bluuebunny/crossref_metadata_2025 huggingface dataset:
Beyond innate immunity: the role of interferon regulatory factor 3 in CD4+ T cell function (IRM15P.462)
Authors: Fares-Frederickson Nancy , Otero Dennis , Macal Monica , Zuniga Elina , David Michael
Published: January 2023

Interferon regulatory factor 3 (IRF3) is a transcription factor found in the cytoplasm of most nucleated cells. Upon pathogen recognition, IRF3 becomes activated and translocates to the nucleus where it mediates the production of type I interferon (IFN). Whereas IRF3 has been studied extensively in the context of type I IFN production and innate immunity, its role in adaptive immunity has remained largely unexplored. The objective of our research is to elucidate the role of IRF3 in CD4+ T cell function. Our findings demonstrate that IRF3-deficient CD4+ T cells show robust proliferation in response to TCR engagement, but are compromised in their ability to differentiate into specialized T helper cell subsets—most notably IL-17-producing Th17 cells. As such, CD4+ T cells lacking IRF3 expression fail to induce disease in the T cell transfer model of colitis. These studies reveal a novel, T cell-intrinsic function of IRF3 and extend its role beyond the innate immune response to pathogens.
Tspan33 is differentially expressed during B cell differentiation (LYM7P.620)
Authors: Perez-Martinez Cesar , Zlotnik Albert , Santos-Argumedo Leopoldo
Published: January 2023

Tetraspanin 33 (Tspan33) is a transmembrane protein highly expressed in activated B cells. Tspan33 is a member of the tetraspanin family, which is characterized by conserved domains and functions. Tetraspanins provide a scaffold for the formation of membrane microdomains, called Tetraspanin Enriched Microdomains. Other members of this family have been implicated in cellular adhesion, motility, invasion and signaling. Tspan33 is important for the Notch signaling pathway, because it is essential for the correct membrane localization of ADAM10, an important sheddase in the Notch pathway. The aim of this study is to describe the expression pattern of Tspan33 in different subsets of human B cells. Peripheral and Umbilical cord blood mononuclear cells were separated using density gradients and cell sorting to separate different B cells subsets based on the markers CD19, CD27, IgD, CD38 and CD24. We then used RT-PCR to measure Tspan33 expression. These results were confirmed by flow cytometry. Our results indicate that Tspan33 is differentially expressed during B cell differentiation, with the lowest expression in Naïve B cells and the highest in the Memory B cell compartment.
Notch signaling regulates antigen sensitivity of naive CD4+ T cells by tuning co-stimulation (LYM7P.613)
Authors: Laky Karen , Evans Sharron , Fowlkes B
Published: January 2023

Adaptive immune responses begin when naïve CD4+ T cells engage peptide+major histocompatibility complex class II and costimulatory molecules on antigen-presenting cells (APC). Notch signaling can influence effector functions in differentiated CD4+ T helper and T regulatory cells. Whether and how ligand-induced Notch signaling influences the initial priming of CD4+ T cells has not been addressed. We have found that Delta Like Ligand 4 (DLL4)-induced Notch signaling potentiates phosphatidyl inositol 3-OH kinase (PI3K)-dependent signaling downstream of the T cell receptor+CD28, allowing naïve CD4+ T cells to respond to lower doses of antigen. In vitro, DLL4-deficient APC were less efficient stimulators of CD4+ T cell activation, metabolism, proliferation, and cytokine secretion. With deletion of DLL4 from CD11c+ APC in vivo, these deficits translated to an impaired ability to mount effective CD4+ T cell-dependent immune responses. These data implicate Notch signaling as an important regulator of adaptive immune responses.
IFNγ restrains Th1 expansion in experimental autoimmune encephalomyelitis (LYM8P.634)
Authors: Sestero Christine , Naves Rodrigo , De Sarno Patrizia , Raman Chander
Published: January 2023

We found that the enhanced experimental autoimmune encephalomyelitis (EAE) severity in Ifngr1-/- mice was associated with elevated numbers of Th1 cells and level of IFNγ in brain and spinal cord. Furthermore, encephalitogenic Th1 polarized T cell cultures generated from Ifngr1-/- mice immunized with MOG35-55 peptide to induce EAE contained several fold greater numbers of IFNγ-expressing Th cells than in similar T cell cultures from WT mice. These observations oppose the dogma that IFNγ signaling has a feed forward activity for Th1 expansion and led us to investigate the possibility that IFNγ functions as a negative feedback for Th1 differentiation and/or expansion. The addition of neutralizing anti-IFNγ mAb during in vitro Th1 polarization of in vivo primed CD4 T cells resulted in the generation of at least a two-fold greater number of IFNγ+ T cells. This effect of anti-IFNγ mAb was not observed with CD4 T cells primed in vitro or with CD4 T cells lacking IFNGR or STAT1. We further determined that IFNγ constrained Th1 expansion by T cell intrinsic expression of iNOS and production of nitric oxide. IFNγ treatment effectively attenuated EAE induced by encephalitogenic Th1 cells but not Th17 cells. Our results reveal that IFNγ has a primary role in restraining Th1 program in vivo. Thus targeting the IFNγ signaling pathway might be effective in a Th1 predominant autoimmune disease. Supported by grants from the IRACDA/NIGMS (CS), NIH (PD, CR), NMSS (CR); and FONDECYT 1140049 (RN).
Tpl2 differentially regulates IFNγ and IL-17 production by CD4+ T cells in a T cell transfer model of colitis. (LYM8P.636)
Authors: Acuff Nicole , Li Xin , Kirkland Rebecca , Nagy Tamas , Watford Wendy
Published: January 2023

