bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloid-KLF2 knockout neonatal mice experience mortality from lipopolysaccharide in an age-dependent and cell-specific manner
Authors: Mukherjee Devashis , Satyavolu Sriram , Thomas Asha , Jain Mukesh , Nayak Lalitha
Published: August 2023

Background

Preterm neonates exhibit increased mortality from Gram negative sepsis as compared to term neonates. The innate immune system is at a critical phase of development during the neonatal period. Mechanisms underlying activation of the preterm neonatal innate immune response are poorly understood. Our prior data has shown that Kruppel-like factor (KLF)-2 is a critical transcription factor to maintain murine myeloid cells in a quiescent state. We hypothesize that myeloid KLF2 plays a critical role in the neonatal immune response in Gram negative sepsis.

Methods

Preterm neonatal (P4) and term neonatal (P12) myeloid KLF2 knockout (MyK2KO) and age matched Cre mice were given 5ug/gm of E. coli O55:B5 lipopolysachharide (LPS) via intraperitoneal (i.p.) injection. For cell depletion studies, antibodies were injected i.p. 24 hours prior to LPS injection, followed by a repeat dose 24h after LPS. Ly6G antibody was used for neutrophil depletion, CD115 for monocyte depletion and equal dose of both for depletion of both cell lines.

Results

P4 MyK2KO mice have significantly increased mortality within 48 h of LPS exposure as compared to P4 Cre mice (81% vs 41%, n=19, p<0.01 χ2 test). At P12, there is 100% survival of both groups of mice. Depletion of either neutrophils, monocytes or both together, decreased the mortality in P4 MyK2KO mice from 81% to 55%.

Conclusion

Preterm neonatal mice lacking myeloid KLF2 have an exaggerated response to LPS as compared to term neonatal MyK2KO or age-matched control mice. In vivo depletion of neutrophils, monocytes or both prior to LPS attenuates this response. Future studies will focus on the mechanism underlying the postnatal age-specific and cell-specific role of KLF2 in the neonatal innate immune response.

Supported by grants from Rainbow Faculty Pilot Grant (Mukherjee PI) and NIH (R01 HL142647 Nayak PI)
Propolis inhibits IgE-mediated mast cell activation by suppressing SYK/MAPK signaling pathway
Authors: Van Nguyen Thi , Jiang Yuna , Yu Zhen Nan , Jin Juan , Han Eui Hyeog , Kim Hyoung Tae , Song Chang Ho , Hur Yong Kap , Chai Ok Hee
Published: August 2023

Mast cells play a critical role in the effector phase of immediate hypersensitivity and allergic reactions. Propolis exerts multiple biological effects and exhibits immunomodulatory and antioxidant properties. However, its role in antidinitrophenyl (DNP) immunoglobulin E (IgE)-activated mast cells has not been investigated. In this study, we examined the mechanism underlying the inhibitory effect of propolis (Unibee powder propolis, Uniquebiotech, Co., Ltd., Iksan) on DNP-IgE-mediated mast cell degranulation, with a specific focus on histamine and cytokines release, using rat peritoneal mast cells. Propolis protected mast cell degranulation and inhibited histamine release triggered by anti-DNP IgE in rat peritoneal mast cells (RPMC) in vitro. Interestingly, propolis also decreased the level of TNF-a, IL-4, and IL-13 released from anti-DNP IgE-activated RPMC. Moreover, the SYK and its downstream-MAPK signaling pathway also were suppressed in the anti-DNP IgE-activated RPMC by the incubation with propolis. Together, these results suggest that propolis extract effectively inhibits IgE-mediated mast cell anaphylaxis-like reactions.

This research was supported by the 2022 Collaborative R&BD Program of The Food Industry Promotional Agency of Korea.
SPA4 peptide suppresses the lipopolysaccharide (LPS)-stimulated lactate secretion in lung epithelial cells through modulation of Toll-like receptor-4 (TLR4)
Authors: Godbole Nachiket Madan , Awasthi Shanjana
Published: August 2023

Lung infection and inflammation can cause severe injury leading to acute respiratory distress syndrome. An increased expression and activity of the TLR4 is associated with inflammatory response. Simultaneous elevated level of lactate is used as the marker of disease severity and is associated with poor outcomes in critically ill patients. Therapeutic treatment with TLR4-interacting SPA4 peptide (amino acid sequence: GDFRYSDGTPVNYTNWYRGE) suppresses inflammation in immune cells and in mouse models of lung infection and inflammation. In this study, we evaluated the effect of SPA4 peptide on lactate production by H441 human lung epithelial cells against Pseudomonas aeruginosa-derived LPS. Briefly, the H441 cells were challenged with LPS (1 μg/ml) and treated with SPA4 peptide (100 μM) after 1 h of LPS challenge. Next day, the cell-free supernatants and cell lysates were harvested. We also transfected the cells with TLR4 siRNA to achieve suppression of TLR4. Levels of protein and lactate were quantitated in cell lysates and in filtered supernatants using biochemical assays. The lactate levels were normalized with cell protein. The unchallenged, vehicle-treated cells served as control. Our results demonstrated reduced secretion of lactate by SPA4 peptide-treated cells (187.5% of control) versus vehicle-treated cells (324.7% of control) against LPS stimuli (p<0.05). The immunostaining and confocal imaging showed knockdown of TLR4 in up to 50% cells. The LPS-stimulated lactate secretion was reduced and SPA4 peptide treatment did not further affect the lactate levels in TLR4 siRNA transfected cells. Together, our results suggest that the SPA4 peptide suppresses lactate secretion through modulation of TLR4 in lung epithelial cells.

