Mouse Spleen Cells Research Articles

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells.

To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action. This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively. GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+ and CD45+CD4+ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05). GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

Intranasal immunisation with recombinant Toxoplasma gondii uridine phosphorylase confers resistance against acute toxoplasmosis in mice.

Toxoplasmosis is caused by Toxoplasma gondii, which infects all warm-blooded animals, including humans. Currently, control measures for T. gondii infection are insufficient due to the lack of effective medications or vaccines. In this paper, recombinant T. gondii uridine phosphorylase (rTgUPase) was expressed in Escherichia coli and purified via Ni2+-NTA agarose. rTgUPase was inoculated intranasally into BALB/c mice, and the induced immune responses were evaluated by mucosal and humoral antibody and cytokine assays and lymphoproliferative measurements. Moreover, the protective effect against the T. gondii RH strain infection was assessed by calculating the burdens of tachyzoites in the liver and brain and by recording the survival rate and time. Our results revealed that mice immunised with 30μg rTgUPase produced significantly higher levels of secretory IgA (sIgA) in nasal, intestinal, vaginal and vesical washes and synthesised higher levels of total IgG, IgG1 and, in particular, IgG2a in their blood sera. rTgUPase immunisation increased the production of IFN-gamma, interleukin IL-2 and IL-4, but not IL-10 from isolated mouse spleen cells and enhanced splenocyte proliferation in vitro. rTgUPase-inoculated mice were effectively protected against infection with the T. gondii RH strain, showing considerable reduction of tachyzoite burdens in liver and brain tissues after 30 days of infection, and a 44.29% increase in survival rate during an acute challenge. The above findings show that intranasal inoculation with rTgUPase provoked mucosal, humoral and cellular immune responses and indicate that rTgUPase might serve as a promising vaccine candidate for protecting against toxoplasmosis.

Transcriptomic analysis of spleen B cell revealed the molecular basis of bursopentin on B cell differentiation

The bursa of Fabricius, the acknowledged humoral immune organ unique to birds, plays a vital role in B cell development. Bursopentin (BP5) derived from the bursa is reported to induce the development and formation of B cells. However, the mechanism of BP5 on B cell differentiation is still unclear. In this paper, total B lymphocytes from mice immunized with H9N2 subtype AIV vaccine were stimulated with BP5. The results show that BP5 at the experimental dosages promoted B cell differentiation, including the total B cells, activated B cells, differentiated B cells, mature B cells and plasma cells. Then, the in vivo immune experiment proved that the percentages of activated and differentiated B cells from mice immunized with AIV vaccine and 0.25 mg/mL BP5 were increased. To investigate the molecular mechanism of BP5 on B cell differentiation, the gene expression profiles of B cells purified from the spleen cells of mice immunized with AIV vaccine and BP5 were detected following RNA sequencing technology. The results show that BP5 at 0.05 and 0.25 mg/mL induced the enrichment of various biological functions, and stimulated five common significant enrichment pathways in B cells from the immunized mice. Additionally, 120 and 59 differentially expressed genes (DEG) represented transcriptional factors in B cells following 0.05 and 0.25 mg/mL BP5 immunization, respectively. In summary, these results suggest that BP5 regulates various gene expression involved in regulation of B cell development, which provides the knowledge required for additional studies on B cell differentiation in response to bursal-derived peptides and also provides an important experimental basis for improving vaccine immunity.

