bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine tuning CD8+ T cell functional avidity by the PD-1/SHP2 axis
Authors: Li Jianping , Sui Yongjun , Berzofsky Jay A
Published: January 2023

T cells of high functional avidity recognize low antigen levels and have the most effective cytotoxic responses against viruses and tumor cells. Understanding the molecular regulations of T cell functional avidity is essential to aid novel vaccine design as well as adoptive immunotherapy by improving the T cell quality. Programmed cell death protein 1 (PD-1) is known to negatively regulate the signal strength of T cell receptor (TCR). Activated PD-1 recruits Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (Shp2), which dephosphorylates kinases involved in TCR signaling. However, it is unclear whether PD-1/SHP2 can modulate T cell functional avidity. To address this question, we treated splenocytes from OT-1 transgenic mice with anti-PD-1 or SHP099 (SHP2 inhibitor), or a combination of both for 3 days in a selective culture condition of APC-pulsed stimulation with 10−6 M or 10−9 M SINNFEKL, respectively. Our preliminary data showed that blocking SHP2/PD-1 enhanced functional avidity and antigen sensitivity of CD8+ T cell in vitro when stimulated with limited SINNFEKL antigen. Blocking of PD1/SHP2 also enhanced CD8 beta expression, which has been shown correlated with high functional avidity CD8+ T cells. These data suggest that PD1/SHP2 is involved in regulating T cell functional avidity.
A subset of mature neutrophils contains the strongest PMN-MDSC activity in blood and tissue of patients with head and neck cancer
Authors: Brandau Sven , Si Yu , Bruderek Kirsten , Merz Simon F , Jansen Philipp , Lang Stephan , Gunzer Matthias , Klode Joachim , Squire Anthony
Published: January 2023

A high neutrophil-to-lymphocyte ratio in the circulation and high frequencies of tumor-associated neutrophils (TAN) in malignant tissue are associated with poor outcome and tumor progression in patients with cancer. It is hypothesized that immunosuppressive neutrophil activity (aka PMN-MDSC activity) contributes to this effect. However, the exact cellular identity of human PMN-MDSC is still under debate. In this study, we sought to identify the neutrophil subset that contained the highest T cell suppressive activity. To this end, we purified different subsets of circulating neutrophils by FACS and performed multi-parameter immunofluorescence together with digital pathology on 2-D and 3-D tumor tissue samples.

We found that a population of circulating, mature, arginase I+ neutrophils that co-purified with mononuclear cells in density gradients, most potently suppressed T cell function in multiple in vitro assays. These PMN-MDSC were also superior to monocytic MDSC in T cell suppression. Using a novel technology of tissue whole mount labelling, clearing and imaging we derived 3-D spatial maps of neutrophil – T cell interaction in human tumors. We found that T cells, which were conjugated to arginase I+, myeloperoxidase+ TAN, had significantly reduced expression of proliferation and cytotoxicity markers. In patients, frequent conjugation of T cells to those PMN-MDSC was associated with poor prognosis. In contrast to circulating PMN-MDSC, tissue PMN-MDSC expressed high amounts of LOX-1 (oxidized low density lipoprotein receptor 1) and a high intratumoral frequency of LOX-1+ PMN-MDSC was associated with poor survival.

We identified and characterized PMN-MDSC activity in human cancer patients.
Glycolysis gene Hif1a negatively regulates the induction of intraepithelial CD4+CD8aa+ T cells
Authors: Sujino Tomohisa , Harada Yosuke , Nomura Ena , Miyamoto Kentaro , Tanemoto Shun , Yoshimatsu Yusuke , Kubota Yoshiaki , Kanai Takanori
Published: January 2023

Intestinal CD4 T cells reside in both lamina propria and intraepithelial lesion. We previously reported CD4+CD8aa+ intraepithelial lymphocytes (DPIELs) exhibit anti-inflammatory function against intestinal inflammation. Recently, metabolic status is important both for induction and for keeping cells in active phase. For example, Tregs use oxphos for cellular metabolism, while Th17 cells mainly use glycolysis. It is still unknown the metabolic status in tissue resident T cells and the effect of metabolic related gene for cell development in gut. By using mito-tracker and seahorse system, we first evaluated the metabolic status of tissue resident CD4 T cells. Then we used mouse model by deletion of the glycolysis related gene to uncover the development of intestinal resident CD4 T cells. The ratio of mitochondrial membrane potentialhipopulation of CD4IELs was increased in Germ free mice than in SPF mice. Most of DPIELs express the mitochondira membrane potentiallo. Although total CD4 positive IELs increased glycolysis compared to the splenic naive CD4 T cells, DPIELs expressed less pS6 and reduced glycolysis related gene, Hif1a. Next, We generated conditional Hif1a and Vhl ko mice (CD4cre: Hif1af/f: Hif1a (ΔCD4), CD4cre: Vhlf/f: Vhl (ΔCD4), CD4cre: Hif1af/f: Vhlf/f (DKO)) respectively to understand whether Hif1 was negatively regulated the induction of DPIELs or not. The ratio of DPIELs was increased in Hif1a (ΔCD4), DKO mice and decreased in Vhl (ΔCD4) mice. These results reveal total CD4IELs uses preferentially glycolysis, but glycolytic related gene Hif1a is negatively regulated the induction of DPIELs.
Discordant requirements for CD11c+ APCs in priming CD8 T cell responses against H-2Kb versus H-2Db restricted TMEV-associated peptides
Authors: Tritz Zachariah Peter , Orozco Robin , Malo Courtney , Ayasoufi Kathy , Yokanovich Lila , Goddery Emma , Khadka Roman , Fain Cori , Hansen Michael , Jin Fang , Johnson Aaron J
Published: January 2023