Supported by grant from NIH (R01HL136325) and 2021 American Association of Immunologists Careers in Immunology Fellowship Program.
The BNT162b2 vaccine’s empty lipid nanoparticle is able to induce an NF-κB response
Authors: Zelkoski Amanda E , Ennis Katherine H E , Lu Zhongyan , Malloy Allison M W
Published: August 2023

Lipid nanoparticles (LNP) are essential components of messenger RNA (mRNA) vaccines that have been a cornerstone of the COVID-19 public health response. These LNPs are used for cell entry and protection of the mRNA. Others have shown that the LNP of the BNT162b2 vaccine is able to augment the immune response against the vaccine target. However, it is unknown how this LNP stimulates innate immune cells such as monocytes that participate in the engagement of the adaptive immune response.

We used a THP-1 monocyte reporter cell line for nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF) activation to understand if empty LNPs could stimulate innate cells in comparison with toll-like receptor (TLR) agonists. With this cell line, we show that the BNT162b2’s LNP is able to initiate activation of NF-κB, but not IRF in a dose-dependent manner. The NF-κB response was similar to that of low doses of R848, a TLR 7/8 agonist, as well as LPS, a TLR4 agonist. Next, we evaluated NF-kB activation in the absence of MyD88 or TRIF using knockout reporter cell lines. We demonstrate that NF-κB activation is reduced in both knockout cell lines. In comparison, LPS primarily stimulated NF-kB activation through MyD88, and R848 relies on both pathways.

Our data show that LNPs can stimulate key innate immune cells through NF-kB pathways but not through IRF suggesting that this master regulator may participate in the immune activation induced by the current mRNA vaccine platform. Moreover, our data suggest that LNPs can use both MyD88 and TRIF signaling cascades to induce activation. Understanding the mechanisms behind the immunostimulatory capacity of LNPs can shed light on modulating these vaccine components to enhance vaccine design.

Defense Health Program, HU00012120094
The transcriptional repressor HIC1 is required for the maintenance of tissue-resident ILC1
Authors: Rodrigues Grace , Doan Anh , Camilleri Tania , Huntington Nicholas , Scheer Sebastian , Schuster Iona , Degli-Esposti Mariapia A , Zaph Colby
Published: August 2023

The group 1 innate lymphoid cell compartment is comprised of conventional natural killer (cNK) and type 1 innate lymphoid cells (ILC1). Within the innate lymphoid cell pool, these cells are delineated by expression of the transcription factor TBET, and both subtypes express cell surface receptors NK1.1 and NKp46. Functionally, ILC1 and cNK cells have been shown to mediate pathogen clearance and tumour control through release of type 1 cytokines, particularly IFN-□. While being closely related, a key feature distinguishing ILC1 from cNK cells is their propensity to reside within a tissue niche rather than enter the circulation. However, the precise mechanisms that regulate tissue residency of ILC1s remain unknown. Here, we show that the transcription factor Hypermethylated in Cancer 1 (HIC1) is a critical control point in the development and maintenance of ILC1. In the liver, HIC1 expression is restricted to the ILC1 compartment. Deletion of HIC1 in NKp46+ cells (Hic1ΔNcr1 mice) resulted in a specific and significant reduction in liver-resident ILC1, while cNK cells were unaffected. Additionally, we show that cNK cells isolated from Hic1ΔNcr1 mice are incapable of restoring the ILC1 compartment in lymphocyte-deficient hosts, unlike wild type donors. Thus, our results identify an important role for HIC1 in the development and maintenance of peripheral ILC1.
The role of MATRIN3 (MATR3) in innate immune response
Authors: Islam Zohirul , Chim Bryan , Suzawa Masataka , Cutrona Nicholas , Smith Patrick T , Foroushani Amir K , Hafner Markus , Muljo Stefan A
Published: August 2023

MATR3, one of the most abundant inner nuclear matrix proteins, is implicated in amyotrophic lateral sclerosis (ALS) and distal myopathy. MATR3 has been reported to have both DNA- and RNA-binding abilities. However, it is not understood mechanistically how mutation of MATR3 leads to ALS pathogenesis. To understand its loss of function effects, we mutated MATR3 in the human HAP1 cell line using CRISPR/Cas9 technology. Using two different RNA sequencing methods, Oxford Nanopore Technologies and Illumina, we discovered the upregulation of many interferon-stimulated genes (ISGs) in MATR3-deficient HAP1 cells. Furthermore, we have validated these results by Nanostring technology. Next, we hypothesized that HAP1 cells may be responding to endogenous double-stranded RNAs. Interestingly, we observed similar amounts of dsRNA in both MATR3 knock-out (KO) and wildtype (WT) cells which suggests that indeed intracellular dsRNAs are present and could be stimulating HAP1 cells; however, it is not the case that MATR3 KO cells are stimulated because they are producing significantly more dsRNAs. Currently, we are investigating whether and which RNA sensors are activated in MATR3 KO cells. Also, we are investigating whether MATR3’s DNA- and/or RNA-binding abilities are responsible for the innate immune response. Accordingly, we plan to perform CUT & RUN and photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) respectively, in HAP1 cells. Altogether, our results illustrate that MATR3 is a novel negative regulator of the innate immune response. This work was supported by the Intramural Research Program of NIAID, NIH.

