bluuebunny/crossref_metadata_2025 huggingface dataset:
ALCAM and VCAM-1 as potential urinary biomarkers in Caucasian lupus patients
Authors: Habazi Deena , Vanarsa Kamala , Stanley Samantha , Gokaraju Sirisha , Ding Huihua , Parodis Ioannis , Gunnarsson Iva , Mohan Chandra
Published: January 2023

Background

Lupus affects millions of people worldwide and is difficult to diagnose early in patients. By utilizing enzyme-linked immunosorbent assays (ELISA), this study seeks to identify urinary biomarkers that may detect lupus in its earlier stages, allowing for better, earlier treatment regimens.

Methods

197 urine samples from a Swedish SLE cohort were divided into four groups, each matched for demographics: 23 active non-renal SLE samples (SLEDAI>0, rSLEDAI=0), 29 active renal SLE samples (SLEDAI>0, rSLEDAI>0), 46 inactive SLE samples (SLEDAI=0), and 99 healthy controls. These samples were screened for 2 human proteins using ELISA, selected because they exhibited biomarker potential in our earlier screens.

Results

VCAM was noted to be significantly elevated (2-tailed t-test, p<0.0001) in SLE patients compared to healthy controls. More specifically, VCAM was elevated in active renal samples when compared to healthy samples (p<0.0001). An ROC curve shows that VCAM can be used to differentiate between active renal samples and control samples (AUC=0.8889, p-value=0.0250). ALCAM was also elevated (p<0.0001) in SLE patients compared to healthy patients. However, ALCAM was not as highly elevated in active renal samples compared to healthy samples (p<0.001). Additionally, an ROC curve does not show ALCAM strongly differentiating between active renal samples and control samples. Based on these results, VCAM is the more promising biomarker of the two.

Conclusions

Urinary VCAM and ALCAM are both potentially viable biomarkers for lupus that warrant further investigation. Studies are in progress to compare the performance of these urinary markers to conventional yardsticks and their relationship to other clinical parameters.
Macrophage heterogeneity correlates with autophagy-dependent anti-mycobacterial activity and ability to present antigen in human macrophages
Authors: Singh Vipul Kumar , Khan Arshad , Papanna Ramesha , Mann Lovepreet , Wan Jon Hak , Canaday David , David Sunil A , Jagannath Chinnaswamy
Published: January 2023

Mycobacterium tuberculosis (Mtb) kills around 1.5 million people each year. Albeit, only 10% of humans develop active disease after aerosol infection, the basis for differential susceptibility of humans remains unclear. To determine if human macrophages are heterogeneous in their ability to kill Mtb, cord blood derived macrophages were treated with either IFN-γ or IL-4 to prepare M1 and M2 phenotype respectively in vitro. Macrophages were infected with a 1:1 ratio of Mtb and days 3 and 5 post infection, macrophage lysates were plated for CFU counts. In addition, intracellular autophagy was measured in gfpMtb infected macrophages using cytoID stains to detect autophagosomes and LC3 lipidation through western blot of macrophage lysates. Mtb infected macrophages were overlaid using CD4 T cells specific for the Antigen-85B epitope to measure antigen presentation. M1 macrophages showed a bacteriostatic ability and restricted the growth of Mtb while IL-4 treated M2 macrophages showed nearly 1 log increase in the growth of Mtb. Thus M2 macrophages were permissive for growth of Mtb. Consistent with this observation, gfpMtb co-localized with autophagosomes more frequently in M1 macrophages suggesting a role for autophagy during growth inhibition. M1 macrophages also presented Ag85B to CD4 T cells better than M2 macrophages. Enhanced antigen presentation by M1 macrophages was also found to be dependent on IFITM3, Atg5 and Atg 16 genes as siRNA knock down of these specific genes resulted in reduced antigen presentation by M1 macrophages. These observations suggest that autophagy pathways within macrophage may govern their heterogeneity into M1 or M2 hence their innate ability to kill intracellular Mtb and trigger immune activation.
Alterations of Specific Lymphocytic Subsets with Ageing and with Aged-Related Metabolic and Cardiovascular Diseases
Authors: Liu Chao Lien , Tram Van Thi Ngoc , Lin Chung Hao , Liao Kuo Chen , Chen Ying Jen
Published: January 2023

