bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of αPD-1 and extended half-life IL-2 is effective against the GL261 glioma and uniquely reverses GBM-associated immunosuppression
Authors: Tritz Zachariah Peter , Ayasoufi Katayoun , Malo Courtney Sarah , Himes Benjamin , Zastrow Alexi , Wolf Delaney , Goddery Emma , Khadka Roman , Fain Cori , Chen Maggie , Yokanovich Lila T , Jin Fang , Hansen Michael , Wang Chensu , Moynihan Kelly , Irvine Darrell J , Wittrup K Dane , Parney Ian F , Johnson Aaron J
Published: January 2023

Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12–18 months post diagnosis. The unique immune environment of the brain and the immunosuppressive features of GBM complicate the use of immunotherapies against these tumors. We aimed to promote the efficacy of αPD-1 against the GL261 murine model of GBM by supplementing this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. Neither αPD-1 nor MSA-IL-2 are effective monotherapies for GL261 tumors, but the combination provides durable tumor clearance and prevents subsequent rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Our combination therapy also reversed multiple key features of GBM-associated peripheral immune suppression, including depressed CD4 T cell counts and dampened MHC class II expression. Our combination therapy’s ability to overcome these immune derangements associated with glioma growth and to function independent of MHC class I restricted antigen presentation enhance the appeal of this therapy for future translational adaptation.

This work was supported by the National Institutes of Health (R01 NS103212) and the Mayo Clinic-Koch Institute Cancer Solutions Team Grant
Monoclonal antibodies displaying mutations in Fc region to prolong protection against fentanyl toxicity
Authors: Khaimraj Aaron J , Hicks Dustin , Baehr Carly , Pravetoni Marco
Published: January 2023

Synthetic opioids including fentanyl are the lead cause of opioid-related deaths in the United States. Current treatments for opioid overdose include the opioid receptor antagonist naloxone; however, naloxone is not as effective against highly potent opioids, has a relatively short half-life, and precipitates withdrawal symptoms. Monoclonal antibody (mAb) therapy offers a promising strategy to combat opioid-related overdoses. Opioid-specific mAb bind to target molecules in the bloodstream and inhibit them from crossing the blood-brain barrier. We produced a lead fentanyl-specific mAb that protects against fentanyl toxicity in rats. The efficacy of mAbs is reliant on maintaining a high concentration in the bloodstream to ensure sufficient binding to the target drug. The neonatal Fc receptor (FcRn) is responsible for the recycling of IgG in circulation, thus increasing their effective half-life. Therefore, to increase the duration of efficacy of a lead chimeric fentanyl-specific mAb, we introduced three different mutations to the Fc region of the mAb. Biolayer interferometry paired with recombinant FcRn showed a 10-fold increase in affinity of the recombinant mAb compared to wild-type. Efficacy of the mutated and WT mAb against fentanyl-induced behavior and toxicity was also tested in mice. Both the mutated and WT mAb reduced fentanyl-induced antinociception, respiratory depression, and bradycardia. Finally, the half-life and long-term efficacy of these mutated mAbs was assessed in transgenic mice expressing human FcRn. Extending the half-life of these mAbs will facilitate more successful clinical approaches in preventing opioid-related overdoses.

This work was funded by NIDA under CounterACT grant U01-DA051658
The effects of dietary zinc supplement on intracellular zinc concentration in bovine immune cells
Authors: Franco Carlos Enrique , Diaz Fabian Esteban , Rients Emma , Smerchek Dathan T , Hansen Stephanie L , McGill Jodi L
Published: January 2023

Zinc (Zn) is one of the most abundant metals in our body and is critical for innate and adaptive immune functions. In humans and mice, Zn deficiency may lead to immunodeficiency, reduced reproductive health, and increased disease susceptibility. However, trace mineral requirements and evidence of Zn dysregulation are poorly defined in cattle. This study aimed to evaluate intracellular Zn concentrations in immune cells from young growing steers with different Zn dietary supplements. Eighteen Angus crossbred steers were stratified by body weight and randomly assigned to two dietary treatments groups: 1) control, no supplemental Zn, or 2) high Zn; 150 ppm of Zn delivered in premixes in total mixed rations. To evaluate the intracellular Zn concentration of immune cells, whole blood cells were loaded with FluoZinTM-3, a fluorescent cell-permeant Zn indicator, then stimulated with Zn pyrithione (100 μM) or TPEN [N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine] (50 μM) to measure maximum and minimum mean fluorescent intensity. Cells were stained with surface antibodies to identify immune cell populations and data were collected by flow cytometry. The intracellular Zn concentrations of circulating CD8 T cells and γδ T cells, but not other populations, differed significantly between treatment groups. These results indicate that intracellular Zn concentration can be influenced by dietary Zn intake, and that certain immune populations are differentially affected by Zn status.