This work was supported by the Intramural Research Program of NIAID, NIH.
BCG component mycolic acid protects against monocyte exhaustion in vitro
Authors: Wu Yajun , Wang Jing , Li Liwu
Published: August 2023

Monocyte exhaustion reflected in pathogenic inflammation and immune suppression underlies the pathogenesis of sepsis. The objective of this study is to define novel approaches in alleviating monocyte exhaustion, based on intriguing clinical reports that BCG components may be beneficial in reducing polymicrobial infection-triggered sepsis severity. We recapitulated in vitro the development of exhausted murine monocytes through prolonged culture with high dose bacterial lipopolysaccharide (LPS), measured by the expansion of immature Ly6Chi population with elevated expression of pathogenic mediator CD38 and immune suppressor PD-L1, and reduced expression of CD86. Intriguingly, we observed that co-culture of murine bone marrow monocytes with Mycobacterium tuberculosis (BCG) component mycolic acid (MA) can significantly alleviate the development of exhausted monocytes. We found that monocytes co-stimulated with MA and LPS exhibited significantly reduced induction of CD38 and PD-L1 as compared to monocytes challenged with LPS alone. On the other hand, monocytes co-treated with MA and LPS exhibited enhanced expression of CD86 as compared to cells treated with LPS. In the monocyte-T cell coculture system, we found that T cells co-cultured with MA and LPS-treated monocytes had a significantly higher proliferation potential as compared to the T cells co-cultured with LPS-treated monocytes. Mechanically, we demonstrated that MA exerts its protective effect by selectively activating P-AKT and inhibiting STAT1/P-STAT1. In conclusion, we demonstrate that the BCG component mycolic acid can potently alleviate monocyte exhaustion and may serve as a future promising agent in treating sepsis.

Supported by grants from NIH (AI136386)
The glucose transporter GLUT4 tethers RIG-I-like receptors to suppress antiviral immunity
Authors: Harrison Andrew G , Yang Duomeng , Cahoon Jason G , Geng Tingting , Karginov Timofey A , Torrance Blake L , Chiari Conner C , Haynes Laura , Vella Anthony T , Witczak Carol A , Bogan Jonathan S , Wang Penghua
Published: August 2023

The current dogma of RIG-I-like receptor (RLR) regulation states that dysregulated interferon (IFN) production is predominantly repressed by reversible posttranslational modifications (e.g., ubiquitination). However, RLR activation has profound outcomes on cellular metabolism, which governs the nature of these inflammatory immune responses. Here, we reveal a novel mechanism of immunometabolic regulation of RLR signaling by the glucose transporter 4 (GLUT4). In addition to its canonical role in maintaining organismal glucose homeostasis, GLUT4 attenuates innate immune signaling through sequestering RLRs into the plasma membrane. Translocation of GLUT4 from intracellular compartments to the plasma membrane is tightly regulated by UBXN9, a ubiquitin-domain containing protein (UBXN). Disruption of UBXN9 releases GLUT4, suppresses RLR signaling/IFN responses and enhances viral replication, while genetic ablation of GLUT4 improves antiviral immunity. Strikingly, these distinct phenotypes are independent of glycolysis, but dictated by the mobilization of GLUT4 to the plasma membrane. Further, GLUT4 colocalizes with RIG-I after insulin treatment (which stimulates GLUT4 trafficking) or viral infection to blunt RLR activation. Fine-tuning of this UBXN9-GLUT4 axis is critical for antiviral immunity, but dysregulation of these components may underlie inflammatory myopathies that are characterized by a hyperactive RLR-IFN pathway. Together, this study reveals a novel link between GLUTs and cytosolic immune sensors, and underscores the intersection of vesicular trafficking, glucose metabolism and antiviral immunity.

Supported by grants from NIH (NIH R01AI132526, R21AI155820)
Upping the “Anti-” for arthritis: Antimicrobial defenses through anti-inflammatory NOD-like Receptor NLRX1 during Lyme disease
Authors: Tupik Juselyn D , McClune Mecaila E , Camp Hailey W , Gregory Julia A , Dressler Jules M , Markov Madanick Justin W , Nagai-Singer Margaret A , Jutras Brandon L , Allen Irving C
Published: August 2023

Lyme disease, caused by the bacterium Borrelia burgdorferi, is an emerging infectious disease of global concern. Roughly 60% of untreated patients will develop synovitis of the joints termed Lyme arthritis, resulting from sustained cytokine and chemokine production by the innate immune system. Currently, there are limited therapeutics for antibiotic-refractory Lyme arthritis, warranting investigation into how our innate immune system can mitigate this inflammation. Here, we studied how the anti-inflammatory NOD-like Receptor (NLR) NLRX1 could play a role in controlling Borrelia infection. Because NLRX1 modulates inflammatory signaling, cell death, autophagy, and metabolism, we hypothesized that NLRX1 could have unique antimicrobial effects against Lyme disease. Using novel Nlrx1−/− mice, we found that NLRX1 significantly decreased arthritis severity in wildtype mice compared to knockouts and modulated bacterial load in vivo. We next determined in Nlrx1−/− murine BMDMs that NLRX1 may control infection by promoting Reactive Oxygen Species (ROS) mediated-cell death and autophagy, while decreasing cell proliferation and modulating inflammasome activation in vitro. Finally, by infecting novel NLRX1 overexpression human monocytes, we found that elevated NLRX1 significantly decreased pro-inflammatory NF-κB-mediated cytokine secretion. Ultimately, these results indicate that NLRX1 plays a protective role in mitigating Lyme arthritis in murine and human models. Further, this protection may occur through NLRX1’s modulation of cell death and attenuation of NF-κB signaling. Consequently, these results warrant further exploration of Borrelia regulation by NLRX1 for development of new treatments for Lyme arthritis.

