bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation-dependent post-transcriptional regulation of CD40L mediates B cell development and survival in germinal centers
Authors: De Maio Diego Javier Prado , Narayanan Bitha , La Porta James , Ganapathi Usha , Xie Ping , Covey Lori R
Published: February 2023

We have described a posttranscriptional pathway of induced mRNA stability that occurs at late times of T cell activation and is engaged by the binding of a polypyrimidine tract-binding protein (PTBP1) complex to the 3′UTR of the CD40L transcript. We explored the in vivo role of this pathway by generating a mouse bearing a deletion of the CD40L stability element (termed CD40LΔ5) and found that Tfh cells harboring the stability defect expressed approximately 60% of WT CD40L. Importantly, this decrease in CD40L corresponded to a poorly elaborated germinal center (GC) response which included significantly decreased levels of switched antibodies, antibody-secreting cells, GL7+ B cells and differentiated GC B cell subsets. In contrast, there was little difference in either somatic hypermutation or affinity maturation between B cells from WT compared to CD40LD5 mice. Notably, both the number and percentage of early memory B cells and plasmablasts were significantly decreased indicating that CD40L expression linked to mRNA stability was critical for B cell differentiation. To understand the impact of this stability pathway on gene expression, CD19+ B cells from CD40LD5 immunized mice were transcriptionally profiled using RNAseq and pathways associated with cell survival and proliferation were downregulated and apoptotic pathways upregulated compared to WT B cells. Importantly, the level of AID mRNA was unchanged whereas c-myc expression was significantly reduced in GL7+ B cells. Together these findings reveal that the CD40L stability element plays a critical role in regulating CD40L expression in Tfh cells, which in turn supports the optimal proliferation and cell survival of high affinity GC B cells and the differentiation of B cell subsets.
HIRA-dependent H3.3 deposition and its modification facilitate somatic hypermutation of immunoglobulin gene by maintaining the proper chromatin context and transcription
Authors: Yu GuoJun , Duan Zhi , Zhang Yongwei , Gupta Varun , MacCarthy Thomas , Scharff Matthew D
Published: February 2023

The histone variant H3.3 and its chaperone HIRA are involved in active transcription but their detailed roles in regulating somatic hypermutation (SHM) in human B cells have not been fully elucidated. Here we found that the knockout (KO) of H3.3 HIRA chaperone significantly decreased SHM in the Ramos human germinal center like B cell line without changing the levels of AID and other major proteins known to be involved in SHM. The HIRA KO was associated with a decrease at the immunoglobulin (Ig) heavy chain locus of many factors related to transcription elongation including H3.3 and this was accompanied by decreased nascent transcription of the Ig heavy chain locus. However, H3K79me2/3 elongation modification was not affected. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the Ig locus was also reduced by the HIRA KO, suggesting that the HIRA loss decreased the level of H3.3K36me3. However, the chromatin accessibility of Ig locus was increased by the HIRA depletion. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit the trimethylation of H3.3K36 reduced SHM in Ramos cells, while the H3.3K79M mutant did not. Consistent with HIRA KO, H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on Ig heavy chain variable and the downstream switching regions. These results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and further confirm the central role of transcription-coupled chromatin accessibility in SHM in human B cells.
Downregulation of key B cell transcription factor Ets1 in response to antigen receptor signaling depends on IKK2 and unconventional downstream effectors.
Authors: Kearly Alyssa , Ottens Kristina , Satterthwaite Anne B , Garrett-Sinha Lee Ann
Published: February 2023

The transcription factor (TF) Ets1 plays a critical role in maintaining B cell quiescence. High Ets1 levels in resting B cells prevent premature plasma cell differentiation and suppress autoimmune responses, but Ets1 must be downregulated to allow for proper B cell responses. Despite this vital role, it remains unknown how the Ets1 expression pattern is coordinated. Upon activation of antigen receptors (ARs), Ets1 is downregulated and quantification of newly transcribed pre-mRNA indicates that a decrease in Ets1 gene transcription is responsible. New protein synthesis is shown to be dispensable for this downregulation, suggesting that it does not rely on upregulation of a repressor but rather changes in abundance or activity of existing TFs. NFκB is activated in response to AR signaling and its binding motifs are found in putative regulatory elements identified near the Ets1 gene. Inhibition of NFκB-activating kinase IKK2 prevents the decrease in Ets1 transcription in response to AR stimulation. However, neither RelA nor cRel downstream of IKK2 are required for proper control of Ets1 expression. Next, we examined potential roles for Foxo3 and Rictor, unconventional downstream effectors of IKK2. Foxo3 has a similar expression pattern to Ets1 and is downregulated upon BCR stimulation, but B cells lacking Foxo3 regulate Ets1 expression in a manner similar to WT B cells. Rictor is a component of the mTORC2 complex, but inhibition of mTOR signaling does not impact Ets1 downregulation in response to BCR stimulation. Overall, these data suggest that Ets1 expression is coordinated by unconventional, but as of yet unidentified, downstream effectors of IKK2 in response to AR signaling.

