Knoop, Kathryn A.

ABSTRACT Cytotoxic T lymphocytes (CTLs) are known to eliminate target cells through perforin-mediated, contact-dependent killing - a process limited by the number of effector T cells despite its serial nature. CTLs likely also possess a mass killing mechanism that can eliminate target cells more efficiently. Using CAR-T cells against B7H3 present on B16 tumor targets, we demonstrate that activated T cells can also kill targets in a perforin-independent manner by releasing diffusible TNF and IFNγ capable of killing nearby targets in a paracrine fashion even if they bear no antigen. Against an unperturbed target, paracrine killing is inefficient but is enhanced by the deletion of TNFR1 signaling molecules such as TAK1, HOIP, or TBK1/IKKε. Notably, these molecules are inhibited naturally by pathogen-encoded antagonists. Expression of a microbial-encoded antagonist, such as the Yersinia -encoded YopJ that antagonizes TAK1 or Ebola-encoded VP35 that antagonizes TBK1/IKKε, alters the target cell response to these cytokines from non-lethal to lethal. We propose that target cells are inherently resistant to killing by TNF and IFNγ, but the presence of a microbial factor alters this sensitivity, providing for the selective elimination of the infected cell while minimizing harm to uninfected bystander cells. We propose the term ‘pathogen-restriction’ to describe this discriminatory mechanism. Potentially, any microbial-derived factor that crosstalks with the TNF and IFNγ signaling pathways can function as a pathogen-restriction element. GRAPHICAL ABSTRACT Short Summary T cells use TNF and IFNγ to kill target cells in a paracrine manner where target elimination is restricted by the presence of pathogen-derived factors.

Key points Staphylococcus aureus showed a significant increase in relative abundance in CRSwNP patients following endoscopic sinus surgery compared to pre‐surgery samples. Other Staphylococcus species were found to correlate positively with S. aureus in patients with nasal polyps; among those, S. caprae correlated strongly while being the most represented in samples. Patients with recurrent nasal polyp growth exhibited a substantially greater postoperative increase in the relative abundance of S. aureus .

Abstract Description Sepsis, the life-threatening organ dysfunction stemming from the severe inflammatory response to systemic microbial infection, can affect humans of any age. One consequence of sepsis is the development of prolonged immunoparalysis that contributes to increased susceptibility to infection. With vaccination being an important means for protecting humans from infection throughout their lifetime, it is surprising that there has been no study performed to date assessing vaccine efficacy in sepsis survivors. We found neonatal and adult mice given an intramuscularly delivered inactivated multivalent influenza vaccine 30 days after sepsis induction had reduced total and class switched Ab titers, compared to non-septic controls. The reduced humoral response was due, in part, to impaired CD4 T cell and B cell responses to the vaccine. Reduced Ab titers were similarly seen in post-septic mice given an intranasal live-attenuated influenza vaccine. ELISpot data demonstrating reduced numbers of antibody secreting cells in the spleens of vaccinated septic mice further support the ELISA results. Moreover, mice vaccinated prior to sepsis induction also showed reduced antibody titers. Importantly, vaccinated septic mice had reduced protection to a lethal influenza challenge, demonstrated by increased mortality and viral burden in the lungs. These data collectively show how sepsis can lead to increased susceptibility to pathogens normally neutralized by vaccine-induced immunity. Funding Sources Supported by NIH/NIAID R35 GM140881; 5R01 GM139046; 1T32TR004385 Topic Categories Translational and Interventional Immunology (TI)

