CFSE Dilution Assay Research Articles

Ketone bodies rescue T cell impairments induced by low glucose availability.

Ketogenic diets are proposed as a therapeutic approach for type 1 and type 2 diabetes due to their low glucose intake. However, their potential effects on the immune system need investigation. This study aims to explore how glucose concentration and beta-hydroxybutyrate (BHB) impact T cell phenotype, metabolism, and function, with a focus on systemic inflammatory response (T2D) and autoimmunity (T1D). T cells from healthy donors were cultured in vitro under varying glucose concentrations with or without BHB. Flow cytometry was employed to analyze changes in T cell phenotype, while proliferation was evaluated through a CFSE dilution assay. Additionally, we used a novel flow cytometry method allowing a direct assessment of T cell metabolism. Culturing T cells in low glucose concentrations revealed their dependency on glucose metabolism, leading to reduced proliferation rates, overexpression of exhaustion markers and increased susceptibility to Treg suppression and the influence of immune-modulating drugs such as rapamycin, FK506, and MMF. Notably, T cells cultured in low glucose concentrations increased the expression of BDH1 to utilize BHB as an alternative fuel source. Finally, the addition of BHB to the culture effectively rescued T cell impairments caused by insufficient glucose levels. T cells display limited capacity to adapt to low glucose levels, resulting in profound functional impairment. However, T cell functions can be efficiently recovered by the presence of 2mM BHB.

Sequential radiation exposure: uncovering the potential of low dose ionizing radiation in mitigating high dose effects on immune cells

Purpose The radioadaptive response refers to a phenomenon wherein exposure to a low dose of ionizing radiation (LDIR) can induce a protective response in cells or organisms, reducing the adverse effects of a subsequent higher dose of ionizing radiation (HDIR). However, it is possible to administer the low dose after the challenge dose. This study was conducted to determine the potential mitigating effect of LDIR administered after HDIR on mice immune cells. Materials and methods Alongside the conventional adaptive response setting, one group of mice was initially exposed to HDIR and subsequently treated with LDIR. Neutrophil activation was done using DHR-reducing assay and cell proliferation was evaluated through CFSE-dilution assay in helper (CD4+) and cytotoxic (CD8+) T cells. Cytokine production by these T cell subsets was also assessed by intracellular staining using flow cytometry. Results The results of this study revealed no change in neutrophil function between any of the mice groups compared to the untreated control group. Although significant changes were not detected in the proliferation of CD4+ T cells, decreased proliferation was observed in stimulated CD8+ T cells in the HDIR group. In contrast to IFN-ɣ, which showed no evident change in either of the T cell subsets after stimulation, IL-4 was rigorously dropped in stimulated CD4+ T cells in the HDIR group. Conclusions In summary, the results of this study indicated that the administration of LDIR to mice before HDIR was not able to reduce the detrimental effects of HDIR in our experimental setting. Instead, we observed a mitigating effect of LDIR when administered after the challenge dose. This suggests that not only the dose and duration but also the order of LDIR relative to HDIR affects its efficacy.

Immunogenicity and In Vivo Protective Effects of Recombinant Nucleocapsid-Based SARS-CoV-2 Vaccine Convacell®.

The vast majority of SARS-CoV-2 vaccines which are licensed or under development focus on the spike (S) protein and its receptor binding domain (RBD). However, the S protein shows considerable sequence variations among variants of concern. The aim of this study was to develop and characterize a SARS-CoV-2 vaccine targeting the highly conserved nucleocapsid (N) protein. Recombinant N protein was expressed in Escherichia coli, purified to homogeneity by chromatography and characterized by SDS-PAGE, immunoblotting, mass spectrometry, dynamic light scattering and differential scanning calorimetry. The vaccine, formulated as a squalane-based emulsion, was used to immunize Balb/c mice and NOD SCID gamma (NSG) mice engrafted with human PBMCs, rabbits and marmoset monkeys. Safety and immunogenicity of the vaccine was assessed via ELISA, cytokine titer assays and CFSE dilution assays. The protective effect of the vaccine was studied in SARS-CoV-2-infected Syrian hamsters. Immunization induced sustainable N-specific IgG responses and an N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys, an N-specific CD4+/CD8+ T cell response was observed. Vaccinated Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower lung weight relative to the body, and faster body weight recovery. Convacell® thus is shown to be effective and may augment the existing armamentarium of vaccines against COVID-19.

