Humoral and Cellular Immune Responses Mucosal Inductive Site

Nasal-Associated Lymphoid Tissue Is a Mucosal Inductive Site for Virus-Specific Humoral and Cellular Immune Responses

This information is current as Adrian W. Zuercher, Susan E. Coffin, M. Christine of September 27, 2021. Thurnheer, Petra Fundova and John J. Cebra J Immunol 2002; 168:1796-1803; ; doi: 10.4049/jimmunol.168.4.1796 http://www.jimmunol.org/content/168/4/1796 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Nasal-Associated Lymphoid Tissue Is a Mucosal Inductive Site for Virus-Speciﬁc Humoral and Cellular Immune Responses1

Adrian W. Zuercher,* Susan E. Cofﬁn,† M. Christine Thurnheer,* Petra Fundova,‡ and John J. Cebra2*

Peyer’s patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of Downloaded from IgA؉ and IgG2a؉ B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract. The Journal of Immu- nology, 2002, 168: 1796Ð1803. http://www.jimmunol.org/

he mucosal immune system consists of two functionally tract. However, the known compartmentalization of mucosal immune distinct types of tissue: 1) inductive sites, where naive B responses depending on the route of Ag administration (8, 9) suggests T and T cells are clonally selected and expanded upon Ag that structures other than PP, e.g., in the respiratory tract, may function contact; and 2) effector sites, where activated B and T cells relocate as mucosal inductive sites. after Ag-priming in inductive sites to express their effector functions. In the murine upper respiratory tract, nasal-associated lymphoid This concept is best established for the components of the intestinal tissue (NALT) is believed to be the equivalent of the Waldeyer’s mucosal immune system. They are organized in the so-called gut- ring of humans (10). It consists of a paired lymphoid tissue located by guest on September 27, 2021 3 associated lymphoid tissue (GALT), and include Peyer’s patches at the floor of the nasal cavity lined by ciliated respiratory epithe- (PP), mesenteric lymph nodes (LN), and dispersed lymphoid cells in lium, and has been postulated in different studies as a possible the epithelial layer and in the gut lamina propria (1). PP are inductive functional equivalent in the upper respiratory tract to PP in the gut sites and have been described as the major location for Ag-specific B (reviewed in Ref. 11). Several findings support this hypothesis. cell activation and isotype switching to IgA (2) and generation of ϩ First, the cellular composition of NALT is similar to PP (12–14); IgA memory B cells (3), as well as for the induction of Ag-specific both tissues contain a major population of naive B cells as well as CTL (4). Generally, these primed B and T cells emigrate from PP, naive (CD45RBhigh) T cells (12). Second, it has been shown in rats undergo terminal differentiation, and eventually home to the lamina that both NALT and PP have overlaying epithelium containing M propria and the intra-epithelial lymphocyte compartment of the gut cells (15, 16) and follicle-associated epithelium (17) that may (5–7). In contrast, little is known about the anatomic location and serve as entry-sites for different pathogens (18, 19). Third, upon functional potential of inductive and effector sites in the respiratory stimulation with Ag, the major isotype of Ab produced by NALT B cells is IgA (12, 13, 20, 21). Salivary glands and tear glands were suggested as possible effector sites for IgA production (22, Departments of *Biology and †Pediatrics, University of Pennsylvania School of Med- icine, Philadelphia, PA 19104; and ‡Division of Immunology and Gnotobiology, In- 23), which is reflected by the presence of IgA Abs in saliva and stitute of Microbiology, Czech Academy of Science, Prague, Czech Republic tear fluid. Despite these similarities, differences between PP and Received for publication June 27, 2001. Accepted for publication December 4, 2001. NALT, such as a markedly diverging expression and function of The costs of publication of this article were defrayed in part by the payment of page homing receptors (24), have been reported. Other hallmarks of charges. This article must therefore be hereby marked advertisement in accordance mucosal inductive sites, like formation of germinal centers as well with 18 U.S.C. Section 1734 solely to indicate this fact. as isotype-switching and expansion of surface IgAϩ B cells upon 1 This work was supported by Grant AI-23970 from the National Institutes of Health (to J.J.C.), and a fellowship from the Swiss Foundation for Medical-Biological Grants Ag-stimulation have not been directly demonstrated in NALT. (Schweizerische Stiftung fuer medizinisch-biologische Studien; to A.W.Z.). The Flow Moreover, even though specific CTLs have been observed in cer- Cytometry Facility of the Cancer Center at the University of Pennsylvania is supported by the Lucille P. Markey Trust. vical or mediastinal LN after intranasal (i.n.) immunization (25, 26), it is unknown whether CTLs are induced in NALT. 2 Address correspondence and reprint requests to: Dr. John J. Cebra, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA, Using i.n. infection with reovirus serotype 1/Lang, an estab- 19104-6018. E-mail address: [email protected] lished mucosal pathogen that infects and elicits mucosal immune 3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; NALT, responses in both the gastrointestinal (4, 27–29) and respiratory nasal-associated lymphoid tissue; i.n., intranasal(ly); PP, Peyer’s patch; LN, lymph tract (30, 31), we have structurally and functionally analyzed mu- node; UEA, Ulex Europaeus agglutinin I; BALT, bronchus-associated lymphoid ϩ ϩ tissue. rine NALT. Germinal centers, as well as IgA and IgG2a B

