bluuebunny/crossref_metadata_2025 huggingface dataset:
The splicing factor SRSF6 controls Mycobacterium tuberculosis replication through its role in maintaining mitochondrial homeostasis
Authors: Wagner Allison , Weindel Chi , West Kelsi , Watson Robert , Patrick Kristin
Published: January 2023

Macrophages are the primary host cell that supports the survival and replication of the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). We are interested in how macrophage proteins are functionalized during Mtb infection. Global proteomics analysis of Mtb-infected primary macrophages found that many RNA binding proteins of the Serine/Arginine rich splicing family (SRSF) can be differentially phosphorylated, suggesting that these factors regulate splicing decisions during Mtb infection. Knockdown of one such SRSF protein, called SR6, results in basal upregulation of interferon stimulated genes (ISGs) in macrophages. We hypothesized that this phenotype was driven by mitochondrial damage that leaks mitochondria DNA into the cytosol and activates cGAS. Consistent with this idea, we measured increased mitochondrial stress and increased cell death in SR6 KD macrophages. Transcriptomics analysis of these cells revealed upregulation of an alternatively spliced (AS) isoform of the apoptotic factor BAX, which contributes to mitochondrial dysfunction, mtDNA release, and basal ISG expression. While BAX isoforms are known to regulate cell death, this is the first report of BAX AS regulating the type I IFN response. Lastly, we found that SR6 KD macrophages are permissive to Mtb replication, suggesting that controlling cell death at the level of BAX isoform abundance is an important but unappreciated component of the macrophage antibacterial repertoire. Together, our data support a model whereby SR6-dependent AS of Bax triggers mitochondria stress causing mtDNA leakage and type I IFN expression, metabolic dysfunction that primes cells to undergo cell death, and increased susceptibility to Mtb infection.
Defects in macrophage galactose-type lectin (MGL) signaling and pathogenesis of Mycobacterium tuberculosis and Human Immunodeficiency virus co-infection
Authors: Browning Sarah , Naqvi Kubra F , Fan Xiuzhen , Bharaj Preeti , Lisinicchia Joshua , Paez Reina N , Chauhan Sadhana , Huante Matthew , Martinez-Martinez Yazmin Berenice , Endsley Mark , Gelman Benjamin B , Endsley Janice
Published: January 2023

Mycobacterium tuberculosis (Mtb) and Human Immunodeficiency virus (HIV) are important causes of death in infected patients worldwide, and act synergistically to worsen disease in those with co-infection. C-type lectin receptors (CLRs) are an important component of innate immunity that can recognize and respond to pathogen-associated signals. The macrophage galactose-type lectin (MGL, CLEC10) has been described as an immunomodulatory CLR associated with M2 macrophages and regulation of inflammation following infection. Our lab previously identified an important antibacterial role of murine MGL-1 in host immunity to Mtb. More recently we observed that human peripheral blood mononuclear cell-derived macrophages, and THP-1 cells, upregulated MGL following in vitro exposure to Mtb, Mycobacterium bovis BCG, and TLR2 agonists. Silencing of MGL in primary human macrophages permitted greater intracellular Mtb replication. Interestingly, we observed that MGL is suppressed by HIV in differential transcriptome analysis of lung from HIV-infected humanized mice, in lung and spleen of autopsy specimens from HIV-infected decedents, and upon in vitro infection of human macrophages. Using a recombinant human MGL/Fc chimeric molecule we observed that Mtb, and not HIV, directly bound MGL. MGL surface expression was reduced in lung of mice with chronic HIV infection compared to those with Mtb, and variable in co-infected animals. These results indicate that human MGL is an important CLR for pathogen recognition and initiation of protective immunity by host macrophages to Mtb. The suppression of MGL due to HIV may increase susceptibility to Mtb in people with HIV or exacerbate disease progression in those with co-infection.

Supported by grants from NIH (R33 AI138328, R61 AI138328, U24 MH100930)
STING activation under pre-existing inflammatory conditions causes severe skin disease
Authors: Pyclik Marcelina , Bagavant Harini , Papinska Joanna , Araszkiewicz Antonina , Deshmukh Umesh
Published: January 2023

The activation of the stimulator of interferon genes 1 (Sting1) pathway causes rapid induction of type I IFNs. Recently, different STING agonists are being proposed as therapeutics for controlling viral infections like those caused by the SARS-CoV-2. Considering that excessive STING activation leads to autoimmunity, this study was undertaken to investigate the effects of STING activation under pre-existing systemic inflammatory conditions induced by a viral infection. Female BALB/c mice were treated (Tx) with TLR3 agonist poly(IC), which was followed 24h later by subcutaneous injection of STING agonist diamidobenzimidazole (diAbZi). Only diAbZi or vehicle Tx mice were used as controls. Mice Tx with poly(IC) and diAbZi showed the highest systemic levels of type I IFNs, IL-6, and TNFα. Within 10 days, the salivary glands showed increased ILC1, monocytes, and CD8+ T cells. However, glandular function was not affected. Five days after Tx, except for the vehicle controls, all mice showed differing extents of redness, hair loss, and ulceration at the injection site. By day 10, diABZi alone Tx mice showed either normal skin or a small scar formation. In contrast, mice Tx with poly IC and diABZi, showed extensive hair loss, erythema, and ulceration. In addition, hematoxylin and eosin-stained skin sections showed extensive neutrophilic infiltration at the site of ulceration, epidermal thickening, and patchy lymphocytic infiltrate that extended into the subdermal layers. Our data demonstrate that STING activation under systemic inflammatory conditions, such as a viral infection, can lead to severe skin disease. Therefore, caution needs to be exercised in employing STING agonists as therapeutics for treating SARS-CoV-2.
High frequency electrical stimulation reduces neuronal HMGB1 release
Authors: Yang Huan , Datta-Chaudhuri Timir B , George Sam J , Haider Bilal B , Wong Jason , Tracey Kevin J , Chavan Sangeeta S
Published: January 2023

