Nasopharyngeal-associated Lymphoreticular Tissue Research Articles

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11. Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

A novel combined adjuvant for nasal delivery elicits mucosal immunity to influenza in aging

Since a combination of flt3 ligand plasmid (pFL) and CpG-oligodeoxynucleotides (ODN)(3) as a dendritic cell (DC)-targeting double mucosal adjuvant elicited ovalbumin-specific secretory IgA (S-IgA) antibody (Ab) responses, we examined whether this double adjuvant could induce influenza-specific protective immunity in aged mice. A double adjuvant plus A/Puerto Rico/8/34 (PR8) hemagglutinin (HA) induced increased numbers of CD11b(+) CD11c(+) DCs and both CD4(+) Th1- and Th2-type responses in the nasopharyngeal-associated lymphoreticular tissue, nasal passages and cervical lymph nodes. Further, increased levels of PR8 HA-specific S-IgA Ab responses were detected in the upper respiratory tact (URT) of aged and young adult mice given nasal PR8 HA with this double adjuvant. Thus, when mice were challenged with PR8 virus via the nasal route, both aged and young adult mice given nasal vaccine exhibited complete protection. Further, IgA-deficient mice nasally immunized with a double adjuvant influenza vaccine failed to provide protection against PR8 challenge. These results indicate that a nasal double adjuvant successfully induces PR8 HA-specific IgA Ab responses in both young adult and aged mice, which are essential for the prevention of influenza infection in the murine URT.

A Combination of Flt3 Ligand cDNA and CpG Oligodeoxynucleotide as Nasal Adjuvant Elicits Protective Secretory-IgA Immunity to Streptococcus pneumoniae in Aged Mice

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.

A combination of Flt3 ligand cDNA and CpG ODN as nasal adjuvant elicits NALT dendritic cells for prolonged mucosal immunity

We explore cellular and molecular mechanisms of nasal adjuvant of a combination of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN). The double DNA adjuvant given with OVA maintained prolonged OVA-specific secretory IgA (S-IgA) Ab responses in external secretions for more than 25 weeks after the final immunization. Further, both Th1- and Th2-type cytokine responses were induced by this combined adjuvant regimen. The frequencies of plasmacytoid DCs (pDCs) and CD8 + DCs were significantly increased in nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given the combined adjuvant. Importantly, when we examined adjuvanticity of pFL plus CpG ODN in 2-year-old mice, significant levels of mucosal IgA Ab responses were also induced. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the development of an effective mucosal vaccine for the elderly.

A Novel Adenovirus Expressing Flt3 Ligand Enhances Mucosal Immunity by Inducing Mature Nasopharyngeal-Associated Lymphoreticular Tissue Dendritic Cell Migration

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.

Nasal Cholera Toxin Elicits IL-5 and IL-5 Receptor α-Chain Expressing B-1a B Cells for Innate Mucosal IgA Antibody Responses

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.

NALT CD11b+ Dendritic Cell Migration Contributes to Induction of Ag Specific Immunity (B111)

Abstract Our previous study showed that targeting nasopharyngeal-associated lymphoreticular tissue (NALT) dendritic cells (DCs) with an adenovirus vector expressing Flt3 Ligand (Ad-FL) induced Ag-specific mucosal immunity that mediated by increased numbers of DCs and CD4+ Th1- and Th2-type cytokine responses. In this study, we examined the kinetics of DC subset formation and intracellular cytokine production in NALT and in cervical lymph nodes (CLNs). C57BL/6 mice were nasally immunized with Ad-FL and OVA three times at weekly intervals. As controls, mice were given Ad expressing the firefly luciferase protein (Ad-Luc). Mononuclear cells were isolated at various time points and the frequencies of DC subsets and intracellular cytokine production by CD4+ T cells were determined by FACS. An Increased frequency in CD11b+ DCs was seen in nasal passages (NPs) and submundibular glands (SMGs) at day 21. On the other hand, the frequency as well as the actual numbers of DCs in NALT was the highest 3 days after the second nasal immunization. Further, peak cell numbers of CD11b+ DCs in CLNs were seen at day 14. Of importance, intracellular cytokine FACS analysis showed highest IFN- γ and IL-4 production in CLNs at day14. These results suggest that NALT CD11b+ DCs migrate into NPs and SMGs via the CLN and play an important role in the induction of both Th1- and Th2-type cytokine responses in mice given Ad-FL as nassal adjuvant. Supported by DE 12242, AI 18958, AI 43197, and AG 025873.

Combination of Nasal Lymphotactin and Macrophage Chemoattractant Protein-1 Expressing Plasmids Elicits Antigen-Specific Salivary S-IgA Antibody Immunity

It has been shown that mucosal application of chemokines with protein antigen (Ag) successfully induces mucosal and systemic antibody (Ab) responses, as well as CD4+ T cell responses. We examined whether plasmids expressing lymphotaction (LTN) and/or macrophage chemoattractant proteine-1 (MCP-1) cDNA (pLTN and pMCP-1) exhibit nasal adjuvanticity when administered with protein Ag. When either pLTN or pMCP-1 was employed as a nasal adjuvant in mice, OVA-specific IgG Ab responses were seen in plasma ; however, both failed to induce OVA-specific secretory IgA (S-IgA) Abs in external secretions except anti-OVA IgG and IgA Abs in nasal washes of mice given pLTN. However, mice nasally immunized with a combination of pLTN and pMCP-1 showed OVA-specific S-IgA Ab responses in both saliva and fecal extracts. Further, that combination of chemokines used as a nasal adjuvant induced the highest levels of CD4+ T cell proliferative and Th2-type cytokine responses in cervical lymph nodes (CLN), as compared with a single chemokine nasal administration. In addition, a significantly increased number of CD11c+ dendritic cells (DCs) which expressed co-stimulatory molecules such as MHC II, CD40 and CD86 noted in the nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given pLTN and pMCP-1. These results show that a combination of pLTN and pMCP-1 given as a nasal adjuvant induces optimal mucosal S-IgA Ab responses in submandibular glands, which are mediated by increased number of DCs in the NALT and Th2-type CD4+ T cells in the CLN.

