bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying Mtb-Specific T Cell Epitopes Across the Spectrum of Tuberculosis Infection: Implications for Diagnostics and Vaccines
Authors: Panda Sudhasini , Morgan Jeffrey , Cheng Catherine , Saito Mayuko , Gilman Robert , Ciobanu Nelly , Crudu Valeriu , Catanzaro Donald , Catanzaro Antonino , Rodwell Timothy , Perera Judy , Chathuranga Teshan , Gunasena Bandu , DeSilva Aruna , Peters Bjoern , Sette Alessandro , Arlehamn Cecilia S
Published: November 2024

The challenge in advancing tuberculosis (TB) diagnostics and vaccines arises from our limited understanding of diverse Mtb antigens triggering distinct T cell responses in spectrum of TB infection. We addressed this with a comprehensive analysis, screening 20,610 Mtb-derived T cell epitopes using IFNγ Fluorospot in active TB (ATB) and BCG-vaccinated individuals, comparing to LTBI. We identified 137 unique epitopes in ATB, 16% recognized by multiple participants, suggesting a broad response, mainly targeting cell wall and cell processes antigens. BCG-vaccinated individuals also showed a heterogeneous response with 85 unique epitopes. There was little overlap between the epitopes found in ATB and BCG-vaccinated individuals, implying distinct antigen recognition in spectrum of TB. From these findings, we developed an ATB-specific peptide pool (ATB116), distinguishing pulmonary ATB with over 60% sensitivity and 80% specificity across diverse locations. We also aligned the identified epitopes with 157 mycobacteria strains to assess their conservation across different Mtb strains and the broader mycobacterium genus. This revealed 17% of the epitopes are 100% conserved across the mycobacterium genus, suggesting that the sensitivity and specificity of the peptide pools can be improved. In summary, our T cell reactivity analysis identified stage-specific Mtb epitopes, offering diagnostic potential, measuring Mtb-specific immune responses, and advancing vaccine development.
FOXO1 inhibitor has the antiviral and anti-inflammatory effects against SARS-CoV-2 infection in in vitro and in vivo models.
Authors: Furukawa Ryutaro , Hara Atsushi , Ouji-Sageshima Noriko , Kitabatake Masahiro , Shichino Shigeyuki , Ueha Satoshi , Matsushima Kouji , Ito Toshihiro
Published: November 2024

COVID-19 treatment aims to inhibit SARS-CoV-2 replication and subsequent hyper-inflammatory responses. Forkhead box O1 (FOXO1) is a transcription factor involved in various physiological processes, including inflammation and immune systems. Here, we investigated the antiviral and anti-inflammatory effects of AS1842856 (AS), a FOXO1 inhibitor, against SARS-CoV-2 infection. We found that AS could suppress viral load and active virus titers in Calu-3, a human airway epithelial cell line, infected with SARS-CoV-2. In addition, the expression of inflammatory-related cytokines was reduced, while that of IL-10, an anti-inflammatory cytokine, was increased by AS. AS also reduced expressions of ACE2 and TMPRSS2, both host cell proteins required for SARS-CoV-2 infection. Moreover, we investigated the effects of AS in animal models. AS administration (50 mg/kg/day, i.p.) into a Syrian hamster model resulted in reduced weight loss, a decrease in viral load in lungs, and histological improvement of pneumonia. We also found AS improved survival rate in mouse COVID-19 models, generated by using mouse-adapted QHmusX strain. Single-cell RNA sequencing using hamster lungs indicated AS suppressed SARS-CoV-2 gene in alveolar epithelial cells and decreased CXCL10+ macrophages, which are related to pathogenesis of severe COVID-19 according to previous studies. Our results suggested AS could attenuate the severity of COVID-19 by modifying immune responses and suppressing SARS-CoV-2 infection.
Extent of immune history shapes the antibody response against the influenza virus in a mouse model
Authors: Lyu Huibin , LYU HUIBIN , Teo Qi Wen , Lee Chang-Chun , Liang Weiwen , Choi Danbi , Mao Kevin , Bruzzone Roberto , Wilson Ian , Wu Nicholas , Mok Chris
Published: November 2024

Antigenic imprinting refers to the effects of prior exposures on subsequent immune responses to antigenically related viral strains and the human immune history influences the magnitude and quality of future antibody responses. However, the impact of age-dependent immune history on the antibody response to the 2009 H1N1 virus remains largely elusive, primarily due to the complexity of human experiences with infection and vaccination. Moreover, how the extent of immune history affects antibody response is unclear. In this study, we investigated the antigenic imprinting effect on hemagglutinin (HA) and neuraminidase (NA) by utilizing a mouse model, sequentially infecting them with pre-2009 seasonal H1N1 viruses and challenging them with 2009 H1N1 virus. We assessed HA- and NA-specific antibodies both before and after subjecting different groups of mice to the 2009 H1N1 viral challenge. Intriguingly, our findings show that the extent of prior infection experience influences the functional antibody response to both HA and NA against antigenically drifted or shifted strains. Furthermore, our additional experiments revealed that the pattern of antigenic imprinting for HA and NA is different across different virus strains. In conclusion, this study underscores how the extent of prior infection experiences can determine the quality and specificity of antibody responses. These findings are vital for developing more effective strategies for vaccination and antiviral treatments.
The Role of Chemokine-Like Receptor-1 in Recruitment and Activation of Myeloid Cells During WNV Encephalitis
Authors: Kwok Keven , Durrant Douglas
Published: November 2024