cells, were induced and expanded in NALT, and potent reovirus- with 0.1 mg/g body weight Avertin (2,2,2,-tribromoethanol; Aldrich, Mil- specific IgA responses were detected in the upper respiratory tract waukee, WI) and 1–2.5 ϫ 107 PFU reovirus was applied to both nostrils ␮ (NALT, palatine salivary glands, and submandibular LN) and gas- with a micropipette in a total volume of 25 l saline/0.5% gelatin. trointestinal tract (PP, small intestine, and mesenteric LN). Impor- Virus titration tantly, virus-specific CTLs were detected in NALT as well as in For determination of viral titers in various tissues, C3H mice (n ϭ 3–4 per mediastinal, submandibular, and cervical LN. Limiting dilution time point) were sacrificed by CO2 asphyxiation and cervical dislocation analysis revealed a 5- to 6-fold higher precursor-CTL frequency in on days 2, 5, 7, and 14 after i.n. infection. After perfusion through the right NALT compared with cervical LN. Thus, NALT is a mucosal ventricle with 20 ml PBS, the upper right lobe of the lungs, the entire inductive site in the upper respiratory tract for specific humoral trachea, one palatine salivary gland, and a 1-cm piece of terminal ileal and cellular immune responses. small intestine were removed, washed, and weighed. The tissues were homogenized in 3 ml saline/0.5% gelatin and serial dilutions incubated on monolayers of L-929 fibroblasts in 6-well tissue culture plates (Costar, Materials and Methods Cambridge, MA) for 45 min at 37°C, and thereafter overlaid with 3 ml of Mice and media 1% Agar in complete M199 medium and cultured at 34°C. Cultures were overlaid with more Agar/M199 after 3 and 6 days. Plaques were counted Male C3HeB/FeJ (referred to as C3H), BALBc/ByJ, and DBA/2 mice were after 7 days incubation. purchased from The Jackson Laboratory (Bar Harbor, ME). C.B-17 (C.B- Igh1b/IcrTac) mice were from Taconic Farms (Germantown, NY). All Analysis of Ab production in organ fragment cultures mice were used at the age of 8–14 wk. Stock mice were housed in the ϭ animal facility of the Department of Biology, University of Pennsylvania On days 0, 4, 7, 11, and 14 after i.n. infection, C3H mice (n 3–4 per time point) were sacrificed and blood collected by heart puncture for serum

