bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-2 mutein with diminished CD25 binding expands regulatory T cells and blocks autoimmunity
Authors: Kim James , Eryilmaz Ertan , Sato Yosuke , Harbison Carole , Shaw Mike
Published: August 2023

Since its identification, IL-2 has been tested as a therapy for both oncology and autoimmunity as it has the ability to expand effector T cells and NK cells as well as regulatory T cells (Tregs). Due to its toxicity, IL-2 has been the focus of molecular engineering to improve its therapeutic properties and expand its therapeutic index. To target CD4+CD25− and CD8+CD25− T cells, we rationally designed a murine and a human IL-2 variant to bind the IL-2Rβγ (IL2Rbg) heterodimer without binding to IL-2Rα (IL2Ra CD25). The mutein demonstrated reduced binding to CD25 and reduced ability to induce phosphorylation of STAT5 in Tregs while maintaining binding to IL-2Rbgc. Despite these results, the IL-2-mutein exhibited significant expansion of CD25+Foxp3+ T cells. To demonstrate biological relevance of these regulatory T cells, with two different dosing regimens, the IL-2-mutein completely blocked incidence of hyperglycemia in NOD mouse. These data suggest the identified IL-2.mutein is a potent anti-inflammatory in vivo.
A transformable three-dimensional simulated solid tumor model reveals the widespread killing potential of natural killer cells with controlled lytic granule positioning
Authors: Li Yu , Pedroza Luis , Orange Jordan S
Published: August 2023

Cancer immunotherapy using engineered natural killer (NK) cells has shown great promise. However, the limited spatial complexity of current models hinders the study of NK cells in three-dimensional, solid tumor-like environments. Previously, we found that NK cells converge lytic granules before degranulation. It helps focus the secretion of cytotoxic cargos to the synaptic cleft, thus preventing the non-specific killing of bystander cells. We had speculated that bypassing this protective mechanism and forcing NK cells to degranulate non-directionally may promote broader destruction of cancer cells and eradicate tumor-resident non-triggering cells.

To evaluate our hypothesis, we designed a novel strategy for creating complex cellular environments with controlled spatial distribution and intercellular dynamics. Using hydrogel-embedded cells as building blocks, we construct 3D models that arrange multiple cell populations within desired inner structures. Notably, these structures can undergo controlled transformations enabled by hydrogels comprised of thermo-responsive polymers. During this transformation, cells experienced rapid but mechanically gentle translocation to pre-designated positions, allowing us to control cell contacts actively. With these systems (which we have termed TheCOS for Thermal Collapse Of Strata), we observed that controlling NK cell lytic granule positioning at a lytic immune synapse was able to promote bystander cell death within a complex multicellular environment. Using quantitative imaging, we calculated an area of damage attributable to radial degranulation, which suggests a potential benefit to this as a strategy for improving cytotoxic cell therapy for solid tumors.
Role of NK cells in circadian regulation of lung inflammation
Authors: Sengupta Shaon , Forrest Kaitlyn , Paul Oindrila , Naik Amruta , Assi Lora , Issah Yasmine
Published: August 2023

Circadian rhythms form an anticipatory system wherein various aspects of cellular physiology oscillate across a 24hrs period; these rhythms have been known to modulate various facets of immunity. We have previously shown that mice infected with Influenza A virus (IAV) at dusk had 3-fold higher mortality than littermates infected at dawn--a hallmark of circadian regulation. This time-of-day specific protection was lost when NK cells were depleted just before the infection, suggesting a role of NK cells in mediating the circadian regulation of IAV infection.

We investigated the mechanism underlying the role of NK cells, both cell-intrinsic or extrinsic in circadian regulation of lung injury. We noted that viral clearance was unaffected, but immunopathology was markedly worse in the NK depleted mice than the controls. Next, we investigated the effect of the loss of the circadian clock on NK cell behavior and found that splenic and pulmonary NK cells in naïve mice with a disrupted circadian clock have a less mature phenotype than control NK cells with intact clock. Our transcriptomic profiling of NK cells sorted from clock-intact versus clock-disrupted animals revealed that several genes that regulate cell cycle progression and functional maturity of NK cells were differentially expressed across the two groups. Pulmonary NK cells from clock mutant mice were less mature than clock-intact mice. Finally, the Ncr1cre+Bmal1fl/fl mice (core clock gene Bmal1 deleted in NK cells) infected with IAV had higher mortality through heightened immunopathology but not delayed viral clearance.

In summary, these data show that NK cell-autonomous clock boost host tolerance to mediate circadian protection from IAV infection.

Supported by grants from NIH (R01HL155934-01A1, R01HL147472, R01AI137062-01)
Inhibitory Receptor TIGIT is a critical regulator of Natural Killer Cell Activation and Survival Following Strong Stimulation
Authors: Lammers Marshall L , Judge Sean , Nielsen Ryan N , Farley Lauren , Iranpur Khurshid , Sheng Michael K , Vick Logan , Murphy William J , Canter Robert
Published: August 2023

