bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined PD-1 and LAG-3 Blockade Together with HLA-A*0201 Epitopes Immunotherapy Restore the Function of CD8+ TRM Cells and Reduce UV-B Induced Recurrent Ocular Herpes in HSV-1 Infected “Humanized” HLA Transgenic Mice
Authors: Benmohamed Lbachir , Coulon Pierre-Gregoire A , Srivastava Ruchi , Roy Soumyabrata
Published: January 2023

Exhaustion of herpes simplex virus type 1 (HSV-1)-specific CD8+ T cells is a notable cause that contribute to the frequency and severity of recurrent herpes. In the present study, we found that functionally exhausted HSV-specific CD8+ T cells, expressing both the programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3), two immune checkpoint receptors that promotes T cell exhaustion, were frequent in symptomatic (SYMP) patients with a history of numerous episodes of recurrent corneal herpetic disease. Similarly, using a novel “humanized” HLA-A*0201 transgenic mouse model of UV-B induced recurrent corneal herpetic disease, we demonstrated that SYMP mice with increased virus shedding and severe recurrent corneal herpetic disease had more exhausted HSV-specific tissue resident PD1+LAG-3+CD8+ TRM cells in both trigeminal ganglia (TG) and cornea. Dual blockade of both PD-1 and LAG-3 pathways restored the number and function of antiviral CD8+ TRM cells and appeared to be synergistically superior to blockade of either pathway alone. Moreover, blockade of PD-1/LAG-3 immune checkpoints in combination with therapeutic vaccination with a mixture of HLA-A*0201-restricted CD8+ T cell epitopes UL9196–204, UL25572–580, and UL44400–408, recently identified from HSV-1 proteins, significantly improved protection against UV-B induced recurrent herpes. This pre-clinical study indicate that dual blockade of Immune checkpoints combined with therapeutic vaccination with human epitopes identified from HSV-1 proteins is a promising strategy to protection against recurrent herpes in human.
High dimensional analysis of the GI tract as a long-term reservoir for chronic viral infection
Authors: Macleod Bethany L , Dickson Russell , Hezaveh Kebria , Elsaesser Heidi J , Guidos Cynthia J , Brooks David G
Published: January 2023

Chronic viruses such as HIV and hepatitis are a major health concern worldwide. As an organ constantly exposed to the external environment, the gastrointestinal tract (GIT) must constantly balance suppressive and inflammatory immunity, making it an ideal organ for virus persistence. Yet despite its importance, chronic virus infection of the GIT and its ramifications towards host immunity remains poorly understood. We recently discovered that the GIT is a long-term reservoir for chronic lymphocytic choriomeningitis virus (LCMV); persisting well after virus is cleared from the blood and other peripheral tissues.

To identify host and microbial alterations that facilitate chronic GIT infection we performed high dimensional CyTOF analysis, 16s rRNA-seq and next generation sequencing. Primary infection induced acute CD4 T cell ablation and chronic infection promoted Treg outgrowth, which was associated with viral persistence. Interestingly, chronic infection led to acute GI pathology and dysbiosis, with long-term infection enhancing susceptibility to IBD. Transcriptional signatures indicated enhanced type I interferon induced inflammation and metabolic changes associated with immune exhaustion. Interestingly, upstream regulators of GIT immunity were shared in chronic LCMV, HIV and SIV, indicating the induction of conserved pathways across multiple viral infections. Finally, we demonstrate the efficacy of immunotherapeutic targets and cell depletions to control the long-lived chronic GIT reservoir. Ultimately our studies define molecular, cellular and microbial impacts of chronic virus infection to alter GI function and identify potential immunotherapeutic strategies to control virus in this long-lived reservoir.
Structural and functional insights of the capsid protein from Zika virus
Authors: Song Hao , Shang Zifang , Qi Jianxun , Gao George F.
Published: January 2023

Zika virus (ZIKV) emerged as a global public health concern, and is distinct from other flaviviruses in many aspects, e.g., causing transplacental infection, fetal abnormalities and vector-independent transmission through body fluids in humans. The capsid (C) protein is a multifunctional protein, since it binds to viral RNA in the process of nucleocapsid assembly and plays important roles in virus infection processes by interacting with cellular proteins, modulating cellular metabolism, apoptosis and immune response. Here we solved the crystal structure of ZIKV C protein at a resolution of 1.9 Å. The ZIKV C protein structure contains four α helices with a long pre-α1 loop, and forms dimers. The unique long pre-α1 loop in ZIKV C contributes to the tighter association of dimeric assembly and renders a divergent hydrophobic feature at the lipid bilayer interface in comparison with the known C structures of West Nile and dengue viruses. We reported the interaction between the ZIKV C protein and lipid droplets through confocal microscopy analysis. Substitutions of key amino acids in the pre-α1 loop of ZIKV C disrupted the interaction with lipid droplets, indicating the loop is critical for membrane association. We also recognized ZIKV C protein possesses broad binding capability to different nucleotide types, including single-stranded and double-stranded RNAs or DNAs. Furthermore, the highly positively charged interface, mainly formed by α4 helix, is proposed to be responsible for nucleotide binding. These findings will greatly enhance our understanding of ZIKV C protein, providing information for anti-ZIKV drug design targeting the C protein.
Can Age-Associated B cells (ABC) Contribute to Anti-Viral Immunity?
Authors: Kugler-Umana Olivia Antonia , Kuang Yi , Zhang Wenliang , Devarajan Priyadharshini , Xia Jingya , Swain Susan L
Published: January 2023