(Philadelphia, PA). After infection with reovirus, mice were kept physi- Downloaded from cally separated from naive stock mice in a Trexler plastic isolator (Standard isolation. After perfusion with 20 ml PBS, the entire small intestine and Safety, McHenry, IL). mesenteric LN were surgically removed. PP were visually detected and L-929 fibroblasts were grown in M199 medium (Life Technologies, excised from the small intestine. After decapitation, submandibular LN were removed. NALT was isolated after removal of the mandible as de- Grand Island, NY) containing 5% FCS (Life Technologies), 2 mM L- glutamine (Life Technologies), 1000 U/ml penicillin, and 0.1 mg/ml scribed elsewhere (13). Palatine salivary glands were isolated after removal streptomycin (Life Technologies). CTLs were grown in RPMI-1640 of the hard palate. For organ fragment culture (35), tissues were sterilized by sequential (Life Technologies) containing 10% FCS, L-glutamine, penicillin, ϫ streptomycin, 50 ␮g/ml gentamicin (Life Technologies), and 50 ␮M washes as described in detail elsewhere (36). Individual PP or LN, 3 3 http://www.jimmunol.org/ 2-ME (Sigma Aldrich, St. Louis, MO). For organ fragment cultures, mm pieces of small intestine from jejunum, individual palatine salivary Kennett’s HY medium (Life Technologies) supplemented with 10% glands, and NALT from one mouse (a pair of NALT still attached to a small piece of nasal epithelium overlaying the palate) were incubated in FBS, L-glutamine, penicillin, streptomycin, and gentamicin was used. wells of 24-well tissue culture plates (Costar) in 1 ml complete Kennett’s Histology, immunohistochemistry, and immunofluorescence HY medium for 7 days under a 90% O2/10% CO2 atmosphere at 37°C. Reovirus-specific IgM, IgA, and IgG2a Abs were measured by RIA. For NALT was frozen in OCT compound (TissueTek; EMSCO, Philadelphia, this purpose, flexible polyvinyl plates (Serocluster; Costar) were coated PA), horizontal 5-␮m cryosections were cut on a Cryocut 1800 cryotome with 2.5 ϫ 109 particles of reovirus per well in 50 ␮l PBS overnight at 4°C. (Leica; Dolbey-Jamison, Norristown, PA) and after air-drying fixed in Plates were blocked with 1% BSA in PBS and incubated with organ frag- ice-cold acetone. Some slides were stained with hematoxylin (Mayer’s ment culture supernatant fluid overnight at 4°C. Thereafter, plates were hematoxylin; Sigma Aldrich) and eosin (Sigma Aldrich). For immunohis- incubated for6hatroom temperature with 125I-labeled anti-IgA, anti- by guest on September 27, 2021 tochemical staining, slides were incubated in 0.3% H2O2 for 30 min, then IgG2a, or anti-IgM Abs (all from Southern Biotechnology Associates). blocked with Superblock (Pierce, Rockford, IL) for 30 min, and with bi- Radioactivity of individual wells was measured using a 1272 Clinigamma otin/avidin block (Vector Laboratories, Burlingame, CA) following the gamma counter (Wallac, Gaithersburg, MD). Total IgA Abs were mea- manufacturer’s instructions. Slides were incubated for 60 min with 50 ␮l sured by RIA as described earlier (35), and a standard curve of purified, of 2.5 ␮g/ml biotinylated anti-IgD (clone AMS9.1; BD PharMingen, San monoclonal IgA was used to convert cpm to nanograms per milliliter. Diego, CA), 2.5 ␮g/ml biotinylated anti-CD4 (clone GK1.5; BD PharM- ingen), 2.5 ␮g/ml biotinylated anti-CD8 (clone 53.6-72; BD PharMingen), In vitro restimulation and analysis of cytotoxic lymphocytes or 4 ␮g/ml biotinylated Ulex Europaeus agglutinin I (UEA; Vector Lab- oratories), followed by an incubation with HRP-conjugated streptavidin Seven days after i.n. infection of C3H mice with reovirus, single cell sus- (1/1000; BD PharMingen). All reagents were diluted in a solution of 10% pensions of NALT, mediastinal, cervical, and submandibular LN pooled Superblock in PBS. 3,3-Diaminobenzidine (Sigma Aldrich) was used as from 16–20 mice were restimulated in vitro by incubation in 96-well round-bottom microtiter plates (2 ϫ 105/well; Costar) in the presence of substrate according to the manufacturer’s instructions, followed by coun- ϫ 4 terstaining with hematoxylin (Gill’s hematoxylin; Fisher, Pittsburgh, PA), 5 10 virus-pulsed, irradiated, thioglycolate-elicited peritoneal exudate cells (4). After 24 h, ConA-conditioned medium (5% (v/v)) mixed with and overlaid with Permount (Fisher). ␮ For immunofluorescence, slides were incubated for 30 min with 50 ␮lof methyl-a-D-mannopyranosid (100 M; Sigma Aldrich) was added. Effector the following FITC-conjugated reagents: PNA (Pierce, coupled to FITC in cells were restimulated the same way after 7 days of culture, and after a ␮ total of 13 days of culture, cellular cytotoxicity was assessed in a standard our laboratory as described in Ref. 32), 10 g/ml anti-IgD (clone 11- 51 ␮ Cr-release cytotoxicity assay (37). L-929 fibroblasts infected with reo- 26c.2a; BD PharMingen), 10 g/ml anti-IgA (Southern Biotechnology As- ␮ 51 sociates, Birmingham, AL), and 10 ␮g/ml anti-IgG2a (Southern Biotech- virus (or uninfected for control) were labeled with 100 Ci Cr (NEN, nology Associates). Sections were overlaid with Vectashield containing Boston, MA) and then incubated with effector cells at different E:T ratios 4Ј,6Ј-diamidino-2-phenylindole (Vector Laboratories). (3000 target cells/well). After5hofincubation in V-bottom microtiter plates (Costar), 100 ␮l supernatant fluids were collected, mixed with 1 ml Flow cytometry scintillation fluid (Cytoscint; ICN Pharmaceuticals, Costa Mesa, CA), and ␤-emission measured on a LS6500 multipurpose scintillation counter Single cell suspensions (2 ϫ 105–106/sample) of NALT or submandibular LN (Beckman Coulter, Fullerton, CA). were stained for 20 min at 4°C with FITC-conjugated anti-CD19 (clone 1D3; BD PharMingen), PE-conjugated anti-CD4 (GK1.5; BD PharMingen), FITC- Limiting dilution of precursor cytotoxic lymphocytes CD8 (53-6.72; BD PharMingen), FITC-PNA, PE-␬ L chain (Southern Bio- Replicate cultures (n ϭ 17–24) containing varying numbers of either NALT or technology Associates), FITC-IgA (Southern Biotechnology Associates), and cervical LN cells (4.7 ϫ 102–1.2 ϫ 105 for NALT, 3.9 ϫ 103–5 ϫ 105 for FITC-IgG2a (Southern Biotechnology Associates). Cells were washed and cervical LN) obtained from C3H mice 7 days after i.n. infection were restim- fixed in 1% paraformaldehyde in PBS and analyzed on a FACScan flow cy- ulated in vitro for 7 days as described above. Thereafter, the contents of in- tometer (BD Biosciences, Mountain View, CA). WinMDI2.8 (The Scripps dividual microtiter wells were harvested and equally divided into two wells of Research Institute, La Jolla, CA) software was used for evaluation. V-bottom microtiter plates containing either reovirus-infected or -uninfected 51 51 Virus preparation and infection L-929 cells (3000 target cells/well, labeled with Cr)fora5hstandard Cr- release cytotoxicity assay as described above. Cultures were considered to Third passage stocks of reovirus type 1/Lang (33) were produced and pu- demonstrate cytotoxicity if the resulting 51Cr release was at least three times rified as described (34). For i.n. infection, mice were lightly anesthetized more than background of spontaneous release. Cytotoxicity was considered 1798 NALT IS A MUCOSAL INDUCTIVE SITE virus-specific if the release from infected targets was at least 20% higher from NALT displays the typical structure and organization of a secondary infected than from uninfected targets. The 95% confidence intervals of the lymphoid tissue and contains epithelial cells that potentially enable it linear regression curves were calculated using the Sigmaplot 2.00 software to efficiently take up Ag from the nasal cavity. (Jandel Corporation, SSPS, Chicago, IL). As shown in Fig. 2, B cells were the most abundant cell population Results in NALT comprising 47–78% of lymphocytes (Fig. 2, upper panel) Structure and cellular composition of naive murine NALT depending on the strain of mice. The ratio of B:T cells varied from 3.8:1 in C.B17 to 0.9:1 in C3H mice, whereas the CD4:CD8 ratio We analyzed the structure of NALT by immunohistochemistry. As varied from 3:1 in C.B17 to 1.6:1 in C3H mice (Fig. 2, lower panel). shown in Fig. 1, NALT in the naive C3H mouse is organized in These data reveal that the cellular composition of NALT is similar to distinct B and T cell areas (Fig. 1). As in PP, B cells reside in follicular ϩ PP. As most studies using reovirus have been done in C3H mice, this areas, whereas T cells, particularly CD8 T cells, predominantly oc- strain was used for all further analyses. cupy the parafollicular spaces between the B cell follicles. NALT is lined with an epithelium that binds the lectin UEA (Fig. 1). UEA is ϩ ␣ Induction of germinal centers and generation of IgA and specific for -L-fucose residues which are typically present on M ϩ cells and follicle-associated epithelium (16, 38). Staining with labeled IgG2a B cells in NALT after infection with reovirus UEA was restricted to the epithelium on the luminal side of NALT To address the function of NALT, C3H mice were infected i.n. (Fig. 1). A detailed view of the luminal epithelium revealed staining with 1–2.5 ϫ 107 PFU of reovirus serotype 1/Lang in a volume of of individual cells within the epithelium. These data demonstrate that 25 ␮l. Replicating virus was detected in the respiratory tract Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Histological analysis of naive murine NALT. Serial horizontal sections of naive mouse NALT were stained with biotinylated anti-IgD and HRP-labeled streptavidin, biotinylated anti-CD4, and HRP-streptavidin, or biotinylated anti-CD8 and HRP-streptavidin. Original magniﬁcation ϫ100. Sections were counterstained with Gill’s hematoxylin. Serial horizontal sections of paired NALT (N) were stained with H&E (ϫ40) or biotinylated UEA and HRP-streptavidin or HRP-streptavidin alone (ϫ200). Staining of the epithelium lining the luminal side of NALT is indicated by the arrows (ϫ40). Staining of individual cells at higher magniﬁcations is shown (ϫ200 and ϫ400). NC, nasal cavity. Sections were counterstained with Mayer’s hematoxylin. The Journal of Immunology 1799

