bluuebunny/crossref_metadata_2025 huggingface dataset:
Myosin 18A is a novel checkpoint regulator in B cell differentiation and antibody-mediated immunity
Authors: Gupta Neetu , Cheung Michael B , Enyindah-Asonye Gospel M , Matsui Ken , Kosik Ivan , Dvorina Nina , Baldwin William M , Yewdell Jonathan W
Published: January 2023

The B cell antibody response is tightly regulated to facilitate pathogen-specific immunity and prevent self-reactivity, with actin cytoskeleton dynamics playing an important role in regulating B cell activation. The newly discovered unconventional myosin family protein Myosin 18A (Myo18A) is most closely related to Myo2A, and regulates important cellular processes in non-lymphoid cells. Myo18A is expressed in both precursor and mature B cells, and interacts with ezrin, Myo2A and tyrosine phosphorylated proteins suggesting that it may regulate physiological functions of B cells. We investigated the function of Myo18A in antibody-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Basally, Myo18A deficiency led to expansion of both bone marrow progenitor B cells, and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia with greater levels of splenic IgM secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing antibodies against the major antigen hemagglutinin, associated with early expansion and persistent accumulation of antigen-specific germinal center B cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into antibody secreting cells, and induce Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, self-tolerance and humoral immunity.

Supported by an NIH grant (R21 AI117350) to Neetu Gupta
Intrinsic B cell TLR-BCR linked coengagement induces mature and protective antibody responses in the absence of T cells
Authors: Rivera Carlos Eduardo , Yan Hui , Fisher Amanda Dee , Chupp Daniel Phillip , Simon Raphael , Zan Hong , Xu Zhenming , Casali Paolo
Published: January 2023

Maturation of the antibody response is critical for the generation of virus and bacteria neutralizing antibodies. It entails CSR and SHM together with plasma cell and memory B cell differentiation, and it is generally dependent on T cell help. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR-BCR coengagement can induce a fully mature antibody response in vivo in the absence of T cells, and such coengagement must entail physical linkage of TLR and BCR ligands. To test this hypothesis, we vaccinated Tcrβ−/− Tcrδ−/− mice with NP-LPS or Salmonella Typhimurium flagellin (coengaging B-cell TLR4 with NP-specific BCR or TLR5 with flagellin-specific BCR). Vaccination of Tcrβ−/− Tcrδ−/− mice with NP-LPS or flagellin induced activation of the B cell NF-κB pathway to elicit NP or flagellin-specific class-switched IgG and IgA antibodies and plasma cells. It also induced formation of germinal center-like (GC) structures, B cell SHM, selective clonal expansion and intraclonal diversification, and generation of memory B cells. Similar antibody responses to NP and flagellin were reproduced in NSG/B mice (by grafting immunodeficient NSG mice with purified C57BL/6 mouse B cells), thereby showing that such responses could be generated in the absence of not only T cells but all other immune cells. Finally, LPS and flagellin induced class-switched and specific antibodies which neutralized E. coli and S. Typhimurium, respectively, and protected mice from lethal infection. Thus, TLR-BCR coengagement can induce a mature and protective antibody response, complete with CSR/SHM, plasma cell and memory B cell differentiation in the absence of T cells.

Supported by NIH grants AI 079705, AI 105813/105813S1, AI138944, AI 167416 and Lupus Research Alliance grant LRA 641363 to Paolo Casali
Lactoferrin-derived Chimeric Peptide (LFch) boosts TGFβ1-induced Treg Differentiation by diminishing of TCR Signal intensity via TβRIII
Authors: Jang Young-Saeng , Yang Seok-Won , Park Sunhee , Kim Yu-Rim , Han Joo-Hee , Kim Pyeung-Hyeun
Published: January 2023

Regulatory T cells (Tregs) are a special subset of T cells to suppress excessive immune response and maintain immunologic tolerance. It is known that lesser TCR signal strength is favorable for Foxp3+ Treg differentiation. We recently reported that LF substantially promoted Foxp3 expression by activated CD4+ T cells and this activity was further enhanced by TGF-β1. In the present study, we explored the effect of lactoferrin chimera (LFch, synthetic LF-derived chimeric peptides) along with TGFβ1 on Foxp3+ Treg differentiation. LFch itself did not induce Foxp3 expression. However, LFch dramatically enhanced TGFβ1-induced Foxp3 expression. LFch little affected the phosphorylation of Smad3, which is a canonical TGF-β1 mediator. Instead, LFch decreased the phosphorylation of S6, IkB, and PI3 kinase (an activation hallmark of TCR signaling). These effects of LFch were completely abolished by pretreatment of LFch with soluble TβRIII. Consistently, the enhancement of TGFβ1-induced Foxp3 by LFch was also abrogated by treatment of soluble TβRIII. Finally, TGFβ1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch potently enhances TGFβ1-induced Foxp3+ Treg differentiation by diminishing TCR signal intensity through TβRIII.
CD6 – a costimulatory receptor at the nexus of Teff / Treg development
Authors: Ampudia Jeanette , Chu Dalena , Connelly Stephen , Ng Cherie
Published: January 2023

The balance of proinflammatory effector T cells (Teff) and anti-inflammatory regulatory T cells (Treg) is associated with autoimmune and inflammatory diseases. CD6 is a costimulatory receptor expressed on T cells with its greatest expression on pathogenic Teff cells, while Tregs are CD6low. Notably T cells from CD6knockout mice fail to differentiate to Th17 cells but were seen to skew to a Treg phenotype more rapidly. Although the role of CD6 in Teff development is well characterized, here we sought to investigate the role of CD6 in the development of Treg cells.