Ageing of the immune system (immunosenescence (ISC)), is a complex subject characterized by a decrease in cell-mediated immunity, particularly with T-cell function. We conducted a clinical study to analyze peripheral blood (PB) for alterations in T-cell subsets among young healthy (YH) controls, elderly healthy (EH) controls, age-matched subjects with metabolic diseases (MDs; E-MDs), and cardiovascular diseases (CVDs; E-CVDs). The frequencies of expressions of CD3T, CD8T, and invariant natural killer T (NKTs; iNKT) cells were reduced in the EH, E-MD, and E-CVD cohorts; in contrast, CD4T and regulatory T (Treg) cell frequencies tended to increase with ageing, whereas they were lower in subjects with aged-related MDs and CVDs. Moreover, subsets including naïve, effector memory T (TEM) cells, effector memory T cells re-expressing CD45RA (TEMRA), as well as the CD28−CD57+phenotype in the CD4T and CD8T subpopulations consistently showed accumulation of senescent T-cell subsets in ageing, and in patients with E-MDs and E-CVDs compared to YH volunteers. Notably, serum cytokines, including Interferon (IFN)-γ, Transforming growth factor (TGF)-β, and Growth differentiation factor (GDF)-15 levels were consistently reflection of the alterations in T-cell subsets for within-group comparisons. Ageing-, E-MD-, and E-CVD-associated changes in PB T-cell subsets were paralleled by alterations in expressions of cytokines involved in lymphopoiesis, which might play important roles in ageing, and aged-related MD- and CVD-mediated dysregulation of immune functions. Furthermore, identifying these altered T-cell subsets can potentially serve as diagnostic markers for preventing ageing problems and treating aged-related MDs and CVDs.
Role of natural killer cell activity in HIV infections, aging, and breast cancer survival
Authors: Williams R Michael , Zuniga Joaquin , Granados Julio , Yunis Edmond J.
Published: January 2023

Natural killer cell activity participates in the elimination of tumors and virus infections. Data presented here include their role in a virus infection (HIV), metastatic breast cancer, and aging per se. We have analyzed two gene segments MHC HLA class I ligands and NK receptors Each have single amino acid differences. Together these alleles can identify long term survivors. Homozygosity of HLA–Bw4 supertype in HIV progression has been described. Since HLA-KIR genetic interactions of HLA-Bw4 with KIR (3DS1) only included Bw4, with the exception of B*27 and B*44 that use different mechanism of protection, on the clinical outcome of HIV infection, NK–ligand interactions (NKG2A-HLA-E with HLA class I leader peptides) explain the long term non-progressors (LTNP) to AIDS. HLA-B alleles have Methionine (Met) or Threonine (Thr) at second position (P2) of their leader peptide. HLA-Bw4 alleles, with exception of B*38, encode leader peptide with Thr at P2 and HLA-Bw6 genes that encode Met at P2 are B*07, B*08 and B*14. All others have Thr at P2. Comparisons of the Thr in LTNP with progressors, up to 15 years. with non-infected Caucasian controls were significant. Also, aged Mexicans demonstrated significant increase of homozygosity of Thr at P2 and Mexican patients with metastatic breast cancer were studied 10 years after treatment with surgery and chemotherapy,

The 39 survivors demonstrated significant increase of homozygosity of Thr at P2 compared to controls without cancer and the 37 non survivors. These findings need to be studied in different malignancies and different ethnicities as well as in mouse models. Our findings support the immunogenetic theory of cancer and aging.
Production of Peanut-Specific IgE in a Food Allergy Model Is Promoted by T Follicular Regulatory Cells Via an IL-10-Dependent Pathway
Authors: Xie Markus Ming , Liu Hong , Yang Kai , Dent Alexander
Published: January 2023

T follicular regulatory (TFR) cells are a specialized subset of Foxp3+ T cells found within the germinal center (GC) reaction and its role remains controversial. Here, we have used a peanut allergy model in mice to examine the role of TFR cells in controlling the production of antigen-specific IgE and IgE-controlled anaphylaxis. Using a mouse model with a specific loss of TFR cells (Foxp3-cre Bcl6-flox mice), we found TFR cells play an essential role in promoting and maintaining GC B cells, IgE production and anaphylaxis. Compared to control mice, TFR-deficient mice lacked circulating peanut-specific IgE four weeks after the priming period and anaphylactic responses were significantly weakened. The loss of IgE in TFR-deficient mice coincided with a marked decrease in GC B cells and peanut-specific IgG1. Similarly, deletion of regulatory T cells using FOXP3DTR mice abolished peanut-specific IgE and IgG1 responses. We identified a positive role for TFR cells in the IgE response using Foxp3-cre Pten-flox mice, which have augmented TFR cell responses, higher peanut-specific IgE and increased GC B cells. Mechanistically, TFR cells require Blimp1 controlled IL-10 to promote GC B cell survival and IgE production. Further studies using mice deficient of IL-10Ra (Mb1-cre Il10ra-flox mice) showed IL-10 signaling was pivotal for GC B and Ab responses. Blocking IL-10 signals in vivo mimicked the loss of IgE levels in TFR-deficient mice and rescued mice from anaphylaxis. Our data unexpectedly show that TFR cells have a critical helper function in the production of GC-dependent IgE, in part by producing IL-10. These studies have provided greater understanding of how allergic immune responses are controlled by the GC reaction.
Activated Plasmodium-specific CD8 T cells find liver stages mostly randomly
Authors: Zenkov Viktor S , Ganusov Vitaly V , O'Connor James , Cockburn Ian A
Published: January 2023