Abstract 4168: BALB/c-CD3EDG HuGEMMTM mouse model for evaluation of CD3-targeted tumor immunotherapy

Abstract Introduction: T cell immune response plays critical roles in cancer immunotherapy. CD3 complex, composed of CD3ε, CD3δ and CD3γ chains, is an essential component of TCR-CD3 complex mediating TCR signaling. Targeting CD3 complex has therefore become a popular strategy for manipulation of T cell function. Clinically relevant animal models for proper evaluation of human CD3 antibodies is in great demand in the field. Currently, adoptive transfer of human CD3+ T cells or transplantation of human hematopoietic stem/precursor cells to immunodeficient mice are usually employed. However, these mouse models are not fully immunocompetent. In this study we have evaluated in vitro CD3 EDG functionality and in vivo therapeutic efficacy, in BALB/c-hCD3EDG knock-in mouse in which all the three components of the CD3 complex — Cd3ε, Cd3δ, and Cd3γ— are replaced by their human counterparts, CD3E, CD3D, and CD3G. Methods: A humanized CD3EDG mouse model (HuGEMM) on BALB/c background was generated by replacing the entire mouse CD3EDG with its human counterpart through ES cell-based gene targeting. The cell surface expression of CD3E and intracellular expression of CD3D and CD3G were confirmed by FACS. Naïve CD3+ T cells were isolated from the splenocytes of homozygous CD3EDG HuGEMM and activated by anti-hCD3 antibody. T cell proliferation was demonstrated by CFSE dilution assay. To validate the TCR functionality of humanized CD3EDG in vivo, we evaluated bispecific antibody anti-hCD3/hEpCAM5 in BALB/c-CD3EDG HuGEMM bearing CT26-hEpCAM5. Results: The expression of human CD3E, CD3D and CD3G were confirmed by flow cytometry. hEpCAM5 antigen expression was confirmed in CT26 cell line in vitro and tumor in vivo by flow cytometry. The TCR functionality was detected by in vitro T cell activation assays using anti-hCD3 antibody. In vivo efficacy was demonstrated using a bispecific antibody that simultaneously binds to human CD3 and human tumor associated antigen EpCAM5 in human CD3 EDG knock-in mice engrafted with CT26-hEpCAM5 tumor. The therapeutic efficacy of the bispecific antibody showed 32% tumor growth inhibition compared to control treatment. No significant body weight loss was recorded in any of the animals. Conclusion: This study showed the successful establishment of humanized CD3 knock-in BALB/c mouse models providing a full immunocompetent model for human CD3-targeted tumor immunotherapies evaluation. Citation Format: Demi Xiaojuan Liu, Kaixia Lian, Xuefei Yan, Jie Lin, Lei Zheng, Annie Xiaoyu An, Xiaoxi Xu, Ludovic Bourre, Jingjing Wang. BALB/c-CD3EDG HuGEMMTM mouse model for evaluation of CD3-targeted tumor immunotherapy. [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 4168.

Isolation and in vitro Expansion of Regulatory T Cells from β-Thalassemia Major Kids and in vitro Evaluation of Their Plasticity and Inhibitory Effects in the Presence of Pro-Inflammatory Cytokines

Introduction: Graft-versus-host disease (GvHD) is a life-threatening syndrome commonly associated with hematopoietic stem cell transplantation (HSCT). Preventing the incidence of GVHD after HSCT along with minimizing long-term immunosuppression is currently under investigation with regulatory T cells (Tregs). As Tregs are a low-frequency population and the yield of all memory Tregs is not sufficient for clinical application, an initial Treg expansion is essential. Methods: Thirty milliliters of peripheral blood from the β-thalassemia major (beta-TM) patients and healthy controls were obtained and Tregs were isolated using MACS. Isolated cells were cultured in the presence of rapamycin and rIL-2 followed by activated with anti-CD3/CD28-coated beads. To evaluate Treg plasticity, expanded Tregs were cultured in a medium containing IL1β, IL6, TGFβ, and IL2, with or without 500 nM rapamycin for 72 h. To assess the functional properties of Tregs, CFSE dilution assays were performed to evaluate the ability of in vivo expanded Tregs from beta-TM patients. Statistical analysis was performed using paired t-test and independent t-test, with the aid of SPSS version 12.0. p-value ≤0.05 was considered significant. Results: The percentage of Tregs isolated from the control group was significantly higher than the Tregs isolated from patients (p-value = 0.01), which is probably due to the iron overload in beta-TM patients as a result of continuous blood transfusion. Also, the percentage of Tregs after 5 days of expansion had a significant increase in both groups compared to before expansion (p-value = 0.03). Our results also showed that the expansion of Tregs after 72 h in the presence of inflammatory cytokines and in the absence of rapamycin led to the increase in the intracellular expression of IL-17 (p-value = 0.01), while intracellular expression of IL-17 remained low following the addition of 100 nM rapamycin to the culture medium (pvalue = 0.073). The results of the functional evaluation of expanded Tregs showed relatively differences in both patient and control groups. Thus, expanded Tregs inhibited the proliferation of responder T cells in a dose-dependent manner in the control group (p-value = 0.028), while in the patient group this inhibitory effect was not significant (p-value = 0.055). Conclusion: Tregs isolated from beta-TM patients have poorer inhibitory performance than Tregs isolated from healthy individuals. Also, we concluded that rapamycin stabilizes the Treg population by inhibiting the production of IL-17, all necessitating the administration of appropriate immunosuppressive drugs in patients receiving Treg therapy.

