bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual regulatory role of c-Rel O-GlcNAcylation in T cell function and Type 1 Diabetes
Authors: Tomalka Jeffrey Alan , Ramakrishnan Parameswaran
Published: January 2023

O-GlcNAcylation is a post-translational protein modification involving the addition of N-acetylglucosamine (GlcNAc) to serine and threonine residues on target proteins. Global O-GlcNAcylation levels rise under hyperglycemia such as that caused by Type 1 Diabetes (T1D). We found that hyperglycemia induces O-GlcNAcylation of the NF-kappaB protein c-Rel at a single serine residue, S350. O-GlcNAcylation of c-Rel enhances its DNA binding and the transcription of c-Rel-dependent pro-autoimmune Th1 cytokines in T cells. This direct link between hyperglycemia and augmented Th1 function imply a key regulatory role for c-Rel O-GlcNAcylation in the pathology of T1D. Interestingly, our recent results show that O-GlcNAcylation inhibits binding of c-Rel to the FOXP3 promoter, leading to reduced FOXP3 expression. Together, these data suggest that c-Rel O-GlcNAcylation has a novel, dual regulatory role in controlling T cell function by: 1) Enhancing pro-autoimmune T cell function and 2) Suppressing Treg function. This creates an immune environment prone to heightened inflammation and autoimmunity that may exacerbate the frequency of autoimmunity in Western countries. Our current work focuses on defining the molecular mechanisms by which c-Rel O-GlcNAcylation regulates transcriptional activation in T cells and Treg cells. We are studying the non-obese diabetic (NOD) mouse model of spontaneous diabetes to examine the pathophysiological role of c-Rel O-GlcNAcylation. Knowledge on the regulation of autoimmunity by c-Rel O-GlcNAcylation will enhance our understanding of the etiology immune-mediated pathologies in diabetes and reveal a new therapeutic target to treat autoimmune diabetes.
HIGH-DIMENSIONAL ANALYSIS OF HUMAN REGULATORY T CELLS USING MASS CYTOMETRY
Authors: Dawson Nicholas , Cook Laura , Pesenacker Anne , Hoeppli Romy , Morishita Kimberly , Broady Raewyn , Levings Megan K
Published: January 2023

Regulatory T cells (Tregs) are critical for maintenance of peripheral tolerance in a variety of tissues. Evidence that Tregs seem to be a phenotypically diverse population that varies between individuals and tissues suggests that there may be functionally-specialized subsets of Tregs which vary depending on location and/or disease state. We developed a new mass cytometry-based method to measure FOXP3 in combination with 20 other parameters to characterize the phenotype of human Tregs in different tissues, and to enable analysis of antigen-specific cells. We first developed a protocol to detect FOXP3, the Treg lineage-defining transcription factor, via mass cytometry. We found this optimized protocol is more sensitive than commercially-available methodology to detect FOXP3 by mass cytometry and is compatible with staining of other intracellular targets such as cytokines and other transcription factors. To ask how Treg populations differ depending on location and disease state, we stained mononuclear cells from adult peripheral blood, pediatric thymus and cord blood, as well as synovial fluid from pediatric subjects with juvenile idiopathic arthritis with our Treg-specific mass cytometry panel. The resulting data were analyzed using several bioinformatics approaches including viSNE and revealed diversity in the heterogeneity and phenotype of Tregs depending on their origin. The ability to stain FOXP3 using mass cytometry will facilitate the further characterization of Tregs in health versus disease and help us understand how Tregs are functionally specialized in the context of different tissues and disease.
Loss of SOCS1 in dendritic cells increases dendritic cell maturation and decreases the CD8 T cell response to Listeria monocytogenes.
Authors: Alice Alejandro , Kramer Gwen , Crittenden Marka R , Bahjat Keith , Gough Michael J
Published: January 2023

The suppressor of cytokine signaling (SOCS) family is a group of structurally related proteins characterized by a central SH2 domain that targets the SOCS proteins to specific molecules within the JAK-STAT pathway, and a SOCS-box domain that promotes degradation of the cytokine-receptor complex. SOCS-1 is induced by cellular activation and serves as a negative feedback mechanism for a range of cytokines. It has been described that silencing SOCS-1 in dendritic cells (DC) enhances antigen presentation, T cell priming, and anti-tumor immunity. Therefore, inhibition of SOCS-1 could profoundly augment the potency of the L. monocytogenes (Lm) based cancer vaccines that we are testing in clinical studies. We questioned the role of SOCS-1 in dendritic cells in vivo during intracellular infection, a scenario where antigen-specific T cells are required for microbial clearance. To test this we characterized the infection and immune response to Lm in CD11c-Cre × SOCS1fl/fl mice that have a SOCS1 deficiency in DC. DC lacking SOCS1 showed increased maturation in vivo at baseline, but showed equivalent responses to vaccination with DC-targeted antigens combined with α-CD40. We found that Lm can be recovered from spleens and livers from mice with SOCS1-deficient DC at similar levels to that of the wild type animals. However, mice with SOCS1-deficient DC showed a dramatic defect in the specific-CD8+ T cell response while the specific-CD4+ T cells response was not affected. The lack of CD8+ T cells response correlated with low levels of IFN-γ and MCP-1 in serum during the first days of infection and decreased NK maturation. Our results demonstrate that SOCS1 expression in DC is critical to generate a strong CD8 T cell response to Lm.
The Nrf2 activator, tBHQ, inhibits IL-2 production by human T cells in a largely Nrf2-independent manner
Authors: Zagorski Joseph Ward , Turley Alexandra E , Maser Tyler P , Rockwell Cheryl Elizabeth
Published: January 2023

