bluuebunny/crossref_metadata_2025 huggingface dataset:
Mincle and TNF signaling crosstalk enhances type 1 and innate immune responses to Orientia tsutsugamushi
Authors: Fisher James Robert , Liang Yuejin , Card Galen , Gonzales Casey A , Soong Lynn
Published: January 2023

O. tsutsugamushi is an obligate intracellular bacterium and etiologic agent of scrub typhus, a life-threatening neglected disease endemic to the South-Pacific. The lungs harbor the greatest burden of infection and display strong type 1 proinflammatory responses. Here, we investigated whether the C-type lectin receptor Mincle contributes to immune recognition and inflammation. Following lethal infection in C57BL/6 mice, we performed pulmonary differential gene expression analysis via NanoString. We found 312 significantly induced genes (adj. p < 0.05) at the terminal phase of disease, of which Mincle (Clec4e) was the 4th most upregulated gene. Mincle signaling partners (Fcgrs), Cxcr3, Ccr5, and their ligands, as well as Il27, were also highly upregulated. To define a role of Mincle in immune recognition, we exposed WT or Mincle−/− bone marrow-derived macrophages (MΦ) to live or inactivated bacteria and analyzed inflammatory markers via qRT-PCR. We observed infectious dose-dependent increases in Mincle transcription along with Cxcl1, Ccl2, Tnf, and Nos2 early in infection. Mincle−/− cells had abrogated responses of Cxcl1 and Ccl2. Blocking TNF signaling prior to infection markedly reduced Mincle, Ccl2, Cxcl1, Cxcl10, and Il27 transcripts. MΦ treated with rTNF-α prior to infection had greater levels of Mincle, Il27, Cxcl10, and Ccl2 expression than treatment with TNF or Orientia alone, suggesting a synergistic effect. Our study provides the first evidence for Mincle sensing O. tsutsugamushi and a role of Mincle-, TNFα-, and IL-27-related crosstalk during infection. Since there are no effective vaccines for scrub typhus, our results help understand innate immune recognition of this understudied bacterium and its disease.

Supported by grants from NIH (R01 AI132674 to LS, T32 AI007526-20 to LS, and R21 AI156536-01 to LS). JF was a recipient of a NIAID Emerging and Tropical Infectious Diseases T32 fellowship.
Palmitic acid reprograms inflammatory responses to microbial ligands in macrophages, and mediates innate immune memory in vivo.
Authors: Seufert Amy Lorraine , Hickman James W , Traxler Ste K , Peterson Rachael M , Lashley Sydney J , Napier Ruth J , Shulzhenko Natalia , Napier Brooke A
Published: January 2023

Dietary saturated fatty acids (SFAs) modulate circulating lipids in mice and humans, which can alter the inflammatory capacity of innate immune cells. The most common SFA in human serum, palmitic acid (PA), is enriched in Western diets, and while it is known to induce an inflammatory response in macrophages, the impact of PA on responses to pathogenic stimuli is unknown. We developed an in vitro model to determine the impact of PA on the macrophage response to lipopolysaccharide (LPS). We find that bone marrow-derived macrophages (BMDMs) exposed to PA for 12–24 hours exhibit significantly enhanced TNF, IL-6 and IL-1β secretion following LPS exposure. BMDMs treated simultaneously with PA and the unsaturated fatty acid oleic acid (OA) show a reversal of this PA-induced hyper-inflammatory response to LPS, suggesting that OA negatively regulates the memory-like feature of PA. While monocytes and splenocytes from mice fed a PA-enriched diet exhibit unaltered basal inflammation, ex vivo LPS treatment leads to significantly enhanced pro-inflammatory cytokine expression. Mice exposed to PA for 9 days, followed by 7 days without PA, exhibit increased LPS-induced endotoxemia severity and mortality, and enhanced mRNA expression of tnf, il-6, and il-1β in blood. Rag−/− mice exposed to PA for 12-hours showed significantly reduced kidney fungal burden after Candida albicans infection. We hypothesize that PA induces a long-lasting innate immune memory program in macrophages, and reprograms their response to subsequent inflammatory challenge via metabolic and epigenetic alterations. Our findings are the first to suggest dietary PA regulates innate immune memory and is detrimental in an acute septic response, yet beneficial for pathogen clearance.

Supported by National Institute of General Medical Sciences (NIGMS) 525 grant 5R35GM133804-02 to B.A.N.
NADase activity found in bacterial TIR proteins may aid in innate immune evasion
Authors: Palm Cheyenne , Koudjra Rachida , Obiorah Trinity , Snyder Greg A , Snyder Michelle , Harberts Erin
Published: January 2023

Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with a mutation known to inhibit NADase activity. We are now using TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing RT-PCR to analyze the effects of these mutant TIRs on innate cell activation. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.