Combine and conquer: manganese synergizing anti-TGF-\u03b2/PD-L1 bispecific antibody YM101 to overcome immunotherapy resistance in non-inflamed cancers

BackgroundOur previous work showed that the anti-TGF-β/PD-L1 bispecific antibody YM101 effectively overcame anti-PD-L1 resistance in immune-excluded tumor models. However, in immune-desert models, the efficacy of YM101 was limited. Bivalent manganese (Mn2+) is identified as a natural stimulator of interferon genes (STING) agonist, which might enhance cancer antigen presentation and improve the therapeutic effect of YM101.MethodsThe effect of Mn2+ on STING pathway was validated by western blotting and enzyme-linked immunosorbent assay. Dendritic cell (DC) maturation was measured by flow cytometry. The synergistic effect between Mn2+ and YM101 in vitro was determined by one-way mixed lymphocyte reaction, CFSE dilution assay, and cytokine detection. The in vivo antitumor effect of Mn2+ plus YM101 therapy was assessed in CT26, EMT-6, H22, and B16 tumor models. Flow cytometry, RNA-seq, and immunofluorescent staining were adopted to investigate the alterations in the tumor microenvironment.ResultsMn2+ could activate STING pathway and promote the maturation of human and murine DC. The results of one-way mixed lymphocyte reaction showed that Mn2+ synergized YM101 in T cell activation. Moreover, in multiple syngeneic murine tumor models, Mn2+ plus YM101 therapy exhibited a durable antitumor effect and prolonged the survival of tumor-bearing mice. Relative to YM101 monotherapy and Mn2+ plus anti-PD-L1 therapy, Mn2+ plus YM101 treatment had a more powerful antitumor effect and a broader antitumor spectrum. Mechanistically, Mn2+ plus YM101 strategy simultaneously regulated multiple components in the antitumor immunity and drove the shift from immune-excluded or immune-desert to immune-inflamed tumors. The investigation in the TME indicated Mn2+ plus YM101 strategy activated innate and adaptive immunity, enhanced cancer antigen presentation, and upregulated the density and function of tumor-infiltrating lymphocytes. This normalized TME and reinvigorated antitumor immunity contributed to the superior antitumor effect of the combination therapy.ConclusionCombining Mn2+ with YM101 has a synergistic antitumor effect, effectively controlling tumor growth and prolonging the survival of tumor-bearing mice. This novel cocktail strategy has the potential to be a universal regimen for inflamed and non-inflamed tumors.

Assessment of commonly used methods to determine myelin-reactivity of T cells in multiple sclerosis

Many studies have analyzed myelin-reactivity of T cells in multiple sclerosis (MS); however, with conflicting results. In this study we compare methods to determine myelin reactivity of T cells and aim to delineate the cause of inconsistency in the literature. Challenging T cells with myelin antigens we found a significant increase in antigen-reactivity of T cells from patients with MS using an ELISpot-assay, in contrast to a CFSE-dilution assay. Comparing the two assays showed that the myelin-reactive T cells detected in the ELISpot-assay originated primarily from effector memory T cells in contrast to the myelin-reactive T cells of the CFSE-assay representing a population of both naïve, central memory and effector memory T cells. This diversity in T cell populations activated in the two assays likely contribute to the discrepancy found in the literature and encourages thorough considerations when choosing an assay to determine antigen-specificity of T cells in future studies.