TIGIT has emerged as a critical regulator of phenotype and function in mouse and human NK cells with key studies showing TIGIT upregulation suppresses NK anti-tumor responses. However, the role of TIGIT in promoting NK survival has received less attention. We hypothesized that TIGIT exerts a dual role on NK cells balancing decreased activation with enhanced survival. Our results showed sorted human NK TIGIT+ and TIGIT-cell subsets upregulate TIGIT to near 100%, post IL-2 cytokine stimulation, suggesting TIGIT’s importance in NK survival following cytokine stimulation. Sorted human TIGIT-NK cells also showed significantly greater apoptosis compared to TIGIT+ NK cells. NK cells from TIGIT KO mice showed greater proliferation and expansion in vitro and in vivo with higher numbers of NK cells and increased Ki67 expression following acute cytokine stimulation. NK cells from TIGIT KO mice also display significantly lower viability and fold expansion (0.2±0.2 vs 2.0±0.7) when compared to C57BL/6 (WT) aged-matched mice. In vivo, there was a greater number of NK cells in the spleen and lung of WT mice following IL-15 exposure, peaking on day 4 but remaining elevated on day 8. NK cells from TIGIT KO mice also showed significantly higher apoptosis (66 ± 6% vs. 20 ± 6%, P<0.001 in vitro and 25±4% vs. 12±2% P<0.01 in vivo). Similar results were observed in reconstituting NK cells of mice from TIGIT KO donors post bone marrow transplant (BMT). Overall, our results indicate that TIGIT plays a critical role in regulating NK cell responses following cytokine stimulation, suggesting that TIGIT blocking strategies may lead to both greater acute expansion and anti-tumor efficacy, but subsequently greater depletion of NK cells long-term following activation.
Anti-HER2 cell specific doxorubicin delivery using synthetic bacterial spores for resistant ovarian cancer treatment
Authors: D’Atri Domenico , Kong Minsuk , Bilotta Maria Teresa , Tanner Kandice , Fitzgerald David , Ramamurthi Kumaran S
Published: August 2023

Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. SSHELs are synthetic spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 μm-diameter silica beads. Via a unique cysteine engineered protein, the surface of SSHELs may be covalently decorated to display HER2 (SSHELsαHER2). SKOV3 and SKBR3 cells’ targeting by SSHELsαHER2 was evaluated by flow cytometry and confocal microscopy. SSHELsαHER2 specifically bound to target cells and were internalized proportionally to their concentration. Subsequently, SSHELs were successfully loaded with doxorubicin (Dox-SSHELsαHER2). Dox-SSHELsαHER2 capabilities to kill tumor cells was evaluated by flow cytometry and caspase assay. HER2 negative cells were used as a control. Dox-SSHELsαHER2’s efficacy was tested in vivo using athymic nude mice injected with SKOV3 ovarian cancer cells and then treated for up to 40 days. Dox-SSHELsαHER2 can reduce SKOV3 tumor growth up to 75% when compared to the free drug. Further, when compared to liposomal doxorubicin (DoxilTM) Dox-SSHELsαHER2 showed similar efficacy in reducing tumor growth, but without any of the typical side effects correlated with DoxilTM injections. With a facile, reproducible manufacturing process that allows for straightforward targeting towards specific cell types, and lower overall toxicity, we suggest that SSHELs may represent a versatile strategy opening new research avenues for targeted drug delivery treatment or immune system stimulation through vaccination.
Engineered live attenuated bacteria producing 5-hydroxyindole remodels heterogeneous tumor microenvironment and promotes antitumor immunity
Authors: Das Jugal Kishore , Song Jianxun
Published: August 2023

The highly immunosuppressive tumor microenvironment (TME) stimulates cancer cells resistance to immunotherapy. We have previously shown that a live-attenuated Brucella melitensis Δ vjbR(BmΔ vjbR) bacterial strain, breaks cancer cell resistance to immunotherapy by remodeling the TME and facilitating infiltration of cytotoxic CD8+ T cells (CTLs) into the tumor. As a result, there was shrinkage of tumor size and significant improvement in survival of these mice. However, BmΔ vjbR was efficient in only controlling colorectal tumors, but was rendered ineffective in B16 melanoma. In this study, we sought to improve the efficacy of BmΔ vjbR in controlling different cancers by engineering it to augment the production of 5-hydroxyindole (HI), i.e., BmΔ vjbR-HI. The effect of BmΔ vjbR-HI in modulating the function and activity of CTLs was assessed by flowcytometry in in vitro assays. The tumor modulating activity of the BmΔ vjbR-HI was also assessed by intravenous injection of this bacteria followed by adoptive CTL therapy in colon carcinoma, melanoma and pancreatic orthotropic murine tumors. We found that BmΔ vjbR- HI increased inflammatory cytokines in CD8+ T cells. The granzyme-B production and cytotoxicity of CD8+ T cells was also enhanced on treatment with the bacteria. BmΔ vjbR- HI remodeled the TME and ameliorated MC32-CEA colorectal cancer, B16-OVA melanoma and Panc-02 orthotropic pancreatic tumors. Moreover, the BmΔ vjbR- HI homed into the TME by uptake in myeloid derived suppressor cells. Overall, our findings demonstrate that BmΔ vjbR- HI remodels heterogeneous TMEs of different cancers to promote CTL-mediated antitumor immunity. These findings have significant translational potential in antitumor microbial immunotherapy.