Funded by NIH R01 AI122720 and the Lupus Research Alliance.
H3K9 dimethyltransferase G9a deficiency modulates B-cell response to LPS
Authors: George-Alexander Lou-Ella , Kania Anna , Mi Tian , Scharer Christopher D. , Boss Jeremy M
Published: February 2023

B cell differentiation is a tightly regulated process coordinated by the timed expression of various transcription factors and chromatin accessibility changes mediated by histone modifying enzymes. The histone methyltransferase (HMT) G9a, dimethylates histone H3 lysine 9 (H3K9) at promoters and inhibits gene expression through recruitment of proteins that impair chromatin accessibility. HMTs are expressed ubiquitously but display distinct enzymatic activities and patterns of chromosomal localization. G9a is most known for co-localizing with Blimp-1 to silence genes associated with the B cell phenotype in plasma cells. However, our findings show a constitutive expression of G9a during B-cell division. In addition, B cell processes that are modulated by G9a mediated dimethylation remain to be elucidated. To study G9a’s involvement at various stages of B-cell differentiation, we crossed G9afl/fl mice onto the CD19Cre/+ background (G9aKO mice). Phenotypic characterization of these mice identified a skewing within the mature B cell population towards marginal zone (MZ) B cells in G9aKO mice. Upon challenge with LPS significantly elevated activated B-cell and plasmablast frequencies were observed in G9aKO mice. Regions with chromatin accessibility and expression changes identified by ATAC-Seq and RNA-Seq elucidated the B cell processes that are modulated in G9a deficient mice. B cell processes that are subject to direct modulation by G9a will be elucidated using CUT&Tag. Together, our data shows the importance of G9a in epigenetic modulation required for B cell function.
Integrative transcriptome and chromatin landscape analysis reveals distinct epigenetic regulations in human memory B cells
Authors: Casali Paolo , Moroney Justin B , Vasudev Anusha , Pertsemlidis Alexander , Zan Hong
Published: February 2023

Memory B cells (MBCs) are long-lived and produce high-affinity, generally, class-switched antibodies. Here we use a multiparameter approach involving CD27 to segregate naïve B cells (NBCs), IgD+ unswitched (unsw) MBCs and IgG+ or IgA+ class-switched (sw) MBCs from humans of different age, sex and race. Conserved antibody variable gene expression indicates that MBCs emerge through unbiased selection from NBCs. Integrative analyses of mRNAs, miRNAs, lncRNAs, chromatin accessibility and cis-regulatory elements uncover a core mRNA-ncRNA transcriptional signature shared by IgG+ and IgA+ swMBCs and distinct from NBCs, while unswMBCs display a transitional transcriptome. Some swMBC transcriptional signature loci are accessible but not expressed in NBCs. Profiling miRNAs reveals downregulated MIR181 and concomitantly upregulated target-genes RASSF6, TOX, TRERF1, TRPV3 and RORa in swMBCs. Finally, lncRNAs differentially expressed in swMBCs cluster proximal to chromosome 14 IgH chain locus. Our findings provide new insights into MBC transcriptional programs and epigenetic regulation, opening new investigative avenues on these critical cell elements in human health and disease.
Deubiquitination of NLRP6 by Cyld critically regulates colonic inflammation
Authors: Poojary Venuprasad K , Mukherjee Sandip , Kumar Ritesh , Mosialos George , Theiss Arianne L , Flavell Richard A
Published: February 2023

The inflammasome NLRP6 plays a crucial role in regulating colonic inflammation and cancer. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation and increased colon cancer. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in colonic inflammation. (Nat Immunol. 2020. 21(6):626–635. PMID: 32424362)
MAPK regulates the immune responses in liver cirrhosis and hepatocellular carcinoma
Authors: Chang Chiung-Fang , Lu Ssu-Jung , Wang Chih-Hsuan , Jeng Kuo-Shyang
Published: February 2023

Liver diseases causes approximately 2 million death per year worldwide. Aberrant apoptosis is associated with the progression of liver fibrosis and cirrhosis, even for hepatocellular carcinoma (HCC). HCC is one of major causes of cancer death worldwide due to high heterogeneity and recurrence. Immune scores could be used to link HCC disease-free survival and to identify several microenvironment-related genes. MAPK signaling plays an important role in liver fibrogenesis and HCC. ERK activation is associated with aggressive HCC, resulting in short overall survival. In this study, the immune responses of liver fibrosis and HCC mediated by ERK signaling were investigated.

Both WT and Erk2 deficient mice were fed with choline-deficient diet to induce liver cirrhosis. Erk2 deficient livers have less degree of liver cirrhosis than WT livers. Our preliminary data suggested there was a significant increasement in the percentages of regulatory T cells in Erk2 deficient splenocytes. The percentages of follicular T cells were similar in WT and Erk2 deficient splenocytes. However, there was no significant differences in Erk2 deficient hepatic CD4 T cells for regulatory T cells and follicular T cells. In addition, Egr1 is one of ERK signaling downstream transcription factors. In HCC, there were about 10-fold differences in gene expression in the Egr1high and Egr1low group and the differential gene expression was identified. In hepatotoxicity, there was about more than 100 genes involved in HCC, 50~70 genes in liver cirrhosis, liver inflammation and liver necrosis/cell death. Therefore, MAPK signaling plays an important role in regulating the immune responses in the liver cirrhosis and HCC progression.
Downregulation of AW112010 upon treatment with 6-formylindolo[3,2-b] carbazole protects mice from fulminant Concanavalin-induced T cell-mediated liver injury
Authors: Cannon Alkeiver S , Wilson Kiesha , Miranda Kathryn , Nagarkatti Mitzi , Nagarkatti Prakash
Published: February 2023