Autoimmune diseases are approaching epidemic levels, estimated to affect 5-8% of the population. A number of autoimmune diseases are driven by autoreactive T cells, specifically T helper 1 (Th1) and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a T cell transfer model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2-/- naïve CD4+ T cells were impaired in IL-17A secretion under traditional Th17 inducing conditions of IL-6+TGF-β. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In vivo, transfer of Tpl2-/- T cells resulted in reduced proportions of CD4+ T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that Th17 cells alternatively induced by IL-6+IL-23 was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1-dependent responses, including Th1-mediated immunopathology during autoimmunity.
Deep sequencing of the TCRB repertoire of human Foxp3+ and Foxp3- T cells demonstrates that they are completely distinct and non-overlapping (LYM8P.638)
Authors: Golding Amit , Darko Samuel , Wylie William , Douek Daniel , Shevach Ethan
Published: January 2023

Maintenance of peripheral tolerance requires a balance between auto-reactive conventional T cells (Tconv) and thymically-derived Foxp3+ regulatory T cells (tTregs). Considerable controversy exists regarding the similarities/differences in TCR repertoires expressed by Tconv and tTregs. We generated highly purified populations of human Tconv and tTregs based on the differential expression of CD25 and CD127. The purity of the sorted populations was validated by intracellular staining for Foxp3 and Helios. We used deep sequencing of entire TCRB sequences to analyze the TCR repertoire of Tconv and tTregs. Our studies demonstrate that Tconv and tTreg cells are, in fact, entirely distinct CD4+ T cell lineages.
Differential regulation of PTEN and Akt by TCR signal strength controls CD4+ T cell fate decisions (LYM8P.627)
Authors: Hawse William , Morel Penelope
Published: January 2023

Akt/mTOR signaling influences CD4+ T cell differentiation; low levels favor Treg induction and high levels favor Th induction. Here, we identify several key elements in this pathway that are differentially regulated by TCR signal strength, including Akt, mTORC2 and the lipid phosphatase PTEN. The control of PTEN is poorly studied in T cells. Here, we identify multiple mechanisms that regulate PTEN. During Th induction, PTEN function is suppressed via lower mRNA levels, lower protein levels and an increase in phosphorylation. During Treg induction, PTEN function is maintained through the stabilization of PTEN mRNA transcription and sustained protein levels. Differential Akt/mTOR signaling regulates PTEN transcription via FoxO1. Additionally, Akt is controlled by the TCR signal. Phosphorylation of FoxO1 by Akt requires that Akt is phosphorylated on Thr308 and Ser473 by PDK1 and mTORC2 respectively. Different TCR signal strengths resulted in alternate phosphorylation on Akt where low TCR signal strength only induces phosphorylation of Thr308 on Akt. Low TCR signals result in the phosphorylation of an inhibitory site on Rictor, a subunit of mTORC2, thus providing a mechanism by which Akt activity is controlled by TCR signal strength. Proteomic data suggests that low and high TCR signal strength induce non-overlapping sets of phosphorylated Akt substrates. This work reveals how differential regulation of the Akt/mTOR pathway drives alternate CD4+ T cell fate outcomes.
ICOS-ICOSL interaction-independent Tfh cell differentiation of Foxp1-deficient CD4+ T cells (LYM8P.629)
Authors: Geng Jianlin , Wei Hairong , Shi Bi , Wang Haikun , Stevens Anna , Sprout Stephanie , Hu Hui
Published: January 2023

CD4+ T follicular helper (Tfh) cells are essential for germinal center (GC) and long-lived antibody responses. Studies have shown that the deletion of the inducible T cell co-stimulator (ICOS) results in severe defects of Tfh cell differentiation and germinal center responses. It has been shown that ICOS-ICOS ligand (ICOSL) interaction is not only critical for Bcl6 induction, but also essential for the follicular recruitment of activated CD4+ T cells by follicular bystander B cells that collectively form an ICOS-engaging field. Previously we have identified transcription factor Foxp1 as a critical negative regulator of Tfh cell differentiation. We now provide evidence showing that naïve CD4+ T cells deficient in both Foxp1 and ICOS still differentiate into Tfh cells at high frequencies with Bcl6 induced. Such results are confirmed in ICOSL-deficient recipient mice, in which Foxp1-deficient CD4+ T cell differentiate into Tfh cells. Taken together, our studies suggest that Foxp1 plays a profoundly dominant role in regulating Tfh cell differentiation.
Differential role of B7 co-stimulation on B cells and dendritic cells for Th1 and Tfh differentiation (LYM8P.631)
Authors: Kamphorst Alice , Watanabe Masashi , Araki Koichi , Rasheed Mohammed Ata Ur , Davis Carl , Yang Shu , Konieczny Bogumila , Hodes Richard , Ahmed Rafi
Published: January 2023