This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2020-06540 from the USDA National Institute of Food and Agriculture.
Characterization of SARS-CoV-2-specific CD8+ T cells in COVID-19 convalescent, vaccinated, and individuals with respect to age
Authors: Choy Cecily , Lu Jian , Li Jiangyuan , Hemani Humza , Chen Joseph , Lustig Ana , McKelvey Julia , Melvin Denise , Ruffolo Jeannie , Zukley Linda , Wallace Tonya , Dunn Christopher , Nguyen Cuong , Fan Jinshui , De Supriyo , Chia Chee , Ferrucci Luigi , Egan Josephine , Weng Nan-ping
Published: January 2023

COVID-19, an infectious disease caused by SARS-CoV-2, has given rise to the current global pandemic. Despite the number of infections nearing 300 million people worldwide, CD8+ T cells against SARS-CoV-2 in COVID-19 convalescent, vaccinated, and healthy adults and their changes with age have not been fully characterized. Here, we report the frequency, phenotype, and in vitro expansion capacity of circulating CD8+ T cells recognizing twenty-two epitopes from five SARS-CoV-2 proteins (S, N, M, ORF1ab, and ORF3a). Through multi-color flow cytometry using antigen-specific tetramers, we found that the frequencies of circulating CD8+ T cells ranged from undetectable to 1% in healthy, convalescent, and vaccinated individuals. Analysis of healthy adults ranging from 17 to 98 years of age revealed that the frequency of CD8+ T cells recognizing the epitope N-LLL (N222–230, LLLDRLNQL) is negatively correlated with age, particularly in the frequency of naive N-LLL-specific CD8+ T cells. Furthermore, the percentage of donors which displayed expansion of N-LLL-specific CD8+ T cells in response to in vitro peptide stimulation was significantly higher in convalescent patients than in healthy donors. These preliminary results suggest that there is an age-associated reduction of N-LLL-specific CD8+ T cells and exposure to SARS-CoV-2 primes these cells for robust expansion in response to stimulation with the N-LLL epitope. This work is still ongoing, and we will present the final results during the meeting.
Sodium Pyruvate Inhalation for COVID-19 Long Hauler Symptoms – An Effective and Inexpensive Treatment
Authors: Nadler Riley A
Published: January 2023

Studies show that 1 in 4 COVID-19 patients are afflicted with chronic, or long hauler, symptoms. This means that approximately 12 million Americans continue to suffer from COVID weeks and months after the active infection subsides. The long hauler symptoms include hypoxemia (low SaO2), fatigue, coughing/sneezing, trouble breathing, body aches and headaches. N115 (EmphyCorp/Cellular Sciences Inc.) is a clinical grade sodium pyruvate nasal spray currently under evaluation by the FDA for the treatment of COPD, Pulmonary Fibrosis and other chronic respiratory diseases. This study examined the effects of N115 treatment on the symptoms associated with COVID-19 long haulers. We have observed significant improvement in SaO2 levels, trouble breathing and fewer headaches indicating that N115 is a potential therapeutic for a major disease with few current options for treatment.
Characterization of pathological IgE-mediated mast cell activation in Lyme disease
Authors: Galloway Sarah D , Shoham Maia , Lee Brandon , Torrez-Dulgeroff Laughing Bear , Irnov Irnov , Lin Athena , Strausz Satu , Hansen Paige , Blacker Grace , Salomon-Shulman Rachel , Potula Hari-Hara Surya Kumar , Markovic Maxim , Nahass George R , Colace Olivia , Raveh Tal , Pollack Beth , Sanders Erin , Ollila Hanna , Wagner Christine Jacobs , Robinson William H , Weissman Irving L , Tal Michal C
Published: January 2023

Lyme disease, caused by the bacteria Borrelia burgdorferi, is the most common and rapidly growing vector-borne infectious disease in the United States and Europe. High variability in disease burden among Lyme patients suggests that individual immune responses may be key drivers of clinical presentation and patient outcomes. Use of high resolution flow-based immunosorbent profiling revealed that a subset of Lyme patients with persistent symptoms were producing high concentrations of IgE specific to B. burgdorferi. Comparing C57B/6 mice, which are tolerant to B. burgdorferi, and C3H/HeJ mice, which are susceptible to disease, we find high levels of IgE specific for B. burgdorferi in C3H/HeJ but not C57B/6 mice. Furthermore, IgE was found to target Borrelia peptidoglycan in both acute and chronic infection models. Histologic analysis of mouse Lyme arthritic ankle tissue showed mast cells, which release highly immunogenic effectors upon activation by bound IgE, degranulating at significantly higher rates compared to uninfected controls. Forced mast cell degranulation exacerbated Lyme arthritis in infected mice. This data suggests that a subset of Lyme patients with persistent symptoms may have developed an allergic response to conserved bacterial antigens from a B. burgdorferi infection, as opposed to an autoimmune type response. Inclusion of IgE reactivity in diagnostic testing and examination of pathological immune responses to bacterial antigens could assist clinicians in patient care and effective treatments.

Research reported in this publication was supported by the Fairbairn family foundation; Bay Area Lyme Foundation; the Younger family foundation; the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the Virginia and D. K. Ludwig Fund for Cancer Research; M.C.T. was supported by Stanford Immunology training grant 5T32AI007290, and the NIH NRSA 1 F32 AI124558-01 award. L.B.T.D. was supported by a Stanford Diversifying Academia Recruiting Excellence fellowship. S.D.G was supported by the California Institute for Regenerative Medicine Bridges 2.0 Training Program grant EDUC2-08397. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Epigenetic immunophenotyping in monitoring of SARS-CoV2 vaccine response and COVID-19 disease course
Authors: Jung Janine Nadine , Hoffmueller Ulrich , Samans Bjoern , Raschke Eva , Chornet Marta Rossello , Schildknecht Konstantin , Lozza Laura , Chornet Araceli Rossello , Zaragoza Lourdes Alos , Laforet Javier Hernandez , Babel Nina , Olek Sven
Published: January 2023

The objective was to explore whether epigenetic immune cell counting can advance efficiency and quality of diagnostic and immune monitoring related to COVID-19. Application areas were monitoring disease course, therapeutic clinical development, and measurements of SARS-CoV2 vaccine responses.