Jugal Das and Jianxun Song are recipients of Careers in Immunology Fellowship 2022.
The radiosensitizing effects of monopolar spindle kinase I in syngeneic models of triple negative breast cancer and its implications on the tumor immune microenvironment
Authors: Jungles Kassidy Mikala , Wang Zhuwen , Bishop Caroline , Jungles Kalli R , Wilson Cydnee , Liu Meilan , Pearson Ashley N , Holcomb Erin A , James Jadyn , Chandler Ben , Huber Amanda , Speers Corey W , Rae James , Green Michael
Published: August 2023

Triple negative breast cancer (TNBC) is an aggressive breast cancer subset with poor outcomes. TNBCs do not express the estrogen, progesterone, or HER2 receptors and are thus not candidates for antiestrogen and anti-HER2 therapies, leaving few effective treatments. One therapeutic strategy is to target molecular components of an individual patient’s cancer. Prior work in our group has nominated monopolar spindle kinase I (TTK) as a gene that is upregulated in cancerous breast tissue compared to healthy tissue and correlates with locoregional recurrence following radiation in breast cancer patients. However, the effects of TTK inhibition and radiation on the immune system is not understood. We hypothesize that the TTK inhibitor empesertib radiosensitizes murine TNBC models to radiation in vitro and in vivo and modulates the immune tumor microenvironment. Single agent empesertib inhibited the growth of murine TNBC cells with IC50 values up to 30nM. Sub-IC50 values of TTK inhibitor radiosensitized 4T1 (radiation enhancement ratio or rER: 2.4 ± 0.4) and Py8119 (rER: 1.5 ± 0.1) cells. TTK knockdown (via shRNA) also resulted in changes in radiosensitization in vitro. Furthermore, we also observed a similar phenotype in vivo. In our 4T1 in vivo model, mice receiving combined treatment had decreased tumor growth compared to mice that receiving single-agent therapies or vehicle (****p<0.0001). Quantities of monocyte derived suppressor cells were altered with radiation and TTK inhibition. This combined therapy suggests that changes in the underlying immune mechanisms due to TTK inhibition and radiation are important in TNBC. Future work will examine the mechanisms of TTK inhibition and radiation on systemic and tumoral immune changes.
Global inhibition of Ly-49/MHC-I interactions by anti-MHC-I augments innate and adaptive immunity against cancer metastasis
Authors: Kazmi Soha M , Sinha Surajit , Natarajan Kannan , Sharma Suveena , Kim Yong-Hee , Boyd Lisa , Hernandez Jonathan , Margulies David H , Shevach Ethan M , Panda Abir K
Published: August 2023

We have identified an pan-anti-MHC-I mAb (M1/42), which blocks the interaction of NK cell inhibitory receptors (Ly-49 antigens) with MHC-I, but does not inhibit antigen presentation by MHC-I. Administration of M1/42 in vivo markedly activated IFNg-producing NK cells. NK cell-derived IFNg stimulated APC to produce IL-12/-15/-18 cytokine cascades that further drove the proliferation of NK cells and memory phenotype (MP) T cells. M1/42 treatments profoundly augmented innate and adaptive immunity against PD-1-sensitive and -resistant transplanted tumors. Given that NK cells are critical to control of cancer metastases, we evaluated the effects of M1/42 therapy in several metastatic cancer models. M1/42 treatments successfully constrained B16F10 lung metastasis by enhancing CD8+ T cell infiltration and inducing melanoma-specific MP CD8+ T cell expansion and activation. In contrast, treatment with conventional checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4 and anti-NKG2A) had no therapeutic effect. scRNAseq analysis of B16F10 lung melanoma tumor infiltrating lymphocytes revealed that M1/42 treatment significantly enhanced the expression of multiple genes associated with NK cell proliferation and activation. M1/42 also enhanced Th1-type innate and adaptive immune responses, and greatly restricted liver and lung metastasis produced by pancreatic adenocarcinoma cell line via intrasplenic or intravenous injection. Thus, the global inhibition of Ly-49/MHC-I interactions by M1/42 results in activation of a potent innate and adaptive immune response that restrains the growth of both transplanted tumors and cancer metastasis produced by intrasplenic or intravenous injection of tumor cells.

This work was supported by the Intramural Research Program of NIAID, NIH.
Hysteresis of human complement C4A to C4B binding in hemolytic activities and inhibition by immune complex aggregates
Authors: Zhou Danlei , Liu Xiaoping , Coss Samantha , Yu Chack-Yung
Published: August 2023

--Human complement C4 is a sophisticated innate immune effector that features two classes of polymorphic proteins with acidic C4A and basic C4B. The thioester carbonyl from Gln-1016 of C4A has high binding affinity towards protein antigens rich in amino groups to form covalent amide bonds; while that of C4B manifests avid preference binding towards carbohydrate antigens rich in hydroxyl groups to form covalent ester bonds. Human subjects with deficiency of C4A are predisposed to increased risk of systemic lupus erythematous (SLE). We performed flow cytometry of activated C4 on red blood cells using monoclonal antibodies against human C4d for both C4A and C4B, and monoclonals specific for C4A. Among human subjects with equal numbers of C4A and C4B genes, the levels of C4d protein from C4B deposited on red cells are consistently 3–10 times higher than that from C4A. We performed in depth kinetic studies of activated C4A and C4B by measuring the time course of Ab-sensitized sheep erythrocyte (EA) binding and cell lysis using plasma C4A or C4B. We tested immune complex (IC) aggregates for inhibition of EA lysis. The results showed both C4A and C4B in plasma can lyse EA but C4A exhibit hysteresis properties by lagging C4B. At dilution to <1% or [C4] < 2 mg/mL, the efficiency of C4B on lysis EA remained significantly higher than that of C4A. Addition of IC aggregates inhibited the course of C4A and C4B on lysis of EA: 1.17 mg/mL IC inhibited 50% EA lysis by 1.20 mg/mL C4A. 5.32 mg/mL IC was required to inhibit 50% EA lysis by 1.01 mg/mL C4B. In conclusion, activated C4B binds quickly to sensitized erythrocytes. Activated complement C4A prefers binding to IC aggregates with hysteresis properties.