Supported by grant from NIH (R25 GM119970 04)
TGF-β and CIS inhibition synergistically enhance natural killer cell-based immunity
Authors: Souza-Fonseca-Guimaraes Fernando
Published: January 2023

Immune “checkpoint” inhibitor antibodies have revolutionized cancer therapy and function by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8 T cells. Recent studies have also shown similar programs in other effector lymphocyte populations such as natural killer (NK) cells. NK cells are key to cancer immunosurveillance, particularly in settings of metastasis. They are dependent on the cytokine IL-15 for their survival, development, and function, with ablation of the IL-15 signaling inhibitor CIS (Cish), shown to enhance NK cell anti-tumor immunity by increasing NK cell fitness in the tumor microenvironment (TME). The TME has also been shown to impair NK cell fitness via the production of immunosuppressive transforming growth factor (TGF)-β, even in the presence of high IL-15 signaling. Here, taking advantage of sophisticated proteomics and transcriptomics methods with computational biology analysis we identified an unexpected interaction between CIS and the TGF-β signaling pathway in NK cells. Independently, for the first time a Cish- and Tgfbr2-double-deficient NK cells were generated by our group, which displayed both hyper-responsiveness to IL-15 and hypo-responsiveness to TGF-β, with dramatically enhanced immunity against multiple mouse models of cancer. Remarkable, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development and this resistance is dependent on NK cell hyper-activity. This study contributes to the understanding on how targeting multiple suppressive pathways unveils a new immunotherapy approach that might have broad therapeutic interventions, particularly by enhancing NK cell effector functions during disease.

This work is supported by project grants from the National Health and Medical Research Council (NHMRC) of Australia (#1140406), the US Department of Defense – Breast Cancer Research Program – Breakthrough Award Level 1 (#BC200025), the ANZSA SRG Grant, and was supported by a grant (#1158085) awarded through the Priority-driven Collaborative Cancer Research Scheme and co-funded by Cancer Australia and Cure Cancer.
BCG vaccination impacts the epigenetic landscape of progenitor cells in human bone marrow
Authors: Sun Sarah J , Dumaine Anne , de Bree L Charlotte J , Netea Mihai G , Barreiro Luis B
Published: January 2023

The BCG vaccine, administered to more than 4 billion people worldwide, is designed to protect against Mycobacterium tuberculosis infection. Interestingly, clinical studies suggest that BCG also provides a degree of protection against heterologous infections, implicating other facets of the immune system apart from adaptive immunity. It has been hypothesized that BCG vaccination can leave immunological “scars” within hematopoietic stem and progenitor cells (HSPCs) that further impact downstream innate immune cell function. This is supported by studies in mice demonstrating that exposure to BCG leads to expansion and differential gene expression within hematopoietic stem cells and multipotent progenitors (HSCs/MPPs). However, very little is known about the impact of BCG vaccination on human bone marrow. Here we performed droplet-based scRNA- and scATAC-sequencing on human bone marrow aspirates from 20 healthy individuals, both before and 90 days after intradermal BCG vaccination or placebo. Over 1000 sites of differential chromatin accessibility across multiple CD34 subpopulations were present 90 days following BCG vaccination. Peaks of differential chromatin accessibility were enriched for binding of lineage determining and stress transcription factors, some of which were upregulated on the gene expression level. Expression levels of a subset of BCG-induced genes were found to significantly correlate with increased Il1b secretion of donor paired PBMCs in response to a C. albicans challenge. These data indicate that BCG vaccination re-wires transcription factor activity in human bone marrow in a way that may be linked to responses of PBMCs to secondary immune challenge with non-mycobacterial pathogens.
Comparison of DNA sensing by non-immune cells and tissues
Authors: Heller Loree , Znida Katarina , Bosnjak Masa , Conniff Amanda Sales , Jesenko Tanja , Markelc Bostjan , Tur Jared , Semenova Nina , Cemazar Maja
Published: January 2023

Pattern recognition receptors (PRRs) are nearly ubiquitous in both immune and non-immune mammalian cells. PRRs detect pathogen invasion as well as internal damage, inducing the secretion of pro-inflammatory molecules and cell death. Members of the PRR subset that detect intracellular DNA are termed DNA sensors. We examined DNA sensing in non-immune cells and their equivalent tissues using electroporation. This physical delivery method mitigates the need for viral or chemical agents that could confound the source of PRR activation. We used RT-qPCR and/or RNA-Seq to quantify mRNAs encoding DNA sensors and their signaling pathways and western blots, ELISAs and bead arrays to quantify related proteins. Several tumor cell types and non-tumor cells, including keratinocytes, fibroblasts, and myoblasts, expressed different DNA sensors at baseline and responded to backbone plasmid DNA delivery with individual DNA sensor signatures. Some similarities were observed among all these cell types, including upregulation of the DNA sensors ZBP1, DDX60, and Ifi204, the mouse analog of human Ifi16. Each cell type secreted a range of pro-inflammatory molecules including Type I interferon. DNA electroporation to B16-F10 melanomas, skin, and skeletal muscle induced unique cytokine and chemokine signatures, paralleling cellular signatures. ZBP1, DDX60, and Ifi204 were upregulated in skin and muscle; however, no significant regulation was observed in tumors. In muscle particularly, this may be due to the homogeneity of the tissue; tumors are inherently heterogeneous, producing an assortment of individual cellular responses.

