bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic effect of IFN-γ on breast cancer targeted therapy
Authors: Nagai Yasuhiro , Tsuchiya Hiromichi , Ji Mei Q , Zhang Hongtao , Greene Mark I
Published: January 2023

ErbB2 is amplified in ~30% of breast cancer patients, and its amplification is associated with poor prognosis and worse survival outcome. We examined the synergistic effect of interferon-γ (IFN-γ) with anti-erbB2/neu mAb on erbB2-positive cancer model. IFN-γ showed marginal effects on its own, indicating that immune therapies mediated by this cytokine alone are unlikely to be beneficial. However, we discovered that treatment of the tumors with anti-erbB2/neu mAb concomitant with IFN-γ led to dramatic inhibition of in vivo tumor growth in the syngeneic tumor model and the preventional MMTV-neu transgenic mouse tumor model. We noted an increase of M1 macrophage accumulation in the tumor tissues as well as diminished myeloid derived suppressor cells, and increased CD8+ T cell cytotoxicity against tumor cells in the IFN-γ plus anti-erbB2/neu mAb treated mice. The tumor cells treated with both mAb and IFN-γ underwent changes in tumor stem cell marker ALDH-1, indicating a loss of stem cell-like properties, while mAb treatment alone did not accomplish this phenotypic change. We found that IFN-γ treatment significantly increase tumor PD-L1 expression, so we included anti-PD-1 or PD-L1 antibody with this combination therapy. Interestingly, anti-PD-L1 antibody further reduced tumor growth, while anti-PD-1 did not affect very much. These results indicate that the treatment of IFN-γ can improve targeted therapy, and further induce PD-L1 expression on tumor cells, creating a new target on the tumor cells for anti-PD-L1 therapy.
Identification of potential immune targets in controlling Endometrioid Endometrial Carcinoma metastatic progression.
Authors: Billaud Jean-Noel , Tugendreich Stuart , Toburen Debra
Published: January 2023

Endometrial adenocarcinoma is a common cause of gynecological cancer death in North America. The most dominant subtype, Endometrioid Endometrial Cancer (EEC) accounts for >80% of this cancer and is estrogen-dependent. At diagnosis, 75% of women have the disease confined to the uterus, which is considered Stage One. Five-year survival for Stage One patients is 80%, however, about 15–20% develop metastasis.

Total RNA extracted from tissues obtained after surgical resection from three women at stage one EEC was subjected to RNA-sequencing. The publicly available dataset (SRP045645) was downloaded directly from the Sequence Read Archive and the FASTQ files were processed with Biomedical Genomics Workbench for secondary analysis including mapping, quantification and differential expression analysis. Through streamlined integration the data was uploaded to Ingenuity Pathway Analysis (IPA) for biological interpretation.

We show how in silico solutions developed by QIAGEN Bioinformatics enable us to analyze the biological wparameters involved in EEC metastatic progression from an early stage. By comparing these patients’ RNA-seq results, we determined that key Canonical Pathways and other biological processes differentiated the three patients from one other. The predicted transcriptional program allowed us to visualize key upstream drivers, three of these drivers are GDF15, FGF3 and EDN1. They have been predicted to be potential master regulators of a Causal Network that increase metastasis, epithelial-to-mesenchymal-transition, and cellular invasion in one of the three patients. Based on these results we propose these three immune molecules as possible therapeutic targets to counteract the metastatic processes in EEC
Extensional stress modulates leukocyte adhesion probability
Authors: Benson Bryan , Ransohoff Richard Milton , Huang Alex Yee-Chen
Published: January 2023

Post capillary venules (PCVs) contribute a tiny fraction of the total vasculature, but are the predominant site of leukocyte adhesion and infiltration. Given the astronomical number of leukocytes and length of the vasculature, this restriction of adhesion to PCV requires tight and redundant regulation: adhesion molecule expression, chemokine presentation, and wall shear stress (WSS) from blood flow converge to promote this phenomenon in PCVs.

To mimic WSS, parallel plate fluidic channels have become the standard of in vitro models to study leukocyte adhesion. These models match the WSS of in vivo PCVs, but vary in their height (h), the smallest dimension that most strongly dictates fluid forces. Surprisingly, in our own experiments and in literature, leukocyte adhesion decreases as h decreases towards the 25 micron diameter of in vivo PCVs, whereas computational studies predict more adhesion with smaller h. This discrepancy suggests that a biological response within leukocytes opposes passive physical forces, or that other mediating factors are at play in vivo.

In the transition from capillary to PCV, leukocytes experience another fluid stress besides WSS: extensional stress, due to the cross-sectional area of the vessels increasing at bifurcations.

We created biomimetic models of the capillary to PCV transition in which we independently vary extensional stress and WSS and assay leukocyte adhesion to adsorbed CXCL12 and ICAM-1. Here, extensional stress has a powerful influence on human peripheral blood mononuclear cell (PBMC) and Jurkat adhesion probability, with higher extensional stress strongly promoting leukocyte adhesion. We are currently investigating the roles of mechanosensitive channels PIEZO1 and TRPV2 in this effect.
IL-36γ prevents immunopathology during influenza infection
Authors: Wein Alexander N , Dunbar Paul , McMaster Sean R , Kumar Nivedha , Hayward Sarah L , Denning Timothy L , Kohlmeier Jacob E
Published: January 2023