Both in vitro T cell proliferation and telomere length are decreased, but CD25 expression and IL-2 production are not affected in aged men.

IntroductionAging is a natural process involving dysfunction of multiple organs and is characterized by increased susceptibility to infections, cancer and autoimmune diseases. The functionality of the immune system depends on the capacity of lymphocytes to proliferate in response to antigenic challenges, and telomere length has an important role regulating the number of cell divisions. The aim of this study was to determine the possible relationship between telomere length, interleukin 2 (IL-2) production, CD25 expression and proliferation of peripheral blood mononuclear cells (PBMCs) in aged men.Material and methodsTelomere length was measured by RT-PCR in PBMCs from young and aged men. IL-2 production and CD25 expression were determined by ELISA and flow cytometry, respectively. Cell proliferation was measured by CFSE dilution assays upon in vitro stimulation with concanavalin A (Con A).ResultsPBMCs from aged men showed a shorter telomere length and a reduced capacity to proliferate in vitro, compared to young men. In contrast, no significant differences in the level of CD25 expression on T lymphocytes, and in vitro production of IL-2 were detected in both groups. In addition, no significant correlation was detected between levels of CD25 expression, IL-2 production, cell proliferation, and telomere length in aged men.ConclusionsIn aged men the telomere length shortening and the reduced T cell proliferation are not related to the capacity of IL-2 production and CD25 expression on T lymphocytes.

Abstract 6209: Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages

Abstract Background: Macrophages are multipurpose phagocytes with various functions. Neutrophil extracellular traps (NETs) are web-like structures ensnare microbes which were composed of dsDNA, histones, and antimicrobial peptides. NETs are generally degraded by DNAse and macrophages, however, it is unknown how the degradation of NETs affects the local immune response thereafter. Here, we investigated the interaction between NETs and peripheral blood monocytes (PBMo). Method: 5 × 105 Polymorphonuclear leukocytes(PMNs), PMNs irradiated with UV for 15 minutes (UV-PMN) and PMNs stimulated by PMA for 15min and extensively washed (PMA-PMN) were placed on 6 well plate and cultured for 2 hours and NETs formation was visualized by SYTOX green. Peripheral blood was obtained from healthy volunteers and CD14+ PBMo were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with those PMNs at 37°C in 5% CO2 conditions for 3 days. The differentiation of macrophage was evaluated by the expression of M1/M2 markers by flowcytometry. Cytokine production was evaluated using the Cytokine Array system. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 mAb and the effect on T cell proliferation was evaluated with CFSE dilution assay. Results: PMA-PMN, but not PMN or UV-PMN, produced massive SYTOX(+) NETs within 2 hours. Co-culture of the NETs with PBMo stained with PKH26 showed NETs are phagocytosed by monocytes within 24 hours. PBMo co-cultured with PMA-PMN showed significantly enhanced expression of CD86, and reduced expression of CD163 as compared to those co-cultured with PMNs or UV-PMN. However, the expression of CD86 was suppressed if PMA-PMN were pretreated with 100 u/ml DNase I. The supernatant of the co-culture with PMA-PMN showed markedly increased levels of IL-1β and TNF-α. PBMo co-cultured with PMA-PMN strongly promote the proliferation of autologous T cells. However, the enhanced T cell growth were significantly suppressed by the pretreatment of NETs with DNase I. Conclusion: Monocytes appear to differentiate to proinflammatory macrophages after the phagocytosis of NETs. In tumor-bearing host, NETs are known to be increased in circulating blood, which may promote tumor associated inflammatory process. Citation Format: Akira Saito, Hideyuki Ozawa, Kazuya Takahashi, Yuki Kimura, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Naohiro Sata, Joji Kitayama. Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6209.

Induction of different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells treated with various doses of 177Lu-Nimotuzumab

Introduction Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. 177Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of 177Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells. Materials and methods Nimotuzumab labeled with 177Lu was characterized by SE-HPLC. Specificity of 177Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of 177Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR. Results 177Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with 177Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of 177Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of 177Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of 177Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of 177Lu-Nimotuzumab dose. Conclusion 177Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by 177Lu-Nimotuzumab with therapeutic gains.