Supported by NIH grant R01CA196796.
Proteomics analysis to identify intracellular inflammatory pathways modulated by Fh15 in macrophages stimulated with LPS-E. coli
Authors: Armina-Rodriguez Albersy , Valdés-Fernández Bianca , Roche-Lima Abiel , Carrasquillo-Carrión Kelvin , Espino Ana M
Published: January 2023

Previous studies of our laboratory have demonstrated that Fh15, the recombinant variant of F. hepatica FABP exhibit powerful anti-inflammatory properties. The administration of a single IV dose (for NHP) or IP dose (for mouse) of Fh15 prior to or after lethal doses of E. coli-LPS, respectively is enough to significantly suppress the pro-inflammatory cytokine storm and prevent the lethal pathologic consequences of septic shock. This suggest that Fh15 has a broad spectrum of action and may modulate pro-inflammatory mechanisms. The present study aimed to identify intracellular inflammatory pathways that could be modulated by Fh15. To achieve this goal, we have applied a quantitative proteomic approach using a Tandem Mass Tag peptide labelling, which has been used to quantified and identified biological macromolecules such as proteins. Samples were analyzed using Liquid Chromatography with tandem mass spectrometry. RAW 264.7 cells were stimulated with LPS and/or Fh15, cells treated with Fh15 or LPS alone were used as control. For the bioinformatics analysis, a fold change of 1.5 and pValue ≤ 0.05, were considered for the enrichment analysis, using the Ingenuity Pathway Analysis. Using these approaches, we identified 257 proteins associated with LPS treatment: 173 were up regulated and 84 were down regulated. Furthermore, we identified 139 proteins associated with Fh15 treatment: 38 were up-regulated and 101 were down-regulated. The IPA analysis identified proteins associated with NFkB, iNOS and Th1/Th2 pathways. We also identified 8 proteins differentially abundant that are associated with inflammatory and/or infectious diseases. Currently we are evaluating these proteins with ELISA, Western Blot and/or Flow cytometry techniques.

Supported by 1SC1AI155439-01 NIAID
Adenosine deaminase-1 (ADA-1) induces maturation and skewing of cytokine and chemokine production in dendritic cells to improve adaptive immune responses.
Authors: Cusimano Gina , Chakhtoura Marita , Taramangalam Bhavani , Connors Jennifer , Kutzler Michele A , Haddad Elias
Published: January 2023

Dendritic cells (DCs) are critical mediators of antigen-specific immunity through their ability to present antigen and drive T cell differentiation into different effector subsets such as follicular helper T cells (TFH). TFH cells provide both physical and cytokine-mediated stimuli to B cells resulting in somatic hypermutation and class-switching of B cell receptors. Molecules that target TFH cells, such as adenosine deaminase-1 (ADA-1), would improve humoral immunity. We have previously demonstrated that co-delivery of plasmid-encoded adenosine deaminase with an HIV-1 envelope and SARS-CoV-2 DNA vaccines in vivo, enhanced both humoral and cellular responses. However, the mechanism by which ADA-1 is acting as an adjuvant remains to be elucidated. To this end, we treated monocyte-derived DCs from healthy human donors in vitro with recombinant ADA-1 protein and evaluated the expression of maturation markers, cytokines and chemokines. ADA-1-treated DCs had a significantly increased expression of CD40 and CD86 as well as HLA-DR compared to their unstimulated, immature counterparts. The level of co-stimulatory marker and HLA-DR expression on ADA-1-treated DCs was similar to that on LPS/IFN-γ-treated DCs, indicating ADA-1-mediated DC maturation. ADA-1-treated DCs also exhibited a significant increase in IL-6, IL-1β and CXCL13 expression. IL-6 is a key pro-TFH cytokine and IL-1β and CXCL13 may play a role in TFH cell differentiation, function, and proliferation. Ongoing studies are aimed to drive ADA-1 overexpression in DCs and evaluate effects on antigen presentation. Overall, elucidating the mechanism of ADA-1’s adjuvanticity will allow for its progression as a clinical adjuvant.

This work was supported by funding to Dr. Elias Haddad from NIH 5RO1AI106482-01A and 1U19 AI128910-01.
dNTP catabolism is a macrophage-intrinsic gatekeeper preventing NLRP3 inflammasome hyperactivation
Authors: Zhong Zhenyu , Huo Shanshan , Liu Danhui , Wang Xiaochen , Liang Shuang
Published: January 2023

Aberrant activation of NLRP3 inflammasome drives the development of many diseases, including atherosclerosis, gout, osteoarthritis, Alzheimer’s disease, macular degeneration, type 2 diabetes and cancer. Our recent work demonstrated that new mitochondrial DNA (mtDNA) synthesis, initiated at the inflammasome priming step, is a prerequisite for producing oxidized mtDNA (ox-mtDNA)—an activating ligand of NLRP3. However, little is known regarding how innate immune cells, such as macrophages, manage to prevent NLRP3 inflammasome hyperactivation under physiological conditions, thereby avoiding the development of immunopathology. Here we show that SAMHD1, a cytosolic dNTP hydrolase, functions as a macrophage-intrinsic gatekeeper that restricts NLRP3 inflammasome hyperactivation. Mechanistically, inflammasome priming activates SAMHD1 to inhibit cytosolic dNTP buildup, thereby preventing their transport into mitochondria. This in turn protects mitochondria from uncontrolled new mtDNA synthesis and ox-mtDNA production, ultimately attenuating the extent of NLRP3 inflammasome activation. Consistent with these findings, SAMHD1-deficient mice exhibited NLRP3 inflammasome hyperactivation and developed exacerbated immunopathology in vivo relative to their wild-type littermates in response to various inflammatory insults. Together, these results establish cytosolic dNTP catabolism as a physiologically important protective mechanism to restrict NLRP3 hyperactivation, thereby linking dysregulation of nucleotide metabolism with the onset of NLRP3-dependent immunopathology.