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Eukaryotic expression and immunogenicity of Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes human and animal intestines, causing malnutrition and iron-deficiency anemia. Calreticulin is a multifunctional protein involved in all stages of parasitic infection. Studies have found that parasites can secret calreticulin to regulate the host's immune response. To explore the immunogenicity of the eukaryotic expression plasmid of Ancylostoma ceylanicum calreticulin (Ace-CRT), we constructed a recombinant Ace-CRT eukaryotic expression plasmid (pEGFP-N3-Ace-CRT). Successful expression of the target protein in Human Embryonic Kidney (HEK) 293T cells was confirmed by indirect immunofluorescence and Western blot analysis. BALB/c mice were immunized with pEGFP-N3-Ace-CRT plasmid. Measuring IgG antibody levels in immunized mice sera by ELISA showed that the recombinant plasmid stimulated IgG antibody production in mice. Spleen lymphocytes were collected from vaccinated mice to determine the proportion of T cell subsets and the expression levels of cytokines. Flow cytometry revealed that the percentage of CD3+CD4+ and CD3+CD8+ T cells in mice spleen in the immunization group was significantly higher than that in the control group. Recombinant plasmid immunization increased IL-4, IL-10, IL-12, and IL-13 expression while decreasing IL-5, IL-6, and INF-γ in mice spleens. These results indicate that the eukaryotic plasmid constructed in this study had good immunogenicity and mainly induced a T helper 2 response in the host, laying a foundation for screening candidate molecules for anti-hookworm vaccines.

Antitumor impact of amygdalin on adaptive immune response in BALB/c mice with breast cancer

Amygdalin is a potential therapeutically target in cancer. The main purpose of this experimental study was to evaluate the therapeutic effect of amygdalin in the mice model of breast cancer. The percentages of CD4, CD8 T lymphocyte, intracellular IFN-γ, and Granzyme B were assessed in spleen cells of tumorized mice treated with 50 and 150 mg/kg of amygdalin (AG 50 and AG 150 ). The expression of caspase 3 and p53 , tumor size, and survival rate of Balb/c mice was determined in tumor tissue after amygdalin administration. No significant difference was observed in the frequency of CD4+ and CD8+ T cells in the three study groups. However, a significantly increased level of granzyme B in CD8+ T cells, as well as a significant decrease in the level of IL-10 in CD4+ T cells was detected in the AG 50 group compared to the AG 150 . There was no significant difference in the expression of caspase 3 and P53 between the two groups. A significant change was seen in tumor size and survival rate of AG 50 and AG 150 groups compared to the controls. Our findings indicated that antitumor effect of amygdalin in vivo was probably due to stimulating the effective immune response, not apoptotic genes induction.

Renoprotective anti-CD45RB antibody induces B cell production in systemic lupus erythematosus based on single-cell RNA-seq analysis

ObjectiveSystemic lupus erythematosus (SLE) is a multisystem autoimmune disease that commonly affects the kidney. Single-cell RNA sequencing (scRNA-seq) technology is a powerful tool for characterizing individual cells and elucidating biological mechanisms at the cellular level. The purpose of this study was to identify the mechanism underlying kidney injury in SLE using scRNA-seq technology.Methods: scRNA-seq data of peripheral blood mononuclear cells (PBMCs) in SLE were retrieved from the GEO database, followed by batch effect elimination, dimensionality reduction, cluster analysis, cell annotation and enrichment analysis. A model of SLE was developed in NZB/WF1 mice. Effects of anti-CD45RB antibody on the SLE-induced kidney injury were evaluated, and we measured the distribution of regulatory T cells and B cells in mouse spleen and kidney tissues, levels of kidney function-related indexes, deposition of IgG and C3 in the glomeruli, and the levels of inflammatory cytokines. ResultsCD45RB was a specific marker gene of B cell clusters and had influence on the B cells. anti-CD45RB antibody treatment induced regulatory B cells and consequently arrested the kidney injury caused by SLE. In addition, depletion of regulatory T cells was found to partially undermine the alleviatory effect of anti-CD45RB antibody on SLE-induced kidney injury. ConclusionCollectively, our data suggest that anti-CD45RB antibody can prevent the SLE-induced kidney injury, pointing to anti-CD45RB antibody as a potential therapeutic strategy in kidney injury-related disease.