Abstract Description Fungal species, such as Candida albicans, are commonly found as part of the normal microbiota in both the skin and intestine. However, these fungal species, including C. albicans, can also become opportunistic pathogens and have been associated with intestinal and allergic disorders. We hypothesized C. albicans colonization could occur in early life when the neonatal microbiota is permissive to pathogens and lacks colonization resistance compared to later times in life. To investigate this question, we sequenced stool of premature and term infants and frequently found C. albicans colonization, suggesting fungal colonization within the human GI tract. To investigate the impact of long-term C. albicans colonization to the immune system, we established colonization by oral administration of C. albicans to neonatal mice. Colonization of C. albicans was associated with increased diversity of bacterial species in the intestinal microbiota at weaning. Additionally, we observed significant increases of inflammatory cytokines IL-17 and G-CSF in the serum. Future research will expand on the impact of C. albicans colonization on the intestinal microbiota and immune systems. Funding Sources Center for Individualized Medicine Microbiome Program Research Team Science Award, Mayo Clinic NIH, NIDDK R01 DK134366 Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Late-onset neonatal sepsis (LOS) is often the result of systemic bacteremia developing between 3-90 days of age, predominantly in prematurely born populations. An initial spike of IL-6 serum levels is a hallmark diagnostic tool and has been implicated as a source for the resulting cytokine storm. Yet, other than antibiotics, there is a lack of effective therapeutics targeting the overwhelming inflammatory response. We performed single-organism infections of pathogenic and commensal clinical E. coli isolates to compare the acute cytokine response in our LOS model in neonatal (postnatal day 5-7) and periweaning (postnatal day 17-20) pups. As expected, neonatal pups had increased bacterial burden and mortality during pathogenic infection compared to periweaning-age and commensal infection. An in-depth, 32-cytokine analysis of serum, liver, and spleen revealed overall elevated and tissue-specific inflammation in the neonates with a positive correlation of IL-6 serum levels to many cytokines (MCP-1, M-CSF, MIP-1β, etc.). Isolate-specific responses were assessed in an in vitro co-culture model with RAW-blue macrophages. While both isolates induced inflammatory NFκB/AP-1 activation, commensal E. coli caused elevated IL-1β while pathogenic E. coli led to significantly elevated IL-6. Both age- and pathogenic-specific inflammatory activation suggest unique signaling from the E. coli infection and the neonatal response, calling for neonatal-specific treatment options. Funding Sources Mayo Clinic Graduate School of Biomedical Sciences R01 DK134366 (Knoop) T32 DK124190-04 (Muske) Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Abstract Description Broad spectrum antibiotics are being increasingly used in obstetric patients without long term follow up on neonatal risks. Previous data have suggested antibiotics for preterm premature rupture of membranes increases neonatal late onset sepsis and nectrozing enterocolitis (NEC), but the mechanism is unknown. We analyzed samples from the Mechanisms of Maternal Immunoglobulin A control over the neonatal microbiota and the development of NEC (MAMI) trial. MAMI enrolled infants born under 32 weeks and collected maternal-infant biospecimens. We performed a detailed chart review for maternal antibiotic exposure and analyzed breast milk samples collected 10-45 days post delivery by ELISA. Data were compared by t test and one way ANOVA. Breast milk IgA was higher than IgG and IgM (p < 0.0001, p < 0.005). When compared by antibiotic exposure, IgA and IgM were decreased with any antibiotic exposure (p = 0.02, p = 0.01). IgA was decreased by length of antibiotic exposure (p = 0.02). We stratified by the antibiotic spectrum of activity into narrow (penicillin, ampicillin) and broad (2nd and 3rd generation cephalosporin, gentamicin, etc) and found that the spectrum of antimicrobial activity was associated with a decrease in maternal IgA with broad spectrum antibiotics with the least amount of IgA (p < 0.004). Our data demonstrate mammary IgA and IgM are decreased with antenatal antimicrobial exposure. These data suggest a mechanism by which antenatal antibiotics may lead to neonatal sepsis and NEC. Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Neonatal sepsis caused by bacteria such as Escherichia coli affects 1.3 million infants a year. The immunological responses activated in neonates during neonatal sepsis are currently thought to overwhelm the infant. Understanding where to intervene in the inflammatory response could lead to the development of therapeutics. Using Nur77-GFP reporter mice and our established animal models of neonatal sepsis, we observed γδ T cells-innate-like lymphocytes- are activated in a TCR-dependent manner in response to pathogenic bloodstream E. coli infection in neonatal mice (post-natal day 5-7). We find a subset of γδ T cells show similar levels of TCR-dependent activation of following infection with either pathogenic or commensal E. coli. In pathogenic bloodstream E. coli infection, there is a significant release of pro-inflammatory cytokines IL-17 by these CCR6+ γδ T cells in addition to IL-6 in the serum compared to serum in commensal infection. We found that in neonatal mice, CCR6+ γδ T cells have high expression of IL-6 receptor, and preliminary data suggests IL-6 blockade reduces IL-17 production. In summary, this suggests that during an E. coli systemic infection, the IL-17 producing γδ T cells are being activated by IL-6 due to higher levels of IL-6 receptor. Further work will seek to understand how γδ T cells may detrimentally contribute to neonatal sepsis outcomes. Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Abstract Description Infants are vulnerable to infectious diseases due to the immaturity of their immune system, including a delay in producing pathogen-targeting antibodies. Maternal immunization strategies have increased in use to protect infants from disease by vertical transfer of protective pathogen-specific antibodies. Late-onset sepsis (LOS) is a substantial contributor to neonatal mortality that results from a bloodstream infection at least 72 hours after birth. The implicated pathogen is often a member of the intestinal microbiome before systemic infection, suggesting translocation from the intestine as the path to the bloodstream. We hypothesized that maternal immunization of animals prior to pregnancy could protect nursing pups in a murine model of LOS. Following maternal immunization against Enterobacteriaceae taxa implicated in sepsis, we observed significantly decreased pup mortality following an oral challenge with Escherichia coli. This protection was associated with increases in antigen-specific IgG and IgA in both the mammary tissue of vaccinated dams and stomach contents of nursing pups. Interestingly, we did not observe differences in the infant microbiota following maternal vaccination, suggesting increased pathogen-specific IgG and IgA did not perturb the developing microbiota. This work suggests maternal immunization may be useful to protect offspring from enteric pathogens, and further supports the role of human milk and human milk factors in protection from LOS. Funding Sources Combating Antibiotic Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Invariant Natural Killer T (iNKT) cells recognize glycolipid antigens presented on CD1d and rapidily respond to direct immune responses. iNKT cells develop in the thymus and migrate to peripheral tissues in what has been presumed to be differentiated/committed states. Accordingly, colonic iNKT cells are established in early life, considered to remain ‘fixed’ post-weaning, and determine life-long colitis susceptibility. Using single cell RNA sequencing (scRNA-seq), we demonstrate that humans and mice contain colonic iNKT populations transcriptionally resembling immature and intermediate thymic iNKT precursors. Contrary to prevailing paradigms, we demonstrate colonic iNKT cell populations expand in adult mice when CD1d expressing colonic goblet cells form goblet cell-associated antigen passages. Expansion preferentially affected intermediate iNKT cells, was durable, and protective in a colitis model. These studies reveal that the adult colon harbors iNKT cells with retained plasticity and uncover a novel role for goblet cells as unconventional antigen presenting cells regulating this axis.

Interaction between breast milk antibodies and gut microbes shape immune responses during weaning