(from 2.5 ϫ 105/mouse in naive mice, n ϭ 16, to 3.0 ϫ 106/mouse at day 7, n ϭ 19) and further to 1.8 ϫ 106/mouse (n ϭ 6) by day 14 postinfection). FACS analysis showed that the percentage of total (CD19ϩ) B cells remained stable during the course of infection (data not shown). Similarly, the CD4:CD8 ratio did not change during the course of infection. Induction of germinal center B cells (PNA-binding, ␬ L chainlow, Ref. 3) was observed in NALT on day 7 and increased through day 14, in parallel with expansion of both surface IgAlow and IgG2alow germinal center B cells, as well as surface IgAhigh and IgG2ahigh memory type B cells (Ref. 3; Fig. 4). Staining with anti-IgG2b Ab gave similar results (data not shown). A similar expansion of PNAϩ, IgAϩ, and IgG2aϩ B cells was observed in submandibular LNs, although with slightly delayed kinetics compared with NALT (Fig. 4). FIGURE 2. Lymphocyte composition of NALT. Single cell suspension These findings were confirmed by immunofluorescence staining of NALT cells (2 ϫ 105–106/sample) isolated from naive DBA/2, C.B-17, of histological sections of NALT. NALT of naive mice was char- BALB/c, or C3H mice were stained with FITC-labeled anti-CD19, PE- ϩ anti-CD4, or FITC-anti-CD8. Numbers represent the percentage of positive acterized by the presence of distinct areas of IgD B cells (Fig. 5). ϩ ϩ cells among gated lymphocytes. One of two identical experiments is No PNA-binding, IgA , or IgG2a cells were detected before shown. infection. Seven days after inoculation with reovirus, germinal Downloaded from centers characterized by the presence of PNA-binding cells were observed preferentially at the periphery of NALT. These PNA- (NALT, palatine salivary gland, lung, and trachea), and to a lesser positive follicles colocalized with IgA-stained and IgG2a-stained extent in the small intestine, and was cleared within 7–14 days after infection (Fig. 3). These data demonstrate that the respiratory tract is the major site of viral infection after i.n. inoculation, but http://www.jimmunol.org/ that coincidental swallowing of virus leads to infection and possible cross-priming of the gastrointestinal tract. Next, we examined the effect of the virus infection on NALT. The average number of cells recovered from NALT increased 54% from 1.3 ϫ 105 (n ϭ 12 mice) before infection to 2.0 ϫ 105 (n ϭ 91) at day 7 ( p ϭ 0.003), and decreased to initial levels by day 14 of infection (1.2 ϫ 105; n ϭ 14). In contrast, cell expansion was more dramatic and prolonged in submandibular LNs where cell numbers increased 11-fold within the first 7 days after infection by guest on September 27, 2021

