CCL20. All images were obtained using a ×10 objective, except for the image in F, which was originally obtained using a ×20 objective. The data are representative of images obtained from at least three individual mice. M, Expression of mRNAs for chemokines and TNF family members was measured by quantitative PCR. The expression of each molecule was first normalized to the expression of GAPDH and was then normalized to the average expression of that mRNA in C57BL/6 mice. RNA was extracted from five mice per group, and gene expression was examined in individual samples.

FIGURE 2.

Impaired chemokine expression in the NALT of Ltα−/− mice. Frozen sections of NALT from C57BL/6 mice (A–C), Ltα−/− mice (D–F), plt/plt mice (G–I), and Cxcl13−/− mice (J–L) were probed with Abs to CXCL13 (A, D, G, and J), CCL21 (B, E, H, and K), and CCL20 (C, F, I, and L). Sections were counterstained with 4′,6-diamido-2-phenylindole hydrochloride (DAPI). Arrows in A, I, and L indicate epithelial expression of CCL20. All images were obtained using a ×10 objective, except for the image in F, which was originally obtained using a ×20 objective. The data are representative of images obtained from at least three individual mice. M, Expression of mRNAs for chemokines and TNF family members was measured by quantitative PCR. The expression of each molecule was first normalized to the expression of GAPDH and was then normalized to the average expression of that mRNA in C57BL/6 mice. RNA was extracted from five mice per group, and gene expression was examined in individual samples.

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We next tested whether the plt/plt mutation and the loss of CXCL13 lead to similar defects in chemokine expression in NALT. As shown in Fig. 2,G, CXCL13 was expressed at nearly normal levels in the NALT of plt/plt mice, whereas CCL21 was not detected (Fig. 2,H). In addition, the expression of CCL20 in the dome epithelium appeared nearly normal (Fig. 2,I). As expected, CXCL13 was not detected in Cxcl13−/− mice (Fig. 2,J). In contrast, CCL21 was highly expressed in Cxcl13−/− mice and was found at high levels in vascular endothelium and at lower levels in reticular cells (Fig. 2,K). Finally, the levels of CCL20 appeared nearly normal in the NALT of Cxcl13−/− mice (Fig. 2 L). Thus, the loss of CXCL13 alone and the mutation of the CCL21/CCL19 locus appeared to recapitulate some (but not all) of the defects in chemokine expression observed in the NALT of Ltα−/− mice.

Because immunofluorescence was not sensitive enough to consistently detect CCL19 or CXCL12 (not shown), particularly after the decalcification of NALT, we also examined the expression of homeostatic chemokines using quantitative PCR. As shown in Fig. 2 M, the expression of CCL19, CXCL13, and CCL20 was substantially reduced in the NALT of Ltα−/− mice, whereas the expression of CCL21 and CXCL12 was not impaired. As expected, the expression of CCL19 and CCL21 was impaired in the NALT of plt/plt mice, whereas the expressions of CXCL13, CXCL12, and CCL20 was essentially normal or slightly reduced (CCL20). Unexpectedly, the expression of CCL19 was considerably reduced in the NALT of Cxcl13−/− mice, even though CCL21 expression was normal. As expected, CXCL13 was not expressed in Cxcl13−/− mice. Interestingly, the expression of CXCL13 correlated with the expression of LTβ and TNF-α, which were reduced in Ltα−/− mice and in Cxcl13−/− mice. In contrast, another TNF family member, LIGHT, was expressed at similar levels in the NALT of all groups (27). Thus, the relative production of chemokine mRNAs correlated with their staining patterns detected by immunofluorescence. In addition, the expression of CXCL13 appears to be codependent on the expression of LTαβ and TNF-α.