CD8 T cell priming after viral infection of the central nervous system (CNS) is incompletely understood. In peripheral infections, T cell priming generally relies upon stimulation by antigen-bearing dendritic cells (DCs) migrating into draining lymph nodes. The parenchyma of the brain, however, is largely devoid of dendritic cells and evidence demonstrating that activated DCs leave the brain parenchyma is lacking. We therefore sought to understand the role of DCs and specific MHC class I molecules in priming CD8 T cells following CNS infection. We employed H-2Db−/− H-2Kb−/− (MHC-I deficient) mice that express a floxed H-2Db or floxed Kb molecule, a unique transgenic mouse system developed by our research program. Crossed with an MHC class I deficient CD11c-Cre expressing animal results in DC-specific ablation of Db or Kb Class I restricted antigen presentation ability. We found that CD11c+ APCs are critical for the early priming of CD8 T cells against the Db restricted VP2121–130 epitope of the model pathogen, TMEV. However, DCs are ultimately dispensable for long-term development of these antigen specific T cells. Discordantly, the loss of MHC class I restricted antigen presentation ability on CD11c+ APCs does not impact the priming of T cells against the H-2Kb restricted OVA257–264 epitope following intracranial TMEV-OVA infection, but does impact the ability of these T cells to successfully occupy the CNS. This work suggests that differing patterns of MHC class I restriction for CNS pathogen-derived epitopes might have functional consequences on the APC requirements for a successful CD8 T cell response against neurotropic viruses.
Comparative evaluation of metabolic, electrophilic, and immunologic effects of itaconate and its ester derivatives on macrophage activation
Authors: Swain Amanda C , Bambouskova Monika , Kim Hyeryun , Duncan Dustin , Auclair Karine , Chubukov Victor , Simons Donald M. , Roddy Thomas P. , Stewart Kelly M. , Artyomov Maxim N.
Published: January 2023

Following the discovery of itaconate’s immunoregulatory properties, several ester derivatives of this dicarboxylic acid were designed to further examine its role. Here, we compare the metabolic, electrophilic, and immunologic profiles of macrophages treated with unmodified itaconate and a panel of itaconate derivatives. Using WT and Irg1-deficient macrophages, we show that neither dimethyl itaconate (DI) nor 4-octyl itaconate (4OI) are converted into intracellular itaconate in either resting or TLR-activated cells, 4-monoethyl itaconate (4EI) yields only small quantities of intracellular itaconate, while exogenous itaconic acid readily enters macrophages and accumulates to physiologically relevant amounts. We find that both DI and 4OI induce a strong electrophilic stress response, in contrast to itaconate and 4EI. This correlated with their differential immunological impact: DI and 4OI both inhibited IκBζ induction and IL-6 secretion. In contrast, itaconate treatment had minimal impact on IκBζ, IL-6 and pro-IL-1β levels, but demonstrated a distinct defect in IL-1β secretion. Altogether, this systematic evaluation stresses the importance of using unmodified itaconate in future mechanistic studies.
MEK1/2 blockade results in altered inflammatory response and delayed alveolar bone repair
Authors: Fonseca Angélica Cristina , Melchiades Jéssica Lima , de Campos Soriani Azevedo Michelle , Colavite Priscila Maria , Mortari Carolina Fávaro Francisconi , Tampanez André Petenuci , Garlet Gustavo Pompermaier
Published: January 2023

M1 and M2 macrophages play an important modulating role in tissue repair. In alveolar bone repair, the determinant these different phenotypes acquisition and its impact in repair process remains undefined, with a current assumption that assuming M2 conversion contributes to repair resolution. Considering MEK1/2 signaling pathway as a determinant for M2 polarization, the objective of this project was to evaluate the effect of MEK1/2 pharmacological inhibition in the local host response and repair outcome. C57Bl/6 (WT) mice, treated with MEK1/2i inhibitor PD0325901 (10mg/kg/24h/IP), which underwent extraction of the right upper incisor and, subsequently analyzed by computed microtomography (uCt) and histomorphometric analysis. uCt analysis, performed at 0,3,7 and 14 days periods revealed an overall smaller bone volume in MEK1/2i group, with a statistical difference in 14 days (p<0,05 MEK1/2i versus Control). These results, when combined with the histomorphometric analysis, reveals a delay in the alveolar bone repair process in the MEK1/2i group, characterized at 7 days by a higher density of connective tissue parameters, specifically collagen fibers, blood vessels, inflammatory cells and blood clot, when compared with control group. Additionally, a lower density of bone tissue parameters, specifically osteoblasts, osteoclasts and bone matrix, where observed upon MEK1/2i inhibition. Therefore, partially, the results show that MEK1/2 inhibitory effect results in delayed alveolar bone repair, initially involving the control of inflammatory cell migration and subsequent bone formation.