Malaria is a major infectious disease, with 500,000 people dying every year. Malaria infection starts when an infected mosquito injects malaria sporozoites during feeding which migrate to the liver and form liver stages. Elimination of liver stages is a major feature of CD8 T cell-based malaria vaccines. It is established that vaccines inducing high numbers of memory CD8 T cells are capable of eliminating all liver stages, preventing clinical malaria. However, how CD8 T cells are capable of doing that in the 48-hour liver stage in mice remains unclear. One possibility is that as sporozoites invade hepatocytes from the blood, they induce local inflammation, acting as an attraction signal for T cells. We performed experiments in which fluorescent-labeled Plasmodium sporozoites and Plasmodium-specific and non-specific CD8 T cells were tracked in mouse livers using intravital microscopy. To analyze the data we used three metrics to measure attraction of T cells to the infection site based either on angles or distances from a T cell to the parasite. We found that for the majority of imaged T cells, there is no evidence of attraction to the parasite, indicating that in this experimental system, vaccine-induced CD8 T cells locate the infection site randomly. By modeling T cells searching for the infection site and utilizing experimentally measured speeds of T cell movement in the liver, we found that if enough memory CD8 T cells are present in the liver, all parasites can be found within 48 hours after sporozoite injection. Taken together, our results suggest that memory CD8 T cells in the liver perform the search for infection randomly, precluding the need to find specific signals and receptors of T cell attraction to the infection site.
Costimulation of type-2 innate lymphoid cells by GITR promotes effector function and ameliorates type 2 diabetes
Authors: Galle Lauriane , Sankaranarayanan Ishwarya , Hurrell Benjamin P , Howard Emily , Lo Richard , Maazi Hadi , Lewis Gavin , Banie Homayon , Epstein Alan L , Hu Peisheng , Rehan Virender K , Gilliland Frank D , Allayee Hooman , Soroosh Pejman , Sharpe Arlene H , Akbari Omid
Published: January 2023

Metabolic syndrome is characterized by disturbances in glucose homeostasis and the development of low-grade systemic inflammation, which increase the risk to develop type-2 diabetes mellitus (T2DM). Type-2 innate lymphoid cells (ILC2s) are a recently discovered immune population secreting Th2-cytokines. While previous studies show how ILC2s can play a critical role in the regulation of metabolic homeostasis in the adipose tissue, a therapeutic target capable of modulating ILC2 activation has yet to be identified.

We found that GITR, a member of the TNF superfamily, is expressed on murine adipose tissue ILC2s and its engagement on activated ILC2s induces Th2-cytokine secretion. Moreover, we showed that GITR engagement on ILC2s improves glucose homeostasis resulting in both protection against insulin-resistance onset and amelioration of established insulin-resistance. Our adoptive transfer studies demonstrated that this protective effect is dependent on ILC2-derived Th2-cytokines, particularly IL-13. Transcriptome analysis demonstrated that GITR agonist activates the NF-κB signaling pathway and inhibits ILC2 apoptosis, altogether favoring ILC2 survival and activation. Finally, we also found that GITR is expressed on human adipose tissue resident ILC2s and GITR engagement robustly induces Th2-cytokine.

Together, these results highlight the critical role of GITR as a novel therapeutic molecule against T2DM and its fundamental role as an immune checkpoint for activated ILC2s.
Natural killer cell inhibition of HIV vaccine responses
Authors: Gyurova Ivayla E , McNally Jonathan P , Rydyznski Carolyn E , Waggoner Stephen N
Published: January 2023

There is an increasing appreciation of the ability of natural killer (NK) cells to inhibit adaptive immunity. We previously found that NK cells inhibit antiviral T-cell responses and antibody production during acute virus infection in mice. NK cells also inhibit follicular helper (Tfh) T-cell and germinal center (GC) B-cell responses following immunization with alum-adjuvanted NP-KLH. Of note, NK-cells are linked to a reduced frequency of immunoglobulin heavy chain mutations and impaired affinity maturation of NP-specific GC B cells, indicating that NK cells are important modulators of both the magnitude and the quality of the humoral responses. As a successful HIV vaccine putatively requires more extensive and efficient affinity maturation of HIV-specific immunoglobulin sequences than has been achieved in vaccine efforts to date, we hypothesize that NK-cell suppression of Tfh and GC responses represents a promising target to enhance HIV-specific vaccine responses. To test this hypothesis, we administered a HIV envelope (Env)-expressing vaccinia virus (VACV-Env) to mice depleted or not of NK cells. The absence of NK cells was associated with increased frequencies of HIV-specific GC B cells and elevated production of Env-specific immunoglobulins. The enhancing effect of NK-cell depletion at both priming (VACV-Env) and boosting (Env protein) immunizations was robust. These results are consistent with recent demonstration of an association between NK-cell dysfunction and development of bnAbs in HIV-infected individuals. Thus, NK cells are important regulators of affinity maturation and of HIV-specific antibody responses, suggesting that interventions targeting this NK cell activity could enhance HIV vaccine efficacy.
The lysophosphatidylcholine transporter, MFSD2A, is essential for CD8+ memory T cell maintenance and secondary response to infection
Authors: D'Cruz Louise M , Piccirillo Ann , Hyzny Eric , Menk Ashley , Wallace Callen , Hawse William F , Buechel Heather Marie , Wong Bernice , Foo Juat Chin , Cazenave-Gassiot Amaury , Wenk Markus , Delgoffe Greg M. , Watkins Simon , Silver David
Published: January 2023

Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. Here we discovered the sodium-dependent lysophosphatidylcholine transporter, Major Facilitator Super Family Domain Containing 2a (MFSD2A), is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of lysophosphatidylcholine (LPC) esterified to long chain fatty acids (LCFAs) into activated CD8+ T cells, and MFSD2A deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, that when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.
PU.1 and IRF8 regulate the transcriptional landscapes differently in naïve and activated B cells
Authors: Wang Hongsheng , Li Peng , Lin Jianxin , Oh Jangsuk , Bolland Silvia , Leonard Warren J , Morse Herbert C
Published: January 2023

The Ets family member PU.1 and the IRF family member IRF8 play crucial roles in regulating hematopoietic cell development including B cells. Both factors are constitutively expressed at high levels in B cells and control plasma cell differentiation. IRF8 and PU.1 function either together as a heterodimer or by pairing with other factors to bind target DNA sequences. Several consensus binding sequences for IRF8 and PU.1 have been identified, including the canonical interferon stimulated response element (ISRE) sequence motif (GAAANNGAAA), the Ets-IRF composite element (EICE) (GGAANNGAAA) and the IRF-Ets composite sequence (IECS) (GAAANN[N]GGAA). Although the target genes of IRF8 and PU.1 have been studied previously by chromatin immunoprecipitation (ChIP), the nature and the importance of the motifs engaging both PU.1 and IRF8 in B cells have not been determined. In this study, we performed ChIP-seq using sort-purified naïve follicular B cells from C57BL/6 mice that were either not stimulated or stimulated with anti-IgM plus anti-CD40, which mimic signals for germinal center B cell differentiation. We also integrated our ChIP-seq data with RNA-seq data from B cells deficient for both IRF8 and PU.1, and found that IRF8 and PU.1 regulate gene expression by preferentially binding to the EICE motifs of target genes known to maintain follicular B cell identity, localization, and survival. In contrast, in activated B cells, the binding landscape of IRF8/PU.1 in target genes shifts to IRF8-dominant ISRE motifs, possibly due to elevated expression of IRF8. These results provide new insights into our understanding of the molecular mechanisms underlying the transcriptional control of late stage B cell development.
Temporal dynamics of the serological compartment of rabbit antibody repertoires
Authors: Patel Anand , Bonissone Stefano R , Castellana Natalie
Published: January 2023

Understanding the serum antibody repertoire is an essential first step in improving vaccine design, understanding autoimmune diseases, and discovering drug candidates. Next-generation sequencing (NGS) of peripheral blood mononuclear cells is a common approach to approximate the repertoire; however it is a poor proxy for circulating antibodies. Sampling serum antibodies with mass spectrometry provides orthogonal data - directly querying the repertoire of interest.

In this study, we integrated next-generation sequencing of the B-cell repertoire with mass spectrometry-based proteomic analysis of circulating antibodies in a hyperimmunized New Zealand white rabbit. During a 14 week immunization with 4 boosts, we sampled the B-cell repertoire of blood at multiple time points, as well as the bone marrow and spleen at week 14. At week 14, serum antibodies were enriched for immunogen-specificity using affinity chromatography and subjected to tandem mass spectrometry analysis. We mapped the tandem mass spectra to the antibody sequences in the repertoire to derive a set of clonal lineages and antibodies that are target specific and abundant in serum. We then tracked those lineages across time points and tissues throughout the immunization.

Finally, we sought to determine the fraction of antigen-specific antibodies present in serum but absent from the B-cell derived repertoires. We employed spectral networking to identify novel clonal lineages, and estimate that at least 1/3 of the abundant clones in serum are missing from the repertoire.
Cooperative roles for Aiolos and STAT3 in TH17 differentiation
Authors: Moon Sol C , Sreekumar Bharath , Rasheed Mustafa N , Read Kaitlin , Powell Michael Duane , Oestreich Kenneth J.
Published: January 2023

TH17 cells are a subset of T helper cells involved in mucosal immunity that are defined, in part, by their production of the proinflammatory cytokine IL-17. Dysregulated Th17 cells have been implicated in several autoimmune diseases, such as multiple sclerosis and inflammatory bowel disease. Therefore, there is substantial interest in understanding the mechanisms underlying their differentiation and function. TH17 development requires signals from IL-6 to activate STAT3, which positively regulates the expression of proinflammatory genes including Il17a and Il17f. Our recent work established that the Ikaros Zinc Finger (IkZF) transcription factor Aiolos cooperates with STAT3 to positively regulate gene expression in T follicular helper cells. Given the role of STAT3 in TH17 populations, we hypothesized that Aiolos and STAT3 may also regulate the differentiation of TH17 cells. Here, we find that Aiolos expression is significantly increased in TH17 cells as compared to other T helper cell subsets. Corroborating previously published work, we find that overexpression of Aiolos results in reduced expression of Il2, which negatively regulates the TH17 gene program. Intriguingly, we find that concurrent Aiolos and STAT3 overexpression leads to further repression of Il2, as well as increased expression of the pro-TH17 gene Il6ra. However, expression of Rorc, Il17a, and Il17f were unaffected in this system, suggesting that Aiolos and STAT3 cooperate to regulate cytokine signaling pathways in TH17 cells, rather than ROR gamma t or TH17 effector functions. Collectively, these data suggest that Aiolos and STAT3 may cooperatively regulate TH17 differentiation through a feed-forward, cytokine signaling-mediated mechanism.
Regulation of Tfh cell development through STAT1 signaling
Authors: Qin Lei , Sahoo Anupama , Zhao Yangzhi , Alekseev Andrei , Nurieva Roza I
Published: January 2023

