bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a liquid tumor potency assay for evaluation of immune cell-mediated cytotoxicity in vitro
Authors: Califano Danielle , Streeter Ben , Sullivan Denise , Passaro Austin , Chvatal Stacie , Millard Daniel 
Published: August 2023

Abstract
The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells—to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, with each representing an important aspect driving clinical efficacy of the cellular product. Previous work has shown that impedance-based potency assays provide a real-time, label-free measure of effector cell-mediated cytotoxicity for adherent target cells. Here, we describe an in vitro potency assay that quantifies effector cell-mediated cytotoxicity of liquid tumor target cells. First, the wells were coated with antibody to tether the liquid tumor target cells (Raji = anti-CD40, K562 = anti-CD71, Daudi = anti-CD40). Tethering the liquid tumor target cells significantly increased the impedance measurement, as compared to untethered controls. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of effector to target ratios. Cytolysis of the target cells was calculated by comparing the impedance in treated wells to no treatment control wells. As expected, the CD19-specific CAR T cells produced significantly higher cytolysis in the Daudi (54.3%) and Raji (70.4%) cells, which express CD19, as compared to the K562 cells (30.5%). These results support a real-time, label-free potency assay with liquid tumor target cells.
Dual targeting of mast cells and TSLP with a bispecific antibody
Authors: Alvarado Diego , Vitale Laura , Murphy Michael B , Wasiuk Anna , Widger Jenifer , Crocker Andrea , Patterson Colleen , Mills-Chen Laura , O’Neill Thomas , Baronas April R , McManus Kelly M , Boyer James M , Montanari Noe Rico , Lillquist Jay , Malenchek Linda , Weidlick Jeff , Goldstein Joel , Thomas Lawrence J , Marsh Henry C , Keler Tibor 
Published: August 2023

Abstract

Rationale
Reduction of mast cells (MCs) through stem cell factor (SCF) neutralization combined with inhibition of the alarmin TSLP in a single molecule may result in broader efficacy in inflammatory disorders.

Methods
Monoclonal antibodies (mAb) separately targeting SCF and TSLP were generated from immunized mice. A potent neutralizing antibody against each target was selected and humanized. Bispecific tetravalent antibodies (bsAbs) with specificity towards SCF and TSLP were generated. Candidate bsAbs were tested in vitro for neutralizing activity and evaluated in pilot non-human primates (NHP) studies.

Results
We discovered a novel anti-SCF neutralizing mAb (SCF-12) that potently inhibits SCF-dependent KIT phosphorylation and SCF-enhanced degranulation of primary human MCs. Separately, a novel anti-TSLP mAb (1D10) inhibits TSLP receptor binding and TSLP-dependent TARC release from human monocyte-derived DCs. bsAbs combining humanized SCF-12 and 1D10 with good expression, biophysical profile and robust neutralizing activity against each target were generated. Following demonstration of MC depletion in NHPs with SCF-12, we conducted a pilot NHP study with two lead candidate bsAbs. Pharmacokinetic, pharmacodynamic and tolerability data from this study will be presented.

Conclusions
SCF x TSLP bsAbs were successfully generated from antibodies that neutralize soluble SCF, leading to MC reduction in NHP, and TSLP, a key alarmin implicated in inflammatory and autoimmune disorders. Combined inhibition of these two pathways using a single molecule may be of broad utility in multiple inflammatory disorders.

Celldex Therapeutics
Analysis of T Cell Immunity Generated by Takeda’s Tetravalent Dengue Vaccine
Authors: Omidi Nazanin , Victor Kaitlin , Gray Lauren , Nadeem Iqra , Bolton Jessica , Robinson Tanisha , Hatch Kristin , Sharma Mayuri , Mandaric Sanja , Tricou Vianney , Currier Jeffrey , Friberg Heather 
Published: August 2023