FIGURE 3. Clearance of reovirus after i.n. infection. Lightly anesthetized naive C3H mice were infected i.n. with 1–2.5 ϫ 107 PFU reovirus FIGURE 4. Generation of germinal center (PNA-binding), sIgAϩ, and serotype 1/Lang in a volume of 25 ␮l. Two, 5, 7, and 14 days postinfection, sIgG2aϩ B cells in NALT and submandibular LN after i.n. infection with groups of mice (n ϭ 3–4) were sacrificed and perfused with PBS. Palatine reovirus: FACS analysis. Lightly anesthetized naive C3H mice were in- salivary glands, one submandibular salivary gland, the upper left lobe of fected i.n. with 1–2.5 ϫ 107 PFU reovirus serotype 1/Lang in a volume of lung, trachea, and a 1-cm piece of terminal ileum were isolated, washed, 25 ␮l. At 0, 5, 7, or 14 days postinfection, groups of mice (n ϭ 3–4) were weighed, and homogenized in PBS/0.5% gelatin. Serial dilutions were used sacrificed and single cell suspensions of NALT cells (2 ϫ 105–106/sample) to infect monolayers of L-929 fibroblasts for a standard virus titration as- were stained with PE-anti-mouse ␬ L chain, FITC-PNA, FITC-anti-IgA, or say. PFU were counted after 7 days incubation. Detection limit was ϳ100 FITC-anti-IgG2a. Numbers represent the percentage of positive cells PFU/g tissue. One of four similar experiments is shown. among gated lymphocytes. One of four similar experiments is shown. 1800 NALT IS A MUCOSAL INDUCTIVE SITE Downloaded from http://www.jimmunol.org/