Upregulation of long noncoding RNA (lncRNA) AW112010 has been observed in multiple inflammatory disease models. In line with implication of this lncRNA as a regulator of inflammation, it has also been shown that AW112010 expression decreases upon treatment with anti-inflammatory compounds. Since lncRNAs show abnormal expression in many inflammatory diseases and reactions, elucidating their role in such ailments is necessary. Data from our lab has shown that treatment with aryl hydrocarbon receptor (AhR) ligands induce epigenetic pathway dysregulation, so this study aimed to determine inflammatory genes dysregulated upon treatment with the endogenous AhR ligand, 6-formylindolo[3,2-b] carbazole (FICZ). A murine model of immune cell-mediated liver injury was employed by intravenously injecting 12.5 mg/kg Concanavalin A (ConA). One hour after challenge, mice were treated with 50 μg/kg FICZ via an intraperitoneal injection. Interestingly, analysis of serum collected twenty-four hours after challenge showed that FICZ was able to reduce the level of alanine transaminase (ALT) suggesting a decrease in disease severity. Single cell RNA sequencing was conducted on infiltrating liver mononuclear cells enriched via a percoll gradient. We observed a reduced percentage of T cells upon FICZ treatment and an increase in the percentage of neutrophils. Overall, we observed downregulation of AW112010 in the FICZ-treated group. Specifically, AW112010 was identified as one of the top 20 genes dysregulated in the NK cell cluster and was downregulated in the B cell, CD8+ T cell, and Kupffer cell clusters. Together, these data suggest AW112010 downregulation as a potential mechanism through which FICZ protects mice from severe liver injury.
The type 1 diabetes-associated A946T single nucleotide polymorphism in IFIH1 results in increased type I interferon signaling
Authors: Taylor Jared P , Tse Hubert Ming
Published: February 2023

Single nucleotide polymorphisms (SNP) in the human IFIH1 gene are associated with autoimmunity and interferonopathies. The rs1990760 SNP, which results in an amino acid change from alanine to threonine at position 946 (A946T), is associated with several autoimmune diseases including rheumatoid arthritis, multiple sclerosis, and type 1 diabetes (T1D). IFIH1 encodes for melanoma differentiation-associated protein 5 (MDA5), a cytoplasmic dsRNA sensor. Viral infections have been implicated as environmental triggers in autoimmune diseases. In T1D, Coxsackievirus B (CVB) infections are associated with disease onset and an IFN gene signature is detected shortly before the development of autoantibodies. Interestingly, MDA5 is responsible for detecting dsRNA replication intermediates formed during CVB replication. The objective of this study is to determine the interplay of a genetic risk factor (A946T SNP) and an environmental factor (CVB infections) in the initiation of T1D. We hypothesize that the A946T SNP results in exacerbated signaling and increased production of type I IFNs and inflammatory cytokines during CVB infections. To test this, we infected peripheral blood mononuclear cells (PBMCs) from healthy donors that were genotyped for the A946T SNP and measured the subsequent IFN response. We found that the expression of type I IFNs was increased at baseline in cells homozygous for the A946T SNP in contrast to without the SNP. Overexpression of the A946T SNP also led to increased basal expression of type I IFNs and IFN-stimulated genes in βLox5 cells and a type I IFN reporter cell line. This increased basal expression of type I IFNs may lead to β-cell stress and hyperreactive immune cells and contribute to the initiation of T1D.
The role of the transcriptional repressor YY1 in immunoregulatory gp96-mediated immune responses
Authors: Sedlacek Abigail L , Binder Robert J
Published: February 2023

Heat shock protein (HSP)-mediated immune responses encompass both antigen-specific pro-inflammatory responses as well as non-antigen specific regulatory responses. The type of immune response primed depends on antigen presenting cell engaged by the HSP. Classical dendritic cells (cDCs) and macrophages elicit inflammatory responses in response to immunogenic HSPs such as gp96, whereas plasmacytoid dendritic cells (pDCs) induce regulatory responses. We have previously shown that NRP1 is in part responsible for the difference between these subsets. Gp96 signaling increases NRP1 expression on pDCs but not cDCs. This increased expression drives pDC-Treg interactions and results in activated Tregs. Increased NRP1 expression in pDCs is dependent on DNA methylation and an area of differential methylation has been identified in intron 2 of NRP1. Within this region of intron 2, bisulfite sequencing was used to look for specific sites of methylation. Intron 2 includes 2 CpG sites which show increased methylation in pDCs following stimulation with gp96. In the same region of these CpG sites, there is a consensus sequence for the transcriptional repressor YY1. We are investigating the role of YY1 as an inhibitor of NRP1 transcription, whose binding is blocked by methylation at intron 2. Using ChIP assays, siRNA knockdowns and cell imaging studies we will determine the role of YY1 in gp96-mediated immunoregulatory responses.
Bystander CD8 T cell memory responses partially protect mice against lethal vaginal HSV-2 challenge
Authors: Arkatkar Tanvi , Dave Veronica , Prlic Martin , Lund Jennifer M
Published: February 2023

Herpes Simplex Virus type-2 (HSV-2) infection is one of the most prevalent sexually transmitted diseases, yet no vaccine is currently available. Detailed analysis of HSV-infected human tissue revealed tissue-resident CD8 T cells (TRM) limit the duration and severity of HSV-2 episodes. Interestingly, recent studies reveal that the sensing and alarming function of CD8 TRMs is not restricted to cognate antigen interaction, but CD8 TRM can mediate protection against antigenically unrelated pathogens, termed “bystander activation.” Here, we extended our findings to determine if antigen non-specific CD8 T cells could provide some degree of protection in a bystander fashion in the context of HSV infection. To test this, we created antigen non-specific memory compartments through immunization of mice with Listeria expressing ovalbumin (OVA) (LM-OVA). Mice were then challenged with wild-type HSV-2 to assess the degree of vaccine-mediated protection. Immunization with LM- OVA-induced intermediate protection from lethal viral challenge, suggesting that bystander CD8 T cells (BA-CTL) may mediate protection despite the absence of antigen-specificity. The bystander functionality could be context-dependent, and the unregulated expansion of these cells can be damaging to the host. A potential explanation of BA-CTL regulation is via the involvement of regulatory T cells (Tregs), in the absence of Treg-mediated regulation, massive expansion of BA-CTLs can be predicted. On the contrary, we found depletion of Tregs resulted in significantly fewer BA- CTLs in the vaginal tract on days 2 and 6 post-HSV-2 challenge. Hence how these cells maintain the delicate balance of protection against pathology necessitates further investigation.
Filling the innate myeloid niche in a 3D human tissue-engineered lung model to study age-dependent immune response to respiratory syncytial virus
Authors: Roe Mandi M , Do Taylor , Turner Sean , Gappa-Fahlenkamp Heather , Kovats Susan
Published: February 2023

