bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms for secretion of antibodies with “anti-self” reactivity by the human obese adipose tissue
Authors: Frasca Daniela , Diaz Alain , Romero Maria , Blomberg Bonnie
Published: January 2023

We have shown that the human obese subcutaneous adipose tissue(SAT)contributes to systemic and B cell intrinsic inflammation and increased secretion of autoimmune antibodies. Immune cells are recruited to the SAT by chemokines released by both adipocytes and infiltrating immune cells. We have identified several mechanisms responsible for the release of “self” antigens in the human obese SAT, induction of class switch and production of autoimmune antibodies, such as hypoxia, NK cell cytotoxicity and DNA damage, which induce cell death and further release of pro-inflammatory cytokines. All these processes, in the context of an inflammatory environment like the obese SAT, stimulate the production of autoimmune IgG antibodies which have been described to be pathogenic. We have identified “self” antigens that stimulate the secretion of autoimmune antibodies in cultures of SAT-derived immune cells, using a Protein G immunoprecipitation protocol to purify the autoantibodies, combined with mass spectrometry. Results show that around 25% of these “self” antigens are found at high frequency in all the individuals evaluated so far in independent experiments. Moreover, >90% of these “self” antigens (lipid and protein antigens, DNA, RNA) are cytoplasmic, nuclear or membrane-associated. These results allow the identification of novel pathways to manipulate in order to decrease pathogenic immune responses during aging.
The unfolded protein response contributes to antiproliferative and proapoptotic effects of IFN-λ4 in human hepatic cells
Authors: Onabajo Olusegun O , Wang Fang , Florez-Vargas Oscar , Obajemu Adeola , Vargas Joselin , Prokunina-Olsson Ludmila
Published: January 2023

IFN-λ4 is a type-III interferon with strong clinical significance in humans. Only individuals who carry the ΔG allele of a genetic variant rs368234815-TT/ΔG are genetically able to produce IFN-λ4 protein. Carriers of the ΔG allele have impaired ability to clear hepatitis C virus (HCV) infection, however the mechanisms are not fully understood. Here, we used RNA-seq to evaluate the effects of IFN-λ4 and IFN-λ3 expression in inducible hepatic cell lines (HepG2) with and without the receptor (IFNLR) required for signaling of both interferons. Pathway analysis showed that despite being poorly secreted, IFN-λ4 expression induced a stronger ISG response compared to IFN-λ3. IFN-λ4 expression was also associated with reduced proliferation and increased expression of genes involved in the unfolded protein response (UPR). Subcellular localization analysis revealed that IFN-λ4 was accumulated in the Golgi and lysosomes, suggesting it is misfolded and targeted for elimination. Furthermore, intrinsic but not extrinsic expression of IFN-λ4 inhibited cell proliferation via cell cycle arrest leading to apoptosis; both indicative of UPR outcomes. In conclusion, our results show that in addition to inducing ISGs, IFN-λ4 expression induces UPR, contributing to inhibition of cell proliferation. This could have important consequences for conditions such as HCV and fibrosis, where IFN-λ4 expression has been implicated.
Plasmacytoid dendritic cells contribute to pro-inflammatory and pro-fibrotic milieu in lung fibrosis
Authors: Singh Ram Raj , Valera Isela
Published: January 2023

Fibrosis, the end-result of tissue injury in a wide range of diseases, contributes to >40% of mortality in the US. Plasmacytoid dendritic cells (pDC) are reduced in the peripheral blood, but increase in the lungs and skin of patients with systemic sclerosis, a prototypic fibrotic disease (JCI Insight 2018). The frequency of pDCs in the lungs of patients correlated with the severity of lung disease and with levels of proteins implicated in inflammation and fibrosis. Importantly, depletion of pDCs ameliorated lung and skin fibrosis in the bleomycin-induced animal model. pDC depletion also altered the expression levels of proteins and genes implicated in chemotaxis, inflammation, and fibrosis in the lungs. To test if pDCs directly contribute to inflammatory/profibrotic milieu in fibrosis, we injected C57Bl/6 mice with bleomycin, harvested lungs, and analyzed lungs for proteins by Western blot, ELISA, and flow cytometry. CD36 that is implicated in platelet-collagen adhesion, oxidative stress and inflammation was increased on pDCs but not on mDCs, other myeloid cells, B cells and T cells in the lungs of bleomycin-injected mice as compared to control mice. TLR7 was also increased more on pDCs than on all other immune cells examined, whereas TLR2 was increased more on mDCs and other myeloid cells but not on pDCs and B cells. TGFβ-latent peptide was higher on all immune cells examined in the lungs of bleomycin-injected animals than in controls. Among chemokine receptors, CCR2, CCR3, CCR6, CCR9 and CXCR4 were higher on pDCs than other cells. In summary, pDCs elicit profibrotic milieu in the development of systemic fibrosis. These observations identify the increased trafficking of pDCs’ to the affected organs as a therapeutic target in fibrosis.
TMEM16F regulates bystander TCR-CD3 membrane binding at the immunological synapse
Authors: Connolly Audrey , Panès Rébecca , Bellemare-Pelletier Angélique , Gagnon Etienne
Published: January 2023

The signaling cascade induced by T cell receptor (TCR) triggering has been extensively studied, but the steps separating peptide recognition from TCR phosphorylation remain elusive. In resting T cells, the TCR CD3ɛ and ζ chains dynamically associate with the plasma membrane (PM) through electrostatic interactions with anionic lipids such as phosphatidylserine (PS). Upon ligand recognition, the CD3 chains dissociate from the PM, enabling robust signaling initiation. However, the mechanisms regulating PM dissociation remain obscure. Recent studies have highlighted the role of TMEM16F, a PS-specific scramblase, in regulating TCR signaling, but the molecular steps involved in this activity have not been identified.