Supported by NIH (K22AI135074, R35GM142654), CPRIT (#RR180014)
UTX is an epigenetic regulator of natural killer cell development and anti-viral effector function.
Authors: Cheng Mandy I , Riggan Luke , Ma Feiyang , Chin Scott , Tafti Rana , Pellegrini Matteo , O’Sullivan Timothy E , Su Maureen A
Published: January 2023

Natural killer (NK) cells are circulating type 1 innate lymphocytes that protect against viral infection and cancer. While it is now clear that NK cells display distinct epigenetic states during development and activation, the factors that control the epigenetic programming of NK cells during these processes are not well understood. Here, we show that the H3K27me3 histone demethylase UTX epigenetically regulates NK cells in a cell-intrinsic and demethylase-independent manner by regulating the chromatin accessibility of gene loci involved in homeostasis and effector function. As a consequence, mice with NK cell-specific UTX deficiency displayed an increase in peripheral immature NK cell cells that express higher levels of Bcl-2. Furthermore, UTX-deficient NK cells produce lower amounts of interferon (IFN)-γ and Granzyme B following MCMV infection, resulting in increased mortality. These findings reveal UTX as an essential regulator of NK cell homeostatic and effector epigenetic programs.

M.I.C. is supported by Ruth L. Kirschstein National Research Service Awards (GM007185 and AI007323), and Whitcome Fellowship from the Molecular Biology Institute at UCLA.
Several active components within MSCs secretome inhibit the IL-2-mediated effector functions of NK cells
Authors: Ko Eunhee , Lee Yoojin , Yoon Taejun , Kim Jongsun , Park Yong-Beom
Published: January 2023

Mesenchymal stem cells (MSCs) are well-known for their immunomodulatory functions in innate and adaptive immunity. Their functional diversity was verified in various disease model through paracrine mechanisms. MSCs-derived secretome consists of a variety of bioactive components such as nucleic acids, cytokines, chemokines, and small molecules. However, the effect of MSCs on natural killer (NK) cells activity is still controversial. In this study, we tried to identify active components within MSCs secretome possessing ability to affect NK cells activity and to investigate their action mechanisms. We found that MSCs secretome had no effect on survival and proliferation of NK-92 cells but specifically diminished cell activity, as evidenced by lower secretion of cytotoxic granules and effector cytokines and also reducing killing ability. This effect was reproduced in human peripheral NK cells equally. Moreover, seven candidates for modulating inflammation within MSCs secretome were identified via LC-MS/MS proteomic analysis. Interestingly, they are totally different factors from those known as MSC’s immunomodulatory factor. After treatment of neutralizing antibodies to each candidate, we observed that only three of the groups with neutralizing antibodies recovered cytokine secretion and cytotoxic ability of NK-92 cells. These findings suggest that MSCs secretome can inhibit the effector functions of NK cells via some particular molecules, which are increasing expression of inhibitory receptor CD96 on NK cells, consequently activating inhibitory signal.

This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2021R1F1A1049630).
Preclinical in vivo assessment of replacing linezolid for spectinamide-1599 in the Nix-TB regimen
Authors: Ali Malik Zohaib , Dutt Taru S , Walz Amanda , Pearce Camron , Lee Richard E , Robertson Gregory T , Hickey Anthony J , Meibohm Bernd , Gonzalez-Juarrero Mercedes
Published: January 2023

Tuberculosis (TB) patients diagnosed as drug-resistant TB have limited treatment options. Nix-TB trial is testing three-oral-drugs, namely Bedaquiline (B), Pretomanid (Pa) and Linezolid (L) (BPaL regimen) against drug-resistant TB. The BPaL regimen shows excellent favorable outcomes, but toxic effects are observed due to the long-term administration of Linezolid. Spectinamide-1599 (1599) is a potent protein inhibitor of Mycobacterium tuberculosis (Mtb) without known adverse effects and cross-resistance with other TB drugs. We hypothesized that inhaled 1599 could replace L in the BPaL regimen and provide similar or higher efficacy as BPaS regimen. Therefore, we tested BPaL and BPaS regimens in Balb/c (TB resistant) and C3HeB/FeJ (TB susceptible) mice infected with low-dose Mtb, and evaluated bacterial burden using colony-forming units (CFUs). Compared to untreated mice, CFUs in lungs of BPaL and BPaS treated Balb/c mice were decreased by >1 and 5 logs after 2- and 4-weeks treatment, respectively. In C3HeB/FeJ mice, 4-weeks of BPaL and BPaS treatment decreased CFUs by >3 logs compared to untreated control. However, no statistically significant differences were observed between treatment groups in both the mice strains. Cellular profiling using flow cytometry showed increased monocytes (CD11bhi CD14hi CCR2hi) and neutrophils (CD11b+ Ly6G+) in the bone marrow of BPaS mice compared to BPaL. These results along with cytokine profiling of the two treatment groups showed enhanced inflammatory response in BPaS mice than BPaL. We concluded that inhaled 1599 is a potential replacement for Linezolid if combined as BPaS regimen, and further studies are warranted.