Immune cell type specific epigenetic assays have been developed over the last decade. They are primarily used in therapeutic clinical research in oncology and autoimmune disease. Due to the high sample stability and low amount requirements for epigenetic measurements and available assay portfolio for key immune cell populations relevant in COVID-19, the method promised to be useful and practical in the pandemic setting.

Epigenetic immunophenotyping using whole blood of hospitalized Covid19 patients was applied and CD3, CD4, CD8 and regulatory T cell populations, NK cells, naïve and memory B cells were quantified, and measurement results show to predict mild or severe COVID-19 disease courses. Furthermore, nasopharyngeal swab and saliva samples were applied demonstrating that epigenetic immune monitoring can measure immune cell content in such non-invasive sample types.

Due to the low sample volume and handling requirements and availability of 35 assays for relevant immune cell populations, epigenetic immune monitoring is suitable for therapeutic COVID-19 clinical trials. Another possibility for sample collection is single drops of capillary blood deposited on filter paper (dried blood spots), which have been collected pre and post SARS-CoV2 booster vaccinations in healthy subjects. Such measurements revealed vaccine response for example in changes of cell populations epigenetically active in the markers CCR7 and TIGIT.
Electromyographic characterization and bioinformatic pathway analysis of spinal cord inflammation in a Tat induced HIV-associated sensory neuropathy model
Authors: Cao Ling , Kohsar Ahmad , Wilson-Braun Peter , Harrison Benjamin
Published: January 2023

HIV sensory neuropathy (HIV-SN) is the most prevalent neurological complication of HIV infection. The contribution of Tat in HIV-SN has not been studied extensively. Previously, doxycycline (DOX) inducible HIV-1 Tat transgenic (iTat) mice have been shown to exhibit neuropathy-like behaviors following Tat induction. To further understand Tat-associated HIV-SN, here, we first characterized the model via electromyographic (EMG) evaluation. EMG testing of the hind leg along the sciatic nerve was conducted before DOX (daily i.p. for 14 days at 100 mg/kg) or vehicle (pH-matched phosphate-buffered saline) injection and at days 3, 7, 14, 21, 28 and 35 post the first injection. Initial analysis showed a slightly increased maximal amplitude in DOX mice along with the decreased hind paw grip strength. Further, we performed a NanoString RNA assessment of lumbar spinal cord tissues using a panel of 200+ inflammation-related genes, which was followed by a series of bioinformatic pathway analyses. Short Time-Series Expression Miner (STEM) cluster analysis identified four significant temporal expression patterns of genes. These four profiles were characterized by continuous downregulation, early-, mid-, and late-upregulation. Using PANTHER GO-pathway analysis we found that three signaling pathways were common amongst the four STEM profiles which were the apoptosis signaling, inflammation mediated by chemokine and cytokine signaling, and the Toll-like receptor signaling pathways. Previous studies highlighted selected genes that belong to these pathways and are also targets of Tat. Future work will focus on investigating how these pathways contribute to sensory neuropathy and identifying biomarkers for HIV-SN.

Supported by NIH/NIDA 1R21DA044886 (Cao)
Choline metabolism underpins macrophage IL-4 polarization in vitro and in vivo
Authors: Ghorbani Peyman , Kim Sang Yong , Alecu Irina , Woo Daniel , Ilijevska Maja , Smith Tyler K T , Nunes Julia R C , Minarrieta Lucia , St-Pierre Julie , Bennett Steffany A L , Nair Meera G , Fullerton Morgan D
Published: January 2023

Choline homeostasis in macrophage biology is important for LPS-polarized inflammation. In macrophages, choline mainly supports phosphatidylcholine (PC) synthesis, which is crucial for membrane production and cytokine secretion. Here, we examined choline metabolism in IL-4 polarized macrophages in vitro and in vivo. Like LPS, IL-4 increased choline transporter-like protein 1 (CTL1) expression, choline uptake and the incorporation of choline into PC. Targeted lipidomics analysis revealed increased PC content in IL-4-polarized macrophages, with an enrichment in low-saturated species. Pharmacological inhibition of choline uptake/choline kinase with hemicholinium-3 or RSM-932a showed no effect on certain hallmark IL-4-induced macrophage genes (Chil3, Mrc1, Arg1) but significantly reduced the transcript and protein expression of RELMα. Consistent with the function of RELMα in wound healing, 3T3-L1 cells healed more slowly in a scratch wound assay with conditioned media from IL-4 polarized macrophages in which choline metabolism was inhibited compared to vehicle-treated conditioned media. In addition, inhibiting choline metabolism completely prevented PD-L2 upregulation and increased PD-L1 expression on IL-4-polarized macrophages, together with suppressed cellular respiration and increased glycolysis. Furthermore, in vivo administration of RSM-932a (3 mg/kg i.p.) in C57BL/6J mice lowered RELMα in macrophages, decreased PD-L2 and increased PD-L1, and resulted in a loss of resident peritoneal F4/80hi macrophages. Choline represents an underappreciated regulator of macrophage immunometabolism and strategies to target macrophage choline uptake or choline metabolism may be therapeutically valuable.
Understanding the temporal requirement for T follicular helper cells for germinal center output of long-lived plasma cells and B memory cells.
Authors: Zilch Tiago , Leddon Scott , Fowell Deborah J
Published: January 2023

T follicular helper cells (Tfh) play a critical role in supplying help to B cells for the selection of high-affinity B cells in germinal centers (GC), that give rise to memory B cells (Bmem) and long-lived plasma cells (LLPCs). The products of the GC reaction appear to be temporally distinct, with Bmem cells produced early and LLPCs late.