Recent increases in West Nile Neuroinvasive Disease (WNND) outbreaks in the US underscore the urgent need to understand CNS viral clearance mechanisms. In response to WNV, neurons secrete chemokines (CCL2, CCL5, and CXCL10), facilitating leukocyte entry into the CNS. CNS-infiltrating macrophages amplify this signal, recruiting and activating neuroprotective leukocytes, limiting harmful accumulation, and aiding repair. CMKLR1 promotes CNS inflammatory responses during infectious and autoimmune diseases via inducing macrophage and DC migration following chemerin activation. However, the specific role of chemerin/CMKLR1 in WNND remains unclear. WT and CMKLR1-/- mice were infected with WNV to investigate this. WNV-infected CMKLR1-/- mice exhibited a slight increase in mortality but elevated clinical scores and reduced recovery compared to WT. We hypothesize that CMKLR1 limits WNV pathogenesis by regulating CNS recruitment and activation of inflammatory myeloid cells during WNV encephalitis. Assessing key inflammatory mediators and activation markers in CNS-resident and infiltrating cells in WT and CMKLR1-/- animals will elucidate how CMKLR1 influences the recruitment/activation of these cells, clarifying processes resulting in infection resolution and neural repair.
Unveiling the immune surveillance role of extracellular vesicles
Authors: Kumari Puja , Vasudevan Swathy , Russo Ashley , Wright Skylar , Fraile-Ágreda Víctor , Krajewski Dylan , Jellison Evan , Rubio Ignacio , Bauer Michael , Shimoyama Atsushi , Fukase Koichi , Zhang Yuanpeng , Pachter Joel , Kailasan Vanaja Sivapriya , Rathinam Vijay
Published: November 2024

Intracellular immune surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here, we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma- and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering noncanonical inflammasome activation of GSDMD and pyroptosis. Mechanistically, the interaction between EV lipid bilayer and LPS’ lipid A underlies EV capture of LPS. Furthermore, the intracellular transfer of LPS by EVs is mediated by CD14-dependent receptor-mediated endocytosis. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic pathogen-associated molecular pattern (PAMPs) and intracellular immune surveillance.
O-GlcNAc transferase plays dual antiviral roles by integrating innate immunity and lipid metabolism
Authors: Dong Hong , Liang Chenxi , Yount Jacob , Liu Shan-Lu , Wen Haitao
Published: November 2024

Challenge of host cells with viral pathogens induces robust metabolic reprogramming; however, whether viral component(s) is detected by metabolic enzyme remains completely unknown. Our previous study identified the O-GlcNAc transferase (OGT), an important glucose metabolic enzyme, as a crucial mediator of MAVS-dependent antiviral response. Here, by studying a mouse model with enzyme-inactive OGT, we demonstrate an enzyme-independent function of OGT in limiting influenza A virus (IAV) infection, in addition to its enzyme-dependent effect on MAVS. Biochemical studies reveal a critical antiviral effect of OGT binding with IAV genomic RNA via its N-terminal tetratricopeptide repeats-4 domain. Binding with viral RNA causes a robust translocation of nuclear OGT to cytosolic lipid droplets (LDs) and subsequently antagonizes LD accumulation via destabilizing perilipin 2. This LD-targeting effect of OGT limits IAV replication without altering MAVS signaling. Our findings reveal OGT as a multifaceted metabolic sensor by integrating MAVS-dependent innate immune signaling and lipid metabolism.
B cell-derived IL-10 is an autocrine survival factor and maintains immune homeostasis in the spleen
Authors: Chen Chuxiao , Ramaswami Bala , Mohib Kanishka , Rothstein David
Published: November 2024

IL-10 is a key anti-inflammatory cytokine and IL-10 KO mice develop colitis. Mice with a global IL-10 KO or constitutive B cell-specific IL-10 KO (CD19-Cre.IL-10fl/fl) have otherwise normal B cells. Surprisingly, we found that within 14-21 days, mice with a tamoxifen (Tam) -induced B cell specific deletion of IL-10R (hCD20-ERT2-Cre.IL-10Rfl/fl) had a 50% decrease in splenic B cell number vs. Cre-controls. B cell frequency was unchanged due to a concomitant fall in both T cells and innate cells resulting in a 58% fall in splenocyte number. Plasma cells, CD4+ and CD8+ T cells, and neutrophils were disproportionately decreased, resulting in a 20-40% reduction in frequency. A similar but somewhat less severe loss of B cells/splenocytes was seen in Tam-treated hCD20-ERT2-Cre.IL-10fl/fl mice. Adoptive transfer competition studies in immunized µMT hosts treated with Tam revealed a selective loss of hCD20-ERT2-Cre.IL-10fl/fl vs. CD19-Cre.IL-10fl/fl B cells. Furthermore, by day 21, acute loss of B cell IL-10 or IL-10R resulted in ~20% weight loss and colon length, consistent with colitis (pathology pending). In conclusion, IL-10 is an autocrine B cell survival factor and acute loss of B cell IL-10 or IL-10R is associated with a marked contraction of B cells and associated collapse in T cells and innate cells in the spleen, resulting in immune dysregulation and colitis. This requirement for B cell IL-10 has not been previously recognized because it is compensated for developmentally.
Loss of UCP2 in CD4+ T cell promotes Th1/2 Pathogenic Phenotype
Authors: Choi Seungho , Chakraborty Paramita , Hedley Zachariah , Oberholtzer Nathaniel , Mills Stephanie , Mehrotra Shihar
Published: November 2024