Influenza infection is a disease of high importance for public health, but the roles of individual cytokines in the pathogenesis of influenza infection remains to be elucidated. The IL-36 cytokine family is a relative of IL-1 and functions and signals in an analogous fashion. Despite their roles in many inflammatory conditions, the impact of IL-36 family cytokines on influenza pathogenesis and immunity remain largely undefined. To better understand the roles of IL-36 cytokines during influenza infection, we investigated IL-36 cytokine expression during low (x31) and high (PR8) pathogenesis influenza infection. We observed that IL-36γ is significantly upregulated during both low (x31) and high (PR8) pathogenesis influenza infection and has immune and non-immune cell sources. Unexpectedly, we observed that deletion of IL-36γ causes greater morbidity and mortality compared to WT mice, suggesting that the IL-36γ has a previously unknown protective function in addition to its pro-inflammatory roles. This increased morbidity correlates with a significant decrease in the number of alveolar macrophages in IL-36γ−/− mice during the early stages of influenza infection, and the rate of viral clearance varies between the WT and IL-36γ−/− mice based on the strain and dose of virus. We are currently performing RNA-Seq analysis to identify the protective immune mechanisms induced by IL-36γ signaling during highly pathogenic influenza infection. Overall, our results suggest a novel, protective role for IL-36γ during influenza infection and may suggest potential therapeutic approaches for ameliorating influenza pathogenesis.
The White adipose tissue represents a major hub for memory T cells endowed with potent effector function
Authors: Han Seong-Ji , Zaretsky Arielle Glatman , Collins Nicholas , Oliviera Vinicius , da Fonseca Denise Morais , Belkaid Yasmine
Published: January 2023

The white adipose tissue represents 15% of healthy mammalian hosts. In addition to serving as a scaffold for the lymphatic and blood vasculature, this compartment plays a fundamental role in the control of host metabolism. To which extent the adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Our studies show that under steady state conditions, the white adipose tissue is home to abundant and diverse effector and memory lymphocyte populations. Following infection, the adipose tissue accumulates large numbers of pathogen-specific memory T cells, including tissue-resident (TRM) cells characterized by a defined phenotypic profile and enhanced proliferative capacity. Thus, the white adipose tissue develops immune responses in parallel with, but distinct from, its connected tissue site. Local reactivation of adipose tissue TRM leads to rapid and broad responses with systemic impact on immune pathways. Together, these results uncover the adipose tissue as a reservoir of memory T cells endowed with long-term protective functions, positioning this compartment as an immunological shield between infected tissues and the periphery.
Candida albicans colonization exacerbates skin inflammation in a murine model of psoriasis
Authors: Nakajima Saeko , Harrison Oliver , Linehan Jonathan L , Belkaid Yasmine
Published: January 2023

In addition to being a primary site of exposure to pathogens, the skin is also home to a complex microbiota that modulate skin immune homeostasis. Notably, our previous work revealed that defined microbes differentially regulate the skin immune landscape. In clinical settings, shifts in skin microbiota composition have been shown in the context of skin inflammatory disorders, such as atopic dermatitis, acne vulgaris and psoriasis. However, the precise role of cutaneous microbiota in the initiation, control or promotion of skin inflammatory states remains unclear. To clarify the mechanisms by which the skin microbiota influence host immunity under inflammatory conditions, we focused on psoriasis, a common inflammatory skin disorder mediated by both innate and adaptive immune systems.

Murine studies have shown that topical application of imiquimod, a TLR7 agonist, can induce psoriasis-like skin inflammation, mediated by the IL-23/IL-17 axis. Using this experimental model, we found that previous skin association of mice with C.albicans but not other skin microbes (e.g., S.aureus), significantly enhanced skin inflammation. Notably, C.albicans topical association promoted IL-17A production from CD4+ effector T cells in the skin and skin draining lymph nodes. Our work currently explores how adaptive immunity directed against C.albicans could contribute to the etiology of this important skin disorder. This work aims to define the causative association between defined microbes and the development of skin inflammatory disorders and may allow for the development of tailored clinical interventions aiming at controlling skin microbiota and/or aberrant responses to these microbes.
Specific interaction between defined skin commensal and dermal IL-17A-producing gamma delta T cells.
Authors: Bouladoux Nicolas , Ridaura Vanessa , Claesen Jaen , Fischbach Michael , Belkaid Yasmine
Published: January 2023

The skin is home to a diverse and complex microbiota that play an important role in tissue homeostasis and local immunity. We have previously shown that Staphylococcus epidermidis promotes IL-17A-producing CD8+ T cells that enhance innate barrier immunity and limit pathogen invasion. However, the ability of other defined skin microbes and/or microbes-derived products to modulate skin immunity is still poorly characterized. T cells expressing low level of gamma delta TCR (gdT) are a dermal population of T lymphocytes that are committed to produce IL-17A and can mediate both innate and adaptive-like immune responses. Here, we show that cutaneous colonization with Corynebacterium accolens, a dominant member of the human and murine skin microbiota, induces IL-17A-producing gdT cells in the skin of specific-pathogen free mice. Intriguingly this ability is conserved amongst other species from the same genus supporting the idea that a canonical constituent shared by members of this taxon may be mediating this effect. Using a mutant strain of C. accolens lacking the corynemycolic acid synthase enzyme, we found that corynemycolic acids, a major component of the cell envelope, are important for the induction of IL-17A-producing gdT cells. Furthermore, mice associated with C. accolens displayed increased skin inflammation in an experimental model of psoriasis, a disease associated with an increase of IL-17A-producing gdT cells. Together these results uncover a novel mode of interaction between the skin microbiota and the skin immune system and highlight the important role that the skin microbiota may have in the pathogenesis of skin inflammatory disorders.
NF-κB-inducing kinase (NIK): A novel modulator of eosinophilic esophagitis (EoE) in mouse models and human patients
Authors: Eden Kristin , McDaniel Dylan , Heid Bettina , Allen Irving C
Published: January 2023

Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus driven by T cell and eosinophil responses to dietary allergens resulting in chronic mucosal inflammation and fibrosis. NF-κB inducing kinase (NIK) is a key signaling molecule of the noncanonical NF-κB pathway, an alternative signaling cascade producing chemokines involved in lymphoid stroma development and leukocyte trafficking. Nik−/− mice have been shown to develop a spontaneous hypereosinophilic syndrome in major organs; however, the gastrointestinal mucosa of these mice have not been evaluated. Here we show that Nik−/− mice develop significant eosinophilic esophagitis that mimics human EoE, including intraepithelial eosinophil accumulation, degranulation, mucosal thickening, fibrosis, and basal cell hyperplasia. Additionally, these mice display mucosal hyperplasia in the cranial glandular stomach surrounding the gastroesophageal junction. Interestingly, the remainder of the gastrointestinal tract including the caudal stomach, small intestine, and colon are unaffected, also similar to human EoE. Esophageal tissue from Nik−/− mice contains elevations in Th2 cytokines as well as TSLP, a gene implicated in human EoE. Retrospective metadata analysis of biopsies from human EoE patients shows many significant changes in gene expression throughout the noncanonical NF-κB pathway that are consistent with our murine model. Given that current models of this disease involve artificial sensitization or infectious agents, we conclude that Nik−/− mice may a useful spontaneous model of eosinophilic disease of the esophageal mucosa with improved physiologic relevance. These findings also highlight a novel role for noncanonical NF-κB signaling in EoE.
Gap Junction Mediated Antigen Transport in Epithelium Promotes Amplification of CD8 T Cell Mediated Bystander Pathology Following Chlamydial Infection
Authors: Murthy Ashlesh K , Manam Srikanth , La Salle Sophie , Holtzman Michael J , Zhang Yong , Ramsey Kyle H , Nicholson Bruce J
Published: January 2023

Chlamydia-specific CD8 T cells play a significant role in oviduct pathology, not bacterial clearance. We hypothesized that gap junction mediated antigen transport (GMAT) from infected to uninfected cells could explain the apparent targeting of uninfected epithelial cells by Chlamydia-specific CD8 T cells. Since gap junction protein connexin 43 is highly expressed in oviduct epithelium, we generated Foxj1Cre-Cx43flox mice with a conditional deficiency of CX43 only in ciliated columnar epithelia in oviducts. Foxj1Cre-Cx43flox mice displayed similar chlamydial infection, but reduced splenic Ag-specific CD8 T cell response and oviduct pathology when compared to WT mice, suggesting that CX43-mediates chlamydial pathogenesis. To evaluate the role of GMAT via CX43 in pathogenesis, HeLa S3 cells were engineered to express functional CX43 or a closed channel point-mutant CX43 (T154A). Chlamydial peptides were observed in neighboring uninfected cells at mid-developmental cycle upon infection of HeLa-CX43, not HeLa S3 or HeLa-CX43-T154A cells. Naïve mouse antigen-presenting cells (APC) optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with Chlamydia-infected HeLa-CX43, when compared to HeLa S3 or HeLa-CX43-T154A cells, demonstrating that GMAT is involved in activation of CD8 T cells. Furthermore, naïve mouse APC optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with a 1:4 mixture of Chlamydia-infected HeLa-CX43 and uninfected HeLa-CX43 cells, when compared to mixtures of infected HeLa-CX43 with uninfected HeLa-S3 or HeLa-CX43-T154A cells. These results demonstrate that GMAT via uninfected epithelial cells amplifies pathogenic Chlamydia-specific CD8 T cell response.
Notch signaling is required for the generation and function of follicular T helper cells in settings of Type-2 inflammation.
Authors: Aringa Mark Dell , Reinhardt R. Lee
Published: January 2023

The presence of IgE and IgG1 antibodies are hallmarks of allergic responses. The generation of high affinity, class-switched antibodies is reliant on interactions of B cells with T follicular helper (Tfh) cells in germinal centers. IL-4 produced by Tfh cells plays a critical role in B cell maturation and survival. The mechanisms controlling IL-4 production by Tfh cells remain largely unknown. Given that Notch signaling is required for the differentiation of Tfh cells and is known to influence cytokine production in T cells, we hypothesized that Notch signaling also plays an important role in regulating the function and maintenance of Tfh cells. Intestinal helminth infection was used to characterize whether persistent Notch signaling is required to maintain Tfh cell fate and function during allergic immunity. The use of intestinal helminths also allows us to assess the role of Notch signaling in lung Th2 cells. Deletion of Notch receptors on T cells of infected mice results in reduced IL-4 production by Tfh and Th2 cells. Despite similar reductions in IL-4 production, Th2 immunity remains intact while Tfh functionality is lost. Overexpression of Notch signaling in CD4+ T cells leads to increased IL-4 production by Tfh cells, but not Th2 cells. While it has been shown that Notch signaling is critical for Tfh differentiation, it is not known if Notch signaling plays a continued role after Tfh differentiation. Inhibition of Notch signaling after Tfh differentiation results in decreased expression of CXCR5 and aberrant localization of IL-4 producing T-cells in the lymph node. These findings suggest that Notch signaling is not only important for Tfh differentiation, but that it is also important for regulating Tfh cell fate, function, and maintenance.
A long-noncoding RNA, Gata3-AS1, is a positive transcriptional regulator of transcription factor Gata3 in TH2 cells.
Authors: Gibbons Hunter Ramsdell , Spurlock Charles F , Aune Thomas M
Published: January 2023

