Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

A broad antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that fails to protect against both seasonal antigenic variants and the next pandemic. However, antibodies directed to conserved sites can confer broad protection. We identify and characterize a class of human antibodies that engage a previously undescribed, conserved, epitope on the influenza hemagglutinin protein (HA). Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal infection in mice. Their breadth of binding extends across most influenza A serotypes and seasonal human variants. Antibodies to the head-stem interface epitope are present at low frequency in the memory B cell populations of multiple donors. The immunogenicity of the epitope warrants its consideration for inclusion in improved or "universal" influenza vaccines.

Primary germinal center-resident T follicular helper cells are a physiologically distinct subset of CXCR5hiPD-1hi T follicular helper cells.

T follicular helper (Tfh) cells are defined by a Bcl6+CXCR5hiPD-1hi phenotype, but only a minor fraction of these reside in germinal centers (GCs). Here, we examined whether GC-resident and -nonresident Tfh cells share a common physiology and function. Fluorescently labeled, GC-resident Tfh cells in different mouse models were distinguished by low expression of CD90. CD90neg/lo GCTfh cells required antigen-specific, MHCII+ B cells to develop and stopped proliferating soon after differentiation. In contrast, nonresident, CD90hi Tfh (GCTfh-like) cells developed normally in the absence of MHCII+ B cells and proliferated continuously during primary responses. The TCR repertoires of both Tfh subsets overlapped initially but later diverged in association with dendritic cell-dependent proliferation of CD90hi GCTfh-like cells, suggestive of TCR-dependency seen also in TCR-transgenic adoptive transfer experiments. Furthermore, the transcriptomes of CD90neg/lo and CD90hi GCTfh-like cells were enriched in different functional pathways. Thus, GC-resident and nonresident Tfh cells have distinct developmental requirements and activities, implying distinct functions.

Continuous Culture of Mouse Primary B Lymphocytes by Forced Expression of Bach2.

Stable, long-term culture of primary B lymphocytes has many potential scientific and medical applications, but remains an elusive feat. A major obstacle to long-term culture is that in vitro mitogens quickly drive B cells to differentiate into short-lived plasma cells (PCs). PC differentiation is governed by opposing teams of transcription factors: Pax5, Bach2, and Bcl6 suppress PC commitment, whereas IFN regulatory factor 4 and Blimp1 promote it. To determine whether transcriptional programming could prolong B cell culture by blocking PC commitment, we generated mouse primary B cells harboring gain- or loss-of-function in the key transcription factors, continuously stimulated these cells with CD154 and IL-21, and determined growth potential and phenotypes in vitro. We found that transgenic expression of Bach2 prohibits PC commitment and endows B cells with extraordinary growth potential in response to external proliferation and survival cues. Long-term Bach2-transgenic B cell lines have genetically stable BCRs [i.e., do not acquire V(D)J mutations], express high levels of MHC class II and molecules for costimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established transgenic cell line causes the cells to secrete large quantities of Ig. This system has potential applications in mAb production, BCR signaling studies, Ag presentation to T cells, and ex vivo clonal expansion for adoptive cell transfer. Additionally, our results provide insight into molecular control over activated B cell fate and suggest that forced Bach2 expression in vivo may augment germinal center B cell or memory B cell differentiation at the expense of PC commitment.

Ideal Vaccines: Balancing B Cell Recruitment and Differentiation.

In this issue of Immunity, Kato et al. show that high-affinity vaccines targeting rare B cells capable of broadly protective antibody responses are not hindered by promotion of terminal plasmacytic differentiation. These findings provide new understanding into vaccine design and offer important insight into B cell fate decisions.

Cross-Reactivity to Kynureninase Tolerizes B Cells That Express the HIV-1 Broadly Neutralizing Antibody 2F5.