Supported by grants from the NIH/NIAID (R21AI159800) and the Cohen Foundation
Biphasic JNK signaling reveals distinct MAP3K complexes licensing inflammasome formation and pyroptosis
Authors: Bradfield Clinton J , Liang Jonathan J , Ernst Orna , John Sinu P , Sun Jing , Ganesan Sundar , de Jesus Adriana A , Bryant Clare E , Goldbach-Mansky Raphaela , Fraser Iain D C
Published: August 2023

Kinase signaling in the tiered activation of inflammasomes and associated pyroptosis is a prime therapeutic target for inflammatory diseases. While MAPKs subsume pivotal roles during inflammasome priming, specifically the MAP3K7/JNK1/NLRP3 licensing axis, their involvement in successive steps of inflammasome activation is poorly defined. Using live–cell MAPK biosensors to focus on the inflammasome triggering event allowed us to identify a subsequent biphasic JNK activation. We find that post–trigger JNK signaling distinctly facilitates both mitochondrial reactive oxygen species generation needed to support core inflammasome formation, and gasdermin–mediated cell permeation leading to release of active IL–1β from human macrophages. We further identify and characterize a xanthine oxidase–ROS activated MAP3K5/JNK2 substrate licensing complex as a novel regulator of GSDMD mobilization preceding pyroptosis. We find that inhibitors targeting this MAP3K5 cascade alleviate morbidity in mouse models of colitis and dampen both augmented IL–1β release and cell permeation in monocytes derived from patients with gain–of–function inflammasomopathies.

This work was supported by the Intramural Research Program of NIAID, NIH.
Characterization of Neuro-Immune Communication in the White Pulp Region of the Spleen
Authors: Keller Alexandra , Ross Ashley
Published: August 2023

Norepinephrine (NE) is a ubiquitous neurotransmitter that facilitates cellular communication in the sympathetic nervous system. Recent advancements have shown sympathetic neurons innervate secondary immune organs, such as the spleen, to control immune physiology. Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique that varies an applied potential at a very fast scan rate with micro-electrodes to detect neurotransmitters with high temporal and spatial resolution. FSCV can be used to detect the sub-second release of NE in an ex vivo murine slice model to study the mechanisms related to neuro-immune communication. Our lab is the first to characterize and quantify this spontaneous transient signaling of catecholamines in the white pulp region of the murine spleen with FSCV. The average concentration of catecholamines released is 581 ± 20 nM, with an average inter-event time of 23.4 ± 0.7 s and a duration of 6.6 ± 0.1 s. Norepinephrine transporter (NET) is known to transport NE back into cells after endogenous release. We blocked NET with the inhibitor nomifensine to provide further evidence of NE release and study the mechanisms involved in NE uptake. To study the mechanisms of NE release in the spleen, we blocked α2-adrenoceptor, a known autoreceptor only found on neurons. Yohimbine is an α2-adrenoceptor antagonist, and when applied to slices, it increased catecholamine concentration and duration, demonstrating that we are detecting neuronal release of NE. Furthermore, we used ELISAs to quantify the extent to which the α2-adrenoceptor affects cytokine production, providing further evidence neuronal-regulated immune signaling in the white pulp region of the spleen.

Supported by grants from NIH (R01AI151552, R01NS121426)
Bi-directional epithelial-immune communication amplifies inflammasome activation and IL-6 release during SARS-CoV-2 infection
Authors: Barnett Katherine Camille , Xie Yuying , Asakura Takanori , Song Dingka , Liang Kaixin , Taft-Benz Sharon A , Guo Haitao , Yang Shuangshuang , Okuda Kenichi , Gilmore Rodney C , Loome Jennifer F , Oguin Thomas , Sempowski Gregory D , Randell Scott H , Heise Mark T , Lei Yu Leo , Boucher Richard C , Ting Jenny P
Published: August 2023

Increased levels of the pro-inflammatory cytokines IL-1β and IL-6 are linked to severe COVID-19. Here, we investigate the contributions of viral host cells, primary human airway epithelia (HAE), and immune bystanders, peripheral blood mononuclear cells (PBMCs), in the production of these cytokines. Despite having functional inflammasomes and supporting viral replication, HAE do not secrete IL-1β upon SARS-CoV-2 infection. PBMCs do not support productive viral replication, but the SARS-CoV-2 envelope (E) protein primes inflammasomes through stimulation of TLR2. To activate inflammasomes in PBMCs, SARS-CoV-2-infected HAE provide a second signal in the form of damage-associated molecular patterns (DAMPs), which includes mitochondrial and genomic DNA. Co-culture of PBMCs with infected epithelial cells stimulates IL-1β release, while addition of benzonase, STING, or caspase-1 inhibitors, but not NLRP3 inhibitors, abrogates this response. In turn, IL-1β stimulates IL-6 secretion from both populations with a robust response from HAE. Thus, while infection in either cell type in isolation does not yield IL-1β or IL-6, immune bystander detection of SARS-CoV-2 infection in the epithelium stimulates both IL-1β and IL-6 production, amplifying pro-inflammatory cytokine production through cell-cell communication. This circuit is reflected in patient data, as COVID-19 autopsy lungs show hallmarks of inflammasome priming and activation in myeloid populations lacking SARS-CoV-2 antigens. Meta-analysis of single cell RNA sequencing (scRNAseq) data reveals discordant, exacerbated priming of inflammasomes in myeloid cells from severe but not mild COVID-19 patients, indicating this circuit is intensified in severe disease.

