Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies.

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Increased cathepsin S in Prdm1-/- dendritic cells alters the TFH cell repertoire and contributes to lupus.

Aberrant population expansion of follicular helper T cells (TFH cells) occurs in patients with lupus. An unanswered question is whether an altered repertoire of T cell antigen receptors (TCRs) is associated with such expansion. Here we found that the transcription factor Blimp-1 (encoded by Prdm1) repressed expression of the gene encoding cathepsin S (Ctss), a cysteine protease that cleaves invariant chains and produces antigenic peptides for loading onto major histocompatibility complex (MHC) class II molecules. The increased CTSS expression in dendritic cells (DCs) from female mice with dendritic cell-specific conditional knockout of Prdm1 (CKO mice) altered the presentation of antigen to CD4+ T cells. Analysis of complementarity-determining region 3 (CDR3) regions containing the ß-chain variable region (Vß) demonstrated a more diverse repertoire of TFH cells from female CKO mice than of those from wild-type mice. In vivo treatment of CKO mice with a CTSS inhibitor abolished the lupus-related phenotype and reduced the diversity of the TFH cell TCR repertoire. Thus, Blimp-1 deficiency in DCs led to loss of appropriate regulation of Ctss expression in female mice and thereby modulated antigen presentation and the TFH cell repertoire to contribute to autoimmunity.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Plasmacytoid Dendritic Cells and Type I Interferon Promote Extrafollicular B Cell Responses to Extracellular Self-DNA.

Class-switched antibodies to double-stranded DNA (dsDNA) are prevalent and pathogenic in systemic lupus erythematosus (SLE), yet mechanisms of their development remain poorly understood. Humans and mice lacking secreted DNase DNASE1L3 develop rapid anti-dsDNA antibody responses and SLE-like disease. We report that anti-DNA responses in Dnase1l3-/- mice require CD40L-mediated T cell help, but proceed independently of germinal center formation via short-lived antibody-forming cells (AFCs) localized to extrafollicular regions. Type I interferon (IFN-I) signaling and IFN-I-producing plasmacytoid dendritic cells (pDCs) facilitate the differentiation of DNA-reactive AFCs in vivo and in vitro and are required for downstream manifestations of autoimmunity. Moreover, the endosomal DNA sensor TLR9 promotes anti-dsDNA responses and SLE-like disease in Dnase1l3-/- mice redundantly with another nucleic acid-sensing receptor, TLR7. These results establish extrafollicular B cell differentiation into short-lived AFCs as a key mechanism of anti-DNA autoreactivity and reveal a major contribution of pDCs, endosomal Toll-like receptors (TLRs), and IFN-I to this pathway.

Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants.

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1-4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.

Peripheral T cell activation, not thymic selection, expands the T follicular helper repertoire in a lupus-prone murine model.

Many autoimmune diseases are characterized by the activation of autoreactive T cells. The T cell repertoire is established in the thymus; it remains uncertain whether the presence of disease-associated autoreactive T cells reflects abnormal T cell selection in the thymus or aberrant T cell activation in the periphery. Here, we describe T cell selection, activation, and T cell repertoire diversity in female mice deficient for B lymphocyte-induced maturation protein (BLIMP)-1 in dendritic cells (DCs) (Prdm1 CKO). These mice exhibit a lupus-like phenotype with an expanded population of T follicular helper (Tfh) cells having a more diverse T cell receptor (TCR) repertoire than wild-type mice and, in turn, develop a lupus-like pathology. To understand the origin of the aberrant Tfh population, we analyzed the TCR repertoire of thymocytes and naive CD4 T cells from Prdm1 CKO mice. We show that early development and selection of T cells in the thymus are not affected. Importantly, however, we observed increased TCR signal strength and increased proliferation of naive T cells cultured in vitro with antigen and BLIMP1-deficient DCs compared to control DCs. Moreover, there was increased diversity in the TCR repertoire in naive CD4+ T cells stimulated in vitro with BLIMP1-deficient DCs. Collectively, our data indicate that lowering the threshold for peripheral T cell activation without altering thymic selection and naive T cell TCR repertoire leads to an expanded repertoire of antigen-activated T cells and impairs peripheral T cell tolerance.

CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy.

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.

Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre­steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5'-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

Hypersensitivity to ferroptosis in chromophobe RCC is mediated by a glutathione metabolic dependency and cystine import via solute carrier family 7 member 11.

Chromophobe (Ch) renal cell carcinoma (RCC) arises from the intercalated cell in the distal nephron. There are no proven treatments for metastatic ChRCC. A distinguishing characteristic of ChRCC is strikingly high levels of reduced (GSH) and oxidized (GSSG) glutathione. Here, we demonstrate that ChRCC-derived cells exhibit higher sensitivity to ferroptotic inducers compared with clear-cell RCC. ChRCC-derived cells are critically dependent on cystine via the cystine/glutamate antiporter xCT to maintain high levels of glutathione, making them sensitive to inhibitors of cystine uptake and cyst(e)inase. Gamma-glutamyl transferase 1 (GGT1), a key enzyme in glutathione homeostasis, is markedly suppressed in ChRCC relative to normal kidney. Importantly, GGT1 overexpression inhibits the proliferation of ChRCC cells in vitro and in vivo, suppresses cystine uptake, and decreases levels of GSH and GSSG. Collectively, these data identify ferroptosis as a metabolic vulnerability in ChRCC, providing a potential avenue for targeted therapy for these distinctive tumors.