Nrf2 is a transcription factor that is activated by cell stress, such as oxidative insult and exposure to xenobiotics. Upon activation, Nrf2 translocates to the nucleus, where it upregulates its target genes, many of which arecytoprotective in function. Nrf2 has been shown to modulate immune function; Nrf2-null mice are more sensitive to many models of inflammatory disease, including experimental autoimmune encephalitis and sepsis induced by cecal ligation and puncture. We have previously shown that activation of Nrf2 in primary mouse CD4 T cells promotes Th2 differentiation, which may have implications on the development of allergy and asthma. However, the role of Nrf2 in human CD4 T cell function remains uncharacterized. Thus, the purpose of the present studies was to determine the role of Nrf2 in the early events following CD4 T cell activation. Primary human CD4 T cells from peripheral blood were treated with varying concentrations of tBHQ (a known Nrf2 activator) prior to activation with anti-CD3/anti-CD28. Although treatment of human T cells with tBHQ suppressed the production of early cytokines, such as IL-2, the model fell short in determining whether these effects were Nrf2-dependent. Toward that end, Nrf2-null T cells were generated by implementation of CRISPR-CAS9 gene editing, in human Jurkat T cells. Treatment of Nrf2-null and Wild-type Jurkat T cells demonstrated that the effects of tBHQ upon cytokine production are largely independent of Nrf2. In contrast, suppression of CD25 expression by tBHQ is partially Nrf2-dependent. The current studies demonstrate that many of the effects of tBHQ on early events following CD4 T cell activation are independent of Nrf2. (This work was funded by NIH grants ES018885 and GM092715)
ADAMTS5: A critical enzyme for the maintenance of influenza-specific CD8+ T cell memory
Authors: McMahon Meagan K , McCulloch Daniel , Stambas John
Published: January 2023

Extracellular matrix (ECM) enzymes, such as the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) family, play a significant role in ECM remodelling. ECM remodelling facilitates immune cell migration and adhesion. ADAMTS5, a member of the ADAMTS family, cleaves ECM proteoglycans, such as aggrecan and versican, and has a well-characterised role in osteoarthritis and embryogenesis. However, ADAMTS5 has an un-identified role throughout the immune response. Our previous studies indicated that ADAMTS5 expression is required to mediate T cell migration during acute influenza virus infection. In this current study, we analysed secondary acute responses (day 7) and primary memory (day 30) following influenza infection of Adamts5−/− mice. Here, we primed Adamts5−/− mice with PR8 influenza virus (1.5×107 pfu/mouse) and challenged 8 weeks later with X31 influenza virus (104 pfu/mouse). Following secondary infection, Adamts5−/− mice displayed greater weight loss and expressed fewer IFNγ+NP366-372+CD8+T cells. Critically, we observed a reduced number of influenza-specific CD8+ memory cells (CD62LhiCD44lo) in Adamts5−/− mice after primary infection, which may result in the lower number of IFNγ+NP366-372+CD8+T cells observed in secondary acute infection. We therefore hypothesize that the ADAMTS5 enzyme, through its ECM remodelling capabilities, is essential for the maintenance of memory and recall of functional immunity following secondary infection.
Specific patterns of self-antigen expression determine the mechanisms by which polyclonal self-reactive CD4+ T cells are tolerized.
Authors: Malhotra Deepali , Linehan Jonathan L , Dileepan Thamotharampillai , Lee You Jeong , Purtha Whitney E , Lu Jennifer V , Nelson Ryan W , Fife Brian T , Orr Harry T , Anderson Mark S , Hogquist Kristin A , Jenkins Marc K
Published: January 2023

Understanding how self-tolerance is maintained requires the ability to track rare, self-reactive T cells of known specificity in normal individuals. However, due to technical limitations much of our knowledge has relied on the study of T cell receptor transgenic cells that recognize model antigens. While this work has identified numerous modes of tolerance, it is unclear which mechanisms operate in normally diverse repertoires and how this decision is made. We studied polyclonal CD4+ T cells specific for an epitope from green fluorescent protein (GFP) in fourteen different mouse strains that express GFP under the control of tissue-specific promoters. We found that clonal deletion was the key tolerance mechanism for self-antigens that were uniformly expressed by thymic antigen-presenting cells. In contrast, self-antigens that were absent from the thymus were ignored. A more complex form of tolerance regulated CD4+ T cells specific for self-antigens with limited expression by thymic antigen-presenting cells. These populations showed partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential of remaining cells. Furthermore, these tolerance mechanisms were also found to regulate CD4+ T cells specific for endogenously expressed self-antigens. Thus, self-antigen expression patterns dictate the tolerance mechanisms by which self-reactive polyclonal CD4+ T cells are regulated.
Tissue-specific antigens induce dual complementary T cell tolerance mechanisms
Authors: Martinez Ryan J , Blanchfield Lori , Barwick Benjamin , Martinez Hunter , Reid Hugh , Evavold Brian D
Published: January 2023

