bluuebunny/crossref_metadata_2025 huggingface dataset:
C-type lectin-like NKC-encoded immunoreceptors NKp80 and NKp65 are selectively expressed by human innate lymphocyte subsets, uniquely signal via hemITAMs and facilitate tissue-specific immunosurveillance via their genetically linked ligands AICL and KACL
Authors: Steinle Alexander Luis , Bauer Björn , Vogler Isabel , Leibelt Stefan
Published: January 2023

NKp80 is an activating C-type lectin-like immunoreceptor (CTLR) on human NK cells. Upon ligation by its cognate ligand ‘activation-induced C-type lectin’ (AICL) up-regulated on PAMP-stimulated myeloid cells and interleukin-experienced NK cells, NKp80 stimulates NK cytotoxicity and cytokine secretion. Unlike other NK receptors, NKp80 uses an atypical cytoplasmic hemITAM for cellular activation. In contrast to “classical” hemITAMs of related myeloid CTLR, we showed that the NKp80 hemITAM does not recruit Syk upon phosphorylation. However, we now provide evidence for specific recruitment of other signaling molecules by NKp80.

Alike its close relative NKp80, NKp65 is a homodimeric CTLR with a cytoplasmic hemITAM and encoded in the Natural Killer Gene Complex (NKC). NKp65 binds with high affinity to its cognate genetically linked ligand KACL almost exclusively expressed by human keratinocytes. We previously showed that NKp65/KACL interaction activates cytotoxicity by NK-92 cells. However, while NKp80 is expressed by virtually all human NK cells, physiological expression of NKp65 remained elusive. Generation of NKp65-specific mAb enabled us to trace NKp65 expression to a minor subset of human innate lymphocytes. Transcriptomics revealed strict correlation between NKp65 expression ILC hallmark genes. Hence, NKp65 expression defines that ILC subset and enables a tissue-specific cross-talk with human keratinocytes. Moreover, we show that NKp80 and NKp65 are mutually exclusive expressed by NK cells and that ILC subset, qualifying NKp80 and NKp65 as markers of these 2 distinct innate lymphocyte lineages and raising questions for their respective subset-associated function.
Hypoxia inducible factor-2α in resting macrophages tempers mitochondrial reactive oxygen species to selectively repress MARCO-dependent phagocytosis.
Authors: Dehn Shirley , Thorp Edward B
Published: January 2023

Hypoxia Inducible Factor α (HIF-α) isoforms promote many key components during wound healing including angiogenesis and glycolytic energy metabolism. In particular, HIF2α is required during proinflammatory cytokine induction within macrophages. However, requirements for HIF2α during inflammation resolution pathways, including signaling during nonphlogistic phagocytosis of apoptotic cells, i.e. efferocytosis, is unclear. While HIF2α was not required for hypoxic efferocytosis, basal HIF2α levels under normoxic conditions in resting bone marrow macrophages were both necessary and sufficient to inhibit the clearance of apoptotic targets, indicating that HIF2α functions as a native suppressor of efferocytosis. A screen of candidate phagocytosis receptors revealed that absence of HIF2α led to selective transcriptional induction of the scavenger receptor MARCO. Antibody blocking or MARCO-deficiency prevented the enhanced engulfment phenotype in HIF2α−/− macrophages. Gene silencing identified the antioxidant NRF2 as the transcription factor responsible for driving elevated MARCO levels. Concordantly, Hif2α−/− phagocytes expressed reduced antioxidants and increased levels of mitochondrial ROS. Inhibition of mROS suppressed NRF2 nuclear translocation, MARCO expression, and efferocytosis. Ex vivo findings were recapitulated in vivo, wherein the enhanced engulfment phenotype was extended to bacterial clearance. Uniquely, HIF2α can act as a phagocytic repressor in resting macrophages in a novel mechanism that maintains cellular quiescence through blockade of a MARCO bacterial response pathway. This implicates HIF2α as a master regulator of cellular oxidative state during macrophage homeostasis.
Characterization of the regulatory functions of lncRNA in Inflammation
Authors: Carpenter Susan
Published: January 2023

The rapid development of deep sequencing technologies has provided us with an unprecedented view of the human genome. One of the most fascinating findings is that less than 3% of the genome codes for protein coding exons, yet more than 85% of the genome is transcribed. Consortium wide efforts such as the ENCODE project and FANTOM are dedicated to the identification of all functional elements present in our genomes. The largest group of RNA produced from the genome is Long noncoding RNA (lncRNA). LncRNA are described as transcripts greater than 200 nucleotides in length that do not code for protein. GENCODE represents the gene set of the ENCODE project and its most recent release GENCODE 24 indicates that there are 15,941 lncRNA present in the human genome. To date there is experimental data available on approximately 1% of known lncRNA. We have developed strategies to enable low to high throughput studies of lncRNA both in vitro and in vivo. In order to study the functions of lncRNA in the inducible inflammatory response we have developed both human and murine NFkB reporter macrophage cell lines. These lines serve as excellent tools for high throughput manipulation of lncRNA, which we are currently testing using whole genome shRNA libraries in addition to CRISPR/Cas9 editing approaches. We know lincRNA-Cox2 is highly inducible following inflammatory stimulation and we have developed a lincRNA-Cox2 transgenic mouse using site-specific TARGATT system and knockout mice using CRISPR/Cas9 technology to study the importance of this lncRNA in host defense against infection. Such approaches provide an efficient workflow for the characterization of any lncRNA in our model system of inflammation.
Human natural killer cell education is dynamically controlled by environmental and cell-intrinsic HLA
Authors: Boudreau Jeanette , Liu Xiao-Rong , Zhao Zeguo , Zhang Aaron , Shultz Leonard D , Greiner Dale L , Dupont Bo , Hsu Katharine C
Published: January 2023

