Cord blood proteomics identifies biomarkers of early-onset neonatal sepsis

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Abstract

BACKGROUND. Symptoms of early-onset sepsis (EOS) in preterm infants are nonspecific, overlapping with normal postnatal physiological adaptations and noninfectious pathologies. This clinical uncertainty and the lack of reliable EOS diagnostics results in liberal use of antibiotics in the first days to weeks of life, leading to increased risk of antibiotic-related morbidities in infants who do not have an invasive infection. METHODS. To identify potential biomarkers for EOS in newborn infants, we used unlabelled tandem mass spectrometry proteomics to identify differentially abundant proteins in the umbilical cord blood of infants with and without culture-confirmed EOS. Proteins were then confirmed using immunoassay, and logistic regression and random forest models were built including both biomarker concentration and clinical variables to predict EOS. RESULTS. These data identified five proteins that were significantly upregulated in infants with EOS, three of which (serum amyloid A, C-reactive protein, and lipopolysaccharide-binding protein) were confirmed using a quantitative immunoassay. The random forest classifier for EOS was applied to a cohort of infants with culture-negative presumed sepsis (PS). Most PS infants were classified as resembling control infants, having low EOS biomarker concentrations. CONCLUSION. These results suggest that cord blood biomarker screening may be useful for early stratification of EOS risk among neonates, improving targeted, evidence-based use of antibiotics early in life. FUNDING. National Institutes of Health, Gerber Foundation, Friends of Prentice, Thrasher Research Fund, Ann & Robert H. Lurie Children’s Hospital, Stanley Manne Children’s Research Institute of Lurie Children’s.

Authors

Leena B. Mithal, Mark E. Becker, Ted Ling-Hu, Young Ah Goo, Sebastian Otero, Aspen Kremer, Surya Pandey, Nicola Lancki, Yawei Li, Yuan Luo, William Grobman, Denise Scholtens, Karen K. Mestan, Patrick C. Seed, Judd F. Hultquist

Pentose phosphate pathway inhibition metabolically reprograms CD8⁺ T cells and disrupts CNS autoimmunity

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Abstract

Multiple sclerosis is characterized by CNS infiltration of auto-reactive immune cells that drive both acute inflammatory demyelination and chronic progressive axonal and neuronal injury. Expanding evidence implicates CD8+ anti-neural T cells in the irreversible neurodegeneration that underlies progression in multiple sclerosis, yet therapies specifically targeting this cell population are limited. CD8+ T cells from patients with MS exhibit increased engagement of the pentose phosphate pathway. Pharmacologic inhibition of the pentose phosphate pathway reduced glycolysis, glucose uptake, NADPH production, ATP production, proliferation, and proinflammatory cytokine secretion in CD8+ T cells activated by ligation of CD3 and CD28. Pentose phosphate pathway inhibition also prevented CD8+ T cell-mediated antigen-specific neuronal injury in vitro and in both an adoptive transfer-based cuprizone model of demyelination and in mice with experimental autoimmune encephalomyelitis. Notably, transcriptional profiling of CNS-infiltrating CD8+ T cells in patients with MS indicated increased pentose phosphate pathway engagement, suggesting that this pathway is involved in CD8+ T cell-mediated injury of axons and neurons in the demyelinated CNS. Inhibiting the pentose phosphate pathway disrupts CD8+ T cell metabolic reprogramming and effector functions, suggesting that such inhibition may serve as a therapeutic strategy to prevent neurodegeneration in patients with progressive MS.

Authors

Ethan M. Grund, Benjamin D.S. Clarkson, Susanna Pucci, Maria S. Westphal, Carolina Muniz Partida, Sara A. Muhammad, Charles L. Howe

CD154:CD11b blockade enhances CD8⁺ T cell differentiation during infection but not transplantation

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Abstract

CD154 is a promising target for immunosuppression in transplantation, autoimmunity, and inflammatory diseases. We previously identified CD11b as a novel alternative receptor for CD154 during alloimmunity. However, the impact of specific CD154:CD11b blockade on immune responses to infection has not been well characterized. Here, we have shown that in contrast with its immunosuppressive effect on graft-specific CD8+ T cells, CD154:CD11b blockade unexpectedly improved both the quantity and quality of murine herpesvirus-68–specific CD8+ T cells as measured by an increase in tetramer-positive KLRG1loCD127hi memory precursor effector cells. The differential effect of CD154:CD11b blockade on graft- versus virus-specific CD8+ T cells was underpinned by differences in phosphorylated S6 downstream of mTOR complex 1; however, differential expression of key transcription factors Eomes and TCF-1 was dictated by the type of antigen stimulus. These data demonstrate that priming conditions play an important role in determining the outcome of immunotherapy and suggest that specific inhibition of CD154:CD11b interactions could be effective for suppressing alloimmune responses while maintaining protective immunity to minimize infectious complications following transplantation.

