bluuebunny/crossref_metadata_2025 huggingface dataset:
Plant derived Nano-11 particle adsorbed with stimulator of interferon genes adjuvant and split influenza virus antigens elicits the cross-protective immunity in pigs
Authors: Gourapura Renukaradhya J , Patil Veerupaxagouda , Hernandez-Franco Juan F , Yadaigiri Ganesh , Bugybayeva Dina , Dolatyabi Sara , Feliciano-Ruiz Ninoshkaly , Schrock Jennifer , Hanson Juliette , HogenEsch Harm
Published: January 2023

We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant (Nano-11) or its combination with a synthetic STING agonist ADU-S100 (NanoS100). Specific pathogen free pigs were vaccinated twice via intramuscular (IM) and intradermal (ID) routes and challenged with a virulent heterologous H1N1. Animals vaccinated IM or ID with H1N2-ADU-S100-Nano-11 had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid. There was a significant increase of IFNγ+ effector memory T-helper/memory cells and late effector cytotoxic T cells (CTLs), IL-17A+ central and effector memory T-helper/memory cells, IL-17A+ early effector CTLs, and CXCL10+ plasmacytoid dendritic cells in the PBMCs of the ID injected H1N2-ADU-S100-Nano-11 compared to H1N2-Nano-11 group. The frequency of IFNγ+ late effector CTLs and effector memory T-helper/memory cells and IL-17A+ late effectorCTLs, as well as in lungs CXCL10+ plasmacytoid dendritic cells was increased in PBMCs of IM injected H1N2-ADUS100-Nano-11 pigs. Increased expression of IL-4 and IL-6 mRNA was observed in tracheobronchial lymph nodes of IM Nano-11 based vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in IM H1N2+ADU-S100-Nano-11 vaccinates by day 6 post infection along with reduced macroscopic lung lesions. In conclusion, despite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs.

Supported by USDA National Institute of Food and Agriculture, AFRI project 2019-67015-29814
B cell selection sites in the nurse shark spleen may represent evolutionary precursors of mammalian germinal centers
Authors: Matz Hanover C , Dooley Helen
Published: January 2023

Sharks are members of the oldest extant taxonomic group (Chondrichthyes) to have immunoglobulin (Ig)-based adaptive immunity. While research has demonstrated that sharks can produce antigen-specific antibody responses, affinity mature these antibodies, and establish immunological memory, histological examination has so far failed to find evidence of germinal centers (GCs). To explore this apparent discrepancy, we immunized several nurse sharks (Ginglymostoma cirratum) with phycoerythrin and visualized the subsequent splenic response using a combination of single nuclei (sn)RNA sequencing and fluorescent microscopy. Our snRNA data reveal the presence of both centrocyte-like and centroblast-like B cells in sharks, as well as a T cell subset expressing several markers found in mammalian T follicular helper cells. RNA fluorescent in situ hybridization experiments show distinct regions of AID expression, polarization of CXCR4 and CXCR5 expression, and association of T cell aggregates with splenic B cell follicles. Fluorescent microscopy clearly demonstrates presentation of nondegraded antigen in the center of B cell follicles. Finally, analysis of Ig variable regions amplified from such B cell follicles reveals evidence of positive selection of clonotypes. Together, our data supports the presence of B cell selection sites in the cartilaginous fish spleen that functionally support the selection of antigen-specific B cells and affinity maturation of the shark Ig repertoire. Further, these sites potentially represent the primordial structures that gave rise to mammalian GCs.

Supported by grants from NIH T32 AI095190, F31 AI147532
Cross-protection by SARS vaccines is improved by booster vaccination
Authors: Dangi Tanushree , Palacio Nicole , Sanchez Sarah , Park Mincheol , Class Jacob , Visvabharathy Lavanya , Ciucci Thomas , Koralnik Igor J , Richner Justin , Macmaster Pablo Penaloza
Published: January 2023

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines have shown extraordinary efficacy against SARS-CoV-2. Recently, we demonstrated that prime-boost coronavirus vaccine regimens can protect against heterologous coronaviruses (Dangi, The Journal of Clinical Investigation, 2021). However, it remains unclear whether boosters are required for such cross-protection. In this study, we show that booster immunizations are critical to elicit cross-protection against heterologous coronaviruses. We first vaccinated BALB/c mice intramuscularly with a poxvirus-based SARS-CoV-1 vaccine developed in 2004 (MVA-SARS-CoV-1) and compared cross-protection following an intranasal SARS-CoV-2 challenge, evaluating cross-protection after a prime-only regimen versus a prime-boost regimen. Interestingly, we show cross-protection only in mice that received boosters. We are currently testing the durability of cross-protection elicited by prime-boost vaccine regimens, and we are also extending these results to humans. Overall, our findings provide a rationale for universal coronavirus vaccines, and highlights the importance of boosters as a strategy to broaden cross-protection to other coronaviruses different than SARS-CoV-2.