To determine whether CXCL13 or CCL21/CCL19 played a role in the differentiation of stromal cells in NALT, we examined the expression of BP3, CD21, and ERTR7. As shown in Fig. 3, A–D, we observed an extensive network of BP3-expressing stromal cells in the NALT of C57BL/6 and plt/plt mice (Fig. 3, A and C), but only a few BP3-expressing fibroblast-like cells were observed in the NALT of Ltα−/− and Cxcl13−/− mice (Fig. 3, B and D). Similarly, the NALT of C57BL/6 and plt/plt mice contained a central B cell follicle with CD21-expressing FDCs (Fig. 3, E and G), whereas CD21-expressing FDCs were never observed in the NALT of Ltα−/− or Cxcl13−/− mice (Fig. 3, F and H). Finally, although fibroblast-like cells expressing ERTR7 were observed surrounding the edge of NALT in all mice (Fig. 3, I–L), reticular networks of ERTR7+ cells (yellow) were only observed in the lymphoid areas of NALT from C57BL/6 and plt/plt mice (Fig. 3, I and K). Thus, the development of stromal-type cells in NALT was impaired in the absence of LTα and CXCL13, but not in the absence of CCR7 ligands.

FIGURE 3.

LTα and CXCL13 are required for the differentiation of stromal cells in NALT. Frozen sections of NALT from C57BL/6 mice (A, E, and I), Ltα−/− mice (B, F, and J), plt/plt mice (C, G, and K), and Cxcl13−/− mice (D, H, and L) were probed with Abs to BP3 (A–D), CD21 and CD11c (E–H), and ERTR7 (I–L). Some sections were counterstained with 4′,6-diamido-2-phenylindole hydrochloride (DAPI) (blue). All images were obtained using a ×10 objective. The data are representative of images obtained from at least three individual mice.

FIGURE 3.

LTα and CXCL13 are required for the differentiation of stromal cells in NALT. Frozen sections of NALT from C57BL/6 mice (A, E, and I), Ltα−/− mice (B, F, and J), plt/plt mice (C, G, and K), and Cxcl13−/− mice (D, H, and L) were probed with Abs to BP3 (A–D), CD21 and CD11c (E–H), and ERTR7 (I–L). Some sections were counterstained with 4′,6-diamido-2-phenylindole hydrochloride (DAPI) (blue). All images were obtained using a ×10 objective. The data are representative of images obtained from at least three individual mice.

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We also examined the role of LTα and chemokines in the differentiation of PNAd-expressing HEVs. As shown in Fig. 4, A and B, well-developed HEVs that express PNAd were easily detected in the NALT of C57BL/6 mice. In contrast, PNAd-expressing vascular endothelial cells were rarely detected in the NALT of Ltα−/− mice (Fig. 4, C and D), and Fig. 4,D is the only example in which PNAd expression was observed. Although PNAd-expressing HEVs were observed in both plt/plt mice (Fig. 4, E and F) and in Cxcl13−/− mice (Fig. 4, G and H), the HEVs appeared to be less well developed. However, PNAd expression on HEVs was still observed in the NALT of plt/plt × Cxcl13−/− mice (Fig. 4 I). Thus, although the loss of CCL21/CCL19 and CXCL13 minimally impaired HEV development, the loss of LTα had a substantial impact and almost entirely prevented the development of HEVs that expressed PNAd, consistent with previous results (25).

FIGURE 4.

LTα is required for the differentiation of PNAd-expressing high endothelial venules. Frozen sections of NALT from C57BL/6 mice (A and B), Ltα−/− mice (C and D), plt/plt mice (E and F), Cxcl13−/− mice (G and H), and plt/plt × Cxcl13−/− mice (I) were probed with Abs to PNAd. Sections in B, D, F, H, and I were counterstained with 4′,6-diamido-2-phenylindole hydrochloride (DAPI) (blue). All images were obtained using a ×10 objective. The data are representative of images obtained from at least three individual mice.

FIGURE 4.

LTα is required for the differentiation of PNAd-expressing high endothelial venules. Frozen sections of NALT from C57BL/6 mice (A and B), Ltα−/− mice (C and D), plt/plt mice (E and F), Cxcl13−/− mice (G and H), and plt/plt × Cxcl13−/− mice (I) were probed with Abs to PNAd. Sections in B, D, F, H, and I were counterstained with 4′,6-diamido-2-phenylindole hydrochloride (DAPI) (blue). All images were obtained using a ×10 objective. The data are representative of images obtained from at least three individual mice.