FIGURE 5. Generation of germinal center (PNA-binding), IgAϩ, and IgG2aϩ B cells in NALT after i.n. infection with reovirus: histological analysis. Lightly anesthetized naive C3H mice were infected i.n. with 1–2.5 ϫ 107 PFU reovirus serotype 1/Lang in a volume of 25 ␮l. At 0, 7, or 14 days after infection, NALT of individual mice was isolated and frozen. Serial horizontal sections were stained with FITC-labeled anti-IgD, FITC-PNA, FITC-anti-IgA, or FITC-anti-IgG2a. Luminal side of NALT is at the bottom of the picture. Original magnification ϫ100. areas, whereas IgDϩ cells predominantly occupied the PNA-neg- was also produced by mesenteric and to a lesser extent by sub- ative regions. By day 14 of infection, PNA-binding cells were mandibular LN which drain the gastrointestinal and upper respi- by guest on September 27, 2021 concentrated at the luminal side of NALT and strongly stained ratory tract, respectively. Compared with IgA, the production of individual IgAϩ and IgG2aϩ cells were scattered throughout virus-specific IgG2a was delayed in NALT, but by day 14 postin- NALT. These findings demonstrate that germinal center reactions fection, NALT was producing considerable amounts of virus-spe- with generation and expansion of IgA- and IgG2a-switched B cells cific IgG2a (Fig. 6B, middle row). Similarly, virus-specific IgG2a are induced in NALT after i.n. infection with reovirus. responses were induced in submandibular and mesenteric LN. In contrast, no appreciable production of virus-specific IgG2a oc- Production of reovirus-specific Abs in organ fragment cultures curred in PP, palatine salivary glands, or the small intestine. Virus- Organ fragment cultures (35) of mucosal and lymphoid tissues of specific IgM was detected in PP, submandibular, and mesenteric the respiratory and gastrointestinal tract were performed to address LN as early as 4 days postinfection, whereas no virus-specific IgM production of total and virus-specific Abs after i.n. reovirus infec- Abs were detected in supernatant fluids of NALT, palatine salivary tion. Marginal amounts of total IgA were produced by NALT and gland, and small intestine fragment cultures (Fig. 6B, bottom row). palatine salivary glands of naive mice, whereas the components of The major isotype of virus-specific Ab induced in serum was GALT produced substantial quantities of IgA before stimulation IgG2a concomitant with production of virus-specific IgM, but only with reovirus (Fig. 6A). After infection, total IgA production was trace amounts of virus-specific IgA (data not shown). stimulated in NALT and palatine salivary glands, and simulta- neously a slight increase was observed in PP and small intestine. Generation of reovirus-specific CTL in NALT However, the total output of IgA was 4- to 150-fold higher (on day It is not known to date whether cytotoxic cellular immune re- 7 or 0, respectively) from PP compared with NALT. Similarly, sponses can be induced in NALT. To address this, 7 days after i.n. production of total IgA was generally higher in small intestine and infection with reovirus, cells from NALT, mediastinal, subman- mesenteric LN compared with palatine salivary glands and sub- dibular, and cervical LN from 16–20 mice were isolated and mandibular LN, respectively. pooled. After in vitro restimulation for 13 days, the presence of Maximal amounts of reovirus-specific IgA were produced by virus-specific CTL was assessed in a standard 51Cr-release cyto- NALT 7 days after infection (Fig. 6B, top row). Whereas produc- toxicity assay. Potent virus-specific CTL were obtained from tion rapidly declined from NALT, secretion of virus-specific IgA NALT, as well as from the LN draining the respiratory tract (Fig. by palatine salivary glands was sustained for at least 2 wk. Only 7). Identical restimulation of NALT and LN cells isolated from marginal amounts of virus-specific IgA were detected in other mu- naive mice did not result in the outgrowth of virus-specific CTL cosal tissues of the oral cavity such as lip or tongue (data not (Fig. 7). shown). Large amounts of virus-specific IgA were detected in su- However, limiting dilution analysis revealed a 5-fold higher fre- pernatant fluid of PP and small intestine fragment cultures indi- quency of precursor CTL in NALT compared with cervical LN cating cross-priming during i.n. inoculation. Virus-specific IgA (Fig. 8). This ratio is similar when the frequencies are adjusted to The Journal of Immunology 1801 Downloaded from

FIGURE 7. CTL in NALT after i.n. infection with reovirus. Lightly anesthetized naive C3H mice were infected i.n. with 1–2.5 ϫ 107 PFU reovirus serotype 1/Lang in a volume of 25 ␮l. After 7 days, mice (n ϭ 16–20) were sacrificed and perfused with PBS. Single cell suspensions http://www.jimmunol.org/ were cultured in microcultures in the presence of virus-pulsed, irradiated APCs and 5% (v/v) ConA-conditioned medium. Cells were restimulated after 7 days of culture with fresh APCs and ConA-conditioned medium. As a control, cells from naive mice were cultured under identical conditions (squares). After 13 days of culture, cells were used in a 5Cr-release cellular cytotoxicity assay with 51Cr-labeled, reovirus-infected (filled symbols) or uninfected (open symbols) L-929 fibroblasts as targets. After5hofincu- ␤ ␮ FIGURE 6. Production of total IgA and reovirus-specific Abs in the bation, -emission of 100 l supernatant fluids was measured on a scin- upper respiratory and the gastrointestinal tract after i.n. infection with reo- tillation-counter. Results show the percentage of specific lysis after sub- virus. Lightly anesthetized naive C3H mice were infected i.n. with 1–2.5 ϫ traction of spontaneous release. Spontaneous release was never above 18% by guest on September 27, 2021 107 PFU reovirus serotype 1/Lang in a volume of 25 ␮l. At day 0, 4, 7, 11 of total release. One of three similar experiments is shown. and 14 days postinfection, groups of mice (n ϭ 3–4) were sacrificed and perfused with PBS. NALT, PP, palatine salivary glands, a 1-cm piece of jejunum, and submandibular and mesenteric LN were isolated and steril- both sites, germinal center reactions with expansion of specific ized by sequential washing. Organ fragment cultures of the tissues were IgAϩ and IgG2aϩ B cells ensue from infection with reovirus. incubated for 7 days. A, Total IgA was measured by RIA using anti-Fab Furthermore, both sites contain similar draining LN, i.e., mes- coated polyvinyl plates. Serial dilutions of organ fragment culture super- enteric LN in the gastrointestinal and submandibular LN in the natant fluid were incubated, and bound Abs detected with polyclonal 125I- labeled anti-IgA Ab. Cpm were converted to ng/ml using a standard curve of monoclonal-purified IgA. B, Supernatant fluids were assessed for reovirus-specific IgA (top panels), IgG2a (middle panels), and IgM (bottom panels) by RIA. Polyvinyl plates coated with 2.5 ϫ 109 particles of reovirus per well were incubated with organ fragment culture supernatant fluid and bound Abs were detected with polyclonal 125I-labeled anti-IgA, anti- IgG2a, and anti-IgM Ab. Results from one of four similar experiments are shown.

