Cell Cytokine Production Research Articles

Abstract C001: Transforming Growth Factor-β Blockade in Pancreatic Cancer Enhances Sensitivity to Combination Chemotherapy in Mice

Abstract Background: TGFβ plays a critical role in promoting pancreatic tumor progression, attenuating CD8 T cell proliferation and cytokine production, enhancing the differentiation of myofibroblasts and the deposition of extracellular matrix. However, single agent TGFβ blockade has shown limited efficacy in mouse models of pancreatic cancer. Methods: We evaluated the TGFβ- blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)- paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment. Results: Blockade of TGFβ with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFβ blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2-/-) mice. TGFβ blockade decreased total α-smooth muscle actin-positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFβ blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFβ blockade increased chemotherapy-induced cell death in vivo. Conclusions: TGFβ regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFβ blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens. This study provided the scientific rationale for the evaluation of TGFβ with both chemotherapy backbones in the clinical setting. Ongoing evaluation of samples from the stopped clinical trials will inform as to the role of TGFb in human PDAC. Citation Format: Li Qiang, Megan Hoffman, Lestat Ali, Jaime Ivan Castillo Silva, Lauren Kageler, Ayantu Temesgen, Patrick Lenehan, Jennifer Wang, Elisa Bello, Victoire Cardot-Ruffino, Giselle Uribe, Annan Yang, Michael Dougan, Andrew Aguirre, Srivatsan Raghavan5, Marc Pelletier, Viviana Cremasco, Stephanie Dougan. Transforming Growth Factor-β Blockade in Pancreatic Cancer Enhances Sensitivity to Combination Chemotherapy in Mice [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C001.

Tyrobp deficiency blocks NLRP3-mediated inflammation and pyroptosis to alleviate myocardial ischemia-reperfusion injury through regulating Syk

PurposeThe present study aims to investigate the biological function of Tyrobp in myocardial ischemia-reperfusion injury (MIRI) and to clarify its potential reaction mechanisms. MethodsAC16 cells were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate the MIRI in vitro. The cell transfection technology was used to downregulate Tyrobp, followed by assessment of cell damage, apoptosis and cytokines production via Cell Counting Kit (CCK)-8 assay, lactate dehydrogenase (LDH) release assay, TUNEL and ELISA assays, respectively. Immunofluorescence assay was performed to assess GSDMD. Corresponding proteins were detected via western blotting, and Co-immunoprecipitation (Co-IP) assay was used to validate proteins interaction. ResultsTyrobp was upregulated in OGD/R-exposed AC16 cells, and Tyrobp deficiency significantly alleviated OGD/R-caused cell viability loss, LDH release and cell apoptosis in AC16 cells. Meanwhile, Tyrobp deficiency inhibited the activation of NLRP3 inflammasome, reduced the production of cytokines and inhibited GSDMD intensity and GSDMD-N expression. Additionally, Tyrobp could interact with Syk and regulate Syk/NF-κB signaling. The rescue experiments showed that the above effects of Tyrobp deficiency on OGD/R-exposed AC16 cells were partly weakened by Syk overexpression. ConclusionTyrobp deficiency alleviated MIRI by inhibiting NLRP3-mediated inflammation and pyroptosis through regulating Syk, providing a novel target for the treatment of MIRI.

Chlamydia muridarum Causes Persistent Subclinical Infection and Elicits Innate and Adaptive Immune Responses in C57BL/6J, BALB/cJ and J:ARC(S) Mice Following Exposure to Shedding Mice.

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, few studies evaluate Cm's effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naïve C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was observed in 15 and 47 of 76 Cm-infected mice, respectively. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). Results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric Cm infection which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.

Intestinal and hepatic benefits of BBR-EVO on DSS-induced experimental colitis in mice.