To study the role of TMEM16F in regulating TCR activation, we knocked-down its expression using shRNA targeting. Our results show that TMEM16F knockdown inhibited PS redistribution, which significantly reduced TCR activation. To showcase TMEM16F activity in relation to TCR signaling, we transduced T cells with a mutant form of the scramblase that constitutively redistributes PS. TMEM16F-mutant T cells showed a significant increase in proximal TCR signaling. This was directly linked with an increase in PS redistribution upon TCR engagement, which enabled the dissociation of bystander CD3ɛ chains and their participation in signaling.

Our results show that PS redistribution by way of TMEM16F activity is critical for TCR signaling. Our results also establish our ability to modulate PS redistribution by targeting TMEM16F activity in order to regulate T cell activation. These findings may ultimately lead to novel approaches enabling the modulation of TCR and other immune receptor activity in T cell-based immunotherapies.
Fibronectin Costimulation of CD40L Expression Integrates Innate and Adaptive Inflammatory Signals
Authors: Ragheb Jack , Zhang Yuxia , Bushar Nicholas , Oh Hyoungbin , Gertz E. Michael , Patnaik Anuja , Chakrabarty Sagarika , Kampen Robert , Kirk Allan D , Roberts David , Schäffer Alejandro
Published: January 2023

CD40 ligand (CD40L) is a well-known regulator of the adaptive immune response that induces and modulates the activation and differentiation of monocytes, dendritic cells, and lymphocytes. Less appreciated is its critical role in promoting the inflammatory response in CD40-expressing cells such as vascular endothelium. Expression of CD40L on TCR activated human CD4+ T cells is biphasic and upregulated by monocyte cell contact early and IL-2R signaling late. Here we demonstrate the existence of additional, distinct pathways that differentially regulate the expression of CD40L. We describe a novel link between vascular endothelial cell adhesion molecule 1 (VCAM-1), extracellular matrix (ECM) proteins, and CD40L expression on human CD4+T cells that constitutes an innate form of T cell costimulation modulated by the type and quantity of ECM protein present, with thrombospondin-1 inhibiting fibronectin-mediated costimulation of CD40L expression. Identification of this previously unrecognized β1 integrin pathway, which promotes CD40L expression on CD4+ T cells independently of monocytes and IL-2, provides new insights into our understanding of the potential role of CD40L in the establishment and progression of inflammatory disease.
NF-kB/mTOR/MYC axis drives PRMT5 protein induction after T cell activation
Authors: Sengupta Shouvonik , Webb Lindsay , Narvaez-Miranda Janiret , Amici Stephanie , Nagy Gregory , Guerau-De-Arellano Mireia
Published: January 2023

Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous system (CNS) mediated by CD4+ T cells. Clinical data has provided evidence that MS patient T cells display an activated or memory phenotype and genome-wide association studies (GWAS) have identified single nucleotide polymorphisms linked to NF-kB complex and MYC genes in MS patients. These data imply a role for TcR (T cell receptor) signaling pathways in MS. PRMT5 is the major type II arginine methyltransferase catalyzing the symmetric dimethylation of histones and other proteins. Our lab has recently reported PRMT5 plays a crucial role in inflammatory T cell expansion and EAE disease. From cancer studies, we are aware of links between NF-kB/MYC signaling and PRMT5 induction. Here, we unravel the impact of NF-kB/MYC pathways in T cell PRMT5 expression and pathogenic T cell responses. We used naïve and memory mouse Th1/Th2 CD4+T cells as models to identify mechanisms controlling PRMT5 protein expression in initial and recall T cell activation. Naïve (initial) mouse T cell activation resulted in NF-kB-dependent transient Prmt5 transcription and NF-kB, mTOR and Myc-dependent PRMT5 protein induction. Recall activation in memory T cells showed rapid induction of PRMT5 protein expression, supported by both transcription and loss of Prmt5-targeting miRNAs. These results highlight the importance of the NF-kB/mTOR/MYC axis in PRMT5-driven pathogenic T cell expansion and may guide targeted therapeutic strategies for MS.
Deciphering liver environmental signaling pathways for Kupffer cell identity
Authors: Sakai Mashito , Troutman Ty Dale , Seidman Jason S. , Ouyang Zhengyu , Spann Nathanael J. , Abe Yohei , Ego Kaori , Bruni Cassi M. , Schlachetzki Johannes C. M. , Nott Alexi , Bennett Hunter , Chang Jonathan , Vu BaoChau T. , Pasillas Martina , Link Verena M. , Texari Lorane , Heinz Sven , Glass Christopher K.
Published: January 2023