Uncoupling protein 2 (UCP2) is an anion carrier protein located in the mitochondrial inner membrane. Mitochondria are the major sites for energy metabolism which is tightly linked with immune cell fate. In the present study, we have investigated UCP2’s function in T cells against murine melanoma model and the experimental autoimmune encephalomyelitis (EAE) model. Loss of UCP2 in CD4+ T cells showed a significant rise in the Th2/Th1 ratio. These cells exhibited an improved Th2 phenotype with increased production of nitric oxide (NO), and Inducible nitric oxide synthase (iNOS). Therefore, CD4+ T cells were treated with NOC-18, a source of NO, or L-NMMA, inhibitor for NOS to demonstrate NO-mediated Th2 polarization. Furthermore, Treatment of Ucp2 siRNA or, Genipin (inhibitor for UCP2) mimicked the effect of UCP2-/- CD4+ T cell with more cytotoxic markers and Th2/Th1 double positive cells. Transcriptomic and metabolomic analysis revealed the lower response of calcium influx by TCR activation and increased NLRF3 activation by uric acid in UCP2 deficient CD4+ T cells. Adoptive transfer of Genipin-treated Tyrp CD4+ cells exhibited superior tumor control as compared to Th1 and Th2, according to the findings. However, in UCP2-/- mice, the severity of EAE worsened, and heterogenic Th2 cells were infiltrated into the spinal cord. Our findings imply that the loss of UCP2 in CD4 increases Th2 polarization with a cytotoxic phenotype and may be relevant to various illnesses.
The TGF-β -- Eomes Axis Maintains Stemness in TCF-1+ CD8+ T Cells by Restricting Tissue Egress From Secondary Lymphoid Tissues.
Authors: Im John , Ma Chaoyu , Srinivasan Saranya , Zhang Nu
Published: November 2024

T-cell exhaustion is characterized by dysfunctional cytotoxic T lymphocytes (CTLs) that express inhibitory and exhaustion markers, such as programmed cell death 1 (PD-1) and CD101, with a reduced capacity to clear antigens. Exhausted CTLs arise from ‘stem-like’ precursors that express T cell factor-1 (TCF-1) and have a unique gene signature similar to hematopoietic stem cells. These self-renewing precursors reside in secondary lymphoid tissues, requiring Transforming growth factor beta (TGF-β) to maintain their residency and mitigate their effector differentiation, partially via controlling the expression of a4 integrins. Downstream of TGF-β, expression of the transcription factor Eomes has been associated with greater levels of exhaustion and is highly upregulated in terminally exhausted cells. Using a chronic lymphocytic choriomeningitis virus (LCMV) infection model in wild-type B6 mice, we found that TGF-β inhibited Eomes expression in TCF-1+ stem-like CD8 T cells. And that deletion of Eomes largely corrected the defects in TCF-1+ stem-like T cells lacking TGF-β receptor signaling. Our data indicate that the TGF-β -- Eomes axis helps to maintain stem-like TCF1+ precursor cells within lymphoid tissues, and that this pathway could be targeted to reinvigorate exhausted CD8+ T cells.
Role of MNK/eIF4E pathway in CD4+ Th2 cytokine-induced lung fibrosis in chronic allergic asthma
Authors: Fattahi Fatemeh , Vallance Laura , Walker Natalie , Holden Julia , Huang Steven , Atasoy Ulus
Published: November 2024

Despite therapeutic advances in allergic asthma, the persistent challenge of airway remodeling still exists, necessitating further exploration. We aimed to investigate the influence of CD4+ Th2 cytokines on fibrosis associated with airway remodeling, with a focus on mesenchymal cell (MC) transformation.

Using both human and mouse lung MCs, we investigated the pivotal role of MNK/eIF4E pathway in regulating fibrous protein expression driven by CD4+ Th2 cytokines. To assess this, we employed western blot and flow cytometry studies, while Luminex assays measured cytokine and chemokine secretion from activated lung MCs responding to Th2 cytokines.

Treatment with Th2 cytokines significantly increased ATX expression in lung MCs, a response mitigated by the specific MNK1/2 inhibitor (eFT-508). Furthermore, this inhibitor not only abolished the phosphorylation of eIF4E but also reduced collagen levels within activated MCs induced by Th2 cytokines. Luminex assay results demonstrated a significant decrease in IL-6, CCL-2, and GM-CSF in the activated MCs upon inhibiting the MNK1/2 pathway. Additionally, eFT-508 exhibited a marked reduction in lymphocyte migration, utilizing the Boyden chamber transwell assay.

Our study reveals that targeting the MNK/eIF4E pathway holds promise in mitigating airway remodeling associated with MCs fibrosis and provides valuable molecular insights for future therapeutic interventions related to fibrosis in the airway remodeling of allergic asthma.
Harnessing temperature-dependent STING signaling to reprogram regulatory T cells and enhance effector T cell immunity.
Authors: Smith Rachael , Heintzman Darren , Fisher Emilie , Voss Kelsey , Ye Xiang , Toudji Yasmine , Chi Channing , Schulman Jacob , Rathmell Jeffrey , Wilson John
Published: November 2024

The cGAS-STING pathway plays a critical role in immune surveillance through its capacity to sense aberrantly localized DNA in the cytosol caused by infection, cancer cell death, or cell stress-induced self-DNA leakage. STING pathway activation ultimately results in the production of Type I Interferons (IFN-I) and other inflammatory cytokines that stimulate T cell immunity. However, the T cell-intrinsic effects of STING agonists have been underexplored, especially in regulatory T cells (Tregs), which have an opposing but crucial role in suppressing excessive inflammation. Unexpectedly, we have found that STING activation repolarizes Tregs to IFN-I producing Th1/Th9-like cells with enhanced antitumor function and reduced oxidative metabolism and suppressive capacity. Inflammation can also coincide with heat (i.e. fever-range temperature, FRT, 39°C), which can cause nuclear or mitochondrial self-DNA leakage, yet the effects of FRT on STING in Tregs are unknown. We were surprised to find that FRT strongly potentiates the effects of STING activation in Tregs and that FRT appears to prime cGAS/STING signaling. Together, our initial data suggest a novel axis linking FRT to STING signaling in inhibiting Treg immunosuppressive function and in promoting Treg transdifferentiation to inflammatory effector-like T cells. These exciting findings motivate further investigation to dissect the mechanisms intertwining STING activation and FRT and their effect on Treg function and metabolism.
BLIMP-1 and CEACAM1 cooperate to regulate human regulatory T cell proliferation and function
Authors: Ding Ying , Yu Aixin , Vujanac Milos , Copsel Sabrina , Moro Alejandro , Nivelo Luis , Tchitchek Nicolas , Rosenzwajg Michelle , Villarino Alejandro , Levy Robert , Koatzmann David , Malek Thomas
Published: November 2024