Itolizumab, a humanized anti-CD6 monoclonal antibody that modulates cell surface levels of CD6, was used to generate CD6low naïve T cells, after which itolizumab was removed and Tregs were differentiated using a standard protocol. Tregs derived from CD6low cells had greater co-expression of FOXP3 and HELIOS (~2-fold) vs. Tregs derived from isotype-treated CD6high. These Tregs possessed greater suppressive function, with increased inhibition of proliferation and cytokine release by stimulated T responder cells by at least 50% more than Tregs derived from CD6high cells; this was verified in three different donors. Similar observations were made when measuring levels of cytokines. CD6low Tregs were able to suppress T responder’s production of proinflammatory cytokines by 60–90% compared to CD6highTregs.

This is the first study to directly characterize the role of CD6 in the development and activity of Treg cells. These data suggest that reduced levels of cell surface CD6 facilitate the development of Treg cells with greater stability and suppressive activity and that modulating the levels of CD6 may decrease the Teff/Treg ratio in patients with autoimmune and inflammatory diseases.
A potential monocyte-regulatory T cell axis in neurorestoration following ischemic stroke
Authors: Rahimpour Shokofeh , Zheng Wen , Monaghan Kelly L , Wan Edwin C K
Published: January 2023

Neurorestoration following ischemic stroke is regulated by a complex interaction between the resident glial cells and the immune cells infiltrated to the brain. Using transient middle cerebral artery occlusion (tMCAO), it was shown that both monocytes and regulatory T cells (Tregs) play a critical role in facilitating neurorestoration following ischemic injury of the brain. However, the underlying mechanisms are not well-understood. Importantly, it is unclear whether the neurorestorative effect of monocytes and Tregs require their cognate interaction. The goal of this study is to investigate the interplay between monocytes and Tregs in facilitating brain recovery following ischemic injury. Using mouse tMCAO model, we found that 1) Monocytes were present in the brain both at the sub-acute (day 3) and chronic phases (day 14) following tMCAO, whereas Tregs were significantly increased in the brain only at the chronic phase. 2) During the chronic phase, monocytes and Tregs interacted within the glial scar. 3) CCR2, which controls monocyte trafficking, was expressed in monocytes but not in Tregs, but the number of both monocytes and Tregs were substantially reduced following tMCAO when CCR2 was deleted. 4) CCR2-deficient mice had a reduced survival rate and a larger area of glia scar during the chronic phase post-stroke compared to CCR2-sufficient mice. 5) Tregs in the cervical lymph nodes expressed CCR4 following tMCAO, which is known to control Treg trafficking. 6) Monocytes express CCR4 ligands, CCL17 and CCL22, during differentiation into monocyte-derived cells (MDCs) in vitro. Thus, our data demonstrate that monocytes may exert their neuroprotective via the regulation of Treg trafficking and/or expansion following ischemic stroke.

This work was supported by NIH grants P20GM109098 and R21NS125056 to Edwin Wan. Kelly Monaghan was supported through The American Association of Immunologists Careers in Immunology Fellowship Program.
Evaluation of the DNA topoisomerase inhibitor irinotecan (CPT-11) as an alternative to irradiation for pretreatment of NK-92s prior to adoptive cell immunotherapy.
Authors: Cruz Amaya Judith B , Navarrete-Galvan Lydia T , Lombardi Vincent C , Isom Matthew , Smith-Gagen Julie , Hudig Dorothy
Published: January 2023

Irradiation damages both proteins and DNA, which reduces the cytotoxicity of NK-92 cells as well as preventing their growth and engraftment when used for adoptive immunotherapies of cancer. We evaluated the DNA topoisomerase inhibitor irinotecan (CPT-11) as an alternative method to 10 Gy irradiation that might better preserve the perforin-granzyme cytotoxicity mediated by NK-92 cells towards tumor cells. Irinotecan is enzymatically converted into an active metabolite SN-38 which promotes its potential to become a delayed inhibitor in comparison to camptothecin which is a structurally similar topoisomerase inhibitor that is immediately active. We pretreated NK-92 cells for 4 hours with different concentrations of CPT-11, and measured NK-92 cell death and the effects on cell-mediated cytotoxicity towards Raji tumor cells (measured by 51Cr release one day after pretreatment). Previous experiments with 10 Gy irradiation were used for comparative purposes. Irinotecan had a dose response on cell death which progressed for 5 days. We found that one day after pretreatment with as little as 80 – 100 uM CPT-11, viable cell recoveries were similar to 10 Gy irradiated with over 50% compared to the counts of control cells. Activity of the viable cells after CPT-11 pretreatment, compared by lytic units, was over 30% conserved whereas irradiated cell activity was 2.9%, compared to controls. This data demonstrate that the negative effects of anti-proliferative treatment are less pronounced with irinotecan, than for irradiation. We conclude that optimization of dosage and exposure times to irinotecan may offer a means to prolong the life and function of NK-92 cells that are used for adoptive immunotherapies.