Supported by grants from NIH R56 AI158314, R01 AI158314, AI029564, AI141333 and CA232109 to J.P.-Y.T; NCI T32CA071341 to K.C.B.; UH3 HL123645, R01 HL136961, P30 DK 065988, P01 HL108808, and from the Cystic Fibrosis Foundation (CFF) BOUCHE19R0 to T.A. and R.C.B; CFF grant BOUCHE19XX0 to R.C.B. and K.O.; CFF grant OKUDA20G0 KO; NC Policy Collaboratory, NIH R01AI157253, and U19 AI100625 to M. T. H.; NSF GRFP 2019255197 to J.F.L. T.A. is a Japan Society for the Promotion of Science Overseas Research Fellow. Y.X. is supported by NSF DMS-191290 and NSF IOS 2107215. Y.X and Y.L.L. are supported by NIH grants R01DE026728 and R01DE031951. Some BSL3 work was performed in the Duke Regional Biocontainment Laboratory (Duke RBL) which received partial support for construction from NIH/NIAID (UC6AI058607).
Scavenger Receptor CD36 Regulates Inflammatory and Type-I Interferon Responses in Tumor-Associated Macrophages
Authors: Xu Ziyan Zoe , Kaech Susan
Published: August 2023

Macrophages are plastic cells of innate immune system with diverse functions in tissue homeostasis. Despite their abundance in the tumor microenvironment (TME), the functions of tumor-associated macrophages (TAMs) and their regulation mechanisms remain largely unknown. Macrophages are also key players in metabolic processes including lipid metabolism. As TME has been known as a “lipid-rich” environment, we are interested in understanding how TAMs’ metabolism and function are regulated by lipid receptors.

In this research, we firstly characterized the lipid metabolic phenotype of TAMs in mouse tumor models. We found the tolerogenic subset of TAM (high expression of F4/80 and PD-L1) is lipid-laden and has greater ability to import lipids. We found F4/80hi TAMs highly express scavenger receptor CD36 which binds to oxidized low-density lipoprotein (oxLDL) in TME. Using myeloid-specific knockout (Cd36flox/flox x Csf1r-Cre) mouse models, we observed that CD36-deficient TAMs lost binding to oxLDL, down-regulated expression of immunosuppressive molecules PD-L1 and CD206, while up-regulated secretion of inflammatory cytokines (TNF, IL-12, IL-1b) and IFNb. Overall, this reprogrammed TAM functional state leads to better anti-tumor immunity. Mechanistically, we conducted single-cell RNA-sequencing and found inflammatory response and type-I interferon response pathways were up-regulated in CD36-deficient TAMs. Furthermore, we showed that IFNb and P38 play critical roles in CD36-dependent immune regulations.

In summary, our data suggests that scavenger receptor CD36 plays critical roles in functional reprogramming of TAMs; and blocking CD36 may have multi-faceted beneficial effects for future cancer immunotherapies.

Supported by fellowship from American Heart Association
Infection with LCMV delays the progression of demyelinating disease in EAE
Authors: Essaghir Ibtissam , Cosby Jennifer , Rase Viva Jeanne , Evavold Brian D
Published: August 2023

Multiple sclerosis is a chronic autoimmune demyelinating disease that affects approximately 2.8 million people worldwide. While several preventative treatments for multiple sclerosis, including Natalizumab and Mitoxantrone exist, there currently is no cure. It is also unknown what provides the initial trigger of disease episodes or exacerbation of the ongoing disease although viral infections might play a role. In contrast to their role as a driver of demyelination, here we investigate whether viral infections play a potential protective role. Using a well-established animal model of chronic-progressive autoimmune disease known as Experimental Autoimmune Encephalomyelitis, we found that infection with lymphocytic choriomeningitis virus (LCMV) infection delayed onset of paralysis as compared to uninfected controls. Consistent with the reduction in EAE disease scores, we identified reduced numbers of myelin-specific CD4 T cells in the CNS. The myelin-specific CD4 T cells were found in the periphery suggesting that the virus impeded their trafficking to the CNS. Additionally, we analyzed that virus-specific CD8 T cells were found in the CNS in preference to the myelin-specific CD4 T cells. Thus, in contrast to the prevailing paradigm of infections driving autoimmune disease progression, we find that viral infection can lessen acute demyelinating disease.

R01NS071518 R01AI147641
Investigating the source of viral-specific memory T cells in glioblastoma
Authors: Kleist Sierra A , Musial Shawn C , Isaacs Jordan F , Degefu Hanna N , Skorput Alexander G , Rosato Pamela C
Published: August 2023

Glioblastoma (GBM) is an aggressive primary brain tumor that is invariably fatal. Immunotherapies have largely failed and a better understanding of the unique brain tumor immune environment is needed. Being historically thought of as immune-privileged, immune cell populations within the brain and brain tumors have been understudied. One such population is resident memory T cells (TRM). In healthy and malignant peripheral tissues, TRM can provide long-term protection against reinfections or cancers, however the role of TRM in the context of the brain and brain tumors is not fully understood. Our lab has discovered that memory T cells specific for common viral infections populate human brain tumors to high frequencies and express markers indicative of tissue residency. We also find these virus-specific memory T cells (vsTM) in mouse models of GBM, despite the absence of infection. Surveillance of tumors by bystander (non-tumor specific) memory T cells is understudied despite their potential to contribute to anti-tumor immunity. Indeed, the source of these cells is unknown; they may originate from circulating memory T cells that infiltrate the tumor, and/or pre-existing TRM that were in the brain prior to tumor formation. To dissect the origin of vsTM in brain tumors we established mouse models with distinct memory T cell compartments. Preliminary data suggests that pre-established vsTRM in healthy brain are the main contributors to tumor vsTM. Future work will assess if vsTM are resident within tumors, and extend studies to virus-specific T cell populations in human brain tumors. Our findings will broaden our understanding of GBM immunosurveillance, and provide valuable insights for the development of novel T cell-based immunotherapies.

