Xiaoping Zhu - Academia.edu (original) (raw)

Papers by Xiaoping Zhu

SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and a... more SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and airborne aerosols. To prevent viral infection and reduce viral spread, vaccine strategies must elicit protective immunity in the airways. FcRn transfers IgG across epithelial barriers; we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized S protein; the resulting S-Fc bound to S-specific antibodies (Ab) and FcRn. A significant increase in Ab responses was observed following the intranasal immunization of mice with S-Fc formulated in CpG as compared to the immunization with S alone or PBS. Furthermore, we intranasally immunize adult or aged mice and hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2, including Delta and Omicron variants. Intranasal immunization also significantly reduced viral transmission between immunized ...

The Journal of Immunology

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against ... more The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal...

Nature Communications, 2019

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity... more Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with β2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune...

Clinical and vaccine immunology : CVI, Jan 12, 2017

A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable ... more A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable and broadly protective antibodies. Many vaccine programs focus on the immune responses to critical epitopes in the gp120 portion of HIV envelope glycoprotein (Env) and seek to improve the quality and quantity of antibodies by altering the sequence, conformation, oligomerization or glycosylation of gp120 to activate appropriate germline B cells and mimic the subsequent maturation pathways seen in infected individuals. As a complement to these strategies, we developed dimeric fusion protein immunogens consisting of HIVBaL gp120 monomer attached to a Gly/Ser linker that is in turn, fused to one half of the dimeric Fc domain from rhesus macaque IgG1 (Env-rFc). We envisioned that Env-rFc may mimic some aspects of immune complexes by binding Fc gamma receptors (FcγRs) on immune cells to increase the strength, breadth, and durability of Env-specific antibody responses. The Env-rFc retained a ca...

Scientific reports, May 16, 2016

CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whet... more CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whether CD23 has any role in B-cell activation remains unclear. We examined the expression of CD23 in different subsets of peripheral B cells and the impact of CD23 expression on the early events of B-cell receptor (BCR) activation using CD23 knockout (KO) mice. We found that in addition to marginal zone B cells, mature follicular B cells significantly down regulate the surface expression level of CD23 after undergoing isotype switch and memory B-cell differentiation. Upon stimulation with membrane-associated antigen, CD23 KO causes significant increases in the area of B cells contacting the antigen-presenting membrane and the magnitude of BCR clustering. This enhanced cell spreading and BCR clustering is concurrent with increases in the levels of phosphorylation of tyrosine and Btk, as well as the levels of F-actin and phosphorylated Wiskott Aldrich syndrome protein, an actin nucleation pro...

Infection and immunity, Jan 26, 2015

Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of men... more Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real-time. Neutrophils were directly seen to chase C. neoformans, and then rapidly internalize it. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in an optimal phagocytosis and subsequent killing of the organisms. Addition of recombinant C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 MAP kinases in neutrophils. Inhibition of p38 MAPK pathway, but not Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils towards C. neoformans, bu...

PLOS Pathogens, 2015

African trypanosomes are extracellular protozoan parasites causing a chronic debilitating disease... more African trypanosomes are extracellular protozoan parasites causing a chronic debilitating disease associated with a persistent inflammatory response. Maintaining the balance of the inflammatory response via downregulation of activation of M1-type myeloid cells was previously shown to be crucial to allow prolonged survival. Here we demonstrate that infection with African trypanosomes of IL-27 receptor-deficient (IL-27R-/-) mice results in severe liver immunopathology and dramatically reduced survival as compared to wild-type mice. This coincides with the development of an exacerbated Th1-mediated immune response with overactivation of CD4 + T cells and strongly enhanced production of inflammatory cytokines including IFN-γ. What is important is that IL-10 production was not impaired in infected IL-27R-/mice. Depletion of CD4 + T cells in infected IL-27R-/mice resulted in a dramatically reduced production of IFN-γ, preventing the early mortality of infected IL-27R-/mice. This was accompanied by a significantly reduced inflammatory response and a major amelioration of liver pathology. These results could be mimicked by treating IL-27R-/mice with a neutralizing anti-IFN-γ antibody. Thus, our data identify IL-27 signaling as a novel pathway to prevent early mortality via inhibiting hyperactivation of CD4 + Th1 cells and their excessive secretion of IFN-γ during infection with African trypanosomes. These data are the first to demonstrate the essential role of IL-27 signaling in regulating immune responses to extracellular protozoan infections.