Chronic pain affects the daily lives of millions of Americans and presents an economic burden of $600 Billion per year. Opiods and other pharmaceutical approaches for the management of chronic pain have notorious detremental side effects. Thus, there is a need for understanding of pain for better therapeutics. Neuroinflammation is closely associated with chronic pain and targeting molecular mediators of neuroinflammation may present a novel opportunity for pain treatment. High mobility group box 1 protein (HMGB1), a key mediator of injury- and infection-elicited inflammation, is involved in the pathology of persistent pain. We recently reported that neuronal HMGB1 is required for mediating inflammation and hyperalgesia following nerve injury (Yang H et al. PNAS, 2021). High frenquency (HF) electrical nerve stimulation has achieved clinical success for chronic pain, but the underlying mechanisms are not clear. Here we assess the effects of HF stimulation in modulating HMGB1 release by sensory neurons. Using microelectrode arrays (MEAs) in cultured dorsal root ganglia (DRG) harvested from transgenic mice that express light-sensitive channel rhodopsin in sensory neurons, we observe that light-evoked HMGB1 release from DRGs is significantly reduced with HF stimulation (HMGB1 levels in unstimulated group = 5.3 ± 0.5 ng/ml; in light exposed group = 25.8 ± 6.0 vs. light + HF stimulation = 8.2 ± 2.1* pg/ml, n=6, *: P<0.01). In agreement, HF stimulation (10 min/per day X 3 days) significantly reduces mechanical hyperalgesia and HMGB1 levels in inflamed paws in C57BL/6 mice subjected to sciatic nerve ligation injury. These studies suggest HF stimulation as a novel therapeutic approach to modulate neuronal HMGB1 release for the treatment of pain

Supported by grant from NIH 2R35GM118182-06, to KJT and SSC
Vagus nerve sensory neurons have distinct neural responses to inflammatory mediators
Authors: Huerta Tomas S , Haider Bilal B , Adamovich-Zeitlin Richard , Chavan Sangeeta S , Tracey Kevin J , Chang Eric H
Published: January 2023

Cytokines are secreted signaling proteins that are important mediators of inflammation. While prior work has demonstrated that the level of cytokines can be regulated by nerve stimulation, the role of the nervous system in sensing these immune mediators is still poorly understood. During periods of inflammation, it has been shown that sensory signals travel up the vagus nerve to the brain. However, it is unclear how individual vagal sensory neurons encode specific immune information. Here we use in vivo calcium imaging of the nodose ganglion to monitor neural activity in individual vagus nerve sensory neurons in response to specific cytokines. Using mice that express the calcium indicator GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in situ using a 1-photon miniature microscope (Miniscope). During imaging, the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor (TNF) were applied directly to the vagus nerve. Fluorescence data was analyzed with a Python-based software CaImAn and a custom pipeline to output the single neuron activity as change in fluorescence: ΔF/F. Our results reveal that specific vagal sensory neurons respond differentially to specific immune mediators. The average amplitude, integral, and delay of TNF-responsive neurons was significantly higher than IL-1β-responsive cells (TNF vs IL-1β, p < 0.01), while having less number of peaks per response (TNF vs IL-1β, p < 0.05). This may suggest different patterns of transient neural activity associated with each particular cytokine. Further investigation into this type of neuro-immune signaling may identify novel neural targets for the treatment of inflammatory disorders.

This study was funded in part by NIH/NIGMS
Structure guided engineering of selective HVEM mutants reveal distinct functions binding to LIGHT and BTLA/CD160
Authors: Chou Ting-Fang , Liu Weifeng , Garrett-Thomson Sarah C , Seo Goo-Young , Fedorov Elena , Ramagopal Udupi A , Bonanno Jeffrey B , Wang Qingyang , Kim Kenneth , Garforth Scott J , Kakugawa Kiyokazu , Cheroutre Hilde , Kronenberg Mitchell , Almo Steven C
Published: January 2023

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM–LIGHT–CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Supported grants from NIH (S10 OD020068, P30CA023100, P30 DK120515, S10RR027366, U01 AI125955, P01 DK46763), U.S. Department of Energy (DE-AC02-98CH10886, DE-AC02-06CH11357), National Center for Research Resources (P41RR012408), National Institute of General Medical Sciences (P41GM103473), Albert Einstein Cancer Center (P30CA013330), Eli Lilly Company, Albert Einstein Macromolecular Therapeutics Development Facility, Price Family Foundation, Albert Einstein Center for Experimental Therapeutics, Pamela and Edward S. Pantzer, and Academia Sinica, Taiwan.
Regulation of humoral immunity in acute vs. chronic viral infection
Authors: Cooper Lucy , Good-Jacobson Kim L
Published: January 2023

