bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial STAT6 O-GlcNAcylation drives type 2 mucosal immunity against helminth infection
Authors: Zhao Ming , Ren Kaiqun , Ruan Hai-Bin
Published: January 2023

The tuft cell-type 2 innate lymphoid cell circuit has emerged as a critical component of mucosal type 2 immunity against helminth, which infects more than 1 billion people worldwide. However, little is known regarding the molecular mechanisms underlying type 2 cytokine-induced tuft cell hyperplasia. O-linked N-Acetylglucosamine (O-GlcNAc), a posttranslational modification on intracellular proteins at serine and threonine residues, plays a central role in many biological processes such as gene transcription, protein stability, and cell signaling. We have previously reported that depletion of intestinal epithelial O-GlcNAcylation by knocking out O-GlcNAc transferase (OGT) leads to epithelial cell dysfunction. Here, we specifically studied the role of protein O-GlcNAcylation in tuft cell development. O-GlcNAcylation in the intestinal epithelium was transiently elevated upon helminth infection, while genetic deletion of OGT impaired tuft cell hyperplasia and type 2 immune responses. Intriguingly, such defects were not observed in mice that OGT was specifically deleted in committed tuft cells, suggesting that O-GlcNAcylation is required for the differentiation of intestinal progenitors toward the tuft cell lineage. Mechanistically, STAT6, a type 2 cytokine-responsive transcriptional factor essential for tuft cell development, is O-GlcNAcylated. O-GlcNAcylation enhanced STAT6 transcriptional activity, while O-GlcNAc-deficient STAT6 blunted its transcriptional activity and tuft cell differentiation. Taken together, our data demonstrate that protein O-GlcNAcylation, by modifying STAT6 signaling, is indispensable for tuft cell-involved mucosal type 2 immune responses against helminth infections.
Differential phenotypes and localization of CD8 T cell responses to acute and chronic viral infections
Authors: Hardman Blair K , Osborne Lisa C
Published: January 2023

Norovirus is the most common viral cause of gastroenteritis globally. Most infections are either acute, symptomatic, and rapidly cleared or persist asymptomatically. However, the mechanisms underlying these outcomes are unknown. MNV-CW3 and -CR6 are murine noroviruses which demonstrate immune correlates of acute and chronic infection respectively. This research interrogates the mechanisms underlying strain-specific differential antiviral CD8 T cell responses to acute or chronic MNV. At days 3, 4, 5 and 8 post-infection, the phenotype and quantity of adoptively transferred MNV specific CD8 T cells in the spleen, mesenteric lymph node (MLN), and intestines are analyzed by flow cytometry. Concurrently, fluorescent microscopy is used to determine the biogeographical distribution of antiviral CD8 T cells throughout the intestine. Flow cytometry shows activated MNV-specific CD8 T cells first accumulate in the MLN following oral infection with either virus suggesting this is the site of immune activation though CR6 infection results in a delayed, reduced CD8 T cell response as shown by CFSE dilution. Supporting the MLN as a primary initiation site of the antiviral CD8 T cell response, prevention of T cell egress from activation sites by FTY720 treatment enriches activated CD8 T cells in the MLN in CW3 infections with a concurrent decrease in the spleen. Furthermore, by day 8 post-infection CD8 T cell populations are skewed to memory precursors or short-lived effectors in CW3 and CR6, respectively. These data suggest these highly related viral strains may induce activation of distinct populations of, or pathways in, APC populations with long-lasting effects on CD8 T cell function and the outcome of infection.
The Wnt-β-catenin-IL-10 signaling axis in intestinal antigen-presenting cells protects mice from colitis-associated colon cancer in response to gut microbiota
Authors: Hussein Mohamed Ahmed Shawky , Swafford Daniel , Shanmugam Arulkumaran , Ranganathan Punithavathi , Manicassamy Santhakumar
Published: January 2023

Loss of immune tolerance to gut microflora is inextricably linked to inflammatory bowel disease (IBD) and colitis-associated colon cancer (CAC). Intestinal antigen presenting cells (APCs) play a pivotal role in regulating intestinal inflammation and CAC, but underlying signaling pathways and molecular mechanisms remain largely unknown. In the present study, using a mouse model of CAC, we show that the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6)-β-catenin-IL-10 signaling axis in CD11c+ APCs suppresses CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of Wnt co-receptors LRP5/6 in CD11c+ APCs in mice (LRP5/6ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of tumor-promoting pro-inflammatory factors and decreased expression of the immunosuppressive cytokine, IL-10. This condition could be improved in LRP5/6ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor promoting inflammatory factors in CD11c+ APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin in CD11c+ APCs or in vivo administration of IL-10 in LRP5/6ΔCD11c mice markedly reduced intestinal inflammation and CAC by suppressing the expression of inflammatory factors. In summary, here we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in regulating CAC in response to the commensal flora and suggest potential means for therapeutic intervention in inflammatory intestinal diseases.
Glucose promotes Treg differentiation and suppresses intestinal inflammation
Authors: Yu Yu , Yang Wenjing , Yu Yanbo , Zhao Xiaojing , Zhou Zheng , Bilotta Anthony J , Yao Suxia , Cong Yingzi
Published: January 2023

