bluuebunny/crossref_metadata_2025_split huggingface dataset:
γc receptor antagonist, EQ102, prevents the NK and T cell-mediated responses driven by IL-15 and IL-21
Authors: Tiet Phoi , Giovannone A J , Al-Mawsawi Laith , Ampudia Jeanette , Marrocco Valeria , Connelly Stephen , Ng Cherie 
Published: August 2023

Abstract
Synergistic signaling of IL-15 and IL-21 is important in driving pathogenic T and NK cell responses in multiple inflammatory diseases. IL-15 and IL-21 are part of the common gamma chain (γc) cytokine family, which use the shared γc receptor subunit in their signaling complexes. IL-15 and IL-21 each promote cytolytic activity and IFNγ production but can also synergistically enhance the function of other cytotoxic cytokines. Consequently, blockade of IL-15 or IL-21 alone is likely insufficient to improve disease outcome in pathologic environments. EQ102 is a multi-cytokine inhibitor that selectively blocks IL-15 and IL-21 signaling while preserving signaling of other γc family members. Here, we sought to investigate the effect of synergistic signaling from both IL-15 and IL-21 on NK and T cell activities and the ability of EQ102 to inhibit these signals and resultant cellular response.
PBMCs from healthy donors or NK-92 cells were incubated with EQ102 for 1 hour and then stimulated with IL-15, IL-21, or in combination. Following stimulation, cellular expression of transcription factors and activation markers were analyzed by flow cytometry. Cell supernatant was assessed for T and NK cell cytokines.
IL-15 stimulation increases proliferation of T and NK cells, where IL-21 had a modest effect. However, co-stimulation of IL-15 and IL-21 enhanced proliferation, activation, and upregulation of IFNγ production over single cytokine IL-15 conditions. EQ102 treatment effectively inhibits the IL-15/IL-21 co-stimulatory cytolytic responses of these cell populations. These results suggest that selective blockade of IL-15 and IL-21 by EQ102 inhibits the synergistic signaling that mediates NK and T cell responses in multiple immune disorders.
None
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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&lt;0.001 in vitro and 25±4% vs. 12±2% P&lt;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

Abstract
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

Abstract
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

Abstract
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&lt;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

Abstract
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

Abstract
--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 &lt;1% or [C4] &lt; 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

Abstract
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

Abstract

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 (&gt; 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.