Chronic infectious diseases caused by viral pathogens such as HIV and Hepatitis C constitute a major global health challenge. Chronic viral infections result in poor humoral immune responses, characterized by the delayed generation of neutralizing antibodies and dysregulation of memory B cell formation. Little is known regarding the mechanisms driving dysfunctional B cell memory during chronic viral infection. We used an in vivo mouse model using acute and chronic strains of LCMV, in conjunction with a B cell tetramer specific for the LCMV nucleoprotein, to understand changes that occur in antigen-specific memory B cell formation in acute vs. chronic viral infection. We performed phenotypic analyses of genetically modified mouse strains, BrdU-labelling studies, antiviral treatment experiments and single-cell RNA-sequencing to examine memory B cell formation, phenotype and function in response to acute vs. chronic LCMV infection. Single-cell RNA-sequencing identified key genes downregulated in chronic memory B cells associated with complement-mediated immune functions. We found that T-bet and CXCR3 modulate multiple aspects of the humoral immune response in chronic viral infection, including GC B cell localisation, memory B cell phenotype, and immune complex expansion. Our data provides novel insight into the cellular and molecular mechanisms driving altered humoral immune responses in chronic viral infection.
Testosterone functions as a tumor suppressor in glioblastoma
Authors: Lee Juyeun , Silver Daniel J , Fodor R’ay , Chung Yoon-Mi , Sharifi Nima , Lathia Justin D
Published: January 2023

A sex difference in the incidence and outcome of patients with glioblastoma (GBM), the most common primary malignant brain tumor, has been well established, as male patients experience a worse prognosis. However, the contribution of sex hormones to anti-tumor immunity has not been fully explored. To test this, we orthotopically implanted syngeneic mouse GBM cells into male mice after castration or sham surgery to assess survival differences. Castrated immune-competent mice succumbed to tumors significantly faster than the sham surgery group, and this was rescued by exogenous testosterone administration. In contrast, immune-deficient NSG castrated mice survived longer than sham animals, suggesting a role for immune cells in the survival difference. Flow cytometry analysis of tumor-infiltrating immune cells further showed decreased cytokine production in T cells from castrated mice, while no difference was found in immune cell infiltration. Flank tumor experiments suggested brain-specific regulation of T cell recruitment, as tumor growth was delayed in castrated mice, with higher infiltration of CD8+ T cells but decreased cytokine production. Surprisingly, mass spectrometry of serum revealed upregulated levels of circulating corticosterone in castrated mice regardless of tumor presence, suggesting that castration may induce systemic immunosuppression via upregulation of glucocorticoids. Taken together, our data demonstrate that testosterone regulates T cell immunity, possibly via glucocorticoids, and ultimately affects GBM tumor growth. Given the decreased level of testosterone in aged males with higher risk of GBM, these findings further suggest the potential therapeutic intervention of testosterone in GBM.

Supported by grants from NIH (P01 CA245705)
The role of Piezo1 mechanosensor in osteoclastogenesis from monocytes
Authors: Rawas-Qalaji Mohamad , Shindo Satoru , Nakamura Shin , Kawai Toshihisa
Published: January 2023

Objectives

Interrelations between bone and the immune system are involved in both homeostatic and pathogenic bone remodeling processes. Although emerging evidence supports that mechanosensitive Ca2+-channel Piezo1 plays a pivotal role in osteoblast-mediated bone regeneration, possible engagement of Piezo1 in the differentiation of osteoclasts from monocytes and resulting bone resorption is largely unknown. In the present study, we sought to establish the role of Piezo1 in RANKL-stimulated monocytes for their differentiation to osteoclasts in vitro.

Methods

Mouse RAW 264.7 monocyte cell line or bone marrow derived mononuclear cells (BMMCs) were stimulated with M-CSF and/or RANKL, in the presence or absence of Yoda1 (Piezo1 activator) or with or without hydrostatic pressure (HP). Ca2+ influx assay was employed to confirm Piezo1 channel activity. To evaluate the level of osteoclastogenesis and bone-resorptive function by mature osteoclasts, TRAP staining and pit formation assays were performed. To monitor the expressions of mRNAs for Piezo1 as well as OC-STAMP, TRAP (ACP5) and MMP9, qPCR were employed.

Results

Yoda1-mediated Ca2+ influx was significantly elevated in RANKL BMMCs and RAW264.7 cells, while significantly suppressing the expressions of osteoclastogenesis-related genes, OC-STAMP, MMP9 and ACP5. Moreover, the number and size of multi-nucleated TRAP-positive cells as well as formation of resorption pits were significantly down-regulated by the treatment with Yoda1 as well as HP.

Conclusions

The present study suggests that mechanosensitive Piezo1 channel expressed on monocytes may play a gatekeeper role in down-modulating over-activation of osteoclastogenesis and function to resorb bone.

Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851
Interferon Gamma Induction of TH1-like Tregs Controls Anti-viral Responses
Authors: Gocher Angela Marie , Cui Jian , Szymczak-Workman Andrea , Vignali Kate , Latini Julianna N , Pieklo Gwen P , Avery Lyndsay , Cipolla Ellyse L , Huckestein Brydie R , Hedden Lee , Meisel Marlies , Alcorn John F , Kane Lawrence P , Workman Creg J , Vignali Dario A A
Published: January 2023

Regulatory T cells (Tregs) are an immunosuppressive cell population that inhibits immune cell function to maintain peripheral tolerance and homeostasis. We have previously shown that Treg-- response to interferon gamma (IFNγ) is required for response to cancer immunotherapy. We aimed to broaden these findings and determine the impact of Treg response to IFNγ and the IFNγ-inducing cytokine IL12, during viral infection. Mice with Treg-restricted deletion of the IFNγ receptor (IFNGR1, Ifngr1L/L Foxp3Cre-YFP) or the IL12 receptor β2 subunit (IL12Rβ2, Il12b2L/L Foxp3Cre-YFP), were infected with the acute (Armstrong), or chronic (Clone 13), strain of lymphocytic choriomeningitis virus (LCMV). Surprisingly, Treg response to IFNγ but not IL12 induced helper T cell 1 (TH1)-like polarization (expression of T-bet, CXCR3 and IFNγ) of Tregs in mice with chronic LCMV infection. Importantly, this effector-like TH1 Treg state was required for adequate suppression of effector T cells during LCMV infection. Additionally, during LCMV infection, Treg-restricted deletion of IFNGR1 not only prevented TH1-like polarization but also reverted these Tregs to a TH2-like state (expression of GATA-3, CCR4 and IL-4). scRNAseq of viral antigen-specific CD8+ T cells from Clone 13 infected mice showed that Ifngr1-deficient Tregs favored CD8+ T cell memory. These findings were confirmed in an Armstrong/Listeria monocytogenes-GP33 memory model. Further, Ifngr1 deletion from Tregs enhanced response to CD8+ T cell-mediated vaccination when challenged with Clone 13. These findings provide fundamental insight on how Tregs sense inflammatory cues from the environment (IFNγ) during viral infection to prevent overt tissue damage and shape the memory response.

Supported by grants from NIH (F32 CA247004-01, T32 CA082084, P01 AI108545, R35 CA263850, R01 CA203689, R01 DK130897, P30 DK120531, P30 CA047904, R01 HL107380, R01 CA206517, R01 AI138504)
TNF-α orchestrates the specific loss of tolerogenic dendritic cells during an acute maturation process in vivo
Authors: Bourque Jessica , Iberg Courtney , Fallahee Ian , Hawiger Daniel
Published: January 2023

The recognition of pathogen-associated molecular patterns (PAMPs) through the corresponding pattern recognition receptors (PRRs) induces activation of individual conventional dendritic cells (cDC) in a process often referred to as maturation that results in a change from tolerogenic to immunogenic functions. In the absence of pro-inflammatory conditions (steady state), tolerogenic cDC that are present in systemic peripheral lymphoid organs promote tolerance that limits subsequent immune responses. However, the fate of such tolerogenic cDC in the presence of specific pro-inflammatory signals remains unclear. Here, we report that instead of reprogramming individual tolerogenic cDC for new immunogenic functions, canonical inducers of maturation such as LPS initiated a burst of TNF-α production in these cDC, which resulted in their acute and specific death mediated by TNFR1. This ablation of individual tolerogenic cDC was enhanced by TNF-α produced by neighboring cDC, amplifying the initial innate stimulus. Overall, our results expand the understanding of the functions of TNF-α and TNFR1 during the maturation process and uncover that immunogenic maturation involves a dynamic remodeling of the cDC functional landscape by selectively ablating the tolerogenic cDC population to prevent an aberrant induction of tolerance. Therefore, our results may also have implications for devising new immunotherapeutic approaches.

This work was supported in part by grants from National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01AI113903) and National Multiple Sclerosis Society (RG-1902-33632), both to DH.
Tregs suppress antigen-specific CD8+ T cells in vivo by depleting pMHC-I complexes from Dendritic Cells
Authors: Mansoori Mohammad Nizam , Kamenyeva Olena , Kabat Juraj , Shevach Ethan M
Published: January 2023

T Regulatory cells (Tregs) regulate antigen-specific immune responses by using a variety of mechanisms. Recent studies have shown that antigen-specific Treg can suppress CD4+ effector T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. It remains unclear as to how Treg suppress CD8+ T effectors, particularly in vivo. To explore Treg-mediated suppression of CD8+ T cells, we generated CD4+ iTregs from OT-II mice specific for Ova323–339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257–264 (SIINFEKL) in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of DCs pulsed with both Ova323–339 and Ova257–264 or with DCs singly pulsed with each peptide. Interestingly, the expansion of OT-I CD8+ T cells in vivo was not suppressed by OT-II iTreg when the two peptides were presented on separately pulsed DCs, but was suppressed when both peptides were presented on the same DCs. OT-II Tregs depleted the Kb-SIINFEKL complexes from the DC surface in vitro by a process of trogocytosis only in the presence of their cognate antigen. The uptake of Kb-SIINFEKL complexes by OT-II Treg was not secondary to leakage of free SIINFEKL from the DC surface as OT-II Treg from MHC-I deficient mice were as efficient in uptake of pMHC-I as Treg from wild type mice. Multiphoton imaging microscopy in vivo reveals a close interaction between Tregs and CD8+ T cells suggesting that suppression of CD8+ T cells in vivo requires close proximity between Treg and responder CD8+ T cell may involve an artificial synapse between Treg and CD8+ T cell.
The activation of self-antigen specific T cell repertoire during acute tissue injury.
Authors: Shin Daniel Seong-Joo , Moon James J
Published: January 2023