Glucose, the most abundant monosaccharide, has been characterized as the most important source of energy in human body, which is also considered as a potential risk factor in varies of diseases. However, the roles of glucose in regulation of intestinal T cell responses and inflammation are still controversial. In this report, we investigated whether and how glucose regulates the CD4+ T cell responses in intestinal inflammation. We treated Rag−/− mice which were transferred with CBir1 CD4+ T cells, which are specific for immunodominant microbiota antigen CBir1 flagellin, with drinking water with or without 0.6% or 6% (w/v) glucose for two weeks. We found that 6%, but not 0.6%, glucose in drinking water promoted regulatory T cells (Treg), but not Th1 and Th17 cells, differentiation in the mesenteric lymph nodes (MLN). In vivo administration of glucose to WT mice at the dose of 6%, but not 0.6%, also increased Treg cells in MLN. Within in vitro assay, glucose promoted Treg differentiation in a dose-dependent manner from 4500ug/ml to 15000ug/ml, but inhibited Treg differentiation with the dose higher than 15000ug/ml. Moreover, glucose-treated T cells cultured under Treg conditions showed enhanced suppressive activity toward naïve T cell proliferation. Mechanistically, glucose upregulated the expression of EGFR, Areg and Gzmb, the genes associated with regulation of Treg suppressive function. Furthermore, glucose at the dose of 6% alleviated the colitis in RAG−/− mice induced by CBir1 T cells, and increased Treg, but not Th1 and Th17, cells in MLN. Collectively, our study indicates that glucose promotes Treg differentiation and function under certain conditions, which plays an important role in intestinal homeostasis.
Indole-3-carbinol prevents colitis and regulates microbial dysbiosis by induction of anti-microbial peptides and mucins in an IL-22 dependent manner
Authors: Busbee Philip Brandon , Wilson Kiesha , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: January 2023

Colitis is an inflammatory bowel disease (IBD) characterized by acute or chronic inflammation within the colon and significant alterations in the microbiome, a term known as microbial dysbiosis. In our previous reports, we showed that a natural indole product and aryl hydrocarbon receptor (AhR) ligand found in cruciferous vegetables (Indole-3-carbinol, or I3C) was able to reduce colitis-induced disease severity and prevent colitis-associated microbial dysbiosis. In the current study, we performed 10× Genomics single cell analysis on colonic epithelial cells (CECs) and lamina propria-derived immune cells from 2,4,6-Trinitrobenzenesulfonic acid solution (TNBS)-induced colitis treated with or without I3C. Single cell analysis showed that treatment with I3C results in alterations in the immune cell profiles, which included increases in both anti-inflammatory regulatory T cells (Tregs), as well as IL-22 producing cells. Additionally, CEC cells in I3C-treated mice showed an increase in host defense responses to microbes, such as mucin (muc2) and anti-microbial peptide (AMP) production. Increased AMP production included mainly beta-defensins (BDs) and regenerating islet-derived protein 3 (Reg3) types. Gene transcriptome array and PCR validation experiments confirmed the results obtained from the single cell analysis. Lastly, as IL-22 is known to play a key role in regulating host defense responses by mucin and AMP production, we neutralized IL-22 in vivo and showed that the ability of I3C to reduce colitis severity and prevent colitis-associated microbial dysbiosis was lost.
Compartmentalized effects of aging on group 2 innate lymphoid cell development and function
Authors: D’Souza Shanti , Shen Xiaofei , Fung Ivan T. H. , Ye Longyun , Kuentzel Marcy , Chittur Sridar V. , Furuya Yoichi , Siebel Christian W. , Maillard Ivan P. , Metzger Dennis W. , Yang Qi
Published: January 2023

The effects of aging on innate immunity and the resulting impacts on immunosenescence remain poorly understood. Here, we report that aging induces compartmentalized changes to the development and function of group 2 innate lymphoid cells (ILC2), an ILC subset implicated in pulmonary homeostasis and tissue repair. Aging enhances bone marrow early ILC2 development through Notch signaling, but the newly generated circulating ILC2 are unable to settle in the lungs to replenish the concomitantly declining mature lung ILC2 pool in aged mice. Aged lung ILC2 are transcriptomically heterogeneous and functionally compromised, failing to produce cytokines at homeostasis and during influenza infection. They have reduced expression of Cyp2e1, a cytochrome P450 oxidase required for optimal ILC2 function. Transfer of lung ILC2 from young mice enhances resistance to influenza infection in old mice. These data highlight compartmentalized effects of aging on ILC and indicate that targeting tissue-resident ILCs might unlock therapies to enhance resistance to infections and diseases in the elderly.
Reduced A20 expression in nasal epithelial cells correlates with disease severity in patients with chronic rhinosinusitis with nasal polyps
Authors: chen cailing , Liu Zheng
Published: January 2023

Objective

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial disorder characterized by heightened and sustained inflammation. A20 is a novel multifunctional protein with immunomodulatory effects. We sought to investigate the expression of A20 in sinonasal mucosa, and its association with local inflammation and disease severity in patients with CRSwNP.

Methods

The expression of A20 in sinonasal samples was assessed by quantitative RT-PCR, IHC, and Western blot. The numbers of local inflammatory cells were counted. CT scan findings and symptom severity were scored. Primary nasal epithelial cells were cultured to explore the regulation of A20 expression.

Results

Compared with control tissues and non-eosinophilic polyps, the mRNA and protein expression of A20 was significantly down-regulated in eosinophilic polyps. A20 was mainly expressed by epithelial cells. The expression of A20 in nasal epithelial cells was significantly reduced in eosinophilic polyps in comparison with those in non-eosinophilic polyps and control tissues. There was a further decrease in A20 expression in nasal epithelial cells in patients with eosinophilic polyps with asthma compared to those without asthma. A20 mRNA was induced by TNF-α, LPS, dsRNA, and dexamethasone, whereas was inhibited by IL-4 and IL-13 in human primary nasal epithelial cells. A20 mRNA levels in nasal epithelial cells negatively correlated with IL-4 and IL-13 levels and eosinophil numbers in sinonasal mucosa. Importantly, mRNA levels of A20 in nasal epithelial cells negatively correlated with disease duration and so on.