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The loss of FDCs and BP3-expressing stromal cells in the NALT of Ltα−/− and Cxcl13−/− mice suggested that the formation of B cell follicles and the ability of B cells to respond to Ag would be impaired in the NALT of these mice. As shown in Fig. 5,A, the frequency of CD19+ B cells was reduced in the NALT of Ltα−/− and Cxcl13−/− mice compared with that in the NALT of C57BL/6 mice, but was normal in the NALT of plt/plt mice. The reduced frequency of B cells in Ltα−/− and Cxcl13−/− mice was observed in all B cell populations, but was most pronounced in CD19+IgD+CXCR5+ mature resting B cells (Fig. 5,B). These differences were reflected in the absolute numbers of CD19+ B cells and CD19+IgD+CXCR5+ B cells (Fig. 5, C and D) and suggested that CXCL13 is important for the recruitment or retention of mature follicular B cells in NALT.

FIGURE 5.

B cell responses are impaired in Ltα−/− and Cxcl13−/− mice. Naive mice (four or five mice per group) were killed, and the NALT was removed and analyzed by flow cytometry. A, B cells were identified using Abs to CD19. The numbers in each panel refer to the percentage and SD of CD19+ cells in the total cell population. B, Follicular B cells were identified using Abs to CD19, CXCR5, and IgD. The cells shown were gated on CD19 expression. The numbers in each panel refer to the percentage and SD of CXCR5+IgD+ cells within the B cell population. C, The total number of CD19+ B cells gated in A is shown. Significance was determined by unpaired t test (ns, not significant). D, The total number of CXCR5+IgD+ follicular B cells gated as described in B is shown. Significance was determined by unpaired t test. The data are representative of five independent experiments. E, Mice were infected with influenza, and germinal center B cells in NALT were identified by the expression of Fas and the binding of PNA on day 10 after infection. The plots shown were gated on CD19+ cells. The numbers in each panel refer to the percentage and SD of PNA+Fas+ cells in the B cell population. F, Mice were infected with influenza, and IgA-expressing B cells in NALT were evaluated on day 14 after infection. The numbers in each panel refer to the percentage of B cells that express IgA. Cells from five NALTs were pooled for this analysis. G, The total number of PNA+Fas+CD19+ germinal center B cells in NALT. Differences were not significant by unpaired t test. H, The total number of IgA+ B cells in NALT. I and J, Mice were infected with influenza, and influenza-specific IgA-secreting cells from the nasal mucosa were analyzed by ELISPOT on day 10 (I) and day 14 (J) after infection. Cells from five NALTs were pooled for this assay. The data are representative of three independent experiments. K, Mice were infected with influenza, and mRNA levels of AID on day 14 after infection were evaluated by quantitative PCR. The expression of AID was first normalized to the expression of GAPDH and was then normalized to the average expression of AID mRNA in C57BL/6 mice. RNA was extracted from five mice per group. Significance was determined by unpaired t test. L, Mice were infected with influenza, and mRNA levels for Iμ-Cα and the housekeeping gene RPL32 were evaluated by PCR in NALT on day 14 after infection.

FIGURE 5.

B cell responses are impaired in Ltα−/− and Cxcl13−/− mice. Naive mice (four or five mice per group) were killed, and the NALT was removed and analyzed by flow cytometry. A, B cells were identified using Abs to CD19. The numbers in each panel refer to the percentage and SD of CD19+ cells in the total cell population. B, Follicular B cells were identified using Abs to CD19, CXCR5, and IgD. The cells shown were gated on CD19 expression. The numbers in each panel refer to the percentage and SD of CXCR5+IgD+ cells within the B cell population. C, The total number of CD19+ B cells gated in A is shown. Significance was determined by unpaired t test (ns, not significant). D, The total number of CXCR5+IgD+ follicular B cells gated as described in B is shown. Significance was determined by unpaired t test. The data are representative of five independent experiments. E, Mice were infected with influenza, and germinal center B cells in NALT were identified by the expression of Fas and the binding of PNA on day 10 after infection. The plots shown were gated on CD19+ cells. The numbers in each panel refer to the percentage and SD of PNA+Fas+ cells in the B cell population. F, Mice were infected with influenza, and IgA-expressing B cells in NALT were evaluated on day 14 after infection. The numbers in each panel refer to the percentage of B cells that express IgA. Cells from five NALTs were pooled for this analysis. G, The total number of PNA+Fas+CD19+ germinal center B cells in NALT. Differences were not significant by unpaired t test. H, The total number of IgA+ B cells in NALT. I and J, Mice were infected with influenza, and influenza-specific IgA-secreting cells from the nasal mucosa were analyzed by ELISPOT on day 10 (I) and day 14 (J) after infection. Cells from five NALTs were pooled for this assay. The data are representative of three independent experiments. K, Mice were infected with influenza, and mRNA levels of AID on day 14 after infection were evaluated by quantitative PCR. The expression of AID was first normalized to the expression of GAPDH and was then normalized to the average expression of AID mRNA in C57BL/6 mice. RNA was extracted from five mice per group. Significance was determined by unpaired t test. L, Mice were infected with influenza, and mRNA levels for Iμ-Cα and the housekeeping gene RPL32 were evaluated by PCR in NALT on day 14 after infection.