Differential effects of periodontal microbiome on the rheumatoid factor induction during rheumatoid arthritis pathogenesis

Association between exposure to periodontal bacteria and development of autoantibodies related to rheumatoid arthritis (RA) has been widely accepted; however, direct causal relationship between periodontal bacteria and rheumatoid factor (RF) is currently not fully understood. We investigated whether periodontal bacteria could affect RF status. Patients with preclinical, new-onset, or chronic RA underwent periodontal examination, and investigation of subgingival microbiome via 16S rRNA sequencing. Degree of arthritis and RF induction was examined in collagen-induced arthritis (CIA) mice that were orally inoculated with different periodontal bacteria species. Subsequently, single-cell RNA sequencing analysis of the mouse spleen cells was performed. Patients with preclinical RA showed an increased abundance of the Porphyromonadacae family in the subgingival microbiome compared to those with new-onset or chronic RA, despite comparable periodontitis severity among them. Notably, a distinct subgingival microbial community was found between patients with high-positive RF and those with negative or low-positive RF (p=0.022). Oral infections with the periodontal pathogens P. gingivalis and Treponema denticola in CIA mice similarly enhanced arthritis score, but resulted in different levels of RF induction. Genes related to B cell receptor signaling, B cell proliferation, activation, and differentiation, and CD4+ T cell costimulation and cytokine production were involved in the differential induction of RF in mice exposed to different bacteria. In summary, periodontal microbiome might shape RF status by affecting the humoral immune response during RA pathogenesis.

Immunogenicity and antigenicity of a conserved fragment of the rhoptry-associated membrane antigen of Plasmodium vivax

BackgroundPlasmodium vivax rhoptry-associated membrane antigen (RAMA) is a glycophosphatidylinositol-anchored membrane protein currently under consideration as a malaria vaccine candidate. Immunoglobulin G (IgG) antibodies induced by P. vivax RAMA (PvRAMA) have been proved to persist over 12 months in the sera of people infected with P. vivax. It has also been shown that through stimulation of peripheral blood mononuclear cells with PvRAMA in vitro, the antigen can induce CD4+ T cells to produce interleukin-10. However, the genetic diversity of the RAMA gene in isolates of P. vivax (pvrama) and the immunogenicity of PvRAMA in animals remain unclear.MethodsGenomic DNA was extracted from blood samples (n = 25) of patients in Jiangsu Province, China with imported infections of P. vivax from endemic countries in South and Southeast Asia. The extract genomic DNA was used as templates to amplify the P. vivax rama gene (pvrama) by PCR, and the PCR products were then sequenced and analyzed by the DnaSP, MEGA, and GeneDoc software packages. Recombinant PvRAMA (rPvRAMA) protein was expressed and purified, and then used to immunize mice. Levels of total IgG and different IgG subclasses of rPvRAMA-immunized mice were evaluated by enzyme-linked immunosorbent assay. Also, spleen cells of rPvRAMA-immunized mice were stimulated with rPvRAMA in vitro and levels of T cells were measured by flow cytometry.ResultsThe average pairwise nucleotide diversity (π) of the pvrama gene was 0.00190, and the haplotype diversity (Hd) was 0.982. The C-terminal of PvRAMA showed lower haplotype diversity compared to the N-terminal and was completely conserved at amino acid sites related to erythrocyte binding. To further characterize immunogenicity of the C-terminal of PvRAMA, mice were immunized with rPvRAMA antigen. The rPvRAMA protein induced antibody responses, with the end-point titer ranging from 1:10,000 to 1:5,120,000. IgG1 was the predominant IgG subclass in rPvRAMA-immunized mice, followed by IgG2b. In addition, levels of CD4+ and CD8+ T cells in the rPvRAMA-stimulated group were significantly higher than those in the phosphate-buffered saline-stimulated group (normal control group).ConclusionsThe high conservation at specific amino acid sites and the high immunogenicity of the C-terminal of PvRAMA indicate the presence of conserved epitopes able to generate broadly reactive humoral and cellular immune responses. These findings support the potential of PvRAMA to serve as a vaccine candidate against P. vivax infection.Graphical

Identification of novel linear epitopes in P72 protein of African swine fever virus recognized by monoclonal antibodies.