247-OR: ADA Presidents’ Select Abstract: Detection of T-Cells Reactive to Hybrid Insulin Peptides in Subjects at Risk for Type 1 Diabetes

Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-gamma ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-gamma ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells in 3 longitudinal samples collected 6 months apart. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative response - but not inflammatory - is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, and our data may indicate that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity. Disclosure R.L. Baker: None. T. Delong: None. M. Rewers: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. K.M. Haskins: None. Funding American Diabetes Association (1-15-ACE-14 to T.D.); JDRF (2-SRA-2017-511-S-B); National Institutes of Health (R21AI133059-01A1)

Detection of T cells Reactive to Hybrid Insulin Peptides in Subjects at Risk for Type 1 Diabetes

Abstract Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-g ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-g ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative (but not inflammatory) response is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, suggesting that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity.

B cell-mediated activation and proliferation of NK cells : crossroads of adaptive and innate immunity

Abstract Belonging to innate immunity, natural killer (NK) cells constitute of circulating lymphocytes which provide a body’s first line of defense against infection and cancer. In the spleen,, NK cells are likely to interact with B cells, but the functional consequence of this interaction remains uncertain. To address this question, we co-cultured primary NK cells with freshly isolated B cells from the spleen and investigated the influence of B cells on NK cells. Here we show that co-presence of B cells in cultures facilitated proliferation of NK cells, as measured CFSE dilution assays. CFSE results showed that proliferation of NK cells by B cells need direct and in direct stimulation. This correlated with microarray data showing that expression of genes involved in the regulation of cell proliferation was significantly increased in NK cells in NK-B co-cultures. NK cells cultured with B cells exhibited higher expression of CD69, B220, CD25 activation markers, suggesting that B cells drastically facilitated NK cell activation. Lack of B cells in mice weakened anti-tumor effect as well as anti-pathogen response, suggesting that NK cells exhibited higher anti-tumor activity in the presence of B cells. The synergistic effect of B cells on NK cell functions also occurred in humans. Given the fact that NK cells can exert a regulatory role in cancer or infection, co-presence of B cells can potentiate the role of NK cells by promoting their proliferation and cytotoxicity.

Cordyceps militaris Induces Immunogenic Cell Death and Enhances Antitumor Immunogenic Response in Breast Cancer.

Cordyceps militaris has been widely used as a traditional medicine in East Asia. Its effects against breast cancer have been reported previously. However, whether C. militaris-induced breast cancer cell death is immunogenic remains unelucidated. This study aimed to determine whether ethanolic extracts of C. militaris (CM-EE) could induce immunogenic cell death (ICD) in breast cancer immunotherapy to improve the efficacy of immune checkpoint inhibitors. Human and mouse breast cancer cells were treated with various concentrations of CM-EE for 72 h, and cytotoxicity was measured using the sulforhodamine B assay. Flow cytometry was used to assess cell death with annexin V/7-AAD staining and measure the surface exposure of damage-associated molecular pattern (DAMP) molecules including calreticulin, HSP70, and HSP90. Western blot for cleaved poly (ADP-ribose) polymerase (PARP) was used to confirm apoptotic cell death. The immunogenicity of CM-EE-induced dead cells was evaluated using the CFSE dilution assay. CM-EE reduced the viability of human (MCF7, MDA-MB-231, HS578T, and SKBR3) and mouse (4T1-neu-HA, TUBO-HA, and TUBO-P2J-HA) breast cancer cells. The IC50 was 25–50 µg/ml in human breast cancer cells and 10–50 µg/ml in mouse breast cancer cells at 72 h. CM-EE-treated breast cancer cells were positively stained by annexin V, cleaved PARP, and cleaved caspase 3/7 which were increased upon CM-EE treatment. Surface exposure of DAMP molecules was increased in dose- and time-dependent manners. The CFSE dilution assay revealed that dendritic cells fed with CM-EE-treated breast cancer cells successfully stimulated tumor-specific T cell proliferation without inhibiting DC function and T cell proliferation. The expression of PD-L1 mRNA and protein level was increased in dose-dependent manners. In addition, CM-EE also potentiated the cytotoxic activity of tumor-specific T cells. CM-EE can induce immunogenic and apoptotic cell death in breast cancer cells, and it is a good candidate for cancer immunotherapy and may improve the efficacy of immune checkpoint inhibitors.

BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection.