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To test whether CXCL13 was also required for B cell responses in NALT, we intranasally infected mice with influenza and assayed the frequency of germinal center B cells on day 10 after infection. As shown in Fig. 5,E, germinal center B cells were not observed in the NALT of influenza-infected Ltα−/− or Cxcl13−/− mice, but were observed at normal frequency in the NALT of plt/plt mice after influenza infection. Similar results were observed on days 14 and 21 after infection. The presence of germinal centers was confirmed by immunofluorescence (not shown). We also tested whether CXCL13 expression in NALT was required for B cells to switch to IgA and differentiate into IgA-secreting cells. As shown in Fig. 5,F, IgA-expressing B cells were observed in the NALT of all groups by day 14 after infection. Surprisingly, the frequency of B cells that expressed IgA was highest in the NALT of Ltα−/− mice (Fig. 5,F), although this was not reflected in the total number of IgA+ B cells (Fig. 5,H) due to the reduced frequency of B cells and the smaller size of NALT in Ltα−/− mice. However, many of these IgA+ B cells were specific for influenza, because we observed the highest number of IgA-secreting, influenza-specific B cells in the nasal mucosa of Ltα−/− mice on days 10 and 14 after infection (Fig. 5, I and J). Interestingly, we observed that the mRNA levels of AID were profoundly reduced in the NALT of Ltα−/− and Cxcl13−/− mice and were also reduced in the NALT of plt/plt mice (Fig. 5,K). Similarly, the levels of Iμ-Cα mRNA transcripts were reduced in the NALT of Ltα−/−, plt/plt, and Cxcl13−/− mice (Fig. 5 L). Because AID is required for isotype switching and because the presence of Iμ-Cα transcripts is a hallmark of recently switched B cells, it appears that isotype switching to IgA is impaired in the NALT of mutant mice and that switching must occur in other locations. Thus, both germinal center formation and switching to IgA in NALT are dependent on LTα and CXCL13.

The poor CCL19 expression in Ltα−/− mice, plt/plt mice, and Cxcl13−/− mice suggested that T cell recruitment and T cell responses may be impaired in these mice. To test this possibility, we first determined the number of T cells in the NALT of the various mouse strains. As shown in Fig. 6,A, CD3+ T cells make up only a minority of cells in the NALT in all mice tested. Surprisingly, the number of T cells in the NALT of Ltα−/− and plt/plt mice was similar to that in C57BL/6 mice, whereas the number of T cells in the NALT of Cxcl13−/− mice was substantially reduced (Fig. 6,C). These observations were consistent between multiple experiments and suggested that CCL21/CCL19 were not instrumental for the maintenance of normal steady-state levels of T cells in NALT. These results were also surprising, because the plt mutation (paucity of lymph node T cells) leads to extremely poor T cell recruitment to the lymph nodes due to lack of CCL21 expression on HEVs of lymph nodes (15). Interestingly, although reductions in the number of T cells in the NALT of Cxcl13−/− mice were observed in both CD4 and CD8 populations, the CD4 population was more severely affected. For example, CD4 cells made up 76 ± 6.2% of T cells in C57BL/6 NALT, 83 ± 4.1% of T cells in Ltα−/− NALT, and 85 ± 1.6% of T cells in plt/plt NALT, but only 60 ± 9.1% of T cells in Cxcl13−/− NALT. The selective loss of CD4 cells in the NALT of Cxcl13−/− mice suggested that CD4 T cells in NALT were primarily CXCR5+ follicular helper cells (11). However, as shown in Fig. 6, B and D, only a minority of the T cells in NALT expressed CXCR5, and this population was maintained in the NALT at a similar frequency in all groups of mice. Thus, although the number of CXCR5+ T cells was reduced in the NALT of Cxcl13−/− mice (Fig. 6 D), the reduced numbers of total T cells in the NALT of Cxcl13−/− mice was not due to the selective loss of CXCR5+ T cells.