African swine fever (ASF) is one of the highly contagious and lethal diseases among domestic pigs and wild boars. The capsid protein P72 of African swine fever virus (ASFV) is very important for the diagnosis and vaccine development. However, the epitope of the protein is not clear. In this study, capsid protein P72 was expressed in Sf9 cells along with its chaperone B602L. A total of ten monoclonal antibodies (mAbs) specific to P72 protein were developed by fusions between SP2/0 cells and spleen cells of mice immunized with the recombinant-P72&B602L proteins expressed in Sf9 cells. Four linear B cell epitopes 31SNIKNVNKSY40, 41GKPDP45, 56HLVHFNAH63 and 185ERLYE189 were identified. Biological information analysis illustrated that epitopes 31SNIKNVNKSY40, 41GKPDP45 and 185ERLYE189 were highly conserved within different ASFV strains. These findings may lead to a better understanding of the antibody-antigen interaction and provide new insights into the vaccine research and serological diagnosis of ASF.

Different serotypes of Escherichia coli flagellin exert identical adjuvant effects

Bacterial flagellin is a potent powerful adjuvant, which exerts its adjuvant activity by activating the Toll-like receptor 5 (TLR5) signaling pathway to induce host pro-inflammatory responses. Flagellin of Salmonella typhimurium (S. typhimurium) has shown strong adjuvant effects for a variety of vaccine candidates, however, the adjuvanticity of different serotypes of Escherichia coli (E. coli) flagellin (FliC) is unclear. To explore the adjuvant activity of different serotypes of E. coli flagellin, FliCH1, FliCH7, and FliCH19 recombinant flagellins were prokaryotically-expressed and purified. The adjuvanticity of three recombinant flagellins was evaluated by analyzing their abilities to induce the IL-8 production in human colorectal adenocarcinoma (Caco-2) cells and the immune responses to co-administrated FaeG antigen in mice. Sequence analysis showed that the N-and C-terminal regions are highly conserved, whereas the central region is hypervariable. The TLR5 recognized site is identical among these three serotypes of flagellins. Coomassie blue staining SDS-PAGE showed the molecular mass of FliCH1, FliCH7, and FliCH19 recombinant flagellin are 66 kDa, 64 kDa, and 68 kDa, which can be recognized by anti-FliCH1, FliCH7, and FliCH19 serum, respectively. Moreover, the flagellin serotypes induced similar levels of IL-8 and TNF-α production in Caco-2 cells, anti-FaeG specific IgG antibodies in mice, and IL-4 production in mice spleen cells. Our results indicated that E. coli flagellins can be an adjuvant for vaccine candidates and that different serotypes of E. coli flagellins possess identical adjuvant effects.

The HMGB1 (C106A) mutation inhibits IL-10-producing CD19hiFcγRIIbhi B cell expansion by suppressing STAT3 activation in mice.

Regulatory B cells have important roles in inflammation and autoimmune diseases. A newly discovered subpopulation of B cells with a CD19hiFcγRIIbhi phenotype inhibits the proliferation of CD4+ T cells by secreting interleukin (IL)-10. The expansion of CD19hiFcγRIIbhi B cells in mouse spleen can be induced by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide stimulation. However, the mechanism of CD19hiFcγRIIbhi B cell expansion and its role in inflammatory diseases are unclear. Here, we report that, under inflammatory conditions, the proliferation and immunosuppressive function of CD19hiFcγRIIbhi B cells were decreased in high mobility group box1 (HMGB1) C106A mutant mice, compared with wild-type mice. The HMGB1 (C106A) mutation in B cells reduced STAT3 phosphorylation, restricting the expansion and suppressive function of CD19hiFcγRIIbhi B cells. Compared with CD19hiFcγRIIbhi B cells from wild-type mice, CD19hiFcγRIIbhi B cells from Hmgb1 (C106A) mice significantly reduced the survival of mice with sepsis. Recombinant HMGB1 promoted the expansion of IL-10-producing CD19hiFcγRIIbhi B cells among LPS-activated B cells in vitro. Furthermore, the percentage of CD19hiFcγRIIbhi regulatory B cells in the peripheral blood was increased in patients with sepsis, compared with healthy controls. These findings implicate the role of HMGB1 in the expansion and immunosuppressive function of CD19hiFcγRIIbhi B cells.