Mucosal immunology, Jan 18, 2015

The epithelial lining of the airway tract and allergen-specific IgE are considered essential cont... more The epithelial lining of the airway tract and allergen-specific IgE are considered essential controllers of inflammatory responses to allergens. The human low affinity IgE receptor, CD23 (FcɛRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains unknown whether the CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo, and whether inhibition of this pathway attenuates allergic inflammation. To this end, we show that in wild-type (WT) mice, epithelial CD23 transcytosed both IgE and ovalbumin (OVA)-IgE complexes across the airway epithelial barrier, whereas neither type of transcytosis was observed in CD23 knockout (KO) mice. In chimeric mice, OVA sensitization and aerosol challenge of WT/WT (bone-marrow transfer from the WT to WT) or CD23KO/WT (CD23KO to WT) chimeric mice, which express CD23 on radioresistant airway structural cells (ma...

Human vaccines & immunotherapeutics, Jan 19, 2015

The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In ... more The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humo...

Proceedings of the National Academy of Sciences, 2014

Significance T lymphocytes are white blood cells that recognize and fight pathogens. Maintenance ... more Significance T lymphocytes are white blood cells that recognize and fight pathogens. Maintenance of sufficient numbers of T cells is essential to prevent susceptibility to infections. Survival of quiescent T cells is maintained, in part, by the interaction between the soluble factor (IL-7 produced by various stromal cells) and the IL-7 receptor (IL-7R) expressed on the surface of T cells. Here, we show that naïve T cells have basal nuclear levels of the transcription factor NF-κB and that is key to maintain IL-7R expression in T cells and for their survival. Our results imply that antiinflammatory therapies targeting NF-κB may affect the pool of naïve T cells required to control infections.

Proceedings of the National Academy of Sciences, 2011

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetratio... more IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin–Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary unc...

Proceedings of the National Academy of Sciences, 2011

IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it pred... more IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization u...

Nature Biotechnology, 2011

Journal of Virology, 2011

Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protec... more Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of...

The Journal of Immunology, 2005

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in nonconvalent association with ... more The neonatal FcR (FcRn) consists of an MHC class I-like H chain in nonconvalent association with ␤ 2-microglobulin (␤ 2 m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the ␤ 2 m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and ␤ 2 m, CNX bound to the FcRn H chain before the FcRn H chain association with ␤ 2 m. However, calreticulin only bound to the FcRn H chain-␤ 2 m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for ␤ 2 m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.

The Journal of Immunology, 2007

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn... more The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-α rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-κB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-κB (IκBα; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-κB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex ...

The Journal of Immunology, 2011

IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune ... more IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune cells, thereby triggering the release of inflammatory mediators as well as enhancing Ag presentations. IgE is frequently present in airway secretions, and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we show that CD23 (FcεRII) is constitutively expressed in established or primary human airway epithelial cells, and its expression is significantly upregulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, human IgE or IgE-derived immune complex (IC) was transported across a polarized Calu-3 monolayer. Exposure of the Calu-3 monolayer to IL-4 stimulation also enhanced the transcytosis of either human IgE or the IC. A CD23-specific Ab or soluble CD23 significantly reduced the efficiency of IgE or IC transcytosis...

The Journal of Immunology, 2011

The FcγRs found on macrophages (Mϕs) and dendritic cells (DCs) efficiently facilitate the present... more The FcγRs found on macrophages (Mϕs) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Mϕs and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8+ T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4+ T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Mϕs and DCs. The presentation of phagocytosed OVA-ICs to CD4+ T cells was considerably enhanced on wild-type versus FcRn-deficient Mϕs, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG–FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Mϕs but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of p...

Journal of Clinical Investigation, 1999

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in ... more The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

Biochemical Journal, 2002

The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated wit... more The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated with β2-microglobulin (β2m). In β2m-/- mice, FcRn functions are greatly impaired. We sought to determine how FcRn HC, particularly its structure and biogenesis, is affected by the absence of β2m. Human FcRn HC, expressed from the β2m-null cell line FO-1FcRn, was present as a monomeric 45-kDa protein under reducing conditions but primarily as a 92-kDa oligomeric protein under non-reducing conditions. Two-dimensional electrophoresis and MS analysis showed that the 92-kDa protein was a dimer of the 45-kDa HC. Immunostaining showed that FcRn HC in FO-1FcRn was co-localized with the endoplasmic reticulum (ER) protein Bip/GRP78 but not with an endosome protein, EEA1. In contrast, FcRn HC in FO-1FcRn+β2m was detected in both the ER and endosome. The dimeric HC in FcRn oligomers was free of β2m association in FO-1FcRn+β2m. Mutation of non-paired cysteine residues at positions 48 and 251 within the ...

SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and a... more SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and airborne aerosols. To prevent viral infection and reduce viral spread, vaccine strategies must elicit protective immunity in the airways. FcRn transfers IgG across epithelial barriers; we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized S protein; the resulting S-Fc bound to S-specific antibodies (Ab) and FcRn. A significant increase in Ab responses was observed following the intranasal immunization of mice with S-Fc formulated in CpG as compared to the immunization with S alone or PBS. Furthermore, we intranasally immunize adult or aged mice and hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2, including Delta and Omicron variants. Intranasal immunization also significantly reduced viral transmission between immunized ...

The Journal of Immunology

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against ... more The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal...

Nature Communications, 2019

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity... more Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with β2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune...

Clinical and vaccine immunology : CVI, Jan 12, 2017

A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable ... more A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable and broadly protective antibodies. Many vaccine programs focus on the immune responses to critical epitopes in the gp120 portion of HIV envelope glycoprotein (Env) and seek to improve the quality and quantity of antibodies by altering the sequence, conformation, oligomerization or glycosylation of gp120 to activate appropriate germline B cells and mimic the subsequent maturation pathways seen in infected individuals. As a complement to these strategies, we developed dimeric fusion protein immunogens consisting of HIVBaL gp120 monomer attached to a Gly/Ser linker that is in turn, fused to one half of the dimeric Fc domain from rhesus macaque IgG1 (Env-rFc). We envisioned that Env-rFc may mimic some aspects of immune complexes by binding Fc gamma receptors (FcγRs) on immune cells to increase the strength, breadth, and durability of Env-specific antibody responses. The Env-rFc retained a ca...

Scientific reports, May 16, 2016

CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whet... more CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whether CD23 has any role in B-cell activation remains unclear. We examined the expression of CD23 in different subsets of peripheral B cells and the impact of CD23 expression on the early events of B-cell receptor (BCR) activation using CD23 knockout (KO) mice. We found that in addition to marginal zone B cells, mature follicular B cells significantly down regulate the surface expression level of CD23 after undergoing isotype switch and memory B-cell differentiation. Upon stimulation with membrane-associated antigen, CD23 KO causes significant increases in the area of B cells contacting the antigen-presenting membrane and the magnitude of BCR clustering. This enhanced cell spreading and BCR clustering is concurrent with increases in the levels of phosphorylation of tyrosine and Btk, as well as the levels of F-actin and phosphorylated Wiskott Aldrich syndrome protein, an actin nucleation pro...

Infection and immunity, Jan 26, 2015

Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of men... more Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real-time. Neutrophils were directly seen to chase C. neoformans, and then rapidly internalize it. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in an optimal phagocytosis and subsequent killing of the organisms. Addition of recombinant C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 MAP kinases in neutrophils. Inhibition of p38 MAPK pathway, but not Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils towards C. neoformans, bu...

PLOS Pathogens, 2015

African trypanosomes are extracellular protozoan parasites causing a chronic debilitating disease... more African trypanosomes are extracellular protozoan parasites causing a chronic debilitating disease associated with a persistent inflammatory response. Maintaining the balance of the inflammatory response via downregulation of activation of M1-type myeloid cells was previously shown to be crucial to allow prolonged survival. Here we demonstrate that infection with African trypanosomes of IL-27 receptor-deficient (IL-27R-/-) mice results in severe liver immunopathology and dramatically reduced survival as compared to wild-type mice. This coincides with the development of an exacerbated Th1-mediated immune response with overactivation of CD4 + T cells and strongly enhanced production of inflammatory cytokines including IFN-γ. What is important is that IL-10 production was not impaired in infected IL-27R-/mice. Depletion of CD4 + T cells in infected IL-27R-/mice resulted in a dramatically reduced production of IFN-γ, preventing the early mortality of infected IL-27R-/mice. This was accompanied by a significantly reduced inflammatory response and a major amelioration of liver pathology. These results could be mimicked by treating IL-27R-/mice with a neutralizing anti-IFN-γ antibody. Thus, our data identify IL-27 signaling as a novel pathway to prevent early mortality via inhibiting hyperactivation of CD4 + Th1 cells and their excessive secretion of IFN-γ during infection with African trypanosomes. These data are the first to demonstrate the essential role of IL-27 signaling in regulating immune responses to extracellular protozoan infections.