Upon activation CD4 T cells differentiate into different subsets that have distinct functions in the immune response. Antigen presentation in the presence of co-stimulation and cytokines is required for optimal CD4 T cell activation and differentiation. Cytokines play a well-described crucial role driving CD4 T cell differentiation, but the role of different antigen presenting cells and co-stimulation remains relatively unexplored. To address this question we developed mice that lack B7-1 expression on B cells or on dendritic cells (DCs), on a B7-2 deficient background. CD4 differentiation was studied in the context of acute lymphocytic choriomeningitis virus (LCMV) infection. We find that mice lacking B7-1 on DCs have a profound defect on virus-specific CD4 T cell expansion that affects Th1 and Tfh numbers, but with the most drastic reduction in Th1 differentiation. In contrast, mice lacking B7-1 on B cells have similar numbers of LCMV-specific CD4 T cells as control mice. However, even though CXCR5 expressing LCMV-specific CD4 T cells numbers appear unaffected by B7-1 deficiency on B cells, there was a reduction in cells expressing high levels of CXCR5, PD-1 and Bcl-6 and a profound defect in LCMV-specific long lived plasma cells and antibodies. We conclude that CD4 T cell differentiation requires cell-type specific co-stimulation, and co-stimulation by B cells is essential to obtain functional Tfh and long-term humoral responses after acute viral infection.
STAT4 promotes atherosclerosis by supporting Tfh and plasma B cell development, T cell activation and CD11b+ cell accumulation in the aortic wall (LYM8P.632)
Authors: Taghavie-Moghadam Paresa , Kaplan Mark , Nadler Jerry , Galkina Elena
Published: January 2023

Atherosclerosis is an inflammatory disease of medium and large size arteries. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor that supports the generation of cells with features of both follicular helper T cells (Tfh) and T helper 1 (Th1) cells. To date, the role of Stat-4 in atherosclerosis and the induction of Tfh cells in this disease has not been defined. To investigate the role of STAT4 in atherogenesis, we generated Stat-4-deficient low density lipoprotein-deficient (Stat4-/-Ldlr-/-) mice. Stat4 deficiency resulted in a significant reduction in atherosclerotic plaque burden within the aortas of Stat4-/-Ldlr-/- mice fed a high fat/high carbohydrate diet with added cholesterol (DDC) diet for 16 weeks compared to age and diet-matched Ldlr-/- control mice (p<0.01). Next we examined spleens and peripheral lymph nodes (PLNs) of Stat4-/-Ldlr-/ mice and found decreased number of CD19+ B cells (p<0.0001) and CD3+ T cells (p<0.05). Interestingly, the number of Tfh cells and plasma B cells (p<0.003) was also diminished in and spleens of Stat4-/-Ldlr-/- mice compared to Ldlr-/- mice. A significant reduction in CD11b+CD11c- cells and MHCII expression was detected in the aortas of Stat4-/-Ldlr-/- vs Ldlr-/- mice. Thus, the atherogenic effects of STAT4 are, at least partially, due to the modulation of the immune response via alterations of Tfh-dependent humoral response. Supported by R01 HL112605 and Suppl.Grant 02S1.
Utilizing CX3CR1GFP/PGRPdsRed reporter mice to monitor myeloid and granulocyte responses during helminth infection (MPF6P.652)
Authors: Barnes Mark , Jang Jessica , Wang Spencer , Lo David , Nair Meera
Published: January 2023

CX3CR1GFP/PGRPdsRed reporter mice have previously been used to monitor trafficking of monocytes and neutrophils, respectively. With this new tool, we examined myeloid cell and neutrophil behavior during murine infection with the helminth, Nippostrongylus brasiliensis (Nb). Nb acutely infects the lungs leading to significant infiltration of PGRPdsRed neutrophils and CX3CR1GFP monocytes compared to naïve mice. Using ex vivo time-lapse confocal microscopy, we discovered that Nb infection increased chemotaxis of CX3CR1GFP monocytes and PGRPdsRed neutrophils. Because CX3CR1GFP expression is a loss of function knock-in mutation, we tested the function of CX3CR1 in the immune response to Nb. After inoculation with infective larvae, CX3CR1GFP mice were less susceptible to infection compared to wild-type mice and had lower worm counts and parasite eggs in the feces. Additionally, ATP cell viability assay revealed that worms recovered from reporter mice were less viable than worms isolated from wild-type mice. Taken together, CX3CR1GFP/PGRPdsRed mice make it possible to monitor myeloid cell and neutrophil behavior, and reveal a previously unrecognized role for CX3CR1 in impairing Nb expulsion.
The fungal secondary metabolite alternariol and Alternaria induced immunity (MPF6P.659)
Authors: Grover Shivani , Howard Brad , Lawrence Christopher
Published: January 2023

Sensitivity to the airborne fungus Alternaria alternata (common mold) is believed to be a common cause of allergic asthma. Epidemiological studies worldwide indicate that Alternaria sensitivity is closely linked with the development and exacerbation of allergic airway disorders such as asthma, allergic rhinitis, and chronic rhinosinusitis. Therapies are limited by the lack of knowledge about the role of individual fungal gene products in airway responses. The A. alternata mycotoxin alternariol is a genotoxic and cytotoxic molecule and a major contaminant of most food, grains and feed products. We hereby present a study where we explored the hypothesis of alternariol having immune-modulatory properties. It led us to the discovery that alternariol is strongly anti-inflammatory. We have investigated alternariol’s dynamic interplay between innate immunity and human bronchoalveolar epithelial cells. Immune assays have provided the key doses needed to suppress LPS-induced inflammation. We have also profiled the response of mutant fungal spores that no longer produce alternariol with normal wildtype spores on lung epithelium. This model has also been used to investigate alternariol’s mechanism of action in relation to aryl hydrocarbon receptor. Greater understanding of the role of alternariol in allergic asthma may lead to improved treatment strategies as well as a potential drug target for inflammatory syndromes.
Comparison of culture-positive and culture-negative bronchoalveolar lavage from mechanically ventilated trauma patients by next-generation sequencing and cytokine analysis (MPF6P.661)
Authors: Smith Ashley , Zhang Yan , Huebinger Ryan , Ireland Sara , Monson Nancy , Allen Michael
Published: January 2023