Conclusions

The decreased expression of A20 might contribute to eosinophilic inflammation and disease progression in patients with CRSwNP.
Early dynamic accumulation of tissue resident memory T cells in the human lung during infancy
Authors: Matsumoto Rei , Connors Thomas , Ural Basak Burcu , Guyer Rebecca , Idzikowski Emma , Poon Maya M.L. , Thapa Puspa , Farber Donna L
Published: January 2023

Infants exhibit increased susceptibility to viral and bacterial pathogens with pulmonary infections representing one of the top causes of infant mortality worldwide. Optimal protection from pathogens is mediated by coordinated functions of different T cell subsets, with tissue resident populations maintaining site-specific immunity in the respiratory tract. Currently, little is known of how tissue localized T cells populate and mediate mucosal immune responses in early life in humans. Here we analyzed T cell populations in the lungs obtained from infant and pediatric organ donors aged 8 days postnatal (PN) to 8 years by imaging and flow cytometry. In the earliest stage of life at 8 days PN, few αβ T cells were present in the lung, while innate-type γδ T cells were present around the airways. During the first two years of life, αβ T cells exhibiting memory phenotypes preferentially accumulated around the central airways and expressed canonical markers of tissue residency (CD69 and CD103), consistent with flow cytometry data. By contrast, the total number of γδ T cells in the lung did not change significantly with age for up to 8 years of life; for example, the ratio of αβ:γδ T cells in neonatal donors was 1.5:1, which increased to 16:1 in lungs of a 3 year old donor. Consistent with the imaging data, the frequency of γδ T cells by flow cytometry was significantly reduced after the early neonatal period. These results show that γδ T cells populate the lung during the early neonatal period and are maintained in relatively constant numbers over childhood, while tissue resident αβ T cells accumulate over first 2 years of life and predominate thereafter. Our results demonstrate the dynamic nature of lung-localized T cell immune responses in a mucosal site.
Deletion of Dnase1L3 from Macrophages results in reduced serum Dnase activity in mice
Authors: Engavale Minal B. , Keyel Peter A
Published: January 2023

Dnase1L3 deficiency leads to 100% penetrance of pediatric onset Systemic lupus erythematous (SLE) while reduce Dnase1L3 levels is seen in adult-onset. Dnase1L3 is an endonuclease primarily secreted by myeloid cells like macrophages into the serum, where it degrades apoptotic bodies and chromatin, which reduces auto antibody formation and prevents SLE in both humans and mice. Importantly, reduction in the Dnase1L3 protein levels occurs both in adult-onset SLE in humans, and in polygenic murine models. This suggests that different levels of Dnase1L3 contribute to SLE progression. Also, decreasing Dnase1L3 levels leads to progressively worse SLE. However, the amount of serum Dnase1L3 needed, or whether the impact of Dnase1L3 is analog or digital remains unknown. In order to reduce Dnase1L3 protein levels, we generated Dnase1L3fl/fl x LysM-cre+/− conditional knockout (cKO) mice on B6 background. We determined the contribution of macrophage Dnase1L3 by measuring the Dnase1L3 protein levels and enzymatic activity by Dnase activity assay. We found that cKO mice showed ~2-log reduction in the serum Dnase activity. These cKO mice represent a cell-specific model to study the impact of Dnase1L3 protein levels on SLE pathogenesis. Together these findings suggest that macrophage specific Dnase1L3 may be a key player in lupus, however there could be other nucleases involved which we plan to inhibit using certain specific inhibitors. Overall, our findings suggest that macrophage specific Dnase1L3 is critical to prevent autoantibody formation in protecting lupus onset
Interferon Genes are Influenced by Sex Hormones (17b-estradiol) in SLE
Authors: Singh Ram Pyare , Hahn Bevra H
Published: January 2023

Environmental factors, genetic defects, miRNA dysregulation, and hormonal factors can influence susceptibility to SLE. Estradiol has been shown to increase the expression of interferon genes and pathways and thus could promote susceptibility to the disease in women. Recent evidence suggests the existence of a nexus between inflammatory pathways and 17b-estradiol, resulting in increased interferon stimulated genes (ISGs), autoantibodies and dysregulation of immune cells in SLE. The mechanisms by which 17b-estradiol interacts with IFN and various immune cells in SLE is poorly understood.

Aim is to determine the role of estradiol in the regulation of candidate target genes and miRNAs and mechanisms of regulation of autoantibody production in SLE.

Women may be more susceptible than men to SLE and other autoimmune diseases in part because many healthy women have higher interferon regulated genes. We found that plasma estradiol levels positively correlated with levels of serum/plasma IL-6, and 21/IL-23 in SLE patients. Plasma 17b-estradiol levels are significantly increased in female SLE patients compared to healthy females (p<0.01). Furthermore, we found that estradiol increases proinflammatory cytokines, and level of estradiol positively correlated with expression of miR21, 25, and 186 in SLE patients. Expression of miR21, miR25 and miR186 are positively correlated with SLEDAI score in SLE.