R21 AR070509, R01 AR073311
Regulatory T cells aid Leishmania major immune evasion by suppressing effector responses against the parasite in an antigen-specific manner
Authors: Zayats Romaniya , Mou Zhirong , Yazdanpanah Atta , Lopez Paul , Koh Wan Hon , Uzonna Jude E , Murooka Thomas
Published: August 2023

Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete and leaves a pool of chronically infected cells. Understanding of the persistence is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells at the site of infection has been proposed to prevent parasite clearance in vivo. Here, we used a novel TCR transgenic mouse model, where CD4+ T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4+ T cell responses and characterize mechanisms which restrain their effector function. We show that PEPCK-specific Tregs are able to suppress nearly 80% of IFNg produced by PEPCK-specific Th1 cells in vitro though the production of IL-10, while decreasing IL-12, TNF, and IL-2. Intravital microscopy studies characterizing PEPCK-specific CD4+ T cell migration dynamics in live mice show a significant recruitment of adoptively transferred cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition, yet augmented at the chronic stage, indicating a fundamentally altered environment. Injection of heat-killed L. major into mice which have already healed a L. major infection, distal to the original lesion, induced a rapid expansion of PEPCK-specific Tregs at the original site of infection, which suppressed endogenous anti-Leishmania response through IL-10 and led to parasite expansion and lesion reactivation. Our results show that antigen recognition by Tregs plays an important role in suppressing effector responses against L. major and thus maintaining concomitant immunity.

Research funded by a grant from the GSK-CIHR Partnered Chair Program
A novel method for studying intercellular networks: quantifying cellular cooperativity and inferring the extracellular signals driving it
Authors: Arazi Arnon , Gierahn Todd , Love Christopher , Hacohen Nir
Published: August 2023

Objective

Develop an integrated experimental-computational approach to determine the functional role played by different cell subsets and extracellular communication signals in shaping the behavior of intercellular networks.

Methods

We present a microwell-based experimental system, that allows isolating and culturing small groups (up to a few dozens) of cells over extended periods of time, while monitoring their interactions and states using fluorescence microscopy. In parallel, we present a computational framework that uses the data generated by this system for estimating the degree of intercellular cooperativity associated with observed behaviors of the interacting cells, and for inferring the contribution of measured extracellular signals in shaping these behaviors.

Results

We used our approach to study the differentiation of mouse Th17 cells. We show that this process requires a relatively large number (> 10) of intermediate intercellular communication steps, in which information processing is carried out by a relatively small percentage (10–20%) of the interacting cells. We correctly predict a correlation between the time in which communication signals are expressed during differentiation, and the degree of intercellular cooperativity associated with their production. Furthermore, we show that we can accurately predict cell-level behaviors. In addition, we correctly identify previously-known effects of cytokines on cell survival, and further predict novel relations, which we validate experimentally.

Conclusions

We present a new and general approach for the study of collective cellular behaviors, identifying the key cell subsets and extracellular communication signals driving them.

This work was supported in part by National Human Genome Research Institute Centers of Excellence in Genomics Science P50 HG006193 (N.H.), by National Institutes of Health Grants P01 AI045757 and DP3 DK09768101 (J.C.L.) and by the U. S. Army Research Office through the Institute for Soldier Nanotechnologies, under contract number W911NF-13-D-0001 (J.C.L.). This work was also supported in part by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. J.C.L. is a Camille Dreyfus Teacher-Scholar.
Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in leukemia samples
Authors: Kim Grace , Papalexi Efthymia , Matulich Peter , Koehler Ryan , Schroeder Sarah , Tran Vuong , Roco Charles , Rosenberg Alexander
Published: August 2023

Single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple research fields, leading to new discoveries. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery which require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required.

Here, we extend our whole transcriptome (WT) split-pool combinatorial barcoding technology to enrich a subset of genes in 16 libraries representing hundreds of thousands of human bone marrow mononuclear cells (BMMCs) from three acute myeloid leukemia (AML) and one acute lymphocytic leukemia (ALL) donors. We used our 1,000 immune gene panel to enrich genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 7% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded high concordance of cell type identities and preserved leukemia-specific signatures such as FLT3, MKI67, and CD19.