Ulcerative colitis (UC), characterized by disrupted intestinal barrier integrity and chronic inflammation, was modeled in mice via dextran sulfate sodium (DSS) induction. This study explored the therapeutic potential of berberine-evodiamine (BBR-EVO), bioactive components of the traditional Chinese medicine Yulian decoction, in DSS colitis. BBR-EVO intervention ameliorated weight loss, diarrhea, colonic shortening, and histopathological damage in colitic mice. The substance increased antioxidant activity while reducing high levels of pro-inflammatory cytokines in the colon, including as TNF-α, IL-1β, and IL-6. BBR-EVO inhibited the DSS-induced decrease in the tight junction proteins ZO-1 and occludin, according to immunohistochemistry. 16S rRNA sequencing demonstrated BBR-EVO partially attenuated DSS-elicited intestinal dysbiosis, reducing opportunistic pathogens and restoring diminished beneficial taxa. Critically, BBR-EVO alleviated secondary hepatic injury in colitic mice, mitigating immune cell infiltration, oxidative stress, cytokine production, and ultrastructural damage, likely by beneficially modulating gut-liver crosstalk. This study reveals BBR-EVO, derived from a traditional Chinese medicine, confers multi-target protective effects in experimental colitis and associated hepatic pathology, warranting further evaluation as a potential therapy for inflammatory bowel diseases like UC. The mechanisms may involve simultaneous augmentation of intestinal barrier integrity, inhibition of inflammation, microbiota regulation, and gut-liver axis optimization.

Phase II trial of multi-kinase inhibitor ESK981 in patients with metastatic castration-resistant prostate cancer.

ESK981 is a potent tyrosine kinase and PIKfyve lipid kinase inhibitor. This phase II trial evaluated the efficacy of ESK981 as a single agent in patients with androgen receptor-positive (AR +) metastatic castration-resistant prostate cancer (mCRPC). Eligible patients had mCRPC with progression on AR-targeted agents and without prior chemotherapy treatment. Each patient received 160mg ESK981 once daily for 5days per week for 4weeks per cycle (except for an adverse event (AE) occurrence). The primary endpoints were a 50% reduction in prostate-specific antigen (PSA50), and safety. Secondary endpoints included the time and the duration of PSA response, PSA progression rates, PSA progression free survival (PFS) and overall survival (OS). Exploratory investigations included whole exome sequencing in patients before treatment, and morphological evaluation of biopsy samples pre- and post-treatment. PSA was evaluated in 13 patients. Only one patient (7.7% two-sided 95% Wilson CI (0.4%, 33.3%)) experienced a reduction in their PSA levels by 50% or more. The most common grade 3 treatment-related AEs were cardiac disorders, diarrhea, hypertension, alanine transaminase and aspartate transaminase elevations. No grade 4-5 events occurred. Median PFS was 1.8months, and median OS was 12.1months. Peripheral immune cells showed increased T cell activation and cytokine production in two patients who received 12-weeks of ESK981. Although relatively well tolerated, ESK981 alone showed no anti-tumor activity in patients with AR + mCRPC and its further evaluation as a single agent in AR + mCRPC is not warranted. (Trial registration: ClinicalTrials.gov, NCT03456804. Registration date: March 7, 2018).

Effects of simulated space conditions on CD4+ T cells: a multi modal analysis.

The immune system is an intricate network of cellular components that safeguards against pathogens and aberrant cells, with CD4+ T cells playing a central role in this process. Human space travel presents unique health challenges, such as heavy ion ionizing radiation, microgravity, and psychological stress, which can collectively impede immune function. The aim of this research was to examine the consequences of simulated space stressors on CD4+ T cell activation, cytokine production, and gene expression. CD4+ T cells were obtained from healthy individuals and subjected to Fe ion particle radiation, Photon irradiation, simulated microgravity, and hydrocortisone, either individually or in different combinations. Cytokine levels for Th1 and Th2 cells were determined using multiplex Luminex assays, and RNA sequencing was used to investigate gene expression patterns and identify essential genes and pathways impacted by these stressors. Simulated microgravity exposure resulted in an apparent Th1 to Th2 shift, evidenced on the level of cytokine secretion as well as altered gene expression. RNA sequencing analysis showed that several gene pathways were altered, particularly in response to Fe ions irradiation and simulated microgravity exposures. Individually, each space stressor caused differential gene expression, while the combination of stressors revealed complex interactions. The research findings underscore the substantial influence of the space exposome on immune function, particularly in the regulation of T cell responses. Future work should focus expanding the limited knowledge in this field. Comprehending these modifications will be essential for devising effective strategies to safeguard the health of astronauts during extended space missions. The effects of simulated space stressors on CD4+ T cell function are substantial, implying that space travel poses a potential threat to immune health. Additional research is necessary to investigate the intricate relationship between space stressors and to develop effective countermeasures to mitigate these consequences.