T follicular helper (Tfh) cells play a key role in providing help to B cells during germinal center (GC) reactions. The generation and function of Tfh cells is regulated by multiple checkpoints including their early priming stage in T zones and throughout the effector stage of differentiation in GCs. Signaling pathways activated downstream of cytokine and costimulatory receptors as well as consequent activation of subset-specific transcriptional factors are essential steps for Tfh cell generation. While to date early signaling events for fate committed differentiation of Tfh cells are sufficiently studied, mechanisms allowing Tfh cells to maintain their commitment/programming are still unclear. Here we report that signal transducer and activator of transcription 1 (STAT1) deficiency in CD4+ T cells leads to a diminished number of Tfh cells at day 7 and 14 days after antigen immunization, while at day 3–4, number of Tfh cells was comparable between STAT1-sufficient and STAT1 deficient mice, suggesting the role of STAT1 in Tfh lineage maintenance rather than in Tfh cell priming. In fact, in both mouse and human Tfh cells interleukin (IL)-21 mRNA and protein expression is dependent on the presence of STAT1. Functionally, STAT1 in cooperation with STAT3, Batf, and IRF4 triggers IL-21 production in Tfh cells by directly binding to and activation of the IL-21 gene locus. Moreover, utilizing gene knockout approach, we have determined that Batf is a crucial connector to form STAT1/STAT3/Batf/IRF4 active complex for triggering IL-21 transcription. Our results thus indicate functional contribution of STAT1 towards regulation of IL-21 expression and Tfh lineage maturation/maintenance.
Regulation of T cell activation and pathogenicity by dimeric pyruvate kinase M2 (PKM2)
Authors: Angiari Stefano , Sutton Caroline , Runtsch Marah C , Palsson-McDermott Eva M. , Mills Kingston H.G. , O’Neill Luke A.J.
Published: January 2023

Pyruvate kinase (PK) catalyses the conversion of phosphorenolpyruvate to pyruvate during glycolysis. Previous studies suggested that the PK isoform PKM2 also plays moonlighting activities different from its enzymatic one, such are regulation of gene transcription and protein phosphorylation. PKM2 was previously shown to control macrophage metabolic remodelling and pro-inflammatory potential, but its role in CD4+ T cell biology is poorly understood. In the present work, we report that PKM2 is upregulated in both murine and human CD4+ T cells following activation in vitro. In particular, PKM2 is present in equilibrium between a monomeric, dimeric and tetrameric isoform in T cells, with the dimeric one appearing exclusively upon activation. Treatment of T cells with TEPP-46, an allosteric activator that induces PKM2 tetramerisation and blocks dimer formation, strongly reduces their activation, proliferation and cytokine production through alteration of intracellular metabolism. TEPP-46 inhibits the generation of both T helper 17 (Th17) and Th1 cells, while inducing regulatory T cell development in vitro. Finally, in vivo treatment with TEPP-46 ameliorates experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. Overall, our results suggest that PKM2 induction and dimerisation are essential steps for T cell activation and pathogenicity, and targeting PKM2 dynamics may have therapeutic utility in T cell-mediated inflammation and autoimmunity.
XL-DNase-seq: Improved footprinting of dynamic transcription factors
Authors: Oh Kyu Seon , Ha Jisu , Baek Songjoon , Sung Myong-Hee
Published: January 2023

As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. Here we introduce an assay termed crosslink (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild crosslinking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various crosslinking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.
A New crucial role for complement receptor CD46 in chromatin packaging and direct gene regulation
Authors: Singh Parul , Kunz Natalia , Le Friec Gaelle , Kazemian Majid , Afzali Behdad , Lavender Paul , Kemper Claudia
Published: January 2023

Chromatin is the condensed form of DNA ‘packaged’ by positively charged histones in the nucleus. Post-translational modifications of histones regulate nucleosome dynamics, chromatin accessibility and gene expression. Signals mediated by autocrine activation of the human-specific complement regulator/receptor CD46 during T cell receptor stimulation are vital to Th1 induction in CD4+ T cells. CD46 is expressed in different isoforms that arise through alternative-splicing of a single gene - with two distinct cytoplasmic tails: CYT-1 or CYT-2. CYT-1 nuclear translocation is critical for the expression of genes that are involved in the metabolic adaptations underlying Th1 activity.

Because CD46-CYT-1 lacks direct DNA-binding domains, we hypothesized that CYT-1 interacts with transcription factor (TF) activator and/or repressor complexes to control gene expression. ChIP-seq analyses of human CD4+ T cells using anti-CYT-1 antibody revealed 327 genes as direct CD46-CYT-1 targets and five conserved TF-binding motifs allowing us to define CYT-1-interacting TFs in T cells. Interestingly, pathway analysis of CYT-1-regulated genes identified broad nucleosome regulation as top ‘CYT-1 activity’. Indeed, we confirmed that CYT-1 directly regulates the expression and post-translational modification of core histones including H3 and H4 at lysine (4, 8, 9, 12, 20 and 27) in a time-dependent manner. Such CD46-driven nucleosome regulation was aberrant in T cells from CD46-deficient patients, explaining now mechanistically their inability to mount Th1 responses.