Supported by the Nevada INBRE NIH GM103440, for student summer research scholarship and the UNR Foundation C.E. Hudig Fund.
Structure of NAD+ consuming Acinetobacter baumannii TIR domain.
Authors: Snyder Greg A , Nallar Shreeram , Snyder Michelle , Krishanu Ray
Published: January 2023

Toll-like and Interleukin-1/18 receptor (TIR) resistance proteins have been identified across biology to function as important multimodal signal transduction and immune regulatory molecules. Select TIR domain containing proteins identified in both eukaryotic and prokaryotic species have also been found to have NAD+ hydrolase activity in bacteria, plants and in mammalian cells. We report the crystal structure of the Acinetobacter baumannii Toll-Interleukin-1 receptor resistance domain protein (AbTIR) determined to a resolution of 2.8 Å, confirm its enzymatic function in NAD+ hydrolysis and map its interaction with NAD+ using HDX-MS. Additionally, we have developed a novel assay for measuring AbTIR’s enzymatic activity in live bacteria using label-free quantitative two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM). These studies provide new insight into TIR protein mechanisms and their varying roles across biology.

Supported by R01 AI-082299, R21 CA191726-01
Host humoral immune suppression by Clostridioides difficile Toxin A and Toxin B
Authors: Ballard-Norman Kaylee M , Ballard Jimmy D , Lang Mark L
Published: January 2023

Gram-positive Clostridioides difficile is the greatest cause of nosocomial enteric disease in the US and is associated with a high rate of morbidity and mortality. During C. difficile infection (CDI), two secreted toxins (TcdA and TcdB) drive pathogenesis. Although T-dependent humoral immunity, specifically high affinity TcdA- and TcdB-neutralizing IgG, is the best correlation for protection against CDI recurrence, this response is commonly lacking following CDI in both mice and patients, leading to a high chance of recurrence. We previously showed that in murine CDI, there was a lack of T follicular helper (Tfh) cell differentiation, reduced IgG class-switching, poor toxin neutralization, and lack of resistance to reinfection. Preliminary data also suggested that TcdB inhibited IgG responses following vaccination against C. difficile. We therefore hypothesize that TcdA and/or TcdB suppress C. difficile-specific T-dependent humoral immunity. Following either CDI or intraperitoneal injection with TcdA or TcdB, immune cells from mesenteric lymph nodes were analyzed for changes in gene expression. Changes in dendritic cell maturation markers were observed in the presence of TcdB but not TcdA. Multi-targeting mRNA profiling showed altered expression of antigen presentation and CD4+ T cell activation genes. Only one of the known TcdB cell entry receptors was expressed by immune cells, specifically by CD4+ T cells and B cells. Efforts are underway to measure the impact of TcdA and TcdB treatment on CD4+ T cell differentiation, Tfh proliferation, and T-B cell interactions. Our data indicate that toxins may hamper neutralizing IgG production by affecting multiple aspects of T-dependent humoral immunity.

This work was supported by NIH grants AI134719 (M.L.L.) and AI119048 (J.D.B.).
Commensal Lactobacillus reuteri tryptophan catabolism promotes host susceptibility to CNS autoimmunity
Authors: Montgomery Theresa L , Eckstrom Korin M , Heney Eamonn R , Wargo Matthew J , Krementsov Dimitry N
Published: January 2023

Members of the Lactobacillus genus have a long-standing history in food processing and probiotic industries, generally being characterized as safe and/or beneficial for the mammalian host. Despite this fact, current literature points to pro- and anti-inflammatory roles, with little consensus as to the mechanisms governing these divergent outcomes. We and others have previously shown that commensal colonization with Lactobacillus reuteri (L. reuteri) enhances host susceptibility to experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. To identify underlying mechanisms, we characterized the genome of commensal L. reuteri isolates, present within our model microbiomes, using whole genome sequencing and comparative genomics. The enzymatic machinery necessary to catabolize dietary tryptophan (Trp) into immunomodulatory indole derivatives, was enriched in the L. reuteri genome. Metabolite profiling of L. reuteri monocultures and serum of L. reuteri-colonized mice revealed active depletion of kynurenines and production of a wide array of Trp-derived AhR ligands, including indole acetate, indole acrylate, tryptamine, and imidazole derivatives, suggesting that Trp catabolism by L. reuteri may drive the enhancement of autoimmunity. Consistently, dietary Trp was required for L. reuteri dependent EAE exacerbation, while depletion of Trp suppressed disease activity and inflammatory T cell responses in the CNS. Our data suggests that Trp catabolism by gut commensals can enhance autoimmunity by production of immunoregulatory indole derivatives and by competition with the host for production of Trp metabolites such as kynurenines.

Supported by NIH R01 NS097596 T32 Training Grant 5T32AI055402-15 F31 Ruth L. Kirschstein NRSA Fellowship 5 F31 NS120381-02
Indispensable roles of TNF signals in protective immunity against Orientia tsutsugamushi infection
Authors: Liang Yuejin , Trent Brandon , Card Galen , Fisher James , Sun Jiaren , Soong Lynn
Published: January 2023

Scrub typhus is an emerging tropical disease caused by obligate intracellular bacteria Orientia tsutsugamushi, with over one million cases reported yearly. Patients may develop acute lung injury and multi-organ failure; the worldwide average mortality rate is 6%; however, little is known regarding key immune mediators during infection. Using murine scrub typhus models, we demonstrated the essential role of TNF signals in bacterial control and host protection. Mice lacking both TNF receptors (TNFR1/R2−/−) or receiving anti-TNF-α treatment were highly susceptible to infection with significantly increased tissue bacterial loads, inflammatory responses, and mortality. Their lung- and spleen-derived leukocytes displayed profound defects in total cell numbers and activation status for both innate immunity (NK cells, neutrophils, and macrophages) and adaptive immunity (CD4+ and CD8+ T cells). Infection of TNFR1−/− or TNFR2−/− mice confirmed the requirement of both receptors in efficient control of disease progression, as lacking either one receptor greatly reduced activated CD4+ and CD8+ T effector cells. Together, this study demonstrates a critical and indispensable role of TNF signals in host protective immunity against O. tsutsugamushi infection and severe scrub typhus.