Supported by grants from FAPESP (2018/10177-9 and 2015/24637-3)
Immunologic outcomes of bidirectional cross talk between gamma delta T cells and dendritic cells is context dependent
Authors: Biradar Shivkumar , Lotze Michael , Mailliard Robbie B
Published: January 2023

T cells expressing the gamma delta (γδ) T cell receptor are a minor cell population, but a critical component of innate immunity. γδ T cells can also influence adaptive immunity through their functional impact on dendritic cells (DC). To investigate the interaction between these cell types, we co-cultured peripheral blood γδ T cells from healthy volunteers with autologous monocyte derived DC. When immature DC (iDC) were co-cultured with resting human γδ T cells in the presence of zoledronate (Zol), drug that activates Vγ9δ2 T cells, DC maturation occurred. Blocking studies determined that γδ T cell-induced DC maturation was partially mediated by TNF-a and IFN-g. Moreover, γδ T cell-activated DC produced enhanced levels of IL-12p70. Interestingly, when activated effector γδ T cells were co-cultured with iDC in the presence of Zol a high degree iDC cell death was noted, suggesting a regulatory role of γδ T cells. When studying the impact of mature DC on γδ T cell function, we found that IL-12p70 deficient PGE-2 matured DC had a higher capacity to expand resting γδ T cells, that expressed higher levels of CD16, and were particularly less sensitive to apoptosis than those co-cultured with high IL-12p70-producing interferon-matured DC. Our findings show that the outcome of bidirectional cross talk between γδ T cells and DC is dependent on the context and activation status of each cell type, and that each regulate and modulate the immune function of the other.
The interaction between group 2 innate lymphoid cells and lipid metabolism
Authors: Zhang Yingyu , Huang Yuefeng
Published: January 2023

Innate lymphoid cells (ILCs) have been found in many tissue sites but are enriched in barrier surfaces such as intestine, lung and skin. They lack antigen-specific receptors yet respond to alarmin signals that are released by tissue, to resemble the cytokine production and effector function of T cells. ILCs play important roles in host defense, tissue repair, autoimmune disorders, inflammation, metabolic homeostasis and more. Recent progress in immunometabolism has highlighted the diverse roles of lipid metabolism in the response and function of immune cells including lymphocytes. But the interaction between ILC2s and lipid metabolism is poorly understood. Our preliminary studies show that, upon activation, intestinal ILC2s significantly upregulate the expression of several genes that are important for cholesterol metabolism. Cholesterol is an essential structural component of mammalian cell membranes and is the precursor for bile acids, vitamin D and steroid hormones. Cholesterol metabolism disorder leads to severe diseases such as hypercholesterolemia, atherosclerosis and diabetes. By using various mouse models and multi-disciplinary experimental approaches, we will study if and how cholesterol regulates the homeostasis, activation and cytokine production of ILC2s and if cholesterol regulation on ILC2s contributes to host defense against helminth infection. We will also investigate if ILC2s in turn modulate lipid, especially cholesterol, metabolism and if ILC2s play any role in hypercholesterolemia or atherosclerosis.
Differential Response of Dendritic Cells and Macrophages to Signals from Airway Epithelial Cells in Humans
Authors: Agrawal Anshu , Agrawal Sudhanshu , Monteiro Clarice , Mohammadi Reza
Published: January 2023

Airway epithelial cells (AECs), dendritic cells (DCs) and alveolar macrophages are the primary cells involved in respiratory immunity and tolerance. Since DCs and macrophages exist in close proximity to AECs, the function of both these cells is influenced by AECs. Using a 2D model to study the interaction of human primary bronchial epithelial cells (representative of AECs) with DCs we have recently demonstrated that AECs enhance the immune surveillance capacity of myeloid DCs (mDCs) from the circulation at homeostasis. Culture of mDCs with AECs resulted in the upregulation of pathogen recognition receptors (PRRs) and downstream signaling molecules which were reflected in the enhanced inflammatory cytokine responses to Klebsiella pneumoniae LPS. In contrast to circulatory mDCs, culture of macrophages with AECs led to a significant inhibition of LPS induced proinflammatory responses. More importantly, AECs induced the secretion of TGF-β and IL-10 from macrophages even in the absence of LPS stimulation. Furthermore, macrophages exposed to AECs induced significantly increased level of T regulatory cells. Thus, it seems AECs induce tolerance in macrophages but enhance the immunogenicity of mDCs suggesting a division of labor between DCs and macrophages in the respiratory mucosa. Investigation into the possible mechanisms for this differential response indicates that exosomes from AECs may be partially responsible for inducing tolerance in macrophages while growth factors secreted by AECs may be involved in priming DCs. These data demonstrate that AECs secrete different mediators that enhance immunogenicity and tolerance in the respiratory tract. These mediators may potentially be used to modulate respiratory immunity.
A metabolic sensor controls pathologic ILC2 function to regulate airway inflammation
Authors: Monticelli Laurel A
Published: January 2023

Employing models of allergen exposure, pathogen infection, and chronic inflammation, research in the Monticelli lab is examining how the innate immune system responds to tissue-specific cues to modulate cellular metabolism in order to coordinately regulate resolution of inflammation and promotion of tissue homeostasis at the body’s barrier surfaces. Our recent findings indicate that subsets of innate lymphoid cells (ILCs) utilize distinct metabolic pathways to fuel their proliferation and activation, and that this bioenergetic re-wiring influences their proinflammatory and tissue protective functions. Ongoing studies are examining how particular cell-extrinsic signals (such as cytokines) and cell-intrinsic factors (such as enzymes and transcription factors) control this metabolic reprogramming.