Long non-coding RNAs (lncRNAs) are RNAs transcribed by the genome, are >200 base pairs long, but are not translated into protein due to presence of multiple translational stop codons. LncRNAs possess a diverse array of regulatory functions including activation and silencing of gene transcription, regulation of splicing, and coordinating epigenetic modifications. Divergent lncRNAs are noncoding genes, which produce a lncRNA in the antisense orientation to an mRNA gene, and may share a promoter region. These lncRNAs may regulate their neighbor mRNA by direct RNA interactions or through the process of their own transcription. Gata3-AS1 is a divergent lncRNA gene in humans neighboring Gata3. The GATA3 transcription factor initiates transcription of genes encoding cytokines IL-4, IL-5, and IL-13, the hallmarks of TH2 polarization. Gata3-AS1 and Gata3 are exclusively expressed in the TH2 lineage of lymphocytes. In this study, we show that GATA3-AS1 is selectively expressed in human TH2 cells, resides solely in the nucleus, and is required for expression of GATA3, IL5, and IL13. Further, GATA3-AS1 RNA is required to establish H3K4-methyation and H3K27-acetylation ‘marks’ at the GATA3 genomic locus under TH2 differentiation conditions and directly binds the WDR5 DNA binding subunit of the MLL methyltransferase. Thus, GATA3-AS1 is a functional lncRNA and positive transcriptional regulator of GATA3 and commitment to the TH2 lineage.
Regulatory action of multiple AP-1 transcription factors balances Th17 lineage stability and plasticity
Authors: Wheaton Joshua D , Carr Tiffany M , Ciofani Maria
Published: January 2023

During an immune response, naïve CD4+ T cells encounter their cognate antigen and are induced to differentiate into one of several distinct T helper (Th) subsets. Although previously regarded as lineage-committed, these differentiated subsets are now appreciated to exhibit a degree of plasticity, whereby certain environmental stimuli permit inter-subset conversion. In particular, Th17 cells have been demonstrated to exhibit a high degree of intrinsic plasticity and are capable of “reprogramming” towards phenotypes typical of both pro- and anti-inflammatory T cell subsets. Despite the suggested importance of this phenomenon in the etiology of autoimmune and inflammatory disorders, the mechanisms underlying Th17 cell plasticity are poorly understood. We have recently identified the AP-1 transcription factor family as a central controller of both Th17 cell stability and plasticity, with different AP-1 family members exhibiting both overlapping and unique control of distinct aspects of the Th17 transcriptional program. Using a combined strategy employing transcriptomics and global analysis of TF occupancy for multiple AP-1 family members, we have identified novel regulatory modules defined by distinct modes of AP-1-mediated activation. These findings provide new insights into the mechanism by which environmentally-regulated transcription factors collaborate for the maintenance of T cell identity.
Unraveling how T cell antigen receptor signaling strength determines T cell differentiation using a combined experimental and computational approach
Authors: Faeder James , Sheehan Robert P , Hawse William F , Gable Matthew J , Lee Robin E C , Morel Penelope A
Published: January 2023

Antigen stimulation of the T cell receptor in a naïve cell triggers a complex network of signaling pathways that collectively determine whether it adopts a tolerogenic, Treg, or immunogenic, Th, phenotype. The complexity of this network presents a major barrier to the development of computational models, but such efforts hold the promise for increased understanding and the development of therapies based on selective T cell differentiation. We previously developed a Boolean model of T cell differentiation that predicted an optimal TCR signal duration for the production of Tregs, a strategy that we implemented and confirmed using an in vitro system. This model suggested that the dynamics of PTEN, a lipid phosphatase that opposes the activation the Akt/mTOR signaling pathway, were predictive of cell fate. Subsequent experiments revealed a critical positive feedback loop for Akt activation acting through the transcription factor FoxO1, a substrate of Akt that regulates PTEN. Here, we present a detailed computational model of Akt signaling during T cell differentiation that tracks the dynamics of numerous protein phosphoforms and complexes. The model is developed using the BioNetGen modeling language and simulated using ordinary differential equations. The calibrated model reproduces dose-dependent kinetics of phosphorylation of Akt at T308 and S473 measured experimentally, and predicts the existence of a sharp threshold in signal strength, which we have confirmed experimentally using Nur77-GFP reporter mice. The model also predicts a reduction in this threshold with reduction in PTEN expression, which we confirmed in mice heterozygous for PTEN deficiency.
Dancing with PKCθ: Fine-tuning T cell fate via cell-penetrating antibodies
Authors: Ozay E. Ilker , Tew Gregory N. , Minter Lisa M.
Published: January 2023