Overall, we demonstrate our modular enrichment strategy preserves biological structure and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments.
Novel Workflow for Activation and Expansion of T Cells Using Microbubble Technology
Authors: Ellis Jason S , Barrett Crisha B , Snow Tiffany , Roussey Jon , Carey Maureen , Ku Chia , Grimley Edward , Wegner Casey J , McNaughton Brandon H
Published: August 2023

Chimeric antigen receptor (CAR) T cell therapy was developed to address the significant hurdles to cancer treatment. In the US, one in four deaths is due to cancer, with treatment costs totaling over $200 billion a year. Traditional treatments, such as chemotherapy and radiation therapy have serious harmful side effects, often with limited success. Despite the immense hurdles to producing a cost effective, efficient, and scalable workflow for manufacturing CAR T cells, thousands s of CAR T cell therapy clinical trials have been initiated around the world. Traditional CAR T cell workflows often result in T cell exhaustion, limiting the effectiveness of the cells produced. At Akadeum, we set out to create a simple workflow to improve researchers’ ability to develop custom cell therapies. Using Akadeum’s microbubbles, we developed a process that combines selection, activation, and expansion into a single workflow. Because of the natural buoyancy of the microbubbles, activation and expansion take place at the top of the culture. As the T cells divide, daughter cells fall to the bottom of the well away from the activation signals. This prevents overstimulation and the resulting exhaustion phenotype that occurs with the industry-standard activation methods (25–30% PD-1+ with Akadeum method compared to >90% with traditional workflow). This method effectively activates T cells, with significant upregulation of standard activation markers, greater than 20-fold expansion, and desirable effector cell ratios. These cells are also highly transducible. Taken together, these data show that Akadeum’s microbubble-based workflow provides all of the critical aspects of CAR T cell generation while eliminating many of the most significant hurdles.
A new automatic gating algorithm replicates expert manual gating of immune cell subsets
Authors: Weit Nicole Yvonne , Bowers Rita , Grazia Giulia
Published: August 2023

Machine learning is increasingly used for the analysis of flow cytometric immunophenotyping data. Many analysis pipelines leverage unsupervised approaches such as dimensionality reduction or clustering, which may lead to populations characterized differently from expert definition.

Here we show a new, supervised algorithm for population identification that faithfully replicates the expert manual gating strategy.

Peripheral blood samples were drawn from 41 different donors, stained with a dry, unitized B cell panel and acquired on two cytometer units. A manual gating strategy was established, and an automatic model trained on a subset of samples (n=31 donors, split into training, validation and test set) from this study. The performance of the automatic model was evaluated on the 10 unused samples.

The automatic identification of populations with differences in median abundance (lymphocytes, CD19+ B-cells and CD19+ CD10+ cells) showed a high level of correlation and lower variability compared to manual gating. The supervised approach presented here retains the gating hierarchy, in contrast to the unsupervised clustering performed by FlowSOM, which splits cells into exclusive metaclusters, leading to metacluster assignments significantly different from expert gating.

By automating the identification of populations, assay reproducibility can be increased by removing inter-operator variability. We show that a supervised machine learning tool that supports user-defined panels and gating strategies can reproduce expert analysis, reduce variability of flow cytometry data analysis, and can provide better fit-for-purpose compared to an unsupervised clustering approach.
Leveraging assay standardization with DURAClone dry antibody panels on six different flow cytometry platforms
Authors: Bowers Rita , Grazia Giulia , Kapinsky Michael , Lacaille Vashti , Schneider Laura
Published: August 2023

The blessing of Flow Cytometry’s versatility and flexibility comes with the curse of human operator dependence, variable or unstable reagent performances and an infinite number of possible assay designs. Aiming at mitigation of the risk of poor data comparability, DURAClone pre-formulated dry antibody panels provide standardized, technically validated assays in single test-unitized format, devoid of antibody pipetting. To demonstrate that DURAClone standardization can be leveraged on different hardware architectures, including different principles for signal detection and processing, we used six different flow cytometry platforms to acquire DURAClone-stained samples. Due to logistic limitations excluding the use of identical samples in all three different participating institutions, only a qualitative comparison was feasible. Here, we show example data from DURAClone-stained samples acquired on a BD Biosciences FACSCanto II, FACSLyric or FACSymphony flow cytometer, a NovoCyte Quanteon flow cytometer, a BioRad ZE5 flow cytometer or a Cytek Aurora flow cytometer. The presented data confirms expected staining patterns obtained with selected DURAClone antibody panels, irrespective of the flow cytometer used to acquire the data.
Insufficient epitope specific T cell clones is responsible for impaired cellular immunity to inactivated SARS-CoV-2 vaccine in the elderly
Authors: Xiao Chanchan , Chen Guobing
Published: August 2023

Aging is a critical risk factor for SARS-CoV-2 vaccine efficacy. Although low humoral immunity in the elderly was observed in mRNA and recombinant protein vaccination, the immune responses to inactivated vaccine in the elderly, and the underlying mechanisms of differences to young, if any, are still unclear. Here, we studied a cohort of 121 young (18–30 years old) and 48 old (60–85 years old) donors vaccinated with inactivated SARS-CoV-2 and demonstrate that the neutralizing antibody response is slower in the elderly, but eventually reached the similar level to day 7 post-vaccination in young. We identified a range of epitopes targeted by CD8 T cells that were underrepresented in immune responses in the old group, especially when measured with tetramers derived from the 13 major SARS-CoV-2 variants. Comparison of the transcriptomes of B, CD4 and CD8 T cells from pre- and post-vaccinated young and elderly revealed genes potentially responsible for low immune responses in elderly, functionally related to antigen processing and presentation. We further built an epitope specific TCR repertoire database using single-cell RNA and TCR sequencing. Statistical and machine-learning based analyses demonstrated that SARS-CoV-2 epitope specific CD8 T cell clones were significantly lower in the old than young, and failed to be boosted after vaccination in elderly. Comparison of BCRs and TCRs from pre- and post-vaccinated young and elderly individuals revealed inadequate receptor repertoire size and diversity might be responsible for low immune response in the elderly. Together, the altered immune cell function and the attenuated antigen specific TCR repertoire were responsible for the impaired CD8 T cell immune response in the elderly.