Self-antigen specific T cells are prevalent in the mature adaptive immune repertoire. At steady state, these self-antigen specific T cells appear to be regulated through a combination of central and peripheral mechanisms of tolerance. Understanding how these self-antigen specific T cells escape tolerance to mediate autoimmune disease remains of significant scientific and clinical interest. Importantly, the mechanisms that determine whether the self-reactivity will exacerbate or regulate the ongoing immune response are unknown. Utilizing an experimental animal model where mice express a complex of three model T cell epitopes (2W, gp66, and OVA) in a tissue restricted manner, we identified and tracked the activation of endogenous polyclonal T cells that recognize these transgenic epitopes as self-antigens. Interestingly, in the context of acute injury to lung tissue of mice expressing the epitopes in the alveolar epithelium, we detect an asymmetric expansion of the self-antigen specific regulatory T cell (Treg) compartment while the conventional T cell populations remained static. These findings suggest that the self-antigen specific T cell repertoire is uniquely programmed to generate a protective response to acute tissue injury.
Disruption of IFNγ, PRF1, GZMB, or LYST results in reduced suppressive function in human CD8+ T cells
Authors: Vemulawada Chakrapani , Renavikar Pranav S , Crawford Michael P , Steward-Tharp Scott , Guo Hui , Jian Zhang , Karandikar Nitin J
Published: January 2023

Human CD8+ T-cells play an important immune regulatory role during autoimmune diseases, characterized by suppressing the activation and proliferation of pathogenic effector cells, mediated in part through cytotoxicity (PRF/GZM), IFNγ secretion or by Fas/FasL interactions. Recent studies from our group demonstrated that IL-12 exposure during in vitro differentiation of naïve CD8+ T-cells induced an immune-suppressive deficit, with Tc1 cells showing significant loss of suppressive ability against CD4+ T-cells. RNAseq analysis revealed that various cytokines, transcription factors, and other regulatory molecules were differentially regulated in these cells. In this study, we further investigated the importance of IFNγ-, PRF1-, GZMB- and LYST-associated pathways in the suppressive ability of CD8+ T-cells. Using the CRISPR-Cas9 RNP optimized transfection system in primary bulk human CD8+ T-cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry when possible, as well confirmation of indels at genomic target sites. We observed that knockout of each of the four molecules, but not the knockout of IL-4 or IL-5, resulted in significantly diminished suppressive ability, as measured through in vitro immune suppression assays. Interestingly, in some of the knockouts, we observed differential expression of non-target molecules indicating potential functional links between these pathways. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T-cell-mediated suppression that could be targeted for immunotherapeutic intervention.

This work was supported, in part, by grant awards to NJK from the NIH (R01 AI121567) and VA.
Multiomic dynamic single-cell profiling of CAR T cell populations associated with efficacy
Authors: Rezvan Ali , Romain Gabrielle , Fathi Mohsen , Heeke Darren , Martinez-Paniagua Melisa , An Xingyue , Bandey Irfan N , Liadi Ivan , Cooper Laurence J N , Singh Harjeet , Bot Adrian , Neelapu Sattva , Varadarajan Navin
Published: January 2023

T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative capacity; interferon responses; and a distinct cluster of pathways related to actin cytoskeleton and migration. We employed a marker-free sorting strategy for enriching T cells with persistent motility. RNA-seq on sorted motile T cells showed an enrichment of the core motility signature. These motile T cells also demonstrated superior in vivo anti-leukemia efficacy in comparison to unsorted T cells. Lastly, we also confirmed the association with increased persistent motility and killing of CAR T cells across diverse CARs. In aggregate, our data identified that independent of CAR design or biomanufacturing, persistent motility serves as a selectable cell-intrinsic biomarker, desired in the bioactivity of expanded CAR+ T cells.
Amino acid F223 in neuroimmune semaphorin 4A is essential for stabilization of human regulatory T cells
Authors: Chapoval Svetlana P , Lee Mariah , Lemmer Aaron , Ajayi Oluwaseyi , Qi Xiulan , Neuwald Andrew F , Keegan Achsah Dorsey
Published: January 2023

Class IV semaphorin family members, Sema4A and Sema4D, displayed opposite effects on human Treg cells in vitro. In PBMC cultures, Sema4A potentiated the CD4+CD25+Foxp3+ cell numbers and increased CD25/Foxp3 expression while Sema4D inhibited them. Sema4A downregulated lung inflammation in allergen-induced experimental asthma whereas Sema4D was necessary for optimal allergic response. Allergen treatment decreased lung local and lymphoid tissue Treg cell numbers in Sema4A−/− mice and increased them in Sema4D−/− mice as compared to similarly treated WT counterparts. Sema4A and Sema4D competitively bind to Plexin B1 in microplate-based binding studies. Using Bayesian Partitioning with Pattern Selection (BPPS) and Structurally Interacting Pattern Residues’ Inferred Significance (SIPRIS), we identified Sema4A residues likely responsible for its functional specificity. We mutated the Sema4A residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. We evaluated their stimulatory activity toward human Treg cells and assayed them for binding to Plexin B1. The F223A mutant was unable to stimulate Treg stability in the in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to WT and M198A proteins. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing novel immunotherapeutics for asthma.