To understand tolerance mechanisms to tissue-specific antigens, we probed the development of polyclonal T cells specific for myelin oligodendrocyte glycoprotein (MOG), a myelin antigen targeted during autoimmune demyelinating disease. We find T cell tolerance occurs via two thymocyte selection processes. First, the presence of tissue-specific antigen intrinsically broadens the TCR repertoire to lower overall reactivity of effector T cells. Second, expression of MOG extrinsically induces a higher frequency of more efficacious antigen-specific Foxp3+ regulatory T cell population that prevents autoimmunity. Interestingly, clonal deletion mediated by thymocyte negative selection of high-affinity T cells was not apparent. Thus, cooperative tolerance exists to prevent autoimmunity towards tissue-specific antigens.
CD4 T cell expression of PTIP is necessary for epigenetic maintenance of Th1/Th17 responses during Cryptococcus neoformans infection
Authors: Eastman Alison J , Gupta Kanav , Neal Lori , Xu Jintao , Zhou Guolei , Malachowski Antoni , Dressler Gregory R. , Kunkel Steven L , Schaller Matthew , Olszewski Michal A
Published: January 2023

Containment and clearance of opportunistic fungal pathogen Cryptococcus neoformans (CN) requires Th1/Th17 polarized CD4 T cells. Studies show that Th1/Th17 T-cell polarization is linked to enriched chromatin activation at histone 3 lysine 4 (H3K4). DNA repair enzyme PTIP forms a complex with MLL3/4 H3K4 methyl transferases, but its role in regulation of immune-related genes remains unknown. We hypothesized that the absence of PTIP in CD4 T cells would result in a defect in generation and/or maintenance of Th1/Th17 driven immune protection against CN infection. CD4Cre+, PTIP knockout mice (CD4-PTIP-KO) and their control Cre- littermates were infected with CN and their fungal burden and T cell polarization assessed at 3 and 6 weeks post-infection (wpi). There were few statistically significant differences at 3 wpi, but no defects in IFNγ and TNFαproduction in these mice. However, by 6 wpi we observed profound defects in fungal clearance and the expression of hallmarks of Th1 and Th17 responses. CD4-PTIP-KO mice had diminished fungal clearance by 1.5-log and showed diminished frequency of IFNγ, TNFα, and IL-17 producing CD4 T-cells as well as suppressed global production of these cytokines. Furthermore, frequency of IFNγ-IL-17 dual-producing CD4 T cells, previously found to correlate with clearance, was diminished in CD4-PTIP-KO mice. Interestingly no T-cell activation defect (frequency of CD44hi and CD62L low) was altered in CD4-PTIP-KO mice. Collectively, we conclude that PTIP is not necessary for induction of Th1 and Th17 polarized T cells but it is crucial for maintaining robust Th1/Th17 cytokine production by polarized T cells throughout C.neo infection, presumably via epigenetic stabilization of CD4 T cells Th1/Th17 programing.
Necrotic liver induces pyroptosis through Caspase-1 in eosinophils
Authors: Macapagal Daphne , Connor Jane , Ramalingam Thirumalai R , Wynn Thomas A , Mustelin Tomas Mikael , Davidson Todd S
Published: January 2023

During sterile inflammatory reactions damage associated molecular patterns (DAMPS) are released from necrotic cells and recruit immune cells to the sites of tissue injury. We found that necrotic liver homogenates, when injected intraperitoneally, can recruit large numbers of immune cells. Among these immune cells were activated eosinophils. Eosinophils are polymorphonuclear leukocytes typically found within tissues of the gastrointestinal and respiratory tracts. While eosinophils are well known to accumulate in the lungs of asthmatics or in response to helminths and parasites, several studies have shown the presence of eosinophils in liver biopsies of drug-induced liver injury, chronic hepatitis C and liver fibrosis. The prevalence of eosinophilic infiltrates in the liver points to a correlation between infiltration and disease outcome. Our results demonstrated eosinophil activation upon exposure to necrotic liver components. Exposure to necrotic liver cells induced eosinophil degranulation as measured by expression of CD107a and eosinophil peroxidase release as measured by ELISA. Further study of eosinophil activation during necrotic liver exposure showed IL-1β and IL-18 release as well as cell death. All of these responses were dependent upon caspase-1 activation. Caspase-1 mediated cell death accompanied by IL-1β and IL-18 release are hallmarks of pyroptosis, an inflammatory cell death pathway. We verified eosinophil pyroptosis in vivo using a liver fibrosis model involving Schistosoma mansoni infected mice. Eosinophils extracted from S. mansoni infected livers showed caspase-1 activation and cytokine release indicating that these cells undergo pyroptosis in vivo in response to hepatocyte injury.
A Novel Role for the 4E-BP/eIF4E Axis in B Cell Antibody Class Switching
Authors: Chiu Honyin , So Lomon , Fruman David A
Published: January 2023