NK cell education is determined by inhibitory KIR and HLA, but how their interactions potentiate reactivity is unknown. Human NK cells derived from CD34+ stem cells transplanted into a novel HLA-B*27:05 transgenic mouse are educated by HLA on both donor human hematopoietic cells and recipient mouse stromal cells. Similarly, NK reactivity in hematopoietic cell transplant patients reflects education by both donor and recipient HLA. Upon transfer to HLA-B*27:05+ mice, mature, uneducated KIR3DL1+ NK cells gain reactive potential, demonstrating that NK cell education can respond to alterations in the HLA available in the local environment. Bone marrow chimera studies reveal that HLA from either hematopoietic or stromal cells is sufficient for this re-education. Strikingly, educated NK cells do not lose reactivity after transfer to HLA-negative mice, implicating a cell-intrinsic contribution of HLA to the maintenance of NK education. We find that HLA knockdown diminishes NK reactivity, and FRET studies reveal that HLA and KIR complex in cis. HLA can be acquired from neighboring cells by trogocytosis, illuminating a mechanism by which ligands can be acquired from trans sources for cis education of NK cells. Altogether, these findings define how KIR and HLA shape NK function and may be applicable in cell therapies to potentiate donor NK reactivity.
The cGAS-STING pathway is present and active in human plasmacytoid dendritic cells
Authors: Deb Pratik , Dai Jihong , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells(pDCs) function as sentinels against viral infection in humans by producingabundant amounts of Type-I and Type-III interferons as well as other cytokines. Detection of viral nucleic acids remains the major initiating factor forinterferon and cytokine production in pDCs. Although the Toll-likereceptor-mediated endosomal pathway of nucleic acid detection has been studiedand documented in great detail, detection of cytoplasmic DNA in human pDCsremains, for the most part, unexplored. Recently, a cytoplasmic pathway of DNArecognition and interferon expression has been discovered in the murine systemand certain human cell lines. This pathway is mediated by the cytoplasmicprotein cGAMP synthetase (cGAS), which initiates Type-I interferon productionvia a cyclic dinucleotide called 2′3′ cyclic GMP-AMP (cGAMP), which in turn,activates the endoplasmic reticulum resident protein Stimulator of InterferonGenes (STING) and leads to cytokine expression. Here, for the first time, wedemonstrate the existence of components of the cGAS/STING pathway present inhuman pDCs. Our data indicate that both cGAS and STING are transcribed and translatedin primary human pDCs. We have also confirmed the functionality of the pathwayby inducing cytokine (IFN-α, IFN-λ, TNF-α, IL-6) production through stimulatingpDCs by exogenous cGAMP. Our study suggests that this cGAS-STING-mediatedcytokine response exists in parallel to the TLR9-mediated DNA recognition andimmune activation in human pDCs.
Identifying the interactome of the RIG-I-like Receptor LGP2
Authors: Thompson Ahna B , Stone Amy EL , Gale Michael J
Published: January 2023

RIG-I-like receptors (RLRs) are DEAD box helicases and cytoplasmic pathogen recognition receptors that recognize and bind to nonself/viral RNA and trigger innate antiviral immunity. RLRs are essential for detection of RNA virus infection, and include RIG-I, MDA5, and LGP2. LGP2 has been implicated as RIG-I or MDA5 cofactor to regulate their activity. We conducted a yeast 2 hybrid (Y2H) screen (using LGP2 as bait) of a human hepatic cDNA library to identify protein binding partners of LGP2. Identified proteins were validated as LGP2 interactors through overexpression and co-immunoprecipitation assays of LGP2 binding. The validated proteins were then submitted to a cell-based interferon beta-promoter-luciferase assay model of Sendai virus (SenV) infection as well as a cell based NFkB-luciferase assay of the same system. Endogenous expression of the validated proteins was then assayed through the Huh7 human hepatoma cell line system with various stimuli including viral infection with SenV and Interferon-beta treatment. Through these studies we will define the LGP2 interactome and identify proteins that act as co-factors in RLR signaling.
SLAP negatively regulates FcɛRI receptor-mediated mast cell response in mice
Authors: Sharma Namit , Ernoult Emilie , McGlade Catherine Jane
Published: January 2023

Mast cells (MCs) are key mediators of innate immune response to pathogens and allergens, etiological factors for drugs, dust and peanut-induced anaphylaxis. Degranulation of MCs in response to binding of high affinity FcɛRI receptor (IgE receptor) to allergen-induced IgE results in the release of preformed mediators including histamine, and de novo synthesis of cytokines causing hypersensitivity symptoms including anaphylaxis. Src-like adapter protein (SLAP), has been shown to regulate signaling downstream of T cell receptor (TCR), B cell receptor (BCR) and receptor tyrosine kinases (RTK) including c-Kit, FLT3 and CSF-1 via recruitment of the Cbl ubiquitin ligase. Here, we show that bone marrow-derived mast cells (BMMCs) from SLAP knockout (SLAP KO) mice displayed significantly enhanced degranulation and de novo synthesis of IL-6 but not TNFα compared to wild type (WT) BMMCs. SLAP KO BMMCs also displayed significantly reduced F-actin polymerization upon stimulation of IgE receptor in comparison to WT BMMCs. In addition, SLAP KO BMMCs maintained enhanced cell surface expression of FcɛRI receptor post stimulation suggesting reduced endocytosis/degradation. While WT and SLAP KO mice have equal numbers of MCs in ear skin, SLAP KO mice showed enhanced passive cutaneous anaphylaxis response. Thus, these results suggest that SLAP negatively regulates MC-induced anaphylaxis reaction via IgE-DNP pathway.
ICOS-dependent PI3K signaling in regulatory T cell development and function
Authors: Mittelsteadt Kristen L , Sullivan Jenna M , Campbell Daniel J.
Published: January 2023