In new unpublished studies, we’ve found that lung-resident ILC2s upregulate expression of the metabolic ‘sensor’ transcription factor aryl hydrocarbon receptor (Ahr) upon activation with select airway allergens. Genetic or pharmacologic manipulation of Ahr signaling in murine models directly regulated ILC2 responses during airway inflammation, suggesting that ILC2-intrinsic sensing of Ahr ligand metabolites is critical for protection versus progression of innate immune lung disease pathogenesis.
PDGFRα ablation leads to alteration of ECM and immune cell composition in the lung
Authors: Park Juwon , Lee Christina , Tallquist Michelle D.
Published: January 2023

Fibroblasts are the major cells responsible for tissue homeostasis, repair, and remodeling. In particular, PDGFRα is expressed by mesenchymal progenitors in the lung that constitute the myofibroblast population which transiently express α-SMA during alveologenesis. In the fibrotic lungs, PDGFRα+ lineages can differentiate into subpopulation of myofibroblasts which regulate tissue repair and remodeling. Although the contribution of PDGFRα+lineage to lung development and fibrosis are particularly well-known, their roles in the lung parenchyma at baseline are less characterized.

To explore how PDGFRα+ fibroblasts affect extracellular matrix in baseline lung, we used genetic ablation of PDGFRα+ fibroblasts using Pdgfrα-CreERT2-driven expression of diphtheria toxin A. Histologically, thin mesenchyme was observed in fibroblast-ablated adult lungs. A majority of PDGFRα+ lineages are Col1a1-GFP+ cells, but PDGFRα-ablated lungs showed a significant decrease in the number of PDGFRα lineage and Col1a1-GFP+ cells. In addition, decreased expressions of ECM genes, such as Col1a1, Col1a2, and fibronectin-1 were observed in PDGFRα-ablated lungs. These data indicated that PDGFRα+ cells are a major source of extracellular proteins and PDGFRα ablation leads to alteration of ECM homeostasis in the baseline lung. Flow cytometry analysis of the lung tissues revealed that PDGFRα ablation resulted in elevated numbers of leukocytes and neutrophils. However, fibroblast loss did not affect macrophage numbers. These data suggested that fewer PDGFRα+ cells or loss of ECM or both can affect the recruitment of immune cells in the lung. In summary, changes in ECM environment by PDGFRα ablation leads to altered immune cell composition in the lung.
Non-cell-autonomous role of myofibroblasts in pulmonary fibrosis through lactate induction of M2 macrophage polarization
Authors: Liu Gang
Published: January 2023

Rationale

There has been growing evidence that metabolic dysregulations play critical roles in the pathogenesis of lung fibrosis, particularly idiopathic pulmonary fibrosis (IPF). Recent studies from our group as well as others’ showed that lung myofibroblasts displayed augmented glycolysis and secreted more lactate than normal fibroblasts. In this study, we examined the effect of lactate, the end product of myofibroblast glycolysis, on the phenotypic alterations of macrophages that has been frequently implicated in lung fibrosis.

Methods

Levels of M2 macrophage markers were determined by real-time PCR and Western blotting. Lactate transportation into cells was inhibited by siRNA knockdown of lactate MCT1 or MCT1 inhibitors.

Results

First, we confirmed that alveolar macrophages (AM) from fibrotic mouse lungs exhibited a M2 macrophage phenotype, as evidenced by increased expression of M2 markers Arginase 1 (Arg1) and vascular endothelial growth factor (VEGF) in these cells. We found that lactate induced M2 polarization of bone marrow derived macrophages (BMDM) and AMs. Furthermore, conditional media from lung myofibroblasts as well as coculture with lung myofibroblasts promoted M2 differentiation of BMDMs and AMs. Lactate induced M2 polarization could be inhibited by incapacitating lactate transporter MCT1 through siRNA knockdown or specific inhibitors.

Conclusions

Our data suggest that lung myofibroblast derived lactate promotes the pro-fibrotic differentiation of lung macrophages. We provide novel insight into the mechanism by which lung myofibroblasts participate in the pathogenesis of lung fibrosis. Targeting lactate secretion or intake by lung macrophages could be novel strategies to treat lung fibrosis.
Role of cardiac macrophages in controlling the infection and homeostasis of steady state heart function in aging
Authors: Saljoughian Noushin , Wu Qian , Kumar Naresh , Ganesan Latha P. , Lafuse William P , Rajaram Murugesan
Published: January 2023

Activation of immune system following cardiac infection is critical for mounting inflammatory and regenerative response to maintain the steady state heart function. Cardiac resident macrophages (CRMs) play a critical role in maintenance of homeostatic functions and tissue repair during infection. There are two subsets of CRMs in the heart CCR2+MHCIIhigh and CCR2−MHCIIhigh/low. We have previously demonstrated that Mycobacterium avium (M.a.) infection causes cardiac arrhythmia and dissemination of bacteria into the heart tissue of old mice. These abnormalities are due to an increased infiltration of myeloid cells and fibrosis. In order understand the role of CRMs in cardiac dysfunction in aged mice, we used the young and old mouse infection model. Cardiac single cells were prepared and analyzed with flow cytometry. The results show that the infiltration of myeloid cells into the heart was significantly increased in young mice during early stage of infection. In particularly, we observed an increased frequency of MHC-IIhigh CRMs in the heart of young mice upon M.a infection. In contrast, the aged mice shows an increase in numbers but fail to maintain the CRM frequency during the course of infection. Interestingly, we also observed the higher expression of MerTK on CRMs in the young mice than old mice. Our in vitro experiments demonstrated that activation of LXR enhances the expression of MerTK in macrophages and efferocytosis function. In conclusion achieved data indicates that the accumulation of MHC-II high CRMs and MerTK positive macrophages in the hearts of young mice provides the mechanism of M.a. clearance and efficient regeneration. Thus, MerTK is a promising therapeutic target in prevention of cardiac damage caused by M.a. in ageing.
Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation
Authors: Watanabe Yasuharu , Nagai Yoshinori , Takatsu Kiyoshi
Published: January 2023

Obesity-associated adipose tissue inflammation contributes to the development of type 2 diabetes. Chronic activation of IL-1β system in adipose tissue on metabolic disorders is well demonstrated. However, a mechanism for its expression and activation in the tissue has remained unexplored.