With age, follicular helper T cell (TFH) dependent B cell responses erode, reducing germinal center formation, follicular B cell antibody responses and memory. Aged mice and humans develop an additional B cell population, termed age-associated B cells (ABC), that lack CD21 and CD23 and may produce TFH independent antibodies. The aged ABC population is heterogeneous, with some expressing transcription factors and adhesion molecules suggesting antigen exposure, and such ABC have been implicated in autoimmunity. Similarly, we found a unique population of responding B cells following influenza A virus (IAV) infection of aged mice that was Fas+/GL7− and we called infection induced-ABC (iABC). We asked if the CD21−CD23− ABC are progenitors of these non-follicular iABC. Upon IAV infection, transferred ABC gave rise to an iABC (Fas+GL7−) population in T and B deficient RAG KO and TFH deficient SAP KO hosts. This ABC-derived iABC population shared the phenotype of iABC in infected aged mice and could become T- cell independent Ab-producing cells. We found that aged ABC can be separated into sIgD+ (putative naïve B cells) and sIgD−(memory-like B cells). When transferred to SAP KO hosts, the sIgD+ ABC efficiently gave rise to iABC, some of which become CD138+ Ab-secreting cells. This suggests that upon T-independent stimulation, the population of sIgD+ABC in aged mice can develop into IAV-specific Ab secreting cells and may thus provide an alternate B cell-mediated pathway of protection against IAV infection. Studies are underway to evaluate the contribution of iABC-produced Ab to IAV protection.
Early Cancer detection in the Pacific
Authors: Sika-Paotonu Dianne , Liligeto Urijah , Guilford Parry
Published: January 2023

Cancer is now the second leading cause of death across the South Pacific Region, and leading cause of death in at least two Pacific nations. As Pacific countries are also operating health systems with constrictive budgets and constrained resourcing, it can be difficult to quantify and obtain precise cancer measures and estimates such as incidence, morbidity, survival and mortality figures over time. It is acknowledged that within this resource constrained environment, the diagnosis, treatment and management of cancer patients is fraught with challenges and difficulties where routine tests and equipment may be unavailable and even vary from hospital to hospital. Circulating tumour DNA (ctDNA) refers to tumour DNA fragments found in the blood circulation. Using a simple blood test, these fragments can be identified by the presence of mutations in cancer genes and can be isolated and quantified to provide information concerning cancer’s size and progression. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer diagnostics and surveillance and also lead to improved cancer health outcomes for Pacific Peoples in the Region. Preliminary engagement efforts with clinical and research persons were undertaken in order to understand the local context and explore the potential utility of ctDNA technology for early cancer detection to support and improve the diagnostics, care and health outcomes of cancer patients in the Solomon Islands. Interest was expressed in the possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth. The potential utility of ctDNA for early cancer detection remains a promising approach for cancer diagnostics.
CXCR5+ST2+memory Th2 cell reduced maturation and fecundity of Nippostrongylus brasiliensis through increase of expression of Major Basic Protein in eosinophils
Authors: Obata-Ninomiya Kazushige , Ishiwata Kenji , Endo Yusuke , Ichikawa Tomomi , Onodera Atsushi , Hirahara Kiyoshi , Okamoto Yoshitaka , Kanuka Hirotaka , Ziegler Steven F , Nakayama Toshinori
Published: January 2023

Once mice experience the infection of helminth, the mice acquired resistance to the helminth. We have studied about memory responses against Nippostrongylus brasiliensis (Nb) in mice. Nb penetrates into skin, passes through the lungs and finally reaches to the gut. It has been reported that IL-33 contributes to the immune responses against re-infection of helminth. However, the IL-33-dependent immunity to Nb has not been fully defined. In this study, we identified subsets of memory Th2 (mTh2) cells; CXCR5+ST2+CD44hiTh2 cells and ST2−CD44hiTh2 cells producing IL-4 and IL-13, and increasing accumulation of eosinophils in the lungs in helminth infection. CXCR5+ST2+mTh2 cells rather than ST2− mTh2 cells strongly expressed IL-5 and increased the expression of Major Basic Protein (MBP) in eosinophils in the lungs, which is important for reduction of maturation and fecundity of Nb in the intestine. Notably, we also found ST2+Treg cells but not ST2− Treg cells can suppress the ST2+mTh2 cell-mediated reduction of fecundity of Nb. Taken together, these findings indicate that we identified a mechanism against helminth elicited by a subpopulation of IL-5-producing Th2 cells through the accumulation of eosinophils strongly expressing MBP in the lungs. This would be helpful to establish the drugs and/or vaccination against helminth,
Structural and Functional Investigations Into B7-1:NGFR
Authors: morano nicholas , Garret Sarah , Almo Steven
Published: January 2023

Although Nerve Growth Factor Receptor (NGFR, TNFSFR16) is traditionally thought of as a receptor that regulates the growth of neurons, it is also widely expressed throughout the immune system in response to inflammation where its function is poorly characterized. In addition to binding the four neurotrophins (NGF, BDNF, NT3, NT4), the Almo lab and others have recently discovered that NGFR can also act as a receptor for B7-1 (CD80). B7-1 is a major costimulatory molecule expressed on antigen presenting cells (APCs) such as dendritic cells, macrophages, and microglia in response to inflammation. B7-1 can bind to CD28 on naïve T-cells, which induces T-cell activation. It can then bind CTLA4 (upregulated on T cells after CD28 activation), which reduces activation. B7-1 also interacts with PD-L1. The structure and function of the B7-1-NGFR interaction is currently unknown. We have recently conducted epitope mapping experiments that suggest the NGFR binds to B7-1 on the same interface that CD28 and CTLA4 bind to B7-1. This interface appears to be independent of the PD-L1-B7-1 binding interface. NGFR epitope mapping data suggests that B7-1 binds to NGFR near the binding site for NGF, and similarly to other TNFRSF receptor-ligand interactions. Furthermore, our data shows that NGFR and CTLA4 can compete for binding to B7-1. In addition, we are investigating the role the B7-1:NGFR binding plays in neurons.
Crispr/Cas9 Gene Editing Targeted to an Intron of a Novel Isoform of the β-Subunit of Thyroid Stimulating Hormone in Peripheral Leukocytes
Authors: Klein John R , Pawar Hitesh N
Published: January 2023