Authors

Katie L. Alexander, Kelsey B. Bennion, Danya Liu, Mandy L. Ford

Pan-H7 influenza human antibody virus neutralization depends on avidity and steric hindrance

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Abstract

H7N9 avian influenza virus is a zoonotic influenza virus of public health concern, with a 39% mortality rate in humans. H7N9-specific prevention or treatments for humans have not been approved. We previously isolated a human monoclonal antibody (mAb) designated H7-235 that broadly reacts to diverse H7 viruses and neutralizes H7N9 viruses in vitro. Here, we report the crystal structure of H7 HA1 bound to the fragment antigen-binding region (Fab) of recombinant H7-235 (rH7-235). The crystal structure revealed that rH7-235 recognizes residues near but outside of the receptor binding site (RBS). Nevertheless, the rH7-235 IgG potently inhibits hemagglutination mediated by H7N9 viruses due to avidity effect and Fc steric hindrance. This mAb prophylactically protects mice against weight loss and death caused by challenge with lethal H7N9 viruses in vivo. rH7-235 mAb neutralizing activity alone is sufficient for protection when used at high dosed in a prophylactic setting. This study provides insights into mechanisms of viral neutralization by protective broadly reactive anti-H7 antibodies informing the rational design of therapeutics and vaccines against H7N9 influenza virus.

Authors

Iuliia M. Gilchuk, Jinhui Dong, Ryan P. Irving, Cameron D. Buchman, Erica Armstrong, Hannah L. Turner, Sheng Li, Andrew B. Ward, Robert H. Carnahan, James E. Crowe Jr.

A CARD9-deficiency mouse model recapitulates human chronic CNS candidiasis, identifying defective monocytic-cell responses in immunopathogenesis

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Abstract

Human Caspase Recruitment Domain Containing Protein 9 (CARD9) deficiency predisposes to invasive fungal disease, particularly by Candida spp. Distinctly, CARD9-deficiency causes chronic central nervous system (CNS) candidiasis. Currently, no animal model recapitulates the chronicity of disease, precluding a better understanding of immunopathogenesis. We established a knock-in mouse homozygous for the recurring p.Y91H mutation (Y91HKI) and, in parallel to Card9-/- mice, titrated the intravenous fungal inoculum to the CARD9-genotype to develop a model of chronic invasive candidiasis. Strikingly, CARD9-deficient mice had predominantly CNS involvement, with neurological symptoms appearing late during infection and progressive brain fungal burden in the absence of fulminant sepsis, reflecting the human syndrome. Mononuclear cell aggregation at fungal lesions in the brain correlated with increased MHCII+Ly6C+ monocyte numbers at day 1 post-infection in WT and Y91HKI mice, but not in Card9-/- mice. At day 4 post-infection, neutrophils and additional Ly6C+ monocytes were recruited to the CARD9-deficient brain. As in humans, Y91HKI mutant mice demonstrated cerebral multinucleated giant cells and granulomata. Subtle immunologic differences between the hypomorphic (p.Y91H) and null mice were noted, perhaps explaining some of the variability seen in humans. Our work established a disease-recapitulating animal model to specifically decipher chronic CNS candidiasis due to CARD9 deficiency.

Authors

Marija Landekic, Isabelle Angers, Yongbiao Li, Marie-Christine Guiot, Marc-André Déry, Annie Beauchamp, Lucie Roussel, Annie Boisvert, Wen Bo Zhou, Christina Gavino, Julia Luo, Stéphane Bernier, Makayla Kazimerczak-Brunet, Yichun Sun, Brendan Snarr, Michail S. Lionakis, Robert T. Wheeler, Irah L. King, Salman Qureshi, Maziar Divangahi, Donald C. Vinh

Fetoplacental extracellular vesicles deliver conceptus-derived antigens to maternal secondary lymphoid tissues for immune recognition