The immune response to respiratory syncytial virus (RSV) is vastly influenced by age. RSV can lead to severe disease in neonates and the elderly. To study human age-dependent innate immune responses, we utilized a 3D Human Tissue-Engineered Lung Model (3D-HTLM) consisting of multiple layers that mimic alveoli, including vascular endothelium, stromal cells, and small airway epithelial cells at air-liquid interface. Our goal is to determine if blood monocytes (Mo) added to the 3D-HTLM will fill the myeloid niche and acquire characteristics of tissue resident cells, including alveolar macrophages (AM), and respond to RSV infection. Based on CD1c and CD14 expression, freshly enriched Mo included small populations of cDC2s, Mo-DCs, and non-classical CD14− Mo, all of which lacked CD206 and CD169 markers typical of innate lung resident cells. The 3D-HTLM was harvested 4 days after Mo addition and 1 day after RSV infection (MOI 10). Independent of RSV, all CD45+ Mo, cDC2s, Mo-DCs, and CD14− Mo had significantly increased expression of CD206, CD169, and HLA-DR, including a minor population resembling AM, identified as CD1c−CD14loHLA-DR+CD169+CD206+CD71+. After seeding the 3D-HTLM, cDC2s had increased expression of activation markers CD86, CD83, and CD40. Mo populations showed few phenotypic changes after RSV infection of the 3D-HTLM, yet cDC2s and Mo-DCs had increased production of IL-12 and TNFα, respectively. These data suggest Mo populations adapt to the 3D-HTLM environment and respond to RSV, but transcriptional profiles will better indicate whether the AM niche is being filled. The 3D-HTLM seeded with myeloid cells from neonates or the elderly will be a significant tool to study age-dependent innate immune responses to RSV.
Chronic ethanol consumption increases susceptibility to influenza A virus infections via alteration of memory CD8 T cell responses
Authors: Zacharias Zeb Ralph , Legge Kevin L
Published: February 2023

Alcoholism is estimated to contribute to approximately 95,000 deaths within the United States per year making it the third leading preventable cause of death. These alcohol-related deaths are due, in part, to the increased risk of severe disease that human alcoholics have during bacterial and viral infections. Our laboratory has previously shown using a mouse model of chronic ethanol consumption that increased disease severity during a primary influenza A virus (IAV) infection is due to deficits in the developing IAV-specific CD8 T cell response. However, unlike mice, human alcoholics have likely been exposed to IAV prior to the onset of alcohol consumption. Therefore, we determined the effects of chronic alcohol consumption on pre-existing IAV immunity. Our results show that mice with established immunity to IAV that then chronically consumed alcohol (IAV-immune➞EtOH) exhibited increased morbidity and mortality to secondary heterologous IAV challenge compared to IAV-immune mice that then consumed water (IAV-immune➞H2O). IAV-immune➞EtOH mice also showed deficits in IAV-specific killing of target cells and a reduction in the number of IAV-specific CD8 T cells compared to IAV-immune ➞H2O mice. Finally, our results show that following a heterologous IAV challenge, the fold-increase of IAV-specific CD8 T cells within the lungs of IAV-immune➞EtOH was reduced compared to IAV-immune➞H2O mice. Interestingly, IAV-immune➞EtOH mice had reduced expression of CXCL10 and CXCL11 within their lungs, suggesting defects in cell trafficking. Overall, these data and our prior studies demonstrate that chronic alcohol consumption negatively impacts both the naïve and memory compartments of the CD8 T cell response to IAV infection.
IL-33 mediated stromal-myeloid cell crosstalk controls intestinal helminth infestation
Authors: De Martin Angelina , Scandella Elke , Lütge Mechthild , Perez-Shibayama Christian , Gil-Cruz Cristina , Engetschwiler Céline , Harris Nicola , Le Gros Graham S , Ludewig Burkhard
Published: February 2023

Interleukin-33 (IL-33) is a nuclear cytokine of the interleukin-1 family and is released upon cell damage thereby acting as an alarmin. IL-33 plays an essential role in promoting host-protective immune responses against helminth parasites at different mucosal surfaces including the intestine. However, early events after gastrointestinal nematode infection are poorly understood and the cellular source of IL-33 in the gut remains ill-defined. Here we show that Cxcl13-Cre-positive fibroblastic stromal cells (FSCs) of the lamina propria are the main source of IL-33 in the small intestine. Within the first days of nematode infection, lamina propria FSCs showed strongly increased IL-33 expression. Ablation of IL-33 in Cxcl-13-Cre-positive FSCs resulted in an increased infestation with the gastrointestinal nematode Heligmosomoides polygyrus bakeri. We found that myeloid cells were recruited to the site of worm invasion early after infection and that the upregulation of a distinct set of inflammatory cytokines was dependent on FSC-derived IL-33. Collectively, these data unveil that IL-33-mediated crosstalk between stromal and myeloid cells is a critical event during the initial immune response against gastrointestinal nematodes.
Immunotargeting of T Follicular Helper (TFH) Cells for SLE Treatment
Authors: Papillion Amber M , Bachus Holly , Ballesteros-Tato Andre
Published: February 2023