Supported by NIDA DP2 Avenir (1DP2DA051912-01) EREEP Grant Northwestern University
Heterogeneity in NK memory stem cells after Zika virus infection using single cell approaches
Authors: Kujur Weshely , Murillo Oscar , Adduri SitaRamaRaju , Mulik Sachin
Published: January 2023

Zika virus infection during pregnancy causes congenital defects such as microcephaly and ocular pathologies. Our lab previously reported that Zika virus infection in mice induces memory like NK cells that express CD27, had higher TCF-1 levels and possessed superior antiviral ability than naïve CD27+ NK cells. Moreover, these cells revealed stem like properties. We termed those NK cells with memory and stem cell like features as “NK memory stem cells (NKSCM). We hypothesized that NKSCM may contain subpopulations that differ in stemness program and antiviral capacity. To determine this, we performed single cell RNA-seq on NKSCM cells. Our analysis revealed 8 distinct clusters in NKSCM. Of these, Cluster 0 showed upregulation of Dusp1, Dusp2, Junb, Fosb while cluster 4 showed downregulation of Dusp1, Dusp2, Junb, Fosb genes. Cluster 5 had upregulation of proliferation associated genes (Mki67, Cdk1 and Pcna). Cluster 3 revealed downregulation of cytotoxic genes (Prf1, Gzma, Gzmb) while gene upregulation of Id3, Myb, Sell, Cd226 and Cxcr3 was evident in this subset. Similarly, other clusters showed differential gene expression profiles. Currently, our analysis is ongoing for single cell ATAC-seq datasets to determine epigenome at single cell level in these NKSCM clusters. Finally, the experiments are underway to functionally characterize these NKSCM subpopulations and to evaluate their role in Zika viral defense.

This work was supported by Department of Pulmonary Immunology, University of Texas Health Science Center at Tyler, TX, USA and American Association of Immunology Careers in Immunology Fellowship
Highly multiplexed, single-cell spatial phenotyping of Epstein-Barr virus infected tissues.
Authors: Nikulina Nadezhda , Ben Cheikh Bassem , Braubach Oliver , Fennell Eanna , Leahy Ciara , Pugh Matthew , Taylor Graham , Murray Paul
Published: January 2023

The Epstein-Barr virus (EBV), a group 1 carcinogen, is a causative factor in nine different cancers that cause ~165,000 deaths globally each year. Almost all adults carry EBV asymptomatically; the virus persisting primarily in rare memory B cells for the lifetime of the host. However, in some infected individuals, for reasons that are unknown, EBV may contribute to the development of cancer. The immune microenvironment in which infected cells reside may influence or trigger cancer development, but we know very little about how this occurs. We have begun to address these critical questions by deploying a spatial biology assay for comprehensive study of the immune microenvironment of EBV-infected tissues. Using AKOYA’s immunofluorescence based Phenocycler assay, we developed an antibody panel that can detect 40 or more different biomarkers in situ at single cell resolution. The panel contains antibodies directed at EBV infected cells at various stages of viral infection, as well as biomarkers for different immune cell lineages, immune cell activation states and tissue structures. This design allows simultaneous identification of infected immune cells within their microenvironment, while, crucially, maintaining spatial relationships between them. We have now set out to produce unbiased and comprehensive spatial phenotypic maps of normal and EBV infected human FFPE tissues. Our data will provide an initial understanding of how the immune microenvironment of virus-infected cells influences tissue pathology and, ultimately, the development of cancer.
Neurological chikungunya virus infection induces corpus callosum degeneration associated with resident immune cell activation and peripheral immune cell infiltration
Authors: Knight Audrey Claire , Nagel Jonathan , Anderson Elizabeth J , Atkins Hannah M , Montgomery Stephanie , Baxter Victoria K
Published: January 2023

Chikungunya virus (CHIKV) is an Old World alphavirus that typically induces arthralgia and rash. However, CHIKV is also capable of infecting the central nervous system (CNS), resulting in encephalitis, myelitis, and peripheral neuropathy. Patients who survive acute infection typically have long lasting neurological effects. To date, an immunocompetent small animal model that recapitulates CHIKV CNS disease has not been established. To develop a mouse model of CNS CHIKV infection, we intracranially inoculated 4–6 week old C57BL/6J mice with 1 of 3 clinical CHIKV isolates: Asian lineage strains SM2013 and AF15561, and ECSA lineage strain SL15649. CHIKV CNS disease is most commonly reported in regions where ECSA lineage strains circulate; in agreement with observations in humans, SL15649 was associated with more severe neurological disease, while SM2013 induced minimal to no disease, and AF15561 induced an intermediate clinical phenotype. At 7dpi when mice demonstrated signs of CNS disease but infectious virus was cleared from the brain, significant corpus callosum degeneration was observed in mice infected with SL15649, and to a lesser extent AF15561. Recruited CD4+ T cells and B cells were significantly elevated in the corpus callosum of mice infected with SL15649 compared to mice infected with SM2013. SL1549 infected mice also had significant micro- and astrogliosis in this area. At 3dpi, prior to neurological signs, the corpus callosum in SL1549 infected mice did not show significant degeneration but did have an influx of peripheral immune cells and increased gliosis. These results suggest that the CNS manifestations of CHIKV are associated with corpus callosum degeneration driven by both peripheral and CNS resident immune cells.