Here we demonstrate that IL-1β transcript was enriched in neutrophils of white adipose tissue (WAT), and the expression of IL-1β was up-regulated by a high-fat diet in the WAT but not in other tissues, including bone marrow, peripheral blood, and spleen. The co-culture of neutrophils with 3T3-L1 adipocytes indicate that contact interaction with adipocytes is critical for the expression of IL-1β via NF-κB activation in adipose tissue neutrophils. Lipolysis of adipocytes accumulated neutrophils prior to macrophages in the WAT, and produced high levels of IL-1β via an ASC-caspase-1-mediated inflammasome pathway. Leukotriene B4 production in the WAT also contributed to neutrophil accumulation. Furthermore, neutrophils and IL-1β contributed to the expression of chemotactic molecules involved in high-fat diet-induced macrophage infiltration into the WAT. We have identified previously unappreciated roles for neutrophils in the development of adipose tissue inflammation: robust IL-1β production and infiltration of macrophages to initiate chronic inflammation.
Slc11a1 gene variants influence over host response patterns and periodontal diseases resistance and susceptibility phenotypes
Authors: Melchiades Jéssica Lima , Cavalla Ian Franco , Colavite Priscila Maria , de Campos Soriani Azevedo Michelle , Fonseca Angelica Cristina , Trombone Ana Paula Favaro , Silva Renato Menezes da , de Faveri Marcelo , Figueiredo Magda Ferez , Garlet Gustavo P
Published: January 2023

Genetic variants in Slc11a1 are shown to be associated with different infectious diseases in humans. This study aimed to correlate the Slc11a1 genetic polymorphisms rs2290708 and rs3731865 with periodontal diseases resistance/susceptibility profiles in humans, and to evaluate impact of Slc11a1 hypo/hyperresponsive variants on experimental periodontitis in mice. Genotypic analysis was performed in 45 patients with chronic periodontitis (CP), 476 healthy individuals (H) and 207 individuals with chronic gingivitis (CG), and subgroups were analyzed for analysis of possible correlations between expression/genotype and in vitro tests. The polymorphisms rs2290708 and rs3731865 were associated with periodontitis risk, with a higher frequency of the genotypes CT+TT and GC+CC and the alleles T and C in the CP group in the control case approach. The analysis of the putative SNPs functionality demonstrated that polymorphic T (rs2290708) and C (rs37371865) alleles were associated with increased TNF-α, IL-1β, IL-6, RANKL and RANKL/OPG ratio in periodontal lesions. In vitro results reinforced the potential hyper-reactive nature of the polymorphic alleles T and C, since macrophages bearing such alleles presented an increased the production the same cytokines cited above. The hyperreactive variants of Slc11a1, prevalent in AIRmax mice when compared with hyporeactive AIRmin strain, were associated with increased alveolar bone loss, leukocyte influx and production of proinflammatory cytokines in experimental periodontitis. Therefore, polymorphisms in the Slc11a1 influence host response patterns and periodontal diseases phenotypes in experimental and human periodontitis.

Supported by grants from FAPESP (2019/12120-7 and 2015/24637-3)
EVALUATION OF CELL MEDIATED IMMUNITY OF PROTECTIVE VZV VACCINATION IN ADULTS BY FLUORESCENT T- AND B-CELL ELISPOT ASSAY ON A SINGLE CELL LEVEL
Authors: Ettischer-Schmid Nicole , Strecker Kevin , Preyer Rosemarie
Published: January 2023

Since decades it is known that immunity against varicella zoster virus (VZV) is mainly mediated by cellular immune reactions, predominantly conveyed by T-cells. Vaccination against VZV is now recommended not only in children, but in adults from 50 years to reduce the risk of herpes zoster (HZ) development, which increases by age. The aim of this work was to evaluate mechanisms and differences of the T- and B-cell mediated immunity against VZV in vaccinated adults and non-vaccinated younger adults by 3-colour fluorescent Enzyme-Linked-ImmunoSpot (EliSpot) Assay.

To evaluate T-cell mediated immunity, IFNy, IL-2 and IL-5 were simultaneously detected in one well. Our underlying hypothesis says that freshly vaccinated donors should express all three cytokines, whereas younger adults with a vaccination and/or infection during childhood should express a different cytokine pattern when stimulated with VZV. Adult donors with a known history of VZV infection showed significantly more spots when stimulated with peptide mixes compared to adult donors with no history of VZV infection.

For evaluating functional B-cells, latent and lytic peptide mixes were used to stimulate B-cells and total antibody-production for IgG and IgM was detected in one well in parallel. This was done for short incubation times to detect antibody-secreting cells (ASCs) in the periphery and for longer incubation times for memory B-cell evaluation.