T follicular helper (TFH) cells promote antibody (Ab) responses by providing both survival and differentiation signals to B cells. As such, the expansion of self-reactive TFH cells contributes to auto-Ab production and correlates with disease severity in SLE. Importantly, blockade of TFH activity prevents auto-Ab production and disease progression in lupus-prone mice. Thus, TFH cells have been suggested as a target for treating SLE. Unfortunately, there are currently no therapies to selectively deplete TFH cells in vivo. We have previously identified IL-2 as a critical factor that suppresses TFH cell responses in vivo. In agreement with its negative role in TFH cell development, we show here that low-dose recombinant IL-2 (rIL-2) treatment prevents self-reactive TFH cell responses in lupus-prone mice. Importantly, this effect is independent of TREGS. Thus, our results demonstrate a novel immunosuppressive function of IL-2 that is independent of its role in regulatory T cell homeostasis. In addition, we demonstrate that rIL-2 can be targeted to TFH cells by adding an IL-6 blockade. Altogether, our data suggest that TFH cell suppression following low-dose IL-2 treatment is a significant mechanism underlying the immunosuppressive effects of low-dose rIL-2 immunotherapies.
Skin delivery of hapten and neurokinin-1 receptor antagonists by microneedle arrays targets neurogenic inflammation and prevents contact dermatitis
Authors: Bandyopadhyay Mohna , Morelli Adrian E , Erdos Geza , Sumpter Tina L , Tkacheva Olga , Shufesky William , Falo Louis D , Larregina Adriana T
Published: February 2023

Contact dermatitis (CD) is a chronic inflammatory disease caused by type-1 immunity. Skin exposure to haptens stimulates the secretion of Substance-P (SP) and initiates the neurogenic inflammation that intensifies CD. Neurokinin-1 receptor (NK1R)-signaling by SP or hemokinin-1 (HK1) amplifies immune responses. Nonetheless, the role and therapeutic implications of the NK1R-SP-HK1 axis in CD remain unclear. We show that SP, HK-1 and the NK1R are required for CD. Specific deletion of the NK1R in keratinocytes decreased the rapid release of IL-1β and IL-6 at the site of contact sensitization which impaired the innate and adaptive immunity of CD whereas deletion of the receptor in dendritic cells (DC) prevented only the adaptive immune response of the disease. Therefore, we hypothesized that blockade of NK1R during sensitization would be a feasible immunosuppressive intervention to treat CD. We developed a system of microneedle arrays (MNA) that co-deliver hapten and NK1R antagonists into mouse skin. This immunosuppressive approach resulted in decreased skin migration and lymph node homing of stimulatory dermal DC transporting the hapten from the sensitization site. Conversely, the immunosuppressive MNA did not affect the migration and lymph node homing of epidermal Langerhans cells (LC), and depletion of LC resulted in loss of the NK1R antagonist beneficial effects. In addition, immunosuppressive MNA caused deletion of hapten-specific T cells and increased T-regulatory cells, which prevented CD-onset and -relapses in a hapten-specific manner. Our findings indicate that immune-regulation by engineering localized skin neuroimmune-networks can be used to treat cutaneous diseases that, like CD are caused by type-1 immunity.
A novel application of chimeric antigen receptor (CAR) T cells for treating severe allergic diseases
Authors: Christie Karen Michelle , Fay Brittany L , Ma Zhengyu (Mark)
Published: February 2023

IgE suppression by Omalizumab has shown to be highly efficacious at controlling severe allergic asthma. The antibody-based drug, however, has a relative short half-life and requires repeated administrations resulting in high costs. To develop a therapy that can suppress IgE with a single treatment, we designed an adoptive T cell therapy (ACT) approach that targets the source of IgE, IgE-expressing B cells. To this end, we constructed chimeric antigen receptors (CARs) that recognize the extracellular membrane-proximal domain (EMPD) of membrane IgE (mIgE), which is exclusively expressed on all IgE-producing B cells. EMPD exists only on mIgE but not on secreted IgE, therefore EMPD-specific CARs cannot be blocked by secreted IgE. Two peptides composing the EMPD region were used to create four hybridomas that produced EMPD-specific monoclonal antibodies. The variable regions were cloned and used to construct single chain variable fragments (scFvs) for second generation CARs including the 4-1BB co-stimulatory domain. EMPD-specific CARs were efficiently expressed on primary human T cells through lentiviral transduction. One of the CARs, 2E3E10, produced potent cytotoxicity and IFN gamma in response to U266 cells and Daudi cells expressing mIgE. The response to U266 cells was especially impressive since the expression level of mIgE is very low. The results show proof of concept that redirecting T cells with EMPD-specific CARs can mediate potent T cell response and eliminate mIgE-expressing B cells. Exploiting the ability of CAR-expressing T cells to continuously eliminate IgE-expressing B cells, this cell-based immunotherapy approach has the potential to control severe allergic diseases with a single treatment.
Generation of PT101, a highly selective IL-2 mutein for treatment of autoimmune diseases
Authors: Visweswaraiah Jyothsna , Sampson Erik R , Kiprono Timothy , Petaipimol Parika , Monsef Adrianne , Kis-Toth Katalin , Otipoby Kevin L , Sundy John , Higginson-Scott Nathan , Viney Joanne L
Published: February 2023