Foxp3+ regulatory T cells (TR) are critical for maintaining immune tolerance and preventing inflammatory disease. Given their immunosuppressive capabilities, manipulation of TR function and/or abundance is a promising therapeutic strategy to either augment or inhibit immune responses in the context of disease. A detailed understanding of the key factors that control TR development, homeostasis, and function is essential for the successful application of TR-based therapies. Recent work suggests that inducible T cell costimulator (ICOS) signaling is required for maintenance of effector TR (eTR) cells, which migrate to peripheral sites of inflammation. ICOS ligation is most notably a potent activator of phosphatidylinositol 3-kinase (PI3K), however the key signaling pathways downstream of ICOS that support eTR have not been investigated. In order to study the role of ICOS-dependent PI3K signaling, we obtained mice that carry a knock-in mutation in the Icos gene that alters the cytoplasmic tail motif of the ICOS protein, thereby specifically abolishing ICOS-mediated PI3K activation (IcosY181F mice). The frequency of TR is reduced in these mice, as is the proliferation of ICOShi TR in secondary lymphoid organs. CD4+ IcosY181F cells have impaired regulation of downstream PI3K targets, including p-AKT, p-S6, and CD62L. IcosY181F mice develop more severe disease earlier than WT controls using a model of experimental autoimmune encephalomyelitis and have lower eTR frequencies at effector sites. Taken together, data from our lab suggest an important role for ICOS-mediated PI3K activation in the development, maintenance, and function of TR.
The microtubule-associated protein Lis1 regulates T lymphocyte homeostasis and differentiation
Authors: Ngoi Soo M , Lopez Justine , Chang John T
Published: January 2023

The microtubule-associated protein Lissencephaly 1 (Lis1) is a key regulator of cell division during stem cell renewal and differentiation. We examined the role of Lis1 in T lymphocyte homeostasis and fate diversification in response to microbial infection. T cell-specific deletion of Lis1 resulted in depletion of the peripheral CD4+ and CD8+ T lymphocyte pool, owing to a selective loss of homeostatic, cytokine-induced proliferation. By contrast, cognate antigen-triggered proliferation was relatively unaffected in CD8+ T cells, enabling Lis1-deficient T cells to differentiate into terminal effector cells in response to microbial infection. Strikingly, however, Lis1-deficient CD8+ T cells failed to develop into long-lived memory lymphocytes. Taken together, these findings suggest that Lis1 plays an important role in T cell homeostasis and the generation of memory T lymphocytes.
The impact of hypoxia on metabolic subsets of bone marrow plasma cells.
Authors: Minges Wols Heather A , Aguilera Alexya N. , Witte Pamela L
Published: January 2023

The metabolic stress of being an antibody-secreting cell is likely great. Indeed, existing literature suggests short-lived plasma cells (SLPC) die due to high metabolic stress. How long-lived (LL) PC maintain longevity and escape the same fate as SLPC is unknown. We hypothesized that LLPC in the marrow may adapt to metabolic stress by employing a different strategy such as glycolysis, which may be triggered by the hypoxic nature of the marrow environment. LLPC reside in hypoxic niches, as evidenced by Hypoxyprobe™-1 (PIM)-positivity. Here, we show that PC are divided into two distinct subsets based on fluorescence following MitoTracker Red CM H2Xros staining: a predominant MitoHI subset, suggestive of aerobic respiration, and a rarer MitoLO subset, suggestive of glycolysis. MitoLO PC have higher lactate levels and less NADH than MitoHI PC. Following immunization and boost with TNP-KLH, Ag-specific PC are detected in both subsets at similar frequencies, indicating newly-made PC are filling both compartments equally. Further, comparable amounts of Ig are detected from 24h supernatants of MitoHI and MitoLO PC. As PC are in hypoxic niches, and at least a subset of PC appears to use glycolysis, we also tested the ability of MitoHI and LO PC to survive in hypoxic versus normoxic conditions. Notably, MitoHI and LO PC are comparable in function following 24h culture at 3 or 20% O2, as detected by ELISPOT and ELISA. However, 24h survival may be slightly enhanced in the MitoLO PC. In all, these PC subsets appear to function similarly despite metabolic differences. It is interesting that most LLPC use aerobic respiration while residing in a hypoxic environment; this is in contrast to HSC, which reside in similar hypoxic marrow niches but use glycolysis.
The role of AP-1 family transcription factor networks in regulating Th17 cell identity
Authors: Ciofani Maria , Carr Tiffany
Published: January 2023