P21-76 Effects of tobacco heating products on viability, proliferation and cytokine production of mesenchymal stem cells isolated from periodontal ligament

P21-76 Effects of tobacco heating products on viability, proliferation and cytokine production of mesenchymal stem cells isolated from periodontal ligament

T cells promote distinct transcriptional programs of cutaneous inflammatory disease in human skin structural cells.

T cells and structural cells coordinate appropriate inflammatory responses and restoration of barrier integrity following insult. Dysfunctional T cell activity precipitates tissue pathology that occurs alongside disease-associated alterations of structural cell subsets, but the mechanisms by which T cells promote these changes remain unclear. We show that subsets of circulating and skin-resident CD4+ T cells promote distinct transcriptional outcomes in human keratinocytes and dermal fibroblasts that correspond with divergent T cell cytokine production. Using these transcriptional signatures, we identify T cell-dependent outcomes associated with inflammatory skin disease, including a set of Th17 cell-induced genes in keratinocytes that are enriched in the skin during psoriasis and normalized by anti-IL-17 therapy, and a skin-resident T cell-induced gene module enriched in scleroderma-associated fibroblasts. Interrogating clinical data using T cell-derived structural cell gene networks enables investigation of the immune-dependent contribution to inflammatory disease and the heterogeneous patient response to biologic therapy.

Sepsis induced dysfunction of liver type 1 innate lymphoid cells

BackgroundSepsis is a life-threatening condition triggered by uncontrolled immune responses to infection, leading to widespread inflammation, tissue damage, organ dysfunction, and potentially death. The liver plays a crucial role in the immune response during sepsis, serving as a major site for immune cell activation and cytokine production. Liver type 1 innate lymphoid cells (ILCs) consist of NK cells and ILC1s. They maintain the local immune microenvironment by directly eliminating target cells and secreting cytokines. However, the specific roles and pathological changes of liver-resident NK cells and ILC1s during sepsis remain poorly understood.ResultsThis study aims to investigate the pathological changes of NK cells and ILC1s, which might contribute the dysfunction of liver. Sepsis mouse model was established by cecal ligation and puncture (CLP). Mouse immune cells from liver were isolated, and the surface makers, gene expression profiles, cytokine response and secretion, and mitochondrial function of NK (Natural Killer) cells and ILC1s (Innate Lymphoid Cell 1) were analyzed. A significant decrease in the number of mature NK cells was observed in the liver after CLP. Furthermore, the secretion of interferon-gamma (IFN-γ) was found to be reduced in spleen and liver NK cells when stimulated by IL-18. Mitochondrial activities in both liver NK cells and ILC1 were found to be increased during sepsis, suggesting an enhanced metabolic response in these cells to combat the infection. However, despite this heightened activity, liver NK cells exhibited a decreased level of cytotoxicity, which might impact their ability to target infected cells effectively. RNA sequencing supported and provided the potential mechanisms for the proinflammatory effects and exhaustion like phenotypes of liver NK cells.ConclusionsSepsis induces dysfunction and exhaustion-like phenotypes in liver NK cells and ILC1, which might further impair other immune cells and represent a potential therapeutic target for sepsis.

Effect of docosahexaenoic acid as an anti-inflammatory for Caco-2 cells and modulating agent for gut microbiota in children with obesity (the DAMOCLE study).