Hashimoto’s disease and Graves’ disease are two forms of autoimmune thyroiditis (AIT). Although various treatments protocols are available for each disease, the specific events involved in the induction of AIT remain largely unknown. We identified a novel splice variant of the β-subunit of thyroid stimulating hormone (TSHβ) in humans and mice, referred to as TSHβv, that is produced almost exclusively by bone marrow cells and peripheral leukocytes. In mice, pituitary-derived TSHβ uses exon 4 and exon 5 to code for the TSHβ polypeptide, whereas TSHβv uses exon 5 and the 3′ end of intron 4. We recently demonstrated that during viral and bacterial infection, peripheral leukocytes traffic to the thyroid and produce large amounts of TSHβv intrathyroidally. We have speculated that although this may have beneficial physiological effects for the host, it also may be a factor in the initiation and/or perpetuation of AIT. To explore this, we made Crispr/Cas9 knockout mice in which the production of immune system TSHβv but not pituitary TSHβ is disrupted by insertion of a stop codon in the terminal portion of intron 4. Of 24 founder mice, 3 had gene editing in intron 4 as determined by PCR and sequence analyses. Those animals were back-crossed with wildtype mice to get F1 heterozygous colonies and clean genomic DNA with deletions at the stop codon insertion site. F1 offspring from two of the founder mice lacked the gene-edited site in intron 4. However, 6 offspring from the third founder animal had the expected editing in intron 4. Those animals are being bred to obtain homozygous mice for experimental use. To our knowledge, this is one of the few examples of gene disruption targeted to an intron with potential functional deletion of a novel isoform.
Epigenetic programming underpins B cell dysfunction in human SLE
Authors: Scharer Christopher , Blalock Emily , Mi Tian , Barwick Benjamin , Jenks Scott , Neary Bridget , Patterson Dillon , Deguchi Tsuneo , Lee Eun-Hyung , Wei Chungwen , Sanz Ignacio , Boss Jeremy M
Published: January 2023

Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naïve cells. Although these cells have distinct markers and phenotypes, their epigenetic architecture and how it contributes to SLE remains poorly understood. To establish if epigenetic mechanisms contributed to pathogenic B cell programming in SLE, we systematically determined the DNA methylation, chromatin accessibility and transcriptome landscape of human B cell subsets from SLE and healthy subjects. To comprehensively cover the B cell lineage, we isolated resting naïve, activated subsets, class-switched memory, plasmabasts, and a newly defined effector B cell subset. These data define a differentiation hierarchy between the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Intriguingly, an SLE epigenetic signature was already established in resting naïve cells and persisted in more differentiated subsets. DNA methylation profiling revealed a set of CpG surrounding IFN response genes whose methylation status were highly predictive of disease activity. The AP-1 and EGR transcription factor networks were highly enriched in the SLE accessible chromatin and dysregulated genes. Together, these transcription factors acted in synergy with TBET to shape the epigenome of expanded SLE effector B cell subsets. These results provide insights into the epigenetic programming of human B cell subsets, identify loci that correlate with the expansion of distinct pathogenic B cell subsets, and suggest that early alterations in the epigenome may predispose B cells towards a disease fate.
In vivo depletion of microRNA-183-96-182 cluster alleviates autoimmunity in B6-lpr lupus mice
Authors: Wang Zhuang , Heid Bettina , Lu Ran , Khan Deena , Ahmed S Ansar , Dai Rujuan
Published: January 2023

We have previously reported a significant upregulation of microRNA-183-96-182 cluster (miR-183C) in splenocytes of several murine lupus models. In this study, we found that the upregulation of miR-183C is inversely related to the expression of Foxo1 and Foxo3a, which play important roles in the regulation of T lymphocyte homeostasis and immune responses. Importantly, we found that inhibition of miR-183C with specific antagomirs significantly reduces lupus-related inflammatory cytokines expression such as IFNg, IL-6, and IL-17 in in vitro activated splenocytes from both MRL and MRL-lpr mice. We further showed that inhibition of miR-183C increases expression of Foxo1 and that inhibition of Foxo1 with siRNA increases IFNg and IL-6 in activated splenocytes from MRL mice. These data suggest that the miR-183C may regulate inflammatory response by targeting Foxo1.