Supported by grants from NIH (R01 AI132674, R21 AI156536 and R21 AI153586).
Early Life Development of Bronchial Associated Lymphoid Tissue Containing Germinal Center in the Human Lung
Authors: Matsumoto Rei , Gray Joshua I , Connors Thomas J , Guyer Rebecca , Rybkina Ksenia , Bradley Marissa , Poon Maya M L , Farber Donna L
Published: January 2023

Early life susceptibility to respiratory pathogens is well established in infants and young children, however the specific adaptations of the infant immune system for responding to multiple respiratory challenges are not known. Here, we examined lung and lung-associated lymph nodes derived from infant and pediatric organ donors (0–13 years) by confocal immunofluorescence and high dimensional flow cytometry. We identified early formation of bronchial associated lymphoid tissue (BALT) where active immune responses are initiated. At 8 days of life, few immune cells were present around the airways. By 5 months old, we start to observe BALT formation. However, BALT lose their structure with aging and are mostly absent by 10 years of age with CD4 memory T cells dominating the population around the central airways. Germinal centers are seen in 50–60% of BALT during the first two years of life. The percentage of BALT with germinal centers decreases over age, while germinal center formation in lung lymph nodes is maintained throughout infancy and childhood. Flow cytometry data shows seeding of transitional and immature B cells in the lung during infancy, which decreases over age. However, proportion of IgG and IgA memory B cells increases significantly over age both in the lung and lung lymph nodes. The frequency of naïve B cells is stable throughout childhood in both sites. Our results suggest that localized tertiary lymphoid structures in pediatric lungs may be an important mechanism for mobilizing rapid in situ protection during the first few years of life when memory T cells and B cells are not fully established.

Supported by grants listed below: “Human Atlas of Neonatal Development and Early Life-Immunity (HANDEL-I)”(award Number 2018PG-T1D071 The Leona M. and Harry B. Helmsley Charitable Trust) “Immunobiology and alveolar physiology of the aging lung” (U01 HL145547, NIH/NHLBI)
Diet induced changes in microbiota composition affect the development of small intestinal T cells over generations
Authors: Barragan Luisa Cervantes , Royer Charlotte , Yaceczko Maddelyn , Rodriguez-Marino Naomi
Published: January 2023

The relationship of microbiota and host is a dynamic and bidirectional process. The microbiota has a strong influence in the development of the host’s immune system, yet the host shapes the composition of the microbiota as well, through the immune response, and importantly, through diet. In the current study we evaluated how modification in the carbohydrate and fiber content of a diet impacts the intestinal immune response. Mice were weaned into a standard natural ingredient diet (Reg) or a purified non-fermentable fiber only diet (NFF) and the immune cell composition was analyzed after four weeks. Animals in the NFF diet had strong changes in their microbiota composition and defects in the development of small intestinal intraepithelial T cells including the lack of CD4+CD8aa+ T cells and TCRab+CD8ab+ T cells. The effect of NFF consumption both in microbiota composition as well as IEL T cell development, was reversible if animals were fed a Reg diet. However, if animals were born from parents fed an NFF diet and kept on NFF diet until weaning, the effect on IEL T cells was non reversible and the offspring of these mice did not develop IEL T cells even if fed a reg diet. Fecal matter transplant from mice fed a Reg diet, together with a Reg diet was able to induce the development of IEL T cells, suggesting the defect observed in the offspring of mice fed a NFF diet is the result of the loss of microbes required for the development of IEL T cells. This study shows how changes in the diet can result in the loss of specific microbiota species over generations that are important for the correct development of the intestinal immune system.

Supported by grant from NIH RO1 DK129950-01
Perturbed function of natural killer cells by inflammatory cytokines in acute (AML) and chronic (CML) myeloid leukemias
Authors: Kuznetsova Valeriya , Patel Sweta , Luca Francesca , Camacho Virginia , Matkins Victoria , Welner Robert S
Published: January 2023