BLIMP-1, encoded by the PRDM1 gene, is a major transcriptional hub distinct from Foxp3 in human Tregs, but its mechanisms of action have not been fully determined. Here, using CRISPR/Cas9-mediated disruption of PRDM1, we show that BLIMP-1 limits Treg proliferation while supports Treg functional activity. PRDM1KO human Tregs are less suppressive and exhibit increased expansion in a humanized mouse model of xenogeneic GVHD. BLIMP-1 restrains Treg proliferation by lowering IL-2R signaling through downregulation of CD25 and by inhibiting responsiveness to TCR/CD28 signaling and mTOR activation through upregulation of CEACAM1, an immune checkpoint. Expression of CEACAM1 is induced by IL-2 in human, but not mouse, Tregs. Prolonged IL-2R signaling leads to optimal BLIMP-1 expression and chromatin opening of human CEACAM1, leading to high cell surface CEACAM1 through a 3’ regulatory region containing motifs for binding PRDM1 and STATs. Correspondingly, CEACAM1 is highly induced on Tregs from autoimmune patients undergoing low-dose IL-2 therapy, and these Tregs showed reduced proliferation. In vivo studies using xenogeneic GVHD mouse model show that CEACAM1KO human Tregs have higher suppressive activities, indicating a potential role of CEACAM1 in Treg function. Collectively, our findings uncover a novel IL-2-dependent regulatory loop whereby BLIMP-1 and CEACAM1 act to limit human Treg proliferation and contribute to Treg suppressive function.
Evaluation of a human peripheral blood NK humanized hIL-15 mouse model lacking murine Fc gamma receptors
Authors: Dubé Philip , Smith Nicholas , Buczek Monika , MacBride Megan , Andino Esther , Sack Emily , Freer Debra , Vedder Michelle , Bott Kathleen , Baskin Louise , Richardson Janell
Published: November 2024

Fc gamma receptors (FcγRs) on residual murine immune cells can interact with human IgG-based therapeutics and confound preclinical results. This is critical for therapeutics with human NK cell mediated antibody-dependent cellular cytotoxicity (ADCC) as their primary mode of action. We compared humanization, kinetics, and persistence of NK cell reconstitution in human IL-15-expressing mice with or without FcγRs using intraperitoneal (IP) or intravenous (IV) administration of cells from multiple donors.

Methods: Negatively selected human NK cells were engrafted into hIL-15 NOG or FcResolv™ hIL-15 NOG (FcγR knockout) with identical protocols. Human reconstitution, immune profile and persistence was evaluated via serial blood sampling and terminal collections for blood, spleen, and bone marrow.

Results: Human NK cell reconstitution and persistence was equivalent between strains. There was a significant difference in the overall chimerism in IV vs. IP administration (IV > IP). There was no significant difference in % body weight, body condition scores, morbidity or mortality across study duration. NK cell persistence and kinetics were consistent between strains, administration, and donors, with a peak at 4-5 WPE and persistence to 12+ WPE in the blood. The dominant circulating NK phenotype was CD56+CD16+.

Conclusions: huNK-engrafted hIL-15 NOG and FcResolv hIL-15 NOG mice provide a 12+ week study window, with equivalent reconstitution, immune profiling and kinetics.
Evaluation of an IgG therapeutic efficacy in a humanized immune system mouse model lacking murine Fc gamma receptors
Authors: Baskin Louise , Smith Nicholas , Buczek Monika , MacBride Megan , Andino Esther , Sack Emily , Freer Debra , Vedder Michelle , Bott Kathleen , Richardson Janelle
Published: November 2024

Fc gamma receptors (FcγRs) on residual murine immune cells in humanized immune system (HIS) mice can interact with human IgG-based therapeutics and confound preclinical results. We assessed impact of murine FcγRs on anti-PD1 efficacy in HIS mice engrafted with lung adenocarcinoma cells treated with pembrolizumab or vehicle. We also present humanization results for the newly generated FcγR knockout NOG-EXL. Methods: HIS NOG (huNOG) or HIS FcγR knockout NOG mice (FcResolv™ huNOG) were made using identical protocols with CD34+ cells (3 shared donors). Reconstitution was evaluated in naïve animals and HCC827 cells were inoculated in remaining animals. From D7, mice were dosed twice weekly for 4 wks then euthanized for blood, spleen, and tumor analysis. Results: Humanization was equivalent between strains. Pembro treatment showed significant tumor growth inhibition in 1 donor in FcResolv huNOG, but not in donor-matched huNOG. Human TILs in pembro-treated mice were significantly different between the strains for all donors, with more CD8+ T cells and fewer TAMs in FcResolv huNOG compared to vehicle-treated mice, and no significant differences in huNOG. Murine TIL analysis showed differences in murine macrophage populations between strains. Conclusions: Anti-PD1-treated FcResolv huNOG mice show expected pharmacodynamic changes and donor-dependent efficacy, whereas pembro-treated huNOG mice showed neither, demonstrating the impact of murine FcγRs on antibody IgG-based therapeutics.
Assessment of SARS-CoV-2 Spike and NCAP antigen-specific memory B cell frequency and Ig class/subclass usage following natural infection and/or COVID-19 mRNA vaccination
Authors: Kirchenbaum Greg , Becza Noemi , Yao Lingling , Liu Zhigang , Chepke Jack , Lehman Paul
Published: November 2024