Previously, we showed that PKCθ signaling is neutralized in human T cells when anti-pPKCθ (against Thr538 phosphorylation) is delivered intracellularly, using a cell-penetrating peptide mimic (PTDM). Using a humanized model of graft-vs-host disease, we further demonstrated these effects were durable and provided a survival benefit when anti-pPKCθ-treated human PBMCs were used to induce disease. Of note, intracellular delivery of anti-pPKCθ altered PKCθ cellular localization compared to mock-treated cells. In the present study, we show that PKCθ localization is modulated in TH1, TH2, TH17, and iTregs following anti-pPKCθ delivery, and this modulation influences TH cell fate in vitro. It has been reported that PKCθ stimulates STAT3 transcription via the NF-κB pathway and has differential effects on STAT4 and STAT6. We found that, through its cellular localization, pPKCθ may differentially regulate the expression and translocation of STAT proteins, as well as master transcription factors, to ultimately control T cell differentiation programs. Altogether, we demonstrate that we can change the subcellular localization of PKCθ using a cell-penetrating antibody and as a result, can redirect the differentiation potential of T helper cells.
Sepsis inhibits pre-existing Ag-specific memory CD4 T cell-mediated immunity
Authors: Griffith Thomas S , Sjaastad Frances V , Murphy Katherine A , Kucaba Tamara A , Badovinac Vladimir P
Published: January 2023

Sepsis strikes 750,000 Americans annually. Patients who survive severe sepsis display prolonged immune dysfunction with deficits in innate and adaptive immune responses. Thus, the most common cause of death of septic patients is the not from the initial septic event, but from a subsequent secondary infection. CD4 T cells support the function of a variety of immune cells needed during a productive and protective immune response, and perturbations in the CD4 T cell compartment can dramatically affect overall immune system fitness. Sepsis patients have defects in the DTH response, marked by a failure to respond to skin testing with Ag to which previous exposure is known to have occurred. Using the cecal ligation and puncture (CLP) mouse model of sepsis, we replicated this observation in CLP-treated mice. DTH is predominately mediated by CD4 T cells, bringing into question to what extent memory CD4 T cells are affected by sepsis. We observed rapid apoptosis of persisting memory Ag-specific CD4 T cells after sepsis induction. In addition to the numerical change in Agspecific CD4 T cells, sepsis altered Ag-specific functions of memory CD4 T cells. Specifically, Ag-specific CD4 T cells from CLP-treated mice produced significantly less effector cytokines (IFNγ, TNF, and IL-2) on a per cell basis after restimulation and their proliferative capacity was significantly decreased compared to the same Ag-specific population in sham-treated mice. These numerical and functional deficiencies in Ag-specific CD4 T cells in CLP-treated mice corresponded to a loss in CD4 T cell-mediated protection. Together, our data show CLP-induced sepsis impairs adaptive cellular immunity (in part) by affecting the number and function of Ag-specific memory CD4 T cells.
Unique Properties of Long-lived Effector CD8 T cells
Authors: Renkema Kristin , Jameson Stephen , Hamilton Sara
Published: January 2023

Long-lived effector CD8 T cells (LLEC) are generated post infection and persist months after the infection has cleared. LLEC retain a “terminally differentiated” phenotype (CD62L-, CD27-, KLRG1+), which distinguishes them from other long-lived memory CD8 T cell populations. Upon pathogen re-challenge, these cells potently clear viral and bacterial pathogens more effectively than other memory CD8 T cell populations. Unlike other circulating effector memory cells, LLEC express many NK cell markers and are capable of produce IFNγ within hours after in vitro exposure to IL-12 and IL-18, suggesting that they uniquely possess innate-like, rapid effector mechanisms that aide in swift pathogen clearance and survival of the host. Interestingly, LLEC-phenotype CD8 T cells are prevalent in humans, and represent the predominant T cell population in “dirty” mice, that have naturally acquired environmental microbes. Our studies, focused on the establishment, maintenance, and functional properties of protective memory CD8 T cells, reveal LLEC as a distinct effector memory population that shares many characteristics with NK cells and suggest that vaccine approaches that expand the LLEC pool may be beneficial for protective CD8 T cell responses.
CD91-Dependent Release of IL-1β by GP96 Involves the Activation of the Inflammasome Complex
Authors: Wang Yifei , Binder Robert J
Published: January 2023

The immunogenic heat shock protein, gp96, binds to CD91 to elite an immune response, characterized by cross-presentation of chaperoned peptides and release of cytokines, including IL-1β. IL-1β is pro-inflammatory cytokine, which is secreted by immune cells upon sensing pathogen associated molecular pattern (PAMP) or damage associated molecular pattern (DAMP). IL-1β is synthesized as a precursor protein, and requires cleavage by Caspase-1 to the matured IL-1β which is released from the cells. Caspase-1 activation requires the inflammasome protein complex. For Inflammasome activation, signal 1 induces pro-inflammatory gene transcription, and signal 2 activates Inflammasome. We have previously shown that gp96 induces NFκB activation and IL-1β secretion via CD91.1 Here we examined the utilization of the inflammasome for IL-1β in this setting. We report that gp96 is able to work as signal 1 to prime production of pro-IL-1β in APCs and in a dose-dependent manner. The kinetics of priming pro-IL-1β differ between gp96 and other PAMPs such as LPS. We will explore the dependence of IL-1β release on the inflammasome.
Neonatal CD8+ T cells provide innate immune protection against unrelated pathogens
Authors: Watson Neva B , Peng Seth , Nzingha Kito , Wang Jocelyn , Grenier Jennifer K , Smith Norah L , Patel Ravi K , Grimson Andrew W , Rudd Brian D
Published: January 2023