FIGURE 6.

T cell responses are impaired in the NALT of Ltα−/− mice. A, T cells in the NALT of naive mice were identified using Abs to CD3. The numbers in each panel refer to the percentage and SD of CD3+ cells in the total population. B, Follicular Th cells were identified using Abs to CD3 and CXCR5. The numbers in each panel refer to the percentage and SD of CXCR5+ cells within the T cell population. C, The total number of T cells gated as described in A is shown. Significance was evaluated by an unpaired t test (ns, not significant). D, The total number of CXCR5+CD3+ T cells is shown. Significance was evaluated by unpaired t test. The data are representative of five independent experiments. E–G, Mice were infected with influenza, and virus-specific CD8 T cells in NALT were enumerated by flow cytometry on day 10 after infection. E, Influenza-specific CD8 T cells in NALT were identified by tetramer binding. The numbers in each panel refer to the percentage of tetramer-binding cells within the CD8 T cell population. F, The total number of H2-DbNP366–374 and H2-DbPA224–233 CD8 T cells in NALT is shown. Significance was evaluated by an unpaired t test. G, The total number of CD8+ T cells is shown. No significant differences were found by unpaired t test. The data are representative of two independent experiments using four or five mice per group. H, Mice were infected with influenza, and cells from the NALT and spleen were pooled from 18–20 mice/group on day 10 after infection. Influenza-specific CD8 T cells were identified by tetramer binding. The plots shown were gated on CD8+ cells. The numbers in each panel refer to the percentage of tetramer-binding cells within the CD8 T cell population.

FIGURE 6.

T cell responses are impaired in the NALT of Ltα−/− mice. A, T cells in the NALT of naive mice were identified using Abs to CD3. The numbers in each panel refer to the percentage and SD of CD3+ cells in the total population. B, Follicular Th cells were identified using Abs to CD3 and CXCR5. The numbers in each panel refer to the percentage and SD of CXCR5+ cells within the T cell population. C, The total number of T cells gated as described in A is shown. Significance was evaluated by an unpaired t test (ns, not significant). D, The total number of CXCR5+CD3+ T cells is shown. Significance was evaluated by unpaired t test. The data are representative of five independent experiments. E–G, Mice were infected with influenza, and virus-specific CD8 T cells in NALT were enumerated by flow cytometry on day 10 after infection. E, Influenza-specific CD8 T cells in NALT were identified by tetramer binding. The numbers in each panel refer to the percentage of tetramer-binding cells within the CD8 T cell population. F, The total number of H2-DbNP366–374 and H2-DbPA224–233 CD8 T cells in NALT is shown. Significance was evaluated by an unpaired t test. G, The total number of CD8+ T cells is shown. No significant differences were found by unpaired t test. The data are representative of two independent experiments using four or five mice per group. H, Mice were infected with influenza, and cells from the NALT and spleen were pooled from 18–20 mice/group on day 10 after infection. Influenza-specific CD8 T cells were identified by tetramer binding. The plots shown were gated on CD8+ cells. The numbers in each panel refer to the percentage of tetramer-binding cells within the CD8 T cell population.