This work was supported by grants from the National Key Research and Development Program of China (2018YFC2002003 to G.C., 2021YFC2009400 to G.Z.), the Natural Science Foundation of China (U1801285 and 92169102 to G.C., 81971301 and 32050410285 to O.J.L., 82201954 to Z.R.)
Antibody response of Comirnaty vaccine in different immunocompromised groups during the COVID-19 pandemic
Authors: Togoo Khongorzul , Batmunkh Burenjargal , Tsogtbayar Nomin-Erdene , Batmunkh Ulziisaikhan , Selenge Enkhtuya , Togtokhbaatar Khuselt-Od , Boldbaatar Gantuya , Jagdagsuren Davaalkham , Gunchin Batbaatar , Sandag Tsogtsaikhan
Published: August 2023

A nationwide vaccination program was initiated on February 23, 2021, in Mongolia by 4 types of vaccines for COVID-19 were offered voluntarily. People at increased risk of severe disease were vaccinated with Comirnaty. In this study, we aimed to compare specific antibody responses after the second dose of the Comirnaty vaccine in different immunocompromised groups. This prospective cohort study was conducted from March–April to July–August of 2021. 225 vaccinees presented an immunocompromised population examined for anti-SARS-CoV-2 RBD-IgG (ELISA, Proteintech, USA) before the first dose of vaccine and RBD-IgG antibodies were measured 14 – 21 days after the second dose of vaccine. An increase of antibody titer more than 3-fold was considered a positive antibody response after vaccination. We could attend to the after the second dose of examination 172 (76.4%) of 225 vaccinees. Antibody response to COVID-19 vaccines was detected in 164 (95.1%) vaccinees that demonstrated positive antibody response. A positive response was found in all (100.0%) immunocompromised (19 patients received anti-cancer chemotherapy, 90 patients received corticosteroids, 63 HIV-infected patients) groups. The mean titer of RBD-IgG antibodies in anti-cancer chemotherapy (185.1±49.1ng/mL, CI95: 161.4–208.8) and corticosteroids group (161.6±70.3 ng/mL, CI95: 147.1–176.2) was significantly higher compared to HIV-infected group (98.8±32.6 ng/mL, CI95: 90.6–107.0). A significant impact on antibody response after the second dose of Comirnaty COVID-19 vaccination was associated with the immunocompromised condition. The study was funded by the Ministry of Health of Mongolia.

None
Understanding Mechanisms of Adjuvancy in Muscle by mRNA/Lipid Nanoparticles
Authors: Majumdar Devdoot , Dowell William , Languon Sylvester , Miller Zachary , Dearborn Jake
Published: August 2023

Vaccines utilizing encapsulated mRNA within lipid nanoparticles (LNPs) have demonstrated unprecedented efficacy in clinical studies of mRNA-1273 and BNT162b2, two prominent vaccines developed against the S protein of SARS-CoV-2. Despite several Phase III trials offering deep clinical intuition about vaccine efficacy, the musculo-immune basis for elicitation of a strong germinal center response from mRNA/LNP vaccine remains unclear. Here, we characterize the trajectory of the immune response to an mRNA lipid nanoparticle vaccine, encapsulated in sm-102 (Moderna). Furthermore, we find that intramuscular administration of empty LNPs elicits a strong neutrophil and dendritic response within 24h. Additionally, we show that skeletal muscle may play a role in the early immune response to mRNA LNPs. Inflammatory LNPs thus contribute to the well-established role of RNA immunogenicity. Using a reporter of transduction, we show that both >10% of muscle-resident myeloid and lymphoid cells are directly transduced with mRNA vaccines, and that these cells play a crucial role in the downstream germinal center response. Transcriptomic measurements of muscle tissue reveal a direct role role of ionizable lipid operating as an adjuvant to induce this response. Using in vitro and molecular tools, these studies advance our understanding of the cellular and molecular basis for LNP-mediated inflammation and adjuvancy underpinning the exceptional clinical efficacy of mRNA/LNP vaccination.
Detection of envelope-dimer epitope-like broadly protective antibodies in dengue-immune children in the Philippines following vaccination and natural infection
Authors: Mpingabo Patrick Ilunga , Ylade Michelle , Crisostomo Ma Vinna , Thiono Devina , Daag Jedas Veronica , Coello Ana E , Rodriguez Guillermo R , Lowman Kelsey , Firdous Saba , Aogo Rosie , Odio Camila , White Laura , de Silva Aravinda , Deen Jacqueline , Katzelnick Leah C
Published: August 2023

The dengue vaccine, Dengvaxia, has been described as eliciting antibody-dependent enhancement (ADE) in dengue-naive individuals. There is an urgent need to develop an effective dengue virus (DENV) vaccine that elicits protective, cross-reactive (CR) neutralizing antibodies (NAbs), which are capable of neutralizing DENV1–4 without triggering ADE such as EDE Abs.

Here, we investigated the CRNAbs profile following DENV natural infection versus vaccination in children with prior DENV infection (n=2,996) from a longitudinal vaccine (study) cohort in Cebu, Philippines. In total, 1,214 of these children remained unvaccinated while 1,782 children received a single dose of Dengvaxia in June of 2017 during a mass vaccination campaign. Serum samples were collected one month before and one year after the vaccine campaign.