Abstract
Dengue is a viral disease transmitted to humans through the bite of infected mosquitoes and is caused by dengue virus (DENV), which has four distinct serotypes, DENV1 to DENV4. Severe dengue is a leading cause of serious illness and death in some Asian and Latin American countries. The Walter Reed Army Institute of Research (WRAIR) has been working to investigate the cell mediated immunogenicity of Takeda’s live attenuated virus tetravalent dengue vaccine (TAK-003). Previously, we have used ELISPOT and Flow-ICS assays to show that TAK-003 induces a robust IFN-γ producing DENV-specific T cell response that is durable to at least 6 months post-vaccination. In addition, this response is mediated by both CD8+ and CD4+ T cells that produce IFN-γ and/or TNFα and IL-2. Using samples from a phase II study, DEN-313 (NCT02948829) conducted in healthy children aged 4 to 16 years in dengue endemic locations, herewith we extend the findings on TAK-003 induced DENV-specific T cell responses to: (1) present data on durability of the CMI response through 3 years post-vaccination; (2) verify the serotype coverage of the T cell response; (3) assess multifunctional capacity of the durable memory T cells, and (4) describe the phenotypic changes that occur among these cells based upon central memory (CD45RA−/CCR7+), effector memory (CD45RA−/CCR7−) and effector memory RA phenotyping (CD45RA+/CCR7−). Our data describe the frequency, functionality, and memory phenotype of T cells induced by TAK-003 up to 3 years post vaccination.
Finding received through corporative research and development agreement with Takeda vaccine company.
Double Negative B cells are decreased in patients with Idiopathic Inflammatory Myopathies
Authors: Reyes Raul Fernando , Torres-Ruiz Jiram , Gómez-Martin Diana , Maravillas-Montero José Luis 
Published: August 2023

Abstract

Background
Idiopathic inflammatory myopathies (IIM) are systemic autoimmune diseases characterized by skeletal muscle inflammation, frequently accompanied by extra muscular features associated with specific autoantibodies. The detailed pathogenesis of IIM remains unclear, but evidence suggests autoreactive B cells may play an essential role in this process. However, little is known about changes in peripheral blood B cell subsets in adult IIM patients.

Objective
This study aims to determine the frequency of B cell subsets among patients with IIM and their clinical correlation with disease activity.

Methods
Using a multiparametric flow cytometric approach, we determined the frequencies of B cell subsets in a cohort of 36 patients with IIM and correlated their values with clinical and laboratory data.

Results
DN B cells (CD27− IgD−), which represent an atypical B cell subset associated with various autoimmune diseases, showed a significant decrease in frequencies and a correlation with the MMT8 score. Within the DN subset, the DN1 subpopulation (CD21+ CD11c−) showed a significant decrease compared to the control group. There was also a significant decrease in the memory B cell subpopulation (CD27+ CD38−) and the non-classic memory B cell subpopulation (CD27− CD38− CD24+). A significant increase in naive B cells (CD27− IgD+) was observed. However, none of the previously described subpopulations correlated with clinical features.

Conclusion
This study demonstrated a decrease in frequencies in the double negative B cell subset in adult IIM patients and showed a correlation with clinical features, which suggests that these B cells could be related to disease activity. UNAM-DGAPA-PAPIIT IN212122
Type 1 interferons promote anti-RBC antibody responses in interferon-dependent lupus mice
Authors: Paul Kausik , Lee June Young , Hernandez-Armengol Rosario , Shibata Tomohiro , Jefferies Caroline Anne , Gibb David R 
Published: August 2023

Abstract
Red blood cell (RBC) transfusion can induce anti-RBC antibodies that promote hemolytic events. Patients with systemic lupus erythematosus (SLE) have an increased frequency of RBC alloimmunization, and two-thirds of patients express a type 1 interferon (IFNα/β) gene signature. We previously reported that IFNα/β induces RBC alloimmunization in the IFNα/β-dependent pristane lupus mouse model. However, it is unclear whether the lupus-like phenotype or IFNα/β directly promoted alloimmunization. Thus, we examined alloantibody responses in MRL/lpr mice, which have an IFNα/β-independent lupus-like phenotype. MRL/lpr mice produced anti-dsDNA lupus antibodies. However, unlike pristane-treated WT mice, IFNα/β-stimulated genes (ISGs) were not elevated in MRL/lpr mice. After transfusion with allogeneic RBCs, untreated WT and MRL/lpr mice produced minimal levels of anti-RBC antibodies, compared to elevated levels produced by pristane-treated WT mice. To determine whether IFNα/β can induce alloimmunization in MRL/lpr mice, WT and MRL/lpr mice were treated with recombinant IFNα (rIFNα) or the IFNα/β stimulus, poly(I:C), prior to transfusion. While rIFNa and poly(I:C) induced RBC alloimmunization in WT mice, they failed to induce alloimmunization in MRL/lpr mice. Thus, MRL/lpr mice are resistant to IFNα/β-induced alloimmunization due to a currently explored mechanism. These findings also illustrate that allogeneic transfusion induces alloimmunization in an IFNα/β-dependent lupus model, but not in an IFNα/β-independent model. Thus, the lupus-like phenotype is not sufficient to induce alloimmunization. Collectively, these findings warrant investigation of the role of IFNα/β in RBC alloimmunization in patients with SLE.
NIH (R01 HL165169-01); NIH (R03 HL158637-01); NIH (K08 HL141446-03)
TLR7 expression increases with disease progression and mediates expansion of distinct B cell subsets in primary Sjögren’s Disease
Authors: Punnanitinont Achamaporn , Kasperek Eileen M , Kramer Jill M 
Published: August 2023