These data define a novel role for CD46 in chromatin packaging and support our notion that intracellular/autocrine complement regulates basic cell-physiological processes.
Investigating the role of plasma membrane order and disorder in antigen-specific responses by CD4+ T lymphocytes
Authors: Bamezai Anil K , Karsalia Ritesh , Sengupta Soumini
Published: January 2023

Model membranes composed of unsaturated and saturated phospholipids segregate into the ordered (Io) and disordered (Id) phases. The addition of cholesterol to the model membranes promotes the formation of ordered phase. The existence of ordered and disordered phases in cellular membranes remains a topic of discussion. However, it is posited that cellular membranes show assemblies of small dynamic nanometer sized ordered phases, which are enriched in cholesterol and saturated phospholipids. Early “T cell activation” events are initiated in and around the lipid microenvironment of the plasma membrane that has co-existing Io and Id-like phases. We have examined the role of membrane order/disorder in antigen-specific CD4+ T cell activation and clonal expansion by first creating membrane disorder by inserting 7-ketocholesterol, an oxysterol, into the membrane, and then reconstituting membrane order of the disordered plasma membrane by inserting cholesterol into it. Membrane anisotropy in T cells was assessed by flow cytometry after labeling with Di-4-ANEPPDHQ, a fluorescent probe, and antigen-specific clonal expansion was examined by MTT assays. Significant revival of antigen-specific CD4+ T cell proliferative response was observed after reconstituting the disrupted membrane order with cholesterol. These findings demonstrate that cholesterol-dependent membrane order (Io) is critical for responses generated by CD4+ T cells and point to the importance of membrane order and the lipid microenvironment in signaling processes through the antigen receptor. We are currently examining the mechanisms underlying the inhibitory responses promoted by the disordered membrane lipid microenvironment.
Pathogenic role of CD4+ T cells in the progression of neurotropic mouse hepatitis virus induced neuroinflammation
Authors: Chakravarty Debanjana , Saadi Fareeha , Kundu Soumya , Bose Abhishek , Khan Reas , Dine Kimberly , Kenyon Lawrence C , Shindler Kenneth S , Sarma Jayasri Das
Published: January 2023

Neurotropic Mouse hepatitis virus (MHV) induced demyelination serves as a model to understand the mechanisms of demyelination and concomitant axonal loss in CNS, which mimics the pathological symptoms of the human neurological disease Multiple Sclerosis (MS). Inflammation triggered by MHV consists of mixed population of inflammatory cells, predominantly microglia/macrophages, which differs from autoimmune model of MS, where infiltrating T cells are the major contributors to the pathology. Though the acute phase of MHV inflammation is controlled by microglia/microglia, the role of T and B cells in viral induced axonal degeneration and demyelination is controversial. In order to explore the role of CD4+ T cells during transition from an early protective innate response to a highly regulated adaptive response following MHV infection, MHV-infected CD4−/− mice were compared with MHV-infected CD4+/+ (wildtype) mice at days 5/6, 10, 15 and 30 post infection. Viral titre estimation and nucleocapsid gene amplification confirm enhanced replication of the virions in the absence of functional CD4 + T cells in brain tissue. Viral anti-nucleocapsid antigen staining further shows the persistence of virus at day 30 p.i. in CD4−/− mice unlike CD4+/+ mice. Moreover, histopathological analysis of liver, brain and spinal cord showed an elevated susceptibility of CD4−/− mice to encephalitis and axonal degeneration with augmented progression of acute poliomyelitis, bulbar vacuolation, and dorsal root ganglionic inflammation rarely observed in CD4+/+ infected mice. In summary, current study suggests that CD4 T cell is critical for viral clearance, resolving microglial activation, and controlling chronic progressive axonal degeneration.
Macrophage subsets determine sex differences between male and female immune responses to Francisella tularensis
Authors: Sanchez Michelle A , Martinez Danielle , Spencer Charles
Published: January 2023

Francisella tularensisis the causative agent of the human disease tularemia. F. tularensisinfects a variety of cells, including macrophages, in order to cause pathogenicity. As few as 10 inhaled microorganisms cause a lethal infection by over-activating the host’s own inflammatory response. Differences in the intensity of the inflammatory response exist between the sexes which leads to differences in sensitivity to autoimmune and infectious disease. Males tend to be more susceptible to infectious diseases whereas females tend to be more susceptible to autoimmune diseases. However, our preliminary data unexpectedly demonstrated that female mice were more susceptible to F. tularensis-mediated disease than male mice. We hypothesize that the elevated inflammatory state of females may make them more susceptible to F. tularensis disease and that the M1 and M2 macrophage subsets may determine this differential inflammatory response. Naïve M1 and M2 macrophages will be purified from male and female mice and infected in vitro to directly compare cellular responses between male and female mice. In parallel, M0 macrophages will be differentiated in vitro prior to infection for similar comparison.
Immunomodulatory capability of VCG in Chlamydia immunity
Authors: Eko Francis , Pais Roshan , Richardson Shakyra , Omosun Yusuf Omosun , Black Carolyn M , Igietseme Joseph
Published: January 2023