This project was funded by National Institute of Health under the funding number NIH-NIAID-Allergy and Infect Diseases AI120670.
The biological mechanisms of metformin effects on aging-associated inflammation in obesity
Authors: Calvo Sara Santa Cruz , Bharath Leena , Nikolajczyk Barbara
Published: January 2023

Obesity prevalence within the 60+ years-old (yrs) population is drastically increasing. This alarmingly high rate of obesity compels comparative studies to identify common players involved in aging-derived and obesity-associated inflammation. Blood CD4+ T cells from lean older humans show an upregulated Th17 signature accompanied by a defective redox system. Treatment of these cells with the anti-diabetic drug metformin (100μM) promoted a more young-like anti-inflammatory/antioxidant phenotype. Here, we investigate whether the protective effects of metformin observed in CD4+ T cells from lean aging adults persist in obesity.

Blood CD4+ T cells from obese older (60+ yrs) donors secrete twice the amount of the Th17 cytokines IL-17F and IL-21 when compared to their younger counterparts (25–35 yrs). Glutamate production, which supports Th17 cytokines secretion, is 1.5-times higher in cells from older compared to younger subjects. Mitochondrial superoxide production in these cells was 25% higher than in younger cells, with no changes in total peroxide.

Contrary to findings from cells of lean subjects, metformin failed to reduce Th17 cytokines in cells from obese older subjects, perhaps due to metformin’s inability to reduce glutamate production. However, metformin reduced hydrogen peroxide production 1.75-fold, with no effect on superoxide, in cells from obese older subjects.

These results collectively indicate that 1) obesity-associated upregulation of Th17 cytokines is mechanistically linked to glutamine metabolism and mitochondrial superoxide production and 2) metformin’s action targets peroxide production rather than superoxide, which is insufficient for reducing age-associated inflammation in obesity.

The Biological mechanisms of Metformin Effects on Aging-Associated Inflammation 1R56AG069685-01
The ZFP36 family of RNA-binding proteins regulate homeostatic and autoreactive T cell responses
Authors: Cook Melissa Erin , Bradstreet Tara R , Webber Ashlee M , Kim Jongshin , Santeford Andrea , Schwarzkopf Elizabeth A , Apte Rajendra S , Blackshear Perry J , Edelson Brian T
Published: January 2023

The zinc finger 36 (ZFP36) family of RNA-binding proteins, consisting of ZFP36, ZFP36L1, and ZFP36L2, is known to negatively regulate mRNA stability and/or inhibit translation of many transcripts, including cytokines. While there are reports of all three family members regulating T cell cytokine production, delineating the functions of these genes is challenging due to spontaneous phenotypes upon global deletion of single genes and potential redundancy in their functions. To overcome this, we generated Cd4-Cre+ Zfp36fl/fl Zfp36l1fl/fl Zfp36l2fl/fl mice. Only upon triple deletion, but not individual or paired deletions, do mice spontaneously develop an inflammatory disease characterized by early lethality, hypercytokinemia, and immune cell infiltration into some peripheral organs, including the central nervous system. As SNPs in human ZFP36L1 and ZFP36L2 have been linked to susceptibility for multiple sclerosis, we tested ZFP36 family member individual and paired T cell-deficient mice in experimental autoimmune encephalomyelitis (EAE). To our surprise, Cd4-Cre+ Zfp36l1fl/fl Zfp36l2fl/fl mice were markedly protected from EAE clinical disease, while no change in disease severity was seen with any deletion strains involving Zfp36. There was a severe impairment in generating antigen-specific CD4+ T cells in Cd4-Cre+ Zfp36l1fl/fl Zfp36l2fl/fl mice during EAE priming. Our findings demonstrate a novel redundancy of the ZFP family members in regulating T cell homeostasis and a shared role for ZFP36L1 and ZFP36L2 to promote clonal expansion. Understanding the individual and shared functions of the ZFP36 family members may lead to opportunities to target them to suppress T cell-driven autoimmunity.

M.E.C. supported by the National Science Foundation Graduate Research Fellowship program (DGE-1745038) and by grant 5T32AI007163 from the NIAID. B.T.E. supported by the NIAID (R01 AI113118).
Copper homeostasis is critical for T cell activation
Authors: Mandarano Alexandra H , McGargill Maureen A
Published: January 2023

Elevated copper levels are associated with autoimmune disease, infection, and tumors in both humans and mice. Modulating copper availability is a promising potential intervention to treat these diseases. However, the mechanisms underlying the beneficial impact of copper modulation in disease are poorly understood. Moreover, the role of copper homeostasis in immune function, including in T cell responses, is not clear. Copper is a required cofactor for proteins involved in essential T cell processes, including extracellular matrix regulation, signaling pathways, mitochondrial metabolism, autophagy, and maintenance of reactive oxygen species. To investigate the impact of copper levels on T cell function, we examined T cell activation during altered copper availability. We found that copper chelation limited T cell proliferation and reduced expression of activation markers following stimulation. We also found that both activation and copper availability altered copper transporter expression in T cells. Additionally, copper modulation substantially impacted the transcriptional profiles of activated T cells, including expression of transcription factors, co-stimulatory markers, cytokines, and metabolic genes. Overall, our data demonstrate that altered copper levels impair T cell activation. Further, our results reveal new pathways impacted by copper homeostasis in T cells, which will be key to both understanding the role of copper in disease and leveraging this role for therapeutic development. Studies to evaluate the role of copper homeostasis in the immune response in vivo are ongoing.