We previously found that neonatal and adult CD8+ T cells adopt different fates after infection with specific pathogens because they are derived from a distinct progenitor cell that is distinguished by expression of Lin28b. In this report, we investigated if CD8+ T cells respond differently to unrelated pathogens in early life. To start, we compared gene expression profiles of neonatal and adult CD8+ T cells after stimulation with innate cytokines (IL-12 + IL-18). Interestingly, we found that neonatal CD8+ T cells are more responsive and express a distinct cytokine profile compared to their adult counterparts. To examine the biological significance of these age-related differences, we next adoptively transferred monoclonal populations of neonatal or adult CD8+ T cells into T cell-deficient recipients and compared their ability to mediate immune protection against an irrelevant strain of Listeria monocytogenes. Surprisingly, we found that neonatal donor cell displayed increased proliferation and effector functions, and the pathogen burden was significantly reduced when compared to recipients containing adult donor cells. We also found that Lin28b may be responsible for the enhanced responsiveness to innate cytokines in early life, as ectopic expression of Lin28b in adult cells enabled them to initiate a program of bystander activation that was analogous to neonates. Collectively, these findings indicate that neonatal CD8+ T cells possess innate-like functions during infections at the cost of generating memory, and these unique behaviors may be driven by high levels of Lin28b. We currently are employing ATAC-seq to determine whether Lin28b changes the program of bystander activation by altering the chromatin landscape.
TGF-β Controls the Formation of Kidney-Resident T Cells via Promoting Effector T Cell Extravasation
Authors: Mishra Shruti , Ma Chaoyu , Demel Erika L , Liu Yong , Zhang Nu
Published: January 2023

Tissue-resident memory T (TRM) cells, a population of noncirculating memory T cells, are one of the essential components of immunological memory in both mouse and human. Although CD69+CD103+ TRM cells represent a major TRM cell population in barrier tissues including the mucosal surface and the skin, CD69+CD103− TRM cells dominate most nonbarrier tissues, such as the kidney. TGF-β is required for the differentiation of CD69+CD103+ TRM cells in barrier tissues. However, the developmental control of CD69+CD103− TRM cells in nonbarrier tissues remains largely unknown and the involvement of TGF-β signaling is less clear. In this study we demonstrated that TGF-β promoted the formation of kidney-resident T cells via enhancing the tissue entry of effector T cells. Mechanistically, TGF-β enhanced E- and P-selectin and inflammatory chemokine-mediated extravasation of effector T cells. Thus TGF-β controls the first developmental checkpoint of TRM cell differentiation in nonbarrier tissues.
Diet-induced Obesity Impairs Murine B cell Development and Antibody Production Accompanied by a Defect in Production of SPMs
Authors: Crouch Miranda J , Guesdon William , Armstrong Michael , Reisdorph Nichole , Shaikh Saame Raza
Published: January 2023

Diet-induced obesity is associated with increased risk for infections and poor responses to vaccinations, which may be due to compromised B cell function. There is limited information about the effects of obesity on B cell function outside of the context of adipose tissue inflammation. Therefore, we studied B cell development and ex vivo B cell antibody production in a murine model of diet-induced obesity. Analysis of the bone marrow revealed that obese mice displayed a two-fold reduction in the number of CD19+ cells, resulting in decreased frequencies of various B cell subsets. Early lymphoid commitment markers such as IL7Rα, IL7R, and STAT5 were significantly decreased at the transcript level. In addition, obese mice had reduced mRNA expression of the B cell lymphopoiesis markers, PAX5 and Oct2, compared to controls. Functionally, splenic B cells from obese mice were hyperstimulated with elevated ex vivo IgM and IgG secretion in the absence of stimulation. When B cells from the obese mice were challenged with anti-TLR4 or anti-BCR/TLR9 in culture, IgM and IgG levels were diminished. Mechanistically, the levels of D-series specialized pro-resolving lipid mediators (SPMs), which are potent regulators of inflammation and adaptive immunity, were lowered in obese mice compared to controls. Furthermore, select enzymes involved in generation of SPMs were also decreased in obese mice. Overall, these findings demonstrate that obesity hinders B cell development and drives a dysregulated antibody response, which could contribute to impaired responses to infections and vaccinations.
The Toll-like receptor ligand CpG-A induces type 1 interferons in B cells contrasting the proinflammatory inducing activity of CpG-B
Authors: Akkaya Munir , Akkaya Billur , Sheehan Patrick , Miozzo Pietro , Rawat Mukul , Pena Mirna , Kim Ann S , Kamenyeva Olena , Kabat Juraj , Qi Chen-Feng , Bolland Silvia , Chaturvedi Akanksha , Pierce Susan K
Published: January 2023

B cells express the innate immune system receptor, Toll like receptor 9 (TLR9), a potent regulator of B cell function, that signals in response to unmethylated CpG sequences in microbial DNA. A dichotomy in the function of the two major classes of CpG-containing oligonucleotides, namely CpG-A and CpG-B, appears to exist with CpG-A restricted to inducing type 1 interferon (type 1 IFN) in innate immune cells and CpG-B to activating B cells to proliferate and produce antibody and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost the innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. We provide evidence that CpG-A activated B cells in vitro in a TLR9 dependent fashion resulting in the production of IL-6, proliferation, and alterations in the expression of a variety of B cell surface markers, albeit in a less robust fashion as compared to CpG-B. Neither CpGB nor CpG-A alone induced B cells to express type 1 IFN. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B induced a robust type 1 IFN response in B cells both in vitro and in vivo. Conversely, CpG-B-DOTAP but not CpGA-DOTAP promoted B cell germinal center responses in vivo and the production of high affinity antibodies. We also provide evidence that difference in the function of CpGs versus DOTAP-associated-CpGs may be related to their intracellular trafficking in B cells since DOTAP enhances the trafficking of CpG-B but not CpG-A to late endosomal compartments. These findings have important implications for the use of CpG-A and CpG-B to augment type 1 IFN production versus B cell responses in vivo.
Exosomes secreted from virus- infected microglia can activate an inflammatory response in the central nervous system
Authors: Olson Julie K , Luong Nhungoc
Published: January 2023