CD4 T cells can adopt one of two opposing fates: a helper T cell (Th) specialized in supporting pathogen clearance, or a regulatory T cell (Treg) that attenuates immune responses. IL-17A-producing inflammatory Th17 cells stand out among Th cells, possessing a high level of inherent plasticity in response to altered environments. The underlying mechanisms controlling such flexibility are largely unknown. In this regard, we have identified the AP-1 family of transcription factors (TFs) as key regulators of Th17 cell identity. Our previous work revealed the pioneering role of BATF in supporting global enhancer accessibility and flexibility in CD4 T cells, and here, we describe an antagonistic AP-1 TF, JunB, that limits Th17 plasticity. Indeed, we find that CD4 T cells deficient in JunB exhibit dysregulated effector cytokine and TF expression signatures, and lack disease potential in a mouse model of autoimmunity. In particular, JunB serves to restrain inappropriate Treg and Th1 differentiation. Here, we (i) use fate-mapping reporter mice to dissect the requirement for JunB in maintaining Th17 cell stability, (ii) define the JunB targets that facilitate subset conversion events; (iii) define novel genomic lineage-restriction elements regulated by AP-1 complexes; and (iv) define activating and repressing roles of JunB that support Th17 lineage stability. Such mechanistic insight is key to guiding future work aimed at exploiting the AP-1 balance in Th17 cells to divert damaging inflammatory responses into favorable regulatory responses.
Mutation of the SH3 binding site within the chB6 alloantigen
Authors: Funk Phillip E , Bhattacharya Rohini
Published: January 2023

It is well established that central tolerance of B cells involves the deletion of large numbers of B cell clones. However, the mechanism of this deletion remains unclear. Chickens are one of the classic models of vertebrate immunity and bursa of Fabricius presents model system to study this process. Our lab has been interested in the role of the chB6 alloantigen (formerly called Bu1) in the developing of B cells within the bursa. We have shown that chB6 can trigger apoptosis when stimulated by anti-chB6 antibody and this apoptosis uses intermediates commonly found in death receptor signaling pathways. Recently, chB6 has been found in close proximity to the BCR on DT40 cells. Others have noted that BCR signals in DT40 lead to apoptosis. Homology analysis places chB6 within the larger CD2/SLAM family of proteins based on the presence of two extracellular Ig domains. CD2/SLAM family members serve to modulate signals in immune cells. How chB6 initiates signals, and how those signals might be coordinated with other components of the BCR complex, remains unclear. Previous work indicated a 15 amino acid stretch in the cytoplasmic domain and critical in initiating apoptosis. Within this region is the PXXP motif indicating a binding site for SH3 domain containing proteins. We have mutated the proline codons within this region and transfected these mutated chB6 cDNAs into BK3a cells to test the hypothesis that this SH3 binding site is important in death signaling via chB6. Preliminary evidence suggests that this region is in fact critical in transducing a death signal. This project furthers our efforts to understand the biology of chB6. This work supported by a grant from the DePaul University Research Council.
Regulation of metabolic pathways in CD4 T memory cell responses of older individuals
Authors: Yanes Rolando E , Goronzy Jorg J
Published: January 2023

Upon activation, T cells enter a developmental program characterized by clonal expansion and effector cell differentiation followed by contraction and survival of long-lived memory T cells. This program is regulated by interplay of activation-induced signaling and metabolic changes. During the proliferative stage, naïve T cells undergo a metabolic switch to glycolysis which provides the molecular substrates to support T cell growth. The subsequent survival of memory T cells is supported by a switch from mTORC activity to AMPK activation and oxidative metabolism. Compared to naïve T cells, memory T cells have increased mitochondrial capacity; activation induced metabolic changes are less well characterized. Recall responses, such as CD4 T responses to influenza vaccination, are compromised in the elderly. Here, we examined whether the regulation of metabolic pathway contribute to this defect. Upon activation, CD4 memory T cells upregulate oxidative phosphorylation as well as glycolysis. Oxidative phosphorylation peaks 48 hours after activation in parallel to increases in mitochondrial mass. Subsequently, it is then sharply downregulated, while increased glycolysis is maintained. While CD4 memory T cell responses in older individuals (>65 years) display appropriate upregulation of glycolysis, they are associated with increased oxidative metabolic activity compared to younger individuals (< 30 years old). Mitochondrial function was superior as documented by increased spare respiratory capacity with normal mitochondrial mass as well as production of ATP and reactive oxygen species. We propose that the increased mitochondrial activity compromises memory T cell function in older individuals.
Membrane sequestration of the mIgG cytoplasmic domain confers a safety trigger mechanism governing the enhanced activation of isotype-switched IgG-B cell receptor
Authors: Chen Xiangjun , Sui Yinqiang , Liu Wanli
Published: January 2023

A hallmark of the adaptive immunity is the memory for previously experienced pathogens. Memory B cells that express the isotype-switched IgG-BCR are in charge of the establishment of the memory IgG antibody response. A trigger safety mechanism is necessary to govern the burst-enhanced activation of a smoking-gun-like IgG-BCR to avoid illegitimate activation. Here, we reported that the specific interaction between positively-charged mIgG cytoplasmic tail (mIgG-tail) and negatively-charged acidic phospholipids of the plasma membrane can sequester the key tyrosine in ITT motif in the membrane hydrophobic core, which help quench mIgG-tail basal level signaling in quiescent B cells. Indeed, we observed that disruption of the interaction in quiescent B cells induced higher basal level phosphorylation of mIgG-tail ITT motif and promoted the survival signal of primary B cell in vivo. Disruption of the interaction also led to excessive recruitment of prominent BCR signaling microclusters into the B cell immunological synapse, more aggressive Ca2+ mobilization and gene expression indicated by Nur77-GFP reporter mice, which ultimately led to hyper-proliferation of primary B cells after antigen engagement. Physiologically, mIgG-tail was released by antigen engagement or Ca2+ mobilization in the initiation of IgG+ B cell activation, which then enhances Ca2+ mobilization, and in turn facilitates more release of mIgG-tail to further enhance memory B cell activation through such a positive feedback mechanism. Conclusively, our findings shed new light on a novel regulatory mechanism of IgG+ memory B cell to balance immunoprotection and immunopathology and provide new perspective of self-tolerance in class-switched IgG+ memory B cells.
Group 3 innate lymphoid cells continuously require the transcription factor GATA-3 after commitment
Authors: Zhong Chao , Cui Kairong , Wilhelm Christoph , Hu Gangqing , Mao Kairui , Belkaid Yasmine , Zhao Keji , Zhu Jinfang
Published: January 2023