Screening, characterization and anti-cancer application of purified intracellular MGL

L-methionine-γ-lyase (MGL) producing bacterial isolates were screened from soil samples that further characterized as ‘Klebsiella oxytoca BLM-1’ by biochemical and 16S rDNA sequencing. Intracellular MGL obtained from K. oxytoca BLM-1 by sonication was purified by Octyl-Sepharose and Sephadex G-200 column chromatography. MALDI-TOF-MS analysis of protein band (Mr ~ 63 kDa) confirmed the PLP-dependence and structural similarity with MGL enzyme. Purified MGL (1.1 μg) exhibited the maximum activity in potassium phosphate buffer (80 mM; with L-met 20 mM pH 7.0) at 37 °C. That further enhanced in the presence of NaCl (2 mM), Tween-80 (1.0 %; v/v) and EDTA (5 mM). Km and Vmax for purified MGL by using L-met as substrate was found to be 5.32 mM and 0.386 U/mL/min. The purified MGL showed PLP dependence and the half-life was 365.59 min. The MGL was effective against breast cancer (MCF7), gastric adenocarcinoma and human glioblastoma (U87MG) cancer cell lines with IC50 values of purified MGL 0.041 U/mL, 0.008 U/mL and 0.009 U/mL, respectively. The U87MG, greatly affected by MGL treatment, when cultured in DMEM medium (10 mL) with PLP, homocysteine and 10 % FCS as compared to control/untransformed mouse spleen cells.

The Multi-Kinase Inhibitor Lucitanib Enhances the Antitumor Activity of Coinhibitory and Costimulatory Immune Pathway Modulators in Syngeneic Models.

Lucitanib is a multi-tyrosine kinase inhibitor whose targets are associated with angiogenesis and other key cancer and immune pathways. Its antiangiogenic properties are understood, but lucitanib's immunomodulatory activity is heretofore unknown. Lucitanib exhibited such activity in vivo, increasing CD3 + , CD8 + , and CD4 + T cells and decreasing dendritic cells and monocyte-derived suppressor cells in mouse spleens. Depletion of CD8 + T cells from syngeneic MC38 colon tumor-bearing mice reduced the antitumor efficacy of lucitanib and revealed a CD8 + T-cell-dependent component of lucitanib's activity. The combination of lucitanib and costimulatory immune pathway agonists targeting 4-1BB, glucocorticoid-induced TNFR (GITR), inducible T-cell co-stimulator (ICOS), or OX40 exhibited enhanced antitumor activity compared with each single agent in immunocompetent tumor models. Lucitanib combined with blockade of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein-1 (PD-1) coinhibitory immune pathways also showed enhanced antitumor activity over the single agents in multiple models. In CT26 tumors, lucitanib, alone or combined with anti-PD-1, reduced CD31 + vessels and depleted F4/80 + macrophages. Combination treatment also increased the number of intratumoral T cells. Gene expression in pathways associated with immune activity was upregulated by lucitanib in MC38 tumors and further potentiated by combination with anti-PD-1. Accordingly, lucitanib, alone or combined with anti-PD-1, increased intratumoral CD8 + T-cell abundance. Lucitanib's antitumor and pharmacodynamic activity, alone or combined with anti-PD-1, was not recapitulated by specific vascular endothelial growth factor receptor-2 (VEGFR2) inhibition. These data indicate that lucitanib can modulate vascular and immune components of the tumor microenvironment and cooperate with immunotherapy to enhance antitumor efficacy. They support the clinical development of lucitanib combined with immune pathway modulators to treat cancer.

Inflammatory Cytokine-Neutralizing Antibody Treatment Prevented Increases in Follicular Helper T Cells and Follicular Regulatory T Cells in a Mouse Model of Arthritis.