We found that expression of interferon genes is influenced by sex hormones in SLE. Understanding the role of 17b-estradiol in the regulation of pro-inflammatory pathways, ISGs, and microRNAs will further advance our understanding of SLE disease pathology and may identify additional novel therapeutic targets.
Augmentation of the Conversion of 25-hydroxyvitamin D into 1,25-Dihydroxyvitamin D Specifically in Inflamed Intestines for the Treatment of Inflammatory Bowel Disease
Authors: Liu Chenfan , Xu Yi , Cheng Yanmei , Baylink David J. , Abdalla Maisa , Huang Mei , Tang Xiaolei
Published: January 2023

An important pathogenic mechanism of inflammatory bowel disease (IBD) is the penetration of intestinal epithelium barrier by luminal microbes, which does not have a clinical solution. In this respect, previous data have shown that Lgr5+ intestinal stem cells (ISCs) can differentiate into all intestinal epithelial cells and that 1,25(OH)2D, i.e. the active vitamin D metabolite, is essential for the development of Lgr5+ ISCs. However, supplementation of native vitamin D, though effectively raising blood 25(OH)D levels, did not reach the expected outcomes in IBD patients. The data suggest that the conversion of 25(OH)D into 1,25(OH)2D cannot meet the demand in intestines. In addition, our recent findings support that direct systemic delivery of the requied 1,25(OH)2D levels to circumvent the insufficient conversion is impossible because of attending hyercalcemia. Here, we propose to augment such conversion specifically in inflamed intestines using engineered macrophages (MAC-CYP cells) that specifically home into inflamed intestines and overexpress 1alpha-hydroxylase (i.e. the physiological enzyme that converts 25[OH]D into 1,25[OH]2D). We showed that intraperitoneally administered MAC-CYP cells homed specifically into inflamed intestines, significantly suppressed ongoing experimental colitis without causing hypercalcemia, and augmented the migration of Lgr5+ ISCs which differentiated into mature intestinal epithelial cells under physiological and colitis conditions. In vitro, we showed that 1,25(OH)2D enhanced the survival of multipotent Lgr5+ ISCs. In conclusion, our data suggest that specific augmentation of 1alpha-hydroxylase activity in inflamed intestines is a promising therapeutic strategy for IBD.
Endogenous antisense regulates CD39 in Crohn’s disease upon interaction with nucleolin and heterogeneous-nuclear-ribonucleoprotein-A1
Authors: Vuerich Marta , Harshe Rasika , Xie Anyan , Abrahão-Frank Luiza , Gromova Barbora , Zhang Haohai , Mukherjee Samiran , Robles Rene’ J. , Moss Alan C. , Robson Simon C. , Longhi Maria Serena
Published: January 2023

The ectoenzyme CD39 hydrolyzes ATP to generate immunosuppressive adenosine. We have shown that CD39 levels and activity are reduced in Treg and Th17-cells from Crohn’s patients. This impairment is linked to heightened levels of endogenous antisense (AS) present at the 3′ end of the human CD39 gene and regulating CD39 at mRNA and protein level.

Here we note that CD39-specific AS is predominantly located in the nucleus of peripheral blood-derived Treg and Th17-cells of healthy subjects (HS) and Crohn’s disease patients. RNA pulldown assay followed by mass spectrometry shows that AS regulates CD39 upon interaction with nucleolin (NUCL) and heterogenous-nuclear-ribonucleoprotein-A1 (HNRNPA1). Silencing of NUCL and/or HNRNPA1 results in higher CD39 levels in both Treg and Th17-cells. Blockade of antisense in vitro using FANA oligonucleotides decreases AS while boosting CD39 in HS and Crohn’s Treg and Th17-cells. AS silencing in vivo ameliorates experimental colitis induced by trinitrobenzene-sulfonic-acid in NOD/scid/gamma mice reconstituted with antisense+ human CD4-cells and concomitantly administered FANA oligonucleotides. Compared to untreated controls, FANA-treated mice show decreased disease activity index, higher colon length, lower histology score and, phenotypically, increased proportions of circulating CD39+CD4+-cells and decreased frequencies of peripheral blood and splenic CD4+IL-17+-cells.

In conclusion, CD39-specific AS dampens CD39 levels in Crohn’s Treg and Th17-cells by interacting with NUCL and HNRNPA1. Inhibition of AS controls disease activity in experimental colitis suggesting that AS blockade might serve as a novel therapeutic tool to restore CD39 and curb inflammation in Crohn’s disease.
Mesenchymal stem cells derived exosomes promote the expansion of Bregs and alleviate the collagen-induced arthritis
Authors: Tian Jie , Zhu Qiugang , Rui Ke , Lu Liwei , Wang Shengjun
Published: January 2023

Olfactory ecto-mesenchymal stem cells (OE-MSCs) are a population of cells which has been recognized as a novel resident stem cell type in the olfactory lamina propria. OE-MSCs have been shown to exert their immunosuppressive capacity by modulating T cell responses. However, it remains unclear whether OE-MSCs possess any immunoregulatory functions on regulatory B cells (Bregs). Exosomes are secreted nanosized membrane vesicles that are increasingly implicated as an important communication tool among various cell types. In this study, exosomes derived from olfactory ecto-mesenchymal stem cells (OE-MSCs-Exo) effectively enhanced the proportions of CD19+IL-10+ Bregs in the spleen and draining lymph nodes, thus suppressing the development of collagen-induced arthritis (CIA). In culture, OE-MSCs-Exo could significantly promote the differentiation and expansion of CD19+IL-10+ Bregs, and the further investigation demonstrated that the expansion of Bregs was orchestrated by Ebi3 secreted by exosomes. Taken together, OE-MSCs-Exo have the potential to alleviate the severity of CIA through inducing Bregs, indicating OE-MSCs-Exo may represent a new therapeutic strategy for the treatment of rheumatoid arthritis.
Peptide fragments of cystatin protein from filarial parasite has potent anti-inflammatory effect on DSS-induced colitis in mouse
Authors: Khatri Vishal , Chauhan Nikhil , Prasanna Kumar S B , Kalyanasundaram Ramaswamy
Published: January 2023

