Th1-type Chemokines Research Articles

The Potential Role of Moringa oleifera Lam. Leaf Proteins in Moringa Allergy by Functionally Activating Murine Bone Marrow-Derived Dendritic Cells and Inducing Their Differentiation toward a Th2-Polarizing Phenotype.

Moringa oleifera leaves are an inexpensive substitute for staple foods. Despite limited data, Moringa oleifera leaf protein (Mo-Pr) may be allergenic in BALB/c mice. In mouse models and allergic patients, dendritic cells (DCs) may be involved in food allergy. In addition, some allergens, including food allergens, can directly activate DCs and induce Th2 polarization. We investigated whether Mo-Pr can modulate the functional profile of murine bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs were obtained from mouse bone marrow cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then treated with lipopolysaccharide (LPS) or Mo-Pr. BMDC phenotypes were evaluated via flow cytometry, cytokine production was assessed using ELISA, the expression of key genes was studied using qRT-PCR, the effects on T-cell differentiation were investigated using mixed lymphocyte reaction (MLR), and transcriptional changes in BMDCs were investigated using RNA-Seq. Mo-Pr-specific IgE was investigated in recipient serum after BMDC transfer. Mo-Pr treatment significantly induced BMDC maturation, increased the expression of CD80/86 and MHC II, resulted in the production of IL-12 and TNF-α, and induced T-cell differentiation. Mo-Pr treatment stimulated BMDCs' expression of the Th2 promoters OX40L and TIM-4, induced the production of the Th2-type chemokines CCL22 and CCL17, and decreased the Th1/Th2 ratio in vitro. Healthy recipients of Mo-Pr-treated BMDCs produced Mo-Pr-specific IgE.

A Critical Study on the Synthetic and Herbal Medication Pattern in the Management of Psoriasis

Abstract:: Psoriasis is a persistent skin illness that causes dense, shimmering flakes, irritable, dry, and red patches as a result of the fast buildup of skin cells. It is an autoimmune skin condition that is incurable, non-contagious, and relapsing-remitting. Typically, a remission phase lasts one to twelve months. Psoriasis is caused by a number of cellular mechanisms, and it has been hypothesised that T lymphocytes, keratinocytes, allergen cells, Langerhans cells, natural killer cells, phagocytes, a number of Th1-type chemokines, in addition to growth factors like endothelial proliferation factor and keratinocytes growth factor, among others, are key players in the development of psoriasis. Numerous synthetic medicinal drugs have been documented to have the side effects of psoriasis. Due to the safety and accessibility, herbal medications may hold promise as possible anti-psoriatic molecules. Before creating a potential herbal drug, it is important to thoroughly examine the key players in the evolution of psoriasis, such as T-cell stimulation, transportation, and cytokinase inhibition. The purpose of this review is to investigate how psoriasis spreads and becomes activated, as well as how certain medications might exacerbate the condition and how certain plant resources may be used to treat psoriasis. To create a potent, secure, and dependable treatment, more scientific research on these herbal resources is required.

Parnassin, a Novel Therapeutic Peptide, Alleviates Skin Lesions in a DNCB-Induced Atopic Dermatitis Mouse Model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease which requires continuous treatment due to its relapsing nature. The current treatment includes steroids and nonsteroidal agents targeting inflammation but long-term administration causes various side effects such as skin atrophy, hirsutism, hypertension and diarrhea. Thus, there is an unmet need for safer and effective therapeutic agents in the treatment of AD. Peptides are small biomolecule drugs which are highly potent and remarkably have less side effects. Parnassin is a tetrapeptide with predicted anti-microbial activity curated from Parnassius bremeri transcriptome data. In this study, we confirmed the effect of parnassin on AD using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical administration of parnassin improved skin lesions and symptoms in AD mice, such as epidermal thickening and mast cell infiltration, similar to the existing treatment, dexamethasone, and did not affect body weight, or the size and weight of spleen. In TNF-α/IFN-γ-stimulated HaCaT cells, parnassin inhibited the expression of Th2-type chemokine CCL17 and CCL22 genes by suppressing JAK2 and p38 MAPK signaling kinases and their downstream transcription factor STAT1. Parnassin also significantly reduced the gene expression of TSLP and IL-31, which are pruritus-inducing cytokines. These findings suggested that parnassin alleviates AD-like lesions via its immunomodulatory effects and can be used as a candidate drug for the prevention and treatment of AD because it is safer than existing treatments.