During an immune response, activated B cells give rise to antibody secreting plasma cells to fight infection. B cells undergo a process known as antibody class switch recombination (CSR) to produce different classes of antibodies with varying effector functions. The mammalian target of Rapamycin (mTOR) signaling pathway is activated during this process and inhibition of mTOR suppresses both proliferation and differentiation into antibody secreting cells. mTOR is a kinase that is found in two distinct complexes, mTORC1 and mTORC2 and B cells deficient in mTOR have impaired survival, proliferation and differentiation. It has been shown that antibody class switch recombination requires B cell division, however it is unclear if there is another regulatory mechanism through mTOR that is independent of proliferation. The mTORC1 inhibitor rapamycin, at either low concentrations or when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Mechanistic investigation of the mTORC1 substrate 4E-BP, an inhibitor of eIF4E and cap-dependent translation, showed that blocking the 4E-BP/eIF4E axis can decrease antibody class switching independently of proliferation. Genetic deletion of 4E-BP1 and 4E-BP2 isoforms partially rescues from the effects of rapamycin on class switching to IgG1. These results uncover a novel role for mTORC1 and the 4E-BP/eIF4E axis in B cell antibody class switching, suggesting that cap-dependent translation regulates key steps in B cell differentiation. Further study of this pathway may provide mechanistic insight into antibody-mediated autoimmune diseases such as lupus and arthritis.
Dynamic imaging of a Tbet-Foxp3 double reporter in the course of Th1 mediated immune response
Authors: Akkaya Billur , Holstein Amanda H , Kamenyeva Olena , Akkaya Munir , Shevach Ethan M
Published: January 2023

T cells can integrate various environmental cues and acquire different transcription profiles through the differentiation processes. The transcription factor T-bet is switched on by CD4+ T cells during type 1 inflammation and acts as the master regulator of Th1 lineage of T cell differentiation and function. Strong Th1 responses are usually elicited by intracellular pathogens and must be counterbalanced by Foxp3+ Treg cells to prevent immunopathology. Recently, it was demonstrated that T-bet-deficient Treg cells fail to rescue Foxp3-deficient mice from Th1 cell-mediated inflammatory disease raising the questions how and why Tregs adapt the unique Th1 type transcription profile. Using a TbetZsGreen-Foxp3-RFP double reporter mouse model, we have addressed those questions by confocal microscopy of the live tissue sections, intravital microscopy of the popliteal lymph node and flow cytometry. We have demonstrated that Tregs in the T cell areas of the lymph node co-localized with Tbet expressing cells forming large clusters especially at the T cell-B cell border. B cell area also contained T-bet expressing cells both at the steady state and during type 1 inflammation, which were more sparse and moved remarkably faster compared to the T-bet expressing cells in the T cell area. Altogether, this suggests that Tregs are localized in close proximity to T-bet expressing cells in the live lymph nodes and may modulate their movement characteristics. The functional relevance of this needs to be further explored.
NR4A3 controls CD8+ T cell metabolism and differentiation
Authors: Odagiu Livia , Boulet Salix , Daudelin Jean-François , Labrecque Nathalie
Published: January 2023

Following an infection, naive CD8+ T cells expand and differentiate into two major populations of effectors: short-lived effector cells meant to die by apoptosis and memory precursor effector cells (MPECs) destined to survive as memory cells that will confer long-term protection. We postulated that the transcription factor NR4A3, a member of the orphan nuclear receptor family, whose expression is induced by TCR signalling, will regulate in vivo CD8+ T cell response. To elucidate the role of NR4A3 during CD8+ T cell response, we have adoptively transferred wild-type and Nr4a3−/− OT-I T cells (specific for the ovalbumin (OVA) peptide in the context of Kb) into naive recipients and analyzed their response following infection with a recombinant strain of Listeria monocytogenes encoding OVA. Although Nr4a3+/+ and Nr4a3−/− OT-I T cells expanded similarly, we observed an increased generation of MPECs at the peak of the T cell response, which led to an enhanced (2–3X) generation of memory T cells. Furthermore, Nr4a3−/− effector and memory T cells produce more cytokines than their wild-type counterpart. The analysis of the early T cell response demonstrated that NR4A3 controls the metabolic activity of activated T cells, as Nr4a3−/− OT-I T cells express less nutrient receptors and have reduced phosphorylation of S6. Altogether, these results suggest that NR4A3 controls effector and memory T cell differentiation by modulating their metabolic activity during the immune response.
Early window of susceptibility of allospecific CD4+ T cells to AhR-induced suppression reflects excess production of IL-2, over-expression of regulatory genes and repression of cell cycle genes.
Authors: Kerkvliet Nancy , Ehrlich Allison , Pennington Jamie , Wang Xisheng , O’Donnell Edmond , Tilton Susan , Rohlman Diana , Funatake Castle J
Published: January 2023

Activation of AhR in CD4+ T cells leads to suppression of Th1-, Th2- and Th17-mediated immune responses. In allograft models, the first 3 days of the immune response represent a critical window for AhR activation during which significant changes in donor CD4+ T cells occur. However, little is known about the mechanisms that underlie this early window of CD4+ T cell sensitivity to AhR activation and whether limiting AhR signaling to this early window is sufficient to suppress the allograft response. We used a rapidly metabolized AhR ligand, 10-Cl-BBQ, to show that early AhR activation alone is indeed sufficient to suppress GVHD. Early activation of AhR was also sufficient to increase the frequency of CD4+ Foxp3+ T cells on day 15 of the GVH response. AhR activation induced an early increase in IL-2 production by donor CD4+ T cells that was responsible for the Tr1-like phenotype on day 2 and the Foxp3+ Treg phenotype on day 15. However, blockade of IL-2 signaling, which prevented the induction of Foxp3+ Tregs, failed to alter AhR-induced suppression of GVHD. Global gene expression analysis of purified alloresponding donor CD4+ T cells on day 2 and 3 of the GVH response revealed up-regulation of many genes associated with regulatory T cells and down-regulation of genes involved in cell cycle progression. Reduced cell cycling was validated by reduced BrdU incorporation on day 4 of the GVH response. These results are consistent with a model of AhR signaling during naïve CD4+ T cell activation leading to induction of unique AhR-Tregs that induce premature cell-cycle arrest in effector CD4+ T cells; the lack of mature CD4+ T helper cells prevents the maturation and differentiation of allospecific CD8+ CTL leading to suppression of GVHD.
Identification of BATF target genes critical to its function in immune responses
Authors: Morman Rosemary E , Poston Stacie , Taparowsky Elizabeth J.
Published: January 2023