Mechanically ventilated trauma patients are at high risk for ventilator-associated pneumonia (VAP). VAP diagnosis relies on several clinical factors including the identification of a specific pathogen by culture-dependent techniques. Often, patients exhibit signs of VAP, yet the hospital lab is unable to culture a pathogen from a bronchoalveolar lavage (BAL) sample. Here, culture-positive and -negative hospital results were tested again by Sanger and next-generation sequencing (NGS) on the Ion Torrent PGM. Sample cytokine levels were determined using a Bio-Plex Pro Human Th17 cytokine panel. No significant difference was seen between the identification methods in culture-positive BAL. However, NGS analysis of culture-negative BAL identified hundreds of bacterial genera, including a group of patients that exhibited similar bacterial composition, diversity, and abundance. This group was significantly different from other culture-negative BAL that were dominated by one or two suspected pathogens. Culture-negative BAL contained significantly less IL-1β, IL-6 and TNF-α than culture-positive BAL. NGS is a valuable method for pathogen identification, particularly for difficult to culture BAL. Culture-positive BAL exhibit less bacterial diversity and increased cytokine production than culture-negative BAL. The grouping of culture-negative BAL with similar characteristics may denote a core lung microbiome.
Immune sensing of fungal cell wall carbohydrates programs immunity to A. fumigatus infection: role of γδ T cells. (MPF6P.662)
Authors: Amarsaikhan Nansalmaa , O`Dea Evan , Tsoggerel Angar , Li Hongtao , Templeton Steven
Published: January 2023

The human fungal pathogen Aspergillus fumigatus is associated with allergic sensitization and invasive infection in susceptible individuals. How the combined recognition of fungal molecular patterns programs host adaptive immunity is not well understood. Previously, we observed that fungal chitin promotes Th2-skewed immunity and eosinophil recruitment that proved detrimental to fungal clearance in invasive infection. In this study, we further characterized the role of early cytokine and effector cells in chitin derived eosinophil activation. We observed that single aspiration of a high-chitin isolate (Af5517) resulted in a marked increase in early transcription of IL-17A. Furthermore, eosinophil recruitment was not increased in RAG1-/- mice, suggesting that γδ T cells are required for early recognition of conidia-associated chitin. Although IL-17A was not required, the absence of γδ T cells (known to be potent sources of innate IL-17A) resulted in a significant decrease in eosinophil recruitment after single and multiple aspirations of Af5517. Also, fungal strains displaying chitin increased exposure or manipulating cell surface carbohydrate ratio by blockage of β-glucan recognition on receptor level resulted in increased airway eosinophil recruitment suggesting that relative high chitin/low β-glucan recognition leads to increased eosinophil recruitment and Th2 responses that are promoted by early responses of γδ T cells.
Killer lymphocytes deploy granulysin and granzymes to induce oxidative death of parasites (MPF7P.712)
Authors: Dotiwala Farokh
Published: January 2023

Killer lymphocytes protect against parasites, but what happens to intracellular parasites when the infected host cell is recognized is unknown. Here we show that the killer cell antimicrobial peptide granulysin delivers death-inducing granzymes into Trypanosoma cruzi, Toxoplasma gondii, and Leishmania major, where they generate superoxide, disrupt the mitochondrial potential and induce death, which shares features of granzyme-mediated bacterial death and mammalian apoptosis. Granzyme B also cleaves key oxidative defense enzymes in T. cruzi. Parasite killing depends on perforin to deliver granulysin and granzymes into the infected host cell, is blocked by superoxide scavengers and by overexpressing oxidative defense enzymes. T. cruzi infection was compared in wild-type mice that lack granulysin and GNLY-transgenic mice. GNLY-transgenic mice survived infection that was lethal to most wild-type mice. In vivo protection required perforin and was mediated by CD8+ cytotoxic T lymphocytes.
Role of IL-33 in chronic pulmonary mycobacterial infection (MPF7P.705)
Authors: Umemura Masayuki , Fukui Masayuki , Yamasaki Masatoshi , Fukui Chiho , Nakae Susumu , Matsuzaki Goro
Published: January 2023

IL-33 is a recently identified IL-1 family cytokine that promotes type 2 immune responses by signaling through the receptor complex consisted of ST2 and IL-1RAcP. Recent study using ST2 KO mice showed that IL-33 mediated signaling is required for the development of innate and adaptive immune responses against pulmonary Cryptococcus neoformans infection, suggesting involvement of IL-33/IL33R system in pulmonary cell-mediated immunity. In contrast, the ST2 KO mice showed normal protective immunity against pulmonary Mycobacterium tuberculosis (Mtb) infection. In this study, we analyzed role of IL-33 in host defense against chronically infected Mtb using IL-33 KO mice. The survival rate of the IL-33 KO mice was similar to that of the wild-type (WT) mice during 1 year observation period. Bacterial burdens of various organs of the IL-33 KO mice on the day 120 were nearly the same as that of the WT mice. In the infected lungs, inflammatory cytokine such as IFN-γ, TNF-α and IL-6, production of the IL-33 KO mice were similar to that of the WT mice. This result indicated that the generation of Th1 cells was not affected in the IL-33 KO mice infected with Mtb. Furthermore, the size of granulomatous lesionss in the lungs of the IL-33 KO mice were similar in comparison to the granulomas that of the WT mice on day 250 of infection. These data strongly support the notion that the lack of IL-33 neither suppress nor enhances protective immunity in the lung after mycobacterial infection.
CD8 T cell mediated disruption of tight junctions is associated with vascular permeability in vital brain regions during experimental cerebral malaria (MPF7P.708)
Authors: Huggins Matthew , Jin Fang , Johnson Aaron
Published: January 2023