The temporal requirements for Tfh help are not well understood and we still do not know when and for how long this help is critical. In the total absence of help supplied by Tfh cells, both GCs and LLPCs are greatly diminished. Our previous work (Schrock, Leddon et al 2019, PNAS) suggested that sustained GC Tfh were critical for LLPC generation, but interestingly not for Bmem generation. We hypothesize that a temporal requirement for GC Tfh regulates LLPC versus Bmem GC output. To test this, we have established a number of models that allow for deletion of Tfh at different times post OVA/CFA immunization. To specifically target Tfh cells, CD4 iCRE Bcl6fl/fl mice were used for time-dependent deletion of Bcl6 upon tamoxifen administration at different stages of the GC reaction. Tfh and GC output are determined using multicolor Flow Cytometry panels, BM B cell Elispot for LLPCs and a Bmem Elispot. With these systems in place, we aim to pinpoint the temporal requirement for Tfh for both Bmem and LLPCs, speculating that timing of Tfh help will differ for these two arms of the GC response.

Understanding this temporal requirement of Tfh help within the GC can not only help us to better design future vaccines that promote LLPCs, but also guide the development of drugs to modulate Tfh longevity in autoimmune disorders, where aberrant Tfh development or maintenance contribute to pathology.
Developmental dynamics and genomic programming of GC-derived precursors that give rise to long lived plasma cells
Authors: Vijay Godhev Kumar Manakkat , Thakkar Kairavee , Chetal Kashish , Hay Stuart , Fan Jingyu , Joglekar Alok V , Salomonis Nathan , Singh Harinder
Published: January 2023

Plasma cell (PC) precursors are generated during a germinal center (GC) response and migrate to the bone marrow (BM) where they undergo terminal differentiation into short-lived (SL) or long-lived (LL) antibody secreting cells. The developmental dynamics and genomic states of PC precursors remain to be elucidated1. We utilize a novel experimental system, involving CD138+ splenocytes isolated from NP-KLH immunized C57Bl/6J mice (21–42 dpi) and their adoptive transfer into B cell deficient mMT mice, to analyze developmental dynamics of antigen-specific PC precursors and their progeny at single-cell resolution. We demonstrate that SLPC precursors are generated earlier (d21), whilst LLPC precursors are generated later (d35) during a GC response. scRNA-seq analyses reveals increased frequency of a novel cluster of transitional cells expressing both B-lineage and plasma cell-specific genes. Coupled analyses using BCR-seq shows that NP-specific transitional cells express cell cycle genes and undergo greater clonal expansion on d35 to give rise to proliferating and quiescent PCs. Antigen-specific clonal tracking in the spleen and BM compartments suggests that the transitional cells emanating from the GC, differentiate into proliferating PCs before migrating to the BM. CITE-seq analyses and reconstitution experiments demonstrate that proliferating PCs are contained within the B220− CD138+ CD44+ CD11a+ subset and are enriched for BMPC precursors. These results reveal novel GC-derived transitional cells that generate precursors of both SLPCs and LLPCs with differing developmental and proliferation dynamics in a humoral immune response.
The PGI2 signaling pathway decreases glycolysis and mitochondria respiration in mouse Th2 cells
Authors: Zhou Weisong , Zhang Jian , Chowdhury Nowrin U , Norlander Allison E , Toki Shinji , Newcomb Dawn C , Peebles R Stokes
Published: January 2023

Prostaglandin I2 (PGI2) is a lipid molecule produced in the cyclooxygenase (COX) metabolic pathway and regulates T cell function and inflammation. We have previously shown that PGI2 inhibited type 2 cytokine production by Th2 cells in vitro and in vivo. To further investigate the mechanism of the inhibitory effect of PGI2 on Th2 differentiation, we determined the effect of the PGI2 analog cicaprost on T cell metabolism under Th2 conditions. Mouse naïve CD4 T cells from the spleen were activated with antibodies against CD3 and CD28 under Th2 polarization conditions and treated with cicaprost or vehicle for 4 days. Seahorse assays measured cellular glycolysis and mitochondria respiration. We found that cicaprost significantly decreased basal and maximum glycolysis in Th2 cells. Furthermore, when we performed Seahorse assays on the cells rested for 2 days after the initial 4 day T cell activation, we discovered that cicaprost not only suppressed basal glycolysis and glycolytic reserve but also dose-dependently inhibited basal and maximum mitochondria respiration. These results suggest that the PGI2 signaling pathway inhibits Th2 cell metabolism, providing a possible mechanism for PGI2-mediated inhibition of Th2 differentiation and Th2 cell-dependent immune disorders.