BATF, an immune specific AP-1 transcription factor, functions together with IRF4 and JUNB to regulate genes important for antibody class switch recombination (CSR) in B cells and for the functional differentiation of distinct CD4+ T helper (Th) subsets, including Th2, Th9, Th17 and Tfh cells. BatfΔZ/ΔZ(KO) mice display phenotypes directly related to deficiencies in the cell types mentioned above. In an attempt to define the gene expression changes responsible for these phenotypes, RNA-seq was performed to profile transcripts in KO versus WT mouse B cells. Bioinformatic analyses identified 47 genes whose transcripts were either over-represented or under-represented in the absence of BATF. Many of the transcripts found to be misregulated in KO B cells were misregulated in KO T cells as well, suggesting that these genes are common molecular targets of BATF transcriptional activity. Retroviral mediated re-expression of BATF in the KO cells resulted in the proper regulation of these target genes. Furthermore, BATF reconstituted KO B cells were able to undergo CSR and naive CD4+ T cells re-expressing BATF could be differentiated into Th2 and Th17 subsets. Our current efforts are employing this rescue strategy with retroviruses expressing BATF proteins modified for their DNA binding, dimerization and transcriptional properties to better understand the molecular parameters that control how BATF regulates gene sets in B cells and T cells. Our long term goal is to exploit the regulation of BATF for in vivo strategies to manage inflammation and autoimmune disorders.
MDM2 ubiquitin ligase in Lupus CD4 T-Cell pathology.
Authors: Titov Anton A , Brusko Todd M. , Baker Henry V , Sobel Eric , Morel Laurence
Published: January 2023

Systemic Lupus Erythmatosus (SLE) is an autoimmune condition of complex etiology involving genetic susceptibility and environmental factors. CD4+ Helper T-Cells provide co-stimulation to B-Cells to facilitate the production of a large repertoire of high affinity autoantibodies against numerous self-antigens such as dsDNA, histones, and snRNPs. Microarray analyses have been utilized previously to interrogate relevant pathways in disease pathology based on mRNA expression differences in T-Cells from healthy donors and SLE patients. Elevated levels of the E3 ubiquitin ligase MDM2 have been reported in immune cells of MRL.lpr mice and linked to resistance to activation-induced cell death (AICD), but the role of MDM2 in CD4+ T-Cells of human SLE subjects remains unclear. Here, we provide evidence for increased expression of MDM2 in CD4+ T-Cells of human SLE CD4+ T cells and a spontaneous genetic murine model of lupus, NZM2410 derived triple congenic (TC). Moreover, our data also support reduced AICD as a direct consequence of high MDM2. MDM2 has potential roles in the proteasome/ubiquitination pathways as well as inhibitory interactions with apoptotic regulators such as p73 and p53. Further work is needed to elucidate the contribution of this proto-oncogene to autoimmunity in humans and its suitability as a target for therapeutic intervention.
Classical and monocyte-derived DCs require distinct transcriptional programs for cross-presentation.
Authors: Briseno Carlos G , Haldar Malay , Kretzer Nicole M , Wu Xiaodi , Durai Vivek , Theisen Derek , Grajales-Reyes Gary E , Bagadia Prachi , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

Classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) cross-present cell associated antigens, but whether these cells use a universal program for this process is unknown. In examining the requirements for cross-presentation by Mo-DCs, we discovered that TremL4 identifies two Ly-6Chimonocyte populations, of which only Ly-6ChiTremL4− monocytes can differentiate into Zbtb46+ Mo-DCs in response to GM-CSF and IL-4 in vitro. By contrast, Ly-6Chi TremL4+ monocytes lost Mo-DC potential and instead were committed to development of Ly-6Clo TremL4+ monocytes. Further, we found that differentiation of monocytes with GM-CSF required addition of IL-4 to generate Zbtb46+ Mo-DCs that cross-presented as efficiently as CD8α+ cDCs in vitro. However, unlike CD8α+ cDCs, Mo-DCs required only IRF4, and not Batf3, to cross-present cell-associated antigens. Further, Irf4−/− monocytes treated with GM-CSF and IL-4 could not differentiate into Zbtb46+ Mo-DCs, and instead developed into macrophages. Thus, distinct transcriptional programs are used by CD8α+ cDCs and Mo-DCs for cross-presentation which may drive different antigen-processing pathways.
Galectin-7 regulates infiltration of alternatively activated macrophages into the central nervous system
Authors: Zangbede Fredice Quenum , Chauhan Arun , Sharma Jyotika , Mishra Bibhuti B
Published: January 2023