total numbers of CD8ϩ cells in the initial NALT or cervical LN populations (NALT: 23.5% CD8ϩ cells, frequency ϭ 962/106; cervical LN: 27.8% CD8ϩ cells, frequency ϭ 165/106; ratio ϭ 5.8:1).

Discussion FIGURE 8. Limiting dilution analysis of precursor CTL in NALT (F) E In this study, we have analyzed the induction of reovirus- and cervical LN ( ). Lightly anesthetized naive C3H mice were infected i.n. with 1–2.5 ϫ 107 PFU reovirus serotype 1/Lang in a volume of 25 ␮l. specific humoral and cellular immune responses in the upper After 7 days, mice (n ϭ 20) were sacrificed and perfused with PBS. Var- respiratory tract. We found a number of striking functional ious dilutions of single cell suspensions were cultured for 7 days as de- similarities between the mucosa-associated lymphoid tissue of scribed above. Contents of individual wells were assessed for cytotoxicity the upper respiratory and the gastrointestinal tract. Both sites against 51Cr-labeled reovirus-infected or uninfected L-929 fibroblasts in a contain organized secondary lymphoid tissue lined by epithe- 51Cr-release assay. Results were evaluated as described in Materials and lium which possibly enables them to efficiently absorb Ag. At Methods. Frequencies (f) of precursor CTL among total cells are shown. 1802 NALT IS A MUCOSAL INDUCTIVE SITE respiratory tract, that may serve to amplify the responses We are currently investigating this in a model of reovirus infection generated in PP or NALT, respectively. of rats. It is well accepted that mesenteric LN serve to amplify immune Several other studies have described humoral immune responses responses initiated in PP. Unlike this, it remains elusive which, and in murine NALT (12, 13, 20, 21). In these studies, Ab production whether at all, LN of the respiratory tract fulfill a similar function was assessed using the ELISPOT technique. In general, the results for NALT. However, some of our findings may implicate such a correlate well with our findings using the organ fragment culture role for the submandibular LNs. Whereas only modest expansion technique. Both IgA- and IgG-producing cells were observed in of cells was observed in NALT after i.n. infection (Ͻ2-fold within NALT after i.n. infection with live influenza virus (21, 21, 44). Our first 7 days postinfection), a more pronounced expansion of cells results show local production of both IgA and IgG2a Abs in NALT occurred in submandibular LN (11-fold) over the same time pe- after i.n. infection with reovirus. This is in line with the reported riod. Furthermore, increased numbers of cells were still found in production of a mixed Th1/Th2-type cytokine profile after respi- submandibular LN 14 days postinfection, although by that time ratory infection with reovirus (45). Others described IgA as the cell numbers in NALT were not significantly different from naive clearly predominating local isotype in NALT (12, 13). In these mice. In contrast, our analysis of precursor CTL frequencies in studies, cholera toxin subunit B was used either as Ag or as an NALT vs cervical LN (and presuming similar numbers for sub- adjuvant in combination with bacterial Ag, which may somehow mandibular LNs) revealed a higher frequency of virus-specific mediate this skewing of the B cell response toward IgA. However, CTL in NALT (Fig. 7). Moreover, kinetic analysis of generation of the use of the fragment culture technique may allow for a more PNAϩ, IgAϩ, and IgG2aϩ B cells showed a delay in the genera- quantitative analysis of Ab production than ELISPOT, as it inte- Downloaded from tion of these cells in submandibular LN compared with NALT grates the number of specific cells with the actual secretion of Ab, (Fig. 4), i.e., whereas percentages of PNAϩ, IgAϩ, and IgG2aϩ yielding information about total Ab production capacity of an en- cells peaked or reached a plateau after ϳ7 days in NALT, the tire organ or tissue. For example, we show that 7 days postinfec- ϳ respective cells appeared later in submandibular LNs. Taken to- tion, NALT produces 4 times less total IgA than PP (Fig. 6A), gether, these findings may suggest that submandibular LN can in- which is probably due to the smaller number of total cells present deed amplify the specific responses generated in NALT, and that in NALT. Nevertheless, equal amounts of virus-specific IgA are these structures are functionally equivalent to PP and mesenteric produced, which likely indicates a higher frequency of Ag-specific http://www.jimmunol.org/ IgA-producing B cells in NALT than in PP. LN, respectively. In this study, we report the first direct demonstration of CTL Despite these similarities, the essentially different physiological generation in NALT. NALT CTL appear to kill infected targets functions of the respiratory tract (gas exchange) vs the gastroin- more efficiently at lower E:T ratios than CTL from the draining testinal tract (uptake of food) result in distinct requirements for the LNs of the same animals (Fig. 7). More strikingly, the 5- to 6-fold immune components present at these sites. GALT largely ignores higher precursor CTL frequency in NALT compared with cervical food Ags to allow uptake of nutrients, and mounts only minimal LN (Fig. 8) clearly shows that NALT is a potent inductive site for immune responses to the components of the resident bacterial specific CTL responses upon i.n. infection. A similarly increased