The therapeutic potential of filarial parasite derived recombinant cystatin was reported previously by our group in the mouse model of colitis. We showed that cystatin-treatment elevated IL-10+FoxP3+Tregs, IgM+B1a cells and AAMs in the colon and peritoneal cavity. In this study, we attempted to identify the therapeutically active peptide(s) of cystatin. Total length of cystatin is 157 aa. We first synthesized three 40-mer peptides of cystatin. 25μg of peptide 1, 2 or 3 was injected i.p. into groups of 10 mice each on days 3–6 following DSS-administration. Control mice received DMSO only. Mice that received whole cystatin protein remained as positive control. Following treatment, clinical and pathological disease parameters were determined. Our results show that treatment with peptide 1 and 2 significantly ameliorated the inflammatory responses in the colitis mice and significantly reversed the gross and histopathological changes in the colon. However, peptide 3 had no effect in alleviating the colitis symptoms. Analysis of the colon tissue showed that the myeloperoxidase activity was significantly (p<0.005) decreased following peptide 1 and 2 treatment. The frequencies of F4/80+TLR-4+CD11c+ peritoneal macrophages were significantly decreased in colitis mice. There was also reduced infiltration of LY6G+ cells and MPO+ cells and increased FoxP3+Tregs counts in the colon tissue of these animals compared to the controls. These attenuative effects of both peptide 1 and 2 in colitis was comparable to the whole cystatin protein treatment. These studies thus demonstrate that peptide 1 and peptide 2 of the parasite cystatin can be potentially developed as a peptide therapy for colitis. Study supported by Blazer Foundation of Rockford.
Effect of Curcumin on pro-inflammatory cytokines in primary Sjögren’s Syndrome
Authors: Jayakanthan Kabeerdoss , Sandhya Pulukkol , Mohan Hindhumathi , Danda Debashish
Published: January 2023

Introduction

Curcumin reduces disease severity and ameliorates lupus-like/ Sjögren’s Syndrome-like disease in mice model. The immunological basis of these effects is unknown. This study examined the effect of curcumin on pro-inflammatory cytokines secreted by salivary gland in patients with primary Sjögren’s syndrome (pSS).

Methods

Minor salivary gland (MSG) tissue was collected from patients undergoing biopsy for suspected pSS. The tissues were treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30μM) and cultured in RPMI 1640 medium for 48 hours at 37°C in CO2 incubator. After the incubation period, culture supernatant and tissue were stored in the freezer (−80 °C). IL-6 levels were measured in supernatant by ELISA kits. Expression of pro-inflammatory cytokines, IL-6, IL-8, TNF-α, IL-1β, IL-4, IL10, IL-17 IL-21, and IFN-γ was done by qPCR. Differences between groups were tested by Student’s t-test.

Results

Forty seven patients were recruited. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glandular inflammation and negative serologies constituted sicca controls. In pSS group, but not in controls, median IL-6 levels in supernatant was less in curcumin treated as compared to PHA alone [5.5 (0.7 – 1.3) vs 18.3 (12–32) ng/ml;p=0.04]. mRNA expression levels of IL-6 and IL-1β were lower in curcumin treated groups as compared to PHA alone in both cases and controls (p= 0.0009 & p=0.04 respectively). There was no difference in other cytokine levels between the treatment groups.

Conclusion

Curcumin reduces secretion of IL-6 levels in salivary gland tissue of patients with pSS. Curcumin suppressed PHA induced mRNA expression levels of IL-6 and IL-1β in MSG tissue of pSS and controls.
Improving Hematopoietic Stem Cell transplantation with Flonase for potential use in treating a novel mouse model of Idiopathic Thrombocytopenic Purpura
Authors: Varady Erika , Inlay Matthew
Published: January 2023

Idiopathic Thrombocytopenic Purpura (ITP) is a rare autoimmune disease which causes the destruction of platelets. Patients who suffer from ITP can have excessive bleeding due to a lack of platelet coagulation. Current therapies for ITP include steroid, and intravenous immunoglobulin treatment, however they do not guarantee lasting remission. Spleen removal is the only therapy that can lead to lasting remission but is very invasive and damaging to the patients. The purpose of this study is to find a more effective and permanent therapy to treat ITP, such as through bone marrow transplantation (BMT), in a novel mouse model of ITP. BMT can be a risky procedure, so we sought to first improve it by upregulating the important chemotactic receptor, CXCR4, on the surface of hematopoietic stem cells (HSCs). We can increase surface CXCR4 on HSCs by treatment with the glucocorticoid drug fluticasone propionate (Flonase), which increases migration towards the chemokine SDF-1. Our data indicate that after Flonase treatment, HSCs significantly increase bone marrow engraftment in mouse competitive transplantations. Second, we have created an inducible ITP mouse model using CRISPR technology to test BMT as a possible therapy for ITP. Our mouse model expresses foreign antigens ovalbumin and hen egg lysozyme on platelets to elicit an immune response and platelet destruction. A novel ITP mouse model can be useful for testing potentially more permanent therapies for ITP.
Anaplastic lymphoma kinase fusions as a target for TCR-directed cellular therapies
Authors: Heather James M , Spindler Matthew J , Cobbold Mark , Gainor Justin F , Johnson David S , Hata Aaron N
Published: January 2023

Chromosomal rearrangement derived gene fusions with the catalytic domain of anaplastic lymphoma kinase (ALK) drive oncogenesis in several cancers, including in ~5% of non-small-cell lung cancers (NSCLCs). While ALK tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of these patients the majority will develop resistance; novel treatments are required. We hypothesize that the ALK kinase domain may be a suitable target for directed immunotherapy. Full length ALK is normally expressed during fetal development but becomes highly restricted in adults, and anti-ALK immune responses are readily detected in other ALK-fusion+ cancers, correlating with good prognosis.