Abstract 3269: Reappraisal of immunologic mechanisms of hypomethylating agents in treatment of high-risk myelodysplastic syndrome and acute myeloid leukemia

Abstract Hypomethylating agents (HMA) have been used for the treatment of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML) of the elderly. In addition to direct cytotoxic effect of HMA on MDS/AML, HMA upregulates effector T-cell function by demethylating T-cell exhaustion-associated genes (Hazem, Cell 2017) and promoting effector T-cells trafficking to tumor microenvironment by Th1-type chemokines reactivation (Peng, Nature 2019). To elucidate dynamic changes of immune cells profile and gene expression after HMA treatment in MDS/AML, we conducted single cell RNA sequencing of three sequential bone marrow (BM) samples from one HR-MDS patient treated with azacitidine. In BM samples at diagnosis (‘diagnosis’), after 2 cycles of azacitidine (BM blasts increased from 6 to 11%; ‘progression’), and after 3 additional cycles of azacitidine (BM blasts decreased to 3.5% with trilineage hematopoiesis; ‘response’), cell clusters were identified according to immune cell types. The relative proportion of the NK-cell cluster showed the most significant increase up to response (1.3% at ‘diagnosis’, 4.3% at ‘progression’, and 11.8% at ‘response’). By contrast, the CD8 T-cells cluster showed modest increase of proportion at the 3 timepoints (2.7%, 3.9%, and 7%, respectively). Differentially Expressed Gene (DEG) analysis showed CXCR4 was overexpressed in the BM NK-cell cluster at the ‘response’ time point. To determine whether the NK cells can be trafficked to BM by HMA treatment in mouse MDS/AML models, mouse myeloid leukemia cells (C1498 cells) were transplanted into the tail vein of 6-to-8-week aged syngeneic C57/BL6 mice. C1498 bearing mice were intraperitoneally treated with HMA (decitabine) or vehicle for 5 consecutive days. While HMA inhibited in vivo AML proliferation, the quantity of NK cells (NK1.1+) was significantly increased in BM of HMA-treated mice than vehicle controls. In addition, CXCR4 expression in NK1.1+ cells from BM of HMA-treated mice were significantly up-regulated, compared with vehicle controls. The NK depletion in HMA-treated AML mice decreased anti-leukemic effect, suggesting that NK cells may be one of immune cell subsets responsible for HMA induced anti-leukemia effect. Taken together, recruitment of NK cells into MDS/AML BM through CXCR4 overexpression and anti-leukemic cytotoxicity exerted by NK cells can be one of the important immunologic mechanisms of HMA in MDS/AML. Treatment strategy of recruiting more NK cells BM by HMA and maximizing cytotoxicity of NK cells should be further investigated. Citation Format: Junshik Hong, Suji Min, Jihyun Park, Carly M. Fielder, Qianni Hu, Sung-Soo Yoon, Tae Kon Kim. Reappraisal of immunologic mechanisms of hypomethylating agents in treatment of high-risk myelodysplastic syndrome and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3269.

NOD2 Agonism Counter-Regulates Human Type 2 T Cell Functions in Peripheral Blood Mononuclear Cell Cultures: Implications for Atopic Dermatitis.

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host-pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.

NMR-Based Metabolomic Imprinting Elucidates Macrophage Polarization of THP-1 Cell Lines Stimulated by Zinc Oxide Nanoparticles.

