Interleukin-2 inhibits germinal center formation by limiting T follicular helper cell differentiation.

T follicular helper (Tfh) cells promote T cell-dependent humoral immune responses by providing T cell help to B cells and by promoting germinal center (GC) formation and long-lived antibody responses. However, the cellular and molecular mechanisms that control Tfh cell differentiation in vivo are incompletely understood. Here we show that interleukin-2 (IL-2) administration impaired influenza-specific GCs, long-lived IgG responses, and Tfh cells. IL-2 did not directly inhibit GC formation, but instead suppressed the differentiation of Tfh cells, thereby hindering the maintenance of influenza-specific GC B cells. Our data demonstrate that IL-2 is a critical factor that regulates successful Tfh and B cell responses in vivo and regulates Tfh cell development.

Live attenuated influenza vaccine (LAIV) impacts innate and adaptive immune responses.

Influenza A infection induces a massive inflammatory response in the lungs that leads to significant illness and increases the susceptibility to secondary bacterial pneumonia. The most efficient way to prevent influenza infection is through vaccination. While inactivated vaccines induce protective levels of serum antibodies to influenza hemaglutinin (HA) and neuraminidase (NA) surface proteins, these are strain specific and offer little protection against heterosubtypic influenza viruses. In contrast, live attenuated influenza vaccines (LAIVs) induce a T cell response in addition to antibody responses against HA and NA surface proteins. Importantly, LAIV vaccination induces a response in a mouse model that protects against illness due to heterosubtypic influenza strains. While it is not completely clear what is the mechanism of action of LAIV heterosubtypic protection in humans, it has been shown that LAIV induces heterosubtypic protection in mice that is dependent upon a Type 1 immune response and requires CD8 T cells. In this study, we show that LAIV-induced immunity leads to significantly reduced viral titers and inflammatory responses in the lungs of mice following heterosubtypic infection. Not only are viral titers reduced in LAIV vaccinated mice, the amounts of inflammatory cytokines and chemokines in lung tissue are significantly lower. Additionally, we show that LAIV vaccination of healthy adults also induces a robust Type 1 memory response including the production of chemokines and cytokines involved in T cell activation and recruitment. Thus, our results indicate that LAIV vaccination functions by inducing immune memory which can act to modulate the immune response to subsequent heterosubtypic challenge by influencing both innate and adaptive responses.

Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus.

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance.

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens.

The omentum is a site of B1 cell lymphopoiesis and immune responsiveness to T cell-independent antigens. However, it is unknown whether it supports immune responses independently of conventional lymphoid organs. We showed that the omentum collected antigens and cells from the peritoneal cavity and supported T cell-dependent B cell responses, including isotype switching, somatic hypermutation, and limited affinity maturation, despite the lack of identifiable follicular dendritic cells. The omentum also supported CD4+ and CD8+ T cell responses to peritoneal antigens and recruited effector T cells primed in other locations. Unlike conventional lymphoid organs, milky spots in the omentum developed in the absence of lymphoid tissue-inducer cells, but required the chemokine CXCL13. Although the lymphoid architecture of milky spots was disrupted in lymphotoxin-deficient mice, normal architecture was restored by reconstitution with lymphotoxin-sufficient hematopoietic cells. These results indicate that the milky spots of the omentum function as unique secondary lymphoid organs that promote immunity to peritoneal antigens.

A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus.

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.

B cells promote resistance to heterosubtypic strains of influenza via multiple mechanisms.

Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.

The effect of B cell receptor signaling on antigen endocytosis and processing.

B cell receptor (BCR)-mediated antigen processing and presentation involves both the BCR-mediated internalization and processing of cognate antigen as well as the formation and expression of antigenic peptide-MHC class II complexes. While BCR signaling is known to result in changes in the biosynthesis and intracellular trafficking of class II molecules, the effect of BCR signaling on the cell biology of antigen endocytosis and processing is less clear. Therefore, the effect of BCR signaling on the cell biology of fluid phase antigen endocytosis, processing and presentation was analyzed in both B cell lines or in normal splenic B cells. The results demonstrate that BCR signaling alters neither the global level of fluid phase antigen endocytosis nor the duration of intracellular persistence of fluid phase internalized antigen. Moreover, while BCR signal does result in an increase in the level of total cell surface MHC class II molecules as well as specific peptide-class II complexes, stimulation failed to alter the fraction of class II molecules loaded with antigen-derived peptide. These results indicate that while BCR-mediated signaling elicits an increase in the expression of antigenic peptide-class II complexes, signaling does not augment antigen presentation by profoundly altering the basic biology of antigen endocytosis and processing. These results also demonstrate that the high efficiency of BCR-mediated antigen processing (when compared to fluid phase antigen processing) is likely to occur independent of BCR signaling-induced global alterations in the biology of endocytosis, processing and presentation. This finding suggests that if BCR signaling augments the efficiency of processing of cognate antigen, it must impact unique aspects of BCR-mediated antigen processing, such as the intracellular persistence of internalized antigen-BCR complexes.

Lipid raft-independent B cell receptor-mediated antigen internalization and intracellular trafficking.

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.

Changes in hormone sensitivity in the ventral prostate of aging Sprague-Dawley rats.

The adult rat ventral prostate, which has been used extensively as a model for hormone-dependent prostate cancer, is composed of hormone-dependent columnar secretory epithelial cells and a mixture of hormone-independent cuboidal epithelial cells, basal epithelial cells, and stromal cells. Androgen ablation causes the gland to regress due to the selective loss of the secretory luminal epithelial cells that undergo apoptosis. Most, if not all, of the studies examining the induction of apoptosis and the mechanism of regression have used young adult males at around 3 months of age. Prostate cancer, however, is a disease of older males, and we have therefore investigated whether age-related changes in hormone sensitivity and apoptosis occur in the ventral prostate of aged animals (12 months old) compared to young animals (3 months old). We have observed distinct differences in the morphology of the prostate between young and old rats prior to castration and a significant slowing in the rate of regression after castration in older animals. These changes are accompanied by changes in lipofuscin accumulation and levels of the antioxidant enzymes catalase and manganese (Mn) superoxide dismutase in the gland.