Abstract
Primary Sjögren’s Disease (pSD) is a systemic autoimmune disorder in which the exocrine tissue exhibits chronic inflammation. Additionally, patients often experience diminished tear and saliva production along with many extra-glandular disease manifestations. The causes of pSD are poorly understood and no curative treatments are available. Previous studies in our lab conducted in a pSD mouse model demonstrated that TLR7 agonism accelerates pSD pathogenesis and drives expansion of age-associated B cells (ABCs). To extend our prior work, we hypothesized that pSD splenocytes show heightened TLR7 expression and activation, and expansion of B cell subsets linked to autoimmunity relies on TLR7 in pSD mice. We harvested the spleens of pSD females at three different disease time points (pre-disease, clinical disease and advanced stage disease) and performed flow cytometry. Parallel experiments were performed using age and sex-matched healthy controls. Splenocytes were also cultured with the TLR7 agonist Imiquimod (Imq) and IL-6 secretion assessed by ELISA. In addition, spleens were isolated from TLR7-deficient pSD females at the clinical disease stage and flow cytometry was performed. We found splenocytes derived from pSD animals at the clinical disease stage secreted higher levels of IL-6 in response to Imq stimulation as compared to those derived from healthy control animals. TLR7+ B cells and ABCs (B220+, CD23−, CD21−, T-bet+) increased with disease progression in pSD mice. In pSD mice lacking TLR7, ABCs and germinal center B cells were decreased compared to the TLR7-sufficient pSD parental strain. Thus, splenocytes from pSD mice show enhanced sensitivity to TLR7 and TLR7 signaling mediates expansion of ABC subset in pSD.
NIH/NIDCR T32 DE023056 NIH/NIDCR R01 DE029472
B cells Regulation by Interferon-gamma in a Neuromyelitis Optica Spectrum Disorder animal model
Authors: Arellano Gabriel , Loda Eileah , Chen Yanan , Podojil Joseph R , Neef Tobias , Popko Brian , Balabanov Roumen , Miller Stephen D 
Published: August 2023

Abstract
Neuromyelitis Optica Spectrum Disorder (NMOSD) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and auto-antibody response against the water channel protein Aquaporin-4 (AQP4). In NMOSD, B cells plays a central role regulating autoreactive T and antibody secreting cells, and the use of anti-B cells therapy is a common practice for patients. Despite this, the mechanisms of B cells response against AQP4 are poorly understood. We have demonstrated that the immune tolerance against the immunodominant AQP4201–220 peptide in mice is maintained by IFN-g, controlling disease incidence, severity, and chronicity. Actively immunization of C57BL/6 (WT) treated with neutralizing anti-IFN-g antibodies, IFN-g knockout (IFNGKO) and IFN-g receptor KO (IFNRKO) mice was induced using the AQP4201–220 peptide, showing an ascendant paralysis starting from the tail to complete hind limb paralysis. CNS histological analysis showed an increased presence of T and B cells, and the complement system in lesion sites, associated with CNS damage. CNS Th17 profile was also observed in the anti-IFN-g treated group. Conversely, IFN-g treatment in IFNGKO mice decrease disease severity with a reduction of CNS CD4+ and B cells. Remarkably, IFN-g regulates the IL-6 expression by B cells in an antigen specific fashion. B-cell depletion significantly reduced the disease incidence, T cells CNS infiltration and anti-AQP4201–220 serum antibodies. Finally, using anti-IL-6R treatment, we showed that IL-6 signaling is necessary for the B cell activation and their induction of Th17 cells. In conclusion, AQP4201–220 tolerance is strictly regulated by IFN-g and its signaling, restraining B cells pathogenicity through IL-6 signaling modulation.
This work was supported by National Institutes of Health grants R21AI151438 and R01 NS099334
Treg cell depletion in adult mice results in activation of antigen-presenting cells prior to fatal autoimmune disease
Authors: Kim Yong-Hee , Panda Abir K , Shevach Ethan M 
Published: August 2023