The Department of Veterans Affairs BX004299 (RSP), NIH AI095227 (RSP), NIH AI111820 (RSP), NIH AI145265 (RSP), NIH AI124456 (RSP), NIH AI145397 (RSP).
Contributions of the PD-1/PD-L1 pathway to Borrelia persistence and inflammation
Authors: Helble Jennifer D , McCarthy Julie E , Starnbach Michael N , Hu Linden T
Published: January 2023

Borrelia burgdorferi, the causative agent of Lyme disease, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. Exhausted T cells are present in many long-term infections and mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Indeed, we have found that CD4+ T cells in the draining inguinal and popliteal lymph nodes, as well as the heart and ankle joint, significantly upregulate the immunoinhibitory molecule PD-1 following B. burgdorferi infection. While PD-1 expression on T cells is a strong indication of CD4+ T cell exhaustion, we have extended our findings by assessing expression of a ligand for PD-1, PD-L1, on antigen presenting cells in vitro and in vivo following B. burgdorferi infection. We have shown that not only is PD-L1 is significantly upregulated on both bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) following B. burgdorferi stimulation, but is also upregulated on macrophages and a subset of conventional dendritic cells (cDC2s) in the draining lymph nodes following in vivo infection. We are currently testing if mice deficient in either PD-1 or PD-L1 exhibit changes in ankle joint or heart inflammation compared to wild-type mice, which would suggest that the PD-1/PD-L1 axis is critical for regulating inflammation during B. burgdorferi infection. Together, these data will allow us to determine how this immune checkpoint regulates inflammation during B. burgdorferi infection.

Supported by grants from the NIH (R01 AI150157), the Harvard Medical School Fairbairn Family Lyme Research Initiative, and the Tufts University School of Medicine Natalie Zucker Research Center for Women’s Scholars Award.
Cutaneous CD4+ T cell subsets promote distinct outcomes in human keratinocytes and fibroblasts
Authors: Morawski Peter A , DeBerg Hannah , Fahning Mitch , Stefani Caroline , Lacy-Hulbert Adam , Gratz Iris , Campbell Daniel J
Published: January 2023

Epithelial, stromal, and immune cells in the skin cooperate to ensure appropriate response to pathogens and environmental damage. Recent single cell transcriptional analyses of healthy and fibrotic human skin revealed a wide range of phenotypes that arise in response to distinct cytokine milieu. Cutaneous T cells promote host defense and the subsequent resolution of inflammation and repair of damaged skin through the production of cytokines, but the precise mechanisms T cells use to impact the differentiation and function of skin cells involved in inflammatory and tissue-repair responses remain understudied. To assess the functional capacity of cutaneous lymphocytes we isolated skin-tropic and skin-resident T helper cells including a novel population of pro-fibrotic migratory tissue resident memory cells we recently discovered. We measured both quantitative T cell cytokine production following TCR stimulation, and the impact of T cell derived cytokines on human keratinocyte and fibroblast transcriptional profile and morphology. We find that phenotypically and functionally distinct circulating and skin-resident T cell subsets have the capacity to promote discrete cytokine-dependent transcriptional and cellular outcomes in the skin including the induction of metabolic, proliferative, and inflammatory gene profiles. Thus, cutaneous T cells support host-protective and tissue-repair responses through direct activity on keratinocytes and dermal fibroblasts.
Lymph node-targeted long-acting butyrate micelles induce regulatory immune modulation
Authors: Cao Shijie , Wang Ruyi , Bashir Mohamed Elfatih H , Su Yanlin , Sabados Matthew , Hesser Lauren A , Nagler Cathryn R , Hubbell Jeffrey A
Published: January 2023

Regulatory T cells (Tregs) play essential roles in maintaining immune homeostasis and preventing autoimmunity and allergy. Peripherally derived Tregs are induced most efficiently in the lymph nodes (LNs), yet current therapies are not effectively delivered to these sites. The short-chain fatty acid butyrate facilitates extrathymic differentiation of Tregs and has been considered a promising therapeutic candidate. However, there are challenges to its use, including its foul odor, quick metabolism, and low efficiency with traditional oral delivery. Herein, we designed butyrate-prodrug micelles that deliver butyrate to peripheral LNs through subcutaneous (SC) administration. We have developed two polymeric micelles, one with a neutral charge (termed NtL-ButM) and one with a negative charge (termed Neg-ButM), which both contain 28 wt% butyrate and have similar sizes of ~40 nm. In a biodistribution study, we observed that Neg-ButM, not NtL-ButM, accumulated in draining LNs for over one month. At the cellular level, the Neg-ButM were taken up mostly by the macrophages and dendritic cells in the LNs and inhibited LPS-induced activation of those cells. In antibiotic-treated mice, three weekly doses of Neg-ButM significantly increased the numbers of CD25+ Foxp3+ CD4+ Tregs in draining LNs compared to either PBS or NtL-ButM treatment. Furthermore, we demonstrated that, when combined with intragastrically administered peanut proteins, the SC-injected Neg-ButM protected from anaphylactic responses in a mouse model of peanut allergy. This approach provides a potential Treg-based therapy against food allergies.

Supported by a seed grant from the Chicago Immunoengineering Innovation Center of the University of Chicago.
Practical Solutions to Improve Animal Study Reproducibility
Authors: Ibsen Eric
Published: January 2023

A 2012 study published by Amgen researchers found that 90% of the in vivo oncology studies were not reproducible. Historically, 95% of post-phase III oncology clinical trial therapies failed to receive regulatory approval due to a lack of demonstrated efficacy, not toxicity. The overwhelming lack of reproducible data underscores the dire need to critically examine our current standard animal study conduct processes for areas of potential improvement. In today’s competitive climate, researchers must identify the causes of poor animal study irreproducibility and implement meaningful and effective solutions. Recently, questions and debates concerning the validity of preclinical science data have resulted in multiple published reviews on the problem. Fortunately, as covered in this presentation, cost-effective technology exists to mitigate this “Crisis of Animal Study Irreproducibility”. This presentation covers:

The factors contributing to poor animal study reproducibility.

Publicly-available and online resources and practically-applicable concepts, such as the ARRIVE guidelines, PHISPS protocols, and the N3R Design Assistant.