Zinc oxide (ZnO) nanoparticles (NPs) have been widely used in industry, cosmetics, drugs, bioimaging, and drug delivery. ZnO NPs have been found to interact and interfere with cellular physiology via macrophages, thereby resulting in macrophage polarization. The functional reprogramming of the cells is synchronized through cellular metabolic adaptations. The current study, therefore, aims to establish crosstalk between ZnO-NP-induced metabolic alterations and macrophage polarization in PMA-activated THP-1 cell lines. We observed moderate to heightened cytotoxic response in terms of cell viability and proliferation. The results also revealed increased Th1-type cytokine and chemokine expression. In order to characterize the changes in metabolite concentration in treatment groups, we employed multivariate data analysis (principal component analysis and partial least-squares discriminant analysis) of 1H NMR spectra. The results revealed biologically relevant patterns and alterations in many metabolic pathways. These alterations and patterns were found to be in line across the immune-cytotoxic axis. Furthermore, the results also implicate the role of carbon metabolism toward the classical activation of macrophage polarization. The omics approach could identify the markers involved in NP-induced toxicity, thus elaborating our vision of cytotoxicity that is currently limited to end-point and cytokine assays. Also, it could be emphasized that metabolic reconfiguration upon NP stimulation could direct macrophage polarization toward classical activation.

Pre-Clinical Autoimmunity in Lupus Relatives: Self-Reported Questionnaires and Immune Dysregulation Distinguish Relatives Who Develop Incomplete or Classified Lupus From Clinically Unaffected Relatives and Unaffected, Unrelated Individuals.

Systemic lupus erythematosus (SLE) is propelled by pathogenic autoantibody (AutoAb) and immune pathway dysregulation. Identifying populations at risk of reaching classified SLE is essential to curtail inflammatory damage. Lupus blood relatives (Rel) have an increased risk of developing SLE. We tested factors to identify Rel at risk of developing incomplete lupus (ILE) or classified SLE vs. clinically unaffected Rel and healthy controls (HC), drawing from two unique, well characterized lupus cohorts, the lupus autoimmunity in relatives (LAUREL) follow-up cohort, consisting of Rel meeting <4 ACR criteria at baseline, and the Lupus Family Registry and Repository (LFRR), made up of SLE patients, lupus Rel, and HC. Medical record review determined ACR SLE classification criteria; study participants completed the SLE portion of the connective tissue disease questionnaire (SLE-CSQ), type 2 symptom questions, and provided samples for assessment of serum SLE-associated AutoAb specificities and 52 plasma immune mediators. Elevated SLE-CSQ scores were associated with type 2 symptoms, ACR scores, and serology in both cohorts. Fatigue at BL was associated with transition to classified SLE in the LAUREL cohort (p≤0.01). Increased levels of BLyS and decreased levels of IL-10 were associated with type 2 symptoms (p<0.05). SLE-CSQ scores, ACR scores, and accumulated AutoAb specificities correlated with levels of multiple inflammatory immune mediators (p<0.05), including BLyS, IL-2Rα, stem cell factor (SCF), soluble TNF receptors, and Th-1 type mediators and chemokines. Transition to SLE was associated with increased levels of SCF (p<0.05). ILE Rel also had increased levels of TNF-α and IFN-γ, offset by increased levels of regulatory IL-10 and TGF-β (p<0.05). Clinically unaffected Rel (vs. HC) had higher SLE-CSQ scores (p<0.001), increased serology (p<0.05), and increased inflammatory mediator levels, offset by increased IL-10 and TGF-β (p<0.01). These findings suggest that Rel at highest risk of transitioning to classified SLE have increased inflammation coupled with decreased regulatory mediators. In contrast, clinically unaffected Rel and Rel with ILE demonstrate increased inflammation offset with increased immune regulation, intimating a window of opportunity for early intervention and enrollment in prevention trials.

Oleanolic Acid Alleviates Atopic Dermatitis-like Responses In Vivo and In Vitro.

Oleanolic acid (OA) is a pentacyclic triterpenoid, abundantly found in plants of the Oleaceae family, and is well known for its beneficial pharmacological activities. Previously, we reported the inhibitory effect of OA on mast cell-mediated allergic inflammation. In this study, we investigated the effects of OA on atopic dermatitis (AD)-like skin lesions and its underlying mechanism of action. We evaluated the inhibitory effect of OA on AD-like responses and the possible mechanisms using a 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD animal model and tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated HaCaT keratinocytes. We found that OA has anti-atopic effects, including histological alterations, on DNCB-induced AD-like lesions in mice. Moreover, it suppressed the expression of Th2 type cytokines and chemokines in the AD mouse model and TNF-α/IFN-γ-induced HaCaT keratinocytes by blocking the activation of serine-threonine kinase Akt, nuclear factor-κB, and the signal transducer and activator of transcription 1. The results demonstrate that OA inhibits AD-like symptoms and regulates the inflammatory mediators; therefore, it may be used as an effective and attractive therapeutic agent for allergic disorders, such as AD. Moreover, the findings of this study provide novel insights into the potential pharmacological targets of OA for treating AD.