To further characterize the role of miR-183C in lupus pathogenesis, we developed B6-lpr mice with conditional depletion of miR-183C in T and B lymphocyte (miR-183C−/− B6-lpr) by serial crossbreeding of B6-lpr with miR-183Cfl/fl and hCD2-iCre mice. To our surprise, there was no significant difference in the production of IFNg and IL-6 in in vitro activated splenocytes from knock out (miR-183C−/− B6-lpr) mice when compared to that from control (miR-183Cfl/fl B6-lpr) mice. Impressively, we found that miR-183C−/− B6-lpr had reduced serum anti-dsDNA levels, spleen and lymphoid node weight when compared to controls. The histopathological analysis also revealed reduced inflammation in kidney in miR-183C−/− B6-lpr mice. Together, our results suggest a potential role of miR-183C in lupus pathogenesis and that miR-183C might be a new potential target for lupus therapy.
Distinctions in gene-expression in PBMC from male and female SLE patients
Authors: Olferiev Mikhail , Fernandez David , Greenman Dina , Peng Mary , Kirou Kyriakos , Crow Mary K
Published: January 2023

The female predisposition for multiple autoimmune diseases is well known. The mechanisms might be related to the female reproductive system, sex hormones, incomplete X-chromosomal inactivation etc. We investigated gene expression in PBMC of quiescent SLE patients and corresponding healthy donors of both genders (10 matching samples per group) using the RNA-seq approach.

Principal component analysis differentiated the 4 donor groups, with PC1 reflecting disease and PC2 reflecting gender. Male SLE patients received the highest score on PC1 and were placed closer to females than healthy males on PC2. We identified 796 and 99 differentially expressed genes in male and female SLE patients compared to HD (p<0.05, logFC=1.5). Fewer genes (20 vs. 27) differed between genders in SLE vs. HD (p<0.05, logFC=1.5). Weighted Gene Co-expression Network Analysis (WGCNA) identified 24 clusters of co-expressed genes. The most intriguing were clusters that differed in male and female HD and were upregulated in SLE of both genders. Those clusters include genes related to EGF receptor ligands (AREG, EREG, HBEGF), transcription factors ATF3, NR4A1, NR4A2, NR4A3, cytokines and chemokines (CCL3, CXCL2, CXCL8, CCL3L3, IL1A, IL1B).

Despite being clinically similar, male SLE patients showed a higher level of affected genes and had a lower divergence from females based on gender-related genes. Male HD had the lowest basal level of pro-inflammatory genes and EGF receptor ligands. EGFR is expressed in pituitary and involved in the reproductive hypothalamic-pituitary axis, possibly providing relative protection of males. We propose that gender-specific differences in gene expression reveal immunopathogenic mechanisms in autoimmune disease.
A non-depleting PC61 antibody allows residual CD25 function that maintains regulatory T cell-mediated immune homeostasis
Authors: Hayes Erika , Hagan Cassidy , Campbell Daniel J.
Published: January 2023

IL-2 is a critical regulator of immune homeostasis through its role in the maintenance and function of regulatory T cells (TR) and its stimulatory capacity on effector cells. Most TR constitutively express the high affinity IL-2 receptor component CD25, which increases their sensitivity to IL-2 and allows them to outcompete NK cells and memory T cells for access to IL-2 in vivo. Historically, the blocking CD25 antibody clone PC61 has been used to examine the role of CD25 in IL-2 signaling in mice. However, interpretation of results has been difficult due to uncertainty of whether the observed in vivo effects are mediated by CD25 blockade or TR depletion. We used an engineered isoform of PC61 (PC61N297Q) that cannot deplete CD25-expressing cells to specifically explore how functional inhibition of CD25 impacts TR mediated immune homeostasis. In vivo treatment of B6 mice for one week with IL-2 neutralizing antibodies (αIL-2) reduced TR frequency and number by half in lymphoid tissues, resulting in immune dysregulation that included enhanced DC activation. By contrast, treatment with PC61N297Q did not decrease TR abundance compared to controls. Although PC61N297Q reduced TR sensitivity to IL-2 by about 100-fold compared to control TR, this was not sufficient to erase their selective advantage over effector cells for IL-2 access in vivo, as normal levels of pSTAT5 were maintained in TR stained directly ex vivo from mice treated with PC61N297Q. Residual IL-2 signaling in PC61N297Q treated TR was CD25 dependent, since it could be blocked with an antibody that directly binds IL-2 at the CD25 binding site. Thus, we conclude that reducing the CD25 function on PC61N297Q treated TR is not sufficient to alter immune homeostasis.
CD2-iCre mice reveal a role for ABCB7 in B cell development and bone marrow homeostasis
Authors: Lehrke Michael Jonathan , McCue Shaylene , Rajcula Matthew , Shapiro Michael , Shapiro Virginia Smith
Published: January 2023

ABCB7 is a mitochondrial transporter that exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Using CD2-iCre ABCB7 cKO mice, we found ABCB7 is required for early B cell development. These mice have a severe block in pro-B cell development at the Fr. C to Fr. C′ transition, with reduced expression of intracellular μ and pre-BCR. CD19-cre and CD23-cre ABCB7 cKO mice have normal B cell development and peripheral B cell populations, indicating ABCB7 is required only during early B cell development. We observed reduced expression of Runx1, Ikaros, Foxo1, E47 (E2A), and Pax5 in CD2-iCre ABCB7 cKO pro-B cells. Interestingly, we also observed decreased BACH2 expression, which indicates elevated heme synthesis is occurring in the absence of ABCB7. We hypothesize that in the absence of ABCB7, developing B cells enhance heme synthesis to dispose of accumulating mitochondrial iron. Using a hen egg lysozyme BCR transgene, we were able to rescue B cell development in CD2-iCre ABCB7 cKO mice. Intriguingly, at the age of 5–8 weeks old, CD2-iCre ABCB7 cKO mice become lethargic and hunched. CBCs reveal an increase in peripheral blood neutrophils. Both the spleen and bone marrow become highly infiltrated by CD11b+Ly6Glow/+Ly6C− cells that are immature and mature neutrophils. As other knockout mice with pro-B cell blocks do not have neutrophilia, we considered if increased iron or heme is responsible for our observation. As heme can stimulate TLR4, we propose that excess heme from developing ABCB7-deficent B cells activates macrophages. In support of this, hemin induced GM-CSF production in BMDM cultures. We are currently exploring the mechanism by which excess heme production can stimulate the production of neutrophils.
MicroRNA-142 Is an Essential Negative Regulator of B Cell Maturation and Malignant Transformation
Authors: Graham Natalie Marie , Wang Wei-Le , Magilnick Nathaniel , Lee Jaewoong , Wang Huafeng , Zhang Bin , Marcucci Guido , Muschen Markus , Reyes Estefany , Boldin Mark P
Published: January 2023