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules. Indeed, mature NK cell counts are associated with favorable prognosis in AML and prolonged TKI-free remission in CML. However, NK cell maturation and anti-leukemic activity are suppressed during the disease, and the environmental drivers of this impairment remain unclear. Given an established role of inflammatory cytokines (particularly IL-1β, IL-6, and TNFα) in the progression of myeloid malignancies, we define their impact on NK cell function in chimeric mouse models of leukemia – Flt3-ITD/TET2, AML/ETO9a, Meis/Hox (AML), and BCR-ABL1 (CML), where NK cells do not carry the oncogene. Reflecting clinical observations, NK cell frequencies and numbers are reduced, implying oncogene-independent mechanisms of NK dysfunction in different leukemic subtypes. Moreover, both AML- and CML-exposed NK cells display immature phenotypes, diminished expression of activating receptors, and impaired ex vivo target-specific degranulation. The single cell RNA-seq analysis of CML-exposed NK cells confirmed decreased expression of NK maturation and cytotoxicity markers and upregulation of inhibitory receptors and genes associated with inflammatory cytokine response. Furthermore, IL-6-STAT3 and IL-1β/TNFα transcriptional signatures are enriched during the disease, which is likely to be triggered by leukemic cytokines. Thus, we next found that leukemic serum dampens NK cell degranulation ex vivo, compared to serum from control mice. These data suggest that soluble leukemic factors contribute to NK cell dysfunction and represent an optimal target for NK-boosting immunotherapies across different types of myeloid malignancies.
Universal protection efficacy against influenza viruses by multi-subtype neuraminidase and M2 ectodomain virus-like particle vaccination in young and older adult mice
Authors: Kim Ki-Hye , Li Zhuo , Bhatnagar Noopur , Subbiah Jeeva , Park Bo Ryoung , Shin Chong Hyun , Kang Sang-Moo
Published: January 2023

Annual influenza vaccination is recommended to update the variable hemagglutinin antigens. [add more scientific justification for why NA + M2e immunity will be important?] Here, we first designed a virus-like particle (VLP) displaying consensus multi-neuraminidase (NA) subtypes (cN1, cN2, B cNA) and M2 ectodomain (M2e) tandem repeat (m-cNA-M2e VLP). Vaccination of mice with m-cNA-M2e VLP induced broad NA inhibition (NAI), M2e antibodies as well as interferon-gamma secreting T cell responses. Mice vaccinated with m-cNA-M2e VLP were protected against multi strains of different subtypes (H1N1, H5N1, H3N2, H9N2, H7N9) influenza A viruses containing substantial antigenic variations. From the T cell depletion and passive transfer experiments, we found that immunization of mice with m-cNA-M2e VLP induced cellular and humoral immune components contributing to broader cross protection. Also, surrogate effector assay indicated antibody-dependent cellular cytotoxicity as an additional protective immune contributor. Furthermore, comparable cross protection by m-cNA-M2e VLP vaccination was induced in aged mice. This study supports a novel strategy of developing a universal vaccine against influenza viruses potentially in both young and aged populations by inducing multi-NA subtype and M2e immunity with a single VLP entity.

Supported by grants from NIH/NIAID (AI093772, AI154656, AI147042)
The effects of metformin on influenza vaccination responses in healthy older adults
Authors: Martin Dominique Elizabeth , Cadar Andreia N , Haynes Laura , Kuchel George A , Bartley Jenna M
Published: January 2023

With aging, there is a progressive decline in the immune system leading to increased risk of infection and diminished vaccination responses. Concurrently, there are age-related changes to systemic and cellular metabolism. Influenza (flu) is a leading killer of older adults despite availability of a seasonal vaccine. Metabolic adjuvants may target metabolic deficits in immune cells and have potential to increase vaccination efficacy. Here we aimed to determine the effects of metformin, an FDA approved diabetes drug that targets AMPK and modulates cellular metabolism, on flu vaccination responses. In this study, healthy older adults (non-diabetic/non-prediabetic, age: 74.4±1.7 years) were randomized to metformin (n=8, 1500mg XR/daily) or placebo (n=7) treatment for 20 weeks. 10 weeks after the start of treatment, subjects were vaccinated with High Dose Flu Vaccine. Blood was obtained prior to treatment, prior to vaccination, and 1, 5, and 10 weeks post vaccination. To evaluate the effect of metformin treatment on vaccination responses, peripheral blood mononuclear cells (PBMCs), serum, and plasma were collected from each visit. Serum antibodies titers were quantified and PBMCs were stimulated with live flu virus to assess cell-mediated responses. Clear responses to vaccination were seen in T and B cell subsets via flow cytometry. There were trending increases in antibody fold changes with metformin, indicating a need to further explore the effects of metformin on humoral responses. Overall, metformin had no negative effects, but requires additional studies to determine if it has favorable effects on flu vaccine responses in healthy older adults.

Supported by American Federation for Aging Research (AFAR Diamond 18155, Bartley) and J. Bartley start up funds
Identification of conserved coronavirus epitopes targeted by antibodies after SARS-CoV-2 infection or vaccination
Authors: Bradley Todd , Geanes Eric , LeMaster Cas , Fraley Elizabeth R , Khanal Santosh , McLennan Rebecca , Grundberg Elin , Selvarangan Rangaraj
Published: January 2023

SARS-CoV-2 is a novel betacoronavirus that causes coronavirus disease 2019 and has resulted in millions of deaths worldwide. Novel coronavirus infections in humans have steadily become more common. Understanding antibody responses to SARS-CoV-2, and identifying conserved, cross-reactive epitopes among coronavirus strains could inform the design of vaccines and therapeutics with broad application. Here, we determined that individuals with previous SARS-CoV-2 infection or vaccinated with the Pfizer-BioNTech BNT162b2 vaccine produced antibody responses that cross-reacted with related betacoronaviruses. Moreover, we designed a peptide-conjugate vaccine with a conserved SARS-CoV-2 S2 spike epitope, immunized mice and determined cross-reactive antibody binding to SARS-CoV-2 and other related coronaviruses. This conserved spike epitope also shared sequence homology to proteins in commensal gut microbiota and antibodies targeting this region reacted with proteins in human fecal protein extracts. This indicated that the composition of the gut microbiota could prime immune responses in humans. Thus, SARS-CoV-2 conserved epitopes elicited cross-reactive immune responses to both related coronaviruses and host bacteria that could serve as future targets for broad coronavirus therapeutics and vaccines.