Docosahexaenoic acid (DHA) is a long-chain omega-3 polyunsaturated fatty acid. We investigated the dual health ability of DHA to modulate gut microbiota in children with obesity and to exert anti-inflammatory activity on human intestinal Caco-2 cells. In a pilot study involving 18 obese children (8-14years), participants received a daily DHA supplement (500mg/day) and dietary intervention from baseline (T0) to 4months (T1), followed by dietary intervention alone from 4months (T1) to 8months (T2). Fecal samples, anthropometry, biochemicals and dietary assessment were collected at each timepoint. At preclinical level, we evaluated DHA's antioxidant and anti-inflammatory effects on Caco-2 cells stimulated with Hydrogen peroxide (H2O2) and Lipopolysaccharides (LPS), by measuring also Inducible nitric oxide synthase(iNOS) levels and cytokines, respectively. Ten children were included in final analysis. No major changes were observed for anthropometric and biochemical parameters, and participants showed a low dietary compliance at T1 and T2. DHA supplementation restored the Firmicutes/Bacteroidetes ratio that was conserved also after the DHA discontinuation at T2. DHA supplementation drove a depletion in Ruminococcaceae and Dialisteraceae, and enrichment in Bacteroidaceae, Oscillospiraceae, and Akkermansiaceae. At genus level, Allisonella was the most decreased by DHA supplementation. In Caco-2 cells, DHA decreased H2O2-induced reactive oxygen species (ROS) and nitric oxide (NO) production via iNOS pathway modulation. Additionally, DHA modulated proinflammatory (IL-1β, IL-6, IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokine production in LPS-stimulated Caco-2 cells. An improvement in gut dysbiosis of children with obesity seems to be triggered by DHA and to continue after discontinuation. The ability to modulate gut microbiota, matches also with an anti-inflammatory effect of DHA on Caco-2 cells.

Sirtuin 6 inhibits group 3 innate lymphoid cell function and gut immunity by suppressing IL-22 production.

Group 3 innate lymphoid cells (ILC3s) are enriched in the intestinal mucosa and play important roles in host defense against infection and inflammatory diseases. Sirtuin 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD+)- dependent deacetylase and has been shown to control intestinal epithelial cell differentiation and survival. However, the role of SIRT6 in ILC3s remains unknown. To investigate the role of SIRT6 in gut ILC3s, we generated SIRT6 conditional knockout mice by crossing Rorccre and Sirt6flox/flox mice. Cell number and cytokine production was examined using flow cytometry. Citrobacter rodentium infection and dextran sodium sulfate-induced colitis models were used to determine the role of SIRT6 in gut defense. RT-qPCR, flow cytometry and immunohistochemistry were used to assess the intestinal inflammatory responses. Here we show that SIRT6 inhibits IL-22 expression in intestinal ILC3s in a cell-intrinsic manner. Deletion of SIRT6 in ILC3s does not affect the cell numbers of total ILC3s and subsets, but results in increased IL-22 production. Furthermore, ablation of SIRT6 in ILC3s protects mice against Citrobacter rodentium infection and dextran sodium sulfate-induced colitis. Our results suggest that SIRT6 may play a role in ILC3 function by regulating gut immune responses against bacterial infection and inflammation. Our finding provided insight into the relation of epigenetic regulators with IL-22 production and supplied a new perspective for a potential strategy against inflammatory bowel disease.

Activation and Regulation of Indirect Alloresponses in Transplanted Patients With Donor Specific Antibodies and Chronic Rejection.

Following transplantation, human CD4+T cells can respond to alloantigen using three distinct pathways. Direct and semi-direct responses are considered potent, but brief, so contribute mostly to acute rejection. Indirect responses are persistent and prolonged, involve B cells as critical antigen presenting cells, and are an absolute requirement for development of donor specific antibody, so more often mediate chronic rejection. Novel in vitro techniques have furthered our understanding by mimicking in vivo germinal centre processes, including B cell antigen presentation to CD4+ T cells and effector cytokine responses following challenge with donor specific peptides. In this review we outline recent data detailing the contribution of CD4+ T follicular helper cells and antigen presenting B cells to donor specific antibody formation and antibody mediated rejection. Furthermore, multi-parametric flow cytometry analyses have revealed specific endogenous regulatory T and B subsets each capable of suppressing distinct aspects of the indirect response, including CD4+ T cell cytokine production, B cell maturation into plasmablasts and antibody production, and germinal centre maturation. These data underpin novel opportunities to control these aberrant processes either by targeting molecules critical to indirect alloresponses or potentiating suppression via exogenous regulatory cell therapy.

Analysis of the immunomodulatory properties of mycobacterium cell wall fraction on the cytokine production of peripheral blood mononuclear cells of healthy dogs.

Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research. To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines. Eight healthy Labrador retrievers. PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta. A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 μg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 μg/dL MCWF only at 72 h (p < 0.05). MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.