Supported by funding from ALSAC.
The phosphatidylinositol-transfer protein Nir3 modulates T cell development and function
Authors: Lu Wen , Helou Ynes A , Au-Yeung Byron B , Shrinivas Krishna , Liou Jen , Weiss Arthur
Published: January 2023

Before emerging as functional T lymphocytes, thymocytes transit through multiple selection stages during which T cell antigen receptor (TCR) signaling controls the survival and subsequent maturation. Hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2) by phospholipase C-γ (PLCγ1) represents a critical step in T cell receptor (TCR) signaling which leads to calcium increases as well as PKC and Ras activation, events that contribute to T cell activation. PIP2 in the plasma membrane (PM) is depleted rapidly upon TCR stimulation and the replenishment of PIP2 levels is dependent on delivery of its precursor phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the PM. It was not clear how the delivery of PI from ER is regulated in thymocytes and T cells or how the process impacts T cell development and function. Here, we show that a membrane-associated PI transfer protein, Nir3 (Pitpnm2), promotes PIP2 replenishment following TCR stimulation and is important for T cell development. The high expression level of the Nir3 gene in thymocytes suggests its role in thymocyte selection. In Nir3 deficient thymocytes, the replenishment of PIP2 following TCR stimulation is significantly slower. Moreover, Nir3 deficiency attenuates calcium mobilization in DP thymocytes in response to weak TCR stimulation. The impaired TCR signaling led to impaired thymocyte development at the beta-selection and positive selection stages in Nir3 deficient mice. These findings reveal the role of Nir3 in TCR signaling and thymocyte development. This study highlights the importance of PIP2 replenishment and the role of Nir3 in PI transfer through regulated ER-PM interactions during TCR signaling.

This work was supported by in part by the Howard Hughes Medical Institute, the National Institutes of Health (NIH), NIAID R37 AI114575, and DRC Center Grant P30 DK063720 (UCSF Parnassus Flow Cytometry Core, UCSF Diabetes Center).
The Ikaros zinc finger transcription factor Eos as a candidate regulator of TH2 differentiation and function.
Authors: Tuazon Jasmine , Sreekumar Bharath , Read Kaitlin , Yaeger Michael , Varikuti Sanjay , Gowdy Kymberly M , Oestreich Kenneth J
Published: January 2023

Although CD4+ T helper 2 (TH2) cells normally defend against parasitic infection, they play a central role in the pathogenesis of allergic asthma. Therefore, understanding the molecular mechanisms governing TH2 differentiation and function is a top priority for improving asthma outcomes. One pathway of interest for controlling TH2-mediated disease is IL-2/STAT5 signaling, which is essential for TH2 polarization yet boasts a complex regulatory network that is incompletely understood. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Eos as a candidate regulator of IL-2/STAT5 signaling underlying TH2 differentiation and function. To start, we show that Eos gene and protein expression is increased in TH2 cells relative to CD4+ T cell subsets that are negatively regulated by IL-2/STAT5 signaling, such as TH17 and TFH cells. Given these findings, we sought to define the functional effects of Eos in TH2 cells using in vitro and in vivo murine house dust mite asthma models. Intriguingly, Eos deficiency in vitro and in vivo results in reduced expression of key TH2 transcription factors (Gata3, Blimp-1), effector cytokines (IL-4, IL-13), and differentiation receptors (IL-2Rα, IL-2Rβ, IL-4Rα). Mechanistically, our data reveal that Eos interacts with and increases the activity of STAT5, suggesting that Eos enhances TH2 differentiation and effector function through direct STAT5 regulation. These findings in proinflammatory TH2 cells are of high significance, as Eos, to date, has largely been associated with the immunosuppressive functions of TREG cells. Taken together, our data reveal a novel mechanism by which Eos positively regulates IL-2/STAT5 signaling to promote TH2 differentiation and function.

Work supported by grants from the NIH (R01AI134972) and The Ohio State University (Susan Huntington Dean’s Distinguished University Fellowship).
Profound abnormalities in thymic epithelial cells in Rag1 hypomorphic mice: implications for immune reconstitution after stem cell transplantation
Authors: Pala Francesca , Oguz Cihan , Corsino Cristina , Martins Andrew , Lack Justin , Tsang John , Notarangelo Luigi , Bosticardo Marita
Published: January 2023

Thymic epithelial cells (TEC) are a heterogeneous population of stromal cells which orchestrate T cell development and selection. Although recent reports highlighted TEC complexity, their developmental origin, hierarchy and differentiation still remain ill-defined. We compared TEC distribution and gene expression in wild-type (WT) mice and in mice carrying Rag1 hypomorphic mutations (Rag1mut) observed in patients with immunedeficiency and immune dysregulation. Single cell RNA-seq analysis of TECs isolated from Rag1mut mice revealed an excess of cortical TECs (cTECs). Medullary TECs (mTECs), albeit reduced in number, showed a similar distribution in previously described mature subsets. To address whether TEC abnormalities in Rag1mut mice might result from a developmental block, we compared them to TECs from neonate WT mice. Indeed, we observed a similar TEC distribution in adult Rag1mut mice and newborn WT mice, although we identified peculiar features in cTEC subsets of Rag1mut mice. Transplantation of WT hematopoietic stem cells (HSC) into Rag1mut mice was able not only to correct thymocyte development, but also to revert TEC phenotype to normalcy. However, in a competitive transplantation setting where 10% of WT and 90% mutant HSCs were used, only partial restoration of thymocyte subsets was observed, associated with incomplete rescue of TEC phenotype. In summary, we show that abnormalities of TEC composition in Rag1 mutant mice correlate with impaired development of thymocytes, highlighting the role of lymphostromal cross-talk in TEC maturation. Moreover, these data point to the need to achieve full donor chimerism after HSC transplantation in order to rescue the thymic compartment and prevent defects of immune tolerance.