The range for an effective vaccination should be confirmed by further investigations with more and well characterized donors. Furthermore, these trials could facilitate the detection of significant differences between vaccinated and non-vaccinated donors and open the possibility to define effectiveness of vaccination.
Targeting shingolipids inhibits alcohol-induced liver disease
Authors: Deng Zhongbin , Chu Shenghui , Sun Rui , Gu Xuemei , Chen Liang
Published: January 2023

Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to pathogenesis of alcoholic liver disease (ALD). We used WT mice, RORγt-deficient mice and sphingosine kinase-deficient mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Ethanol-fed and control mice were treated by SK1 inhibitor. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. We found that alcohol intake induces an increase in Th17 cells in the gut. Increased Th17 cells were due to upregulation of SK1 activity and Rorc activation. The pathogenic role of S1P/S1PR1 signaling in ALD was attributable to the migration and activation of Th17 cells. Importantly, deletion of SK1 markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. In conclusion, our findings suggest that S1P signaling was crucial in the pathogenesis of alcoholic liver disease in a Th17 cell dependent manner.
Nexus between CNS resident microglia and migrating peripheral T cells pave the way for host immunity against neurotropic virus infection
Authors: Sarma Jayasri Das , Chakravarty Debanjana , Saadi Fareeha , Khan Reas , Burrows Amy , Kundu Soumya , Sharma NIkhil , Bergmann Cornelia , Shindler Kenneth S , Sen Ganes C
Published: January 2023

Neurotropic virus induced neuroinflammation initiates with the activation of brain resident immune cells which causes a heightened release of chemokines/cytokines leading to a compromise in the blood brain barrier integrity. This accentuates the upregulation of regulatory molecules like antiviral Interferon stimulating gene induced tetratricopeptide repeats protein, Ifit2, and augments infiltration of peripheral immune cells. Among the early infiltrating immune cells, a large population is composed of neutrophils and monocyte/macrophages which, along with brain resident microglia, orchestrate the full panoply of innate immune responses. Ifit2 has been shown to be beneficial in protecting mice from lethal neurotropic virus infection by restricting viral replication and amplifying antiviral responses. Using a murine neurotropic coronavirus infection in Ifit2−/− mice, we report that in the absence of Ifit2, viral replication is significantly increased and mice develop severe neuropathy. Despite the enormous viral load, Ifit2-deficient mice are impaired in microglial activation and recruitment of peripheral T cells into the CNS. To further confirm the role of migratory T cells in viral-induced neuroinflammation and neurodegeneration, studies were conducted in CD4−/− mice. Results showed no significant change in acute stage pathogenesis, whereas CD4−/− mice showed accelerated loss of axon-myelin coherence during the chronic stage. Together, studies in Ifit2 and CD4 knockout mice reveal that the interplay between brain resident microglia and migratory helper T cells is important for strengthening host immunity against neurotropic virus infection.
A novel and human-specific CD46-KLF/SP interaction mediates gene expression required for successful Th1 induction
Authors: Singh Parul , Kunz Natalia , Le Friec Gaelle , Wang Luopin , Lavender Paul , Kazemian Majid , Kemper Claudia
Published: January 2023

Signals mediated by autocrine activation of the human-specific complement receptor CD46 during T cell receptor (TCR) stimulation are vital to Th1 induction in human CD4+ T cells, but how exactly CD46 in a molecular level mediates this role is currently undefined. CD46 is expressed in different isoforms that can bear either one of two distinct cytoplasmic tails: CYT-1 or CYT-2. Nuclear translocation of CYT-1 is a critical requirement for the expression of genes coding for nutrient-influx-channels and mTORC1 activity that mediate metabolic adaptations needed for Th1 responses. The lack of a DNA binding domain in CD46-CYT-1 precludes it from acting directly as a transcription factor (TF) and we hence hypothesized that CYT-1 regulates gene expression via direct interaction with specific TF activator and/or repressor complexes. Indeed, CUT&RUN experiments performed using our novel antibody raised against cleaved CYT-1 identified key members of the KLF/SP TFs gene family as potential interacting partners of CD46-CYT-1. Subsequent ELISA and MST experiment confirmed strong, dose-dependent CYT-1/KLF/SP TFs interactions and also demonstrated that CYT-1 fostered KLF/SP TFs binding to appropriate DNA motifs. Genome-wide comparison of the KLF/SP TFs and CYT1 bound genes in T cells revealed their enrichment in crucial basic cell-physiological pathways. Moreover, the CUT&RUN data in conjunction with ATAC-seq analyses indicated that this novel CYT-1/KLF/SP axis may control general chromatin remodeling – a notion we are currently exploring. These data define a novel and critical human-specific pathway of gene regulation and further underpin the vital role of intracellular/autocrine complement in the regulation of normal cellular activity.
Identification of MLL1 as a novel regulator of Tfh cell differentiation
Authors: Belanger Simon , Getzler Adam J , Fung Kai , Greenbaum Jason , Pipkin Matthew E. , Crotty Shane
Published: January 2023

Transcription factors controlling T follicular helper (Tfh) cell differentiation have been extensively studied in recent years. However, there is limited knowledge of the different chromatin regulators involved in the control of Tfh differentiation. Most transcription factors control gene expression with the help of chromatin regulators, which modify chromatin structure to regulate gene expression. We hypothesize that chromatin modifying enzymes are involved in the control of Tfh differentiation. To uncover novel chromatin modifying enzymes essential for Tfh differentiation, we screened a shRNA library targeting all known chromatin regulators using our in vivo RNAi screen. Utilizing this screen, we identified the histone methyltransferase MLL1 as a positive regulator of Tfh differentiation after viral infection.