Non-toxic Stx derivatives from Escherichia coli possess adjuvant activity for mucosal immunity

Both B subunit of Shiga toxin 1 (Stx1-B), which mediates the binding of toxin to the membrane, and mutant Stx1 (mStx1), which is a non-toxic double-mutated Stx1 harboring double amino acid substitutions in the A subunit, possess potent mucosal adjuvant activity. Nasal immunization of mice with ovalbumin (OVA) plus the Stx1-B or mStx1 induced OVA-specific serum IgG and mucosal IgA responses. IgG subclass analysis revealed that mStx1 and Stx1-B as mucosal adjuvants supported Ag-specific IgG1 followed by IgG2b Abs. The co-administration of either mStx1 or Stx1-B with OVA enhanced the production of IL-4, IL-5, IL-6 and IL-10 with low IFN-γ, by OVA-specific CD4 + T cells. To better elucidate the mechanisms underlying mStx1’s and Stx1-B’s adjuvant activity, we next sought to examine whether or not dendritic cells (DC) residing in the nasopharyngeal-associated lymphoreticular tissue (NALT) were activated by nasal administration of Stx1-B or mStx1. We found that mice nasally administered with Stx1-B or mStx1 showed an up-regulation in the expression of CD80, CD86 and especially CD40 on NALT DCs. Taken together, these results suggest that non-toxic Stx derivatives could be effective mucosal adjuvants for the induction of Th2-type, CD4 + T cell mediated, antigen-specific mucosal IgA and systemic IgG Ab responses, and that they likely owe their adjuvant activity to the up-regulation of co-stimulatory molecules including CD80, CD86 and CD40 on NALT DCs.

New generation of mucosal adjuvants for the induction of protective immunity.

Invasion of infectious agents through mucosal surfaces can be prevented by use of the common mucosal immune system (CMIS), which interconnects inductive tissues, including Peyer's patches (PPs) and nasopharyngeal-associated lymphoreticular tissue (NALT), and effector tissues of the intestinal and respiratory tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant has been shown to be essential. When vaccine antigen is administered together with mucosal adjuvant, antigen-specific T-helper (Th) 1 and Th2 cells, cytotoxic T lymphocytes (CTLs) and IgA B cell responses are effectively induced by oral or nasal routes via the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium (FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying dendritic cells (DCs). Elucidation of the molecular/cellular characteristics of M cells and mucosal DCs will greatly facilitate the design of a new generation of effective mucosal adjuvants and of a vaccine delivery vehicle that maximises the use of the CMIS. Our recent efforts at mucosal vaccine development have focused on nasal administration of vaccine antigen together with nontoxic mutant-based or cytokine-/chemokine-based adjuvant for the induction of the protective immunity. To this end, a chimeric form of a nontoxic adjuvant combining the merits of mutant cholera toxin A subunit (mCT-A) and heat labile toxin B subunit (LT-B) was created as the second generation of detoxified toxin-based mucosal adjuvant. When a vaccine antigen was coexpressed together with an immune stimulatory/delivery molecule in crop seed, this edible vaccine is not only effective but also extremely practical in that it can be produced in huge quantities and preserved and shipped over long distances at room temperature without altering the quality of the vaccine. Because such qualities would greatly facilitate global vaccination, this new generation edible vaccines with a built-in adjuvant and/or M cell-targeted edible vaccine promises to be a powerful weapon for combating infectious diseases and bioterrorism.

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue.

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.

Sendai virus fusion protein mediates simultaneous induction of MHC class I/II-dependent mucosal and systemic immune responses via the nasopharyngeal-associated lymphoreticular tissue immune system.

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4(+) Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.

Nasopharyngeal-Associated Lymphoreticular Tissue (NALT) Immunity: Fimbriae-Specific Th1 and Th2 Cell-Regulated IgA Responses for the Inhibition of Bacterial Attachment to Epithelial Cells and Subsequent Inflammatory Cytokine Production

To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 micrograms) and CT (1 microgram) induced Ag-specific Th1/Th2-type response in CD4+ T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P. gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.

気管，食道周辺領域の粘膜免疫反応 咽頭・へん桃の感染に対する粘膜免疫応答

Adenoids and palatine tonsils are known to function as nasopharyngeal-associated lymphoreticular tissue for mucosal immune responses in the upper aerodigestive tract. In the present study, we investigated the immune responses of tonsillar lymphocytes in response to nontypeable Haemophilus influenzae (NTHi). Adenoids, palatine tonsils and nasopharyngeal secretions were obtained from patients who had undergone adenoidectomy or palatine tonsillectomy, and the numbers of NTHi in the samples were quantitated. The antibody activities against P6, one of the outer membrane proteins of NTHi, were determined by ELISA and the numbers of P6-specific antibody-producing cells were determined by ELISPOT. Production of cytokines from CD4+ T cells incubated with P6 was determined by ELISA and RT PCR. The results showed an increase of P6-specific IgA antibody activity and a decrease of NTHi in nasopharyngeal secretions with older age. The numbers of NTHi in the adenoids and the palatine tonsils decreased in samples having a large number of P6-specific IgA antibody-producing cells. CD4+ T cells more frequently produced Th2 type cytokines than Th1 type cytokines. The findings suggest that the adenoids and the palatine tonsils play an important role in regulating mucosal immune responses and the colonization of NTHi in the nasopharynx.