This work was supported through internal institutional funds from Children’s Mercy Research Institute and Children’s Mercy Kansas City.
Robust antibody responses in children after Pfizer vaccination
Authors: Gupta Sneh Lata , Anderson Evan J , Rostad Christina A , Suthar Mehul S , Wrammert Jens
Published: January 2023

SARS-CoV-2 is a devastating global pandemic which has resulted in mass loss of life. It is essential to vaccinate children to achieve herd immunity and prevent community transmission. In addition, the importance of vaccinating children is further illustrated by increasing COVID-19 cases among this age group and the associated Multisystem Inflammatory Syndrome in Children (MIS-C). Children possess a relatively naïve immune system compared to adults. With reopening of schools, relaxation of mask mandates and new emerging viral variants, it is imperative to better understand the magnitude and quality of vaccine induced responses in children, especially in terms of breadth of immunity against emerging viral variants.

In the current study we have assessed the humoral response after vaccination in children 11–17 years of age. We found robust responses in children, with Spike and RBD specific antibody titers that were significantly higher than adult vaccinees. In addition, these titers were also much higher than those seen in both convalescent hospitalized pediatric COVID and MIS-C cases. Compared to adults, the pediatric vaccinees showed a similar breadth against current viral variants. Finally, using a live neutralization assay we similarly found that children exhibited higher neutralization titers than adults and that the neutralization titers positively correlated with their RBD binding titers. Overall, our data show that vaccination induces more potent antibody responses in children as compared to adults, with similar breadth against emerging viral variants.
Durability of B and T cell responses against a spike protein of SARS-CoV2 elicited by mRNA vaccines
Authors: Hamilton Lauren Taylor , Koguchi Yoshinobu , Christie Tanisha , Shimada Takashi , Iwamoto Noriko , Fox Bernie , Redmond William L , Piening Brian D
Published: January 2023

The pandemic of SARS-CoV2 had resulted in over 266 million infections and over 5.2 million deaths throughout the world as of early-December 2021. Pfizer and Moderna mRNA vaccines were approved for Emergency Use Authorization by the US FDA. Even though 8.2 billion doses of vaccine have been administered, the world continues to see wide-spread SARS-CoV2 infection. Therefore, it is important to understand the durability of anti-SARS-CoV2 immune responses. Serum and PBMC samples were collected from vaccinated volunteers (N=40, Age 22–67, median age 41, Male/Female = 14/26, Moderna/Pfizer = 37/3) over 8 months (pre-vaccination baseline: TP1, 2 weeks-, 3 months-, and 8 months-post full vaccination: TP2, TP3, and TP4, respectively). First, we found robust induction of anti-S IgG and IgA for all subjects at TP2, which was higher than those from Covid-19 patients. Follow up samples (TP3 and TP4) showed steady decay of antibody levels over 8 months post-vaccination. Next, we evaluated memory B cell response using flow cytometry. We detected S-protein specific B cells by using flag-tagged S-protein. The frequency of S-protein specific B cells varied among donors and was weakly associated with the level of serum anti-S IgG antibody. Lastly, we assessed T cell responses. PBMCs were stimulated with an overlapping 15-mer peptide mix that covers the entire S-protein. Antigen-specific activation of T cells were detected based upon IFN-γ and TNF-α production. We found induction of robust CD4 T cell response at TP2 but a clear reduction at TP3. TP4 showed equivalent levels of T cell responses as TP3. Together, we found that B and T cell responses against the spike protein of SARS-CoV2 induced by mRNA vaccines are not durable.

This study was supported by funding from Shimadzu Corporation.
Oncolytic Viral Therapy Mediates its Effectiveness by Inducing and Enhancing CD4 cytotoxic T-cells
Authors: Grimes Jeffrey M , Murchison Charles , New James S , Crossman David , Markert James M , Leavenworth Jianmei Wu
Published: January 2023

Malignant glioma remains a fatal disease with a dismal survival rate. To address this urgent medical need, we pioneered the concept of engineered oncolytic viruses for cancer. Using the herpes simplex virus (HSV)-1 as an experimental virus, our published Phase I trials have demonstrated the safety and efficacy of such oHSV-based therapy for treating brain tumor patients. In searching for biomarkers for better responses to this therapy, our preclinical studies using intracranial glioma models and an oHSV secreting murine IL-12 (M002) revealed that a subset of CD4+ T-cells expressing the cytolytic signatures, including granzyme B, were increased in M002-treated mice compared to vehicle-treated controls. Correspondingly, CD4+ T-cells isolated from brain tumors of M002-treated mice killed tumor cells better than cells isolated from control tumors. However, the factors and temporal requirements that direct the development of this CD4 cytotoxic T-cell (CTL) subset are not fully understood. To address this question, we performed single-cell RNA sequencing analysis of CD4+ T-cells isolated from brain tumors of M002-treated and control mice. The results revealed a complex change of the CD4+ T-cell compartment with CD4 CTLs being induced from two different precursors. The first CTL subset was derived from T helper-1 (Th1) cells and retained Th1 gene signatures while expressing high levels of CTL associated molecules. The second CTL subset was a reprogrammed regulatory T-cell population capable of producing high levels of cytokines and granzymes. Both CD4 CTL subsets were more prominent in M002-treated mice, suggesting that M002 therapy could induce CD4 CTLs with specialized functionality, which may account for its improved outcome.