Supported by grants from the Dartmouth Cancer Center
CD8+ T Cells Differentiate into a Potent CD43+ Effector Population During Acute Allograft Rejection
Authors: Krummey Scott , Cohen Gregory , Kallarakal Melissa
Published: August 2023

Although acute rejection threatens allograft function, little is known about the functional properties of graft-specific effector CD8+ T cells. Using the Balb/c H-2L(d) into C57Bl/6 H-2(b) fully allogeneic skin graft model, we identified host CD8+ T cells specific for the L(d) QL9 epitope using MHC tetramers. L(d) QL9+ T cells increased in number and differentiated into CD44+ effector populations by day 10 post-graft with Balb/c skin, but not third-party C3H skin. Using dimensionality reduction analysis, we found that a population of CD62Llo effector CD8+ T cells expressing the surface receptor CD43 formed during graft rejection in the draining lymph nodes, spleen, and blood. The frequency of CD43+ effectors peaked from day 10–14 post-graft, indicative of a short-lived population. CD43+ CD8+ T cells displayed potent effector functions relative to CD43− cells, including Granzyme B expression, IFN-γ/TNF-α co-production, and accelerated Balb/c graft rejection (p<0.01 for each). Adoptive transfer experiments revealed that only 31% of the L(d) QL9+ T cells divided on day 14 post-graft, versus 95.6% of OT-I T cells after mOVA skin grafting. CD43 expression was only expressed on cells in their final division. Finally, we rechallenged flow cytometry purified CD45.1 CD43+ and CD43− effector CD8+ T cells with a Balb/c skin graft, and found that approximately half of the CD43− population became CD43+, while all of the CD43+ population remained CD43+ on day 21 (49% vs 96%, p<0.001). Together, these data demonstrate that CD8+ T cells differentiate into a CD43+ population after grafting that undergoes weak proliferation and yet is functionally potent.
Mechanisms of aberrant tissue residency programming of uterine natural killer cells in uterus transplantation
Authors: Porrett Paige , Greene Morgan E , Asiimwe Rebecca , Bell Markayla , Wright Emma , Gunn Deidre , Richter Holly
Published: August 2023

Uterine natural killer cells (uNK) are the most common human decidual lymphocytes, but the origin and ontogeny of uNKs remain unclear. Studies of human uterus transplant recipients (UTx) suggest that uNKs originate in the blood, but how these cells become tissue resident and differentiate into decidual NK cells is unknown. We studied the molecular mechanisms governing uNK tissue residency in human endometrium using single-cell RNA-seq and flow cytometry analyses. trNK cells with a CD49a+ CD16− phenotype were the most abundant CD56+ cells in the endometrium of 5 healthy control volunteers (HCs) [27±17% trNK vs. 4±2.7% CD49a− CD16+ conventional NK cells (cNK); p<0.02]. trNK cells differentially expressed genes associated with T cell tissue residency, such as ITGAE and ZNF683, compared to cNK. Although peripheral blood NK cells can express CD103 when incubated with TGF-β, trNKs upregulated CD103 expression in response to IL-15 alone in vitro. Given the dependency of CD103 expression on NFAT signals, we studied CD103 expression on trNKs by incubating human endometrium (n=7) with the calcineurin inhibitor FK506, which decreased IL-15-induced upregulation of CD103 (81% CD103+ [IL-15 alone] vs. 70% CD103− [IL-15 + FK506]; p=0.02). We next tested the in vivo relevance of these findings by comparing the frequency of trNKs in endometrial biopsies of five HCs with two biopsies of one UTx on FK506 immunosuppression. The UTx recipient had significantly fewer trNKs among CD45+ cells in two biopsies (HC: 27±17% vs. UTx: 4.3±2%; p=0.04). Altogether these findings suggest that IL-15-induced calcium signals influence uNK tissue residency, with significant implications for transplant recipients.

This work was supported from UAB AMC21 and support of the uterus transplant program from the University of Alabama at Birmingham. P. Porrett is additionally supported by NIH R01 AI145905.
Water channel Aquaporin 4 is required for T cell activation
Authors: Nicosia Michael , Beavers Ashley , Kish Danielle , Farr George W , McGuirk Paul R , Pelletier Marc F , Fairchild Robert L , Valujskikh Anna
Published: August 2023

Aquaporins are a family of ubiquitously expressed transmembrane water channels. We have previously reported that Aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. The goal of this study was to identify mechanisms of prolonged graft survival following AQP4 blockade.

Stimulation of murine WT T cells + AER-270 or AQP4−/− T cells resulted in decreased expression of T cell activation markers CD25 and CD44, and reduced cell volume when compared to control WT T cells. In addition, the absence or inhibition of AQP4 also limited IL-2 production by T cells following stimulation. Intact AQP4 was also required for optimal IFNg production by T cells. Analogously, AQP4 inhibition significantly limited the ability of human T cells to proliferate following TCR stimulation. We then examined the role of AQP4 in TCR signal transduction and found that AQP4 was required for nuclear translocation of NF-KB and NFAT and for optimal calcium flux following TCR stimulation. Either the absence or inhibition of AQP4 impaired TCR signal transduction as early as phosphorylation of Lck (pY394), ZAP70 (pY319) and LAT (p171). AQP4 was previously shown to associate with the actin cytoskeleton in other cell types, we next tested its involvement in TCR polarization/capping. We found that loss of AQP4 resulted in significantly impaired TCR capping and dysfunctional post-TCR stimulation actin remodeling kinetics.