BackgroundFollicular T helper (TFH) and follicular regulatory T (TFR) cells play important roles in humoral immunity. Nevertheless, their significance in rheumatoid arthritis (RA) pathogenesis has not been fully elucidated. As an important treatment strategy, the effect of inflammatory factor-neutralizing antibodies on TFH and TFR in RA remains unclear.MethodsWe used the collagen-induced arthritis (CIA) mouse model to illustrate the quantity and functional changes in TFH and TFR cells. The changes of plasmablast, TFH and TFR cells in the spleen and peripheral blood of CIA mice were analyzed by flow cytometry. The levels of TFH and TFR and their functional subsets in the spleen after anti-inflammatory antibody treatment were analyzed and compared. The functional changes of TFH and TFR in CIA mice before and after treatment were detected by in vitro culture experiments.ResultsPlasmablast levels were increased in CIA spleen and peripheral blood and both TFH and TFR cell levels were upregulated. TFH and TFR cells were decreased significantly after the anti-inflammatory antibody treatment. TIGIT+ and TIGIT+CD226− TFH cells in CIA mouse spleen were elevated and PD-1 and ICOS expression on spleen TFH and TFR cells was increased. Both the ability of TFH cells to secrete IL-21 and aid B cells and the ability of TFR cells to secrete IL-10 and inhibit TFH cells were enhanced in the CIA mice. After antibody treatment, the cell subsets and functions were recovered.ConclusionGerminal center TFH and TFR cells were increased and their functions were enhanced. With inflammatory factor-neutralizing antibody treatment, TFH and TFR subsets and their functions returned to normal. These findings provide important information on the dynamics of humoral immune-related cell subsets in RA and the effects of treatment on them.

Antitumor properties of nuclear erythroid cells

Purpose. To study suppressor and/or cytotoxic activity of the nuclear erythroid cells (NEC) against tumor cells of various origins. Material and Methods. C57Bl/6 mice and P815, L1210, B16 and L929 tumor cells were used. “Phenylhydrazine” NECs were obtained from mice with induced hemolytic anemia. “Erythropoietin” NECs were isolated from the “phenylhydrazine spleen” and further cultured in the presence of erythropoietin. Another source of NEC was neonatal mouse spleen, human and mouse fetal liver cells, and mouse bone marrow cells cultured with erythropoietin. The cytostatic effect of NEC or their supernatants was recorded by reducing proliferation of P815, L1210, B16, LLC, L929 lines. Results. The presence of pronounced direct antitumor activity was found in both NEC and their culturing products in relation to cells of various tumor lines. The suppressor effect was not specifc. Conclusion. We know about the signifcant numerical predominance of NEC during the embryo development over all other hematopoietic cells and their high suppressive potential. Therefore, it can be assumed that erythroblasts are involved in process of creating antitumor protection of a fetus during this period of life.