Alternatively activated macrophages (M2 MΦ) function to dampen host inflammatory responses and promote wound healing and tissue repair. As the blood brain barrier (BBB) restricts the flow of cells into the brain, understanding how M2 MΦ extravagate into the central nervous system (CNS) is of great interest. In a mouse model of neurocysticercosis (NCC) we previously demonstrated that M2 MΦ are essential in containing neuropathology in helminth infected CNS. Analysis of helminth infected mice revealed an abundant expression of galectin-7 that recognize galactose moieties in brain endothelial cells. The Galectin-7−/− NCC mice displayed a reduced accumulation of M2 MΦ in parasite infected brain. This reduction in M2 MΦ numbers in the CNS was correlated with an increased neuropathology and shortened survival. The use of adoptive transfer of fluorescent labeled cells demonstrated a decreased influx of M2 MΦ into the CNS of helminth infected NCC brains. The use of in-vitro transmigration assay conclusively demonstrated a key function of Galectin-7 expressed on endothelial cells in centrally regulating M2 MΦ entry into the CNS.
ENU-induced C-terminal truncation of Runx3 in mice enhances maintenance of memory T cells through upregulation of IL-15 expression
Authors: Su Yu-Chia , Chen Yi-Ting , Chang Mei-Ling , Cheng Bing-Ho , Kung John T
Published: January 2023

Immunological memory provides faster and stronger immune responses to protect host from infectious pathogens and tumor cells. T cell memory is a major part of immunological memory, and plays an important role to eliminate tumor cells or cells infected by intracellular pathogen. Cytokines including IL-15, IL-7 and IL-4 have been reported to enhance the generation or maintenance of memory T cells. IL-15 enhances homeostatic proliferation and survival of memory CD8+ T cells. An ENU-affected mouse pedigree carried a Tyr301stop point mutation of Runx3, which resulted in a truncated form of Runx3. These mice started to show spontaneous diarrhea after weaning (spontaneous colitis, SC). SC mice also expressed CD4-derepression in the CD8+ T cells, and loss of memory phenotype CD8+ T cells. SC CD8+ T cells showed proliferation deficiency and decreased memory CD8+ T cell precursors after activation and also had poor response to IL-15-induced proliferation. However, SC host mice enhanced in vivo long-term survival of activated WT CD8+ cells through improvement of homeostatic proliferation. This improvement of homeostatic proliferation was due to elevated serum IL-15 levels. In summary, C-terminal truncated Runx3 in mice results in upregulation of IL-15 expression and provides enhanced homeostatic environment for memory CD8+ T cells; however, CD8+ T cells have intrinsic memory defect. Thus, the SC mouse model is a valuable tool for investigations on the effect of immune system upon spontaneous colitis.
TCR signal strength and antigen affinity modulate CD8+ memory T Cell development
Authors: Solouki Sabrina , Huang Weishan , August Avery
Published: January 2023

CD8+ T cells play a critical role in adaptive immunity by maintaining the ability to differentiate into CD8+ memory T cells. During an intracellular infection, CD8+ T cells pass through several characteristic phases before becoming mature memory cells. Initial antigen stimulation causes naïve CD8+ T cells to clonally expand and differentiate into short-lived effector cells (SLECs). Subsequently, SLECs undergo a contraction phase to give rise to memory precursor effector cells (MPECs); 5–10% of these MPECs survive the initial contraction phase from SLECs to further develop into CD8+ memory T cells. We have shown that the non-receptor tyrosine kinase Itk, which regulates TCR signal strength, can significantly suppress CD8+ memory T cell development. We hypothesize from that TCR signal strength may function synergistically with antigen affinity to regulate development of memory CD8+ T cells. We are testing this hypothesis using Itk deficient OT-1 TCR transgenic mice, in which CD8+ T cells are engineered to be specific for the Ovalbumin (Ova) protein but exhibit reduced TCR signaling, adoptive transfer of donor naïve CD8+ T cells into marked recipient mice, followed by an infection with recombinant Listeria monocytogenes expressing Ova peptide variant epitopes for CD8+ T cells. Using this model, we can quantitatively determine how TCR signal strength intersects with TCR affinity to control memory development. Our findings suggest that TCR signal strength and TCR affinity separately regulate CD8+ memory development. This data holds societal impact and promise for the advancement of rational vaccine development.
Treatment with rifampicin ameliorates atopic dermatitis-like skin disease in NC/Nga mice through mast cell inactivation
Authors: Kang Tae Jin , Kim Seung Hyun , Lee Ki Man , Lee Geum Seon , Seong Ju-Won
Published: January 2023

Atopic dermatitis (AD) is a common inflammatory skin disorder mediated by inflammatory cells such as macrophages and mast cells. Rifampicin is a major drug for treatment of tuberculosis. Recently it was reported that rifampicin has anti-inflammatory and immune suppressive activity. We herein investigated the effect of rifampicin on atopic dermatitis in NC/Nga mouse model. AD was induced by treatment of 1-chloro 2, 4-dinitrobenzene (DNCB). And then mice were treated with rifampicin by oral administration and were observed to relieve the diverse symptoms. The severity score and scratching behavior were alleviated in rifampicin-treated groups. Serum IgE and IL-4 levels were also decreased in rifampicin-treated groups. We next examined if rifampicin has anti-atopic activity via the suppression of mast cell activation. Rifampicin suppressed the release of β-hexosaminidase and histamine from human mast cell (HMC)-1 cells stimulated with compound 48/80. Treatment of rifampicin inhibited secretion of inflammatory mediators, such TNF-α and PGD2, in mast cells activated by compound 48/80. The mRNA expression of COX-2 was reduced in the cell treated with rifampicin at a dose-dependent manner. These results suggest that rifampicin can be used to treat atopic dermatitis.
The neuropeptide, neurokinin A, has divergent effects on mast cell function
Authors: Sumpter Tina L , Falo Louis D , Larregina Adriana T
Published: January 2023