To measure baseline status and change in CRNAbs level following vaccination versus natural infection, we ran Focus Reduction Neutralizing Test (FRNT) using mature DENV1–4 virions and Blockade of binding (B0B) assay against EDE Abs on a random subset of these DENV-immune children’s samples (n=231). Furthermore, we also measured IgG binding against the DENV1–4 E protein monomer and DENV2 E protein dimer.

Both vaccinated and naturally infected groups elicit CRNAbs that recognize the quaternary epitopes of EDE antibodies. We also see a significant increase in antibody response in both groups, but CRNAbs had a stronger correlation to mature FRNT in naturally infected compared to vaccinated populations, indicating differences in antibody quality. These results highlight the presence of EDE-like broadly NAbs that could possibly protect against the four DENV serotypes and provide an insight for future vaccine candidates.

This work was supported by the Intramural Research Program (DIR) of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, the Philippine Department of Health and the University of North Carolina (PO1: P01AI106695).
PD-1 immune checkpoint blockade can synergize with PSGL-1 inhibition to reinvigorate exhausted T cells
Authors: Viramontes Karla M , Neubert Emily N , DeRogatis Julia M , Henriquez Monique , Tinoco Roberto
Published: August 2023

P-selectin glycoprotein ligand-1 (PSGL-1) is an immune checkpoint regulator that is highly expressed on T cells. PSGL-1- deficient mice infected with chronic lymphocytic choriomeningitis virus (LCMV) were shown to control chronic viral infection due to increased effector function by anti-viral T cells. However, the cell-intrinsic role of PSGL-1 expression on CD4+ and CD8+ T cell exhaustion is not fully known. Using adoptive transfers of WT or PSGL-1-deficient TCR transgenic CD4+ and CD8+ T cells in mice infected with chronic LCMV, we characterized the differentiation and effector response. We found increased expansion and effector function by PSGL-1-deficient T cells early during infection, however, at later stages of infection, PSGL-1-deficient T cells were functionally exhausted. We found that exhausted CD4+ and CD8+ PSGL-1-deficient T cells were reinvigorated more effectively than WT cells after PD-L1 blockade due to their increased proliferation, cytokine production, and accumulation. We also observed increased responses of CD8+PSGL-1-deficient T cells in melanoma tumors. Our findings showed that PSGL-1 expression was required for the maintenance of both CD8+ and CD4+ exhausted T cells and that PSGL-1 deletion synergized with PD-1 blockade to reinvigorate these cells. These findings highlight an important cell-intrinsic role for PSGL-1 expression in the maintenance and reinvigoration of exhausted T cells.

T32 AI 141346 R01 AI13723
B cell dysfunction during aging is not dependent on stem cell aging
Authors: Greer Braxton D , Maul Robert W , Gearhart Patricia J
Published: August 2023

Humoral immune responses to vaccination are a critical component of protection from infectious diseases. Older humans simultaneously have reduced antibody production in response to vaccination and an increased burden of mortality when infected with viral respiratory infections such as influenza or SARS-CoV-2. Therefore, it is critical to understand molecular drivers of immunosenescence in order to develop vaccination strategies that better protect the elderly. By adoptively transferring naïve B cells from old or young mice into young B cell-depleted recipients, our lab has characterized the germinal center dynamics and antibody production in response to a model antigen, nitrophenyl-chicken gamma globulin (NP-CGG). B cells from young mice were found to be antigen specific in the germinal center and produced specific anti-NP antibodies. B cells from old mice failed to produce anti-NP antibodies and had a large proportion of non-specific germinal center B cells. We sought to uncover the origin of this age-related deficiency. We hypothesized that age-induced changes in hematopoietic stem cells (HSCs) might be passed on to mature B cells and disrupt their function. We transplanted HSCs from young or old donors into young, irradiated recipients and analyzed the ability of the progeny B cells to mount a specific antibody response. To our surprise, HSC age did not encode B cell function, and B cells from both HSC donor ages could mount an NP-specific response, implicating an alternative source of aging dysfunction. Further study into the cause of age-related B cell dysfunction will guide the development of vaccine additives that improve protective immunity in the elderly.

Supported by NIA intramural funding.
Heme augments proliferation and epigenetic programming during B cell differentiation
Authors: Padilla-Quirarte Herbey O , Kania Anna , Hicks Sakeenah , Maurer Jeffrey , Boss Jeremy M , Scharer Christopher D
Published: August 2023

Heme is an iron-containing porphyrin ring that is a key part of the cytochrome proteins that function in the electron transport chain and facilitate OXPHOS metabolism. Recently, our group found that many iron-metabolism related genes are highly expressed in B cells, particularly in memory B cells (MBC) in both mice and humans. In ex vivo cultures, naïve (nB) and MBC respond to heme addition by augmenting the frequency of plasmablast formation. In this work, we further characterized the role of heme in augmenting B cell differentiation and function. To begin addressing the question of how heme modulates B cell differentiation, proliferation, or both, we first conducted ex vivo cultures of CellTraceYellow (CTY)-labeled B cells stimulated with CD40, IL-4 and IL-5, treated with and without heme. We found that the first division occurs within 28 hours post-stimulation for nB cells and nB and MBC that were treated with heme divide earlier than those that were treated with vehicle.. When the number of cells in each division was analyzed, we found more cells in later divisions in heme-treated than in vehicle-treated ones, suggesting that heme is important for B cell proliferation. To further characterize the molecular effects of heme on B cell differentiation we preformed RNA-seq and ATAC-seq on activated B cells and plasmablasts. The addition of heme augmented both the accessible chromatin and gene expression of hundreds of genes leading to differences surrounding key metabolic and differentiation regulators. These data further our understanding for how heme modulates B cell differentiation and promotes the formation of antibody secreting plasmablasts.
PAD4-dependent neutrophil extracellular traps dagame intestinal barrier integrity through histone protein components
Authors: Chiang Hao-Sen
Published: August 2023