An adaptive humoral response can encompass multiple Ig classes/subclasses, each capable of contributing to host defense through different effector functions. Which Ig class/subclass are elicited in response to an antigen encounter is the consequence of context, such as site of antigen contact, presence of immunostimulatory motifs and the cytokine secretion signature of helper T cells. Moreover, the number of antigen encounters and duration of the ensuing immune responses can further diversify the memory B cell compartment’s epitope specificity, affinity and Ig class/subclass usage. Beyond characterization of Ig usage in blood, whether differences also exist in the Bmem compartment following multiple SARS-CoV-2 infections and/or COVID-19 vaccinations is still not well understood. Leveraging multiplexed B cell ImmunoSpot assays, the scope of this work is to characterize SARS-CoV-2 S- and N-specific Bmem and their respective Ig class/subclass usage in the context of complex immune exposure histories. In agreement with recent observations detailing the antibody response elicited following COVID-19 mRNA vaccination and/or breakthrough infection, alterations in the affinity, breadth and IgG subclass usage of S-specific Bmem was also observed. Collectively, our findings demonstrate the feasibility of performing in-depth characterization of Bmem in a streamlined pipeline that requires minimal cell material and serves to motivate usage of the multiplexed FluoroSpot platform.
Label-free morphometric characterization of T cells for cell and gene therapy research and development.
Authors: Teranishi Kazuki , Wagatsuma Keisuke , Uematsu Mika , Tsubouchi Asako , An Yuri , Tamoto Ryo , Yanagihashi Yuichi , Kawamura Yoko , Murata Yuri , Akai Satoru , Komoriya Kaoru , Schneider Greg , Nomaru Hiroko , Ota Sadao
Published: November 2024

Identification, characterization, and minimally invasive isolation of T cells is critical for developing cellular therapies. Here we used VisionSort, an artificial intelligence (AI)-driven cellular analysis and sorting platform, to characterize and isolate human T cell subsets with therapeutically relevant phenotypes, label-free. By capturing single-cell digital phenotypes, we characterized human T-cells and generated ‘ground truth’ functional profiles for 1) glycolysis level, 2) exhaustion state 3) activation or resting profiles and 4) viability. A set of machine-learning derived classifiers was generated to identify these phenotypic classes label-free. The classifiers showed area under the curve (AUC) performance ranges for detecting specific, phenotypically defined T-cell populations between 0.923 and 0.995. Using a combination of supervised and unsupervised machine learning approaches, we also show identification of follicular helper T (Tfh) cells from other CD4+ T cells based on morphology alone. When we analyzed the morphological profiles of T cell subsets by unsupervised machine learning using uniform maniform projection and approximation (UMAP), we found that both gamma delta (γδ) T cells were distinct from alpha beta (αβ) T cells and CD8+ T cells were distinct from CD4+ T cells. Here we report on the use of AI-based cytometry to characterize and isolate phenotypically defined T cell subsets label-free, with applications in basic life science and cell therapy R&D.
Advancements in Research Grade Biosimilars Enable the Study of Therapeutic Antibodies
Authors: Cantor Joseph , Gao Guo-Jian , Rigaud Stephanie , Wei Ai-Li , Bazirgan Omar , Verma Shilpi
Published: November 2024

Biosimilars for therapeutic antibodies contain identical variable sequences to FDA-approved

antibodies. Recombinant BD research-grade biosimilars can be used to model several important aspects of major antibody therapies in use today. Our biosimilars with the huIgG1 (N297A) Fc mutation exhibit greatly reduced background binding and clarify the identification/study of therapeutic antibody target cell subsets. These flow cytometry reagents are available with many fluorochrome options to facilitate deeper phenotyping using large panels. Secreted targets (e.g., TNF-α) of therapeutic antibodies can either be bound in solution or in a membrane/cell-associated state; biosimilars may also be useful in studying this context-dependent target engagement. Alternatively, wildtype huIgG1 Fc biosimilars in no-azide/endotoxin-free (NA/LE) formats permit comparative mechanism-of-action (MOA) studies. Finally, biosimilars can be used in pre-blocking experiments to identify non-competing and competing clones used to study membrane target regulation, or gate on cell subsets defined by these targets in models of pre-exposure to therapeutic antibodies. In sum, our research-grade biosimilar antibodies can empower multiple avenues of research on therapeutic antibodies, which in turn are having major clinical impacts across medicine.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD is a trademark of Becton, Dickinson and Company. © 2023 BD. All rights reserved. NPM-2940
Ranked abundance of 115 proteins following simultaneous measurement by multiplex immunoassay in serum, plasma, and cell culture supernatant samples.
Authors: Droll Deborah , Saporita Anthony , Gilliam Brooke , Grana Paula , Qiang Xiao
Published: November 2024