Functional specialization of tissue resident macrophages occurs through environmental signals controlling activity and/or expression of transcription factors. Kupffer cells are resident macrophages in the hepatic sinusoids and have critical roles in the innate immune response and iron metabolism. Here, we characterize transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. Nr1h3 encoding LXRα is rapidly and highly induced in repopulating liver macrophages, suggesting its induction plays a crucial role in Kupffer cell differentiation. Restricted deletion of Nr1h3 in Kupffer cells reveal that it is required for shaping the Kupffer cell-specific enhancer landscape. Further, we obtain evidence that combinatorial interactions of DLL4 and TGF-β/BMP produced by sinusoidal endothelial cells and endogenous LXR ligands are required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of RBPJ through Notch signaling plays a key role in activating poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogram the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Using molecules which mimic these liver environment signals, we show that it is possible to induce Kupffer cell-specific genes in mouse bone marrow progenitor cells and human monocytes in vitro. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.
Loss of miR-146a skews the dendritic cell milieu in favor of CD8a+CD103+ cross-presenting migratory dendritic cells and reduces production of IL-27
Authors: Flynn Patrick A , Lind Evan F
Published: January 2023

miR-146a is a micro-RNA that acts on transcripts upregulated in response to LPS induced TLR4 signaling. Activation of TLR4 results in maturation and survival of dendritic cells (DCs) to activate T cells in the spleen. Interestingly, little is known how miR-146a regulates DC phenotype/function and whether it has direct effects on the regulation of effector T cell function. To probe this question, we utilized a miR-146a knockout mouse model to study both DCs and lymphocytes. Splenocytes were analyzed by multi-parameter flow cytometry and we found that miR-146a mice had a greater population of cross-presenting CD8a+CD103+ DCs compared to wild-type mice. T cells from miR-146a mice had increased CD44 and CD69 expression, and decreased CD62L expression. Bone marrow derived DCs (BMDCs) were sorted (via multi-parameter flow cytometry) and subjected to RNA isolation and transcript analysis. BMDCs lacking miR-146a had 2.5-fold higher IL1R2 and 1.3-fold higher STAT1 transcript levels in addition to 13 increased genes. Supernatants from control and miR-146a knockout BMDCs were analyzed in vitro for a cytokine profile. In the presence of LPS, miR-146a knockout BMDCs had decreased IL-27 protein concentration compared to control mice. Our data indicate that the lack of miR-146a results in a skewing of DC subsets to favor CD8a+CD103+ DCs who are known to excel at cross-presentation and migrate through tissues. RNA transcript analysis suggests spontaneous maturation of the DCs from miR-146a knockout mice. IL-27 signaling in DCs has been shown to limit TH1 responses in mice. Our data suggest that miR-146a is important for regulation of IL-27 signaling in DCs. We are now using a collagen induced arthritis model to further understand the role of miR-146a in DCs.
Serum amyloid A promotes LPS clearance and attenuates LPS-induced tissue injury
Authors: Ye Richard Dequan , Cheng Ni , Liang Yurong , Du Xiaoping
Published: January 2023

Lipopolysaccharide (LPS) is a major microbial mediator of inflammation and is responsible for sepsis resulting from Gram-negative bacterial infections. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute-phase proteins in vertebrates, but the relationship between SAA expression and LPS-induced tissue inflammation and injury remains unclear. We constructed transgenic mice expressing human SAA1 under an inducible promoter. These mice were partially protected against lung inflammatory response caused by systemic LPS administration and by cecal ligation and puncture (CLP). Interestingly, transgenic SAA1 expression did not attenuate TNFα-induced lung inflammation and injury. Further experiments identified SAA1 binding to LPS to form a complex that promoted LPS uptake and clearance by macrophages. This effect could be disrupted by a peptide derived from SAA1, and blockade of SAA1-LPS interaction markedly reduced the protective effect of SAA1 and exacerbated inflammation in both the transgenic mice and wildtype mice treated with LPS or CLP. These findings demonstrate that acute-phase SAA expression provides innate protection against LPS-induced inflammation and tissue injury in mice.
B cell recruitment to the intestine critically depends on GATA3- and RORgt-mediated development of NKp46 negative innate lymphoid cells
Authors: Zheng Mingzhu , Mao Kairui , Li Dan , Fang Difeng , Lyu Jun , Zhu Jinfang (Jeff)
Published: January 2023

The isolated lymphoid follicles (ILFs) enrich for B cells, most of which are conventional B cells, however, how they are recruited into and remain in intestine is not fully understood. Here, we show that in the small intestine lamina propria (siLP) of the Gata3f/f-VavCre mouse strain, in which no T cells and very few innate lymphoid cells (ILCs) are present, B cells were also largely absent, despite B cells were normal in number in the spleen of these mice. Mixed bone marrow chimera experiments showed that Gata3-deficient B cells were able to migrate to the siLP. Normal intestinal B cells were found in Tcra−/− mice excluding the possibility of T cell contribution to B cell recruitment. Bone marrow reconstitution of various donors into Gata3f/f-VavCre mice indicated that ILCs were involved in B cell migrating into small intestine LP. Further immunofluorescent analyses using RorcGFP/GFPand Gata3f/f-Rorc-Cre mice implicated that deficiency in NKp46 negative ILC3s, presumably the lymphoid tissue inducers, was responsible for impaired B cell recruitment. In summary, our findings suggest that NKp46 negative ILC3-mediated formation of tertiary lymphoid structures such as ILFs is required for B cell recruitment and/or retention in the small intestine.
IL-12 signaling drives the differentiation and function of a TH1-derived TFH1-like cell population
Authors: Powell Michael Duane , Read Kaitlin , Sreekumar Bharath , Oestreich Kenneth J.
Published: January 2023