Attendees receive first-hand experiences with a variety of animal study conduct approaches and commercially-available animal study workflow software applications, along with a summary of each tool’s direct and indirect impacts on the causes of animal study irreproducibility.

Exploration of how these concepts are applied in one Animal Study Workflow Software to solve problems, improve animal study reproducibility and faithfully preserve detailed study results for future access.
Development of a serum-free, chemically-defined workflow solution for T cell culture
Authors: Lopes Vanda , Quintero Zayda , Ni Jessie
Published: January 2023

Cancer immunotherapy has become one of the most promising fields in the treatment of oncologic related disorders, by harnessing the power of T-cells. Expansion and/or differentiation of cells with the right therapeutic profiles, in a consistent and safe manner, requires the use of defined cell culture reagents. One major cause of variability is serum, with its undefined composition. The presence of undefined reagents can lead to the inability to reproduce results between labs, and stop the progression from discoveries to clinical applications. Here we introduce a chemically-defined serum substitute and a low DMSO cryopreservation solution specially designed for T cells. After identifying the essential cell property parameters, and establishing appropriate cell culture assays, we initiated formulation optimization. We were able to identify the critical components required for appropriate cell expansion and cryopreservation. We were able to develop a chemically-defined solution, comparable in performance to serum, while bringing the benefits of being fully defined. We were also able to develop a low DMSO cryopreservation solution, tailored for T cell applications, comparable in performance to home brew or other commercially available solutions containing 10% DMSO. Our development work was allowed to gain in depth knowledge of the impact of each component in T cell biology, allowing us at BioLegend to provide custom tailored solutions. The development of defined reagents provides a smooth transition and reproducibility between research and clinical development.
Alloantigen-specific Chimeric Antigen Receptor Regulatory T cell therapy in non-human primate islet transplantation
Authors: Ellis Gavin I , Coker Kimberly E , Winn Delaine W , Deng Mosha Z , Shukla Divanshu , Bhoj Vijay , Milone Michael C , Wang Wei , Liu Chengyang , Naji Ali , Duran-Struuck Raimon , Riley James L
Published: January 2023

Though islet transplantation has emerged as a promising therapeutic for type 1 diabetes, the immunosuppressive agents that prevent rejection carry significant risk of morbidity. Adoptive transfer of chimeric antigen receptor (CAR) Tregs has the potential to protect allografts and transform transplantation medicine in the way that CAR T cells have revolutionized the treatment of cancer. However, there lacks a large animal model in which to optimize the safety and efficacy of CAR Treg therapy. Therefore, we developed methods to sort, transduce, and expand Cynomolgus macaque CAR Tregs recognizing the human/non-human primate cross-reactive alloantigen Bw6. Our novel pan-primate artificial antigen presenting cells expanded Tregs to clinical sized doses of >500 million cells from as few as 25k sorted cells. Expanded Tregs expressed protein and epigenetic markers indicative of stable CAR Tregs and displayed suppressor function without cytotoxicity. CAR Tregs transferred into an antigen negative recipient could be detected for 7 days in peripheral blood and 1 month in bone marrow. When transferred to a diabetic recipient simultaneously with allogeneic Bw6+ islets, islets were partially protected from rejection as measured by increased control of blood glucose with insulin treatment. Transferred Tregs persisted in peripheral blood for >34 days while displaying markers of stable, activated Tregs including CAR, FoxP3, and Helios. Importantly, we could not detect CAR+ FoxP3− effector T cells which would target the graft and expedite graft rejection. Our results provide a proof of concept for CAR Treg adoptive therapy in a large animal model and establish a framework for optimization of therapy before a clinical trial in humans.
Paired Immunoglobulin-Like Receptors Impact The Differentiation of Reparative Macrophages Following Allogeneic Challenge
Authors: Warunek Jordan , Lucas Anna , Mathews Lisa , Ossart Jason , Oberbarnscheidt Martin H , Lakkis Fadi G , Turnquist Heth R
Published: January 2023

Monocytes differentiate into CD206+ CD301+ reparative macrophages (MΦs) that orchestrate tissue repair in response to damage signals and cytokines. Yet, transplanting allogeneic materials produces a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses. B6 monocyte paired immunoglobulin-like receptor (PIR)-A3 recognizes BALB/c H2-Dd to generate pro-inflammatory dendritic cells that promote kidney and heart transplant rejection. Conversely, monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shape MΦ differentiation after Tx was not understood, so we tested the hypothesis that recognition of self-versus-allogeneic MHCI by PIRs divergently modulates MΦ differentiation. Monocyte-derived MΦ differentiation in wild type (WT), Rag2−/− γc−/−, Pira−/− and Pirb−/− B6 mice was assessed by flow cytometry 3 days after exposure to 20×106 radiation-damaged BALB/c allogeneic (allo) or B6 syngeneic (syn) splenocytes administered intraperitoneally. Syn materials predominantly stimulated monocyte differentiation into phenotypically reparative MΦs. Allo cells instead transformed monocytes exclusively into pro-inflammatory Ly6chi CD86hi MΦs. Rag2−/− γc−/− mice behaved similarly. IL-33 released from injured cells metabolically reprograms infiltrating monocytes and MΦs to support their differentiation into CD206+ CD301+ MΦs. Interestingly, Pirb−/− mice display greatly reduced C206+ CD301+ MΦs, which correlated with their decreased expression of the IL-33 receptor. Our data provide important insights into how innate allorecognition by PIR-A disrupts the typical generation of reparative MΦs in response to injured self that is supported by PIR-B.