This work was supported by NIH grant R01AI 122631 and the Merit Review Award Number I01 BX001850 from the US Department of Veterans Affairs Biomedical R&D Service (both awarded to A.D.K.) and by NIH-NIGMS grant R01GM125878 to A.F.N.
Mettl14-dependent m6A modification controls iNKT cells development and function
Authors: Cao Liang , Morgun Eva , Genardi Samantha , Visvabharathy Lavanya , Huang Haochu , Wang Chyung-Ru
Published: January 2023

N6-Methyladenosine (m6A), the most common form of RNA modification, controls CD4+ T cell homeostasis by targeting the IL-7/STAT5/SOCS signaling pathways. The role of m6A modification in unconventional T cell development remains unknown. Using mice with T cell-specific deletion of RNA methyltransferase METTL14 (T-Mettl14−/−), we demonstrated that m6A modification was indispensable for iNKT cell homeostasis. Loss of METTL14-dependent m6A modification led to the upregulation of p53-mediated apoptosis in double positive thymocytes, which in turn decreased distal Va-Ja gene rearrangements, including the invariant Va14-Ja18 TCR, resulting in a drastic reduction of iNKT numbers in the thymus and periphery. Residual T-Mettl14−/− iNKT cells exhibited increased apoptosis, impaired maturation, and decreased responsiveness to IL-2/IL-15 and TCR stimulation. Furthermore, METTL14 knockdown in mature iNKT cells diminished their cytokine production, correlating with increased Cish expression and decreased TCR signaling. Collectively, our study highlights a critical role for METTL14-dependent-m6A modification in iNKT cell development and function.

This work was supported by NIH grants R01 AI41083 and R01 AI057460 to C.-R.W.
The Role of HnRNPA1 in T Cell-Mediated Gut Tolerance
Authors: White Tristan L , Gable Matthew , Jin Ye , Morel Penelope A
Published: January 2023

Our previous research showed that Akt phosphorylation of the RNA-binding protein, (RBP) heterogeneous nuclear ribonucleoprotein (hnRNP) A1, is dependent on TCR signal strength, and occurs under conditions of induced T regulatory (Treg) cell differentiation. We have shown hnRNPA1 is required for optimal Treg differentiation by performing knockdown experiments, and our present research is focused on identifying a role for Akt phosphorylation in hnRNPA1 function. HnRNPA1 is phosphorylated by Akt at S199 and our lab has generated a new mutant mouse model, hnRNPA1-S199A (mA1), in which hnRNPA1 will no longer be Akt-phosphhorylated. Our initial characterization of mA1 mouse model failed to reveal major abnormalities in immune cells populations at steady state except for changes in the Treg proportion in the mesenteric lymph nodes. To investigate the effect of the mA1 on the development of oral tolerance we used an adoptive transfer model in which OT-II mA1 or OT-II WT naïve T cells were transferred into congenic strains and then the mice were given OVA food. Results indicate that mA1 OT-II T cells have a proliferation defect in vivo and we also observed a significant reduction in the induction of OVA-specific Treg in the mA1 T cells. Similar results were obtained when adoptively transferred OT-II mA1 T cells were challenged with limiting doses of specific peptide, given iv. RNASeq analysis of the transferred mA1 and WT T cells is being performed. These results suggest that Akt phosphorylation of hnRNPA1 is necessary for T cell response to low dose antigen and Treg induction in vivo. Our studies identify a new mechanism by which RBP influence T cell proliferation and Treg differentiation which has implications for tolerance to food antigens in the gut.

Supported by NIH (F31-AI152320-02) NIH (R01-AI125513-03)
Role of transcription factor RUNX1 in the maintenance of T helper cell epigenome and rapid recall response
Authors: Korinfskaya Svetlana , Kotliar Michael , Katko Alexander , Hildeman David , Miraldi Emily R , Barski Artem
Published: January 2023

Memory T cells exhibit an accelerated response after a second encounter with the same antigen. In our previous studies, we showed that the ability to produce effector molecules within minutes of the secondary exposure to antigen (“rapid recall response”) is associated with the epigenetic state of the rapid recall genes. This memory epigenome is established during the primary T cell encounter with its cognate antigen. To maintain a “rapid recall response” even when antigen is eliminated, resting memory T cells would have to maintain open chromatin signature for years despite episodic homeostatic T cell proliferation. However, the mechanisms underlying the long-term maintenance of this epigenetic pattern remains unclear. Here, we used 10X Multiome (scRNA-seq and scATAC-seq obtained from the same cells) analysis to show that the association between the chromatin state of memory cells and their cytokine expression upon activation is Th lineage specific.

Bioinformatic analysis showed that open chromatin specific for memory but not naïve CD4 T cells is enriched with motifs of constitutively expressed transcription factors RUNX, SP1, and ETS. Our preliminary Cut&Tag data in total human resting CD4 T cells demonstrates that RUNX1 binds DNA close to T helper cytokines genes (IL3, IL4, IL9, IL10, IL13, IFNG) at “memory-specific” ATAC-seq regions, suggesting the role of RUNX1 in maintaining this “rapid recall” epigenome. Together with the dynamic model of transcriptome and epigenome rearrangements this data could help us to understand how memory T cells establish a platform that enables and maintains their unique ability to respond more rapidly to subsequent encounters with antigen.