Exosomes are membrane-bound nanovesicles released from cells that can be taken up by other cells, thus mediating intercellular communication. Exosomes contain miRNA, mRNA, and proteins that are specific to the cells from which they were released. Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating disease is a mouse model for multiple sclerosis. TMEV infection of susceptible mice leads to a persistent viral infection of microglia in the central nervous system (CNS) which contributes to the development of a chronic progressive demyelinating disease associated with an inflammatory immune response. We have previously shown that TMEV infection of microglia activates an innate immune response with expression of type I interferons, cytokines, chemokines, and effector molecules associated with development and progression of demyelinating disease. Next, we wanted to determine whether microglia infected with TMEV secrete exosomes and whether these exosomes could influence uninfected CNS resident cells, such as microglia, astrocytes, and neurons. Our studies show that exosomes secreted from virus-infected microglia contain viral RNA that can activate an innate immune response in uninfected bystander cells which leads to increased expression of pro-inflammatory cytokines. Most interestingly, exosomes secreted from microglia in the brain of TMEV- infected mice could be transferred to uninfected mice and activate an inflammatory immune response in the brain of uninfected mice. These results suggest that exosomes secreted by virus-infected microglia can activate the immune response in bystander uninfected cells which may contribute to the inflammatory response associated with demyelinating disease.
The role of coinhibitory pathways in allergen-specific CD4+ T cell responses
Authors: Rosskopf Sandra , Jahn-Schmid Beatrice , Bohle Barbara , Battin Claire , Schmetterer Klaus , Pickl Winfried F , Steinberger Peter
Published: January 2023

More than 25% of the population suffers from IgE-mediated immune reactions and its related symptoms. T lymphocytes have a crucial role in initiating and promoting allergies and their responses are tightly regulated by numerous activating and inhibitory signals provided by APCs. Currently, there is limited knowledge regarding the role of inhibitory pathways in allergen-specific CD4+ T cell responses. To address this issue, patients allergic against house dust mites, birch pollen, mugwort pollen and grass pollen were recruited. PBMCs were isolated and then stimulated with allergenic extracts to evaluate coinhibitory pathways using blocking antibodies (e.g. nivolumab, ipilimumab) and to assess the expression of coinhibitory receptors on allergen-specific T cells. Allergenic extracts were analyzed for presence of TLR triggering molecules using NF-κB-eGFP reporter cells expressing TLR1/2, TLR2/6, TLR4 or TLR5. Furthermore, allergen-specific T cell reporters, T cell lines or T cell clones from allergic patients were cocultivated with engineered APCs that present allergenic peptides on MHC class II molecules and express different coinhibitory molecules. Blockade of the PD-1/PD-L1 axis strongly enhanced proliferation of allergen-specific CD4+ T cells in response to allergenic extracts. No correlation was found between IgE concentration and T cell proliferation. Preliminary results show a distinct expression pattern of coinhibitory molecules on allergen-specific T cells. In a next step Th1/Th2 cytokine patterns will be analyzed. Research was performed in accordance with the Declaration of Helsinki and approval of the local ethics committee was obtained (EK1538/2014).
House dust mite allergen induces inflammasome activation and NFkB signal transduction in monocytes
Authors: Yu Sheng-Jie , Yen Hung-Rong
Published: January 2023

Over the past few decades, allergic diseases have become increasingly prevalent worldwide. House dust mite (HDM) is the most important domestic source for allergic diseases such as allergic rhinitis, asthma and atopic dermatitis. Dermatophagoides pteronyssinus (Der p) is the major environmental allergen in southeast Asia because of the humid and warm environment is suitable for its growth. Der p has been identified which could increase proinflammatory cytokine expression through activating innate immune response. Inflammasomes activation was reported which could help host defense pathogens and allergen in the recent years. However, the correlation between HDM induced allergic inflammation and inflammasomes activation remains unclear. The aim of this study was to investigate the effect of HDM allergen on inflammasomes activation and downstream signal transduction. We found that THP-1 cells, stimulated with HDM crude extract, activated NALP1, NLRC4, NLRP3 and AIM2 inflammasomes in a time-dependent manner. The expression of adaptor protein ASC and its caspase effector, Caspase-1, as well as proinflammatory cytokines, IL-1β, IL-6, IL-8 and TNF-α were significantly upregulated after HDM stimulation. Mechanistic study revealed that HDM stimulation increased the expression of phosphorylated NFkB, JNK and AKT. Our study suggests that HDM allergen could induce inflammatory response through NFkB signal transduction pathway and inflammasomes activation. Thus, we could use these data for developing specific target immunotherapy in the future.
A cross species CD200R1 immune checkpoint agonist with potent anti-inflammatory properties
Authors: Gariepy Jean , Khatri Ismat , Gorczynski Reginald , Young Ashley , Woo Lindsay , Chow Chung-Wai , Cydzik Marzena , Prodeus Aaron
Published: January 2023