We found MLL1 to be functionally important for Tfh differentiation. Knockdown of MLL1 utilizing multiple shRNAs impaired Tfh differentiation during LCMV infection. The requirement for MLL1 for proper Tfh differentiation after LCMV infection was further confirmed using a CRISPR/Cas9 approach. Additionally, ablation of MLL1 expression selectively impaired production of IL-21 by Tfh cells. To further understand the role of MLL1 in regulating the Tfh differentiation network, we performed RNA-Seq of MLL1-deficient Tfh as well as MLL1-sufficient Tfh. Analysis revealed that the expression of several genes critical for the regulation of Tfh differentiation is dysregulated in the absence of MLL1. Specific examples will be discussed.
The transcriptional coactivator OCA-B re-wires metabolism and promotes CD4 T cell memory
Authors: Tantin Dean R , Williams Matthew A , Hale J Scott , Kim Heejoo
Published: January 2023

OCA-B (Pou2af1/OBF-1/Bob.1) is a transcriptional coregulatory protein that is not expressed in naïve CD4+ T cells, but becomes stably induced following antigen exposure and T cell activation. We have previously shown that the transcription factor OCA-B interacts with, Oct1 (Pou2f1) is a potent regulator of metabolism. Oct1 dampens oxidative metabolism of amino acids in mitochondria and counteracts reactive oxygen species production. Possible roles of OCA-B in metabolism have not been tested. Our prior work also indicates that both OCA-B and Oct1 are critical for the generation of functional CD4 memory T cells in response to pathogen infection. By co-transferring SMARTA TCR transgenic T cells ectopically expressing OCA-B and empty vector controls into recipient mice, and infecting the recipients with LCMV, we show that OCA-B does not affect primary effector responses, but is sufficient to promote formation of long-lived memory T cells. Although there are no differences in T cell numbers or function at peak response, changes in the expression of proteins such as CD62L and ICOS are consistent with preferential entry into the memory pool and stronger response upon rechallenge. Transcriptomic profiling of cells at the same peak pathogen response timepoint identifies ~80 genes significantly differentially expressed by ectopic OCA-B expression. These genes encode metabolic regulators and transcription master regulators indicative of metabolic rewiring of these cells. This prediction was confirmed by profiling glycolytic and oxidative metabolism in OCA-B conditional knockout and control naïve CD4 T cells stimulated and rested in vitro. These results indicate that expressing OCA-B may be useful as a method of enhancing CD4 memory.
Single cell sequence analysis reveals mouse MAIT cell diversity at steady state and after infection
Authors: Chandra Shilpi , Ramirez Ciro , Ascui-Gac Gabriel , Seumois Gregory , Seo Goo-Young , Simon Hayley , Hartmann Nadine , Murray Mallory Paynich , Vijayanand Pandurangan , Kronenberg Mitchell
Published: January 2023

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that recognize vitamin B metabolites. We have performed single-cell RNA (sc-RNA) sequencing of MAIT cells from thymus, liver, spleen and lung using 10× single-cell genomics, where we sequenced more than 7,000 cells and identified 10 clusters by unbiased clustering. MAIT cells from the different organs are distributed in most of the clusters; except two clusters were lung MAIT cell specific and another consisted of cells exclusively from thymus. The lung specific cluster reveals a tissue residency gene signature. We compared the gene expression profile of MAIT cells with iNKT (invariant natural killer T) cells. Our data reveal that most mouse MAIT cells have a Th17/NKT17–like gene signature (MAIT17), although there are some with Th1/ NKT1-like transcriptomes (MAIT1), particularly in liver and spleen. A Th2/NKT2 gene expression profile was not observed. The thymus specific cluster showed a gene expression profile similar to the most immature or progenitor iNKT cells (NKT0), consistent with other data suggesting a unique thymus differentiation pathway for mouse MAIT that is similar to iNKT cells. We also performed scRNA sequencing of MAIT cells after bacterial infection in the lung. Our results indicate that after infection the transcriptome of lung MAIT cells changes significantly at day 6 and remains altered at day 40. Therefore, our study reveals that although mouse MAIT cells are predominantly Th17 cells and to a lesser extent Th1 cells, there is some heterogeneity and there are profound and relatively long-term changes in transcriptional signatures after infection.
Type 2 cytokines trigger eomesodermin-associated pathway conferring robust interferon-g expression in human NK cells
Authors: Ohayon David E , Donmez Omer , Parameswaran Sreeja , Kottyan Leah C. , Weirauch Matthew T. , Waggoner Stephen N
Published: January 2023

Asthma encompasses a spectrum of chronic respiratory symptoms, generally associated with aberrant expression of type 2 cytokines. As many as 10% of patients are classified as having severe asthma, characterized by poor control of disease symptoms and a paradoxical mixed milieu of both type 2 (IL-4 and IL-33) and type 1 (IFN-γ) cytokines. The source of IFN-g and mechanisms driving expression of this cytokine, which can exacerbate airway hyper-responsiveness, remain poorly defined. Here, we show that in human NK-cell cultures, combined IL-4 and IL-33 stimulation triggers high expression levels of IFN-g, even in the absence of STAT4 activation by prototypical type 1 stimuli like IL-12. At picomolar concentrations, IL-4 enhanced the expression of eomesodermin (Eomes), a critical transcription factor for IFN-γ expression. We performed anti-Eomes ChIP-seq in NK cells, generating a high-quality genome-wide map of Eomes binding with robust peak counts, strong overlap with T cell datasets, and strong enrichment for the Eomes DNA binding motif. These data support a model in which Eomes interacts with the IFNG locus to promote enhanced IFNG expression in NK cells. We also performed ATAC-seq and ChIP-seq following IL-4/IL-33 stimulation to reveal changes in chromatin accessibility and Eomes binding resulting from cytokine exposure. Collectively, our results reveal a novel pathway of IFN-γ induction in type 2 diseases that could contribute to exacerbated pulmonary dysfunction in severe asthma.
Mst1 regulates Foxo1 transcription to differentially control memory CD8 T cell differentiation and survival
Authors: Abdullah Leena , Hills L. Benjamin , Lust Hannah E , Huang Yina H
Published: January 2023