Supported by grants from DoD W81XH-18-1-0315 and UAB Start up Funds
Studies on Antibodies
Authors: Falk K. George , McGuire Grace , Valentine Eugenia , Whitney Elinor
Published: January 2023

Introduction. The preparation of dried antipneumococcus serum, tests of its protective action, and a number of its properties will be described in this paper.

The chemical nature of the active antipneumococcus material is unknown, whether this active material be considered to be a molecule, or a grouping in a complex protein or other molecule. The strength or potency of a preparation is determined at present by animal tests, agglutination and precipitation reactions. The animal tests used here were confined to mouse protection. As the significance of the experiments, such as the potency of a preparation after different treatments, would be determined by such mouse experiments, a critical discussion of this method of testing will first be given. It is evident that practically every treatment to which a labile material such as serum or plasma is subjected will modify its properties in one way or another.
Studies on Antibodies
Authors: Valentine Eugenia , McGuire Grace , Whitney Elinor , Falk K. George
Published: January 2023

Introduction. A method of drying antipneumococcus serums and antibody preparations, and some of the properties of these dried preparations were described in the preceding paper (1). The mouse protection, agglutination, and precipitin titers of dried antiserums when tested immediately and within two months after drying showed, in the majority of instances, a close correlation between the dried and untreated serums with respect to these antibody tests.

In this paper a more extended study of the correlation of agglutination, precipitin and mouse protection tests of dried antipneumococcus serums when stored at different temperatures and for different periods of time and the comparisons with the untreated materials will be described.

Experimental Methods and Results. Detailed results will be given for one antiserum, although similar results were obtained with several other antiserums. A dried powdered, antiserum was divided into two portions immediately after drying. One portion was kept in the ice-box and the other at room temperature.
Skin Reactions to the Colon Bacillus and Its Toxic Products
Authors: Steinberg Bernhard , Wiltsie Catherine Orth
Published: January 2023

Summary and Conclusions

Normal persons give a positive reaction to intradermal injections of B. coli toxic filtrate and washed organisms. Children show a more marked reaction than adults. Patients with a long standing colon bacillus infection react slightly and a small percentage of the patients do not react at all. Negative reactions to the vaccine are more numerous than to the soluble toxic substance. Immunization with B. coli soluble toxic substance reduces but does not obliterate the reaction. Absence of B. coli agglutinins cannot be used as a criterion for absence of other neutralizing antibodies since the agglutinin titer was either nil or slight in persons with negative reactions.

Because normal persons give positive reactions and patients with long standing colon bacillus infection show negative or reduced reactions, the phenomenon is assumed by us to be one in which antibodies neutralize the colon bacillus toxic substances, both soluble and that derived from the bacterial bodies. Our work thus far does not justify either an acceptance or a refutation that thephenomenon may be of an allergic nature as suggested by some workers. It is further concluded that under the conditions of these experiments, the skin reaction for the determination of the presence of colon bacillus infection is of uncertain value.
Diphtheria Antibodies Transmitted from Mother to Child
Authors: Neill James M. , Gaspari Emidio L. , Richardson Lurline V. , Sugg John Y.
Published: January 2023

Measurements were made of the diphtheria antibodies in the blood of a mother and baby during the eighteen months following the birth. Tests were made for antibodies of the antibacterial sort as well as for the antitoxin. The antibacterial antibodies were found in the serum of the baby over the period in which placentally transmitted antibodies could be expected to be retained, but owing to their passive origin they were absent in later bleedings. Control tests showed that the antibacterial antibodies were absent in the sera of babies whose mothers did not possess them. The data of the antitoxin tests owe their interest to the high degree of immunity of the mother and to the length of time over which the blood of both baby and mother was studied.
An Improved Diluent for Diphtheria Toxin in the Schick Test
Authors: White Benjamin , Bunney W. E. , Malcolm Wilbur G.
Published: January 2023

An improved method is described for preparing diphtheria toxin ready diluted for the Schick test. Diphtheria toxin diluted to Schick strength (1/5 m.l.d. per cubic centimeter) with an aqueous solution of Witte peptone 0.25 per cent, sodium chloride 0.85 per cent and phenol 0.5 per cent and passed through a Berkefeld filter retains its toxicity even after ninety-six hours' incubation at 37.5°C. The reactions following the injection into human beings of this ready-diluted toxin and of toxin freshly diluted, as in the previous Schick outfits, are entirely comparable. The use of 0.25 per cent Witte peptone is preferable to 2 per cent Difco-Proteose peptone because of its failure to produce early or diffuse reactions.
Lung Resident Memory T Cells Activated by Oral Vaccination Afford Comprehensive Protection against Pneumonic Yersinia pestis Infection
Authors: Singh Amit K , Majumder Saugata , Wang Xiuran , Song Renjie , Sun Wei
Published: January 2023