Abstract 1706: Hydrogel encapsulated TLR9 agonists show sustained tumor growth inhibition and prolong survival of CT26 tumor-bearing mice

Abstract CpG-oligodeoxynucleotides (CpG ODNs) are known ligands of innate immune receptor, toll-like receptor 9 (TLR9). Activation of TLR9 by its ligands leads to stimulation of an immune cascade represented by secretion of Th1-type cytokine and chemokine milieu, including IFN-α and activation of immune cell surface markers. TLR9 agonists have a number of therapeutic applications. Currently, several TLR9 agonists are in late stage clinical trials in combination with checkpoint inhibitors (CPI) in cancer patients. In these trials, TLR9 agonists are commonly administered once a week for several weeks, suggesting that they may be degraded in the local tissues or dissipated from the site of action rapidly requiring once a week dosing. To improve the stability, delivery, and durability of TLR9 agonist activity with reduced frequency of administration, we encapsulated TLR9 agonists in injectable hydrogels containing poloxamer. TLR9 agonist/hydrogel formulations can be used to slow release of TLR9 agonist following peritumoral/intratumoral or by other routes of administration. In here, we have studied TLR9 agonists with and without encapsulation in hydrogels for tumor growth inhibition and toxicity in syngeneic tumor models in mice. In CT26 colon cancer model, peritumoral and intratumoral administration of TLR9 agonist in five doses in 12 days without hydrogel encapsulation showed potent tumor growth inhibition and increased survival of tumor bearing mice compared with mice injected with vehicle. Peritumoral administration of encapsulated TLR9 agonist in two different types of hydrogels, one that releases the TLR9 agonist at slower rate or the other that releases little faster at the same total dose in single or two doses, respectively, showed similar levels of tumor growth inhibition up to 11 days as that of the TLR9 agonist dosed five times without hydrogel formulation. Both hydrogel formulations prolonged survival of tumor bearing mice to a similar extent. Moreover, the mice administered with hydrogel encapsulated TLR9 agonist showed lower body weight loss compared with unformulated TLR9 agonist. Benefit of hydrogel encapsulation in tumor bearing mice has been confirmed with a second TLR9 agonist as well. Taken together, the current results demonstrate that hydrogel encapsulated TLR9 agonists are released slowly over a time, resulting in sustained tumor growth inhibition with minimal effect on body weight. The encapsulation of TLR9 agonist in hydrogels may permit less frequent dosing in patients. The hydrogel encapsulation can also be expanded to other therapeutic modalities of oligonucleotides such as antisense, siRNA and gene editing technologies. Citation Format: Ekambar R. Kandimalla, Jun Jiang, Lokendrakumar C. Bengani, Xinyu Huang, Jason Zhang. Hydrogel encapsulated TLR9 agonists show sustained tumor growth inhibition and prolong survival of CT26 tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1706.

Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

BackgroundAmplification of the MYCN oncogene is a molecular hallmark of aggressive neuroblastoma (NB), a childhood cancer of the sympathetic nervous system. There is evidence that MYCN promotes a non-inflamed and...

Global inhibition of the interaction of NK inhibitory receptors with MHC-I augments coordinated innate and adaptive immunity against cancer metastasis.