This research was supported by the Intramural Research Program of the NIAID, NIH
Age-associated changes to lymph node fibroblastic reticular cells
Authors: Lancaster Jessica N , Kwok Tina , Medovich Shannon , da Silva Ildefonso Alves
Published: January 2023

The decreased proportion of antigen-inexperienced, naïve T cells is a hallmark of aging in both humans and mice, and contributes to reduced immune responses, particularly against novel and re-emerging pathogens. Naïve T cells depend on survival signals received during their circulation among the lymph nodes by direct contacts with stroma, in particular fibroblastic reticular cells. Gross changes to the architecture of the lymph nodes have been described, but it is unclear how lymph node stroma are altered by aging, and whether these changes contribute to reduced naïve T cell maintenance. Here, we visualized naïve T cell migration in the aged lymph node using 2-photon microscopy and determined that T cell motility is impaired when in proximity to fibrotic deposition. Adoptive transfer of young naïve T cells into young or aged hosts revealed reduced homeostatic turnover in the aged host, supporting the role of T cell-extrinsic mechanisms that regulate their survival. Further, we determined that development of early fibroblastic reticular cells of the lymph node stroma is impaired, which can contribute to declining levels of naïve T cell survival factors. Thus, our study addresses the controversy as to whether aging impacts the composition lymph node stroma and supports a model in which impaired differentiation of lymph node fibroblasts inhibits the interactions necessary for naïve T cell homeostasis.
Spatially Restricted T-cell Activation in Inflamed Tissues
Authors: Bala Noor , Prizant Hen , Hughson Angela , McGurk Alexander , Fowell Deborah J
Published: January 2023

Pathogen control requires T-cells to locate and make direct contact with antigen-presenting cells (APCs) for peripheral reactivation and delivery of effector molecules. The signals that orchestrate T-cell-APC interactions are poorly understood in inflamed peripheral tissues. Using intravital imaging (IVI), we have found that Th1 cell entry into the inflamed dermis is nucleated by perivascular CXCL10+ cellular clusters enriched for APCs. Th1 cells form more stable interactions with APCs within these clusters compared to outer regions. CXCL10+ clusters are amplified in a IFNγ-dependent manner, where CD4+ T-cell IFNγ release increases APC recruitment and CXCL10 expression. We hypothesize that these chemokine clusters serve as CXCL10 Peripheral  Activation (PAC-10) niches, where Th1 cells achieve early peripheral activation away from the pathogen modulated milieu. We seek to determine the relationship between these spatially restricted PAC-10 niches, infection foci, and pathogen clearance. Using a combination of IVI, flow cytometry, and transcriptomics we characterized niche specific Th1 cells and niche cellular composition to determine if PAC-10 niches modulate Th1 cells for pathogen control. Initial studies reveal that niche specific Th1 cells have increased CD69 and IFNγ expression compared to non-niche counterparts. PAC-10 niches appear to have a spatially distinct innate cell population, likely with enhanced ability to support Th1 activation. Future studies will characterize the functional status of niche specific Th1 cells and evaluate the necessity of PAC-10 niches for pathogen control. These studies will inform therapeutic strategies to boost T-cell activation in infection or hinder activation in autoimmunity.

Supported by NIH NIAID P01 AI02851 NIH NIAID R01 AI070826
Strong tonic TCR signaling is associated with negative regulation of naive CD4+ T cells
Authors: Au-Yeung Byron B , Eggert Joel , Scharer Christopher , Zinzow-Kramer Wendy
Published: January 2023

Naive T cells constantly experience tonic or basal TCR signals in response to self-peptides presented by MHC (pMHC) in vivo. We aimed to define how tonic TCR signaling impacts naive CD4+ T cells at the functional, transcriptional, and epigenetic levels. The transgenic Nur77-GFP reporter gene is responsive to tonic TCR signals. Naive CD4+ T cells can express a broad range of Nur77-GFP transgene expression at a single cell level, even if they express identical transgenic TCRs. Immature T cells that express high levels of Nur77-GFP following selection in the thymus tend to sustain high Nur77-GFP expression in the periphery. Naive CD4+ T cells with a Nur77-GFPHI Ly6C− phenotype experience the strongest tonic TCR signaling and in the OT-II TCR transgenic system, exhibit less sensitivity to stimulation with low doses of cognate pMHC or to weaker affinity pMHC. Analyses of gene expression in Nur77-GFPHI Ly6C− cells suggest a transcriptional program consistent with T cell activation, but also of negative regulation. Analyses of chromatin accessibility by ATAC-seq revealed over 3000 differentially accessible chromatin regions in Nur77-GFPHI Ly6C− cells, including several loci within or near genes encoding negative regulators such as Ctla-4 and Tim3. Nur77-GFPHI Ly6C− cells exhibited increased accessibility at multiple loci in the gene encoding Tox. A subset of these loci was previously reported to be more highly accessible in exhausted T cells. These findings suggest that persistent strong tonic TCR signaling may induce changes similar to those that occur during exhaustion. We propose that naive CD4+ T cells adapt to strong tonic TCR signaling and as a result, exhibit decreased sensitivity to subsequent TCR stimulations.