CD4+ T follicular helper (TFH) cells play key roles in the generation of humoral immunity, as they provide T cell help to B cells that ultimately results in the production of pathogen-specific antibodies. A subset of TFH cells that shares features with TH1 cells, referred to as (TH1)-biased TFH (TFH1) cells, has recently been shown to play key roles in responses to chronic infections including HIV and malaria, as well as the onset of some autoimmune diseases. Despite the apparent importance of these cells to comprehensive immune responses and human health, many questions remain regarding their origin, including the molecular mechanisms that regulate their differentiation. Here, we describe a population of TH1-derived TFH1-like cells that produce both the TH1 cytokine interferon-γ and the TFH-associated cytokine interleukin-21 (IL-21). Interestingly, this TFH1-like population exhibits increased B cell helper activity compared to IL-6 derived TFH-like cells. We further demonstrate that IL-12 signaling is required for both the differentiation and functional properties of the TFH1-like cell population. Specifically, IL-12-dependent activation of STAT4, and unexpectedly STAT3, result in both the upregulation of the TFH lineage-defining transcription factor Bcl-6, as well as the increased production of IL-21. Finally, we show that IL-12 signaling is required for TFH1-like cell expression of Icos, a receptor that is critical for the ability of T cells to provide help to B cells. Taken together, our findings provide potential insight into the genesis and regulatory requirements of recently described TFH1 cells with emerging roles in human health and disease.
Gene Expression Profiling of TRIM Family in Individuals with Latent versus Active Tuberculosis and Reveals Potential Biomarkers for Diagnosis
Authors: Li Chuanyou
Published: January 2023

Tripartite motif (TRIM) protein family plays important regulatory roles in innate immune and inflammatory responses. Their dysregulation causes several diseases classified as virus infective diseases, cancers, immunological diseases, neuropsychiatric disorders, or developmental disorders. Nevertheless, the role and function of many members of TRIM family in tuberculosis (TB) remains largely unknown. Our study employed qRT-PCR to first profile the mRNA expression levels of 72 members of TRIM family in patients with active TB, latent tuberculosis infection (LTBI) and healthy individuals. Our results revealed a panel of TRIM genes markedly downregulated in patients with active TB when compared to LTBI subjects and healthy controls. Subsequently, in vitro cell infection experiment identified that some differentially expressed TRIM genes were specifically linked to Mtb infection. More importantly, we established the potential use of these dysregulated TRIM genes as biomarkers for the diagnosis of active TB from LTBI or healthy controls.
Incorporation of genetic diversity provides protection from cerebral malaria in Mus musculus
Authors: Cornwall Douglas , Evavold Brian D
Published: January 2023

Since their creation over a century ago inbred mouse strains have been the workhorse of immunology and due to their reduced biological variability have led to countless biological breakthroughs. However, this reduced variability by its very nature is not necessarily representative of a genetically diverse (outbred) population. Here we try to understand how increased genetic diversity in the Mus musculus system may lead to more protective immunity, through a heightened but not over reactive response. Breeding together wild derived mice, that have been maintained for genetic diversity, and C57Bl/6 mice we are able to look at how the incorporation of diversity into the host genome can alter known C57Bl/6 immune function. The F1 mice from the cross are infected with the well characterized pathogen system Plasmodium bergehi ANKA, which induces cerebral malaria and death of C57Bl/6 animals in ~6 days post infection. In contrast, we show a large reduction of virulence in the F1 mice (<5% morbidity) that corresponds with differential immune response allowing for suppression of disease. Furthermore, F1 mice appear to produce a more controlled reaction that promotes the differentiation of more memory-like cells and fewer effector-like cells. These data suggest that the incorporation of genetic diversity into the C57Bl/6 inbred animal model system drastically alters Plasmodium infection outcome.
Short- and long-term impact of prebiotic supplementation during infancy on immune activity in Wistar rats
Authors: Jeffrey Michael P , Clarke Sandra T , Krysa Nicole , Brooks Steve P.J. , Kalmokoff Martin L , Green Judy , Matias Fernando , Green-Johnson Julia May
Published: January 2023

The aim of this study was to examine short-term and long-term effects of early administration of two dietary oligosaccharide supplements, fructooligosaccharide (FOS) and galactooligosaccharide (GOS) on innate immune parameters in infant Wistar rats. A second aim was to compare the impact of these supplements between sexes. Infant rats were gavaged orally on post natal days (PND) 6–16 with either FOS or GOS solution (0.075 g/50 μL), or with glucose (0.375 g/50 μL) or no gavage as controls while being maintained on an AIN-93G diet post-weaning (n = 8 per treatment/sex). Immune impacts at PND-16 and PND-70 were determined by immunophenotyping via flow cytometry and analyses of whole blood ex vivo TLR ligand responses at PND-70. At PND-16, percentages of CD103+ Dendritic cells (DC) were higher in MLN (P < 0.003) and spleens (P < 0.008) of FOS-treated rats (P < 0.003). At PND-70, CD103+ were unaffected but FOS supplementation had differential effects on MLN and spleen γδ-T cell populations in females versus males. Unstimulated whole blood cultures from rats fed GOS produced less CINC-1 than other treatments (P < 0.02, 57.5 ± 21.6 pg/mL). GOS-fed rats also had lower CINC-1 production in response to TLR2 (Pam3Cys, P < 0.02) and TLR4 (lipopolysaccharide, P < 0.01) stimulation, and produced less IL-1β after TLR4 stimulation (P < 0.05) than rats from all other treatments. Males produced more IL-10 than females after TLR4 stimulation regardless of treatment (P < 0.001, 72.6 ± 11.6 vs. 40.9 ± 8.2 pg/mL). Overall, these results suggest limited impact of early administration of prebiotic supplements FOS and GOS on immune development, with GOS having some long-term potential to modulate TLR2 and TLR4 ligand responses.
Immunization with Leishmania major centrin knock-out (LmCen
−/−
) parasites induces skin resident memory T cells that plays a role in protection against wild type infection (LmWT)
Authors: Ismail Nevien , Karmakar Subir , Bhattacharya Parna , Dey Ranadhir , Nakhasi Hira L
Published: January 2023