Regulatory T cells (Treg) are vital for immune homeostasis and are dysfunctional in autoimmunity. Interleukin 2 (IL-2) drives the proliferation and function of Tregs via its heterotrimeric receptor (CD25/CD122/CD132). Low dose IL-2 is being evaluated for treatment of autoimmune diseases and has been shown to expand Tregs, yet it has a small selectivity window over conventional T cells (Tconv) and natural killer (NK) cells. To enhance IL-2 selectivity, mutations can be introduced to reduce its CD122/CD132 affinity thus creating a CD25 dependency for signaling through CD122/CD132 upon IL-2 facilitated CD25/CD122/CD132 trimer formation. Using structure guided approach, we introduced mutations in IL-2 that significantly decreased CD122 affinity in addition to mutations that increased CD25 affinity. Finally, we explored other mutations, orientation, and linkers to generate a potent, selective molecule with drug-like manufacturability. These structure activity relationship efforts culminated in the generation of PT101, a mutant IL-2 Fc fusion that was selective in activating and expanding Tregs. PT101 selectively induced STAT5 phosphorylation in human and non-human primate (NHP) Tregs in vitro. In humanized mice and NHPs, administration of PT101 dose-dependently and selectively expanded Treg without significant effects on other immune cell types, nor eliciting proinflammatory cytokine production. In a Phase 1a clinical trial, PT101 was well-tolerated and selectively expanded total Tregs by up to a mean maximum of 3.6-fold. There was no evidence of expansion of NK cells nor pro-inflammatory Tconv at any dose studied. Clinical trials in patients with ulcerative colitis and systemic lupus erythematosus are planned with PT101.
IRAK4 degradation abrogates cytokine release and improves disease endpoints in murine models of IL-33/36- as well as Th17-driven inflammation
Authors: Hubeau Cedric J , Campbell Veronica , Lee Albert , Lurier Emily , Skouras Stephanie , Mayo Michele , Fitzgerald Michael , Wang Amy , Chen Dapeng , Rong Haojing , Zheng Xiaozhang , Chesworth Richard , Gollob Jared , Mainolfi Nello , Slavin Anthony
Published: February 2023

Interleukin-1 receptor associated kinase 4 (IRAK4) plays a critical role in TLR and IL-1R mediated inflammation through its catalytic and scaffolding functions, making it an attractive target for the treatment of immune-inflammatory diseases. Kymera has developed oral heterobifunctional molecules that selectively target IRAK4 for degradation by the ubiquitin-proteasome pathway. These degraders have broad and potent activity in-vitro against LPS + IL-1β-induced proinflammatory cytokine and chemokine production that is superior to IRAK4 kinase inhibitors. We evaluated their efficacy in comparison to kinase inhibition in-vivo in both mechanistic, and disease models of skin and CNS inflammation. IRAK4 degraders were administered to mice in models of skin inflammation induced by either IL-33 or IL-36, as well as in an EAE model of Th17-mediated CNS inflammation. In IL-33- and IL-36-induced skin inflammation, IRAK4 degradation resulted in dose-dependent decreases in ear thickness as well as local and systemic cytokines/chemokines that was superior to kinase inhibition. In MOG-induced EAE, IRAK4 degradation was superior to kinase inhibition and significantly reduced disease scores to levels comparable to a S1P receptor agonist. These data demonstrate the broad activity of IRAK4 degradation and its superiority over kinase inhibition in alleviating inflammation induced by IL-1 family cytokines as well as antigen-dependent Th17-mediated inflammation in models relevant to human disease.
PLXDC2 activation by PX-69 ameliorates rheumatoid arthritis through activation of novel immunometabolic mechanisms
Authors: Tubau-Juni Nuria , Leber Andrew , Maturavongsadit Panita , Chauhan Jyoti , Hontecillas Raquel , Bassaganya-Riera Josep
Published: February 2023

Rheumatoid arthritis is a chronic inflammatory disorder that affects up to 1% of the population worldwide characterized by the increased presence of infiltrating immune cells, activation of inflammatory cytokine cascades, oxidative stress, abnormal proliferation of fibroblast-like synoviocytes (FLS) and neovascularization at the joint synovium. Plexin domain containing 2 (PLXDC2) is a transmembrane receptor with antioxidant, antiangiogenic and immunomodulatory properties that provide therapeutic activity in animal models of autoimmune disease, including rheumatoid arthritis. PLXDC2 activation suppresses glycolytic metabolism, while decreasing cytokine and chemokine secretion and production of superoxide species, in immune cells and FLS. In a rat model of collagen-induced arthritis, oral administration of the lead PLXDC2-binding agonist, PX-69, ameliorates disease severity, resulting in decreased clinical scores and paw size. Similarly, hind ankles presented structure preservation, reduced cartilage damage and decreased immune cell infiltration and number of blood vessels in the PX-69-treated group relative to vehicle. Immunologically, PX-69 treatment resulted in a 2-fold decrease of TNFα-producing cells while upregulating the proportion of IL-10+ myeloid cells and regulatory T cells in draining lymph nodes. In synovial tissue, pharmacological activation of PLXDC2 provided greater than 50% reduction in expression of inflammatory mediators (TNFα, IL1β, IL6, Cxcl1) and markers of synoviocyte activation (c-myc, c-fos). These results support the role of PLXDC2 signaling in controlling rheumatoid arthritis and validate PLXDC2 as a novel target for the treatment of this disabling inflammatory disorder.
BET inhibitors synergize with anti-PD1 in rescuing T cell exhaustion in acute myeloid leukemia by increasing TCF1 expression
Authors: Romine Kyle Alexander , Cho Hyun-jun , Kosaka Yoko , Flynn Patrick , Coy Jesse , Byrd Kaelan , Newman Matthew , Lind Evan
Published: February 2023