Background: Previously, we have found that blockade of PD-1/PD-Ls pathway could enhance CD4+ T cells-mediated protective immunity in patients with active tuberculosis (ATB). However, the mechanism of PD-1/PD-Ls pathway involved in negative regulation of anti-TB immunity has been still unclear. Recently, the study of human immunodeficiency virus (HIV) infection demonstrated that PD-1 could induce the expression of basic leucine zipper ATF-like transcription factor (BATF) to inhibit CD8+ T cell function. While the mechanism of immune regulation of BATF in Mycobacterium tuberculosis (M. tb) infection has not yet been elucidated.Methods: We enrolled 104 participants including ATB patients (n = 66), latent tuberculosis infection (LTBI) (n = 16) and healthy control (HC) (n = 22). The expressions of BATF in peripheral blood CD4+ and CD8+ T cells from enrolled subjects were determined using flow cytometry. Intervention with PD-1/PD-Ls pathway was performed by using blocking antibodies or human PD-L1 fusion protein. Silencing BATF in peripheral blood mononuclear cells (PBMCs) by electroporation with siRNA. Real-time quantitative PCR, CFSE dilution assay and enzyme linked immunosorbent assay (ELISA) were employed to test T cell functions after BATF knockdown.Results: The percentages of BATF+CD4+ (P = 0.0003 and P < 0.0001, respectively) and BATF+CD8+ (P = 0.0003 and P = 0.0003, respectively) cells were significantly increased in ATB patients compared with LTBI and HC. BATF-expressing PD-1+ T cells in CD4+ and CD8+ T cells were much higher in ATB group than those in LTBI group (P = 0.0426 and 0.0104, respectively) and HC group (P = 0.0133 and 0.0340, respectively). There was a positive correlation between BATF expression and PD-1 expression in ATB patients (for CD4+ T cells, r = 0.6761, P = 0.0158; for CD8+ T cells, r = 0.6104, P = 0.0350). BATF knockdown could enhance IL-2 and IFN-γ secretions (P = 0.0485 and 0.0473, respectively) and CD4+ T cells proliferation (P = 0.0041) in vitro.Conclusions: In the context of tuberculosis, BATF mediates negative regulation of PD-1/PD-Ls pathway on T cell functions. BATF knockdown can improve cytokine secretion and cells proliferation in vitro.

Abstract 516: Metformin inhibits monocyte differentiation to tumor associated macrophage (TAM) and may restore anti-tumor immunity

Abstract Background: Tumor-associated macrophages (TAMs) play pivotal roles to regulate tumor cell behavior in tumor microenvironment and a high density of TAMs is associated with poor prognosis in various cancers. Recent studies have demonstrated that metformin, the first-line medication for the treatment of type 2 diabetes, have suppressive effects on tumor growth. Here, in this study, we investigated the effect of metformin on monocyte differentiation to TAMs. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and CD14+ peripheral blood monocytes(PBMo) were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with Panc-1, a human pancreas cancer cell, with metformin (10μM~10mM) using double chamber inserts of 0.4-μm sized pores at 37°C in 5% CO2 conditions for 5 days. Separately, PBMo were cultured with M-CSF for 5 days and then with IL-10 (20ng/ml)+TGF-β(20ng/ml) for addition 2 days with or without metformin. The differentiation to TAM were evaluated by the expression of M1/M2 markers as well as PD-L1/L2 by flowcytometry. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 and anti-CD3 and anti-CD28 microbeads and the effect on T cell proliferation was evaluated with CFSE dilution assay. Results: PBMo co-cultured with Panc-1 showed significantly enhanced expression of CD163, CD206, as well as markedly increased side scatter, suggesting the differentiation to M2-type macrophages. The macrophage also highly expressed PD-L1 and strongly inhibited the proliferation of autologous T cells. However, the expression of these antigens and inhibition of T cell growth were significantly suppressed by the presence of metformin with dose dependent manner. Metformin was effective at 100μM and almost nullified the effects of Panc-1 at 1mM. Metformin did not affect the antigen expression pattern during the treatment by M-CSF, but did downregulate the expression of CD206 and PD-L1 if added together with IL-10 +TGF-β. Conclusion: During the co-culture with tumor cells, metformin suppresses the monocyte differentiation to TAM at relatively late stage. Metformin can restore T cell mediated-tumor immunity in tumor microenvironment, at least partly, through the effects on TAM, that may result in tumor inhibition. Citation Format: Akira Saito, Hideyuki Ohzawa, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Satomi Shiba, Homare Ito, Naohiro Sata, Joji Kitayama. Metformin inhibits monocyte differentiation to tumor associated macrophage (TAM) and may restore anti-tumor immunity [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 516.