Mucosal immunology, Jan 18, 2015

The epithelial lining of the airway tract and allergen-specific IgE are considered essential cont... more The epithelial lining of the airway tract and allergen-specific IgE are considered essential controllers of inflammatory responses to allergens. The human low affinity IgE receptor, CD23 (FcɛRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains unknown whether the CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo, and whether inhibition of this pathway attenuates allergic inflammation. To this end, we show that in wild-type (WT) mice, epithelial CD23 transcytosed both IgE and ovalbumin (OVA)-IgE complexes across the airway epithelial barrier, whereas neither type of transcytosis was observed in CD23 knockout (KO) mice. In chimeric mice, OVA sensitization and aerosol challenge of WT/WT (bone-marrow transfer from the WT to WT) or CD23KO/WT (CD23KO to WT) chimeric mice, which express CD23 on radioresistant airway structural cells (ma...

Human vaccines & immunotherapeutics, Jan 19, 2015

The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In ... more The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humo...

Proceedings of the National Academy of Sciences, 2014

Significance T lymphocytes are white blood cells that recognize and fight pathogens. Maintenance ... more Significance T lymphocytes are white blood cells that recognize and fight pathogens. Maintenance of sufficient numbers of T cells is essential to prevent susceptibility to infections. Survival of quiescent T cells is maintained, in part, by the interaction between the soluble factor (IL-7 produced by various stromal cells) and the IL-7 receptor (IL-7R) expressed on the surface of T cells. Here, we show that naïve T cells have basal nuclear levels of the transcription factor NF-κB and that is key to maintain IL-7R expression in T cells and for their survival. Our results imply that antiinflammatory therapies targeting NF-κB may affect the pool of naïve T cells required to control infections.

Proceedings of the National Academy of Sciences, 2011

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetratio... more IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin–Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary unc...

Proceedings of the National Academy of Sciences, 2011

IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it pred... more IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization u...

Nature Biotechnology, 2011

Journal of Virology, 2011

Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protec... more Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of...

The Journal of Immunology, 2005

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in nonconvalent association with ... more The neonatal FcR (FcRn) consists of an MHC class I-like H chain in nonconvalent association with ␤ 2-microglobulin (␤ 2 m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the ␤ 2 m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and ␤ 2 m, CNX bound to the FcRn H chain before the FcRn H chain association with ␤ 2 m. However, calreticulin only bound to the FcRn H chain-␤ 2 m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for ␤ 2 m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.

The Journal of Immunology, 2007

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn... more The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-α rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-κB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-κB (IκBα; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-κB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex ...

The Journal of Immunology, 2011

IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune ... more IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune cells, thereby triggering the release of inflammatory mediators as well as enhancing Ag presentations. IgE is frequently present in airway secretions, and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we show that CD23 (FcεRII) is constitutively expressed in established or primary human airway epithelial cells, and its expression is significantly upregulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, human IgE or IgE-derived immune complex (IC) was transported across a polarized Calu-3 monolayer. Exposure of the Calu-3 monolayer to IL-4 stimulation also enhanced the transcytosis of either human IgE or the IC. A CD23-specific Ab or soluble CD23 significantly reduced the efficiency of IgE or IC transcytosis...

The Journal of Immunology, 2011

The FcγRs found on macrophages (Mϕs) and dendritic cells (DCs) efficiently facilitate the present... more The FcγRs found on macrophages (Mϕs) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Mϕs and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8+ T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4+ T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Mϕs and DCs. The presentation of phagocytosed OVA-ICs to CD4+ T cells was considerably enhanced on wild-type versus FcRn-deficient Mϕs, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG–FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Mϕs but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of p...

Journal of Clinical Investigation, 1999

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in ... more The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

Biochemical Journal, 2002

The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated wit... more The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated with β2-microglobulin (β2m). In β2m-/- mice, FcRn functions are greatly impaired. We sought to determine how FcRn HC, particularly its structure and biogenesis, is affected by the absence of β2m. Human FcRn HC, expressed from the β2m-null cell line FO-1FcRn, was present as a monomeric 45-kDa protein under reducing conditions but primarily as a 92-kDa oligomeric protein under non-reducing conditions. Two-dimensional electrophoresis and MS analysis showed that the 92-kDa protein was a dimer of the 45-kDa HC. Immunostaining showed that FcRn HC in FO-1FcRn was co-localized with the endoplasmic reticulum (ER) protein Bip/GRP78 but not with an endosome protein, EEA1. In contrast, FcRn HC in FO-1FcRn+β2m was detected in both the ER and endosome. The dimeric HC in FcRn oligomers was free of β2m association in FO-1FcRn+β2m. Mutation of non-paired cysteine residues at positions 48 and 251 within the ...