Supported by 5T32AI074490-14
Vista blockade reprograms tumor myeloid cells and relieves adaptive resistance
Authors: Lines J Louise , Schaafsma Evelien , Croteau Walburga , Nowak Elizabeth C , ElTanbouly Mohamed , Sarde Aurélien , Cheng Chao , Noelle Randolph J
Published: January 2023

treatment. Significantly, VISTA shows broad expression in many cancer types, with enhanced expression upon development of adaptive immune checkpoint resistance. In the CT26 colon cancer model, growth of small tumors (40mm3), are retarded by anti-VISTA monotherapy, and are readily rejected following anti-CTLA4/anti-PD-L1 (CP)therapy. In contrast, very large tumors (>600mm3), are refractory to CP therapy. However, combinatorial therapy of CP with anti-VISTA leads to rejection of about half the tumors. Both in monotherapy and combination therapy the mechanisms of enhanced anti-tumor immunity were investigated using scRNAseq, multiplex image analysis and flow cytometry of the tumor immune infiltrate. In both treatment models, anti-VISTA upregulated stimulated myeloid antigen-presentation pathways and reduced myeloid mediated suppression. Multi-spectral imaging revealed an anti-VISTA stimulated increase in contacts between CD8+ T cells and myeloid cells, further supporting the notion of increased antigen presentation upon anti-VISTA treatment. Transcriptional analysis of antigen-specific CD8 T cells showed that anti-VISTA therapy induced T cell pathways highly distinct from the anti-exhaustion effects of CP. These data suggest the non-redundancy of CP and anti-VISTA treatment, and support the use of these treatments in combination to overcome the adaptive resistance seen in contemporary treatments involving PD-1 and/or CTLA-4.

Research was supported by NIH grants R01AR070760 (R.J.N.), R01CA214062 (R.J.N.), and 1R21CA227996 (C.C.) and the Cancer Prevention Research Institute of Texas (CPRIT) (RR180061 to C.C.). C.C. is a CPRIT Scholar in Cancer Research.
Glycogen synthase kinase (GSK-3) synergizes with PD-1/PDL1 blockade to generate super-armed CD8 killers against tumors
Authors: Issa Mark E , Krueger Janna , Kazanova Alexandra , Taylor Alison , Rudd Christopher E
Published: January 2023

Immune checkpoint blockade (ICB) of negative co-receptors on T-cells such as programmed cell death-1 (PD-1) is promising for the treatment of cancer. Despite success, the poor prognosis for most patients highlights the need for novel clinical interventions. We have shown that the kinase, glycogen synthase kinase-3 (GSK-3) negatively regulates T-cell activation due to altered PD-1 and LAG-3 expression (Taylor et al., 2016 Immunity; Rudd et al., 2019 Cell Reports). GSK-3 inhibition (GSK-3i) is as effective as anti-PD-1 in controlling the growth of melanoma (Taylor et al., 2017 Can Res; Krueger and Rudd, Immunity 2017; Stelle et al., 2021 iScience). GSK-3i increases Tbet (Tbx21) transcription, which inhibits PD-1/LAG-3 transcription, while increasing granzyme B (GZMB) and interferon gamma (IFNγ). Here, we show that Gsk3−/− mice and small molecule inhibitors (SMIs) synergize with anti-PD-1 to eliminate melanomas that are resistant to anti-PD-1 monotherapy. Transcriptomic profiling showed that GSK-3 × PD-1 cooperativity was characterized by a specific increase in a family of different granzymes (7/12 GZM genes out of a data base of 20,500 potential genes). Some GZMs have been characterized and others not, but as a family, this increased armory of GZMs in CD8+ T-cells is expected to greatly enhance tumor killing. Overall, our data shows the PD-1 × GSK-3 synergy in limiting tumor growth is due to a specific set of cytolysis mediators needed for tumor killing.
Pro-tumoral role of Granzyme B to aid in invasion and metastasis.
Authors: Tibbs Ellis J , Cao Xuefang
Published: January 2023

The granule exocytosis pathway has long been known to be involved in potent responses to pathogens and cancers. This is executed by the action of Perforin and a family of granule-associated serine proteases known as Granzymes (Gzm). Many studies have shown that these proteases exert their effects by killing affected cells, executing the major function by Cytotoxic T cells (Tc) and Natural Killer cells expressing GzmB. Studies have found that the tumor cells ability to create a suppressive environment is dependent on Gzm expression by Regulatory T lymphocytes (Treg). Bird et al published data showing GzmB aids in the transmigration of Tcs, however the ability of GzmB to aid in the migration of non-T cells has not been investigated. We hypothesize that Granzyme B secretion may cause extracellular matrix remodeling in the tumor microenvironment, aiding in the outgrowth of the tumor cells via promoting invasion or metastasis.

We test our hypothesis by measuring tumor invasion in vitro utilizing the xCelligence transmigration system. We found that culturing B16 cells with recombinant GzmB from Biolegend or Peprotech on Matrigel resulted in a 31% and 47% increase in cellular invasion, respectively. Also, the rate of invasion resulted in a 35% and 61% increase, respectively. We also tested our hypothesis in vivo with a preliminary study using Treg-specific GzmB KO mice (Foxp3cre GzmBfl/fl). We depleted CD8+ T cells, injected B16 cells intravenously, and activated Tregs. We found a 48% decrease in metastatic nests in mice deficient of GzmB in Treg cells after 8 days and a 37% decrease after 16 days compared to WT. These findings shed light on the pro-migratory ability of secretory GzmB and helps us understand an unassuming role of GzmB as a pro-tumoral factor.