Supported by the following grants: K01 OD026529 (VKB) T32AI0075151 (ACK) AAI Careers in Immunology Fellowship (VKB, ACK) UNC SOM Junior Investigator Development Award (VKB) UNC Department of Pathology and Laboratory Medicine (VKB)
Combined blockade of TNF-a and IFN-I receptors in experimental RSV infection: analysis of STAT protein modulation
Authors: Morris Dorothea R , Qu Yue , de Mello Aline Haas , Ivanciuc Teodora , Ansar Maria , Casola Antonella , Garofalo Roberto P
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory illness in young children. Two cytokines key to RSV pathogenesis are tumor necrosis factor (TNF)-a and type-I interferon (IFN-I). Using a dual receptor antibody blockade of IFNAR1 with either TNFR1 or TNFR2, we previously reported that IFN-I and TNF-a synergistically contribute to clinical disease while also controlling peak lung viral replication in RSV-infected Balb/c mice. The objective of the current study was to investigate the possible effect of TNF-a/IFN-I on prolonged viral shedding and to dissect canonical signaling pathways that would likely mediate the above-described activity. We found that viral titer in the lung of all treatment groups was comparable to the RSV control mice at days six and seven post-infection, indicating that lack of these cytokine receptor signaling was not involved in prolonged viral shedding. We then determined expression levels of total lung STAT1, STAT2, STAT3 and their phosphorylated forms as well as IRF1, pIRF3, IRF7, and IRF9 by western blot. Though RSV/IFNAR1, RSV/TNFR1, and RSV/TNFR2 demonstrated variations in protein expression for several of these signaling molecules, none of these proteins were uniquely modulated by the dual receptor blockade. These findings collectively demonstrate that while TNF-a/IFN-I receptor signaling controls peak viral replication in the lung, they have no effect on extended viral shedding and that STAT1, STAT2, and STAT3 signaling appears to be dispensable for the enhanced viral replication observed in the dual receptor blockade treated mice.

Supported by grants from AI062885, AI25434, Clinical and Translational Science Award [(NRSA (TL1) Training Core TL1TR001440)],Supported by UTMB McLaughlin Fellowship
Understanding innate immune cell-mediated inflammation in UV-induced skin injury in lupus-prone mice.
Authors: Maz Mitra Padideh , Shi Hong , Wolf Sonya , Estadt Shannon Nicole , Reddy Alayka , Kahlenberg J Michelle
Published: January 2023

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by sensitivity to ultraviolet (UV) light and overproduction of type I interferons (IFNs), in which skin is affected in up to 70% of total SLE patients. While UV light is one of few well-known triggers of SLE, precise mechanisms governing UV-mediated inflammation are incompletely understood. Our lab recently performed single-cell analysis of cutaneous lupus lesions, showing an increase in activated monocyte-derived CD16+ dendritic cells (DCs) in both lesional and non-lesional skin of lupus patients compared with healthy controls. With the goal of understanding the functionality of this subset, we have turned to lupus-prone murine models. RNA sequencing of UV-exposed skin samples from lupus-prone NZM2328 (NZM), iNZM (lupus-prone mice lacking the type I IFN receptor) and wildtype Balb/c mice demonstrated upregulation of monocyte chemoattractants like CCL2 in NZM greater than iNZM and Balb/c, which was verified at the transcript and protein level of UV-irradiated human lupus keratinocytes compared with healthy controls. We further verified recruitment and differentiation of monocytes into monocyte-derived DCs (moDC) in UV-irradiated skin of NZM, iNZM, and Balb/c mice, as well as increased MHC class II expression in skin-recruited moDCs of chronically UV-exposed NZM mice greater than iNZM and Balb/c, suggesting an importance of type I IFN signaling in this process. Future studies will be directed at understanding mechanisms of monocyte recruitment and differentiation at sites of UV-mediated inflammation, as well as type I IFN effects on moDC antigen uptake and presentation.

Supported by grants from NIH (T32-GM007863, R01-AR071384, and K24-AR076975)
Pathogenic Tissue-Resident Memory T Cells in Exocrine Gland Chronic Graft-Versus-Host Disease
Authors: Costa da Silva Ana C , Sharma Rubina , Kim Clara H , Javaid Ayesha , Martin Daniel , Mays Jacqueline W
Published: January 2023

Salivary glands are frequently affected in chronic graft-versus-host disease (cGVHD), an autoimmune-like disease that occurs after allogeneic hematopoietic stem cell transplant resulting in tissue sclerosis and gland dysfunction. Alloreactive T cells have been identified as disease drivers, yet the function/pathogenicity of tissue-resident memory cells (Trm) in target organs remain unclear. In this study, we examined glandular Trm populations and tissue-specific signals that regulate Trm differentiation and functionality. scRNAseq analysis of human labial minor salivary glands (MSG) revealed enrichment of immune cell clusters in MSG of oral cGVHD patients. Focusing on T CD8 cells, we identified 4 clusters, one of them unique to affected patients which had low expression of CD69, but expressed other markers of Trm, including CD103, BLIMP1 and PD1. Additionally, this cluster was enriched for genes associated with cytotoxicity (IFNG, PRF1 and GZMB) and exhaustion (TIM3, LAG3 and TIGIT). Interestingly, this population showed high expression of CXCR6 and ligand-receptor analysis predicted CXCL16 an important signaling ligand that targets T CD8 cells. Further analysis implicates macrophages as the main source of CXCL16, suggesting their role in local accumulation of CXCR6+ Trm in the gland. Flow cytometry confirmed increased number of CD69+CD103±CD8+ Trm cells in cGVHD patients. Lymphocytic infiltration in MSG using IHC, spatially related the elevated frequency of CD8+IFNg+ T cells within interacinar stroma and damaged acini. The importance of Trm in site-specific cGVHD still needs further investigation. Based on current data, site-specific Trm cells may represent a druggable target in the therapy of refractory local cGVHD.