Abstract Natural Killer (NK) cells are innate lymphocytes involved in the first line of immune defense and T cell adaptive immunity against viral infection and cancer, including metastasis. While on patrol, NK cells contact other cells and recognize MHC-I Ags via stochastically expressed MHC-I–specific inhibitory receptors (Ly49s in mice and KIRs in humans) that prevent NK cell activation via cytoplasmic ITIM. The binding site on MHC-Class-I for Ly49 inhibitory receptors is distinct from that for TCRs. The loss of MHC-I expression on tumor cells (“missing self”) abrogates inhibitory signals, resulting in NK activation. Global inhibition of the NK inhibitory receptor interactions in vivo by a pan-anti-MHC-I monoclonal antibody markedly activated IFNg-producing NK cells, independent of Fc receptors. NK cell-derived IFNg, along with other cytokines (MCSF &amp; FLT3L), primed APC to induce IL-12/-15/-18/-21 cytokine cascades and enhanced levels of MHC-I and MHC-II expression that further drove the proliferation of NK cells and memory phenotype (MP) T cells. The global disruption of NK cell/MHC-I interactions significantly enhanced Th1 type signature transcription factors (Tbet &amp; Eomes), cytokines (IFNg &amp; Granzyme B), and chemokine receptors (CXCR3) on NK and MP T cells. Administration of pan-anti-MHC-I to unmanipulated mice profoundly augmented innate and adaptive immunity against viral infection, PD1-resistant transplanted tumors, and successfully constrained lung and liver metastasis, and protected the animals from tumor burden. Moreover, in vitro enhancement of human NK cell proliferation by a pan-anti-HLA Mab suggests that pan-anti-HLA could be a promising therapeutic strategy against chronic viral infection and cancer metastasis.

14402 Serum levels of thymus and activation-regulated chemokine in patients with psoriasis: Higher in patients with generalized pustular psoriasis

Thymus and activation-regulated chemokine (TARC) is designated a TH2 type chemokine. Serum TARC levels sharply reflect the disease activity of atopic dermatitis. The previous studies reported that serum TARC levels in patients with psoriasis vulgaris (PsV) were comparable with those in healthy controls, however, we sometimes encounter psoriasis patients with high serum levels of TARC. The association of clinical severity of psoriasis with serum TARC levels has not been investigated yet. Furthermore, to date, serum TARC levels in patients with psoriatic arthritis (PsA) or generalized pustular psoriasis (GPP) have never been reported. Thereby, we investigated the association of serum TARC levels with psoriasis by types of psoriasis, and examined correlations of serum TARC levels with clinical severity scores and other results of blood tests. Psoriasis patients visiting our hospital from April 2013 to April 2018 were included in this study. All data were collected retrospectively from the patient charts. Data on 75 patients (51 men and 24 women; PsV 30 patients, PsA 29 patients, GPP 16 patients) were analyzed. The serum TARC levels in patients with GPP were significantly higher than those in PsV and those in PsA. There was significantly positive correlation between serum TARC levels and PASI scores. In conclusion, our study revealed that serum TARC levels were high in patients with GPP. Furthermore, serum TARC levels can potentially be one of biomarkers reflecting the severity or systemic inflammation caused by psoriasis in patients with psoriasis, not so much as in patients with atopic dermatitis.

Histone demethylase UTX/KDM6A enhances tumor immune cell recruitment, promotes differentiation and suppresses medulloblastoma.

The deregulation of epigenetic pathways has been implicated as a critical step in tumorigenesis including in childhood brain tumor medulloblastoma. The H3K27me3 demethylase UTX/KDM6A plays important roles in development and is frequently mutated in various types of cancer. However, how UTX regulates tumor development remains largely unclear. Here, we report the generation of a UTX-deleted mouse model of SHH medulloblastoma that demonstrates the tumor suppressor functions of UTX, which could be antagonized by the deletion of another H3K27me3 demethylase JMJD3/KDM6B. Intriguingly, UTX deletion in cancerous cerebellar granule neuron precursors (CGNPs) resulted in the impaired recruitment of host CD8+ T cells to the tumor microenvironment through a non-cell autonomous mechanism. In both mouse medulloblastoma models and in human medulloblastoma cells, we showed that UTX activates Th1-type chemokines, which are responsible for T cell migration. Surprisingly, our results showed that the depletion of cytotoxic CD8+ T cells did not affect mouse medulloblastoma growth. Nevertheless, the UTX/chemokine/T cell recruitment pathway we identified may be applied to many other cancers and may be important for improving cancer immunotherapy. In addition, UTX is required for the expression of NeuroD2 in precancerous progenitors, which encodes a potent proneural transcription factor. Overexpression of NEUROD2 in CGNPs decreased cell proliferation and increased neuron differentiation. We showed that UTX deletion led to impaired neural differentiation, which could coordinate with active SHH signaling to accelerate medulloblastoma development. Thus, UTX regulates both cell-intrinsic oncogenic processes and the tumor microenvironment in medulloblastoma. Our study provides insights into both medulloblastoma development and context dependent functions of UTX in tumorigenesis.