Acute myeloid leukemia (AML) is an aggressive myeloid lineage white blood cell cancer with an extremely poor prognosis that occurs most frequently above 60 years of age. The current standard of care consists of an intensive induction chemotherapy regimen. Many patients cannot withstand this intensive treatment regimen and rely on other targeted therapies such as small-molecule inhibitors, which inhibit specific oncogenic proteins, but these are not durable. Immunotherapies, which block stimuli that inhibit T cell functions, have had great success in achieving durable remissions in metastatic melanoma. Studies have shown that AML patients exhibit markers of immune suppression by the tumor microenvironment such as increased levels of PD1/LAG3+ exhausted T cells as well as regulatory T cells and myeloid-derived suppressor cells. In addition, inhibitors targeting the BET proteins family, which are histone readers, potently target leukemia cells by reducing myc but also increase IFNy and IL-2 production in T cells by inhibiting BATF, a negative regulator of TCF1. TCF1 is a critical factor in effector memory T cells and TCF1+ T cells are the primary expanders with aPD1. We therefore hypothesized that BETi and aPD1 would synergize in treating AML by modulating both tumor intrinsic and extrinsic factors (enhanced T cell activity). T cells derived from an immune competent FLT3-ITD/TET2/Lys-Cre AML mouse model show significant proliferative dysfunction and enhanced exhaustion markers, but are rescued by ex vivo treatment with BETi + aPD1. Further, In vivo treatment with this novel drug combination showed enhanced reduction of leukemic blasts, increased T cell proliferation, and in vitro expansion of TCF1+ effector memory CD8 T cells.
STING agonism combined with arginase and PTGES/COX2 inhibitors for improved anti-tumor immunotherapeutic benefit
Authors: Filderman Jessica , Chelvanambi Manoj , Storkus Walter J
Published: February 2023

Tertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation. Recent studies have shown that the presence of TLS in human tumors predicts extended survival and superior response to interventional immunotherapy. Our lab has recently demonstrated that treating mice bearing established tumors with agonists of STING, a cytosolic dsDNA sensor, leads to an inhibition in tumor growth in association with tumor vascular normalization, immune cell recruitment, and local formation of non-classical TLS within the tumor microenvironment. However, STING agonism also results in the upregulated expression of compensatory immune regulatory molecules, including ARG2 and enzymes involved in the production of immunosuppressive prostaglandins (i.e. PTGES and PTGS2/COX2), yielding an overall sub-optimal therapeutic paradigm. We are currently determining if combined treatment with STING agonists along with pharmacologic inhibitors of ARG2 and/or PTGES/COX2 results in improved control of tumor growth, increased formation of TLS, and more robust anti-tumor immune responses in vivo in murine melanoma models.
Targeting cancer cell kinase PERK incites antitumor immunity by inducing immunogenic cell death and reprograming tumor-linked immunosuppressive myelopoiesis
Authors: Mandula Jessica , Mohamed Eslam , Sierra-Mondragon Rosa , Chang Shiun , Jimenez Rachel , Satyajit Das , Rodriguez Paulo
Published: February 2023

The tumor microenvironment incites sustained endoplasmic reticulum (ER) stress in infiltrating T and myeloid cells, which impairs their antitumor potential through intrinsic activation of PKR-like endoplasmic reticulum kinase (PERK). Nonetheless, the immunomodulatory role of PERK activation in the tumor cell compartment remains unknown. Using multiple melanoma models, we established that PERK ablation (PERKKO) in cancer cells culminates in caspase-independent immunogenic cell death (ICD) in response to ER stress. In vivo, PERKKO cells underwent ICD and released immunostimulatory factors including HMGB1, ATP, calreticulin and type I interferons which provoked a dramatic anti-tumor response and induction of the abscopal effect. Immunologically, PERKKO tumor cells undergoing ICD spurred influx of CCR2-dependent monocyte-derived dendritic cells (MoDCs) and tumor infiltration of activated antigen-specific T cells. Moreover, PERK deletion in tumor cells triggered systemic expansion of myeloid precursors and promoted their differentiation into MoDCs via type I interferon signaling. Notably, MoDC-induced T cell responses in PERKKO tumors impaired B16-F10 tumor progression and enabled complete rejection of BrafV600E-SM1 tumors. Substantiating the translational potential of our work, inhibition of PERK in melanoma-bearing hosts resulted in significant anti-tumor effects. Also, TCGA analyses of melanoma patients showed that PERK signaling or repressed ICD marker expression coincided with shorter overall survival. Collectively, these results demonstrate that targeting PERK in tumor cells can offer the dual benefit of inducing ICD and reprogramming myelopoiesis to drive protective antitumor immunity.
A DNA nanorobot targeted release aTIGIT improves the immunosuppressive state and synergizes the anti-tumor effect of aPD-1
Authors: hou yuchen , Feng Hao , zuo xiaolei , Xia Qiang
Published: February 2023