Using peptide pool expansions and T cell activation assays, we have detected numerous anti-ALK T cell responses from normal donors and ALK+ NSCLC patients. These donors express a variety of HLA alleles, and some donors display reactivity to multiple pools spanning the kinase domain, suggestive of common T cell responses to multiple ALK epitopes.

Leveraging a high-throughput microfluidic technique to clone and functionally express natively-paired T cell receptor (TCR) genes from these ALK-reactive T cell populations, we are screening for both the exact antigenic peptides and the HLA restrictions of these epitopes. TCRs thus isolated will then be tested against a panel of ALK+/− patient derived and cancer cell lines using in vitro and in vivo models, to assess potential utility in cytotoxic TCR-directed immunotherapies. These patient derived models include those with acquired TKI resistance: by comparing these mutational hotspots with putative epitope sites we expect to find TCRs capable of killing ALK+ cancer cells irrespective of prior treatment.
Unbiased analysis of all possible neoepitopes of MC38-FABF tumor reveals a new universe of cancer neoepitopes with unexpected properties
Authors: George Mariam M , Brennick Cory A , Moussa Marmar M , Hagymasi Adam T , Seesi Sahar Al , Shcheglova Tatiana V , Englander Ryan P , Keller Grant L.J , Balsbaugh Jeremy L , Baker Brian M , Mandoiu Ion I. , Srivastava Pramod K
Published: January 2023

Cancer neoepitopes are the only truly tumor-specific antigens and therefore, the most suitable as cancer vaccines. Current methods to predict neoepitopes, based on studies of viral epitopes, emphasize high affinity MHC-peptide interactions. Increasing evidence in human and murine models indicates that the present neoepitope prediction methods are mostly inaccurate in predicting true MHC I-restricted cancer neoepitopes. Here, in a completely unbiased approach, all possible neoepitopes in a mouse tumor model were tested for their ability to mediate tumor rejection and also CD8+ T cell responses. These studies show that the true tumor rejecting neoepitopes have different properties from those of viral epitopes. Further, CD8+ T cell responses elicited by these neoepitopes possess a more plastic chromatin phenotype with stem-like properties that is known to be associated with anti-viral and anti-tumor immune responses. Such cancer neoepitopes can be exploited for generation of personalized cancer vaccines.
Establishment and Preclinical Therapy of Patient-derived Hepatocellular Carcinoma Xenograft Model
Authors: wu yuwei , Tian Zhigang , Sun Rui
Published: January 2023

Hepatocellular carcinoma (HCC) is a world-wide health problem. Poor and delayed diagnoses as well as high recurrence rate resulting in high mortality rate. In this study, we established a patient-derived xenograft (PDX) model from HCC patients, and continuously maintained with subcutaneous passage more than 20 times. This HCC PDX tumor exhibited the same histological characteristics with the HCC patient and could be used to verify therapeutic effect of liver cancer. We further evaluated this PDX model by experimental chemotherapy, demonstrating that this HCC PDX model was sensitive to sorafenib treatment. Further, the potential of natural killer cell-based immunotherapy for HCC was tested using this model. We found that NK92 cells effectively suppressed the tumor growth in vivo and prolonged the survival time of HCC-bearing PDX mice. This study indicates that HCC PDX model is a good platform to testify the efficacy of preclinical chemotherapy and immunotherapy.
Unraveling tumor-specific iNKT cell plasticity
Authors: Cortesi Filippo , Konczalla Leonie , Sturmheit Tabea M , Siracusa Francesco , Gagliani Nicola
Published: January 2023

Despite the overall success of T-cell based immunotherapies, the number of patients that benefit is still lower than expected. Invariant Natural Killer T cells (iNKTs) offer an alternative and appealing therapeutic platform owing to their intrinsic features and independence from MHC restriction. In two mouse models, mirroring prostate or colon cancer, iNKTs play strikingly opposite roles, either promoting a TH1-skewed anti-tumor response, or fostering tumor progression with TR1-oriented status. The tepid results of iNKT cell-based therapies could reflect a thus far unappreciated plastic conversion of inflammatory iNKTs into regulatory NKT10 within the tumor’s microenvironment.

This study aims to elucidate the occurrence and the dynamics of the inflammatory-to-regulatory iNKT cell plasticity in solid tumors, to clarify the environmental cues triggering their conversion and the molecular mechanisms that govern it. By employing innovative multi-cytokine reporter and fate mapping mice, we reliably detected iNKTs actively switching from producing IFNγ (exNKT1) to producing IL10, in the context of colon polyposis. Further analyses aim to unravel the molecular mechanisms of iNKT cell plasticity and to investigate the contribution of the tumor-specific microbial component.