We have previously reported that the transcription factor GATA-3 is critical for the development of all interleukin 7 receptor α-chain (IL-7Rα)-expressing innate lymphoid cells (ILCs) from their progenitor. After ILC lineage commitment, GATA-3 is essential for the maintenance and function of group 2 ILCs (ILC2 cells). Moreover, GATA-3 also maintains low expression in other mature ILCs, such as ILC3 cells, but its function is still elusive. We found that GATA-3 regulated the homeostasis of ILC3 cells by controlling IL-7Rα expression. In addition, GATA-3 served an indispensable function for the further development of the NKp46+ ILC3 subset. GATA-3 bound directly to the Rorc locus in ILC3 cells, repressed RORγt expression, and, thus, regulated the balance between the transcription factors T-bet and RORγt during NKp46+ ILC3 development. Whole transcriptome comparison of CCR6+ lymphoid tissue-inducer (LTi) ILC3 and NKp46+ ILC3 subsets from Gata3 sufficient and deficient mice showed that, among NKp46+ ILC3 cells, GATA-3 not only positively regulated genes specific to the NKp46+ ILC3 subset, but also negatively regulated genes specific to LTi ILC3 cells. Furthermore, GATA-3 also bound to the Il22 promoter locus and was required for IL-22 production in both ILC3 subsets. Mice deficient for Gata3 in ILC3 cells succumbed to Citrobacter rodentium infection due to defective IL-22 production. In conclusion, despite its low expression, GATA-3 was required for the homeostasis, development and function of ILC3 subsets.
Toxoplasma gondii infection triggers a long-term defect in the generation and function of naive CD4+ T lymphocytes
Authors: Jankovic Dragana , Kugler David G. , Flomerfelt Francis A. , Costa Diego L. , Laky Karen , Kamenyeva Olena , Mittelstadt Paul R. , Gress Ronald E. , Ashwell Jonathan D. , Sher Alan
Published: January 2023

Since Ag-stimulated naïve T cells either die as effectors or enter the activated/memory pool, continuous egress of new T lymphocytes from thymus is essential for maintenance of peripheral immune homeostasis. Unexpectedly, we found that systemic infection with the protozoan Toxoplasma gondii triggers not only a transient increase in activated CD4+ Th1 cells, but also a profound and persistent reduction in the size of the peripheral naïve CD4+ T cell pool. We further showed that the resulting perturbation in T cell homeostasis is mechanistically associated with parasite-induced thymic atrophy and more specifically with a loss in the architectural integrity of the thymic epithelium. This structural degeneration is accompanied by impaired thymic selection as evidenced by decreased CD5 expression on the few recent thymic emigrants that reach the periphery. The resulting quantitative and qualitative deficiency in naïve CD4+ T cells leads to an immunocompromised state that both promotes chronic toxoplasma infection and leads to decreased resistance to challenge with an unrelated pathogen. Interestingly, the changes in thymic structure and function induced by toxoplasma closely resemble those associated with the thymic involution that occurs during aging suggesting a possible influence of infection-induced alterations in the thymus on immunologic senescence.

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious diseases.
New neutrophil models for studying host-fungal pathogen interactions
Authors: Mansour Michael K , Dagher Zeina , Negoro Paige , Martinelli Michelle , Maxcy Katrina , Feliu Marianela , Alashghar Shaden , Carver Alexa , Sykes David B
Published: January 2023

Neutrophils are terminally differentiated phagocytic cells that represent the first line of defense against pathogens. The study of neutrophils in vitro is difficult owing to a short life span. Immortalized cell line models fail to recapitulate all the effector functions of primary cells. Furthermore, the study of murine neutrophil function in vivo is limited by the lack of a model system that can be genetically manipulated (e.g. gene knockout or overexpression) ex vivo. We describe here the development and use of conditionally immortalized granulocyte/macrophage progenitor cells (GMP) capable of unlimited expansion, genetically tractable, and that can be transplanted into mice to study neutrophil function in vivo. These GMP cell lines were derived from UBC-GFP mice by the transduction of an MSCVneo ER-HoxB8 retrovirus such that factor-dependent cell lines emerge in the presence of stem cell factor and b-estradiol (E2). Upon removal of E2 in vitro or transplantation into irradiated recipients, these GMP precursor cell lines differentiate normally to fully functional neutrophils over the course of 4–5 days. Terminal effector function was confirmed by a) flow cytometry, b) phagocytosis, c) superoxide generation, d) transepithelial migration, and e) NET formation. More importantly, these GMP-derived neutrophils were also capable of fungicidal activity. Of particular note, transplantation of these cells into neutropenic mice challenged with lethal dose of the pathogenic yeast C. albicans, showed prolongation of survival. Overall, this model closely recapitulates primary neutrophils, while providing an inexhaustible and genetically tractable supply of cells, a much-needed tool for the study of neutrophil function.
Cellular Basis for Reduced Resistance in DBA/2 Mice to Babesia microti
Authors: Lamisere Hymlaire , Wortis Henry H
Published: January 2023