MILLIPLEX™ PLEXpedition, a customizable multiplex immunoassay kit, was developed to simultaneously screen up to 115 targets including cytokines, chemokines, growth factors, matrix metalloproteinases, and biomarkers of bone health, metabolism, and cardiovascular disease. Two Luminex™ instruments, the xMAP™ INTELLIFLEX™ and the FLEXMAP 3D™, can accommodate all 115 bead regions with comparable sensitivity and sample correlation observed between them. The analyte selection incorporates many biomarkers critical to the field of immunology, using assays developed for two MILLIPLEX™ 48-plex human cytokine, chemokine, and growth factor kits (Cat. No. HCYTA-60K and Cat. No. HCYTB-60K). Sample correlation in serum, plasma, and cell culture supernatants demonstrated that this discovery biomarker panel with a matrix-free format can be used to transition seamlessly to our highly qualified panels, MILLIPLEX™ Human Cytokine Panel A and B, which have optimized serum matrix and lot-to-lot verification for absolute quantification of protein concentration. To probe the relative abundance of each analyte in typical sample matrices, the 115-plex assay was run with a set of 8 cell culture supernatants and 24 healthy and disease serum and plasma samples. Additionally, analytes were ranked by their response to stimulation by LPS or ConA in PBMCs. Our results highlight the utility of the novel PLEXpedition screening kit for evaluating relative abundance in biological samples.
Deciphering the metabolic regulation of immunosuppressive CD11b+Gr1+ myeloid cells in epithelial ovarian cancer.
Authors: Udumula Mary Priyanka , Singh Harshit , Rashid Faraz , Hijaz Miriana , Gogoi Radhika , Philip Philip , Munkarah Adnan , Giri Shailendra , Rattan Ramandeep
Published: November 2024

Immunosuppressive CD11b+Gr1+ myeloid cells are key drivers of immunosuppression and decrease immunotherapy efficacy in epithelial ovarian cancer (EOC). We showed that EOC enhances CD11b+Gr1+ myeloid cell immunosuppression capacity by upregulating αKGDC (ketoglutarate dehydrogenase complex) expression, resulting in increased TCA cycle- glutamine anaplerosis causing enhanced oxidative phosphorylation (OXPHOS), increased immunosuppression markers and T cell suppression. Inhibition of TCA-glutamine anaplerosis by CPI-613 decreased OXPHOS and CD11b+Gr1+ immunosuppressive ability resulting in restored anti-tumor T cell response and inhibition of EOC. Targeted metabolomics of CPI-613 treated CD11b+Gr1+ cells showed a profound decrease in TCA metabolite α-ketoglutarate (αKG). Among all TCA metabolites, only αKG supplementation reversed the OXPHOS inhibition, T cell immunosuppression by CPI-613 and increased immunosuppressive markers in ex-vivo conditions. In vivo treatment of αKG alone replicated the ex-vivo effect and enhanced CD11b+Gr1+ myeloid cell OXPHOS and immunosuppressive ability but did not affect survival. In combination with CPI-613, αKG was able to reverse the inhibitory effect of CPI-613 on immunosuppression, OXPHOS and decreased survival. Our study shows that αKG generated by TCA- glutamine anaplerosis is crucial for supporting an immunosuppressive phenotype of CD11b+Gr1+ myeloid cells in EOC, and targeting αKG may enhance anti-tumor immune responses.
Single-cell transcriptomics of colorectal cancer metastases identifies enrichment for a therapeutically targetable population of TREM2+ macrophages in peritoneal metastases.
Authors: Cottam Matthew , Mirza Muhammad , Arner Emily , Hatem Zaid , Shameer Muhammad , Beckermann Kathryn , Idrees Kamran
Published: November 2024

Colorectal cancer (CRC) is recognized as the third most common cancer and the second leading cause of cancer-related mortality globally. The primary driver behind this mortality is metastatic disease, which significantly worsens the prognosis for CRC patients. Over the past decade, there has been a modest improvement of only 2% in the 5-year survival rate for metastatic CRC (mCRC). To improve our understanding of the cellular composition in mCRC tumors, we performed single-cell RNAseq (scRNAseq) on matched primary colorectal tumors, metastases to the lung, liver, and peritoneum, and malignant ascites from 14 patients. Tumor-associated macrophages (TAMs) were found to be one of the most abundant populations across all samples and TAMs from metastases had increased expression of M2-like alternative activation markers relative to primary tumors, suggesting that TAMs in mCRC are immunosuppressive. Gene co-expression analysis identified a signature of lipid handling containing genes linked to lipid transport, such as APOE, FABP5, PLTP, and CD9, and genes coding for the scavenger receptors CD36, MSR1, MARCO, and TREM2. Consistent with transcriptomics, the majority of CD11b+CD68+ TAMs from resected liver and peritoneal metastases had high TREM2 expression by flow cytometry. In a mouse model of peritoneal mCRC, treatment with anti-TREM2 decreased CD11b+F4/80+ TAMs and increased CD8+ effector T cells, suggesting that TREM2 may be an effective target for immunotherapy in mCRC.
Expanding the split-pool combinatorial barcoding toolkit to characterize the effects of aging in mouse TCR diversity.
Authors: Papalexi Efthymia , Hariadi Bryan , Suyama Alexa , Curca Crina , Roco Charles , Rosenberg Alexander
Published: November 2024

We recently released a flexible and high throughput single-cell sequencing method that leverages split-pool combinatorial barcoding to simultaneously profile the whole transcriptome and T cell receptor (TCR) sequences of up to 1 million human T cells from up to 96 samples. This technology is able to recover reproducibly paired TCR sequences in the majority of assayed T cells across different sample types (CAR-Ts, culture activated T cells, etc).

Here, we report on the extension of our method to be able to recover the immune repertoire and gene expression profiles from mouse T cells. We use our assay to characterize TCR diversity changes during the mouse lifecycle. Specifically, we profile hundreds of thousands of mouse spleen-resident T cells at different stages of life. We perform transcriptome-based clustering and demonstrate that this approach identifies all major T cell populations (CD4+, CD8+, Tregs, etc.) while efficiently detecting paired TCR sequences. Furthermore, consistent with previous findings, we show differences in clonotype diversity that correlate with age.