Leishmaniasis is a vector-born disease transmitted through sand fly bite with no available vaccine. Vaccination through leishmanization with Leishmania major has been used successfully but is not safe. Recently, we have demonstrated immunization with live attenuated LmCen−/− parasite protects against Leishmaniasis via induction of host cellular immunity. Resident memory T cells (TRMs) are considered the first line of defense against infections invading the host through epithelial barrier. We evaluated the generation and function of skin TRMs post LmCen−/− immunization, compared to that generated by LmWT healed mice. We examined chemokine receptors controlling the generation and survival of skin TRMs, as well as effector and recruitment function of those cells post challenge with WT parasites. We observed that expression of CXCR6 and CXCR3 was significantly higher on T cells from spleens and lymph nodes of immunized mice, compared to those from healed mice. At 15 weeks post immunization, the skin of immunized mice had significantly larger population of TRMs compared to healed mice. In addition, epithelial cytokine production, such as IL-15, IL-33 and TNFa was significantly higher in the skin of immunized mice. Upon virulent challenge, immunofluorescent labeling of skin sections showed rapid recruitment of more T cells in the immunized mice compared to healed mice. IFNγ production by skin TRMs post challenge was significantly higher in immunized mice compared to healed mice. Taken together, these results show that immunization with live attenuated parasites is safer than Leishmanization. In addition, it generates large functional population of skin TRMs which play an important role in protection against the Leishmania infection.
Acute congenital Toxoplasma gondii infection alters fetal hematopoiesis via interferon-gamma
Authors: Apostol April , Jensen Kirk , Beaudin Anna E
Published: January 2023

In adulthood, infection drives cytokine-mediated inflammation that can directly influence hematopoietic stem and progenitor cell (HSPC) function and differentiation, but the effects of maternal infection on the trajectory of fetal hematopoietic and immune cells have not been elucidated. Toxoplasma gondii is a TORCH pathogen that can be vertically transmitted upon acute infection during pregnancy, with dire consequences for the developing fetus. Maternal IFNγ production during congenital infection is required to prevent vertical transmission and promote parasite clearance, but at the cost of lowered birth weights and premature abortion. Here, we investigated the effects of acute congenital toxoplasmosis on fetal hematopoiesis. Our examination of fetal hematopoiesis in response to maternal T. gondii infection of varying virulence revealed that discrete HSPC populations, including subsets of fetal hematopoietic stem cells, B1 precursors, and myeloid progenitors are fundamentally altered in response to congenital toxoplasmosis. To assess the ability of IFNγ to mediate these observed changes and directly cross the placental barrier, we performed epistatic crosses of mice deficient for IFNγ receptor. Our results indicate that maternal IFNy produced in response to T. gondii infection may drive observed changes to the fetal HSPC compartment and that fetal HSPCs respond directly to maternal sources of IFNγ in a manner distinct from adult HSPCs. Ongoing experiments attempt to resolve the mechanisms by which vertical transmission and maternal inflammation perturb the establishment of the fetal immune system, and the direct role of IFNγ in mediating these phenomena.
Investigating Epidermal T Cell Function by C-C Chemokine Receptor 6
Authors: Bshara-Corson Savannah , Gonzalez Isaac , Miranda Nadia , Jameson Julie
Published: January 2023

The skin is the largest organ on the body and is responsible for providing protection from foreign pathogens. When skin tissue damage occurs, immune cells aid in wound repair. Epidermal T cells play a key role in maintaining skin homeostasis by their secretion of cytokines and chemokines to facilitate inflammatory responses following tissue damage. During obesity and type 2 diabetes, epidermal T cells exhibit reduced production of select cytokine and chemokine receptors one of which is C-C Chemokine receptor 6 (CCR6). The principle ligand for CCR6 is C-C Chemokine Ligand 20 (CCL20). Upon CCL20 binding, CCR6 induces lymphocyte migration however, little is known about the regulatory role CCR6 plays in cellular functions such as cytokine production and cell surface receptor expression. For this study, murine epidermal T cells were harvested and stimulated in the presence or absence of CCL20. Epidermal T cell production of cytokines and chemokines in response to CCL20 were characterized via Luminex assay. Consequently to being stimulated with CCL20 epidermal T cells upregulate IL-2R and downregulate their T cell receptor both of which signify cell activation. Additionally, CCL20 costimulates TCR stimulation to activate epidermal T cells. Using immunofluorescence microscopy, we show that CCR6 has a pivotal role in activation causing morphological changes to skin resident T cells. Future studies will investigate CCR6 activation and receptor expression of αβ and γδ in human skin.
Does TNF-α regulate thymic T cell developmental block in mice with dysregulated expression of Ly-6A?
Authors: Bamezai Anil K , Moxham Sarah , Patel Akshay
Published: January 2023