IL-7-induced proliferation of peripheral Th17 cells is impaired in HAART-controlled HIV infection.

Th17 cells are key regulators of functional immunity in mucosal tissues, including the gut-associated lymphoid tissue (GALT), an important site of immune impairment in HIV infection. During HIV infection, Th17 cells are lost in large numbers from the GALT. Despite the recovery of peripheral CD4 T cells that accompanies suppression of viral replication with HAART, Th17 cells in GALT are not completely restored. IL-7 is essential for the survival and proliferation of T cells, but its signaling through its receptor IL-7Rα (CD127), is impaired in CD8 T cells and thymocytes during HIV infection. We set out to determine if decreased CD127 expression or impaired CD127 signaling may be the cause of Th17 impairment in HAART-controlled HIV infection. Healthy and HIV donors on HAART were selected for this study of Th17 cell function in HIV. Peripheral CD4 T cells and Th17 cells were isolated using magnetic beads, then stimulated with IL-7. CD127 expression and the phosphorylation of signaling molecules was determined using flow cytometry. Proliferation was determined with a CFSE dilution assay. CD127 was not decreased on Th17 cells from HAART-controlled HIV individuals, in fact, the percentage of Th17 cells that express CD127 was increased in treated HIV individuals. Furthermore, Th17 cells from HAART-controlled individuals, have normal IL-7-induced STAT5 and Bcl-2 responses, but vastly decreased proliferative responses. This reduced IL-7 responsiveness may explain the lack of Th17 cell recovery and ongoing systemic immune activation that persists despite well treated HIV infection.

Novel Methods to Quantitate and Identity Dividing Platelets in Culture Conditions

Background: Unlike dividing cells, platelets are usually regarded as unproductive in that they are anuclear fragments of megakaryocyte and incapable of the canonical cell cycle. However, recent evidence showed that platelets could form progeny or at least undergo fission. The observation indicated the existence of a non-hematopoietic mechanism to determine platelet count in peripheral blood. The assessing the kinetic aspects of platelet division is not easy because automated cell counters often give equivocal results after culture not supporting those previous findings. Manual chamber counting is impractical because of the difficulty in differentiating singlets from any small sized clumps.Objective: We examined platelet division under in-vitro culture conditions to assess how many platelets and how fast they divide.Methods: We devised a method to track platelet division based on flow cytometry and fluorescent microscopic image. Briefly, buffered human platelet suspension was prepared and stained with cell tracking dyes followed by sorting and resuspension in cell culture medium and incubation. Platelet division was assessed quantitatively by observing a decrease in fluorescent intensity by flow cytometry after culture in a liquid medium or counting the number of single colored platelet doublets after culture in hydrogel medium.Results: After 6-hour culture of CFSE labelled platelet in M199 medium, we observed the appearance of a separate cluster of platelets with reduced CFSE intensity adjacent to the original platelet cluster, as is the typical finding of CFSE dilution assay for lymphocyte proliferation. We continued the culture for 20 hours. The division fraction of strongly labeled platelets was 48.7 % (range: 27.5 - 70.4) after 6 hours and 85.6 % (79.1 - 92.0) after 20 hours. For weakly labelled platelets, the division fraction after 6 hours was 35.0 % (24.6 - 43.4) and 75.5 % (34.5 - 97.0) after 20 hours. The division fractions at the two time points were much higher than expected and the effect of external stimuli such as shearing force exposure during the sorting procedure was to be considered. In another assay, we divided platelet suspension into two and stained with labelling dyes of different colors (CFSE and FarRed). The two fractions were mixed and cultured in hydrogel medium. At the beginning of the culture and after 6 hours, we counted platelet singlets and doublets in ten randomly selected fields of con focal microscopy. Single colored doublets can form both by division and contact. Those with two colors can form only by contact. The count of single- and two-colored doublets can be estimated by binary probability prediction if the platelets don't divide. Initially, the doublet counts follow the binary probability prediction (P: 0.65 - 0.88), but after 6 hours, single colored doublets were observed more frequently than two colored doublets (P<0.01, in the order of 10-6). The division was suppressed by treatment with taxol (P =0.26), nocodazole (P=0.38), and cytochalasin D (P=0.61). From the counted numbers of doublets and singlets of the two colors, we could derive a formula for the platelet division fraction. The division fraction of FarRed stained platelets were 8.3 % (95% CI: 2.2 - 14.4), and that of the CFSE stained platelets were 11.9 % (5.4 - 18.4).Conclusion: We introduce novel methods to assess platelet division credibly in culture conditions which can be easily combined with functional studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Andrographolide inhibits breast cancer through suppressing COX-2 expression and angiogenesis via inactivation of p300 signaling and VEGF pathway