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We also wanted to know whether homeostatic chemokines played a role in the development of influenza-specific CD8 responses in the NALT. To test this possibility, we intranasally infected mice with influenza and assayed the number of CD8 cells in NALT that were responding to influenza NP366–374 and influenza PA224–233 presented in H2Db. As shown in Fig. 6, E and F, both NP-specific and PA-specific CD8 cells were observed in the NALT of wild-type and Cxcl13−/− mice, but were not observed in the NALT of Ltα−/− or plt/plt mice at this time. The reduced frequencies of influenza-specific CD8 cells were reflected in the reduced numbers of influenza-specific CD8 cells in the NALT of Ltα−/− and plt/plt mice (Fig. 6,F). This was not due to a general defect in the recruitment of CD8 cells to NALT, because similar numbers of CD8 cells were found in the NALT of all groups of mice at this time (Fig. 6,G). We also assessed the CTL activity of NP-specific CD8 cells in NALT in an in vitro CTL assay. Given the limited number of cells obtained from NALT, particularly in the mutant mice, we pooled cells from NALT of 18–20 mice/group (shown in Fig. 6,H). However, CTL activity was minimal in all groups, even at an E:T cell ratio of 50:1 (C57BL/6, 8.7%; Ltα−/−, 6.1%; plt/plt, 6.8%; Cxcl13−/−, 9.3%). The nonspecific killing of target cells in the absence of peptide averaged 6.8%. The poor killing activity of cells from NALT is explained by the low frequency of NP-specific CD8 cells in NALT. For example, the NALT of C57BL/6 mice contains 13% T cells, 20% of which are CD8 cells, and only 11% of those are NP specific. Thus, NP-specific CD8 T cell make up only 0.28% of the NALT, resulting in an adjusted E:T cell ratio of 0.14:1. Regardless of the poor CTL activity in NALT, however, it appears that the spleen and NALT of both Ltα−/− and plt/plt mice are impaired in their ability to rapidly generate influenza-specific CD8 T cells (Fig. 6 H). However, in data not shown we found that all groups of mice eventually make CD8 responses, but that influenza-specific CD8 responses are delayed by 1–2 days in the NALT of plt/plt mice and are delayed by an additional day in the NALT of Ltα−/− mice. Thus, the rapid generation of influenza-specific CD8 cells in NALT is dependent on CCL21/CCL19 and LTα, but is independent of CXCL13.

The activities of influenza-specific B and T cells are both responsible for clearing influenza from the airways. To test whether the clearance of influenza was impaired in the upper airways of mice lacking LTα or the homeostatic chemokines, we intranasally infected mice with influenza and determined the viral titers in nasal mucosa on day 10 after infection. As shown in Fig. 7, viral titers were significantly higher in the nasal mucosa of Ltα−/− mice, but were similar in all other groups at this time. In fact, virus was cleared from the nasal mucosa of two of four C57BL/6 mice, two of five plt/plt mice, and two of five Cxcl13−/− mice at this time, whereas virus was still detectable in five of five Ltα−/− mice. Thus, viral clearance is delayed in Ltα−/− mice, but occurs with normal kinetics in mice lacking homeostatic chemokines.

FIGURE 7.

Viral clearance is impaired in Ltα−/− mice. Mice were intranasally infected with influenza, and viral titers in the nasal mucosa were determined on day 10 after infection. There were four mice in the C57BL/6 group and five mice in all other groups. Significance was evaluated using an unpaired t test.

FIGURE 7.

Viral clearance is impaired in Ltα−/− mice. Mice were intranasally infected with influenza, and viral titers in the nasal mucosa were determined on day 10 after infection. There were four mice in the C57BL/6 group and five mice in all other groups. Significance was evaluated using an unpaired t test.

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The data presented in this report are consistent with a model in which LTα is important for the normal expression of homeostatic chemokines in NALT. In turn, homeostatic chemokines, particularly CXCL13, play an essential role in maintaining the lymphoid architecture of NALT. For example, the overall structure of NALT is similarly compromised in the absence of either LTα or CXCL13 (Fig. 1). In addition, both LTα and CXCL13 are necessary for the differentiation of FDCs as well as BP3+ and ERTR7+ stromal cells in NALT (Fig. 3). This is consistent with previous data showing that CXCL13 is necessary for primary follicle formation and FDC differentiation under steady state conditions in the spleen (17). Our data are also consistent with the idea that CXCL13 is involved in a positive feedback loop with LTαβ and TNF-α (17), because the expression of these cytokines is reduced in the NALT of Cxcl13−/− mice (Fig. 2 M). In fact, the stromal cell defects in the NALT of Ltα−/− and Cxcl13−/− mice are most likely due to the interruption of this loop and the failure of LTαβ and TNF-α to promote the differentiation of stromal cell precursors. Thus, the structural defects in the NALT of Ltα−/− and Cxcl13−/− mice appear to be linked by the codependent expression of CXCL13 and LTαβ.