The protozoan parasites Babesia bovis, divergens and microti infect humans and other mammals via the tick Ixodus Scapularis. Babesia causes a malaria-like hemolytic disease with the most severe cases seen in those who are immunocompromised or over the age of 50. Mice offer the opportunity to understand genetic and age-related differences in resistance to infection. Previously, we showed that two month old DBA/2 mice are less resistant to Babesia than BALB/cByJ. Further, unlike BALB/cByJ mice, 18 month old DBA/2 mice are not able to clear their infections. Cell transfers established that these differences were at least in part due to CD4 T cell intrinsic differences. We hypothesized that DBA’s relative susceptibility might be due to an inability to produce sufficient cytokines. We harvested the splenocytes of infected young BALB/c and DBA/2 mice at days 7 to 24 post infection and measured the percentage and total number of cytokine-producing T cells. BALB/c mice have more CD4 and CD8 T-cells than infected DBA/2 mice. They also have many more cells with cytoplasmic IFN-y, IL-21 and IL-17A. However, the median fluorescence intensity of cytokine staining and the frequencies of cytokine positive cells did not differ between strains. Thus, the absolute difference in cytokine-positive cell numbers was due to the expansion of the activated T cell populations. In vitro activation revealed that in response to anti CD3 antibody, T cells of DBA/2 mice were less responsive. A similar phenotype might explain why individuals over the age of 50 are more susceptible to severe babesiosis.
ICOS:ICOSL suppresses an excessive T cell response in the CNS during chronic T. gondii infection
Authors: O’Brien Carleigh , Harris Tajie H
Published: January 2023

Regulatory T cells have potent suppressive capacity capable of controlling effector T cell responses and limiting immune-mediated pathology. While the importance of Tregs during the immune response to multiple infections has been demonstrated, the specific mechanisms by which these cells suppress immunopathogenic effector responses in the inflamed brain during infection are not yet known. Tregs are present in the brain during toxoplasmic encephalitis, where effector T cells are required to limit parasite replication. Using multiphoton imaging, we find that Tregs associate extensively with CD11c+ APCs in the brain, and we are currently exploring the importance of this interaction for the maintenance of Treg suppressive function. Inducible T cell costimulator (ICOS) is a costimulatory molecule that has been shown to play an important role in generating effector T cell responses to several pathogens. However, the role of the ICOS-ICOS ligand (ICOSL) interaction between Tregs and APCs in modulating an immune response in the CNS remains poorly understood. We have shown that Tregs in the brain express higher levels of ICOS than effector T cells, and blocking this interaction during chronic T. gondii infection leads to increases in effector T cell responses and immune-mediated pathology. Through in vivo imaging, we have also seen that ICOS-ICOSL blockade changes the behavior of Tregs in the brain, suggesting that ICOS is important for maintaining Treg-APC interactions and potentially allows for control of effector immune responses in the CNS. The excessive immune response seen with ICOSL blockade indicates a shift towards immunopathology, suggesting an important role for ICOS for local Treg suppression in the CNS during chronic infection.
Induction and role of elevated extracellular glutamate during chronic Toxoplasma infection in the brain.
Authors: Wilson Emma H. , David Clement N , Landrith Tyler A , Frias Elma S , Fiacco Todd A , Binder Devin K
Published: January 2023

Toxoplasma is a prolific parasite found in a third of the world’s population that resides primarily as cysts within the brain of the infected host. Infection is for life and continuous recruitment of T cells into the brain parenchyma is required to prevent reactivation of the parasite and fatal encephalitis. Disease can therefore occur in immune compromised individuals including HIV/AIDS, transplant recipients and during congenital transmission which can lead to a broad spectrum of neurological abnormalities. T cells that have migrated into the infected brain parenchyma are heterogenous in function in that they exhibit characteristics of effector, effector memory and tissue resident memory phenotypes. The cell intrinsic and the tissue properties that drive such heterogeneity are unknown. We demonstrate that the extracellular environment of the infected brain includes significantly elevated concentrations of glutamate and hypothesize that this acts to drive infiltrating T cell phenotype and function. Our data show T cells in the brain are capable of responding to the presence of extracellular glutamate either by directly using glutamate following uptake or due to its ability to initiate downstream signaling cascades. By manipulating glutamate concentrations in vitro and in vivo we will test the role of glutamate signaling on the phenotype and function of T cells in the infected brain. Decreasing extracellular glutamate concentrations may be a valuable therapeutic tool to limit neuronal pathology under many circumstances including disease caused by Toxoplasma. It is therefore critical to understand the role of glutamate as a signal to infiltrating protective immune cells.
The Candida albicans quorum-sensing molecule, farnesol, remodels the peritoneal cavity microenvironment to promote innate inflammatory responses in mice.
Authors: Hargarten Jessica C , Atkin Audrey L. , Brown Deborah M.
Published: January 2023

Candida albicans is a polymorphic fungus that causes mucosal candidiasis and life threatening, systemic and intra-abdominal disease in immunocompromised and transplant patients. In normal healthy adults, C albicans is maintained as a commensal mainly through the actions of innate cells. Secretion of the quorum-sensing molecule, farnesol, acts as a virulence factor for C. albicans during systemic infection, but is protective in oral models of infection. Despite the clinical importance of intra-abdominal candidiasis with a mortality rate as high as 65%, the role of farnesol in its pathogenesis has not been studied. In ongoing experiments, we found that introduction of farnesol into the peritoneal cavity (PC) of mice remodels the peritoneal microenvironment to promote innate inflammatory responses. Early following intraperitoneal injection, farnesol stimulates the rapid depletion of B cells followed by recruitment of inflammatory macrophages and neutrophils into the PC compared to thioglycollate and control mice. Kinetic analyses of the transcriptional profile of farnesol-elicited cells reveal an early increase in chemokines followed by increased proinflammatory cytokines consistent with macrophage recruitment and activation. In addition, farnesol-elicited macrophages and dendritic cells demonstrate hallmarks of innate immune activation through high surface expression of class II and co-stimulatory molecules.