Functional aptamers displaying agonistic or antagonistic properties are showing great promise in terms of modulating immune responses. Our group has recently developed pegylated DNA aptamers that can either block inflammatory responses (CD200R1agonist) or restore tumor-directed immune responses (PD-1 antagonist) in vivo. Here, we report the derivation and design of a cross species mouse/human CD200R1 DNA aptamer agonist that blocks inflammatory responses in mouse models of skin graft rejection and asthma. This DNA aptamer was discovered by performing NGS and comparing the resulting aptamer motifs derived from independently screening mouse and human CD200R1 as targets. Importantly, this m/hCD200R1 agonistic aptamer does not suppress cytotoxic T-lymphocyte (CTL) induction in 5 day allo-mixed lymphocyte cultures (MLCs) derived from CD200R1 knockout mice, indicating that its mode of action is directly linked to CD200R1 activation. This study suggests that one can derive agonistic DNA aptamers that can be verified as immunomodulators in mouse models with outcomes translatable to the treatment of human conditions.
Class switch recombination in the absence of the IgH 3′ regulatory region
Authors: Yu Kefei , Kim Ahrom , Han Li
Published: January 2023

The ~28 kb 3′-regulatory region (3′RR) which is located at the most distal 3′ region of the immunoglobulin (Ig) heavy (H) chain locus has multiple regulatory functions that control IgH expression, class switch recombination (CSR) and somatic hypermutation. Here, we report that deletion of the entire 3′RR in a mouse B cell line that is capable of robust cytokine-dependent CSR to IgA results in reduced but not abolished CSR. These data suggest that the 3′RR is not absolutely required for CSR, and thus is not essential for targeting activation-induced cytidine deaminase to switch regions as has been suggested. Moreover, replacing the 3′RR with a DNA fragment including only its four DNase I hypersensitive sites (lacking the large spacer regions), restores CSR to a level equivalent to or even higher than wild type cells, suggesting that the four HS sites contain most of the CSR-promoting functions of the 3′RR. Stimulated cells express abundant germline transcripts, with the presence or absence of the 3′RR, providing evidence that the 3′RR has a separate role in promoting CSR, that is unique from enhancing S region transcription.
Visualization of binding of RAG2 to H3K4me3 regulated by its noncore domain.
Authors: Rodgers William A , Byrum Jennifer , Rodgers Karla K
Published: January 2023

Association of RAG2 with the epigenetic marker H3K4me3 through a PHD domain in its noncore region is important for activating the RAG1 component of the RAG recombinase complex for V(D)J recombination. However, immunofluorescence staining of RAG2 expressed in thymocytes suggests RAG2 is localized principally to the nuclear periphery, away from sites of H3K4 modification. To further test for interactions between RAG2 and separate epigenetic markers, we used confocal microscopy to measure full length RAG2 labeled with GFP at its N-terminus (GFP-FL) and expressed in RAG2−/−pro-B cells. Cross-correlation analysis of cells immunostained with antibodies to H3K4me3 and H3K9me3 showed that RAG2 colocalization with H3K4me3 was significantly greater than with H3K9me3. Furthermore, blocking histone demethylation by treating cells with 2,4-pyridinedicarboxylic acid increased colocalization of GFP-FL with H3K4me3, but not its colocalization with H3K9me3. Interestingly, a GFP-labeled T490A mutant of full length RAG2 (GFP-T490A) showed significantly greater colocalization with H3K4me3 than the labeled wild-type RAG2. GFP-labeled RAG2 core alone (GFP-core) exhibited the poorest colocalization with H3K4me3 of the constructs that we measured. Finally, nanoscale resolution using structured illumination microscopy showed that RAG2 was unevenly distributed in the nucleus, containing dispersed RAG2-rich and RAG2-poor regions. Furthermore, RAG2 interactions with H3K4me3 were restricted to interfaces between H3K4me3-enriched puncta and the RAG2. Altogether, these data provide visualization of RAG2 binding to the epigenetic marker H3K4me3, and that Thr490 of RAG2 regulates this interaction.
The microRNA processing subunit DGCR8 is required for immune response and germinal center formation
Authors: Daum Patrick , Meinzinger Julia , Pracht Katharina , Côrte-Real Joana , Wittmann Jürgen , Jäck Hans-Martin
Published: January 2023

MicroRNAs (miRNA) are a family of single stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. The microprocessor complex subunit DGCR8, which is pivotal for the canonical processing of precursor transcripts into mature miRNAs, has been shown to be indispensable for the pro-B to pre-B cell transition in early B cell development (Brandl et al., 2016). To understand the role of DGCR8 and miRNA processing during the antigen-dependent activation of mature B cells, we deleted DGCR8 during the development of immature IgM-positive B cells into mature splenic B cells by crossing a transgenic CD23-Cre mouse with a mouse homozygous for floxed DGCR8 alleles.

Analysis of LPS-activated DGCR8-deficient splenic B cells from these mice revealed a reduced abundance of mature miRNAs, impaired in vitro survival, and reduced class switch to IgG1. Furthermore, frequencies of follicular B cells in the spleen, as well as B2 B cells in the peritoneal cavity were significantly altered. Finally, mice with a B cell-specific DGCR8-deficiency failed to produce antigen-specific serum IgG antibodies and adequate numbers of GL7-positive germinal center B cells upon immunization with a thymus-dependent antigen.

In summary DGCR8, and thus, most likely miRNAs are required to mount effective and specific T cell-mediated immune responses.