Our findings reveal novel mechanisms underlying the importance of AQP4 in optimal T lymphocyte activation and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation in solid organ transplantation and other diseases.
Solid organ and vascularized composite allotransplantation have distinct requirements for localized delivery of enhanced costimulation blockade to control rejection
Authors: Raimondi Giorgio , Bodine Amy , Nambiar Monessha , Calderon-Colon Xiomara , Tiburzi Olivia , Hou Shenda , Morrissey Molly , Foley Brendan , Zhang Yichuan , Patrone Julia , Schneider Joel
Published: August 2023

We recently demonstrated, in a mouse heart transplant model, the therapeutic effect of Enhanced Costimulation Blockade: the transient combination of CTLA4-Ig with the JAK inhibitor Tofacitinib (Tofa). In the same model, we also proved the efficacy of a graft-localized delivery by means of a Tofa releasing hydrogel (Tofa-Hydro). In this study, we implemented a mouse orthotopic hind-limb VCA model to delineate any differential drug delivery requirement. Moreover, we explored the potential of Tofa-containing lipid nanoparticles (LNp) in achieving selective targeting of immune cells and its therapeutic effect. Local injections of Tofa-Hydro combined with CTLA4-Ig promoted VCA survival, although with a smaller therapeutic benefit (MST=23 days vs CTLA4-Ig-only MST=14 days). We identified a LNp formulation with negligible toxicity and favorable Tofa encapsulation efficiency. Live animal imaging and flow analysis indicated these particles have the unique property of accumulating in draining lymphoid tissues, with minimal distribution to other tissues, and deliver their cargo to multiple immune cells. Four post-transplant SQ injections of Tofa-LNp (with CTLA4-Ig) promoted graft survival longer than Tofa-Hydro (MST>60 days). Our results reveal that SOT and VCA differ in the topography of inflammatory events that contribute to rejection. This novel observation suggests that different activatory mechanisms contribute to allopriming in the two systems, although they share utilization of JAK signaling pathways. Nevertheless, our study demonstrates the efficacy of localized actuation of enhanced costimulation blockade in achieving safer regulation of rejection.

DOD Award # W81XWH-18-1-0789
Type-1 regulatory T cells are critical for curative immunotherapy outcomes
Authors: Zhang Ping , Gartlan Kate H , Minnie Simone A , Yeh Albert , Takahashi Shuichiro , Atilla Erden , Nemychenkov Nicole S , Boiko Julie R , Schmidt Christine R , Legg Samuel R W , Sekiguchi Tomoko , Ensbey Kathleen S , Koyama Motoko , Furlan Scott N , Hill Geoffrey R
Published: August 2023

IL-10 producing CD4+ type-1 regulatory T cells (Tr1) mediate immune tolerance during chronic infection, autoimmunity, and transplantation. We previously demonstrated that eomesodermin (Eomes) promotes Tr1 cell (Eomes+IL-10+) development after allogenic bone marrow transplantation (alloBMT). Here we define the differentiation trajectory of Tr1 cells and the functional states therein. Eomes−IL-10−, Eomes+IL-10− and Eomes+IL-10+ subsets (defined by mCherry and GFP expression respectively) were FACS sorted after alloBMT and transferred into secondary recipients. Eomes+IL-10+ T cells were stable in phenotype after transfer while the Eomes+IL-10− T cell subset differentiated into Eomes+IL-10+ Tr1 cells. Tr1 differentiation was associated with progressive enrichment for cytotoxicity (Perforin, Nkg7, Granzyme K, Tnfrsf9), immune regulatory (IR) (Tim-3, Lag3, Tox, Nr4a2) and chemokine (Ccl3, Ccl4, Ccl5) RNA expression. Equivalent IR, cytotoxic and cytokine/chemokine expression was confirmed at a protein level. The conditional deletion of Eomes in CD4 T cells prevented Tr1 differentiation and resulted in impaired cytotoxicity in vivo, the accumulation of donor CD11c+ dendritic cells, mixed donor chimerism and leukemia relapse, consistent with a critical role for this lineage in tolerance, engraftment and graft-versus-leukemia effects. The deletion of Eomes from CD19-targeted chimeric antigen receptor (CAR) T cells, reduced cytotoxic molecule expression (perforin, granzyme A/B) in CD4 CAR T cells and resulted in immune escape due to CD19 antigen loss in leukemia cells. In sum, Tr1 cells mediate critical Eomes-dependent cytotoxic and regulatory function that are critical for curative immunotherapy outcomes.
TIGIT agonism alleviates costimulation blockade-resistant CD8 memory T cell responses in transplant
Authors: Hartigan Christina R , Wagener Maylene , Ford Mandy L
Published: August 2023

Tcell Immunoreceptor with Igand ITIM domains (TIGIT) is an inhibitory molecule that has been well characterized in cancer and autoimmunity. We recently published that TIGIT agonism prolongs graft survival in a mouse model of transplantation and showed that this was in part dependent on TIGIT-expressing Tregs. However, we have also observed that TIGIT expression is upregulated on memory T cell subsets that mediate costimulation blockade-resistant rejection in transplantation. In the present study, we aimed to determine the cell intrinsic role of TIGIT agonism on memory CD8 T cells in a murine model of transplant. To do this, we induced immunological memory by performing an adoptive transfer of OT-I T cells prior to infecting animals with Listeria monocytogenes that expresses the model antigen OVA. 30-days after infection, we performed OVA-expressing skin grafts and treated with the costimulation blockade therapeutic CTLA-4Ig or CTLA-4Ig + TIGIT agonist. TIGIT agonist + CTLA-4Ig prolonged graft survival (MST= 14d) compared to CTLA-4Ig treatment alone (MST= 11d, p=0.077) and reduced antigen-specific CD8 T cell infiltration into the graft (4-fold reduction, p=0.017). TIGIT agonist also led to the sequestration of CXCR3 expressing OT-I cells in the draining lymph nodes and impaired trafficking of CXCR3+ OT-I cells to the graft compared to CTLA-4Ig alone. Additionally, TIGIT agonism in vivo led to a decrease in the frequency of TIGIT+IFNg+OT-I cells during ex vivo restimulation (2-fold decrease in CTLA-4Ig + TIGIT agonist vs. CTLA-4Ig alone, p=0.028). TIGIT agonism suppresses memory CD8 T cells in transplant by inhibiting their trafficking and decreasing cytokine production to alleviate costimulation blockade-resistant rejection.