One surprising feature of NALT in Cxcl13−/− mice is the impaired expression of CCL19 (Fig. 2 M). This is similar to the impaired expression of CCL19 in the spleens of B cell-deficient mice (28). However, the NALT of Cxcl13−/− mice clearly has B cells, albeit at reduced numbers, particularly in the follicular B cell compartment. Furthermore, the expression of CCL21 is not impaired in the NALT of Cxcl13−/− mice as it is in the spleens of B cell-deficient mice (28). Interestingly, the NALT of Cxcl13−/− mice also has reduced numbers of T cells. We do not know why this occurs, because it is not a selective defect in CXCR5-expressing T cells and does not appear to be strictly related to the levels of CCL19 or CCL21, because the expression of these molecules is similar to that in Ltα−/− mice, which have apparently normal numbers of T cells in NALT.

Another surprising result is that although LTα is important for the expression of CCL19 in NALT, we consistently observed CCL21 expression in the NALT of Ltα−/− mice using quantitative PCR and immunofluorescence. This contrasts with a previous study that used in situ hybridization to show a complete absence of both CCL19 and CCL21 in the NALT of Ltα−/− mice (25). One caveat to our PCR analysis is that CCL21 expression is normalized to GAPDH, which does not take into account the dramatically reduced size of the NALT in Ltα−/− mice. However, the immunofluorescent analysis also shows that CCL21 is expressed in the NALT of Ltα−/− mice, albeit at reduced levels. One possibility is that CCL21 expression in NALT is controlled by another TNF family member, such as LIGHT (29), which is expressed normally in the NALT of Ltα−/− mice (Fig. 2 M). Alternatively, the expression of CCL21 in NALT may not require signaling through the LTβR or other TNFR family members. Regardless of whether the absence of LTα leads to a partial or a complete loss of CCR7 ligands, however, the loss of CCL19 and CCL21 expression in the NALT of plt/plt mice does not dramatically alter its structure or noticeably change T cell numbers, even though the genetic defects in plt/plt mice appear to disrupt the expression of CCR7 ligands in NALT more completely than the lack of LTα signaling in Ltα−/− mice. In addition, DC numbers are normal in plt/plt mice, but are reduced in Ltα−/− mice (not shown) (30). Thus, the loss of CCL19 and CCL21 expression in the NALT of plt/plt mice does not impact the structure of NALT to the same extent as it does in lymph nodes.

The differentiation of HEVs is also impaired in the NALT of Ltα−/− mice. However, PNAd-expressing HEVs appear nearly normal in the NALT of Cxcl13−/−, plt/plt, and plt/plt × Cxcl13−/− mice. These results are similar to those published by Ying et al. (25), who showed that LTα and LTβ are required for the expression of PNAd on the luminal side of HEVs in NALT. This study also showed that LTαβ is required for the expression of the sulfotransferase HEC-6ST and the protein GlyCAM-1, which is modified on sialyl Lewis rats by HEC-6ST to generate PNAd (25). Our results extend this observation and show that the effect of LTα signaling is probably mediated directly rather than indirectly via the LTα-dependent expression of chemokines. However, the HEVs in Cxcl13−/− and plt/plt mice appear somewhat smaller than those in normal mice, possibly due to reduced trafficking of cells through the HEVs.