Overall, we present an extension of our TCR technology that allows for the characterization of mouse TCR sequences and showcases its utility in studying the effects of aging in TCR repertoire diversity. We envision our method will help researchers gain additional insights in mouse models of autoimmune and infectious diseases, as well as cancer.
Blocking of NLRP3 inflammasome activity alleviates iron-induced adverse effects in murine models of colitis
Authors: Saha Piu , Yeoh Beng San , Vijay-Kumar Matam
Published: November 2024

One serious extra-intestinal manifestation of Inflammatory Bowel Disease (IBD) is iron deficiency anemia (IDA). The first choice for IDA therapy is oral iron supplementation; yet the rhetoric that IDA can be ‘treated’ with iron is far from accurate, given the high incidence of severe side effects. We herein investigated the extent to which NLRP3 (NOD-like receptor 3) inflammasome could influence iron-induced adverse effects in IBD. Wild-type (WT) and NLRP3-deficient (Nlrp3KO) mice were treated with dextran sodium sulfate (DSS) for 7 days and then oral FeSO4 (Fe2+) was administered for 3 days. Fe2+ augmented colonic inflammation in WT, but not in Nlrp3KO mice. Histological analysis revealed that iron+DSS treated WT colitic mice displayed more inflammation than Nlrp3KO mice. Interestingly, oral administration of β-hydroxybutyrate (BHB, inhibitor of NLRP3) significantly reduced the Fe2+ induced inflammation in WT mice. Next, to evaluate the role of endogenous iron in colitis, we used homeostatic iron regulator protein (HFE)-deficient mice (HfeKO), which were noted to be highly susceptible to IL-10R neutralization-induced colitis. Intriguingly, deficiency of NLRP3 in HfeKO mice (Hfe-Nlrp3-DKO) significantly protected them from IL-10R neutralization-induced colitis. In conclusion, our study demonstrates that excess iron could exacerbate inflammation during IBD and that BHB could be used as a therapeutic to mitigate iron-induced inflammation in IBD.
The Role of Invariant Natural Killer T (iNKT) Cells in Early Responses to Influenza A Virus (IAV)
Authors: Luczak Justyna , Milek Oliwia , Carter Hannah , Alberola-Ila Jose
Published: November 2024

iNKT cells are a CD1d-restricted innate-like lineage of ⍺ßT cells that are prevalent in mucosal tissues, where they influence early immune responses. iNKT cells differentiate in the thymus into—iNKT1, iNKT2, and iNKT17 analogous to Th1, Th2, and Th17 cells. We have shown in ET-2;CD4cre mice, that sustained E protein activity results in changes in iNKT subset composition, with a shift from iNKT1s towards iNKT2/NKT17 cells and other subsets. To understand the physiological relevance of these changes, we tested the response of ET2 mice to IAV. ET2 mice showed less weight loss, reduction in lung-infected areas, decreased neutrophil infiltration, lower activation markers in inflammatory monocytes, reduced levels of CCL2 and interferon-responsive genes in ET2. We observed increased levels of IFNλ in infected ET2 mice vs. elevated IFNα in WT mice 24 hours post-infection. scRNA-seq analysis of sorted iNKT from WT and ET2 mice showed that ET2 profiles are closer to conventional naïve T cells, and that, after infection, their response is marked by overexpression of Ccr7, -Lselectin, IL1R1. WT response is predominantly type 1. Gene Ontology pathway analysis of infected WT show enrichment of cytokine production and immune cell activation pathways, while pathways enriched in infected ET-2 are associated with adhesion and repair. These experiments suggest that early type 1 responses driven by NKT cells during IAV infection can be deleterious, intensifying inflammation, and neutrophilia.
A clonally restricted subset of exhausted CD8+ T cells maintains the capacity to form memory
Authors: Raposo Colin , Hiam-Gálvez Kamir , Satpathy Ansu
Published: November 2024

Chronic stimulation of CD8+ T cells leads them to enter a state of restrained functionality known as exhaustion. Removal of chronic stimulation leads exhausted T cells to take on a memory phenotype that retains an epigenetic imprint of previous chronic stimulation. However, the fate of individual exhausted T cell clones upon antigen removal remains unclear. Our group has recently shown that CD8+ T cell responses to chronic infection are comprised of many T cell clones with unique T cell receptors that each take on unique distributions of fates. To study the impact of antigen clearance on individual exhausted T cell clones, we analyzed T cell repertoires of mice infected with chronic lymphocytic choriomeningitis virus (LCMV) during infection and after peripheral viral clearance. We show that individual LCMV-specific clones have differential capacity to persist past viral clearance and take on clonally and phenotypically distinct cell states. These states include exhausted phenotypes and central memory-like cells that are characterized by minimal epigenetic imprinting of chronic stimulation. Furthermore, chronic memory cells have altered ability to differentiate upon rechallenge, which enables their preferential survival compared to acute memory cells when rechallenged with chronic LCMV. Together, these data illustrate that a subset of clones within the exhausted T cell pool is primed to form memory and protect the host from a secondary infection.
The role of dendritic cell subsets in Invasive Pulmonary Aspergillosis
Authors: Dhruva Pari
Published: November 2024