Mast cells (MCs) play a critical role in initiation of cutaneous type 2 immune responses. In the cutaneous microenvironment, sensory nerve fibers form a cohesive unit with MCs. In this unit, MC function is likely affected by neuropeptides, such as neurokinin A (NKA). NKA is abundant in the skin, but the affects of NKA on cutaneous MCs have not been extensively reported. NKA signals preferentially through the neurokinin-2 receptor (NK2R). We hypothesized that NKA/NK2R interactions influence cutaneous MC function. The effect of NKA on MCs was evaluated in vivo using the MC/IgE-specific passive cutaneous anaphylaxis (PCA) model. NKA reduced IgE-initiated changes in ear thickness. Likewise, NK2R antagonism enhanced IgE-initiated ear thickness in the PCA model. Intracellular pathways mediating the effects of NKA were delineated using bone marrow derived MCs (BMMCs). BMMCs expressed NK2R protein constitutively and NK2R expression was up-regulated by IgE. Functionally, NKA increased the percentage of IgE-activated MCs releasing granules but repressed transcription and release of the type 2 cytokines, IL-4 and IL-13. Mechanistically, IgE-mediated granule release has been attributed to increased STAT3 activation, whilst IL-4 and IL-13 transcription in MCs is attributed to STAT5 activation. NKA augmented STAT3 phosphorylation in IgE-activated MCs but reduced STAT5 phosphorylation. In sum, NKA modifies IgE-initiated STAT3 and STAT5 activity, granule release and cytokine transcription in MCs in vitro and inhibits MC function in vivo. These data demonstrate a role for NKA in the regulation of MC processes involved in type 2 mediated allergic disorders.
Pyruvate dehydrogenase has a major role in mast cell function and its activity is regulated by mitochondrial MITF
Authors: Sharkia Israa , Erlich Tal Hadad , Landolina Nadine , Assayag Miri , Motzik Alex , Rachmin Inbal , Kay Gillian , Porat Ziv , Tshori Sagi , Berkman Neville , Levi-Schaffer Francesca , Razin Ehud
Published: January 2023

Background

We have recently observed that OXPHOS mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream to the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number and function of mast cells. The localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored.

Objective

To explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and is involved in the regulation of PDH activity.

Methods

Experiments were performed in vitro using human and mouse mast cells and RBL cells and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunological activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined.

Results

PDH is essential for immunologically mediated degranulation of mast cells. Following activation PDH is serine dephosphorylated. In addition, we show for the first time that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity.

Conclusion

The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.
Dendritic cell-based vaccine treated with polysorbitol transporter enhanced antigen-specific CD8+ T cell immune responses through cross-presentation
Authors: Kim Cheol Gyun , Kye Yoon Chul , Kim Narae , Kim Han Wool , Kim Girak , Han Seung Hyun , Yun Cheol-Heui
Published: January 2023

Cross-presentation of epitope from exogenous antigen onto MHC I molecule enhances CD8+ T cell activity is important in host protective immune responses against virus infections and cancer. We previously reported that polysorbitol transpoter (PST), prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine, induces a long-term antigen specific B cell response and phagolysosomal escape of antigens in macrophages via mechanism called ‘proton-sponge effect’. Therefore, we hypothesize that PST could enhance the cross-presentation and antigen-specific CD8+ T cell immune responses.

In the present study, we tried to determine whether PST could enhance cross-presentation of DC using model antigen Ovalbumin (OVA). PST formed a nano-sized complex with OVA (OVA-PST) enhanced SIINFEKL peptide loading onto MHC I molecules. The results further showed that it enhanced OVA-specific CD8+ T cell proliferation and activation. However, PST was not able to upregulate co-stimulatory molecules and pro-inflammatory cytokines, which are necessary to induce adequate protective immune response. Together with OVA-PST, Monophospholipid A (MPLA), a nontoxic TLR4 agonist which is analog of lipid A also used as co-delivery agent in nanoparticle peptide vaccine, upregulated the effects of OVA-PST complexes. Finally, when PST-OVA and MPLA applied to DC-based vaccine for tumor therapy, survival enhanced in mouse engrafted with tumor. Collectively, these results suggested that the PST enhances cross-presentation in DCs together with antigen-specific CD8+ T cell immune responses. Therefore PST with MPLA could act to improve DC-based vaccine.
Peptidome Diversities of human leukocyte antigen (HLA-B) allotypes
Authors: Zaitouna Anita J , Basrur Venkatesha , Foster Leonard J , Carrington Mary , Raghavan Malini
Published: January 2023