A growing body of evidence has shown that resident memory T (TRM) cells formed in tissue after mucosal infection or vaccination are crucial for counteracting reinfection by pathogens. However, whether lung TRM cells activated by oral immunization with Yptb1(pYA5199) play a protective role against pneumonic plague remains unclear. In this study, we demonstrated that lung CD4+ and CD8+ TRM cells significantly accumulated in the lungs of orally Yptb1(pYA5199)-vaccinated mice and dramatically expanded with elevated IL-17A, IFN-γ, and/or TNF-α production after pulmonary Yersinia pestis infection and afforded significant protection. Short-term or long-term treatment of immunized mice with FTY720 did not affect lung TRM cell formation and expansion or protection against pneumonic plague. Moreover, the intratracheal transfer of both lung CD4+ and CD8+ TRM cells conferred comprehensive protection against pneumonic plague in naive recipient mice. Lung TRM cell–mediated protection was dramatically abolished by the neutralization of both IFN-γ and IL-17A. Our findings reveal that lung TRM cells can be activated via oral Yptb1(pYA5199) vaccination, and that IL-17A and IFN-γ production play an essential role in adaptive immunity against pulmonary Y. pestis infection. This study highlights an important new target for developing an effective pneumonic plague vaccine.
Heredity of the Agglutinogens M and N of Landsteiner and Levine
Authors: Wiener Alexander S. , Rothberg Sidney , Fox S. A.
Published: January 2023

The bloods of 461 mothers with 495 children were examined for the agglutinogens M and N of Landsteiner and Levine. Not once were the combinations, M+N- mother with M-N+ child, or M-N+ mother with M+N- child found. The results fully confirm the theory of Landsteiner and Levine. The medicolegal application of the agglutinogens M and N is fully justified at present.
The Agglutinin Response to Typhoid-Paratyphoid Vaccination
Authors: Mudd Stuart
Published: January 2023

Recent claims that prophylactic vaccination against typhoid and paratyphoid fever is not effective because of the absence of somatic antibodies in the sera of vaccinated persons have not been substantiated; somatic antibodies could be detected with proper reagents after administration of a proper vaccine. A vaccine which failed to prevent small outbreaks of typhoid and paratyphoid B infection in a state hospital was found to be deficient in production of somatic agglutinins and its bacterial count was far below specifications. A convenient detector of H agglutination, namely chloroform-treated bacilli, is described.
Some Factors Influencing the Rate of Thermal Destruction of the Tetanus Antitoxin of Antitetanic Horse Plasma at 60° to 66°C.
Authors: Gerlough Tillman D. , White Woodford
Published: January 2023

Data are presented to show the influence of ammonium sulphate, phenol and pH on the heat destruction at 60° to 66°C. of tetanus antitoxin of antitetanic horse plasma. The results may be summarized as follows: Addition of neutral salts increased the rate of destruction of tetanus antitoxin up to an ionic strength of 0.6 to 0.9 where a maximum is reached. With further addition of salt the rate decreased.The pH of maximum stability is at or near neutrality.Phenol increases markedly the rate of destruction of tetanus antitoxin. Its accelerating action is approximately proportional to the square of the phenol concentration.Between 60° and 66°C. an increase in temperature of 1°C. increases the rate of destruction of tetanus antitoxin 1.85 times.
Schick Reactions and Serum Antitoxin Titrations on Children Injected with Diphtheria Formol Toxoid
Authors: Leach Charles N. , Pöch Georg
Published: January 2023

The customary method of determining the effectiveness of diphtheria immunization by various antigens is the well known Schick skin test. This has been considered an indirect means of estimating a certain amount of antitoxin in the blood of individuals before and after injection with the antigen employed. Schick and Michaels (1913) stated that a negative Schick test should indicate an amount of antitoxin present in the blood serum of the subject under investigation not below 0.03 of a unit per cubic centimeter of serum (0.02 units per cubic centimeter of blood). This lower level of protection was later placed at 0.005 unit per cubic centimeter of serum (Schick, von Gröer, and Kassowitz, 1924) and in recent years 0.01 unit per cubic centimeter of serum has been considered sufficient to protect against diphtheria (Dudley, Surg. Capt. S. F., 1934). The so-called “Schick level” is now placed between 0.03 and 0.01 units.
Neutralization of the Bacteriophage
Authors: Clifton C. E. , Mueller Elizabeth , Rogers W.
Published: January 2023

Summary and Conclusions

The time required for an anticoliphage to lyse an actively growing culture of Esch. coli is proportional to the initial concentration of the phage. Other conditions being constant, the concentration of phage in different samples can be determined by noting the relative length of time required for the reduction of a constant initial concentration of bacteria to an arbitrary turbidity endpoint. The time required for a given initial concentration of phage to reduce the concentration of bacteria to this arbitrary end-point is increased in the presence of an antilytic serum. Quantitative measurements, under the dynamic conditions of these experiments, indicate that the neutralization of phage over a wide range of concentrations of phage and of antiserum, goes almost to completion within a few minutes. Observations are presented which suggest that the preliminary union between phage and antiphage is an adsorption process.
Procedure and Apparatus for Preservation in “Lyophile” form of Serum and Other Biological Substances
Authors: Flosdorf Earl W. , Mudd Stuart
Published: January 2023

Procedure and apparatus for the preservation of biological products in lyophile form have been described in detail. The method is one of rapid freezing and rapid dehydration from the frozen state. The product is vacuum sealed in the final container in which it is to be stored and distributed.

The apparatus is simple in operation. Models have been described for use in research laboratories, for board of health and hospital work and for industrial scale production.

Normal and convalescent human sera, animal antisera and complement, miscellaneous proteins, enzymes, viruses, bacteria and other materials have been preserved. Complement may be preserved without detectable deterioration for at least ten months, and indications are that many types of antisera may probably be preserved unaltered for an indeterminate number of years.