Supported by R01AI153442-01A1 from NIAID, NIH
Epigenome accessibility changes before and after activation reveal distinct and progressive differentiation for human memory T cell subsets
Authors: Rose James R , Rahmberg Andrew R , Powell Michael D , Scharer Christopher D , Boss Jeremy M
Published: January 2023

Memory T cells (MTC), an indispensable part of adaptive immune memory, have historically been categorized by the cell surface proteins CCR7 and CD45RA into specialized subtypes (TCM, TEM, and TEMRA), each with unique functions. However, the epigenetic characteristics that distinguish the MTC subsets as well as the gene regulatory networks governing each MTC subsets’ response to activation remain poorly understood. Here we define and categorize the transcriptional and epigenetic differences of MTC and their respective primary subsets found in human blood, both in a resting state and after ex-vivo stimulation. Resting TCM were found to be relatively more similar to naïve cells in both CD4 and CD8 lineages, while TEM exhibited greater numbers of differentially expressed genes (DEG) and alterations to chromatin accessibility. Differentially accessible regions (DAR) discerning memory subsets contained binding motifs for factors thought to regulate memory formation from the bZIP, T-box and HMG families, as well as sites for novel bHLH factors MSC and AHR that may function in MTC development. Examining the effect of stimulation on MTC gene expression and chromatin identified unique DAR and DEG modules, some of which may have initially been primed by previous activation events in naive progenitors. Primed DAR were correlated with augmented expression of important genes in MTC after stimulation, suggesting an epigenetic mechanism of regulation. Ultimately these results describe the accumulation of epigenetic alterations during primary activation and during memory development that distinguish MTC from naïve T cells, enable differentiation into distinct subsets, and influence how MTC respond to secondary activation.

Supported by grants from NIH (RO1 AI113021,T32 GM0008490)
Human T cells in barrier sites exhibit site-specific characteristics and clonal compartmentalization
Authors: Caron Daniel P , Poon Maya M L , Wang Zicheng , Meng Wenzhao , Lam Nora , Szabo Peter A , Wells Steven B , Thapa Puspa , Dogra Pranay , Lee Brian , Kubota Masaru , Matsumoto Rei , Rahman Adeeb , Luning Prak Eline T , Sims Peter , Shen Yufeng , Farber Donna L
Published: January 2023

The skin, gut, and lung are critical physical and immune barriers that protect from pathogen entry. Within these sites, T cell memory is largely maintained by populations of tissue resident memory T cells (TRMs), capable of rapid protective responses. The degree of interconnection between these TRMs from various sites of residence is poorly understood, particularly in humans. Here, we analyze how tissue specificity affects transcriptional heterogeneity and the T cell receptor (TCR) repertoire across 9 tissue sites within individual donors, including barrier sites (skin, gut, and lung) and associated lymph nodes, as well as blood and lymphoid organs (blood, bone marrow, spleen). Cytometry by time-of-flight (CyTOF) and single cell RNA sequencing reveal phenotypic and transcriptomic features unique to skin or gut T cells, which are distinct from T cells in lymphoid sites or blood. These results were reflected in both bulk sequencing of TRBV gene rearrangement and single cell TCR sequencing, where skin and gut T cells exhibited site-specific clonal expansions that were not present in other sites. Conversely, lung T cells showed clonal overlap and transcriptional similarity to T cells in lymphoid and blood rich sites. Site-specific expansions were mediated by TRMs, while clones disseminated across tissues exhibit large clonal expansions and a CD8 terminal effector (TEMRA) phenotype. Together, these results reveal that TRMs in barrier sites are maintained in situ, while memory T cells in the lung, blood-rich and lymphoid sites are more interconnected. Blood T cells are not representative of barrier T cell clonal space. These results have important implications for monitoring and promoting barrier immunity through site-specific targeting.

Supported by grants from NIH (P01 AI106697)
Altered Trained immunity in AID−/− versus wild type mice following infection with Pneumocystis murina
Authors: Curran Shelly , Kovacs Joseph
Published: January 2023

Pneumocystis pneumonia continues to be a life-threatening infection in immunocompromised individuals. Dendritic cells are key antigen presenters to CD4+ T cells, which are critical to controlling Pneumocystis infection along with B cells. β-1,3 glucans are found in the cell wall of the cyst form of Pneumocystis. Glucans derived from other fungi have been shown to induce trained immunity. Therefore, we explored if Pneumocystis infection can induce a trained immunity response in C57Bl/6 (wild type) mice following exposure via a natural route of infection. We also tested activation-induced cytidine deaminase knockout mice (AID−/−), a deficiency that affects B cells, in the same infection model to determine whether a mutation known to affect the adaptive immune response can also alter the manifestation of trained immunity. We found that bone marrow derived dendritic cells (BMDCs) from C57BL/6 Pneumocystis exposed mice respond differently to homologous and heterologous antigen stimulation compared to BMDCs from naïve mice. Furthermore, although the differential response between cells from AID−/− exposed and naïve mice exists, specifically the TNFα response is opposite to that from C57BL/6 mice. In addition, when trained immunity was induced in BMDCs derived from naïve C57Bl/6 mice and AID−/− mice, the cytokine response was distinctive between the cells derived from C57BL/6 mice and AID−/− mice. These results suggest that not only can Pneumocystis infection induce trained immunity in BMDCs, this response is alterable by gene mutations traditionally thought to affect the adaptive immune response.