Current experiments aim to determine the impact of this innate immune recruitment on antifungal defenses and clearance in the PC. Defining the immunostimulatory properties of farnesol will inform development of more appropriate therapeutic strategies for treatment of intra-abdominal Candida infection.
Characterization of the type I IFN response to liver stage infection with genetically attenuated malaria parasite vaccines.
Authors: Minkah Nana K , Sack Brandon K , Miller Jessica L , Vaughan Ashley , Kappe Stefan H
Published: January 2023

Malaria, a disease caused by Plasmodium parasites, kills nearly 600,000 people annually. Inoculated by an infectious mosquito bite, Plasmodium sporozoites travel to the liver and infect hepatocytes. Here, they develop into merozoites that are released into the blood to cause malaria associated mortality and morbidity. No fully protective malaria vaccine exists, but immunizations with genetically attenuated parasites (GAPs) that arrest in the liver confer sterile protection from challenge in mice. Moreover, using a novel super-infection assay, we observed that GAP immunization induces a potent type I IFN response to control liver stage infection. However, the factors required for the induction and propagation of the IFN response, and the influence of this response on adaptive immunity had not been elucidated. To identify the upstream inducers of IFN signaling, we immunized mice with either mosquito debris or GAPs and measured gene expression in hepatocytes by RNA-Seq. 800 genes were significantly upregulated upon GAP immunization, including transcripts for cytosolic RNA and DNA sensors. To examine the influence of the IFN response on adaptive immunity, WT, IRF3−/− and IFNAR−/− mice were immunized with a suboptimal dose of GAP, and challenged 4 weeks later with WT parasite. The immunization regimen elicited sterile protection in 20% of the WT mice, while 100% protection was achieved in IRF3−/− or IFNAR−/− mice. Taken together, the data suggest that hepatocytes utilize RNA and DNA sensors to induce a type I IFN response that is necessary for early control of liver stage infection, yet detrimental to adaptive immunity. Current studies are examining the mechanisms behind the IFN-mediated suppression of adaptive immunity.
TGF-β and NFAT control T cell receptor restimulation-induced cell death of CD4+CD25+Foxp3+ regulatory cells
Authors: Pandiyan Pushpa
Published: January 2023

One of the key questions in the area of immunomodulation is how CD4+CD25+Foxp3+ regulatory cells (Tregs) are controlled. Here we studied how their survival is regulated in the context of T cell Receptor re-stimulation apoptosis. We show that both mouse and human Tregs and iTregs exhibit increased resistance to T cell Receptor restimulation-induced cell death (RICD) compared to conventional CD4+ cells. The enhanced survival was dependent on TGF-β signaling that results in reduced nuclear translocation of nuclear factor of activated T-cells (NFAT), as well as up-regulation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) in Tregs. Tregs also showed significantly decreased susceptibility to α-CD3 antibody mediated apoptosis compared to the effector cells in vivo. While ionomycin increased Treg apoptosis, NFAT activation inhibitors reduced the apoptosis levels of effector cells almost to Treg levels, demonstrating that reduced NFAT activity contributes to decreased RICD in Tregs. Finally, RICD resistance in Tregs manifests itself as increased protection of these cells from cell death during mouse oropharyngeal candidiasis (OPC) infection and inflammatory bowel disease (IBD). Protection from cell death was abrogated in the absence of TGF-β signaling in Tregs. Taken together, our data unravel the previously unrecognized role of TGF-β in promoting Treg survival during TCR restimulation, through regulation of multiple pathways involving NFAT and cFLIP in Tregs. This novel mechanism that offers a selective survival advantage to Tregs may have broader ramifications in immune homeostasis during inflammation.
Evaluation of a newly developed assay designed to assess the interaction between α4β7 and MAdCAM
Authors: Doyle Allison , Cicala Claudia , Jelicic Katija , Van Ryk Donald , Ansari Aftab Ahmed , Fauci Anthony S , Arthos James
Published: January 2023

HIV infection is characterized by high-level replication in gut-associated lymphoid tissues (GALT). This occurs in part because α4β7 +/CD4+ T cells are a preferred target of HIV infection. α4β7 functions as a gut homing receptor. Homing is mediated through a specific interaction with MAdCAM that is expressed on the surface of the high-endothelial venules that line the gut. In addition, α4β7 binds to the V2 domain of HIV gp120. In an SIV model of HIV mucosal transmission, an antibody specific for α4β7 +was found to prevent infection, but the underlying mechanism for this protection is not fully understood. We have proposed that signaling through α4β7 + facilitates HIV transmission, and that the protection from infection that we observed may reflect the capacity of the anti-α4β7 mAb we employed to interfere with this signaling. In order to investigate the role of α4β7+ signaling in mucosal transmission, we have employed a newly developed adhesion assay (Peachman and Rao) that measures the interaction between either the V2 domain of HIV gp120 or MAdCAM with cells expressing α4β7. We have used this assay to evaluate small molecules and antibody antagonists of these interactions. We have found that small molecule antagonists that bind to the active site in α4β7 abrogate binding of cells to both MAdCAM and the V2 domain of HIV gp120. In addition, we determined that retinoic acid increases the adhesion of MAdCAM and V2 to α4β7 expressing cells. This assay should aid in the development of novel strategies designed to interfere with the trafficking of α4β7 +cells to GALT and to mucosal transmission of HIV.
Maternal control of dietary and gut microbial antigen exposure promotes enhanced tolerance and prevents allergic responses
Authors: Knoop Kathryn A , McDonald Keely , Newberry Rodney D
Published: January 2023