2F5 is an HIV-1 broadly neutralizing Ab that also binds the autoantigens kynureninase (KYNU) and anionic lipids. Generation of 2F5-like Abs is proscribed by immune tolerance, but it is unclear which autospecificity is responsible. We sampled the BCR repertoire of 2F5 knock-in mice before and after the first and second tolerance checkpoints. Nearly all small pre-B (precheckpoint) and 35-70% of anergic peripheral B cells (postcheckpoint) expressed the 2F5 BCR and maintained KYNU, lipid, and HIV-1 gp41 reactivity. In contrast, all postcheckpoint mature follicular (MF) B cells had undergone L chain editing that purged KYNU and gp41 binding but left lipid reactivity largely intact. We conclude that specificity for KYNU is the primary driver of tolerization of 2F5-expressing B cells. The MF and anergic B cell populations favored distinct collections of editor L chains; surprisingly, however, MF and anergic B cells also frequently expressed identical BCRs. These results imply that BCR autoreactivity is the primary determinant of whether a developing B cell enters the MF or anergic compartments, with a secondary role for stochastic factors that slightly mix the two pools. Our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1 and supports activation of anergic B cells as a potential method for HIV-1 vaccination.

Minding the gap: The impact of B-cell tolerance on the microbial antibody repertoire.

B lymphocytes must respond to vast numbers of foreign antigens, including those of microbial pathogens. To do so, developing B cells use combinatorial joining of V-, D-, and J-gene segments to generate an extraordinarily diverse repertoire of B-cell antigen receptors (BCRs). Unsurprisingly, a large fraction of this initial BCR repertoire reacts to self-antigens, and these "forbidden" B cells are culled by immunological tolerance from mature B-cell populations. While culling of autoreactive BCRs mitigates the risk of autoimmunity, it also opens gaps in the BCR repertoire, which are exploited by pathogens that mimic the forbidden self-epitopes. Consequently, immunological tolerance, necessary for averting autoimmune disease, also acts to limit effective microbial immunity. In this brief review, we recount the evidence for the linkage of tolerance and impaired microbial immunity, consider the implications of this linkage for vaccine development, and discuss modulating tolerance as a potential strategy for strengthening humoral immune responses.

Extracellular Processing of Lysyl Oxidase-like 2 and Its Effect on Amine Oxidase Activity.

Overexpression of lysyl oxidase-like 2 (LOXL2) is associated with several hepatic and vascular fibrotic diseases and tumor progression in some aggressive cancers. Secreted LOXL2 promotes extracellular matrix cross-linking by catalyzing the oxidative deamination of peptidyl lysine. A great deal remains to be learned about the post-translational modifications of LOXL2, including whether such modifications modulate enzymatic and disease-promoting activities; such knowledge would inform the development of potential therapies. We discovered that upon secretion in cell culture, LOXL2 undergoes proteolytic processing of the first two of four scavenger receptor cysteine-rich domains at the N-terminus. A similar pattern of processing was also evident in tissue extracts from an invasive ductal carcinoma patient. Processing occurred at 314Arg-315Phe-316Arg-317Lys↓-318Ala-, implicating proprotein convertases. siRNA-mediated knockdown of proprotein convertases (furin, PACE4, and PC5/6), as well as incubation with their recombinant forms, showed that PACE4 is the major protease that acts on extracellular LOXL2. Unlike LOX, which requires cleavage of its propeptide for catalytic activation, cleavage of LOXL2 was not essential for tropoelastin oxidation or for cross-linking of collagen type IV in vitro. However, in the latter case, processing enhanced the extent of collagen cross-linking ∼2-fold at ≤10 nM LOXL2. These results demonstrate an important difference in the regulatory mechanisms for LOX and LOXL2 catalytic activity. Moreover, they pave the way for further studies of potential differential functions of LOXL2 isoforms in fibrosis and tumor progression.

Poly- and autoreactivity of HIV-1 bNAbs: implications for vaccine design.

A central puzzle in HIV-1 research is the inability of vaccination or even infection to reliably elicit humoral responses against broadly neutralizing epitopes in the HIV-1 envelope protein. In infected individuals, broadly neutralizing antibodies (bNAbs) do arise in a substantial minority, but only after 2 or more years of chronic infection. All known bNAbs possess at least one of three traits: a high frequency of somatic hypermutation, a long third complementarity determining region in the antibody heavy chain (HCDR3), or significant poly- or autoreactivity. Collectively, these observations suggest a plausible explanation for the rarity of many types of bNAbs: namely, that their generation is blocked by immunological tolerance or immune response checkpoints, thereby mandating that B cells take a tortuous path of somatic evolution over several years to achieve broadly neutralizing activity. In this brief review, we discuss the evidence for this tolerance hypothesis, its implications for HIV-1 vaccine design, and potential ways to access normally forbidden compartments of the antibody repertoire by modulating or circumventing tolerance controls.