Foxo1 is an essential transcription factor required for the differentiation and survival of memory CD8 T cells. Foxo1 activity is heavily regulated by post translational modifications, yet it is unclear whether memory T cell differentiation is regulated in the same manner as survival. Two S/T kinases, Akt1 and Mst1 phosphorylate Foxo1. While Akt1 mediated phosphorylation is known to inhibit Foxo1 transcriptional activity, the effect of Mst1 phosphorylation remains poorly understood. To determine the impact of Mst1 regulation on Foxo1-dependent CD8 T cell differentiation and survival, we generated Foxo1 variants that mimic Mst1 phosphorylation (SD-Foxo1) or disrupt Mst1 phosphorylation (SA-Foxo1). Reconstitution of Foxo1-null T cells with SA- or SD-Foxo1 allowed us to evaluate their effects on early response genes in vitro and memory differentiation and survival in vivo following Listeria monocytogenes infection. We observed significantly higher expression of canonical Foxo1 target genes, including Il7r and Sell when CD8 T cells expressed SA-Foxo1. In contrast, CD8 T cells expressing SD-Foxo1 were defective in inducing canonical Foxo1 targets and in generating memory precursor effector cells post Listeria infection. However, SD-Foxo1 expression promoted survival of memory CD8 T cells as compared to the SA-Foxo1 in vitro. RNAseq analysis revealed that SD-Foxo1 induces an altered Foxo1 transcriptional program including the upregulation of Purinergic Receptor P2X7 (P2RX7), which was recently shown to be required for memory CD8 T cell maintenance and survival. Together, our data suggest that Mst1 regulates a distinct mode of Foxo1 transcription to balance Foxo1-dependent differentiation and survival in CD8 T cells.
Altered ligand-interaction contributes to functional insufficiency due to CTLA-4 gene mutation
Authors: Zhao Zhishan , Lu Linrong
Published: January 2023

CTLA-4 is one of the key inhibitory immune receptors and plays a critical role in maintaining immune tolerance. Both loss of CTLA-4 gene in mice and mutations in humans result in systemic autoimmune diseases with a similar phenotype. However, the precise mechanisms of CTLA-4 have not yet been entirely clarified. Structural and functional analysis of the reported autoimmune cases revealed that pathogenic CTLA-4 mutations occur frequently in ligand binding and dimerization regions. Through genetic mutation screening study, we have discovered a new CTLA-4 point mutation in a patient with recurrent autoimmune inflammation. We used steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, as well as functional assays to compare the function of this CTLA-4 mutant to the WT molecule. We demonstrated that, mechanical force induces conformational changes in CTLA-4 to enhance pre-existing contacts and activates new interactions at the CTLA-4-CD80 and CD86 binding interface. Although the mutant CTLA-4 can function to induce downstream signals, its ability in trans-endocytosis of CD80 and CD86 is impaired due to altered ligand interaction. The conclusions from this study will help us elucidate the immunoregulatory mechanisms of CTLA-4 and provide new strategies for more effective targeting of CTLA-4 in autoimmune diseases and tumors.
Intravenous tolerance induces T cell anergy/apoptosis via IFN-γ/IL-27/PD-L1 axis
Authors: Casella Giacomo , Rasouli Javad , Thome Rodolfo , Alvarez Jorge Ivan , Hunter Christopher A , Zhang Guang-Xian , Ciric Bogoljub , Rostami Abdolmohamad M
Published: January 2023

The induction of peripheral tolerance by intravenous injection (i.v.) of auto-Ags has shown to suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis, by eliciting tolerogenic APCs, Tregs, and suppressing self-reactive T cells. The programmed cell death protein (PD)-1 and its ligands, PD-L1 and PD-L2, regulate the balance between T cell activation and immune tolerance. Although PD-L1 and PD-L2 are expressed on a variety of cell types, however, the mechanisms related to peripheral tolerance that induce these molecules are still unclear. Here we show that PD-L1, but not PD-L2, is critical for inducing peripheral tolerance in EAE, and that PD-L1-dependent tolerance relies on both IFN-γ and IL-27. I.v. administration of auto-Ag(s) attenuates the progression of EAE, by inducing PD-L1 expression in monocyte-derived DCs (moDCs) in the CNS, via IFN-γ/ IL-27-dependent mechanism. Indeed, when PD-L1 is blocked, autoantigen(s)/i.v treatment becomes ineffective. Following IFN-γ−/− CD4+ T cell transfer into Rag1−/− mice, i.v. tolerance treatment failed to suppress EAE. Moreover, loss of IFN-γR signaling in classical DCs type I (cDCs I) compromised IL-27 production and consequent PD-L1 expression by moDCs. We also show that IL-27 stimulates moDCs to over-express PD-L1, in a STAT-3-dependent way. Indeed, in CCR2−/−/WSX−/− (CD45.2) → CD45.1 radiation-induced bone marrow chimera mice, auto-Ag(s)/i.v. treatment failed to suppress EAE. Similarly, EAE mice that expressed mutant STAT-3 (muSTAT-3) in CCR2+ cells were also insensitive to i.v. tolerance induction. Our data reveal a mechanism whereby IFN-γ and IL-27 are the key factors in PD-L1 induction and subsequent peripheral tolerance restoration.