The currently incomplete understanding of iNKT cell plasticity is dampening the appropriate use of these powerful cells in therapeutic settings. As such, clinical protocols employing iNKTs for treating cancer have so far achieved only modest results. By addressing the molecular mechanisms underlying NKT10 conversion, we are likely to find pathways that can be targeted to fully unleash the anti-tumor power of these innate-like T cells in immunotherapy.
A New Cellular Cancer Immunotherapy Platform Technology Using Autologous Human Adipose-Derived Mast Cells
Authors: Fereydouni Mohammad , Motaghed Mona , Ahani Elnaz , Moorman Whitney , Lee Sung Hyun , Kafri Tal , Kepley Christopher L.
Published: January 2023

Background

Autologous immune cell-based therapies to re-activate the immune system have been developed to treat patients with cancer. For example, chimeric antigen receptor T cell (CAR T) therapy is an FDA approved strategy that uses autologous T cells as a cancer treatment. Mast cells (MC) are important immune sentinels and widely recognized for their role as mediators of Type I hypersensitivity. However, they also secrete anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) which are being investigated in clinical trials for cancer treatment.

Methods

We generated adipose-derived mast cells (ADMC) and sensitized with anti-CD20 IgE antibodies and challenged with B-lymphoma cells. In addition, immunocompromised mice were examined for the maximal tolerated dose using ADMC. The ability of the ADMC to be transduced with lentiviral vectors carrying green fluorescence protein was examined.

Results

It is shown that CD20 IgE-sensitized ADMC bind to and are activated by CD20 positive B-lymphoma cells. The ADMC injected into mice do not display signs of anaphylaxis by the systemic injection of up to 1 × 106 ADMC/mouse. It is also shown for the first time that ADMC and their adipose-derived stem cell precursors can be transduced with a lentiviral vector carrying green fluorescence protein without affecting function.

Conclusion

This discovery indicates the ADMC could be transduced with other tumor killing molecules to create a “super killing” cell with potent and diverse anti-tumor activity. Using tumor targeting IgE’s, autologous ADMC could be used as a “Trojan Horse” to deliver these anti-tumor mediators to tumors and serve as a new cancer immunotherapy platform.
The importance of CD4+ Tumor-Infiltrating Lymphocytes (TIL) in Adoptive Cell Transfer
Authors: Hall MacLean , Joerger Autumn , Scott Ellen , Schachner Ben , Weber Amy M. , Nagle Luz , Blauvelt Jamie , Smeltzer Shayna , Morse Jennifer , Kidd M. Scott , Wiener Doris , Richards Allison , Rich Carolyn Jeani , Kodumudi Krithika , Beatty Matthew S. , Sarnaik Amod A. , Pilon-Thomas Shari
Published: January 2023

Immunotherapy, including adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) predominantly targets improvement in MHC Class I-mediated anti-tumor immune responses. The primary objective of this study is to better understand the role of CD4+ TIL in ACT as a complementary avenue for therapeutic efficacy. Briefly, CD4+ TIL were isolated by negative selection from metastatic melanoma patients who received TIL therapy at Moffitt Cancer Center on IRB approved protocols. Individual T cell clones were tracked by TCRbeta sequencing to quantify clonal persistence in patients. CD4+ TIL clones were found to be decidedly persistent in a candidate patient who achieved a complete response (CR) after infusion of 88% CD4+ T cells. CD4+ TIL from additional patients were stimulated with anti-CD3/CD28 in vitro and those who were clinical responders demonstrated a pleiotropic cytokine profile marked by an elevated ratio of Th1:Th2 cytokines (p=0.07, n=13). When cultured with APCs loaded with autologous tumor (AT), CD4+ TIL produced high levels of IFN-gamma in an MHC Class II-dependent manner. Induction of MHC Class II on melanoma cell lines and AT determined that CD4+ TIL secreted IFNg and TNFa directly in response to AT. ACT of tumor-reactive CD4+ TIL in immunodeficient (NSG) mice provided significant control of AT growth when compared to non-reactive CD4+ TIL. Preliminary data in syngeneic mouse models also suggests that antigen-specific CD4+ T cells aid in initial tumor rejection, memory formation and epitope spreading, resulting in an overall increased therapeutic efficacy during ACT. This data supports the conclusion that CD4+ TIL are tumor-reactive and instrumental to an effective anti-tumor immune response in cancer patients.
Super2-expressing CAR T cells generate improved anti-tumor responses against solid tumors
Authors: Brog Rachel A , Kumar Prathna , Sentman Charles , Huang Yina
Published: January 2023

Chimeric antigen receptors (CAR) augment tumor specific immune responses by redirecting all T cells towards recognition of tumor antigen. Although CAR T cell treatment shows promising therapeutic effects in leukemia patients, current CAR therapies are inadequate against solid tumors, in part from exclusion or functional exhaustion in the suppressive microenvironment. Exogenously administered Interleukin 2 (IL-2) improves efficacy and expansion of CAR T cell therapy. However, IL-2 administration is limited by high toxicity and low half-life. Super2 is an engineered IL-2 variant that binds with high affinity to the beta and gamma chains of the IL-2 receptor even without IL-2R alpha chain expression. We found that Super2 leads to increased phosphorylation of STAT-5 and more effectively activates lymphocyte populations compared to endogenous IL-2 in vitro. We hypothesized that engineering CAR T cells to express Super2 will improve their functionality within the tumor microenvironment. CAR T cell-intrinsic Super2 may also increase activation of the endogenous tumor-reactive T cells resulting in enhanced tumor control and the establishment of long-term memory even after loss of CAR T cells. To test this hypothesis, we designed CAR T cells that produce Super2 constitutively or in response to CAR stimulation. We found that Super2-expressing CAR T cells increased tumor infiltrating leukocytes (TILs) compared to CAR T cell treatment alone in immune competent murine models. Additionally, Super2-expressing CAR T cells delayed B16F10 melanoma growth in comparison to CAR T cell alone. We conclude that Super2-expressing CAR T cells provide improved protection against solid tumors.
Nutrient partitioning in the tumor microenvironment and FDG-PET imaging
Authors: Madden Matthew Z , Reinfeld Bradley I , Wolf Melissa M , Cohen Allison S , Manning H Charles , Rathmell W Kimryn , Rathmell Jeffrey C
Published: January 2023

