Huong L. Vu

IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1

Aberrant glycosylation of IgA1 plays an essential role in the pathogenesis of IgA nephropathy. This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1. Biosynthesis of these glycans occurs in a stepwise fashion beginning with the addition of N-acetylgalactosamine by the enzyme N-acetylgalactosaminyltransferase 2 and continuing with the addition of either galactose by beta1,3-galactosyltransferase or a terminal sialic acid by a N-acetylgalactosamine-specific alpha2,6-sialyltransferase. To identify the molecular basis for the aberrant IgA glycosylation, we established EBV-immortalized IgA1-producing cells from peripheral blood cells of patients with IgA nephropathy. The secreted IgA1 was mostly polymeric and had galactose-deficient O-linked glycans, characterized by a terminal or sialylated N-acetylgalactosamine. As controls, we showed that EBV-immortalized cells from patients with lupus nephritis and healthy individuals did not produce IgA with the defective galactosylation pattern. Analysis of the biosynthetic pathways in cloned EBV-immortalized cells from patients with IgA nephropathy indicated a decrease in beta1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2,6-sialyltransferase activity. Also, expression of beta1,3-galactosyltransferase was significantly lower, and that of N-acetylgalactosamine-specific alpha2,6-sialyltransferase was significantly higher than the expression of these genes in the control cells. Thus, our data suggest that premature sialylation likely contributes to the aberrant IgA1 glycosylation in IgA nephropathy and may represent a new therapeutic target.

Heterosubtypic Immunity to Influenza A Virus Infection Requires B Cells but Not CD8+ Cytotoxic T Lymphocytes

Heterosubtypic immunity (HSI), defined as protective cross-reactivity to lethal infection with influenza A virus of a serotype different from the virus initially encountered, is thought to be mediated by cross-reactive cytotoxic T lymphocytes (CTL). This study provides direct evidence for the role of effector CTL versus B cells in HSI in mice with a targeted disruption in the alpha chain of CD8 molecule (CD8(+) T cell deficient) or the immunoglobulin mu heavy chain (B cell deficient), respectively. CD8(+) T cell-deficient mice developed complete HSI. These mice displayed normal humoral immune responses, as determined by titers of subtype cross-reactive antibodies and virus-neutralizing antibodies specific for the immunizing influenza strain. In contrast, HSI was not observed in B cell-deficient mice, although these mice could mount cross-reactive CTL responses. These results show that B cells are required for HSI and provide new insight into the mechanisms of HSI, with significant implications in vaccine development.

Mucosal IL-8 and TGF-β recruit blood monocytes: evidence for cross-talk between the lamina propria stroma and myeloid cells

The lamina propria of the gastrointestinal mucosa contains the largest population of mononuclear phagocytes in the body, yet little is known about the cellular mechanisms that regulate mononuclear cell recruitment to noninflamed and inflamed intestinal mucosa. Here, we show that intestinal macrophages do not proliferate. We also show that a substantial proportion of intestinal macrophages express chemokine receptors for interleukin (IL)‐8 and transforming growth factor‐β (TGF‐β), and a smaller proportion expresses receptors for N‐formylmethionyl‐leucyl‐phenylalanine and C5a, but, surprisingly, they do not migrate to the corresponding ligands. In contrast, autologous blood monocytes, which express the same receptors, do migrate to the ligands. Blood monocytes also migrate to conditioned medium (CM) derived from lamina propria extracellular matrix, which we show contains IL‐8 and TGF‐β that are produced by epithelial cells and lamina propria mast cells. This migration is specific to IL‐8 and TGF‐β, as preincubation of the stroma‐CM with antibodies to IL‐8 and TGF‐β significantly blocked monocyte chemotaxis to the stromal products. Together, these findings indicate that blood monocytes are the exclusive source of macrophages in the intestinal mucosa and underscore the central role of newly recruited blood monocytes in maintaining the macrophage population in noninflamed mucosa and in serving as the exclusive source of macrophages in inflamed mucosa.

Gamma Interferon Is Not Required for Mucosal Cytotoxic T-Lymphocyte Responses or Heterosubtypic Immunity to Influenza A Virus Infection in Mice

ABSTRACT Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-γ) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-γ, we have investigated the mechanism of HSI by analyzing the role of IFN-γ for HSI in IFN-γ gene-deleted (IFN-γ−/−) mice. It has been reported that IFN-γ is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-γ is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-γ−/− mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-γ−/− mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-γ does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.

Oral and Nasal Sensitization Promote Distinct Immune Responses and Lung Reactivity in a Mouse Model of Peanut Allergy

Despite structural and functional differences between the initial sites of contact with allergens in the gastrointestinal and nasal tracts, few animal models have examined the influence of the mucosal routes of sensitization on host reactivity to food or environmental antigens. We compared the oral and nasal routes of peanut sensitization for the development of a mouse model of allergy. Mice were sensitized by administration of peanut proteins in the presence of cholera toxin as adjuvant. Antibody and cytokine responses were characterized, as well as airway reactivity to nasal challenge with peanut or unrelated antigens. Oral sensitization promoted higher levels of IgE, but lower IgG responses, than nasal sensitization. Both orally and nasally sensitized mice experienced airway hyperreactivity on nasal peanut challenge. The peanut challenge also induced lung eosinophilia and type 2 helper T-cell-type cytokines in orally sensitized mice. In contrast, peanut challenge in nasally sensitized mice promoted neutrophilia and higher levels of lung MAC-1(+) I-A(b low) cells and inflammatory cytokines. In addition, nasal but not oral, sensitization promoted lung inflammatory responses to unrelated antigens. In summary, both oral and nasal peanut sensitization prime mice for airway hyperreactivity, but the initial mucosal route of sensitization influences the nature of lung inflammatory responses to peanut and unrelated allergens.

Heterosubtypic Immunity to Influenza A Virus Infection Requires a Properly Diversified Antibody Repertoire

ABSTRACT Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT−/−) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (DH) with an altered DH segment (namely, ΔD-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT−/− mice compared to those in ΔD-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.

Mechanism of heterosubtypic immunity to influenza A virus infection

Abstract Heterosubtypic immunity (HSI) is defined as protective cross-reactive immune responses to lethal infection with influenza A virus of a different serotype than the virus initially encountered, and is thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL). These CTL recognize conserved epitopes of internal proteins, such as nucleoprotein (NP) or matrix (M) protein shared by influenza A virus subtypes. Despite extensive studies, the precise effector mechanism for HSI remains elusive. For example, our recent studies and those of others reported HSI in T cell-depleted, β2-microglobulin-deficient, and CD8 cell-deficient mice. The role for humoral immune responses in HSI is also unclear. Passive transfer of heterosubtypic immune serum did not provide protection against lethal heterosubtypic challenge, while B cell-deficient mice failed to develop HSI. Our recent findings and those of others now allow us to suggest a two-tiered HSI. Early after heterosubtypic challenge, a number of factors including subtype-specific CTL as well as antibody (Ab) responses and other as yet not well characterized host factors are able to minimize temporarily the virus spread, but are unable to clear the infection. In the later phase, the development of virus-neutralizing (VN) antibodies is important for virus clearance resulting in complete host recovery.

Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes.

The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2-4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12-16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.