Immune checkpoint inhibitor (ICIs) therapies have become the prominent breakthrough in the treatment of hepatocellular carcinoma (HCC). Dual blockade of TIGIT and PD-1 have promising results achieving synergistic antitumor effects for ICI-resistant HCC. However, immune-related adverse events (irAEs) and immune cell depletion in tumor microenvironment affect the clinical use of ICIs therapies. DNA nanobots have shown excellent potential as targeted release drug delivery systems in vivo. Here, using DNA origami we constructed an autonomous DNA robot to transport TIGIT antibodies to tumor site. The Pb (2+) dependent DNAzyme GR5 was used to achieve the aTIGIT free-loaded in DNA cargoes. The nucleolin-targeting aptamer serves both as a targeting domain and as a molecular trigger for the open of the DNA cargoes in tumor microenvironment. The CpG oligodeoxynucleotides (ODNs) responsible for immunostimulatory in tumor site by activation of TLR9, producing high-level secretion of various pro-inflammatory cytokines including tumor necrosis factor (TNF)-R, interleukin (IL)-6, and IL-12. DNA gel electrophoresis and TEM showing the structure of the nanobots. FRET efficiency verified the open of the cargoes regulated by nucleolin aptamer. Confocol microscopy showing the targeting of the nanobots to nucleolin-positive HCC cells. Cy5-modified oligonucleotides were conjugated to aTIGIT and the UV-Vis spectrometry results showed that aTIGIT was free-loaded and protected by the DNA cargoes. Tumor-bearing mouse model will be established to evaluate the synergistic antitumor effect of the DNA nanobots for aPD-1 in vivo. Our DNA nanorobot provide a safe and high efficient immunostimulatory delivery system for ICIs therapies.
Dengue virus induces human immune cells to secrete factors capable of tumor cell killing and activation of effector cells against treatment-resistant tumors
Authors: Goldufsky Josef William , Chen Tony , Singh Geeta , Zloza Andrew , Marzo Amanda Lee , Lyday Bruce
Published: February 2023

The use of oncolytic viruses (OVs) as immunotherapies has reinvigorated interest in microbial-based cancer therapies (MBCTs). However, thus far only one OV is FDA-approved and improvement in overall survival is experienced by a small proportion of treated patients. Seeking to take a bold approach, we pursued using Dengue virus (DV) as a novel cancer virotherapy. Thus far, DV has had little to no exploration in cancer, despite being well-studied in the vaccine context, where it demonstrates an outstanding safety profile. In our studies, we identified DV as having the ability to cause tumor cell death. Importantly, co-culture of human peripheral blood mononuclear cells (PBMCs) with DV resulted in cytokine production (including IL-2, IL-6, IL-7, IL-12, TNFα, and IFNγ) rather than cell death. We observed that supernatants from such co-cultures at 72 hours (m.o.i. of 2.0) were able to stimulate T cell- and NK cell-mediated killing of 624.28 melanoma cells, which are resistant to killing due to deletion of the HLA A2.1 gene. Surprisingly, these supernatants alone were also able to cause 624.28 cell death without effector cells or DV present. To determine whether DV could likewise reduce in vivo tumor growth, we utilized our DV plus dendritic cell (DC) platform, in which bone marrow-derived DCs deliver a relevant tumor antigen. C57BL/6 (B6) mice were challenged with B16 cells (5×104) via tail vein injection, resulting in lung melanoma foci. DV treatment (106 pfu, subcutaneous) with gp100-loaded DCs (6×106 via infusion) reduced melanoma foci in the lung by 51% (p<0.05). Future research will focus on mechanisms underlying DV-driven anti-tumor immune responses and tumor cell killing, with the intent of translating these findings to the clinic.
Toward Sézary Syndrome immunotherapy
Authors: Marin Ana V. , Megino Rebeca F. , Viñuela Marcos , Popova Olga , Real-Arevalo Irene , Sanchez-Majano Jose L. , Ortiz-Romero Pablo L. , Castro-Panete Maria J. , Mancebo Esther , Talayero Paloma , Paz-Artal Estela , Paciello Maria L. , Martinez-Lopez Joaquin , Subiza Jose L. , Reche Pedro A. , Lopez-Bigas Nuria , Marcilla Miguel , Paradela Alberto , Moral Manuel Gomez del , Martinez-Naves Eduardo , Serrano Alvaro , Marina-Zarate Ester , Ramiro Almudena R. , Engel Pablo , Dominguez Mercedes , Moreno Inmaculada , Cortegano Isabel , de Andres Belen , Gaspar Maria L. , Garcia-Peydro Marina , Balas Antonio , Moreno Miguel A. , Alenda Raquel , Vicario Jose L. , Luescher Immanuel F. , Toribio Maria L. , Alarcon Balbino , Regueiro Jose R.
Published: February 2023

Sézary syndrome (SS) is a leukemic form of cutaneous mature T-cell lymphoma characterized by circulating malignant CD4 T lymphocytes (Sezary cells). Patients with SS have a poor prognosis and current treatment options show high rates of relapse, morbidity or mortality. Thus, there is an unmet need for an efficient and safe treatment. Sézary cells have unique clonal potentially targetable epitopes, including their TCR, and TCR- and neoantigen-derived HLA-restricted peptides.

Our general aim is to design a patient-tailored two-pronged strategy against SS. The specific aims are 1) to target SS clonal TCR B cell epitopes using mAb and/or CAR T cells, 2) to target SS HLA-restricted T-cell epitopes using TCR peptide- and/or neoantigen-specific human T cells, and 3) to validate efficacy in vitro and in mouse models.

For the generation of mAb, apheresis-purified SS cells or SS TCR CDR3beta peptides were used for immunizations, and screening was done on SS vs non-SS CD4 cells as defined by flow cytometry using CD26 and/or PD-1. For in vitro expansion of SS peptide-specific T cells, SS patient-derived non-SS PBMC were stimulated in 96-well plates with IL-2 and pooled HLA class I+II SS peptides, 10 μM each, defined by SS WGS, WES and RNAseq-based predictions or peptidome studies. After one week, cells were exposed to autologous DC pre-loaded with peptide pools, and cytokine production was analyzed by flow cytometry.

We have obtained preliminary data on aims 1 and 2 studying two SS patients with monoclonal T cell lymphomas, including potential mouse antibodies against a clonal SS TCR using cell and peptide immunization and T-cell hits that seem to be specific of a SS TCR HLA class-I-restricted CDR3beta sequence.