The tumor microenvironment is composed of multiple cell types, including malignant cancer cells and tumor-infiltrating leukocytes (TIL). A possible mechanism of immunosuppression in the tumor microenvironment (TME) is cancer cells outcompeting anti-cancer TIL for nutrients such as glucose. 18F-deoxyglucose (FDG) positron emission tomography (PET) imaging is a staple of diagnosing and monitoring many types of cancer and is based on the Warburg model in which cancer cells within the tumor utilize glucose for growth and proliferation. Here, we use magnetic bead sorting to fractionate FDG-avid murine tumors and measure tumor cell-specific glucose uptake. We find that CD45+ immune cells are more FDG-avid than CD45− cancer cells. We further fractionate immune cell subsets into CD4/8+ T cells and CD11b+ myeloid cells to demonstrate that TIL T cells take up more glucose than CD45− cancer cells and resting splenic T cells. Strikingly, CD11b+ myeloid cells are the most FDG-avid cells in the TME. In MC38 colorectal cancer tumors, we show that CD11b+ F4/80 hi macrophages have high glucose uptake, and by extracellular flux analysis demonstrate higher metabolic activity than tumor T cells and CD45− cancer cells. Intriguingly, while glucose uptake is low in CD45− cancer cells, 18F-glutamine uptake is higher in cancer cells than immune cells. Our results illustrate a novel approach to measuring nutrient uptake in the TME and suggest that TIL are not starved of nutrients in the TME. Future work will determine the effects of immunotherapy and metabolism-targeted therapeutics on cell-specific glucose and glutamine uptake in the TME, as well as determine the immune contribution to cancer PET scans in the context of immunotherapy response.
Arid5a orchestrates immune evasion of pancreatic cancer via the Ido1-–mediated tryptophan metabolism
Authors: Parajuli Gyanu , Hashimoto Shigeru , Kishimoto Tadamitsu
Published: January 2023

Although pancreatic ductal adenocarcinoma (PDAC) is known to be “immunologically cold” tumors, in which the presence of immune cells and their activity are limited by the tumor microenvironment, the immunological nature of this cancer is still largely unknown. AT-rich interaction domain-containing protein 5a (Arid5a) was identified as an RNA-binding protein that plays significant roles in the development of inflammatory diseases via the augmentation of IL-6 signaling. However, the role of Arid5a in cancer remains elusive. To clarify the role of Arid5a in cancer, we used the well-established mouse PDAC model KPC cells, bearing KRAS/TP53 mutations. We found that although KPC cells lacking Arid5a (Arid5a-KO) had similar growth rates as wild-type (WT) KPC cells when subcutaneously inoculated into immunodeficient mice, Arid5a-KO cells had remarkably impaired their growth in immunocompetent mice. Here, we show that Arid5a promotes immunosuppressive tumor microenvironment by inducing the infiltration of regulatory T cells and granulocytic myeloid-derived suppressor cells; and inhibiting the activation of CD8+T lymphocytes. Interestingly, we observed that Arid5a-KO KPC cells had significantly lower expression of indoleamine 2,3-dioxygenase 1 (Ido1) compared with WT KPC cells. Mechanistically, we identified that Arid5a stabilizes Ido1 mRNA through binding to its 3′-untranslated region, and augments the production of kynurenine, which is an immunoregulatory metabolite of Ido1-mediated tryptophan catabolism. Our findings indicate Arid5a as a crucial molecule involved in the immune evasion of PDAC which can be exploited for immunotherapy in malignant pancreatic cancer.
Agonistic-CD40 therapy alters the spatial localization of immune cells in the tumor microenvironment: implications for therapeutic responsiveness
Authors: Maltez Vivien , Byrne Katelyn , Germain Ronald N
Published: January 2023

Despite major advances in immune checkpoint blockade therapies, tumor therapeutic responsiveness remains difficult to predict. This is especially true for pancreatic ductal adenocarcinoma, where total T cell numbers in the tumor proper do not predict responses to immune checkpoint blockade. Here, we use a novel congenic KPC pancreatic mouse model-derived tumor clone library that recapitulates the spectrum of tumor heterogeneity seen in patients, enabling us to perform head-to-head comparisons between therapeutically responsive and refractory tumors. Previous flow cytometry-based work with these clones revealed that tumor-derived CXCL1 is a major determinant in establishing a therapy-refractory environment, and that CXCL1 ablation enables therapeutic responses. Strikingly, we found that refractory tumors contain numerous myeloid clusters not present in responsive tumors, and that CXCL1 ablation abolishes this cluster formation. We hypothesized that these immunosuppressive myeloid clusters could critically hamper the spatial distribution of tumor infiltrating immune cells. In support of this, the spatial organization of multiple immune cell subsets, including regulatory T cells, dendritic cells, and CD8 T cells, is vastly different between responsive and refractory tumors. Upon therapeutic intervention, the spatial organization is altered in both tumor environments, with responsive tumors developing clusters of activated dendritic cells and T cell subsets, and refractory tumors developing an increased number of immunosuppressive myeloid clusters. These data suggest that therapeutic efficacy is determined by foci of local immune activity, a phenomenon that could function as a positive diagnostic biomarker.