The incidence of allergic disorders is rapidly increasing in children in Western societies, and is linked to allergen avoidance diets and decreased exposure to microbes and microbial antigens in early life. Exposure to dietary antigens and the microbiota via the gastrointestinal tract early in life is associated with a reduced risk of allergic disorders, however the optimal time to introduce these antigens, where the immune system encounters these antigens, and what controls the immune systems exposure to define this time in early life is largely unknown. We identified a window of time in early life in which luminal antigens are shunted to the colonic lamina propria (LP) immune system to induce enhanced tolerogenic responses. Delivery of luminal antigens to the colonic immune system during this time in life was mediated by the formation of goblet cell-associated antigen passages (GAPs), which were regulated by goblet cell (GC) intrinsic sensing of breast milk derived epidermal growth factor (EGF) and the blooming gut microbiota. Disruption of GAP formation and antigen delivery during this time in early life abrogated tolerance to dietary antigens, decreased regulatory T cells, and induced a skewing of the immune system toward systemic Th2 responses. We propose that maternal control of antigen delivery during infancy allows for the proper education of the immune system against dietary and microbial antigens, and induction of regulatory T cells necessary to suppress inappropriate Th2 responses later in life.
A unique population of IFNγ-producing innate lymphoid cells promotes Campylobacter-induced colitis
Authors: Muraoka Wayne T , Xia Qingqing , Halperin Sydney , Koroleva Ekaterina , Tumanov Alexei
Published: January 2023

Inflammatory bowel diseases (IBD) are thought to arise from an overly aggressive immune response to the intestinal microbiota. Campylobacter jejuni is a human enteric pathogen that is a major cause of gastroenteritis worldwide. C. jejuni infection also increases the risk of developing IBD-like sequelae. Prior studies showed IL-10 deficient mice develop fulminant colitis when infected by C. jejuni. However, the roles of adaptive and innate immune cells in promoting colitis remain unresolved. Here, we show that T cells are not necessary for C. jejuni-induced colitis. In vivo depletion of Thy1+ cells or neutralization of IFNγ in T cell deficient mice significantly ameliorated C. jejuni-induced colitis, indicating a pathogenic role for IFNγ-producing innate lymphoid cells (ILC1s). In mice, ILC1s are currently identified as NK1.1+NKp46+ cells and prior studies reported that depleting NK1.1+ cells ameliorated ILC1-driven colitis. Unexpectedly, despite effective depletion of NK1.1+ cells in mice lacking T cells, this treatment did not impact IFNγ production or colon pathology after C. jejuni infection. Phenotyping analysis revealed IFNγ-producing ILCs (Thy1+Lin−Tbet+Eomes−GnzA−RORγtlow/−) that accumulated in the colon during C. jejuni-induced colitis did not express NK1.1 or NKp46. Further, mice deficient for the transcription factor RORγt possessed markedly fewer NK1.1−NKp46− ILC1s in the intestine and exhibited reduced severity of C. jejuni-induced colitis. Based on the lack of NK and natural cytotoxicity receptors (NCR) and in accord with the current ILC nomenclature, we propose the tentative designation of NCR−ILC1. Together, these results identified a unique population of ILC1s that promote colitis.
Generation of colonic T-cell mediated protective immunity after rectal infection with LCMV
Authors: Trapecar Martin , Cohen Benjamin , Khan Shahzada , Woodruff Erik , Luong Julie , Sanjabi Shomyseh
Published: January 2023

How viral-specific immunity is generated in the colon is not well understood, leaving a major gap in our knowledge of defining the parameters needed to develop vaccines against rectally transmitted pathogens. Clearance of most intracellular pathogens requires CD8 T cells, and since unlike the small intestine, the colon lacks organized lymphoid aggregates such as Peyer’s patches, generating protective immunity requires a series of orchestrated events to mobilize CD8 T cells to this mucosal organ. Complicating the generation of protective immunity is the fact that the colon naturally serves as an inductive site for tolerance against many commensal organisms and food antigens. Our central hypothesis, supported by our data, is that the tolerogenic nature of the colon limits induction of robust inflammatory responses needed to activate CD8 T cells for their efficient and timely recruitment to the colonic mucosa. We are using our newly established lymphocytic choriomeningitis virus (LCMV) rectal infection model to determine why CD8 T-cell priming is sub-optimal after rectal viral transmission of LCMV and why the activated CD8 T cells are unable to control the virus from spreading systemically. We are employing several genetic tools to ablate various monocytic and dendritic cell populations to determine their specific role in promoting CD8 T cell priming, activation, differentiation, and migration after rectal LCMV infection. This knowledge is required to break down barriers that have prevented development of successful T-cell-mediated vaccines against sexually transmitted RNA viruses, such as HIV, and will ultimately support the development of vaccines against sexually transmitted infections.