One of the most severe complications of Plasmodium falciparum infection in humans is cerebral malaria. Cerebral malaria causes coma, neurological deficits, and potentially death. The disease is characterized by blood-brain barrier (BBB) disruption leading to vascular permeability. In this study, we investigated cerebral endothelial cell tight junction protein alterations and BBB breakdown in the Plasmodium berghei ANKA (PbA) model of experimental cerebral malaria. PbA infected C57BL/6 mice displayed significant areas of vascular leakage within the central nervous system at six days post infection. Regions of permeability co-localized with disruption of the BBB tight junction proteins claudin-5 and occludin on microvasculature as shown using confocal microscopy. Additionally, 3D MRI analysis revealed that PbA infected mice exhibited vascular leakage specific to the hypothalamus, thalamus, and brain stem. In contrast, PbA infected perforin deficient mice retained tight junction integrity and displayed significantly reduced vascular leakage. We propose that CD8 T cells initiate vascular permeability through disruption of cerebral endothelial cell tight junctions. In turn, the resulting imbalance of homeostasis in vital brain regions likely contributes to the pathology and morbidity associated with PbA infection.
Serum-derived bovine immunoglobulin increases peripheral and mucosal CD4 T cell count (MUC8P.736)
Authors: Asmuth David , Somsouk Ma , Min Ma Zhong , Hunt Peter , Miller Christopher , Dong Li Xiao , Hinkle John , Shaw Audrey , Weaver Eric , Klein Gerald
Published: January 2023

A multi-center trial in HIV-enteropathy was conducted to evaluate the impact of serum-derived bovine immunoglobulin (SBI) on markers of peripheral and mucosal immunity as previously reported. Participants (pts) with HIV enteropathy were randomized to SBI 2.5 vs 5.0 g BID or placebo (PBO) during a 4-wk lead-in phase followed by SBI 2.5 vs 5.0 g BID for 20 wks. 103 pts were enrolled with a mean duration of HIV, ART, and enteropathy over 15, 5 and 5 years, respectively. While there was no evidence for a median change in peripheral CD4 counts in all pts from baseline to wk 24 (681 to 661 cells/mL), significant increases were observed among pts in the lowest quartile (n=24)(311 to 366 cells/mL, p=0.002). In this subgroup the SBI pts had increased CD4 counts at wk 4 vs PBO pts (median +42 vs ‑16 cells/mL, p=0.02) which was maintained through wk 24. The mean plasma zonulin levels increased from 358(±317) to 812(±423) ng/mL (p<0.001) for pts receiving SBI through 24 weeks. Duodenal CD4 densities increased from 217 to 329 cells/mm2 (median increase of 145 cells/mm2 [p=0.02]) in biopsies obtained from 8 pts, confirming earlier findings. Duodenal crypt cells expressing Ki67 decreased in 6/7 pts from 41% to 24% (p=0.08) which correlated with the decreased number of Paneth cells per crypt (p=0.048). SBI may be a novel therapy to restore mucosal immunity and impact systemic immune reconstitution among pts who have not achieved normal CD4 counts despite prolonged suppressive ART.
TRAF6 expression in dendritic cells is essential for tolerance to dietary antigens (MUC8P.723)
Authors: Han Daehee , Walsh Matthew , Choi Yongwon , Surh Charles
Published: January 2023

TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). Recently, by studying mice DC-specific deletion of TRAF6 (TRAF6ΔDC) we have reported that TRAF6 expression in DCs is important to prevent a spontaneous Th2-associated gut inflammatory disease. We also found amelioration of TRAF6ΔDC disease by treatment with broad-spectrum antibiotics, suggesting a role for commensal microbiota. To formally demonstrate an involvement of commensal microbiota, we have now generated TRAF6ΔDC bone marrow chimeras under germ-free (GF) conditions. Unexpectedly, the Th2-associated disease in GF TRAF6ΔDC chimeras was exacerbated compared with TRAF6ΔDC chimeras generated under conventional conditions, indicating a beneficial role of commensal microbiota. Moreover, treatment of GF TRAF6ΔDC chimeras with broad-spectrum antibiotics also ameliorated the Th2-associated disease, suggesting a previously unrealized direct immunomodulating effect of antibiotics. Finally, to test whether the Th2-associate disease is driven by dietary antigens (Ags), we established TRAF6ΔDC bone marrow chimeras in “Ag-free” mice, in which GF mice are raised with a chemically-defined elemental diet devoid of macromolecules. Strikingly, the induction of a spontaneous intestinal Th2-associated inflammatory disease was dramatically averted in “Ag-free” TRAF6ΔDC chimeras, indicating that TRAF6 expression in DCs is essential to establish and/or maintain oral tolerance to dietary Ags.
A defect in a small-intestinal lymphoid structure affects colonic B cell development (MUC8P.726)
Authors: Surana Neil , Kasper Dennis
Published: January 2023