Lactobacillus johnsonii-activated chicken bone marrow-derived dendritic cells exhibit maturation and increased expression of cytokines and chemokines in vitro

Lactobacillus species are typical members of gut microflora that immunomodulatory effects and can regulate a variety of immune cells, such as dendritic cells (DCs). Notably, DCs possess the unique ability to initiate primary immune responses. Notably, DCs possess the unique ability to initiate primary immune responses. In this study, we investigated the effects of Lactobacillus johnsonii (L. johnsonii) on the maturation and activation of chicken bone marrow-derived dendritic cells (chBM-DCs). The chBM-DCs generated from chicken bone marrow monocytes were stimulated using lethally irradiated L. johnsonii. L. johnsonii-stimulated chBM-DCs upregulated the expression of major histocompatibility complex class II (MHC-II), CD40, and CD86, decreased phagocytosis, and increased the ability to induce the proliferation of allogeneic T cells, which displayed a mature phenotype and function. Upon maturation with L. johnsonii, the expression of Th1-type cytokines [interleukin (IL)-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)], a Th2-type cytokine (IL-10), pro-inflammatory cytokines (IL-1β and IL-6), and chemokines (CXCLi1 and CXCLi2) greatly increased; however, a high expression of IL-10 was only observed at mid-late time points for chBM-DCs stimulated with high doses of L. johnsonii. Moreover, L. johnsonii upregulated the mRNA levels of TLR2 and TLR5. These results reveal that L. johnsonii plays a potentially important role in modulating the immunological functions of chBM-DCs, suggesting that it influences and mediates immune responses in vitro.

LncRNA SATB2-AS1 inhibits tumor metastasis and affects the tumor immune cell microenvironment in colorectal cancer by regulating SATB2

BackgroundEmerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in colorectal cancer (CRC). Here, we report a lncRNA, SATB2-AS1, which is specifically expressed in colorectal tissue and is significantly reduced in CRC. We systematically elucidated its functions and possible molecular mechanisms in CRC.MethodsLncRNA expression in CRC was analyzed by RNA-sequencing and RNA microarrays. The expression level of SATB2-AS1 in tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). The functional role of SATB2-AS1 in CRC was investigated by a series of in vivo and in vitro assays. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), chromatin isolation by RNA purification (ChIRP), Bisulfite Sequencing PCR (BSP) and bioinformatics analysis were utilized to explore the potential mechanisms of SATB2-AS1.ResultsSATB2-AS1 is specifically expressed in colorectal tissues and downregulated in CRC. Survival analysis indicates that decreased SATB2-AS1 expression is associated with poor survival. Functional experiments and bioinformatics analysis revealed that SATB2-AS1 inhibits CRC cell metastasis and regulates TH1-type chemokines expression and immune cell density in CRC. Mechanistically, SATB2-AS1 directly binds to WDR5 and GADD45A, cis-activating SATB2 (Special AT-rich binding protein 2) transcription via mediating histone H3 lysine 4 tri-methylation (H3K4me3) deposition and DNA demethylation of the promoter region of SATB2.ConclusionsThis study reveals the functions of SATB2-AS1 in CRC tumorigenesis and progression, suggesting new biomarkers and therapeutic targets in CRC.

Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy.

Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates interferon γ (IFNγ)-JAK-STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration and cancer immunity. IFNγ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased TH1-type chemokines and tumor-infiltrating lymphocytes (TILs) and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and TILs, enabling tumors to evade anti-tumor immunity and to resist anti-PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its co-factor, ascorbate/vitamin C, increased chemokine and TILs, leading to enhanced anti-tumor immunity and anti-PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFNγ-JAK-STAT-TET signaling pathway that mediates tumor response to anti-PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy and patient response to anti-PD-1/PD-L1 therapy, and stimulating TET activity as an adjuvant immunotherapy of solid tumors.