Ly-6A is a 12 kD GPI anchored member of the Ly-6 superfamily. Ly-6A and other gene family members show broad tissue expression, including expression on T lymphocytes. Ly-6A has been characterized as a cell adhesion molecule and shown to regulate cell signaling in T cells; specifically cytokine secretion. Published work indicates that this protein’s role in signaling regulation extends as far as mediated cell death pathways, resulting in growth arrest and ultimately cell death in T cell lines. Expression of Ly-6A on T cells in the thymus is developmentally regulated. Peak expression occurs during early stages of development, diminishes during maturation, and reappears at low levels on mature, CD4+ T cells. T lymphocytes in the thymus of Ly-6A transgenic (overexpressing) mice have been observed to experience increased instances of cell death and arrested early stage development. However, the underlying mechanisms of reduced cellularity in Ly-6A transgenic mice is unknown. I hypothesized that TNFα, a cytokine known to have cell death inducing properties, contributes to inhibited T cell development, lower cellularity and extensive cell death of developing T cells in thymi of Ly-6A transgenic mice. To address my hypothesis, I investigated the levels of TNFα in normal and Ly-6A transgenic mice. I find that there is decrease in cellularity in the developing thymus of Ly-6A transgenic mice compared to Ly-6A wildtype mice, consistent with published reports. On a per cell basis, this finding corresponds with increased TNFα release from Ly-6A transgenic T-lymphocytes. We plan to investigate development of T cells in Ly-6A transgenic mice that lack TNFα and specifically look for reversal of block in thymic T cell development.
TCR signal strength shapes functional imprinting during CD4 T cell development
Authors: Verstichel Greet , Thiault Nicolas , Kakugawa Kiyokazu , Chen Angeline , Iwaya Hitoshi , Takazawa Ikuo , Cheroutre Hilde
Published: January 2023

Naïve CD4 and CD8ab T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals install a quiescent program that guides naïve T cells to the lymph nodes where they differentiate upon encounter of cognate antigen. Thymocytes expressing self-specific TCRs can be agonist-selected in response to a strong TCR signal and differentiate in the thymus to functionally committed double negative T cells, NKT cells or regulatory T cells. Although the role for strength of TCR activation is clear in instructing these different outcomes, the impact of self-recognition in the thymus on “naïve” T cells is unclear. Previous work demonstrated that negative selection of self-specific T cells is not all encompassing and that affinity for self could even enhance immune protection against pathogens.

We have used Stim1fl/fl Stim2fl/fl conditional deletion mice that fail to sustain calcium entry into the cytosol upon TCR activation to assess the role of TCR strength during selection. We confirmed that agonist selection is drastically affected in these mice whereas numbers of conventional T cells are not. Interestingly, viSNE representation of multi-parameter flow cytometry revealed single cell heterogeneity among the CD4SP thymocyte subset that is controlled by Stim1/Stim2. This indicates that differential TCR activation levels give rise to phenotypically different CD4 T cells. Purification of a strongly signaled (Stim1/2-dependent) subset of CD4 thymocytes showed functional differences in terms of their ability to proliferate in response to TCR activation. Our work points to a role for self-recognition during T cell selection that shapes the response of individual CD4 T cells to TCR activation in the periphery.
CTL-derived exosomes enhance the activation of CTLs stimulated by low-affinity peptides
Authors: Xiao Zhengguo , Wu Shu-Wei , Li Lei , Wang Yan
Published: January 2023

Cytotoxic T cells (CTLs) of low TCR affinity play an important role in the control of intracellular pathogens and cancers; however, the mechanisms by which lower-affinity CTLs are activated and maintained are not well understood. We recently discovered that fully activated CTLs stimulated by strong-affinity peptides in the presence of IL-12 are able to secrete exosomes (Exo) that can stimulate bystander CTLs without requiring the presence of antigen. We hypothesized that the exosomes secreted by high-affinity CTLs could strengthen the activation of low-affinity CTLs. To test this hypothesis, naive OT-I CD8+ cells were stimulated with altered SIINFEKL peptides of different affinities in the presence or absence of Exo. The presence of Exo preferentially increased cell proliferation and enhanced the production of IFNγ in CTLs stimulated by low-affinity peptides. The expression of granzyme B (GZB) was augmented in all affinities, with higher GZB production in low-affinity stimulated CTLs than in high-affinity stimulated ones. Exosomes promoted the rapid activation of low-affinity CTLs, which remained responsive to exosomes for a prolonged duration. Unexpectedly, exosomes could be induced quickly (24 hours) following CTL activation and at a higher quantity per cell than later (72 hours). While exosome protein profiles vary significantly between early exosomes and their later-derived counterparts, both appear to have similar downstream functions. These results reveal a potential mechanism for fully activated CTLs in activating lower-affinity CTLs that may have important implications in boosting the function of low-affinity CTLs in immunotherapy for cancers and chronic viral infections.
Teaching hemolytic disease of the newborn (Erythroblastalis fetalis) to undergraduate immunology students through the use of an interactive simulation
Authors: Costabile Maurizio
Published: January 2023