The HLA class I genes encode proteins that present short peptide antigen to CD8 T cells, thereby alerting the immune system to the presence of intracellular pathogens and cancers. The HLA class I genes are highly polymorphic, and the polymorphisms influence their peptide-binding specificities. Each HLA variant binds to a diverse array of peptides, the peptidome. Much remains to be understood about HLA class I peptidome diversity variations and their influencing factors. Here we validate the use of Shannon entropy (SE) plots to quantify and compare the diversities of HLA class I peptidomes derived from multiple independently-derived mass spectrometric (MS) data sets. The intrinsic peptide-binding preferences of HLA class I molecules and/or their intracellular assembly characteristics could influence their peptidome diversities. In particular, tapasin is an assembly factor that edits and optimizes the peptide repertoire of many HLA class I molecules, but is non-essential for several other HLA class I molecules. It has been suggested that a tapasin-independent assembly pathway could result in a broader, more diverse peptide repertoire. However, MS-based comparisons indicate that, under cellular conditions of tapasin sufficiency, the peptidome of HLA-B*4405 (a prototypic tapasin-independent allotype) is not more diverse than that of HLA-B*4402 (a prototypic tapasin-dependent allotype). Rather, the intrinsic peptide-binding specificity of HLA-B4402 results in greater length and C-terminal sequence diversity of its peptidome. Together, these studies indicate that structural constraints imposed by MHC class I peptide binding grooves are key determinants of their peptidome diversities.
α-Galactosylceramide and all trans-retinoic acid work together to promote antigen presentation and antibody production
Authors: Chen Qiuyan , Wei Cheng-Hsin , Ross A. Catharine
Published: January 2023

α-galactosylceramide (αGalCer) and all trans-retinoic acid (RA) are both known as potent immune regulators on various steps of the immune responses. Our previous studies have shown that αGalCer and RA could regulate B cell differentiation in vitro and increase antibody production in vivo. To investigate the underlying mechanisms related to the antibody response, animal experiments (in 8-wk old female Balb/c mice) were performed to determine the antigen presentation and B cell response during primary immunization. In a one-day experiment, dextran-FITC or KLH-FITC was injected intravenously with or without αGalCer (2 μg in 100 μl PBS with antigen, iv) or RA (37.5 μg in 10 μl canola oil, oral dose); in a 10-day experiment, KLH was injected (100 μg/100 μl PBS) with or without αGalCer/RA treatment. Spleens were analyzed by flow cytometry and immunofluorescent staining. We observed that Dextran and KLH after 24h of injection were localized at the spleen marginal zone (MZ) area, with only KLH colocalized with the metallophilic macrophages; in the presence of αGalCer and RA, the band of metallophilic macrophages became wider. While dextran moved markedly toward the follicular area after treatment, KLH signal was not seen within the follicle, and also became weaker at the MZ area, suggesting the different presentation and processing mechanisms of the antigens. At day 10, substantially more active germinal centers were formed in the presence of αGalCer and RA, with more focused staining of CD23+GL-7+ cells, markers of germinal center B cells. B cell numbers were increased at both time points. Our study suggests that αGalCer and RA together may promote antigen presentation and induce B cell differentiation that ultimately increases antibody production.
Illuminating T cell – APC interactions using multispectral imaging flow cytometry.
Authors: Pugsley Haley Renee , Friend Sherree L. , Kong Raymond K. , Morrissey Philip
Published: January 2023

Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response. One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging by fluorescence microscopy. However, immunological synapses are rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis brand ImageStream to objectively collect imagery of large numbers of cells. In this study, Raji B cells loaded with Staphylococcal enterotoxin B (SEB) were incubated with human T cells to create T cell-APC conjugates. Cells were stained in various combinations for CD3, CD19, Actin, LFA-1, Lck and NFkB. Using multispectral imaging flow cytometry we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized immunological synapse, evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex and measure the nuclear localization of NFkB in the T cell-APC conjugates.
In vivo characterization of the immune response towards the pathogenic Escherichia coli antigen SslE and modulation of the intestinal microbiota
Authors: Naili Ilham , Baudner Barbara C , Bernalier-Donadille Annick , Pizza Mariagrazia , Desvaux Mickael , Jubelin Gregory , Buonsanti Cecilia
Published: January 2023

Pathogenic E. coli, both intestinal (InPEC) and extraintestinal (ExPEC), account for a wide range of diseases, which can be fatal across developing and developed countries. They can acquire a vast array of virulence factors which may dramatically increase the severity of the infection. Emergence of resistant strains often renders antibiotics inefficient; in the case of Shiga toxin-producing E. coli, antibiotics therapy can even be detrimental. Hence, an E. coli vaccine with broad coverage could be a promising alternative to prevent the spread of such diseases, while offering the potential for protection against several InPEC and ExPEC at once.

Using the «reverse vaccinology» approach on ExPEC strains, nine antigens were identified as protective against a murine sepsis model. With 82% protective efficacy, SslE (Secreted and Surface-associated Lipoprotein of E. coli) was the most potent candidate. Additional models showed SslE to also be cross-protective against other ExPEC strains. Functional assays have demonstrated in vitro and ex vivo that SslE is a mucinase which plays an important role in colonization and virulence of E. coli.

To better understand the mechanism behind such protection, we are investigating the intestinal and systemic immune responses obtained after immunization with SslE using different routes of administration. In-depth analysis of each regimen will allow us to determine the most efficient method of immunization for SslE to be protective. Further, using this selected model of immunization, we will evaluate the potential perturbation caused by SslE immunization on the murine intestinal microbiome. These findings will be important to help us optimize the development of a potential broad spectrum E. coli vaccine.