Supported by NIDCR (1ZIADE000747-05)
Responses of injured nerve in CD137L knockout mice following sciatic nerve crush-induced neuropathic pain
Authors: Cao Ling , Di Nucci Bruno Souza , Wakley Alexa , Ohara Kinuyo
Published: January 2023

Previously, we showed that CD137L, a costimulatory molecule is involved in the development of neuropathic pain following sciatic nerve crush (SNC). CD137L knockout (KO) mice displayed reduced SNC-induced pain-like behaviors and faster recovery following SNC. To determine the responses at the nerve crush site, we further examined the phenotypic and inflammatory changes of injured sciatic nerve in both CD137L KO mice and wild type (WT) C57BL/6 mice. Both CD137L KO and WT mice underwent either SNC or sham surgery. Sample collection occurred at days 0 (naïve), 3, 7, 14, 28, and 56 post-surgery. First, the femur-level segment of sciatic nerve containing the injury site was analyzed via whole mount immunohistochemistry. At the baseline, CD137L KO mice showed higher staining intensity for IB4 (non-peptidergic c-fibers) compared to WT mice. In WT mice but not CD137L KO mice, SNC significantly increased staining intensity for CGRP (all times; peptidergic c-fibers) and IB4 (Days 3 and 28). These increases were not observed in the corresponding CD137L KO mice. Second, selected inflammatory markers were measured via real time qRT-PCR. At baseline, CD137L KO mice expressed significantly lower levels of TNFα, yet higher levels of IL-10. SNC induced higher levels of elevation of IL-6, CCL3, CCL4, and Arg-1 (all peaked at day 3) in CD137L KO mice compared to WT mice. WT but not CD137L KO mice, showed significant up-regulation of CCL5 (throughout the time course) and CXCL1 (peaked at day 14). Therefore, we propose that CD137L-mediated local inflammatory responses may contribute to SNC induced pain-like behaviors in part by affecting the phenotype of injured nerve.

Supported by NIH/NINDS R01NS098426 (Cao), COBRE Histology and Imaging Core funded by NIH/NIGMS P20GM103643 (Meng), and the Kahn Family Foundation through the Carmen Pettapiece Student Research Fellowship (Souza Di Nucci).
MicroRNA control of inflammatory and regulatory T cells in CNS autoimmunity
Authors: Gopal Murugaiyan , Fujiwara Mai , Raheja Radhika , Garo Lucien , Ajay Amrendra , Chitnis Tanuja , Weiner Howard L , Gandhi Roopali
Published: January 2023

A disequilibrium between immunosuppressive regulatory T cells (Tregs) and inflammatory interleukin (IL)-17-producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, molecular mechanisms underlying Treg and Th17 imbalance in the central nervous system (CNS) autoimmunity remain largely unclear and thus identifying factors which drive this imbalance is of high clinical interest. Recently, we found a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. MiR-92a is elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuates EAE. Mechanistically, miR-92a mediates EAE susceptibility in a T cell-intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses by directly repressing the transcription factor, Foxo1. Correspondingly, miR-92a inhibitor therapy ameliorates EAE by modulating the balance between Tregs and Th17 cells. Analogous to mice, miR-92a is elevated in MS patient CD4+ T cells, and miR-92a silencing in patient T cells promotes Treg development while limiting Th17 differentiation. Together, our results identify a previously unknown function by which miR-92a drives CNS autoimmunity via sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.

Supported by NIH R01 R01AI127853 NMSS RG-1507-05164
Secretome of adipose-derived mesenchymal stem cells establish anti-inflammatory milieu by developing M2b/c macrophage
Authors: Yoon Taejun , Ko Eunhee , Park Yong-Beom
Published: January 2023

Mesenchymal stem cells (MSCs) express immunomodulatory and anti-inflammatory effect toward immune system with several mechanisms such as secreting bioactive molecules. Distinct mechanisms of their immune actions, however, are not fully understood. As one of the important cells in immune system, macrophages play a vital role in innate immunity with two functional phenotypes, classically activated (M1) and alternatively activated (M2) macrophages. In this study, immunomodulatory ability of adipose-derived MSCs (AD-MSCs) secretome toward peritoneal macrophage was studied. Secretome was concentrated from human AD-MSCs using 3K TFF membrane. Murine peritoneal macrophages were cultured for 24 hours with secretome of AD-MSCs and 4 hours with LPS. Macrophages were analyzed by flow cytometry, ELISA, and real-time PCR. LC-MS/MS proteomics analysis of secretome was performed and acquired protein sets were analyzed by DAVID Bioinformatics. Secretome of AD-MSCs increased cell viability of peritoneal macrophages. Secretome treated peritoneal macrophages resulted in reduced M1 markers such as CD86, iNOS, and TNFα and increased M2 markers such as CD206, Arginase-1, and IL-10. Also, peritoneal macrophage expressed upregulation of mRNA level of LIGHT and MerTK, M2b and M2c markers, respectively. 83 proteins were identified in secretome according to the proteomics analysis. Several proteins among them were selected as candidates after confirmation of inhibiting the effect of secretome with neutralizing antibodies. In conclusion, secretome of AD-MSCs induced polarization of peritoneal macrophages from M1 macrophages to M2 macrophages and made them create anti-inflammatory milieu by several key secreted proteins.

Supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2021R1F1A1049630) and the Ministry of Health and Welfare, Republic of Korea (HI14C1324)
The long noncoding RNA LUCAT1 promotes immune gene expression in human macrophages
Authors: Vergara Kevin E , Lal Sneha , Cattley Richard , Hawse William F , Atianand Maninjay
Published: January 2023

Long noncoding RNAs (lncRNA) are transcripts larger than 200 nucleotides that mediate their molecular function in an RNA-dependent manner by regulating gene expression at either the transcriptional or post-transcriptional level. lncRNAs play vital roles in many biological processes; however, their functions in immune cells are largely unexplored. Here we describe the expression, function and the mechanism of action of LUCAT1 (Lung cancer associated transcript 1; SCAL1) in human macrophages. While elevated LUCAT1 expression is observed across many cancers, its cellular function in physiological contexts remains unknown. We show that Toll-like receptor 4 (TLR4) and PMA upregulates LUCAT1 expression in human monocyte-derived macrophages and THP1 cells through ERK1/2 signaling. Using 3’-RACE, long-range PCR and RNA FISH, we demonstrate that the LUCAT1 gene transcribes an abundant, 2562-nucleotides long nuclear-localized RNA. Transient perturbation of the nuclear LUCAT1 using antisense oligonucleotides followed by RNA sequencing and protein assays revealed that LUCAT1 promotes the expression of cytokines and chemokines (CSF3, IL24 and CXCL5), transcription factors (NR4A1 and NR4A3), matrix metalloproteinases (MMP1, MMP3 and MMP19) and others in human macrophages. Using siRNA-mediated knockdown of LUCAT1-interacting nuclear proteins, we show that RBMX contributes to the molecular function of LUCAT1, whereas SFPQ controls the biogenesis of the mature LUCAT1. Further, the nuclear LUCAT1 is chromatin-associated, and acts at the level of gene transcription. Collectively, in this study we have uncovered the function of LUCAT1 in macrophages, underscoring the critical roles of lncRNAs in the immune system.
Aging alters murine peritoneal macrophages in a sex-dimorphic fashion
Authors: Lu Ryan , Wang Eric , Kim Minhoo , Sampatkumar Nirmal K , Benayoun Bérénice A
Published: January 2023

Aging is accompanied by general immune dysfunction, which is hypothesized to underlie the pathogenesis of many age-related diseases. In addition, there is accumulating evidence that aging is highly sex-dimorphic. However, the interactions of age and sex on the genomic and functional landscape of immune cells remain largely unexplored. To address this question, we have interrogated cells of the peritoneal cavity, an easily accessible immune niche, during aging in both sexes of C57BL/6 mice using single cell RNA-seq and flow cytometry. Interestingly, we found that macrophages were the most affected by age and sex among peritoneal immune cells, and that their frequency significantly dropped with aging. Macrophages are important for the initiating and resolving of inflammation, and dysfunction can contribute to inflammaging. Thus, we decided to further investigate the potentially sex-dimorphic impact of aging on the genomic landscape and function of peritoneal macrophages. We generated bulk RNA-seq data from purified peritoneal macrophages and identified genes modulated with aging as a function of sex. Complementing this ‘omic’ dataset, we performed an array of functional assays using purified peritoneal macrophages. We observed sex-dependent functional alterations, such as female-specific decrease in phagocytic ability. From our RNA-seq analysis, we identified potential regulators of phagocytosis that are significantly downregulated with age only in females. We are currently validating whether the potential regulators may indeed drive the observed female-specific decrease in phagocytic capabilities. Ultimately, this project will help delineate sex dimorphic mechanisms modulating immune aging.

This work was supported by a Diana Jacobs Kalman/AFAR Scholarships for Research in the Biology of Aging and NIA T32 AG052374 (to R.J.L.), GCRLE-2020 post-doctoral fellowship from the Global Consortium for Reproductive Longevity and Equality at the Buck Institute, made possible by the Bia-Echo Foundation, (to M.K.), and NIA R00 AG049934, Pew Biomedical Scholar award #00034120, an innovator grant from the Rose Hills foundation, and the Kathleen Gilmore Biology of Aging research award (to B.A.B). This work was also partially supported by NCI Cancer Center Support Grant P30 CA014089 through the use of shared resources.
Do adipose macrophages remember obesity? Innate immune memory in weight cycling
Authors: Caslin Heather L , Cottam Matthew , Winn Nathan , Hasty Alyssa
Published: January 2023

Weight loss improves obesity-associated metabolic disease; however, it is difficult to accomplish and even more difficult to maintain. Thus, many individuals regain weight within a few years. Unfortunately, weight cycling worsens the risk of developing diabetes beyond obesity itself. Adipose immune cells contribute to inflammation and diabetes risk with obesity, but less is known about adipose immune cells in weight loss and weight cycling.

New data from a mouse model in our lab suggest that weight loss rescues glucose tolerance, but the immune landscape is not restored to a lean state. Adipose memory T cells, lipid handling macrophages, and exhausted T cells remain elevated with weight loss and further increase with weight regain suggesting that immune cells remember the state of obesity. Because immune memory can occur in the myeloid lineage, this project aims to determine if weight cycling induces innate memory in adipose macrophages.