Peyer’s patches (PPs) are small-intestinal secondary lymphoid organs that represent an important inductive site for intestinal immunity. In this study, we prevented formation of PPs to examine its influence on intestinal immunity. Pregnant dams were injected at E14 with an IL-7Rα antibody, resulting in inhibition of PP formation in the pups. Flow cytometric analysis revealed no change in the numbers of immune cells in the spleen, lymph nodes, or small-intestinal lamina propria (SI LP). Surprisingly, the colon of PP-deficient mice was lymphopenic, with a specific decrease in CD19+ B cells, including colonic plasmablasts. We extensively looked for other defects (e.g., lymphoid structures, changes in cecal or peritoneal B cells, microbiota-related effects) that might explain this colonic phenotype, but everything else was normal. In control animals, the number of B cells present in PPs was strongly correlated to the number of B cells in the colon but not the SI LP. Moreover, if pregnant dams were injected with the IL-7Rα antibody at E18, the resulting pups had normal-appearing PPs and the normal number of colonic B cells. Interestingly, PP-deficient mice have more severe disease in infectious and chemically induced models of colitis, demonstrating that these colonic B cells are functionally important. Taken together, our data strongly suggest that a portion of colonic B cells is regulated by PPs, indicating a link between the small- and large-intestinal immune systems.
Ocular microflora modulates IL-17 and neutrophil recruitment within the conjunctiva associated lymphoid tissue (MUC9P.739)
Authors: St. Leger Anthony , Caspi Rachel
Published: January 2023

Previous studies have suggested a critical role for the functionality of conjunctiva‑associated lymphoid tissue (CALT), a mucosal immune tissue of the ocular surface, to maintain anterior ocular health. In this study, we developed a working animal model to study the functional properties of CALT in WT B6 and interleukin (IL)-22 KO mice, which are known to have impaired barrier function at gut mucosal surfaces. Aged IL-22 KO mice spontaneously developed CALT neutrophilia resulting in opacification of the cornea (keratitis). Neutrophilia in the CALT depended on in situ production of IL-17A/F, and was significantly reduced 48 hours after a single subconjunctival injection of αIL-17A/F. Innate lymphoid cells (ILCs) as well as αβ and γδ T cells isolated from CALT produced IL-17 in response to IL-1β made by activated CD11b+ dendritic cells, suggesting a role for normal ocular microflora in modulating mucosal immune responses at the ocular surface. Indeed, wild type mice treated with topical or oral antibiotics as well as germfree mice had reduced IL-17 and neutrophils in CALT. Our data uncover an interdependence between the CALT and normal ocular microflora for regulating mucosal immune function at the ocular surface and demonstrate its importance for maintaining long-term ocular surface homeostasis.
Pentobra: A novel antibiotic with potent antimicrobial activity against Propionibacterium acnes (TECH2P.901)
Authors: Agak George , Schmidt Nathan , Deshayes Stephanie , Yu Yang , Blacker Alyssa , Champer Jackson , Kao Stephanie , Xian Wujing , Kasko Andrea , Kim Jenny , Wong Gerard
Published: January 2023

Although antibiotics are a common form of treatment against acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in skin are recently compounded by increasing resistance of Propionibacterium acnes clinical isolates. To address these problems, we engineered Pentobra, a peptide-aminoglycoside molecule which has multiple mechanisms of antibacterial action, and investigated whether it can be a potential candidate for the treatment of acne. Pentobra combines the potent ribosomal activity of aminoglycosides with the bacteria-selective membrane-permeabilizing abilities of antimicrobial peptides. Pentobra demonstrated potent and selective killing of P. acnes, but not against human skin cells. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5-7 logs) against multiple P. acnes strains. Moreover, EM studies showed that Pentobra also had robust membrane activity, as treatment with Pentobra killed P. acnes cells. Pentobra may also have potential anti-inflammatory effects. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in microcomedone extracts isolated from human donors. Our work demonstrates that equipping aminoglycosides with selective membrane activity is a viable approach for developing new antibiotics against P. acnes that are effective in cutaneous environments.
Microfluidic device platform for T cell fate analysis (TECH2P.907)
Authors: Falcon Jessica , Conway Daniel
Published: January 2023

Microfluidic systems have enormous potential as investigational devices in biomedical research, in particular immunology. Microfluidic devices can be fabricated with the precise regulation of any number of parameters including controlled surface chemistries, geometrical dimensions, signal input and output and timing. The adjustable nature of microfluidic devices makes them an ideal platform to recreate cellular microenvironments for studying cell-cell interactions, migration, antibody and cytokine production, and differentiation in vitro. Investigations with customized microfluidic devices have the potential for elucidating the mechanism behind T lymphocyte commitment to either the αβ or γδ lineage. Both types of T cells arise from immature CD4-CD8- precursors in the thymus but diverge during development. Recent publications indicate that γδ T cell receptor (TCR) works in concert with ligand to direct progenitors to the γδ fate. We propose to develop a microfluidic device in which immature T lymphocytes can migrate through a thymus-like microenvironment allowing for αβ or γδ lineage commitment. As of yet, the microfluidic device fabrication protocol has been developed and preliminary cell culture experiments with the commonly used murine stromal cell line OP9 have success. Such a device would allow for analysis of specific developmental points in T cell development.
Human regulatory T cell isolation in 55 minutes using EasySep™ Releasable RapidSpheres™ (TECH2P.912)
Authors: Kokaji Andy , Sun C. , MacDonald G. , Clarke Samuel , Fairhurst Maureen , Eaves Allen , Thomas Terry
Published: January 2023