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Abstract

Pregnancy is an immunological paradox where despite a competent maternal immune system, regulatory mechanisms at the fetoplacental interface and maternal secondary lymphoid tissues (SLTs) circumvent rejection of semi-allogeneic concepti. Small extracellular vesicles (sEVs) are a vehicle for intercellular communication; nevertheless, the role of fetoplacental sEVs in transport of antigens to maternal SLTs has not been conclusively demonstrated. Using mice in which the conceptus generates fluoroprobe-tagged sEVs shed by the plasma membrane or released from the endocytic compartment, we show that fetoplacental sEVs are delivered to immune cells in the maternal spleen. Injection of sEVs from placentas of females impregnated with Act-mOVA B6 males elicited suboptimal activation of OVA-specific CD8+ OT-I T cells in virgin females as occurs during pregnancy. Furthermore, when OVA+ concepti were deficient in Rab27a, a protein required for sEV secretion, OT-I cell proliferation in the maternal spleen was decreased. Proteomics analysis revealed that mouse trophoblast sEVs were enriched in antiinflammatory and immunosuppressive mediators. Translational relevance was tested in humanized mice injected using sEVs from cultures of human trophoblasts. Our findings show that sEVs deliver fetoplacental antigens to the mother’s SLTs that are recognized by maternal T cells. Alterations of such a mechanism may lead to pregnancy disorders.

Authors

Juliana S. Powell, Adriana T. Larregina, William J. Shufesky, Mara L.G. Sullivan, Donna Beer Stolz, Stephen J. Gould, Geoffrey Camirand, Sergio D. Catz, Simon C. Watkins, Yoel Sadovsky, Adrian E. Morelli

Unique characteristics of autoantibodies targeting MET in patients with breast and lung cancer

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Abstract

The presence of B cells in tumors is correlated with favorable prognosis and efficient response to immunotherapy. While tumor-reactive antibodies have been detected in several cancer types, identifying antibodies that specifically target tumor-associated antigens remains a challenge. Here, we investigated the antibodies spontaneously elicited during breast and lung cancer that bind the cancer-associated antigen MET. We screened patients with lung (n = 25) and breast (n = 75) cancer and found that 13% had antibodies binding to both the recombinant ectodomain of MET, and the ligand binding part of MET, SEMA. MET binding in the breast cancer cohort was significantly correlated with hormone receptor–positive status. We further conducted immunoglobulin sequencing of peripheral MET-enriched B cells from 6 MET-reactive patients. The MET-enriched B cell repertoire was found to be polyclonal and prone to non-IgG1 subclass. Nine monoclonal antibodies were cloned and analyzed, and these exhibited MET binding, low thermostability, and high polyreactivity. Among these, antibodies 87B156 and 69B287 effectively bound to tumor cells and inhibited MET-expressing breast cancer cell lines. Overall, our data demonstrate that some patients with breast and lung cancer develop polyreactive antibodies that cross-react with MET. These autoantibodies have a potential contribution to immune responses against tumors.

Authors

Michal Navon, Noam Ben-Shalom, Maya Dadiani, Michael Mor, Ron Yefet, Michal Bakalenik-Gavry, Dana Chat, Nora Balint-Lahat, Iris Barshack, Ilan Tsarfaty, Einav Nili Gal-Yam, Natalia T. Freund

High affinity CD16 polymorphism associated with reduced risk of severe COVID-19

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Abstract

CD16 is an activating Fc receptor on natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC), a key mechanism in antiviral immunity. However, the role of NK cell-mediated ADCC in SARS-CoV-2 infection remains unclear, particularly whether it limits viral spread and disease severity or contributes to the immunopathogenesis of COVID-19. We hypothesized that the high-affinity CD16AV176 polymorphism influences these outcomes. Using a novel in vitro reporter system, we demonstrated that CD16AV176 is a more potent and sensitive activator than the common CD16AF176 allele. To assess its clinical relevance, we analyzed 1,027 hospitalized COVID-19 patients from the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC), a comprehensive longitudinal dataset with extensive transcriptomic, proteomic, and clinical data. The high-affinity CD16AV176 allele was associated with a significantly reduced risk of ICU admission, mechanical ventilation, and severe disease trajectories. Lower anti-SARS-CoV-2 IgG titers were correlated to CD16AV176; however, there was no difference in viral load across CD16 genotypes. Proteomic analysis revealed that participants homozygous for CD16AV176 had lower levels of inflammatory mediators. These findings suggest that CD16AV176 enhances early NK cell-mediated immune responses, limiting severe respiratory complications in COVID-19. This study identifies a protective genetic factor against severe COVID-19, informing future host-directed therapeutic strategies.