flora, but at the same time serves to exclude potential pathogens. In by guest on September 27, 2021 precursor CTL frequency in PP over peripheral LN was observed contrast, the immune components of the respiratory tract, in par- after local gastrointestinal reovirus infection (4). It will be inter- ticular the lower respiratory tract, are not permissive and attempt esting to pinpoint effector sites of these CTL to establish whether to keep the noncolonized lungs sterile. These functional differ- they specifically emigrate to mucosal effector sites of the respira- ences among a number of subtle differences might be best reflected tory tract through a homing-receptor mediated process, or whether in the predominance of different isotypes at these sites. Clearly, a more random distribution to different nonlymphoid tissues oc- IgA is the major isotype secreted in the gut, and its limited in- curs, as shown recently by Masopust et al. (46) in systemically and flammatory effector potential is the ideal “low-key” isotype for this orally primed mice. Furthermore, we demonstrate the generation site. In contrast, there is increasing evidence that the lower respi- of germinal centers in NALT after i.n. infection along with expan- ratory tract is mainly controlled and protected by IgG Abs that sion of IgAϩ and IgG2aϩ B cells, and local production of reovirus- have broader effector potential than IgA. Studies in influenza-in- specific IgA and IgG2a Abs. Altogether, these findings provide fected SCID mice after transfer of neutralizing mAbs of different direct evidence that NALT is an inductive site for humoral muco- isotypes (39), and more recently in influenza-infected IgA knock- sal immune responses. Thus, tracking patterns of distribution of out mice (40) have provided elegant evidence for this notion. IgG CTL and specific B lymphoblasts after induction in local mucosal Abs may originate from the systemic circulation and enter the lung inductive sites (e.g., in NALT vs PP) may represent a promising by transudation (41), or be produced locally in bronchus-associ- approach to address cross-priming and communication between ated lymphoid tissue (BALT). Indeed, BALT in rodents has been distant mucosal sites, and a way to experimentally assess questions shown to mount specific immune responses to Ags present in the concerning the regulation and functional characteristics of the lung lumen (reviewed in Ref. 42), as opposed to a more discrete common mucosal immune system. function in humans (43). It may be postulated that NALT may serve an “intermediate” function between BALT and GALT; al- Acknowledgments though it is in physical connection with the alimentary tract and We thank Haruka Hishiki for help with NALT isolation, and Alec McKay not sterile, NALT also serves as an important “gate-keeper” for the for running the FACS and preparing radiolabeled Abs. We also thank Dr. well-protected lower respiratory tract. This intermediate status Chris Cuff for his continuous support and helpful comments. may be reflected in the mixed IgA/IgG2a isotype-pattern prevalent at this site. As shown in Fig. 6, NALT not only produces IgA, but References also considerable amounts of IgG2a Abs (Fig. 6B, middle row). 1. Kato, T., and R. L. Owen. 1999. Structure and function of intestinal mucosal epi- Similarly, IgG2a was produced in larger quantities than IgA in thelium. In Mucosal Immunology, 2nd Ed. P. L. Ogra, J. Mestecky, M. E. Lamm, submandibular LN, whereas IgA predominated over IgG2a in mes- W. Strober, J. Bienenstock, and J. R. McGhee, eds. Academic Press, San Diego, p. 115. enteric LN. A more detailed comparison of the functions of BALT 2. Craig, S. W., and J. J. Cebra. 1971. Peyer’s patches: an enriched source of pre- vs NALT in comparison to GALT will be particularly interesting. cursors for IgA-producing immunocytes in the rabbit. J. Exp. Med. 134:188. The Journal of Immunology 1803

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