Supported by a grant from NIH (K01AR065481)
Naive CD4+ T cell heterogeneity
Authors: Sajani Alia , Schaafsma Evelien , ElTanbouly Mohamed , Lines Janet , Noelle Randolph J
Published: January 2023

Although naive T cells comprise about 90% of circulating and 20% of tissue restricted T cells, most studies on T cells emphasize effector, regulatory or memory functions. The consensus that all naive cells comprise a functionally monomorphic population is challenged by our recent findings. A redefinition of the naive T cell state shows that these cells are heterogeneous, with pre-programed differentiation trajectories. VISTA (V-domain Ig suppressor of T cell activation) is an immune checkpoint restricted to the hematopoietic compartment. Here, using single cell sequencing, we demonstrate unexpected loss of quiescence in VISTA deficient CD4+ T cells compared to Wild Type CD4+ T cells. The quiescence module, defined by Klf2, Klf6, Btg1, Btg2 expression, is lost in VISTA deficient mice. Conversely, there is an expansion of the stem-cell memory-like cluster, defined by Tcf7, Bcl2, and Il7r. This altered subset make-up due to VISTA deficiency demonstrates that naive T cell heterogeneity is tightly regulated. Consequently, we identified 5 subsets in healthy adult mice using single cell sequencing of naive CD4+ T cells. These include a quiescence cluster, memory-like cluster, TCR reactive cluster, IFN responsive cluster and T cell undifferentiated cluster. This heterogeneity is imprinted in the thymus, maintained in the periphery, and is not influenced by the TCR repertoire. Tracking the differentiation trajectory of these naive CD4+ T cells shows biased lineage commitment dependent on progenitor cluster identity. Thus, thymic imprinting and peripheral maintenance of varied naive T cell identities determines T cell fate.

Supported by 5R01AR070760-05
The development of humoral immunity to common dietary antigens in human infants is accompanied by the accumulation of specific plasma cells in the thymus
Authors: Cordero Hector , Hess Jacob , Nitschki Elio , Shihab Ronzon , Roy Poulomi , Kalfa David M , Bacha Emile A , Zorn Emmanuel
Published: January 2023

We have reported on the accumulation of plasma cells (PCs) in the human thymus, starting several months after birth. This thymic PC niche includes clones specific to common viruses and vaccination antigens presumably generated through peripheral responses. Humans acquire humoral immunity to dietary antigens during the first 3 years of life. Here we investigated whether such immunity also resulted in the homing of specific PC to the thymus. Using a combination of dietary antigen microarrays, ELISA and ELISPOT assays, we tested for the presence of serum IgG and thymic antibody-producing cells specific to common food antigens in discarded blood and thymus specimens from 1 day to 2 year-old subjects (N=20). Our studies first confirmed cow’s milk proteins, including casein and lactoferrin, as prevalent targets of humoral responses during infancy. Remarkably, subjects with the highest serum IgG titer to cow’s milk antigens also had high-frequency PC in the thymus specific to the same antigens. Our study demonstrates for the first time the presence of antibody-secreting cells specific to dietary antigens in the thymic of human infants. Their possible contribution to tolerance mechanisms warrants further investigation.

This work was supported by a NIH-NIAID grant U01-AI131339. Dietary antigen arrays (ImmunoCap) used in this study were a gift from Thermo Fisher Scientific.
The role of microRNA-142 in B cell activation and effector functions
Authors: Graham Natalie Marie , Wang Wei-Le , Reyes Estefany , Boldin Mark P
Published: January 2023

Control of gene expression by microRNA (miRNA) has recently emerged as a critical mechanism that regulates B cell activation and function. However, the role of specific miRNAs in this process is unclear. Using germline knockout (KO) mice, we have previously shown that miR-142 ablation impairs humoral immune responses despite significant expansion of the B cell compartment, suggesting that miR-142 is critical for B cell effector function and terminal differentiation. To more precisely dissect the role of miR 142 in B cell effector responses, we developed an activated B cell-specific miR-142 KO mouse by breeding conditional miR-142 KO (miR-142fl/fl) mice with mice expressing Cre recombinase driven by activation-induced cytosine deaminase (AIDcre). To label activated B cells in miR-142fl/fl  AIDcre mice, we bred them to mice carrying a loxP-flanked STOP cassette, allowing for the expression of fluorescent Zsgreen1 reporter protein in the presence of Cre recombinase. As expected, B cell development was largely unperturbed in miR-142fl/fl AIDcreZsgreen+/− mice. In contrast, FACS analysis of naive and antigen-challenged miR-142fl/fl AIDcre Zsgreen+/− mice revealed a significant decrease in germinal center (GC) B cells and plasma cells (PCs). RNA sequencing of GC B cells suggests dysregulation of transcription factor networks such as miR-142-3p targets BACH2 and IRF4 may contribute to the phenotypes observed in miR 142fl/fl AIDcre Zsgreen+/− mice. Future experiments will investigate molecular pathways governing terminal B cell differentiation and effector function in miR-142fl/fl AIDcre Zsgreen+/− mice, thereby elucidating the role of miR-142 in promoting a healthy response to antigen.