microRNA-142 (miR-142) is expressed predominantly in cells of hematopoietic origin and plays a vital role in the regulation of innate and adaptive immunity. We have previously shown that miR-142 ablation in mice results in a marked expansion of the immature and mature B cell compartments. To better understand the role of miR-142 in B cell ontogenesis, we examined the early B cell development in miR-142 null mice using Hardy fraction analysis. Our findings indicate that miR-142 deletion significantly upregulates the frequency of pro-B cells, large pre-B cells, and immature B cells (Fractions B, C, and E), while decreasing the relative number of the earliest B cell progenitors (pre-pro-B cells; Fraction A). Our data links accumulation of pro-B and large pre-B cells in miR-142−/− bone marrow to an increase in their survival capacity. Moreover, we established miR-142 as a negative regulator of interleukein-7 receptor (IL7R), a cytokine receptor which plays a critical role in B cell maturation from pro-B to pre-B cell stage. In agreement with this notion, miR-142-deficient pro-B cells displayed elevated IL7R levels on their surface and proliferated more vigorously than WT cells in response to IL-7 stimulation in vitro. To test the effect of miR-142 dysregulation on malignant B cell transformation, we bred miR-142−/− mice with Eμ-myc transgenic mice, which spontaneously develop a mix of B cell lymphoblastic leukemia and lymphoma. Interestingly, miR-142 haploinsufficiency dramatically accelerated development of the disease in Eμ-myc transgenic mice. Collectively, our results establish miR-142 as an essential negative regulator of primary B cell development, whose dysregulation may contribute to malignant B cell transformation.
TCR-ligand interactions are required for murine epidermal Vγ3Vδ1 T cell development
Authors: Witherden Deborah , Garijo Olivia D , Kelly Ryan , Komori H. Kiyomi , Havran Wendy L
Published: January 2023

Dendritic epidermal T cells (DETC), which bear the Vγ3Vδ1 T cell receptor (TCR), are the exclusive T cell population resident in the murine epidermis where they act as sentinels for neighboring keratinocytes. γδ T cells differentiate from lymphoid precursors largely within the thymic microenvironment in a highly organized manner. Cellular selection processes are also believed to be involved and indeed several lines of evidence suggest that proper development and homing of the Vγ3Vδ1+ subset does require positive selection events in the fetal thymus. To evaluate the role of TCR interactions with cognate ligand in Vγ3Vδ1 T cell development and maturation, we used a soluble DETC TCR tetramer to characterize ligand expression in the fetal thymus and skin. We show that temporal expression of ligand in the thymus closely mimics the window of DETC intrathymic development. Ligand expression is independent of Skint1, a molecule shown to be important for thymic maturation of DETC and their subsequent residence in the epidermis. Furthermore, using the soluble DETC TCR tetramer as a ligand blocking reagent, we show that in fetal thymic reaggregation cultures, TCR-ligand interactions are essential for DETC development and maturation. Current experiments are investigating the nature of the ligand-expressing thymic epithelial cell population. Understanding TCR-ligand interactions involved in DETC development may provide important insight into identifying the enigmatic DETC antigen.
Atrophied thymus creates tTreg repertoire holes altering an antigen-specific population in the periphery
Authors: Thomas Rachel Ruth , Oh Jiyoung , Wang Weikan , Su Dongming
Published: January 2023

We have shown that there is an increased ratio of total thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells generated by the atrophied thymus related to aging. This observation, coupled with the accumulation of peripheral Treg (pTreg) cells with age, poses the difficult question of why Tregs in the periphery of the elderly are unable to suppress amplified self-reactivity-induced inflammaging. We utilized a chimeric mouse model with immune system reconstitution in which lethally irradiated rat insulin promotor-driven (RIP) mOVA host mice received mixed OT-II TCR transgenic and wild-type bone marrow, each expressing distinct congenic identifiers (CD45.1 vs CD45.2). In this system, OVA serves as a mock self-antigen, which is expressed mainly in the pancreas of the host mice. Further, our mOVA host mice carried a FoxN1-floxed gene, for induction of conditional FoxN1 knock-out, resulting in thymic atrophy analogous to age-related thymic atrophy. We observed that chimeric mice with induced thymic atrophy exhibited significantly decreased OVA-specific (OT-II) Tregs, but not total (pan) Tregs, in the spleen and pancreas. The specific Treg cells of mice with thymic atrophy were also more instable, showing relatively decreased FoxP3 expression, and had greater plasticity, showing an increased Th1-like (IFNγ+FoxP3+) Treg phenotype. Overall, our preliminary results suggest that thymic atrophy creates changes in the antigen-specific repertoire during tTreg generation resulting in potential “holes” that may contribute to inflammaging in the elderly and negatively impact Treg cell-mediated regulation in the aged immune system. (Supported by NIH/NIAID 1R01AI121147)
Neurokinin A renders mast cells responsive to autocrine IL-10
Authors: Sumpter Tina L , Li Yuting , Rao Mekhala , Gohel Dhruv , Falo Louis D , Larregina Adriana T
Published: January 2023