BackgroundAndrographolide (Andro), a diterpenoid lactone, has been used for treatment of various cancers with less adverse effects. However, the underlying mechanisms regarding its anti-tumor mechanism still remain unclear.MethodsCell viability and proliferation were measured by CCK8 and CFSE dilution assay. The localization of p50/p65 or cytochrome c was determined using confocal immunofluorescence. Streptavidin-agarose pulldown or ChIP assays were used to detect the binding of multiple transactivators to COX-2 promoter. The promoter activity was examined by a dual-Luciferase reporter assay. The functions of Andro on COX-2-mediated angiogenesis were also investigated using human HUVEC cells through tube formation and spheroids sprouting assay. The in vivo anti-tumor efficacy of Andro was analyzed in xenografts nude mice.ResultsThe results indicated that Andro could significantly inhibit the proliferation of human breast cancers, and suppress COX-2 expression at both protein and mRNA levels. Furthermore, Andro could dose-dependently inhibit COX-2-mediated angiogenesis in human endothelial cells. We have also found that Andro significantly promoted the activation of cytochrome c and activated caspase-dependent apoptotic signaling pathway. Our further explorations demonstrated that Andro inhibited the binding of the transactivators CREB2, C-Fos and NF-κB and blocked the recruitment of coactivator p300 to COX-2 promoter. Moreover, Andro could effectively inhibit the activity of p300 histone acetyltransferase (HAT), thereby attenuating the p300-mediated acetylation of NF-κB. Besides, Andro could also dramatically inhibit the migration, invasion and tubulogenesis of HUVECs in vitro. In addition, Andro also exhibited effective anti-tumor efficacy as well as angiogenesis inhibition in vivo.ConclusionIn current study, we explore the potential effects of Andro in suppressing breast cancer growth and tumor angiogenesis, as well as the precise mechanisms. This work demonstrated the potential anti-cancer effects of Andro, indicating that Andro could inhibit COX-2 expression through attenuating p300 HAT activity and suppress angiogenesis via VEGF pathway, and thereby could be developed as an antitumor agent for the treatment of breast cancer.

Subchondral bone derived mesenchymal stem cells display enhanced osteo-chondrogenic differentiation, self-renewal and proliferation potentials.

Rabbit mesenchymal stem cells (MSCs) are important seed cells in regenerative medicine research, particularly in translational research. In the current study, we showed that rabbit subchondral bone is a reliable source of MSCs. First, we harvested subchondral bone (SCB) from the rabbit knee-joint and initiated the MSC culture by cultivating enzyme-treated SCB. Adherent fibroblast-like cells that outgrew from SCB fulfill the common immuno-phenotypic criteria for defining MSCs, but with low contamination of CD45+ hematopoietic cells. Interestingly, differentiated SCB-MSCs expressed osteogenic and chondrogenic markers at significantly higher levels than those in bone marrow cell suspension-derived MSCs (BMS-MSCs) (P<0.05). No differences in the expression of adipogenic markers between SCB-MSC and BMS-MSC (P>0.05) were observed. Moreover, the results of the colony forming unit-fibroblast assay and sphere formation assay demonstrated that the SCB-MSCs had increased self-renewal potential. SCB-MSCs expressed higher levels of the stemness markers Nanog, OCT4, and Sox-2 compared to in BMS-MSCs (P<0.05). Furthermore, the results of both the CCK-8-based assay and CFSE dilution assay showed that SCB-MSCs exhibited enhanced proliferative capacity. In addition, SCB-MSCs exhibited higher phosphorylation of extracellular signal-related kinase/mitogen-activated protein kinase signaling, which is closely related to MSC proliferation. In conclusion, we identified SCB-MSCs as a novel stem cell population that met the requirements of MSCs; the unique properties of SCB-MSC are important for the potential treatment of tissue damage resulting from disease and trauma.