Immunization with UV-inactivated Chlamydia trachomatis elementary bodies (UV-EB), the infectious form of Chlamydia, does not protect against infection with live Chlamydia. Vibrio cholerae ghosts (VCG), which are empty V. cholerae cell envelopes derived by genetic inactivation, constitute an effective delivery vehicle for cloned vaccine antigens and promotes the induction of protective immunity in the absence of added adjuvants. In this study, we sought to investigate the ability of VCG to enhance the innate and adaptive immune responsesinduced by UV-EB. Thus, bone marrow-derived dendritic cells (BMDC) were pulsed with UV-EB in the presence or absence of VCG and the induction of innate immune responses, T cell proliferation and ability to afford protection against Chlamydia following adoptive transfer into susceptible mice was evaluated. The results show that VCG efficiently modulated the immunostimulatory properties of DCs as indicated by increased secretion of proinflammatory cytokines and expression of toll-like receptors and co-stimulatory molecules associated with DC maturation. Also, UV-EB-pulsed DCs that were exposed to VCG and adoptively transferred into mice resulted in effective chlamydial antigen presentation and protective immunity. The results demonstrate that VCG activate the maturation of DCs leading to enhancement of innate and adaptive immunity to an otherwise non-protective antigen, suggesting their utility as adjuvants for enhancement of protective immunity to inactivated vaccine antigens.
Analysis of mGBPs in host defense against the obligate parasite Toxoplasma gondii
Authors: Steffens Nora , Legewie Larissa , Beuter-Gunia Cornelia , Hanisch Philipp , Weidtkamp-Peters Stefanie , Hänsch Sebastian , Degrandi Daniel , Pfeffer Klaus
Published: January 2023

Toxoplasma gondii (T. gondii) is an obligate, intracellular parasite. During invasion of target cells, T.gondii creates a parasitophorous vacuole (PV). Members of the mGBP (murine guanylate binding protein) family assemble at the cytoplasmatic side of the PV and interact with membrane compartments via yet uncharacterized mechanisms. This analysis aims at unravelling the molecular mechanisms by which mGBPs impair the vital functions of T.gondii. Therefore stable cell lines expressing fluorescent mGBPs were generated and infected with GFP- or mCherry-expressing T.gondii to analyze the dynamics of mGBPs via Confocal Live Cell Imaging. Additionally, super-resolution microscopy technologies were used to analyze the doubly transduced cell lines during infections in detail. Giant unilamellar vesicles were used to investigate the binding capacity of mGBP2 and mGBP7 to lipids and their modulation capacities on membrane integrity. Furthermore, cytosolic compartments of mGBPs and the T.gondii PV are in progress to be characterized by electron microscopy. The different mGBP7-domains will be analyzed to determine the responsible domain for membrane binding. The first results suggest that mGBP7 colocalizes with mGBP3 in vesicle like structures, but only partially with mGBP6 and not with mGBP2. However, mGBP2 influences mGBP7 recruitment to the PV. Additionally, mGBP7, mGBP3, mGBP6 and mGBP2 are able to accumulate at the plasma membrane of T.gondii inside the PV. With unique properties for membrane binding, mGBP2 and mGBP7 interact with specific lipids whereby modulating membrane integrity. Elucidation of the functions of mGBPs will help to counteract toxoplasmosis and to possibly find new treatment opportunities in the long term.
Differential effects of glycerophospholipids versus glycosphingolipids on dendritic cell subsets
Authors: Singh Ram Raj , Halder Ramesh
Published: January 2023

Self-glycerophospholipids, such as phosphatidic acid (PA), bind CD1d, and activate phospholipid-reactive T cells (PLT) (Eur J Immunol 2018). Glycosphingolipids, such as α-GalCer (αGC), activate iNKT cells and exhibit many immune effects, including dendritic cell (DC) maturation. Here, we investigated the effect of glycerophospholipids on DC. Immunization with PA led to a rapid and persistent increase in the proportions and total numbers of plasmacytoid DC (pDC, CD11c+PDCA1+B220+ cells). In contrast, pDCs were initially reduced after αGC injection, but the total numbers recovered later. PA also caused an increase, albeit delayed as compared to increase in pDC, in myeloid DC (mDC)/non-pDC (CD11c+PDCA1− cells). mDC were increased at later timepoints with αGC. Further, PA increased expression of MHCII, CD86 and CD40 on pDC, but only a minimal increase in these on mDC. In contrast, αGC caused a minimal increase in these on pDC, but increased CD80 and CD86 expression on mDC. Finally, only pDC, but not mDC, produced IFNα after αGC or PA. Intriguingly, αGC that caused a less profound pDC maturation than PA induced more IFNα by pDC than PA. αGC, but not PA, also increased TNFα production by pDC. Overall, the two lipid Ag elicited different, complex effects on DC subsets: PA led to rapid expansion and maturation of pDC but did not increase their production of pro-inflammatory cytokines. In contrast, αGC that had little effects on pDC maturation and expansion increased IFNα and TNFα production by pDC. mDC were increased with both PA and αGC, but their costimulatory molecules’ expression increased more with αGC than with PA. Such differential effects of different lipids on DC subsets may have important immune and pathogenetic implications.
CD4+ T cells regulate Mouse Hepatitis Virus induced activation of microglia
Authors: Saadi Fareeha , Chakravarty Debanjana , Sarma Jayasri Das
Published: January 2023