The preferred marker for identifying regulatory T cells (Tregs) is FOXP3 but its intracellular localization currently precludes it from being used for isolating viable cells. Human FOXP3+ Tregs are characterized as CD4+ T cells expressing high levels of CD25 and low levels of CD127. We have developed a novel immunomagnetic approach for the isolation of magnetic-particle-free human Tregs from PBMCs in as little as 55 minutes. This offers a vast improvement in speed over other immunomagnetic Treg isolation approaches, which typically require more than 2 hours. The standard approach for isolating Tregs combines CD4+ T cell negative enrichment followed by CD25 positive selection. By contrast, our novel approach is to first perform the CD25 positive selection step with EasySep™ Releasable RapidSpheres™, a new type of magnetic particle that can be rapidly released from the isolated cells with a no-incubation, no-wash protocol performed at room temperature. Subsequent depletion of CD127high and non-CD4+ T cells is achieved by negative enrichment with EasySep™ Dextran RapidSpheres™. Starting with 5x107 PBMCs, a purity of 87.6% ± 3.1% and recovery of 2.7x105 ± 8.2x104 Tregs (CD4+CD127lowCD25+FOXP3+) can be achieved (n=6). This is the first EasySep™ kit to incorporate the EasySep™ Releasable RapidSpheres™ and it exemplifies the potential of this technology for use in rapidly isolating magnetic particle-free cell populations possessing complex cell surface phenotypes.
Host-influenza proteomics leads to metabolic drug repurposing that reduces viral burden while significantly improving survival after lethal infection (TECH2P.917)
Authors: Smallwood Heather , Morfouace Marie , Duan Susu , Wang Ruoning , Zink Erika , Shulkin Barry , Green Douglas , Pasa-Tolic Ljiljana , Thomas Paul
Published: January 2023

A retrospective survey of pediatric patients infected with respiratory pathogens identified increases in glucose metabolism in the lungs of several individuals. After viral infection the host cell mounts an energetic and resource intensive antiviral response, while the virus exploits the host to generate progeny. Using absolute quantification with Host-Influenza Proteomics (HIP) we sought to characterize changes in host cell homeostasis that contribute to these competing processes. Like in the patients, we found gross metabolic changes occurring after influenza infection in primary human respiratory cells as well as dendritic cells. This new metabolic phenotype is distinct from immune activation and involves increases in glycolysis and glutaminolysis, decreased fatty acid β-oxidation concomitant to non-canonical TCA activity. Based on these findings we developed a metabolic drug repurposing screen in human bronchial epithelial cells and high-throughput viral tittering system. We screened over 160 metabolic substrates and small molecules to identify responsive prophylactic and therapeutic pathways (e.g. lipid and glucose metabolism respectively). One compound known to alter glucose sensitivity, NVP-BEZ235, reduced viral titers and significantly increased survival in vivo after lethal influenza challenge. Human PET scans followed by HIP analysis elucidated a unique metabolic signature in influenza-infection exposing targets for therapeutic intervention.
iRock 2.0 and the iR-Processor: an automated immune repertoire library preparation platform for NGS (TECH2P.919)
Authors: Byrne-Steele Miranda , Pan Wenjing , Brown Brittany , Alleyne Jessica , Caudle David , Yang Qunying , Wang Chunlin , Han Jian
Published: January 2023

Immune repertoire amplification of T and B cell receptors coupled to Next Generation Sequencing (NGS) provides detailed sequence-level insight into the immune system. Previously, we described the automation of a patented, nested multiplex PCR method (arm-PCR) to amplify an immune repertoire, inclusively and semi-quantitatively, on the iR-Processor, an instrument developed by iCubate, Inc. The iR-Processor is an open development platform for diagnostic panels consisting of a closed-cassette and the iR-Processor. The cassette is pre-loaded with the reagents necessary for library preparation, and the iR-Processor instructs the cassette to perform the necessary liquid handling steps and PCR for library generation. Template RNA is added to the cassette with the RT enzyme. iRock 2.0 represents an improved method in which all of the first round cDNA is rescued prior to library generation with a pair of universal primers. Once PCR is complete, residual primer-dimers are removed by magnetic selection of the primary product in the cassette. The amplified library is eluted from the magnetic beads by the user and is directly ready for sequencing using the Illumina MiSeq. The sequencing results from repeat amplification experiments of the TCRβ chain from the same individual and between individuals are compared. The results demonstrate high repeatability among shared CDR3 sequences and an improvement in the percentage of shared CDR3 discovery upon repetition.
Deep profiling of single T cell receptor repertoire and phenotype with targeted RNA-seq (TECH2P.927)
Authors: Ji Xuhuai , Lyu Shu-Chen , Spindler Matthew , Bacchetta Rosa , Goncharov Igor , Han Arnold , Glanville Jacob , Wang Weiqi , Roncarolo Maria , Meyer Everett , Nadeau Kari , Maecker Holden
Published: January 2023

Analysis of TCR repertoires is useful for monitoring T cell responses. Integrating TCR sequencing with expression of targeted genes at the single cell level allows comprehensive analysis of T cell function and specificity. RT-PCR reactions of single T cells were performed using 76 primers from VJC regions and 136 primers for targeted T cell genes. A series of nested PCR reactions incorporated with barcodes and adapters for pooled amplicons sequencing. A bioinformatics pipeline was used data analysis. 2880 single T cells were collected and tested. TCR sequences were obtained in up to 90% of the wells. Based on the CDR3 region, multiple dominant TCR clones were identified. 52 of 68 target genes were validated with sorted target-specific T cells. FOXP3, IL10, PRF1, IL13, and RUNX3 were highly expressed in memory activated cells. The specific CD8+ T cells had high frequency of CCR9, LAG3, CD8, CD62L, and IFNG expression. TGFB, TNFA, IL12, FOXP3, PD1, TBET, CTLA4, MKI67, RUNX1, and GATA3 were major biomarkers in the specific CD4 cells. The gene expression patterns were often associated with TCR repertoire CDR3 variations and/or the TCRβ and TCRα chain usage although the same TCRα/β sequences may have different target genes expressed. We expanded a method enabling sequencing of TCR repertoire and multiple functional genes in single, sorted, T cells through targeted RNA-seq technology. This approach could reveal important insights into T cell functions and clonal development.
High-throughput pairing of T cell receptor alpha and beta sequences (TECH2P.930)
Authors: Sherwood Anna , Howie Bryan , Berkebile Ashley , Berka Jan , Emerson Ryan , Williamson David , McCormick Michael , Kirsch Ilan , Vignali Marissa , Rieder Mark , Carlson Christopher , Robins Harlan
Published: January 2023