Interleukin (IL)-10 is produced by skin-resident mast cells (MCs). However, little is known about mechanisms regulating IL-10 production by MCs. In this study, we investigated the capacity for tissue-derived factors, such as neuropeptides, to regulate IL-10 release from MCs. Neurokinin A (NKA), a tachykinin family member, is highly expressed in the skin, but few studies have addressed its impact on immune cells. In this study, we specifically hypothesized that NKA would affect IL-10 levels in MCs. We investigated the effects of NKA in vitro using bone marrow (BM)MCs, loaded with IgE then activated with cross-linking antigen. Neurokinin A affected three levels of IL-10 regulation in IgE-activated BMMCs: 1) NKA augmented IL-10R expression, 2) NKA increased IL-10 transcription 3) NKA decreased release of an IL-10-degrading protease, without affecting degranulation. Neurokinin A potently reduced IL-4, IL-6 and IL-13 transcription and STAT5 phosphorylation. Neutralization of IL-10 reversed the effects of NKA on transcription and on STAT5 phosphorylation. In the skin, intradermal administration of NKA into MC-sufficient mice upregulated expression of IL-10 RNA, while NKA had no effect on IL-10 RNA when administered into mice lacking cutaneous MCs. We used the murine passive cutaneous anaphylaxis (PCA) model to investigate the relationship between NKA and IgE-initiated MC activation in the skin. Neurokinin A reduced early and late PCA in a manner requiring MC-derived IL-10. Collectively, we have identified a novel mechanism through which the neuropeptide, NKA, acts on MCs to regulate IL-10, broadening our understanding of the role of neuropeptides in MC function.
Ceramide-CD300f interaction inhibits Mrgprb2-mediated mast cell activation and pseudo-allergic drug reactions in mice
Authors: Takamori Ayako , Ando Tomoaki , Kaitani Ayako , Maehara Akie , Izawa Kumi , Okumura Ko , Kitaura Jiro
Published: January 2023

We have previously identified ceramide as a ligand for an inhibitory receptor CD300f (also called LMIR3) and demonstrated that ceramide-CD300f interaction inhibits IgE- and mast cell-dependent allergic responses. According to a recent report, basic secretagogues including inflammatory peptides and drugs associated with allergic-type reactions (c. g., compound 48/80, Ciprofloxacin, or Icatibant) activated connective tissue-type mast cells via Mrgprb2 in mice or via MRGPRX2 in human. To investigate a role of CD300f in pseudo-allergic reactions in mice, compound 48/80, Ciprofloxacin, or Icatibant together with Evans blue dye was intradermally injected into wild-type or CD300f-deficient mice. The results showed that CD300f deficiency enhanced vascular permeability in skin treated with basic secretagogues. Interestingly, ceramide-CD300f interaction inhibited compound 48/80-stimulated degranulation of peritoneal mast cells. Moreover, pretreatment with anti-ceramide antibody or ceramide vesicles enhanced or inhibited vascular permeability in compound 48/80-stimulated skin of wild-type mice. On the other hand, the same pretreatment failed to influence vascular permeability in compound 48/80-stimulated skin of CD300f-deficient mice. Thus, ceramide-CD300f interaction inhibits IgE-independent Mrgprb2-mediated pseudo-allergic reactions as well as IgE-dependent allergic reactions.
A review of the evidence for the existence of and relationship between hypermobile Ehlers-Danlos syndrome (hEDS), postural orthostatic tachycardia syndrome (POTS), and mast cell activation syndrome (MCAS)
Authors: Kohn Alison , Chang Christopher
Published: January 2023

The association of hypermobile Ehlers-Danlos syndrome (hEDS), mast cell activation syndrome (MCAS) and postural orthostatic tachycardia syndrome (POTS) is unclear. We conducted an extensive review of the literature using 2 different search strategies. A narrower strategy included 88 searches of various combinations of terms for each of the three conditions, yielding 19 unique papers. A broader search included 136 searches of various combinations of terms but included all forms of EDS and yielded 40 unique papers. Of these, only 4 and 9 papers from the narrower and broader search strategies were original research articles. None of these papers resulted from a combination of the search terms for the three conditions. All three clinical entities are controversial in either existence or pathogenesis. MCAS is a poorly defined clinical entity and many studies do not adhere to the proposed criteria when establishing the diagnosis. Patients previously diagnosed with EDS hypermobility type may not meet the new, stricter criteria for hEDS, but may for a less severe hypermobility spectrum disorder (HSD). The pathophysiology of POTS is still unclear. An evidence-based, common pathophysiologic mechanism between any of the two, much less all three conditions, has yet to be described. Our review of the literature shows that current evidence is lacking on the existence of MCAS or hEDS as separate or significant clinical entities. Studies proposing a relationship between the three clinical entities are either biased or based on outdated criteria. The reason behind the purported association of these entities stems from an overlapping pool of vague, subjective symptoms, which is inadequate evidence to conclude that any such relationship exists.
α-MSH Induces CD4+CD25+FOXP3+ Cells and Suppress Activation of PBMCs from Pediatric Patients with Allergic Conjunctivitis
Authors: Nieto Jane Eyre , Jiménez Martínez María C. , Fest Scarlett , Casanova Israel , Ayala Julio , Salazar Luis Alberto , Velázquez Henry
Published: January 2023