Multiple Sclerosis (MS) is a human neurological disease characterised by neuroinflammatory demyelination and axonal loss. Studies in MS are focussed to understand the process of demyelination driven by B/T lymphocytes, but we have limited knowledge about direct virus-induced neural cell dystrophy and the role of innate immunity in MS. Our studies employ Mouse Hepatitis Virus (MHV) induced neuroinflammatory model to understand microglia-mediated myelin stripping as a mechanism of demyelination. Microglia are the CNS resident macrophages that lie at the intersection of immune response and neurodegenerative process. They control the initiation, progression and resolution of neuroinflammation in the CNS. One of our studies in MHV model demonstrated that in the absence of CD4+ T cells, mice are more susceptible to acute encephalitis and chronic demyelination. In order to understand the mechanism of anti-viral immune response, we have investigated the role of microglia upon CD4+ T cell modulation. Results from our studies showed that a higher number of Iba1+ microglial cells persist in the brain parenchyma of CD4−/− mice at the chronic infection stage in comparison to the WT mice. Real-time qPCR screening of inflammatory markers like IL6, IL10, IL12, CXCL10, CCL5, CD86 and CD163 presented a consistent proinflammatory microenvironment in the CNS and comparative lower expression of Btk1 and Arg1 in the brains of CD4−/− mice. In conclusion, our findings propose that further in vitro and in vivo studies are necessary to understand the role of CD4+ T cell activation and maturation which might help in favouring a shift towards adaptive immunity, differential microglial activation, viral clearance and maintaining neural homeostasis.
STAT2 is a determinant of yellow fever virus host tropism
Authors: Morrison Juliet , Miorin Lisa , Laurent-Rolle Maudry , Pisanelli Giuseppe , Co Pierre , Albrecht Randy , García-Sastre Adolfo
Published: January 2023

The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015 serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable small animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to establish infection and subsequent dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFNα/β) for its engagement with human Signal Transducer and Activator of Transcription 2 (hSTAT2). We now report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFNα/β-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication in vivo. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.
The role of Blimp-1 in adipose Treg differentiation and effector function
Authors: Beppu Lisa , Frias Adolfo , Hyzny Eric , Poholek Amanda , D’Cruz Louise
Published: January 2023

Visceral adipose tissue regulatory T cells (VAT Tregs) protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory Th1 cells and M1 macrophages, and by preserving insulin sensitivity and glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function. B lymphocyte-induced maturation protein-1 (BLIMP-1) is a transcriptional regulator known to be involved in the development, polarization, and maintenance of various immune cells including CD4+ T cells. Using BLIMP-1 reporter mice, we have discovered that BLIMP-1 is constitutively expressed in a subset of VAT Tregs compared to lymphoid Tregs, and that BLIMP-1+ VAT Tregs are phenotypically distinct from their BLIMP-1-counterparts. Blimp-1 is not required for VAT Treg development, however Treg-specific deletion of BLIMP-1 led to unique changes in VAT Treg markers in lean versus obese adipose tissue. Based on these preliminary data, our project aims to elucidate the role of BLIMP-1 in shaping the VAT Treg compartment and to explore a possible role for BLIMP-1+ VAT Tregs in metabolic co-morbidities such as obesity and Type II diabetes.
Immunodominant and cryptic tumor neoantigen-specific immune responses activated by an armed oncolytic virus expressing a PD-L1 inhibitor
Authors: Wang Guan
Published: January 2023

Tumor neoantigens are exclusively expressed in malignant cells representing an ideal target for tumor immunotherapy. Oncolytic virus selectively infects and lyses tumor cells releasing a full-array tumor antigens and danger factors. Combined with PD1 checkpoint blockade, oncolytic virus triggered tumor neoantigen-specific T cell immune responses through local treatments. The present study developed a novel therapeutic regimen that combines ddVV, GM-CSF, and a PD-L1 inhibitor into a single therapeutic agent by engineering ddVV to co-express GM-CSF and a PD-L1 inhibitor. The novel therapeutic regimen has triple functions: ddVV-mediated oncolysis, GM-CSF-mediated enhancement of dendritic cell recruitment and function, and PD-L1 inhibitor-mediated interruption of PD-1/PD-L1 interaction. As a result, the double-armed oncolytic virus eradicated primary tumor and prevented tumor recurrence more efficiently after local administration. Analyses of underlying mechanisms revealed that the double-armed ddVV possessed a superior capability of improving tumor environment and triggering systemic T cell immune responses against not only dominant but also cryptic tumor neo-antigens in tumor mouse models.