Strong capacity of differentiated PD-L1 CAR-modified UCB-CD34+ cells and PD-L1 CAR-modified UCB-CD34+-derived NK cells in killing target cells and restoration of the anti-tumor function of PD-1-high exhausted T Cells

BackgroundUsing natural killer (NK) cells to treat hematopoietic and solid tumors has great promise. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells provide an “off-the-shelf” solution.MethodsIn this study, we developed two CAR-NK cells targeting PD-L1 derived from lentiviral transduction of human umbilical cord blood (UCB)-CD34+ cells and UCB-CD34+-derived NK cells. The transduction efficiencies and in vitro cytotoxic functions including degranulation, cytokine production, and cancer cell necrosis of both resultants PD-L1 CAR-NK cells were tested in vitro on two different PD-L1 low and high-expressing solid tumor cell lines.ResultsDifferentiated CAR‑modified UCB-CD34+ cells exhibited enhanced transduction efficiency. The expression of anti-PD-L1 CAR significantly (P < 0.05) enhanced the cytotoxicity of differentiated CAR‑modified UCB-CD34+ cells and CAR-modified UCB-CD34+-derived NK cells against PD-L1 high-expressing tumor cell line. In addition, CAR-modified UCB-CD34+-derived NK cells significantly (P < 0.05) restored the tumor-killing ability of exhausted PD-1 high T cells.ConclusionConsidering the more efficient transduction in stem cells and the possibility of producing CAR-NK cell products with higher yields, this approach is recommended for studies in the field of CAR-NK cells. Also, a pre-clinical study is now necessary to evaluate the safety and efficacy of these two CAR-NK cells individually and in combination with other therapeutic approaches.Graphical abstract

Development and optimization of a diluted whole blood ELISpot assay to test immune function

Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis (‘SPIES’) is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.

The combination of sitagliptin and bee honey extract potentiates the anti-proliferative properties of 5-fluorouracil on Caco-2 cell line without detectable inflammatory events

BackgroundColon cancer originates in the colon, specifically the large intestine. It carries a poor prognosis and high mortality rate due to late diagnosis and migration. ObjectiveHere our objective was to evaluate the anticancer effects of sitagliptin (Sita) in colon cancer using Caco-2 cells. Additionally, we examined the role of bee honey extract in modulating cancer cell division and necrotic events commonly observed during drug treatments. MethodsWe monitored cell viability rate to evaluate the effect of bee honey extract compared to 5-fluorouracil (5-Fu), Sita, and their combinations. To gain further foresights into the implicated molecular interaction, we assessed the expression outline of Raf-1 and MEK, as proliferation effectors. Additionally, we examined the expression outline of p53 and Caspase 3, which are associated with programmed cell death (PCD), through western blot analysis. ResultsWe identified the Raf-1 expression pattern as a likely target for the drug combination and bee honey extract (HE), which effectively controlled colon cancer cell proliferation. Our study demonstrates that honey extract, either alone or in combination with drugs, can induce PCD by restoring the p53 and CASP-3 proteins. This was accompanied by a synergistic effect on the production of apoptotic cytokines, particularly interlukine-6 (IL-6) and IL-8, in cancer cells. Moreover, the treatment modulated the levels of pro-inflammatory cytokines, including IL-1α and IL-1β and anti-inflammatory cytokines, including IL-4 and IL-10. ConclusionOur findings shed light on honey extract and its combinations with 5-Fu and Sita in stimulating PCD and modulating cytokine production in Caco-2 cells.

Conditioned media from human adipose tissue-derived mesenchymal stem cells: potential effect on peripheral blood mononuclear cells in co-culture with HeLa cell line.

The use of mesenchymal stem cells (MSCs) for the treatment of various diseases is being investigated, however, their use in cervical cancer has not been well-studied. Here, we examined the impact of collected MSC-conditioned medium (CM) on 1 to 5 days on apoptosis, proliferation, and cytokine production of peripheral blood mononuclear cells (PBMCs) when co-cultured alongside the HeLa cell line for 24, 48, and 72h by CFSE assay, flow cytometry, and real-time PCR, respectively. We found that CMs collected on the third day of MSCs culture significantly increased the proliferation of PBMCs and decreased the proliferation of HeLa cells after 48h. CMs showed no significant effects on cell death, whereas it significantly increased the apoptosis of HeLa cells. Real-time PCR analysis showed that the presence of CM collected on the third day of MSCs culture caused a significant increase in the gene expression of IL2, IFN-γ, and TGF-β in PBMCs after 48h co-culture with HeLa cells. The data mentioned earlier demonstrate that MSC-CM can induce the growth and endurance of PBMCs while concurrently culturing HeLa cells. This observation indicates their promising potential as immunomodulatory therapies for cervical cancer cells. Nevertheless, additional investigation is imperative to comprehensively comprehend the fundamental mechanisms and refine therapeutic strategies involving PBMCs and mesenchymal stem cells.