Abstract 1490: Novel microbiome-derived peptides activate the host innate immune system by regulation of TLR signaling

Abstract The gut microbiome is a key contributor to the maintenance of host physiology. At the same time, increasing evidence implicates microbiome dysbiosis as a key determinant of numerous metabolic and inflammatory disorders. It is, therefore, of paramount importance to understand the interactions between the host and gut microbiota. Second Genome has developed a proprietary, bioinformatics-driven discovery platform to identify novel microbiome-derived peptides with the potential to modulate host immune responses. We have leveraged this platform to evaluate immunomodulatory functions of novel, secreted peptides derived from gut-resident bacterial species Bifidobacterium longum and Bifidobacterium breve. Previously, the microbial genus Bifidobacterium (‘Bifido’) has been shown to be associated with enhanced dendritic cell functions and antitumor effects. Here, we identify Bifido-derived peptides that are putative agonists of certain murine and human Toll-like receptors (TLRs). These peptides induce secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by in vitro-generated mouse dendritic cells and peritoneal macrophages in a TLR4 and CD14-dependent manner. One of the peptides, SG-A, also induces secretion of Th1-type cytokines and chemokines such as IL-21, GRO-α, and IP-10 by human monocytes and monocyte-derived dendritic cells. Moreover, in vivo administration of SG-A in mice elicited a systemic pro-inflammatory immune response in the absence of any secondary stimulus. Collectively, our results demonstrate the ability of a novel bacterial-derived peptide to engage the host innate immune system. Moreover, our findings validate the potential of the Second Genome discovery platform for identification of novel microbiome-derived agents that: i) have the ability to modulate host cell functions; and ii) may be developed into therapeutic agents for immune and inflammatory disorders. Citation Format: Dhwani Haria, Helena Kiefel, Yuliya Katlinskaya, Sunit Jain, Thomas Weinmaier, Shoko Iwai, Todd DeSantis, Toshi Takeuchi, Karim Dabbagh, Kareem Graham. Novel microbiome-derived peptides activate the host innate immune system by regulation of TLR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1490.

CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth.

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells.

Dual Synthetic Peptide Conjugate Vaccine Simultaneously Triggers TLR2 and NOD2 and Activates Human Dendritic Cells.

Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells, and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam3CSK4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam3CSK4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNγ, IL-2, and TNFα. Together, these data indicate that the dual MDP-SLP-Pam3CSK4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.

Cancer epigenetics and effector T cells in immunotherapy and chemotherapy.

17 Background: The tumor microenvironment is comprised of immune cells that have been reprogrammed by active tumor-mediated processes to defeat tumor-specific immunity and promote tumor growth in a highly effective manner. Previous studies have helped define the nature of immune responses in the tumor microenvironment, and provide new insights into designing novel immune therapies to target the immune suppressive mechanisms including Tregs and inhibitory B7 family members (e.g. PD-L1/PD-1) and treat patients with cancer. However, the mechanisms controlling tumor immune phenotype and biological phenotype, and their relevance in cancer therapy are not well understood. Methods: Using human colon and ovarian cancer as our models, we have studied the cross-talk between immune cell subsets and tumor/stem cells, and between immunogenic and oncogenic pathways in the tumor microenvironment, and its impact on tumor immunity and therapy. Results: In patients with colon and ovarian cancer as our models, we demonstrate that Th22 cells and myeloid derived suppressor cells (MDSCs) regulate tumor stemness via epigenetic mechanisms. Furthermore, we found that histone modification and DNA methylation repress the tumor production of Th1-type chemokines, and subsequently determine effector T cell trafficking to the tumor microenvironment, affect tumor progression, and therapeutic efficacy of checkpoint blockade and adoptive T cell transfusion, chemotherapy and patient outcome. Conclusions: Epigenetic silencing of Th1-type chemokine is a novel tumor immune evasion mechanism. Selective epigenetic reprogramming alters T cell landscape in cancer and may enhance clinical efficacy of cancer therapy.