Supported by funds through NIH (RO1 HL135325, T32 AI095190, P30 CA134274) and Maryland Department of Health’s Cigarette Restitution Fund Program.
Transcriptomic and epigenetic profiling of tumor-associated monocyte function
Authors: Contreras Cristina F , Kaczanowska Sabina , Kaplan Rosandra N
Published: January 2023

Monocytes are innate immune cells recognized for their ability to play both tumor permissive and surveillant roles in cancer. Circulating classical monocytes (CD14+ CD16−) can home to the tumor and suppress other immune cells through various mechanisms, including the production of arginase and the release of reactive oxygen species (ROS). Conversely, patrolling nonclassical monocytes (CD14− CD16+) have been shown to employ processes such as phagocytosis and presentation of tumor antigens to prevent metastasis. This heterogeneous monocyte function is influenced by tumor-derived factors that are released during cancer development and progression. Phenotypic and transcriptional alterations in circulating monocytes and other myeloid cells in patients with solid tumors have been reported and associated with poor clinical outcomes. However, perturbations of specific monocyte functions in the setting of solid tumors have not been well explored. Here we present a characterization of monocytes by coupling flow cytometry-based functional assays with sequencing (Func-seq). Healthy donor primary monocytes and monocytic cell lines were used to examine the production of ROS and arginase in response to osteosarcoma-conditioned media and monocyte-mediated phagocytosis of osteosarcoma cells. Bulk RNA-seq and ATAC-seq were performed on FACS-sorted populations to compare differentially expressed genes and establish transcriptomic and epigenetic signatures associated with monocyte-mediated immunosuppression and tumor-cell phagocytosis. The incorporation of functional selection into -omic characterization provides insights into monocyte behavior and potential therapeutic targets to alter their activity in solid tumors.

Funding: US National Institutes of Health grants ZIA BC 011332 and ZIA BC 011855 and NCI cancer moonshot.
HIV-specific CD8 T cells from elite controllers have an epigenetic imprint that preserves effector functions
Authors: Frias Adolfo B , Rutishauser Rachel L , Sharma Ashish Arunkumar , Mi Tian , Abdelsamed Hossam Aly , Zebley Caitlin , Constantz Christian M , Stone Mars , Busch Michael , Deeks Steven , Sekaly Rafick , Youngblood Ben A
Published: January 2023

CD8 T cells from HIV infected ‘elite controllers’ maintain long-lived protective properties including enhanced cytokine production and proliferative capacity over many years. With the advent of adoptive cellular therapy to treat chronic diseases it has become even more vital to understand the intrinsic cellular mechanisms of elite controller HIV specific CD8 T cells that enable lasting antiviral functionality. To identify epigenetic programs that regulate their functional capacity we performed genome-wide DNA methylation analysis of MHC Class I multimer+ CD8 T cells sorted from aviremic elite controllers compared to aviremic non-controllers on suppressive ART. Principle component analysis of the CpG sites broadly distinguished EC and ART HIV specific CD8 T cells, while deeper investigation of the differentially methylated region analysis revealed enrichment of pathways that support a multipotent differentiation state, cytokine signaling, and a long-lived effector cell fate in HIV-specific CD8 T cells from elite controllers. We also observed DNA methylation programs at the transcription factor binding sites of the stem-associated factors TCF-1 and LEF1 that delineate HIV-specific CD8 T cells from elite controllers versus ART-treated individuals. These findings show that HIV-specific CD8 T cells from elite controllers have DNA methylation programs that maintain developmental potential and in turn enable long-term survival, proliferative potential, and effector capacity. These data also provide new insights into the relationship between stem-associated transcription factors and stable epigenetic restriction of T cell developmental capacity.

Supported by grants from NIH (R01AI114442 and R01CA237311 to BY, K23AI134327 to RLR), loan repayment program and National comprehensive Cancer Network Young Investigator Award (CZ), ASSISI foundation support (BY) and American Lebanese Syrian Associated Charities (ALSAC to BY)
A unique gammadelta T cell population in the brain during viral infection
Authors: Berger Jennifer , Monogue Brendan , Beckham J David , Berg Leslie
Published: January 2023

Historically, the central nervous system (CNS) was considered to be an immune privileged site with limited immune responses. Now, it is known that the CNS presents a unique immunological environment with restricted infiltration by immune cells from the periphery or meningeal compartments. There remains limited understanding of which peripheral cells enter the brain during infection, how these cells enter the brain, and whether their function is detrimental or vital to eliminating infectious insult. Here, we utilize the attenuated Venezuelan equine encephalitis virus vaccine strain TC83 (VEEV-TC83) to study neuroimmunology during viral infection. Intracranial (IC) infection with VEEV-TC83 results in high viral titers in the brains of wild-type mice, without lethality, enabling study of immune responses in the brain. During CNS VEEV-TC83 infection, a large number of T cells infiltrate the brain rapidly after infection and, are thus, not antigen-specific T cells. After five days of infection, we found unusually high levels of infiltrating gammadelta T cells (γδ T cells) into VEEV-TC83-infected brains (upwards of 40% of the CNS-infiltrating T cells). The infiltrating γδ T cells express the uncommon surface markers CD11b and IL-23R. In this study we investigated the role of this unique γδ T cell population how these cells infiltrate the brain during viral infection. We found that the γδ T cells in infected brains are a unique subset expressing noncanonical surface markers and cytokines.

Supported by the Training Program in Immunology T32 AI007405