Supported by grants from NIH (5R01AI073707-10, 1F31AI161911 - 01A1)
The adaptation model of immunity: allograft rejection versus allograft adaptation
Authors: Manjili Masoud H
Published: August 2023

Rejection of allografts and acceptance of grafts between identical twins appear to be associated with allogeneity or nonself entity of allografts as suggested by the self-nonself (SNS) model of immunity. Nevertheless, such guilt by association does not necessarily follow a cause-effect direction. The adaptation model proposes that graft rejection or tolerance is determined by the expression of the tissue-specific adaptation receptors (AdRs) which are linked to anti-apoptotic pathways for tolerating the recipient’s alloreactive immune response induced by surgical procedure during organ transplantation. A better engraftment of less matched living-donor organs compared with matched cadaver-donor organs is because of a severely altered AdRs in the latter. To this end, tissue adaptation to an anti-graft immune response suggests that AdRs can be restored in allografts if treated properly. Re-transplantation studies demonstrated that second fresh skin allograft rejection while the first adapted allograft from the same donor remaining viable in the presence of all-reactive T cells. Also, constitutive expression of a bidirectional AdR, B7-H1 linked to Bcl-xL anti-apoptotic pathway, in the cornea and retina protects them from rejection while block-ade of B7-H1 accelerates allograft rejection. Also, severe GVHD following autologous stem cell transplantation could be due to chemotherapy-induced alterations in the AdRs in target tissues. This is supported by demonstrating that autologous TRM cells that are involved in local tissue homeostasis but not alloreactive TEM cells are involved in GVHD. Interestingly, restoration of the putative AdR, B7-H1, expression by parenchymal tissues could prevent or ameliorate GVHD in humans.

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense. This work was also supported by multi-investigator pilot funding from the VCU Massey Cancer Centre, supported in part with funding from NIH/NCI Cancer Centre support grants P30 CA016059.
Tapinarof ameliorates rejection in a human skin grafted model of face transplantation
Authors: Crisler William J , Win Thet Su , Gehad Ahmed , Barrera Victor , Ho-Sui Shannan , Clark Rachael A
Published: August 2023

T cell mediated immune rejection is the primary barrier to broader implementation of face transplants (vascularized composite allografts, VCA). Our bulk and single cell transcriptional profiling of human face transplant rejection demonstrated that VCA rejection is an INFγ driven cytotoxic process with unique induction of skin specific immunoregulatory pathways including SOCS1, KIR3DL2, and LY9. Tapinarof is a novel small-molecule topical aryl hydrocarbon receptor agonist and antioxidant that is effective in treating psoriasis and atopic dermatitis. Patients who cleared on once a day topical tapinarof remained clear for an average of four months after discontinuation, suggesting it has durable immunoregulatory effects in skin. We modeled VCA by grafting NSG mice with freshly excised, living, immunologically intact adult human skin. Once healed, 5×106 allogeneic PBMCs from a second unrelated human donor were infused via tail vein. The inflamed skin mimicked VCA rejection after 21 days, with grafts showing epidermal injury, T cell infiltration, dyskeratosis, and genetic signatures of cytotoxicity, antigen presentation, and INFγ signaling. Topical tapinarof ameliorated this inflammation, with fewer T cells and reduced antigen-specific T cell activation. Induction of immunoregulatory pathways was also noted, including upregulation of SOCS1 signaling, TREX1 mediated reductions in cytosolic nucleotide sensing, and IL1RN inhibition of IL-1 mediated inflammation. Tapinarof could be a novel therapy for VCA rejection that does not require systemic immune suppression. Our results suggest that strengthening immunoregulatory networks in skin is a viable approach to combat inflammation and promote tolerance in VCA rejection.

NIH T32 Training Grant 2T32AR007098-47 Dept. of Dermatology, Harvard Medical School RT170025 (P.I. Rachael A. Clark) Department of Defense - congressionally Directed Medical Research Programs; "T cells and Rejection in Vascularized Composite Allografts).
Mapping the T cell repertoire to a complex defined gut bacterial community
Authors: Nagashima Kazuki , Fischbach Michael A
Published: August 2023

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response. Previous reports have profiled strains from the gut microbiome under artificial conditions of mono-colonization. While this approach can identify strains that have a potential to modulate immune cell function, it does not reveal how a strain behaves in the physiological context of a complex community. Here, we colonize germ-free mice with a complex defined community (>100 bacterial strains) and profile T cell responses to each strain individually. Unexpectedly, the pattern of T cell responses suggests that many T cells in the gut repertoire recognize multiple bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all of the bacteria-specific TCRs exhibit a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that are polyspecific for 18 Firmicutes in the community. By screening three pooled bacterial genomic libraries against 13 pooled hybridomas, we discover that they share a single target: a conserved substrate-binding protein (SBP) from an ABC transport system. Treg and Th17 cells specific for an epitope from this protein are abundant in community-colonized and specific-pathogen-free mice. Our work reveals that T cell recognition of Firmicutes is focused on a widely conserved cell-surface antigen, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

This work was supported by the Stanford Microbiome Therapies Initiative, the Human Frontier Science Program LT000493/2018-L (K.N.), a Fellowship from the Astellas Foundation for Research on Metabolic Disorders (K.N.), a research grant from Kanae Fundation for the Promotion of Medical Science (K.N.), an HHMI-Simons Faculty Scholar Award (M.A.F.); the Leona M. and Harry B. Helmsley Charitable Trust (M.A.F.); NIH grant DK110174 (M.A.F.); the Chan Zuckerberg Biohub (M.A.F.); Stand Up to Cancer (M.A.F.); and the MAC3 Impact Philanthropies (M.A.F.).