Abstract
Studies in mice expressing the diphtheria toxin receptor (DTR) exclusively on regulatory T (Treg) cells (Foxp3-DTR mice) demonstrated the critical importance of Treg cells in maintaining normal immune homeostasis. Adult Foxp3-DTR mice injected with DT begin to die from day 10 after DT treatment. Marked expansion of almost all immune cell types was observed prior to death. The purpose of the present studies was to examine in-depth changes in lymphocytes and antigen-presenting cells at early time points after Treg depletion to determine the most critical cell types controlled by Treg in the steady state. Complete depletion of Treg was observed 2 days after treatment, but profound activation of CD4+ and CD8+ T cells as measured by induction of CD44 expression and vigorous proliferation as measured by Ki-67 incorporation were not seen on day 2 but seen on day 6 of DT treatment. Curiously, most of the CD44hi Ki-67+ T cells were CD25− and plasma levels and intracellular levels of IL-2 were not increased suggesting that early activation of CD4+ and CD8+ T cells was not the result of a burst of IL-2 production. Increased expression of CD80, CD86, CD40, and PD-L1 could be detected on CD11c+ dendritic cells (DCs) on day 6. Anti-CD80/CD86 mAbs reversed the activation of T cells provoked by depletion of Treg cells, while anti-IL-2 or anti-IL-2Rb mAbs had no effect. Taken together, these studies suggest that disruption of the regulation of Treg-mediated control of DC activation represents the first step in the complex autoimmune disease seen after Treg depletion.
This work was supported by the Intramural Research Program of NIAID, NIH.
This work was supported by the Intramural Research Program of NIAID, NIH.
Chaperone-mediated autophagy modulates regulatory T cell differentiation and function
Authors: Harrison Ranee , Botbol Yair , Sidoli Simone , Cuervo Ana-Maria , Macian Fernando 
Published: August 2023

Abstract
Autophagy has been shown to regulate many key functions of the immune system including important roles in effector and memory T cell maintenance and function. Our group showed that chaperone-mediated autophagy (CMA), a selective type of autophagy that declines with age, plays a key role in T cell activation. In this work, we have analyzed the role of CMA in generation and function of regulatory T cells (Tregs). We found that resting Tregs display high basal CMA activity, which further increases upon Treg activation. We generated Treg specific CMA-deficient mice (Foxp3:Cre-LAMP2Afl/fl) and found that they have significantly reduced body weight due to reduced fat mass. We propose that the CMA status of adipose tissue infiltrating Tregs may have a direct impact on adipose tissue inflammation state. In fact, as the Treg-specific CMA-deficient mice age, we found marked inflammation and reduced survival, suggesting a role for CMA in maintaining Treg self-tolerance under homeostatic conditions. We confirmed that CMA-deficient Tregs have reduced suppressive activity in vivo and have identified the Treg sub-proteome regulated by CMA using comparative quantitative proteomic analysis of control and CMA-deficient Tregs. This has allowed us to identify cellular pathways behind the mechanism by which CMA regulates Treg function. The proteomic analysis hits will help in identifying potential targets of CMA that could be used therapeutically to modulate Treg function, to prevent autoimmunity or boost anti-tumor immune responses.
Supported by grants from NIH
P01-AG031782
R01-AI113919
Social Status is Associated with Impaired Anti-Inflammatory Response in Free-Ranging Male Rhesus Macaques
Authors: Sanchez Rosado Mitchell R , Newman Laura , Watowich Marina , Pavez-Fox Melissa , Valle Josue Negron-Del , Phillips Daniel , Skelton Macaela , Siracusa Erin , Higham James , Brent Lauren , Lea Amanda , Sariol Carlos , Snyder-Mackler Noah 
Published: August 2023