Germinal center responses to complex antigens.

Germinal centers (GCs) are the primary sites of antibody affinity maturation, sites where B-cell antigen-receptor (BCR) genes rapidly acquire mutations and are selected for increasing affinity for antigen. This process of hypermutation and affinity-driven selection results in the clonal expansion of B cells expressing mutated BCRs and acts to hone the antibody repertoire for greater avidity and specificity. Remarkably, whereas the process of affinity maturation has been confirmed in a number of laboratories, models for how affinity maturation in GCs operates are largely from studies of genetically restricted B-cell populations competing for a single hapten epitope. Much less is known about GC responses to complex antigens, which involve both inter- and intraclonal competition for many epitopes. In this review, we (i) compare current methods for analysis of the GC B-cell repertoire, (ii) describe recent studies of GC population dynamics in response to complex antigens, discussing how the observed repertoire changes support or depart from the standard model of clonal selection, and (iii) speculate on the nature and potential importance of the large fraction of GC B cells that do not appear to interact with native antigen.

WITHDRAWN: PACE4 Proteolytically Processes LOXL2 with little impact on its catalytic activity.

This article has been withdrawn by the authors. Figs 1C, 2A, and 2E contained some inadvertently mislabeled data. The authors state that the mislabeling does not affect the conclusions of the article.

Human lysyl oxidase-like 2.

Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu(2+)- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2.

Human copper-dependent amine oxidases.

Copper amine oxidases (CAOs) are a class of enzymes that contain Cu(2+) and a tyrosine-derived quinone cofactor, catalyze the conversion of a primary amine functional group to an aldehyde, and generate hydrogen peroxide and ammonia as byproducts. These enzymes can be classified into two non-homologous families: 2,4,5-trihydroxyphenylalanine quinone (TPQ)-dependent CAOs and the lysine tyrosylquinone (LTQ)-dependent lysyl oxidase (LOX) family of proteins. In this review, we will focus on recent developments in the field of research concerning human CAOs and the LOX family of proteins. The aberrant expression of these enzymes is linked to inflammation, fibrosis, tumor metastasis/invasion and other diseases. Consequently, there is a critical need to understand the functions of these proteins at the molecular level, so that strategies targeting these enzymes can be developed to combat human diseases.

MCF-7 cells expressing nuclear associated lysyl oxidase-like 2 (LOXL2) exhibit an epithelial-to-mesenchymal transition (EMT) phenotype and are highly invasive in vitro.

LOXL2 is a copper- and lysine tyrosylquinone-dependent amine oxidase that has been proposed to function both extracellularly and intracellularly to activate oncogenic signaling pathways leading to EMT and invasion of breast cancer cells. In this study, we selected MCF-7 cells that stably express forms of recombinant LOXL2 differing in their subcellular localizations and catalytic competencies. This enabled us to dissect the molecular functions of intracellular and extracellular LOXL2s and examine their contributions to breast cancer metastasis/invasion. We discovered that secreted LOXL2 (~100-kDa) is N-glycosylated at Asn-455 and Asn-644, whereas intracellular LOXL2 (~75-kDa) is nonglycosylated and N-terminally processed, and is primarily associated with the nucleus. Both forms of LOXL2 can oxidize lysine in solution. However, we found that expression of intracellular LOXL2 is more strongly associated with EMT and invasiveness than secreted LOXL2 in vitro. The results indicate that nuclear associated LOXL2 contributes to the stabilization of Snail1 transcription factor at the protein level to induce EMT and promote invasion in vitro, through repression of E-cadherin, occludin, and estrogen receptor-α, and up-regulation of vimentin, fibronectin, and MT1-MMP.

Post-translational modifications of recombinant human lysyl oxidase-like 2 (rhLOXL2) secreted from Drosophila S2 cells.

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2-6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.