We find that adipose macrophages retain their obese phenotype; with increased glycolysis and cytokine production in response to LPS. Interestingly, adipose macrophages from weight cycled mice are even more metabolically active and secrete more basal TNF. We can replicate this phenotype in a cell culture model of innate memory with palmitate or adipose conditioned media, which may be a useful model to determine mechanisms for metabolic memory. Current work is investigating metabolic and epigenetic regulation of both the in vitro and in vivo systems using ATAC-sequencing and metabolic flux analysis. Future studies will explore the contribution of adipose macrophage memory to weight cycling-accelerated metabolic disease and aim to uncover new therapeutic targets to improve metabolic dysfunction.

This work was funded by a Veterans Affairs Merit Award (5I01BX002195) to AH and a T32 Training Grant (DK07563) and AHA Postdoctoral Fellowship (20POST35120547) to HC.
Choline metabolism promotes M2 macrophage polarization in intestinal infection with helminth Heligmosomoides polygyrus
Authors: Kim Sang Yong , Ghorbani Peyman , Woo Daniel , Fullerton Morgan D , Nair Meera G
Published: January 2023

Choline is an essential nutrient functioning as a precursor for membrane phospholipid. In previous studies, LPS polarization augmented both the expression of the choline transporter CTL1 and choline uptake, while specific inhibition of choline transport led to increased TNFα and IL-6. However, whether choline metabolism regulates M2 macrophage polarization or Th2 cytokine inflammation in vivo is unclear. In vitro, CTL1 and phospholipid synthesis was induced in IL-4 polarized M2 macrophages. To determine the function of choline metabolism in M2 polarization in vivo, mice were infected with Heligmosomoides polygyrus, intraperitoneally injected with choline kinase α inhibitor, RSM-932A or vehicle, and sacrificed at day 17 post-infection. RSM-932A treatment impaired weight gain and peritoneal exudate cell numbers in both naïve and infected mice. Within the peritoneal cavity of infected mice, macrophages and B-1 lymphocytes were depleted by RSM-932A treatment, while monocytes and neutrophils were increased. Flow cytometric and intestinal immunofluorescence staining revealed that RSM-932A treatment prevented M2 macrophage polarization in H.polygyrus-infected mice with a significant reduction in expression of PD-L2 and CD206, and conversely increased expression of CD86 and PD-L1. Additionally, RELMα protein in the serum and peritoneal fluid was downregulated by RSM-932A treatment. The impaired M2 polarization was associated with some loss in optimal immunity to H.polygyrus with increased parasite egg burden but no differences in intestinal worm count. This study indicates that choline metabolism is required for M2 macrophage polarization and an optimal immune response against intestinal helminth infection.
NK cell-derived IL-10 enhances Plasmodium-specific CD4+ Tfh differentiation and IL-27 production in mice
Authors: Burrack Kristina S , Harris Morgan , Dileepan Thamotharampillai , Jenkins Marc K , Frosch Anne
Published: January 2023

Malaria, which results from infection with Plasmodium parasites, remains a major public health problem with over 200 million cases and nearly 500,000 deaths annually. Although long-lived, sterilizing immunity does not develop in humans following repeated exposure to Plasmodium parasites, protection against symptomatic disease correlates with the acquisition of humoral immunity. Despite the established role antibodies play in protection to malaria disease, excessive inflammation is thought to contribute to the ineffective immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously showed that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death by dampening the fatal inflammatory response. Interestingly, IL-15C treatment promotes Tfh differentiation at 7 days post-PbA infection (dpi), and genetic deletion of NK cell-derived IL-10 or antibody blockade of IL-10R prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment enhances CD4 T cell-derived IL-27p28 production at 7 dpi and results in increased anti-PbA IgG antibody levels at 42 dpi. These data suggest that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the inflammatory milieu to promote Tfh differentiation, IL-27 production, and PbA-specific antibody generation. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.

Supported by grants from NIH (K22 AI143969-01A1, K08 AI141761-01)
Cell-specific roles for miR-155 during neuroinflammation
Authors: Thompson Jacob W , Huffaker Thomas , Hu Ruozhen , O’Connell Ryan M
Published: January 2023

The pro-inflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). The whole-body deletion of miR-155 in mice confers resistance to a mouse model of MS, EAE, by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. Here we utilize single-cell RNA sequencing and cell-specific conditional miR-155 knockouts to determine the importance of miR-155 expression in distinct cell populations. Time course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells in whole-body miR-155 knockout mice compared with wild-type controls at day 21. Deletion of miR-155 in T cells, driven by CD4 cre, reduced disease severity similar to whole-body miR-155 knockouts. CD11c cre-driven deletion of miR-155 in dendritic cells also significantly reduced EAE disease score, with both T cell and dendritic cell-specific knockouts showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in macrophages during EAE, deletion miR-155 by LysM cre in did not affect disease severity.
Classification of novel conditional POSH knockout murine models
Authors: Guldenpfennig Caitlyn E , Guan Yue , Teixeiro Emma , Daniels Mark
Published: January 2023