Abstract
Exposure to social adversity can influence the onset and progression of disease. One possible mechanism through which social adversity may impact health outcomes is by altering immune homeostasis. Recently, evidence has connected social adversity to changes in gene regulation in the immune response of both humans and nonhuman primates. We investigated how social adversity (quantified as low social status) affected the Th2 immune response in free-ranging male rhesus macaques (n=128). We stimulated peripheral blood mononuclear cells (PBMCs) in vitro with Dexamethasone (Dex), an anti-inflammatory synthetic glucocorticoid (GC), and measured how social status influenced the gene regulatory response to Dex stimulation using mRNA-seq. We detected 2,923 differentially expressed genes (FDR&lt;0.1) in response to Dex in PBMCs. After Dex stimulation, glucocorticoid responsive genes, such as FKBP5 and TSCD22, were induced and inflammatory genes, such as CSF1 and TNF, were repressed. Low social status animals had significantly higher mean expression of inflammatory genes (p=1.2×10−20) compared to high social status animals. Many of these genes, such as CD38 and NKAP, are known to be repressed by Dex. After Dex stimulation, low social status animals also had a higher mean expression (p=1.9×10−4) of genes related to the GC resistance-associated c-Myc/MAPK pathway. For instance, low social status animals had significantly higher expression (FDR&lt;0.2) of signature genes in this pathway including AKT3, MAPK1 and PIK3CD. Our study suggests that the anti-inflammatory immune response in rhesus macaques can be negatively affected by low social status, with animals having a blunted response to Dex and presenting signatures of GC resistance.
R01-AG060931 R00-AG051764 P40-OD012217
Host-targeted self-attenuated influenza virus as an effective nasal spray vaccine in aged population
Authors: De La O Perez Rodrigo , Chen Yanping , Zeng Mingtao 
Published: August 2023

Abstract
The elderly are at high risk for influenza infection and its complication. However, current inactivated influenza vaccines are often modestly immunogenic and show low protective immunity in the elderly, due to immunosenescence. New approaches are urgently needed for developing effective vaccines against influenza for the elderly. In current study, we have constructed new recombinant influenza viruses, named host-targeted self-attenuated influenza viruses (SAIVs), which can express functional mammalian species-specific artificial microRNAs (amiRNAs). The expression of these amiRNAs can inhibit expression of some host factors critical for influenza replication, and therefore the resultant recombinant influenza viruses are replication restricted and attenuated in the host cells. One of these SAIVs, which can express an amiRNA that inhibits expression of the host cellular Cdc2-like kinase 1 (CLK1), was produced in embryonic chicken eggs and evaluated in an aged mouse model of influenza infection. It elicited robust antibody and T cell responses against influenza virus and demonstrated significantly protective efficacy against lethal infection with wild type influenza virus H1N1 PR8 after single dose of intranasal vaccination. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further used as an effective vaccine against influenza in aged population.
Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).
Engineered cytokines target atherosclerotic plaques and locally suppress inflammation
Authors: Volpatti Lisa R , Reda Joseph , Borjas Gustavo , Zhou Zhengjie , Fang Yun , Hubbell Jeffrey 
Published: August 2023

Abstract
Chronic inflammatory diseases are often treated with corticosteroids or TNF blockers to suppress overactive immune responses. However, these immunosuppressants also dampen healthy immune responses to opportunistic pathogens (e.g., respiratory viruses) and are associated with adverse effects. Targeted immunotherapies are needed to provide local immunosuppression without systemic effects. In the context of atherosclerosis, the anti-inflammatory cytokine IL-10 has been shown to suppress vascular inflammation, but its poor pharmacokinetic profile and pleiotropic effects have limited its therapeutic potential. To overcome these challenges, we engineered IL-10 to specifically target atherosclerotic plaques. We accomplished this goal by constructing fusion proteins in which one side is IL-10 and the other side is an antibody fragment (Fab) that binds to protein epitopes of low-density lipoprotein (LDL). In murine models of atherosclerosis, we show that systemically administered Fab-IL-10 constructs bind circulating LDL and hitchhike a ride to atherosclerotic plaques. In a biodistribution study, we observe elevated levels of IL-10 in the aorta but not the lungs of hyperlipidemic mice, indicative of targeted delivery. The targeted Fab-IL-10 constructs significantly reduce aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that Fab-IL-10 constructs are preferentially taken up by macrophages to exert an anti-inflammatory effect. This platform technology can be applied to a variety of cytokines and shows promise as a potential targeted anti-inflammatory therapy.
Supported by the Chicago Immunoengineering Innovation Center, the NIH National Heart, Lung, and Blood Institute, and the American Heart Association.