Intramural Research Program of the NIH Clinical Center
Mice infected with Mycobacterium tuberculosis are resistant to secondary infection with SARS-CoV-2
Authors: Mejia Oscar Rosas , Gloag Erin S , Li Jianying , Ruane-Foster Marisa , Claeys Tiffany A , Farkas Daniela , Farkas Laszlo , Xin Gang , Robinson Richard T
Published: January 2023

Mycobacterium tuberculosis (Mtb)and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease; however, little is known regarding the immunological environment in the Mtb/CoV2 coinfected lung. The goal of this study was to use a mouse model of Mtb/CoV2 coinfection to determine if CoV2 affects Mtb bacterial burden and/or alters the lung immune profile. Using lung collected from human ACE2 transgenic (K18-hACE2) mice infected with both pathogens (Mtb only, SARS-CoV-2 only, and Mtb/SARS-CoV-2 co-infected), we evaluated immune gene expression, cytokine production, and bacterial burden. Surprisingly, these data show that Mtb suppresses CoV2 -related weight loss and lung viral burden in the human ACE2 transgenic mouse model. We also report a reduction in lung interferon gamma production and expression in coinfected mice compared to the Mtb only infected group, suggesting a possible altered T cell profile in the coinfected group. To determine whether Mtb-induced resistance to CoV2 was specific to the ACE2 transgenic model of COVID19, we performed the same set of experiments using a second mouse model of COVID19: Mouse Adapted SARS-CoV-2 (MACoV2) infection of C57BL/6 (B6) mice.

In both model systems, Mtb-infected mice were resistant to secondary CoV2 infection and its pathological consequences, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

Supported by OSU Advancing Research in Infection and Immunity Fellowship Award
HIV compromises Th17 and Th22 immunity in a humanized mouse model of Tuberculosis and HIV co-infection
Authors: Martinez-Martinez Yazmin Berenice , Huante Matthew B , Files Megan , Gelman Benjamin B , Endsley Mark , Endsley Janice J
Published: January 2023

Tuberculosis (TB) kills an estimated 1–2 million people per year and is the leading cause of death in people with HIV (PWH). The CD4+ T helper (Th) populations play significant roles in protective immunity to Mycobacterium tuberculosis (Mtb), and are also important host cells for HIV proliferation and persistence. Emerging evidence in PWH suggests that Th17 and Th22 cells may be preferentially depleted during HIV infection, an outcome that could pose a risk for Mtb containment during co-infection. We employed our humanized mouse model of co-infection to assess pulmonary and peripheral changes in Th17 and Th22 frequency and function due to infection with HIV, Mtb, or both. As determined with multi-plex ELISA, co-infection with HIV suppressed the cytokine response to Mtb including IL-17A, IL-22, IL-6, and IL-1β. Multi-parameter flow cytometric analysis revealed that Mtb and HIV/Mtb infection led to increased Th17+ cells in lung and decreased Th17+ cells in spleen. Following HIV or HIV/Mtb infection, Th22+ cells were significantly decreased in both lung and spleen. Importantly, HIV preferentially infected Th17 cells compared to Th22 and other Th subpopulations. Greater HIV viral load was also observed in the lung, but not spleen, of animals with Mtb/HIV co-infection compared to those with HIV mono-infection. Overall, these results suggest that HIV may compromise Th22 immunity, and exploit Th17+ cells to promote viral pathogenesis, in the setting of Mtb and HIV co-infection.

Y.B. Martinez-Martinez is supported by Conacyt-I2T2 in Mexico, Contex, and the James W. McLaughlin Fellowship Fund. Grant support was provided by NIH R01AI147948 award to J. Endsley
Scrub typhus weakens humoral immune responses through suppression of germinal center and B cell activation
Authors: Gonzales Casey A , Liang Yuejin , Card Galen , Fisher James , Walker David H , Soong Lynn , Sun Jiaren
Published: January 2023

Scrub typhus is a poorly studied, but life-threatening, disease due to uncontrolled growth of Orientia tsutsugamushi bacteria within endothelial cells and phagocytes, in addition to dysregulated Th1 immune responses and inflammation. Cellular and humoral immunity in infected patients and experimental animals are short-lived, and wane within 1–2 years; however, the underlying mechanism of this transient immunity is unclear. No reports have examined splenic germinal center (GC) or B cell activation during this infection. The study objective is to fill this knowledge gap by using endothelium-targeted C57BL/6 mouse models that mimic pathological features of human scrub typhus. Through NanoString, qRT-PCR, and flow cytometry, we found significant repression of key markers crucial for B cell development (CCR7, BAFFR, PAX5) and GC formation (SLAMF1, ICOS-L, CXCR5, BCL-6) in both mild and severe disease outcomes. The widespread impairments in B and T follicular helper (Tfh) cell activation correlated with excessive Th1 cell activation during disease progression. Immunofluorescence staining (B220, CD3, GL7) revealed evidence of GC formation with sublethal infection, but the near absence of organized GCs at late stages of lethal infection, which was consistent with results from our splenic multi-color flow cytometry and serum antigen-specific ELISA assays for IgG isotypes. Ongoing RNA-seq studies for splenic B cells isolated from sub-lethally or lethally infected mice will provide in-depth, B cell-specific insight during infection. In conclusion, this is the first study to show profound alterations in B cell activation and GC formation in murine models, which helps understand “poor” humoral immunity seen with human scrub typhus.

Supported by grants from NIH (R01 EY028773 to JS, R01 AI132674 to LS, R21 AI156536 to LS)