Plenty of SH3 Domains (POSH) is a critical scaffold protein in neuronal and lymphocyte signaling involved in survival, development, and effector function. Our lab has shown a sharp decline in survival and activation in T cells and decreased survival of lymphocyte leukemias upon inhibition of POSH function in vitro. However, the in vivo function of POSH in lymphocyte survival and function has not been examined. We hypothesized that POSH deletion in vivo impacts murine lymphocyte development and effector function. We used the Cre/Lox system to conditionally knockout POSH in T cells (CD4-Cre) or B cells (CD19-Cre) in C57B6 mice. POSH deletion in lymphocytes was examined through Western Blot and RNA extraction, and the effect of the loss of POSH on development and function was assessed via flow cytometry. We found that CD4-Cre T cells showed a developmental block at the maturation stage in the thymus. Upon stimulation, CD4-Cre T cells showed reduced proliferation and cytokine production. Additionally, CD19-Cre B cells showed developmental defects, including more BP-1 expression and a higher frequency of Pre-B early cells. This study demonstrated that the in vivo deletion of POSH impacts development, proliferation, and effector function of lymphocytes. These data corroborate previous in vitro results with our inhibitor. Thus, these novel murine models will enable us to assess the role of POSH in developing a fully functional immune compartment and its role in supporting leukemia oncogenesis and survival.

Supported by grants from NIH-MBARC, NIBIB (EB026560), NIGMS (GM135744), DoD (RT160149)
Mediator complex maintains peripheral T cell tolerance through enforcement of the quiescence module
Authors: Chaudhuri Shuvam Mohan , Zhang Yana , Weinberg Samuel E , Fang Deyu
Published: January 2023

T cell tolerance has traditionally been thought to be maintained at stages after T cell receptor mediated activation either through anergy, exhaustion, contraction or T Regulatory cell mediated dominant suppression. Prior to these mechanisms, however, T cell quiescence is the main factor maintaining T cell tolerance. We have identified Mediator Complex Subunit 1 (Med1), as a novel factor responsible for enforcing T cell quiescence. Mice with T cell specific deletion of Med1 display increased levels of T cell activation despite having a normally functional T Regulatory cell compartment. Med1-deficient T cells were more responsive to T cell receptor mediated activation. Bone marrow chimeras revealed Med1 controls T cell quiescence in a cell intrinsic manner. Bulk and single cell transcriptome sequencing revealed Med1’s transcriptional control over key quiescence factors. By analyzing previously published chromatin immunoprecipitation sequencing we found Med1 controlled a transcription factor network rather than direct quiescence molecules. Med1 is a novel factor responsible for actively enforcing the transcriptional network maintaining T cell quiescence.

Supported by T32DK007169 R01CA257520 R01CA232347
Overcoming inhibitory constraints to TCR signaling by targeting the pH-sensitive phosphatase suppressor of T-cell receptor signaling 1 (STS1)
Authors: Tsai Yuan-Li , Badia Marcel Arias , Kadlecek Theresa A , Shah Neel H , Fong Lawrence , Weiss Arthur
Published: January 2023

T cells are critically important for adaptive immune responses. Therefore, maintaining their proper homeostasis an d controlling the magnitude of their responses is critical. To control T cell responses, T cell activity is negatively regulated by inhibitory molecules. The Cbl protein family are ubiquitin E3 ligases that suppress TCR signaling by mediating the degradation of TCR signaling molecules. However, studies and our results suggest that Cbl proteins down-regulate phospho-proteins without modulating the total level of target proteins, but the mechanisms are unclear. Here, we show that Cbl-b serves as an adapter to recruit STS1, an unconventional tyrosine phosphatase, to dephosphorylate target proteins and inhibit TCR signaling. STS1 binds to a Cbl-b PPVPP motif via its SH3 domain, and the interaction requires Cbl-b phosphorylation by ZAP70. Elimination of the PPVPP motif compromises Cbl-b suppressive activity, and deletion of STS1 leads to T cell hyper responsiveness to TCR activation which phenocopies Cbl-b deficiency. Furthermore, STS1 uniquely relies on histidine to catalyze dephosphorylation, making it hyperactive in acidic environment. As a result, STS1 and Cbl-b deficient T cells remain hyper responsive and insensitive to low pH whereas WT cells are largely suppressed. Animals with STS1 or Cbl-b deletion show delayed tumor growth and improved survival. Together, these results demonstrate Cbl-b partners with STS1 whose activity is responsive to pH changes to modulate TCR signaling. Moreover, they suggest the pH sensitivity of STS1 is uniquely suited to suppress T cell responses that occur in vivo, such those that occur within inflammatory settings and within tumors, where lower pH conditions may be encountered.

Supported by fellowship from Cancer Research Institute (A133417)
Absence of the fatty acid binding protein5 (FABP5) inhibits the establishment of resident memory T cells after a secondary Listeria monocytogenes infection
Authors: Aviszus Katja , Giron Aaron , El Kharbili DiLisio Manale , Zhao Xiaoyun , Gally Fabienne
Published: January 2023