Conventional high-throughput immunosequencing methods profile TCR alpha (TRA) and/or TCR beta (TRB) repertoires, but do not enable pairing of the cognate subunits comprising functional TCRs. We developed a method called pairSEQ that can pair hundreds of thousands of TRA and TRB sequences in a single experiment. Our approach works by allocating random samples of T cells to a micro-titer plate, sequencing amplified cDNA from TRA and TRB genes, and DNA bar-coding the wells. Most TCR sequences uniquely identify clonal cell populations, so pairs can be assigned based on TRA and TRB sequences that occupy the same wells. To validate our method we identified cognate pairs from a sample of cells mixed from two people. The cross-sample pairs directly measure false pairing rates, which closely matched predictions from our statistical model. To show our method can be targeted to any clonal frequency range of interest, we designed a 3-plate experiment to capture pairs with repertoire frequencies from 0.5% to less than 1 in 10 million. These experiments yielded over 300,000 confident TCR pairs with one 96-well plate alone providing over 200,000 pairs. This technology is not restricted by material; in a single experiment we used this method to identify 6,349 TCR pairs from tumor infiltrating lymphocytes in 9 tumor samples. Our TCR pairing method is highly scalable, and should be broadly applicable to all somatically rearranged immune receptor loci, including immunoglobulins.
Rapid and cost effective immunoassay development and fit-for-purpose validation of a biomarker assay using Spatial Proximity Analyte Reagent Capture Luminescence (SPARCL) (TECH2P.760)
Authors: Cameron Mark
Published: January 2023

A novel detection technology was used for rapid immunoassay development and validation. SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) is a proximity dependent and homogeneous chemiluminescent detection method. In a SPARCL assay, a chemiluminescent substrate (acridan) is brought into the proximity of an oxidative enzyme (horseradish peroxidase) through the specific antigen/antibody interaction. A flash of light is generated and is proportional to the amount of IL-8 bound. Assay development was completed in less than 3 days. The validated assay features a wide dynamic range (2 - 4,000 pg/mL), low MRD, short assay run time (60 min.), and sensitivity (2 pg/mL). Quality control samples (QC’s) run in plasma resulted in statistics that show the assay is precise and accurate, with a total error of all QC levels of under 12%. Matrix spiked with IL-8 diluted in a linear manor. The SPARCL technology enables rapid assay development, delivers an assay with desirable performance characteristics and allows for considerable savings in labor, disposables and capital equipment compared to popular platforms. Existing software and luminometers may be used for a SPARCL data acquisition. As a result, there may not be a need to validate new software or plate readers already running in existing Laboratory Information Management Systems or in a regulated environment. The SPARCL assay is microtiter plate-based and is suitable for use with robotics and in HTS applications.
PrimeFlow™ RNA assay as a method to elucidate the effect of protein transport inhibitors on cytokine gene expression in whole blood (TECH3P.941)
Authors: Frank Richard , Scott Bradley , Lai Chunfai , Sias Leslie , Funatake Castle
Published: January 2023

Intracellular antibody staining followed by flow cytometric analysis is commonly used to study cytokine expression in leukocytes. Detection of cytokines typically requires that cells be treated with protein transport inhibitors such as Brefeldin A and Monensin in order to accumulate sufficient levels of intracellular protein. However, it is not known whether the use of these inhibitors affects gene expression. PrimeFlow™ RNA is a new flow cytometric assay that allows the simultaneous interrogation of RNA and protein levels within single cells. We have used this assay to investigate the effect of protein transport inhibitors on the expression of human IL-2, TNF alpha and IFN gamma during the course of a 5 hour stimulation of whole blood with PMA and ionomycin. This represents first time that the effect of protein transport inhibitors on gene expression in whole blood has been examined in single cells.
Identification of predictive protein biomarkers for treatment efficacy and clinical relapses of multiple sclerosis (THER7P.950)
Authors: Raphael Itay , Forsthuber Thomas
Published: January 2023

Despite extensive research, MS remains a disease that lacks a definitive prognostic test to predict imminent disease relapses. Thus, patients may undergo years of unnecessary treatments. Additionally, current treatments for MS can produce dramatically different outcomes in different individuals and therefore there is a critical need to develop biomarkers for treatment efficacy and resistance. We have recently developed a novel quantitative proteomics method to measure changes in proteome expression over the course of experimental autoimmune encephalomyelitis (EAE). Our statistical analyses indicate a strong correlation to EAE severity and/or clinical-phase. Interestingly, we revealed characteristic CNS-specific protein expression waves prior to the onset of clinical symptoms. We are currently testing whether these protein expression waves allow us to predict the onset of clinical episodes and forecast the severity of the disease to guide treatment strategies. Additionally, we have identified changes in the CNS proteome that can be measured in serum during EAE that correlate with the therapeutic efficacy of glucocorticoid treatment. Our studies will provide proof-of-principle for developing homologous human biomarkers that may be useful to predict disease onset and treatment efficacy. Finally, the detected changes in the CNS proteome may provide insights into key mechanisms that contribute to the disease pathology and may be useful to develop new therapeutic targets for MS.