Authors

Anita E. Qualls, Tasha Tsao, Irene Lui, Shion A. Lim, Yapeng Su, Ernie Chen, Dylan Duchen, Holden T. Maecker, Seunghee Kim-Schulze, Ruth R. Montgomery, Florian Krammer, Charles R. Langelier, Ofer Levy, Lindsey R. Baden, Esther Melamed, Lauren I.R. Ehrlich, Grace A. McComsey, Rafick P. Sekaly, Charles B. Cairns, Elias K. Haddad, Albert C. Shaw, David A. Hafler, David B. Corry, Farrah Kheradmand, Mark A. Atkinson, Scott C. Brakenridge, Nelson I. Agudelo Higuita, Jordan P. Metcalf, Catherine L. Hough, William B. Messer, Bali Pulendran, Kari C. Nadeau, Mark M. Davis, Ana Fernandez-Sesma, Viviana Simon, Monica Kraft, Christian Bime, Carolyn S. Calfee, David J. Erle, Joanna Schaenmann, Al Ozonoff, Bjoern Peters, Steven H. Kleinstein, Alison D. Augustine, Joann Diray-Arce, Patrice M. Becker, Nadine Rouphael, IMPACC Network, Jason D. Goldman, Daniel R. Calabrese, James R. Heath, James A. Wells, Elaine F. Reed, Lewis L. Lanier, Harry Pickering, Oscar A. Aguilar

IL-1β Signaling modulates T follicular helper and regulatory cells in human lymphoid tissues

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Abstract

Dysregulation of T follicular helper (Tfh) and T follicular regulatory (Tfr) cell homeostasis in germinal centers (GCs) can lead to antibody-mediated autoimmunity. While interleukin-1β (IL-1β) modulates the GC response via IL-1R1 and IL-1R2 receptors on follicular T cells in animal models, its role in humans remains unclear. We analyzed Tfh and Tfr phenotypes in human secondary lymphoid organs (tonsils, spleen, and mesenteric lymph nodes) using flow cytometry, single-cell transcriptomics, and in vitro culture, comparing findings with samples from autoimmune patients. We observed organ-specific Tfh/Tfr phenotypes according to activation status and IL-1 receptor expression. An excess of IL-1R1 over IL-1R2 expression promoted a unique activated Tfr subset with Treg and GC-Tfh features. IL-1β signaling via IL-1R1 enhanced follicular T-cell activation and Tfh-to-Tfr differentiation, while IL-1β inhibition upregulated IL-1R1, indicating a tightly regulated process. In autoimmune patients, high IL-1β and circulating Tfr levels correlated with increased autoantibody production, linking inflammation, IL-1β signaling, and Tfr/Tfh balance. Our findings highlight the critical role of IL-1β in follicular T-cell activation and suggest that targeting IL-1β signaling in Tfh and Tfr cells could be a promising strategy for treating antibody-mediated autoimmune diseases.

Authors

Romain Vaineau, Raphaël Jeger-Madiot, Samir Ali-Moussa, Laura Prudhomme, Hippolyte Debarnot, Nicolas Coatnoan, Johanna Dubois, Marie Binvignat, Hélène Vantomme, Bruno Gouritin, Gwladys Fourcade, Paul Engeroff, Aude Belbézier, Romain Luscan, Françoise Denoyelle, Roberta Lorenzon, Claire Ribet, Michelle Rosenzwajg, Bertrand Bellier, David Klatzmann, Nicolas Tchitchek, Stéphanie Graff-Dubois

Rheumatoid arthritis synovial fluid induces JAK-dependent intracellular activation of human sensory neurons

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Abstract

JAK inhibitors (JAKi) are widely used anti-inflammatory drugs. Recent data suggest JAKi have superior effects on pain reduction in rheumatoid arthritis (RA). However, the underlying mechanisms for this observation are not fully understood. We investigated whether JAKi can act directly on human sensory neurons. We analysed RNA sequencing datasets of sensory neurons and found they expressed JAK1 and STAT3. Addition of cell-free RA synovial fluid to human induced pluripotent stem cell (iPSC)-derived sensory neurons led to phosphorylation of STAT3 (pSTAT3), which was completely blocked by the JAKi tofacitinib. Compared to paired serum, RA synovial fluid was enriched for the STAT3 signalling cytokines IL-6, IL-11, LIF, IFN-alpha and IFN-beta, with their requisite receptors present in peripheral nerves post-mortem. Accordingly, these recombinant cytokines induced pSTAT3 in iPSC-derived sensory neurons. Furthermore, IL-6+sIL-6R and LIF upregulated expression of pain-relevant genes with STAT3-binding sites, an effect which was blocked by tofacitinib. LIF also induced neuronal sensitisation, highlighting this molecule as a putative pain mediator. Finally, over time, tofacitinib reduced the firing rate of sensory neurons stimulated with RA synovial fluid. Together, these data indicate that JAKi can act directly on human sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.

Authors

Yuening Li, Elizabeth H. Gray, Rosie Ross, Irene Zebochin, Amy Lock, Laura Fedele, Louisa Janice Kamajaya, Rebecca J. Marrow, Sarah Ryan, Pascal Röderer, Oliver Brüstle, Susan John, Franziska Denk, Leonie S. Taams