Infiltrating neutrophils and neutrophil extracellular traps (NETs) accumulate in the inflamed epithelium to induce mucosal inflammation in patients with inflammatory bowel diseases. The formation of NETs is initiated by peptidyl arginine deiminase 4 (PAD4), which facilitates NET generation through chromatin decondensation. We recently demonstrated that NETs impair intestinal epithelium integrity, permeability, and viability in mice with colitis. In addition, disruption of NET formation by administering DNase I or PAD4 inhibitors attenuates intestinal inflammation in colitic mice. However, the effect of genetic loss on PAD4-mediated NET formation and intestinal inflammation has not been determined in chemical-induced colitis models. Moreover, the specific NET components responsible for the damage and impaired barrier integrity in the intestinal epithelium are also unknown.

Here we demonstrated that mice lacking PAD4 failed to produce NETs in the intestine in response to dextran sulfate sodium (DSS)-induced colitis. This impairment in PAD4-deficient mice ameliorated intestinal inflammation, barrier function, and integrity. Mechanistically, NETs, particularly the histone components, altered the integrity of junctional proteins and induced cytotoxicity, resulting in increased gut epithelium permeability. In contrast, inhibition of histone activity in NETs restored the expression and localization of junctional proteins and cell viability, thus enhancing intestinal barrier functions. Collectively, our findings indicate that extracellular histones of NETs have a detrimental role in regulating intestinal barrier function in Caco-2 monolayers and in vivo.
Bile acid conditioned Dendritic cells through FXR and specialize T cell function in the ileum
Authors: Huang Xiangsheng , Sundrud Mark S , Eltzschig Holger , Moore David
Published: August 2023

The accumulation of locally-reabsorbed bile acids (BAs) is suggested to contribute to the development of terminal ileitis. Our previous data showed nuclear receptor Constitutive Androstane Receptor (CAR) can directs T cell adaptation to BAs in the ileum by promoting the IL-10 producing Tr1 cells to prevent ileitis. Still, the mechanisms by which BA activate CAR in ileal T cells is not known. As CAR is not recognized as a BA sensor, its activation in ileal T cells may involves indirect pathways, such as dendritic cells (DCs). Farnesoid X receptor (FXR), a direct primary BA sensor, is highly expressed in both human and mouse DCs. Therefore, we hypothesized that BA-sensing by mucosal DCs through FXR may condition mucosal T cells in the ileum to upregulate CAR and to enforce anti-inflammatory T cell functions. Our result indicated WT DCs exposed to direct FXR agonist, chenodeoxycholic acid (CDCA), displayed increased production of IL-23a and decreased IL-6. While, CD4+ T cells cocultured with CDCA-treated DCs displayed increased CAR/Nr1i3, and its targets, cMyc and MDR1. Importantly, these observations were diminished in DCs from FXR−/− mice. In addition, when FXR is depleted in DC (CD11cCreFXRfl/fl, FXRΔDC), the DC migration in the intestine is deficient, with low expressions of CCL19, CCR9 and less CD11c+CD11b−CD103+MHCII+ migratory DC population in the mesenteric lymph nodes. Meanwhile, the FXRΔDC mice failed to promote CAR/Nr1i3 as well as IL10 in T cells. Furthermore, FXR−/−RAG2−/− mice developed severe ileitis after engrafted with WT naïve T cell. These results suggest that FXR specializes intestinal DC and T cell function in the ileum. Collectively, these studies will shed new light on the understanding and treatment of ileitis in patients.
Fibroblasts participated in mucosal pathology in the oral disease periodontitis
Authors: Ikeuchi Tomoko , Rodriguez Belmaliz Cardona , Chen Zuojia , Wild Teresa Greenwell , Wu Chuan , Moutsopoulos Niki M
Published: August 2023

The oral cavity is a unique stricture; it combines mucosal and bone tissue microenvironments with their respective immunological responses. In fact, oral mucosal inflammation leads to bone destruction in the prevalent human disease, periodontitis. In periodontitis, immune cells, in particular Th17 and neutrophil mediated responses have been linked to pathogenesis, however it is not yet understood how the stromal tissue microenvironment may participate in disease pathology. To date, studies in humans reveal inflammatory signatures in oral mucosal stromal cells of periodontitis, linked to neutrophil recruitment. To understand fibroblast responses and function in periodontitis, we performed scRNA-seq in mouse oral mucosa during a time course in the experimental periodontitis model. We characterize an early responding fibroblast subpopulation with a distinct inflammatory signature, characterized by induction of the IL33- ST2 cytokine axis. We confirm induction of soluble ST2 in oral mucosal fibroblasts during periodontitis and uncover an immune-regulatory role for ST2 within the oral microenvironment. Our work highlights the nature of oral mucosal immune-responsiveness and provides insights into a common human disease.

Supported by NIH intramural funding