Chemokines are also important for the function of NALT. For example, our results show that LTα and CXCL13 are both required for the formation of germinal centers in NALT and for the expression of AID and Iμ-Cα mRNAs. Because AID is required for isotype switching (31), and the expression of Iμ-Cα transcripts is indicative of B cells that recently switched to IgA (32), these data suggest that isotype switching to IgA should be highly impaired in the NALT of Ltα−/−, Cxcl13−/−, and even plt/plt mice. However, despite these local defects, IgA+ B cells accumulate in the NALT of all groups after influenza infection, albeit at lower levels in the NALT of Ltα−/− and Cxcl13−/− mice (Fig. 5 H). Similarly, IgA-secreting influenza-specific plasma cells are found in the nasal mucosa of all groups and are found at highest numbers in Ltα−/− mice. These data contrast with those recently reported by Ying et al. (25), who showed that OVA-specific serum IgG is reduced ∼250-fold in Ltα−/− mice and that OVA-specific vaginal IgA is undetectable after intranasal immunization with OVA and cholera toxin. Other studies also demonstrate that the production of IgA in the intestine is highly impaired in Ltα−/− mice (33). However, our published data showed that influenza-specific IgA in serum is normal in Ltα−/− mice after infection (34). One possible explanation for these results is that isotype switching occurs in other locations, such as the inducible bronchus-associated lymphoid tissue (35), and that IgA-secreting cells subsequently home to the NALT and nasal mucosa after switching. However, switching to IgA is thought to occur only in lymphoid tissues, such as NALT, Peyer’s patches, and isolated lymphoid follicles (5, 36), despite previous studies suggesting that switching to IgA can occur during the development of B cells in bone marrow (37), outside of lymphoid organs (33, 38), and independently of cognate interactions with T cells (39, 40). Thus, switching to IgA may occur via unusual mechanisms in Ltα−/− mice, possibly due to delayed viral clearance and prolonged inflammation in the respiratory tract. Thus, despite apparently inefficient isotype switching in the NALT of Ltα−/−, Cxcl13−/−, and plt/plt mice, IgA+ B cells eventually accumulate in the nasal mucosa of these mice.

Our previous results show that influenza-specific CD8 T cell responses are impaired in the NALT of Ltα−/− mice (7, 34). We now show that these defects correlate with impaired expression of CCL21/CCL19 in the NALT of Ltα−/− and plt/plt mice. This is consistent with previous results showing that T cell responses to contact Ags or s.c. Ags are delayed in plt/plt mice (41). However, T cell responses to other viruses are not significantly delayed in plt/plt mice (42), suggesting that it may be the nature of the Ag that drives the response. Interestingly, influenza-specific CD8 responses in the NALT of Ltα−/− mice are consistently more delayed than responses in the NALT of plt/plt mice, suggesting that the loss of LTα results in more than just the loss of CCL19 and CCL21 expression. We suspect that the impaired expression of CCL20 in the dome epithelium of Ltα−/− NALT may disrupt the recruitment of subepithelial DCs to the follicular dome of the NALT (43, 44, 45). Therefore, the process of Ag acquisition from the nasal lumen could be severely impaired in the NALT of Ltα−/− mice as a consequence of a reduced CCL20 production by epithelial cells. In addition, it was recently demonstrated that LTα is required for homeostatic proliferation of DCs in the spleen (30). Thus, the delayed activation of influenza specific CD8 T cells in the NALT of Ltα−/− mice may result from a combination of deficiencies in DC activation and Ag retrieval from the dome epithelium of the NALT.

Together, these data are consistent with the concepts that LTα is important for the normal expression of homeostatic chemokines in NALT and that the aberrant expression of these chemokines is partly responsible for the structural and functional defects in the NALT of Ltα−/− mice. Structural defects in primary and secondary B cell follicles and stromal cells can be attributed to the lack of CXCL13 expression in the NALT of Ltα−/− mice, whereas defects in HEV development can be directly attributed to the lack of LTα, rather than to impaired expression of CXCL13, CCL19, and CCL21. In contrast, the delayed CD8 T cell responses observed in the NALT of Ltα−/− mice appear to be related to both the lack of CCR7 ligands as well as the lack of LTα itself.

We thank Drs. Frances Lund and Reina Mebius for helpful discussions, and Dr. Jason Cyster for providing the Cxcl13−/− mice.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Trudeau Institute and National Institutes of Health Grants HL69409 and HL63925 (to T.D.R.).

3

Abbreviations used in this paper: NALT, nasal-associated lymphoid tissue; AID, activation-induced cytidine deaminase; DC, dendritic cell; FDC, follicular DC; HEV, high endothelial venule; LTα, lymphotoxin-α; NP, nucleoprotein; PA, acidic polymerase; PNA, peanut agglutinin; PNAd, peripheral node addressin.

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