Do Oral and Silent Word-Reading Fluency Rely on the Same Cognitive-Linguistic Skills? Evidence from a Cross-Sectional Study in Greek.

Researchers tend to use oral- and silent-reading fluency measures interchangeably and to generalize research findings across reading modes, especially from oral to silent reading. In this study, we sought to examine if oral and silent word-reading fluency rely on the same cognitive-linguistic skills. Three hundred and forty-five Greek children (80 from Grade 2, 85 from Grade 4, 91 from Grade 6, and 89 from Grade 10) were assessed on measures of general cognitive ability, speed of processing, phonological awareness, rapid automatized naming, orthographic knowledge, articulation rate, and word-reading fluency (oral and silent). Results of hierarchical regression analyses revealed that phonological awareness was a unique predictor of both reading outcomes in Grade 2 and orthographic knowledge was a unique predictor of both reading outcomes in Grades 4, 6, and 10. However, rapid automatized naming predicted only oral word-reading fluency. These findings suggest that silent and oral word-reading fluency do not necessarily rely on the same cognitive-linguistic skills at the same grade level and we need to exercise some caution when we generalize the findings across reading modes.

Enzymatic depletion of l-Met using an engineered human enzyme as a novel therapeutic strategy for melanoma.

Many cancers, including melanoma, have a higher requirement for l-methionine in comparison with noncancerous cells. In this study, we show that administration of an engineered human methionine-γ-lyase (hMGL) significantly reduced the survival of both human and mouse melanoma cells in vitro. A multiomics approach was utilized to identify global changes in gene expression and in metabolite levels with hMGL treatment in melanoma cells. There was considerable overlap in the perturbed pathways identified in the two data sets. Common pathways were flagged for further investigation to understand their mechanistic importance. In this regard, hMGL treatment induced S and G2 phase cell cycle arrest, decreased nucleotide levels, and increased DNA double-strand breaks suggesting an important role for replication stress in the mechanism of hMGL effects on melanoma cells. Further, hMGL treatment resulted in increased cellular reactive oxygen species levels and increased apoptosis as well as uncharged transfer RNA pathway upregulation. Finally, treatment with hMGL significantly inhibited the growth of both mouse and human melanoma cells in orthotopic tumor models in vivo. Overall, the results of this study provide a strong rationale for further mechanistic evaluation and clinical development of hMGL for the treatment of melanoma skin cancer and other cancers.

Improving Antibody Therapeutics by Manipulating the Fc Domain: Immunological and Structural Considerations.

Interactions between the crystallizable fragment (Fc) domain of antibodies and a plethora of cellular Fc receptors (FcRs) or soluble proteins form a critical link between humoral and innate immunity. In particular, the immunoglobulin G Fc domain is critical for the clearance of target cells by processes that include (a) cytotoxicity, phagocytosis, or complement lysis; (b) modulation of inflammation; (c) antigen presentation; (d) antibody-mediated receptor clustering; and (e) cytokine release. More than 30 Fc-engineered antibodies aimed primarily at tailoring these effects for optimal therapeutic outcomes are in clinical evaluation or have already been approved. Nonetheless, our understanding of how FcR engagement impacts various immune cell phenotypes is still largely incomplete. Recent insights into FcR biology coupled with advances in Fc:FcR structural analysis, Fc engineering, and mouse models that recapitulate human biology are helping to fill in existing knowledge gaps. These advances will provide a blueprint on how to fine-tune the Fc domain to achieve optimal therapeutic efficacy.

Serial and discrete naming and reading in Chinese first graders: Testing predictions from the cascaded processing hypothesis.

Recent studies have suggested that-beyond automaticity and prosody-reading fluency involves parallel processing of adjacent items presented in a sequence, termed "cascaded processing." To date, most studies examining cascaded processing have been conducted in alphabetic orthographies. Thus, the purpose of this study was to examine the cascaded processing hypothesis in Chinese. A total of 119 Grade 1 Chinese children (61 boys and 58 girls; Mage = 7.30 years, SD = 0.31) were assessed on serial and discrete naming of digits as well as on serial and discrete naming of high-frequency one- and two-character words and low-frequency one-character words presented with pinyin. Results of hierarchical regression analyses showed, first, that serial digit naming was a unique predictor of discrete naming of low-frequency one-character words and two-character words, but not of high-frequency one-character words. Second, serial digit naming was a unique predictor of reading of high-frequency one- and two-character word reading after controlling for discrete word reading. These findings suggest that Chinese first graders process high-frequency characters holistically (similar to simple digits), which then facilitates parallel processing of multiple stimuli when they are presented in a sequence.

Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.