Introduction

Allergic conjunctivitis (AC) is one of the most frequent ophthalmological diseases. AC is characterized by ocular surface inflammation and systemic immune dysregulation with low frequency of CD4+CD25+FOXP3+ regulatory T cells and higher frequency of circulating effector T cells. It has been reported that α-melanocyte stimulating hormone (α-MSH) constitutes one of the principal mechanisms of immune regulation in the anterior chamber of the eye and the ocular surface, promoting the differentiation of Tregs in a murine model of experimental autoimmune uveitis and suppressing autoreactive T cells in a murine model of experimental autoimmune encephalomyelitis. This work aimed to evaluate the involvement of α-MSH in Tregs induction in cells obtained from patients with AC.

Methods

Peripheral blood mononuclear cells (PBMC) were obtained from blood sampling after assent consent was obtained from pediatric patients sensitized to house dust mite (HDM) and diagnostic with AC. PBMC were cultured with HDM and/or α-MSH. After 72h of culture, PBMC were harvested and stained with anti-CD4-AmCyan, anti-CD25-PE, anti-FOXP3-FITC and anti-CD69-APC-Cy7 and analyzed by flow cytometry. IL-2, IL-4, IL-6, IL-10, IL-17, TNF-a and IFN-γ were determined by cytometric bead arrays.

Results

A significant increase in the frequency of CD4+CD25+FOXP3+ T cells and a significant decrease in the frequency of CD4+CD69+ cells were observed in HDM+α-MSH stimulated cells, when compared with HDM. Remarkably, IL-4 showed a significant decrease in cultured cells stimulated with HDM+α-MSH.

Conclusion

These results suggest that a-MSH inhibit the IL-4 production, reestablishing the development of Treg cells and control pathogenic CD4+CD69+ T cells.
Regulation of CD8+T-lymphocytes by eEF-2K
Authors: Das Jugal Kishore , Song Jianxun
Published: January 2023

Eukaryotic elongation factor-2 kinase (eEF-2K) is a conserved protein kinase which associates multiple upstream signals with protein synthesis and cellular metabolism. Reprogramming of the cellular metabolic microenvironment in T-lymphocytes is a hallmark of several immune-pathophysiological conditions. However, the role of eEF-2K in T-cell metabolism and signaling remain unclear. The aim of the present study was to unravel the critical role of eEF-2K in regulation of cytotoxic CD8+T-lymphocytes (CTLs).

Primary CTLs from wild type (WT) and eEF-2K knock-out (KO) C57BL/6 mice were stimulated with anti-CD3 and CD28 antibodies and examined for their survival, proliferation and cytokine production. The cellular metabolism was analyzed using Seahorse Extracellular Flux analyzer. A combination of western blotting and flowcytometric analysis was used to assess the key mediators of the central metabolic Akt-PI3k-mTOR pathway.

Following activation, eEF-2K KO CTLs had significantly reduced survival on Day 4 (4-fold change; p<0.05) but proliferated more rapidly as compared to WT CTLs. The oxidative metabolism of CTLs from eEF-2K KO mice was higher leading to their early senescence. Our western-blotting analysis showed the dysfunctional metabolism of eEF-2K KO CTLs was orchestrated by enhanced activation of Akt-PI3k-mTOR signaling pathway. Correlatively, eEF-2K KO CTLs had dysfunctional IL-2, PD-1, CD 62L, CD27 and CD226 expression which was restored by treatment with rapamycin.

Collectively, our results identify that eEF-2K not only plays a crucial role in CTL activation and signaling, but also regulates senescence. These findings may be useful in designing novel strategies by targeting eEF-2K-based immunotherapy.
You are what you eat: CD46 regulated amino acid usage dictates T cell function
Authors: West Erin E , Freeley Simon , Kaminski Marcin M , Veenbergen Sharon , Lee Duck-Yeon , St. John-Williams Lisa , Thompson J. Will , Green Douglas R. , Scholl-Buergi Sabine , Karall Daniela , Huemer Martina , Kemper Claudia
Published: January 2023

Engagement of the complement receptor CD46 on T cells drives Th1 responses and upregulates nutrient and amino acid channels, such as GLUT1 and LAT1. We found that CD46 stimulation upregulates both the arginine transporter SLC7A1 (CAT-1) on human CD4 T cells and, unexpectedly, Arginase 1 expression in these cells, indicating that CD46 may regulate arginine metabolism in T cells. While Arginase 1 has been well characterized in macrophages where it is associated with the IL-10 secreting M2 type, its function in T cells has not been described. During contraction human Th1 cells switch from IFN-γ to IL-10 production and to a self-regulatory phenotype, thus we assessed the role of Arginase 1 in Th1 induction and/or contraction. Surprisingly, CD4 T cells isolated from four patients with rare Arginase 1 deficiency initially mount Th1 responses but display significantly increased IL-10 switching with earlier cellular collapse when compared to healthy control cells. Importantly, although T cells from patients with Arginase 1 deficiency have increased arginine levels, as expected, they produce normal levels of polyamines and NO. Metabolic profiling of the patients’ cells demonstrates that they shunt into a compensatory ‘glutamine usage pathway’ for their critical ornithine and polyamine generation. Analysis of changes in glutamine metabolites and their corresponding downstream pathways, along with in vivo influenza infections of Arg1fl/fl CD4-cre+ mice validate and further define the in vivo role of this novel T cell modulating pathway. Overall, these data demonstrate an unexpected intrinsic role for Arginase 1 and unveil an important compensatory mechanism used to maintain protective Th1 function in the absence of normal arginine catabolism.
The costimulatory molecule ICOS promotes establishment of resident memory CD8+ T cells
Authors: Peng Changwei , da Silva Henrique Borges , Wang Haiguang , Jameson Stephen
Published: January 2023