Use of a novel proteomimetic nanoplatform for simultaneous delivery of antigens with adjuvants to impact tumor growth in multiple tumor models.

40 Background: Patient-specific cancer vaccines derived from tumor antigen have been explored as a promising therapeutic strategy, however, challenges delivering vaccine components in a coordinated fashion to elicit antitumor responses remain. To overcome these, we utilize a novel nanoplatform called the Protein-Like Polymer (PLP), which allows for sustained and targeted delivery of tumor antigens in conjunction with adjuvants. Methods: PLPs containing peptide antigens were synthesized via ROMP and characterized. A library of compounds with different sidechain linkage chemistries, degrees of polymerization (DP), and inclusion/exclusion of Oligo-ethylene glycol (OEG) were made to determine design rules for immune activation. Cell uptake and functional assays using payload-specific T Cells were conducted. Immunization in three independent tumor models was done to show generalizability. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling small molecule STING agonist, 2’3’ cGAMP. Results: Conjugating antigens to the polymer via a cleavable disulfide linkage, which reduces intracellularly in APCs, resulted in increased endosomal localization, higher levels of induced T cell proliferation, cytokine production, and expression of activation markers in CTLs and APCs. Incorporating a diluent amount of OEG side chains reduced enzymatic degradation while increasing immunogenicity and uptake. Additionally, increasing the DP, and therefore the density of antigen side chains, further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates enhanced dendritic cell activation and T-cell response only when paired with cells from their cognate system, with no activity in immune cells not expressing receptors for the payload demonstrating antigen-specificity. Mice bearing established B16F10, MC38, or TC-1 tumors treated with PLPs containing gp100, adpgk, or E7 respectively all showed increased survival times, reduced tumor burden with corresponding changes in immune cell profiles, and immunological memory upon rechallenge. Impressively, mice treated with STING-PLP complexes had significantly smaller tumors vs control at day 14 (0.038g vs 0.76g; p &lt; 0.0001) and allowed for subcutaneous administration of 2’3’ cGAMP, which traditionally requires intratumoral injection. Studies on the effects of vaccinating with pools of neoantigens multiplexed onto one PLP are ongoing. Conclusions: This work validates the ability of PLPs to overcome major limitations in cancer vaccine development. The modularity of the platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology.

NKG2D-bispecific enhances NK and CD8+ T cell antitumor immunity

BackgroundCancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required.MethodsWe identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo.ResultsHER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation.ConclusionWe demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.

AYA22A Aptamers Mitigate Peanut Allergenicity: Insights from Degranulation Assays and Modulating Immune Responses

Food allergy, particularly peanut allergy (PA), is a growing health concern affecting millions globally. PA can lead to severe reactions, including fatal anaphylaxis. Despite the availability of FDA-approved therapies like Palforzia, a cure remains elusive. Current immunotherapies show promise but lack a definitive cure. This study applies an established computational biology tool to design aptamers targeting Ara h1 and Ara h2. The in silico design aims to streamline the selection process, enabling cost-effective and rapid identification of aptamer candidates. The developed aptamers (AYA22A, including AYA22AR321, AYA22AR211, and AYA22AR524), demonstrated efficacy in inhibiting degranulation of RBL-2H3 cells (rat basophilic leukemia cell line) in vitro. They showed promise in neutralizing peanut allergen-induced immune responses. The selected aptamers inhibited degranulation in RBL-2H3 cells, addressing concerns in raw peanuts. Moreover, these aptamers demonstrated stability and effectiveness in peanut plant seeds and commercial products. Our aptamers exhibited potential in modulating immune responses associated with peanut allergy. They influenced Th1/Th2 balance, indicating a role in cytokine regulation. In vitro studies also showed the aptamers’ impact on immune cell expression and cytokine production, resembling responses observed with established immunotherapies. The findings suggest AYA22A aptamers as a potential therapeutic option for peanut allergy, providing a basis for further in vivo investigations.