Fatty acid binding protein 5 (FABP5) is a member of the family of fatty acid binding proteins involved in fatty acid uptake, transport, and metabolism. FABP5 is ubiquitously expressed, including in T cells. During the course of an immune reaction, T cells undergo multiple changes to their metabolism. Once naïve T cells are activated, expansion is triggered, and cells differentiate to become effector cells accompanied by a metabolic change from fatty acid oxidation to glycolysis. Once the pathogen is eliminated, effector cells differentiate into memory cells, with central memory (TCM) and effector memory (TEM) cells surveying the circulation and resident memory (TRM) cells establishing a tissue memory pool that can quickly react to localized reinfection. These TRM cells are thought to rely on lipid uptake and fatty acid oxidation for survival. We wanted to investigate the effect of FABP5 on the development of CD8+ TRM cells in the context of a Listeria monocytogenes (Lm) infection. We had previously shown that the lack of FABP5 leads to a resistance to Lm in mice after a single infection, while the overexpression of FABP5 showed increased mortality and bacterial burden. Here, we used WT and FABP5−/− mice in an infection model with Lm-Ova to analyze the innate and antigen-specific adaptive immune responses. To this end, WT and FABP5−/− mice were infected i.v. with Lm-Ova and their immune responses analyzed at various days after. We show that while the primary immune response is improved in FABP5−/−, there was a profound negative effect on the secondary response. This was based on a severe reduction of Ova-specific TRM in the liver while TCM and TEM populations were unaffected. Flow analyses and functional assays taken throughout will be presented.

Supported by NHlDI 1R01HL141264

Supported by NHlDI 1R01HL141264
TCR-enriched microvesicles provide antigen-specific help for class-switched antibody production
Authors: Li Fenglei , Choudhuri Kaushik
Published: January 2023

Extracellular vesicles have shown essential roles in various biological processes, mainly through mediating cell-cell communications by its surface and internal cargo. Recently it was found that activated T cells release TCR enriched microvesicles (Tc-MV) from immunological synapse, but the function of Tc-MV is still a mystery. By analyzing proteomics and miRNA profiles of Tc-MV derived from mouse OT-II CD4 T cells (OVA323–339 specific), we found Tc-MV are enriched with proteins related to T cell helper function, including TCRs, CD40L, CD28, ICOS, OX40 and LFA-1, and also contained B cell function involved miRNAs, such as miR-21a-5p, miR-28a-3p, miR-155-5p and miR-210-3p. Accordantly, by in vitro coculture, OT-II Tc-MV could promote NP-specific B cells to secret IgG when cognate antigen NP-OVA was present, similarly Tc-MV from SMARTA T cell (LCMV GP61–80 specific) could promote B cells with antigen NP-GFP-GP61–80, but switching either the MV or the antigens in these two systems dampened the antibody production, suggesting Tc-MV help B cells in an antigen specific manner. The helper function of the Tc-MV was also confirmed in vivo by serum antibody production of Rag1−/− mice transferred with Tc-MV and B cells and followed by immunization, importantly the endogenous Tc-MV were found in the serum of the WT mice post-immunization, demonstrating a fundamental role of Tc-MV in the physiological condition. In conclusion, our findings revealed a critical role of Tc-MV in promoting antigen specific antibody production of B cells, which can be exploited for vaccine design.

Supported by University of Michigan Startup Funds and NIH: R01AI134999.
Age-dependent changes in the regulatory program of CD8+ Regulatory T cells (CD8+ Tregs)
Authors: Srinivasan Saranya , Chaoyu Ma , Mishra Shruti , Wang Liwen , Fan Kenneth , Zhang Nu
Published: January 2023

Regulatory T cells (Tregs) help maintain the immune homeostasis. Any defect in the Tregs causes dysregulated immune responses that leads to risk of infection, autoimmunity, and cancer. Increased morbidity in the elderly patients is due to age-related changes in the immune system called immune aging. Despite several age-related studies in immune cells, immune aging in CD8+ Tregs are poorly characterized. With the growing number of evidence for the presence of various CD8+ Treg subset, our group has recently discovered that mice with knock out of specific CD8+ Treg subset characterized as CD8+ CD122+ Helios+ developed lethal autoimmune phenotype. To our surprise, the autoimmune phenotype is CD4+ Treg independent which underscores the important role of CD8+ CD122+ Helios+ Tregs in autoimmunity. This study investigated the age-associated changes in the CD8+ CD122+ Helios+ Tregs. We measured the quantitative and phenotypic changes of CD8+ Tregs at homeostasis in young (2 months) and older (12–24 months) mice. Our results demonstrate that at homeostasis, frequency of CD8+ Tregs in total lymphocytes is higher in older mice. Of note, older CD8+ Tregs upregulated the expression of Natural Killer group 2-member protein like NKG2A and NKG2D. We used acute infection model to test the age-related effect of infection/inflammation in the CD8+ Treg. We found that Treg numbers further expanded in older mice during acute infection and functionally superior to young mice. In addition, older CD8+ Tregs exhibited altered metabolic profile which might contribute to the immune senescence. In summary our findings support that CD8+ Treg may contribute to the weakened immune response in the elders.
T cell fate and central tolerance: using iTregs to elucidate the persistence of thymic development on a T cell’s behavior
Authors: White Jason , Ishihara Kazumi , Sakaguchi Shimon
Published: January 2023

Evidence continues to mount in the T cell field that a cell’s affinity for self-peptide MHC predisposes the T cell to particular outcomes when challenged with certain stimuli (whether that stimuli be self or foreign antigen). This aspect of the system suggests that there is, perhaps, some amount of thymic “programming” that occurs during the development of the T cell which serves as another tolerance mechanism; in this case, bridging the central/peripheral tolerance paradigm. In this project, using an induced Treg model and controlling potential disparities in signalling and TCR strength whenever possible, we attempt to determine to what extent the fate of a naive T cell can remain plastic, and to what extent it still reflects the circumstances of its development.