Aspergillus fumigatus is a ubiquitous mold that causes many lung diseases, the most severe of which is invasive pulmonary aspergillosis (IPA). Innate immunity is paramount for controlling IPA. Here, we examined the role of dendritic cells (DCs). Global depletion of DCs using CD11c-DTR mice results in impaired lung clearance of A. fumigatus, suggesting that one or more of these DC subsets plays a protective role. Unexpectedly however, we found that mice deficient in BATF3 (Batf3-/-), which lack type 1 conventional DCs (cDC1s), cleared A. fumigatus more efficiently than WT control mice at early time points (12 h and 24 h), suggesting that cDC1s play an inhibitory or non-protective role during A. fumigatus lung defense. Mechanistically, Batf3-/- displayed a hyper-type 17 response, which we have previously shown to be critical for elimination of A. fumigatus from the lung. This correlated with differential IL-33 (negative for type 17, lower in Batf3-/-) vs. PGE2 (positive for type 17, higher in Batf3-/-) production, an axis we have reported that regulates type 17 responses. Overall, our data indicates that both protective and non-protective DC subsets are present in the lung during IPA.
M2-polarized macrophages prevent preterm birth and improve neonatal survival and immunity
Authors: Garcia-Flores Valeria , Liu Zhenjie , Romero Roberto , Xu Yi , Miller Derek , Galaz Jose , Winters Andrew , Farias-Jofre Marcelo , Theis Kevin , Gomez-Lopez Nardhy
Published: November 2024

Preterm birth (PTB), commonly preceded by preterm labor, is the leading cause of neonatal morbidity and mortality worldwide. Most cases of preterm labor are associated with sterile intra-amniotic inflammation (SIAI), an inflammatory condition without detectable microorganisms. To date, no successful strategies to treat SIAI have been developed. Herein, we present mechanistic proof that treatment with M2-polarized macrophages (M2 MΦ) can effectively prevent PTB [(HMGB1, n = 28 vs. HMGB1+M2 MΦ, n = 29) (p<0.05)] and neonatal mortality [(HMGB1, n = 20 litters vs. HMGB1+M2 MΦ, n = 14 litters) (p<0.001)] induced by the ultrasound-guided intra-amniotic injection of the alarmin HMGB1 in mice. M2 MΦ halt the premature pathway of labor by infiltrating maternal and fetal compartments, where they inhibit NLRP3 inflammasome activation triggered by HMGB1. Furthermore, M2 MΦ dampen the HMGB1-induced inflammatory response in the amniotic cavity and fetal lung. Notably, neonates exposed to HMGB1 in utero display a reduced capacity to clear bacterial infection and gut microbiome dysbiosis, which are restored by M2 MΦ treatment [(HMGB1, n = 10 vs. HMGB1+ M2 MΦ, n = 10) (p<0.001 and p<0.01, respectively)]. Our findings provide cogent evidence that M2 MΦ can serve as a cellular strategy to mitigate PTB and decrease neonatal mortality.
T cell-specific P2RX7 favors lung parenchymal CD4+ T cell accumulation in response to severe lung infections
Authors: Santiago-Carvalho Igor , Almeida-Santos Gislane , De Gois Macedo Bruna , Barbosa-Bomfim Caio Cesar , Moreira Almeida Fabricio , Cione Marcos Vinicius , Lassounskaia Elena , D'Imperio-Lima Maria Regina , Borges da Silva Henrique
Published: November 2024

In the lung there is a fine line between effective or harmful immune responses. A dysregulated lung inflammatory response can cause severe tissue damage and compromise respiratory physiology. CD4 T cells are key components of the immune response during lung infections and can mediate protection against tuberculosis (TB) or influenza. However, CD4 T cells can also promote lung pathology during these infections, making it unclear how these cells control such discrepant effects. Using mouse models of hypervirulent TB and influenza, we observe that exaggerated accumulation of parenchymal CD4 T cells promotes lung damage. Through adoptive transfer models for CD4-KO mice and antibody-mediated CD4 T cell depletion, we reported that low numbers of lung CD4+ T cells, in contrast, are sufficient to protect against hypervirulent TB. The lung damage caused by TB and influenza leads to increased levels of extracellular ATP (eATP). In both situations, lung CD4 T cell accumulation is mediated by CD4 T cell-specific expression of the eATP receptor P2RX7. P2RX7 upregulation in lung CD4 T cells promotes expression of the chemokine receptor CXCR3, favoring parenchymal CD4 T cell accumulation. Our data were validated using CD4 T cells from mycobacterial-infected patients who have or not polymorphism that confers P2RX7 loss of function. Our findings suggest that direct sensing of lung eATP by CD4 T cells is critical to induce tissue CD4 T cell accumulation and pathology during lung infections.
PD-1 endocytosis unleashes the cytolytic potential of check-point blockade in tumor immunity
Authors: Oroya Andres , Ben Saad Elham , Bachais Meriem , Rudd Christopher
Published: November 2024

PD-1 immune checkpoint blockade (ICB) has revolutionized cancer treatment. Present models indicate that anti-PD-1 operates by preventing PD-1 from binding to its ligand PD-L1. Surprisingly, ICB-mediated internalization of PD-1 and its importance have not been fully explored. Here, we demonstrate that PD-1 internalization is associated with increased cytolytic activity during cancer immunotherapy. Anti-hPD-1 and -mPD-1 antibodies induced internalization of PD-1 generating a PD-1int subset, resistant to further internalization. Moreover, anti-PD-1 downregulation was more effective in CD8+ T cells than CD4+ T cells. ICB also induced PD-1 degradation in a proteosome-dependent manner. Interestingly, Nivolumab outperformed Pembrolizumab with both antibodies exhibiting distinct effects on CD8+ effector and effector memory T-cells. Importantly, PD-1 downregulation was dependent on receptor crosslinking by bivalent but not monovalent antibodies. Consistent with this, B16-PD-L1+ tumor-bearing mice treated with bivalent antibodies showed better anti-tumor response relative to monovalent antibodies. While mono and bivalent blockade increased granzyme B expression, only bivalent antibody induced perforin in CD8+ T-cells. Our findings unveil a mechanism beyond steric blockade and underscore the significance of PD-1 endocytosis in optimizing checkpoint blockade. Targeting PD-1 internalization holds promise for enhancing anti-tumor immunity and improving the efficacy of PD-1 ICB.