Hemolytic disease of the newborn (HDN, Erythroblastalis fetalis) is a potentially fatal condition. It is initiated due to Rhesus (Rh) antigens on fetal red blood cells (RBCs) being seen as foreign by the mothers Rh− immune system. In the first pregnancy, exposure of the maternal immune system to fetal Rh+ RBCs leads to immune activation, IgM production and memory cell formation. A subsequent pregnancy with another Rh+ fetus, activates memory cells, leading to class switching and IgG production, which is placentally transferred to the fetus. Through multiple effector functions, the IgG leads to hemolysis, causing anemia and in severe cases even death of the fetus. The role of the immune response is key to this disease, hence it is taught to undergraduate immunology students at the University of South Australia. Topics include the primary and secondary immune responses, placental transfer of IgG, deletion of self reactive cells and use of mAbs as therapeutic agents are addressed. Experience has shown that students find this topic challenging. In an attempt to enhance understanding, I developed an interactive simulation to teach these principles which was trialed in 2018. The simulation gave students the opportunity to blood group expecting parents and then discuss possible HDN issues and the treatment option. As with other simulations I have generated, (e.g. flow cytometry and mAbs), I suggest this approach is a new approach to teach complex immunological concepts. This approach can enhance student understanding as well as provide a new means of information transferal. The HDN simulation is freely available to all teaching staff who may wish to use it as part of their teaching approach.
HVEM signaling in pulmonary epithelial cells is required for protection against Streptococcus pneumoniae
Authors: Chou Ting-Fang , Hitomi Kaori , Shui Jr-Wen , Kakugawa Kiyokazu , Cheroutre Hilde , Kronenberg Mitchell
Published: January 2023

The most common cause of community-acquired pneumonia in the world is Streptococcus pneumoniae (S. pneumoniae). It is a serious and urgent issue to develop new therapies including non-antibiotic treatments for this pathogen. The herpes virus entry mediator (HVEM), a member of the tumor necrosis receptor super family 14 (TNFRSF14), has been reported to provide host innate immune defense in intestinal mucosal barriers against Citrobacter rodentium infection. Here we show using C10-Cre transgenic mice crossed to mice with a floxed Hvemallele that mice with pulmonary bronchiolar non-ciliated Clara cell-specific deletion of HVEM (HvemdelCC10) display higher bacterial burden and increased mortality after S. pneumoniae infection. Injection of an HVEM neutralizing antibody had a similar effect. This indicates that epithelial HVEM is also critical for pulmonary mucosal protection during the infection. In response to S. pneumonia, HVEM-deficiency led to reduced CXCL1 secretion, accompanied by impaired neutrophil recruitment to the lung. Likewise, deletion of part of the cytoplasmic tail of HVEM (HvemdelCT), causing loss of the ability of HVEM to signal, showed a similar susceptibility to S. pneumonia, indicating the importance of the intracellular signaling of HVEM in epithelial cells for the bacterial clearance. Taken together, these results reveal the protective role of HVEM in epithelial cells against S. pneumonia infection and suggest that activated HVEM signaling in epithelial cells could be used for the treatment of S. pneumoniae infection.
The transcription factor TCF1 is a critical target of ID2 that limits natural killer cell maturation
Authors: Kee Barbara L , Morman Rosemary , Sun Mengxi , Li Zhong-Yin
Published: January 2023

ID2 is an inhibitor of the E protein transcription factors that is expressed in all innate lymphoid cells. In natural killer (NK) cells, ID2 prevents immature cells from acquiring a naïve T cell – like gene program that restricts NK cell maturation and function. Here we show that Tcf7 (encoding the transcription factor TCF1) is the critical E protein target gene in immature NK cells that maintains NK cell progenitors. TCF1 binds to a subset of key “naïve T cell” genes that are normally expressed at low, but detectable, levels in immature NK cells and whose expression is deregulated in the absence of ID2. Importantly, combined deficiency of TCF1 and ID2 restores expression of these naïve T cell genes to near wild-type levels. Moreover, in the absence of TCF1, ID2-deficient NK cells mature and acquire functional capacity but fail to expand to wild-type numbers. Our data indicate that ID2 is essential for NK cell maturation because it controls TCF1, whose heightened expression arrests NK cell maturation and activates a naïve – like gene program.
Spatial distribution of antigen-specific CD8+ T cells in virally infected skin
Authors: Wynne-Jones Erica , Shannon John P , Reynoso Glennys V , Hickman Heather D.
Published: January 2023

CD8+ T cells play a critical role in the elimination of viral infections. After epicutaneous infection with vaccinia virus (VACV), virus replicates in skin keratinocytes and antigen drains to the lymph node, priming antigen-specific CD8+ T cells. Once activated, CD8+ T cell migration into the skin occurs independent of cognate antigen recognition. In laboratory mice, which are immunologically naive prior to viral infection, only antigen-specific CD8+ T cells are activated and can migrate into the skin. However, as humans are exposed to many more pathogens than laboratory mice, activated CD8+ cells present in the circulation can be recruited to the site of infection, regardless of their antigen specificity. To model this phenomenon in mice, we adoptively transferred OT-I CD8+ T cells, infected in the footpad with non-replicating virus to activate these T cells, and then infected epicutaneously with recombinant VACV expressing or lacking SIINFEKL (the cognate antigen for OT-I cells). This system allows the robust recruitment of both antigen-specific and non-specific antiviral CD8+ T cells into the skin. Using a combination of flow cytometry and confocal and intravital microscopy, we are currently investigating the spatial distribution of antigen-specific and non-specific CD8+ T cells in the skin. Additionally, we are quantifying cytokine production by these CD8+ T cells under various conditions, including after infection with viruses expressing distinct antigens, in close proximity. These studies should provide additional clarity on the role of antigen in CD8+ T cell spatial distribution in infected peripheral tissues.
Swift protection from Flu infections in a systemic lupus mouse model
Authors: Abboud Georges , Choi Seung-Chul , Li Wei , Titov Anton , McKinstry Karl Kai , Strutt Tara M , Salek-Ardakani Shahram , Morel Laurence
Published: January 2023