Resident memory T cells (TRM) are critical for localized protective immunity in non-lymphoid tissues, but the signals involved in generating TRM are incompletely defined. Inducible T-cell costimulator (ICOS) is a costimulatory molecule expressed on activated T cells, and is highly expressed on TRM compared to recirculating memory CD8+ T cell subsets. While the role of ICOS has been well elaborated in T follicular helper cells, its function in CD8+ T cells is largely unexplored. Using both mixed bone-marrow chimeras and adoptive co-transfer of antigen-specific CD8+ T cells with acute infection models, we found ICOS deficient CD8+ T cells showed minimal defects in the initial effector expansion and the generation of recirculating memory population. However, consistent with its expression pattern, ICOS deficiency significantly compromised the establishment of CD8+ TRM cells in diverse non-lymphoid tissues. We further confirmed that the interaction between ICOS ligand (ICOSL) and ICOS is essential for CD8+ T cells tissue residency, and the defect of Icos−/− CD8+ T cells tissue residency can be detected even at early time points post infection, indicating that ICOS signaling affects initial retention of activated CD8+ T cells in non-lymphoid tissues. To gain a better understanding of the ICOS signaling, we used shRNA to knockdown KLF2, a transcription factor that we showed previously inhibits generation of CD8+ TRM and which is negatively regulated by ICOS stimulation in CD4+ Tfh. Our data support the model that ICOS-mediated down-regulation of KLF2 is important for the establishment of CD8+ TRM. Further investigations into the mechanistic basis by which ICOS signaling promotes CD8+ T cells tissue residency are ongoing.
Antigen presentation by CD301b+ dermal dendritic cells dictates CD4+ T cell fate
Authors: Tatsumi Naoya , Iwasaki Akiko , Kumamoto Yosuke
Published: January 2023

Different subsets of dendritic cells (DCs) induce different types of effector CD4T cells. Naïve mouse skin is populated with three major DC subsets, including epidermal Langerhans cells, CD103+ dermal DCs and CD301b+ dermal DCs, all of which migrate to the draining lymph nodes (dLNs). We showed previously that CD301b+DCs are required for the differentiation of antigen specific T helper type 2 (Th2) cells upon immunization with ovalbumin (OVA) and papain in the footpad. However, the mechanism by which CD301b+DCs induce Th2 differentiation is unclear. Here, we examined the role of antigen presentation by CD301b+DCs in the priming and Th2 differentiation of antigen-specific CD4T cells. As in CD301b+DC-depleted mice, mice lacking MHC class II (MHCII) specifically in CD301b+ cells (CD301bΔMHCII mice) showed significantly reduced IL-4 production from OVA-specific OT-II CD4T cells 7 days after immunization, suggesting that cognate interaction between CD301b+DCs and CD4T cells is required for the development of Th2 cells. In addition, although the overall cell cycle progression of OT-II cells was unaffected, their entry into the cell cycle was significantly delayed. In both CD301b+DC-depleted and CD301bΔMHCII mice, this reduction of OT-II cells was associated with impaired retention of OT-II cells in the dLN at much earlier timing. Furthermore, CD301b+DCs were required for retaining and activating wild-type polyclonal CD4T cells in the dLNs in an MHCII-dependent manner. These results indicate that CD301b+DCs are required for effective priming of CD4T cells and Th2 differentiation through their cognate interaction with antigen-specific CD4T cells.
Human Tc17 cells harbor potent immune suppressive potential, whereas Tc1 cells lack suppressive ability
Authors: Renavikar Pranav S , Crawford Michael P , Sinha Sushmita , Karandikar Nitin J
Published: January 2023

Regulatory T-cells keep untoward immune responses under control. In several autoimmune diseases, there is a substantial regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that acute relapses of Multiple Sclerosis are characterized by a deficit of CD8+ T-cell mediated immune-suppression. Similar to CD4+ T-cells, lineages for CD8+ cytotoxic T-cells (Tc) are predominantly determined by the cytokine milieu during differentiation. We hypothesized that distinct Tc lineages may harbor differential ability to suppress effector CD4+ T-cell responses. To test this hypothesis, we obtained purified human naïve CD8+ T-cells and activated them in different in-vitro conditions to differentiate them toward Tc0 (control), Tc1, Tc2 and Tc17 lineages. We then tested their ability to suppress various types of CD4+ T-cells in flow cytometric suppression assays. We observed that Tc0 controls and Tc2 cells had comparable suppression. However, Tc1 cells showed significant loss of suppressive ability. Interestingly, Tc17 cells exhibited significantly greater suppression of effector cells of various lineages (including bulk ex-vivo CD4+ T-cells as well as in-vitro differentiated Th1 and Th17 cells). Tc17 cells also showed the greatest proportion of CD8+ T-cells with a terminally differentiated phenotype (CD27−/CD45RO−), which we have previously shown to harbor the highest suppressive ability in a neuroantigen-specific setting. These studies illustrate that Tc17 cells, known to play a vital role in infection, tumor and autoimmunity, may have an important immune-regulatory function against effector CD4+ T-cells that could be harnessed in the design of CD8+ T-cell based immune-modulatory therapy.