Patients with systemic lupus erythematosus (SLE) are more susceptible to infections but it is unclear whether this is due to disease or treatment. Using mice expressing three SLE susceptibility loci (triple congenics hereafter called TC mice including genes that affect both innate and adaptive immunity) we surprisingly found that these mice are resistant to respiratory Flu PR8 (H1N1) infection. TC mice lost 5–10% of their initial body weight (IBW) and promptly inhibited viral replication in the lungs compared to B6 mice that lost up to 30% of their IBW, pointing to a protective role of the innate immune system. Indeed, naïve TC lungs showed hyperactivated neutrophils, monocytes and natural killer cells (NK) compared to B6 lungs. Additionally, NK cells were found to constitutively produce IL-22 capable of enhancing the mucosal barrier. Nevertheless, RAG mice on a TC background could not recover from flu infection just like RAG.B6 mice, suggesting a role for adaptive immunity. Interestingly, depleting CD8 T cells, major players in primary flu infections, did not hinder the recovery of WT TC mice from primary infection. Lastly, depletion of both NK and neutrophils led to a reduced weight loss indicating that these cells do not contribute to early or late protection of TC mice from PR8 infection. In conclusion, systemic lupus in mice creates a favorable environment that impairs Flu virus replication and neither classical innate nor cytotoxic cells are singly responsible for this protection.
High-dimensional immune atlas of second trimester human intestinal immunity
Authors: Konnikova Liza , Stras Stephanie , Werner Lael , Toothaker Jessica , Oldham Austin , Shouval Dror
Published: January 2023

It has long been postulated that newborn’s immune system is immature. To this end, a third of neonatal deaths are due to infections. Similarly, newborns tend to have reduced immune responses to vaccines. Nevertheless, most newborns are healthy. There is limited direct human studies focusing on utero fetal mucosal immune development. Therefore, there is a critical need to investigate fetal mucosal immunity. Using cytometry time of flight (CyTOF) and next-generation sequencing of B and T-cell receptor (B/TCR) repertoires of small (S) and large intestines (LI), we show that intestinal immunity is diverse and functional as early as 16 weeks gestation (GA). Similarly, BCR and TCR repertoires are as diverse in early fetal as in full term samples, though an increase in CDR3β length and distance-from-germline correlates with advanced GA. Innate immunity is dominated by antigen presenting cells, including CD103+DCs, innate lymphoid cells and natural killer cells. B-cells are also present in all tissue examined, with follicular and transitional B-cells enriched in the fetal and CD69+IgM+B-cell in neonatal tissue. Finally, there is high complexity in T-cells, representing >50% of immune cells with similar frequencies at all developmental stages and locations studied. Contrary to our assumption, the majority of T-cells are of central and effector memory subsets. Even more surprisingly, functional tissue resident memory T-cells are abundant and functional in fetal and neonatal samples. Our data provide the foundation for a mucosal immune atlas of second trimester human development and challenge the paradigm that the neonatal immune system is immature, suggesting that development occurs significantly earlier than previously reported.
Alpha-synuclein (αS) acts as an alarmin to promote dendritic cell activation and proinflammatory immune response
Authors: Alam Md Masud , Yang De , Trivett Anna L. , Li Xiao-qing , Oppenheim Joost J
Published: January 2023

Alpha-synuclein (αS) protein is involved in the development of neurodegenerative disorders including Parkinson’s disease. Recently, it has been shown that αS expression by enteric neurites in the inflamed tissues of the gut correlates with the degree of inflammation and αS is chemotactic for phagocytes. However, it is not clear whether αS plays any roles in immune responses. Here, we report that αS has potent immunopromoting activity. Treatment of human monocyte derived dendritic cells (Mo-DCs) by αS resulted in upregulation of costimulatory (CD80, CD83, & CD86) and MHC (HLA-ABC & HLA-DR) molecules and production of proinflammatory cytokines such as IL-12 and TNFα. αS activated NF-κB signaling pathways in DCs and conditioned DCs to potentiate Th1 and Th17 polarization of CD4+ T cells in an allogeneic MLR. αS also induced mouse bone marrow derived (BM)-DCs and macrophages in a Th1-polarizing and M1 direction, respectively. αS activation of APCs is mediated by the TLR4-MYD88 signaling pathways. In an in vivo setting, administration of αS induced the recruitment of APCs at the site of injection. Furthermore, αS enhanced the induction of OVA-specific antibody and T cell responses when mice were immunized with a mixture of OVA and αS. Based on the ability of αS to recruit APCs to the site of administration and to promote antigen-specific immune responses, αS can be considered an alarmin capable of promoting the development of innate and adaptive immune responses. Thus, αS, in addition to acting as an inducer of neurodegenerative diseases, exerts a potent previously unappreciated proinflammatory and immunostimulating effect.