P597: HIGH DOSE IRON IMPAIRS MALIGNANT B-CELL VIABILITY IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Identification of new strategies to improve Chronic Lymphocytic Leukemia (CLL) outcome is fundamental since the disease remains incurable. Malignant B-cells show high levels of reactive oxygen species (ROS), which favors adaptation and survival but also enhances sensitivity to pro-oxidant therapeutics. Redox homeostasis is affected by iron balance since iron excess works as pro-oxidant, and cancer cells rearrange iron trafficking proteins to promote iron uptake. This adaptation might be exploited by exposing malignant cells to iron excess to cause ROS generation, lipid peroxidation, and eventually ferroptosis. We previously demonstrated the feasibility of this strategy in multiple myeloma and prostate cancer pre-clinical models. Aims: We aim at exploiting high dose iron to negatively affect malignant cell survival and increase therapeutic efficacy of current target therapies (BTK and BCL2 inhibitors). We aim at dissecting the molecular mechanisms underlying iron toxicity and at testing whether iron can improve immune dysfunctions. Methods: CLL cell lines (MEC-1, MEC-2 and PCL-12), patients peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) and B lymphocytes purified from patients PB were treated with 300 µM ferric ammonium citrate (FeAC) alone or in combination with the BTK inhibitor ibrutinib (IBR, 5-10 µM) or the BCL2 inhibitor venetoclax (VNTX, 1.25-2.5 nM). We evaluated cell viability by annexin PI staining, lipid peroxidation and ferroptosis by measuring malondialdehyde (MDA) levels and antioxidant gene expression levels by qRT-PCR. We additionally evaluated T cell activation by measuring CD25 expression. MEC-1 xenografts generated in immunodeficient RAG2-/-yc-/- mice were treated with 20 mg/Kg ferric carboximaltose. Results:In vitro, iron treatment impaired cell proliferation in all CLL cell lines analyzed, inducing accumulation of MDA and cell death (p<0.01). In vivo, iron supplementation reduced the amount of MEC-1 cells in bone marrow and spleen of xenograft mice as compared to control saline-treated mice. In patients primary samples, iron induced cell death of leukemic lymphocytes (CD19+CD5+) either as purified cells or within bulk PBMC (p<0.01). Moreover, combination of iron with IBR or VNTX induced cell death at a higher extent compared to each single drug or to iron alone (p<0.01). Leukemic cells in BMMC samples were also iron sensitive and the effect became more evident upon combination with IBR or VNTX (p<0.05). Neither iron nor iron-drug combinations induced cell death in non-malignant cells. Iron also increased CD25 expression in CD8+ T cells and we are currently exploring whether this might indicate that iron can also improve cytotoxic mediated immune response against leukemic cells. As single patient analysis revealed heterogeneity of response, we investigated whether we might predict iron sensitivity by analyzing the antioxidant gene expression profile. All CLL cell lines analyzed showed a lower basal expression of NFE2L2, HMOX1, and GPX4 antioxidant genes than typical iron-resistant cell lines, such as prostate cancer PC-3 cells, suggesting that differences in antioxidant gene expression levels may mediate iron response and that this association is worth to be explored in CLL primary samples to explain heterogeneous iron response. Summary/Conclusion: Our pre-clinical studies suggests that exploiting iron toxicity might be a valuable strategy to improve current target therapies in CLL. Additional studies aimed at distinguishing iron sensitive patients from poor responders will improve the translational potential of this therapeutic approach.

Abstract 5201: Enhanced production of IL-2 from anti CD3-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis

Abstract Objectives: It has been reported that Brazilian green propolis (BGP) possesses anti-tumor and chemo-preventive properties against prostate cancer (Szliszaka E, 2011). And bioactive components from propolis have been shown to have anti-cancerous effects upon hepatocellular carcinoma (Liu X, 2021). These observations revealed that propolis can be an anti-cancer drugs, however the immunological effects upon tumor microenvironments of these naturally occurring components has not been elucidated. In this report, we attempt to clarify immune modulatory effects of BGP and the major component, artepillin C, by observing cytokine production from activated T cells. Methods: The mouse spleen cell stimulated by anti-CD3 monoclonal antibody were co-cultured with BGP, artepillin C and HC030031, an antagonist of TRPA1 Ca2+ channel. The productions of IFN-γ, IL-6, IL-17, IL-4, IL-10 and IL-2 was assayed by ELISA. Results: Our results demonstrated the production of IL-2 was markedly enhanced, while the production of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6 and IL-17 were markedly reduced, in the highly viable stimulated spleen cells treated with BGP. These effects were also reproduced in the cells treated with artepillin C, a major component of BGP. The enhancement of IL-2 production by artepillin C was significantly alleviated by adding 10 HC030031, an antagonist of TRPA1 Ca2+ channel, suggesting the enhancement of the cytokine production by artepillin C could be mediated by Ca2+ influx. Conclusions: The artepillin C, a major component of BGP, is an important regulator of activated T lymphocytes and specifically augmented the expression of IL-2 vie Ca2+-permeable cation channel, TRPA1. These effects may exert immunomodulatory effects upon tumor microenvironments. Citation Format: Nobuo Kondoh, Masako Kamiya, Hanemi Tsuruta, Moe Takahashi, Kumiko Ikeno, Kyohei Ueno, Naoki Umemura, Eiji Takayama, Harumi Kawaki, Genjiro Nakamura, Toru Nikaido, Yasunori Muramatsu, Megumi Ando. Enhanced production of IL-2 from anti CD3-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5201.

Abstract 1665: Humanized mice for preclinical efficacy evaluation of drugs targeting CD73

Abstract CD73 expressed in Treg surface facilities the hydrolysis of AMP into adenosine which inhibits the activation, expansion and homing of T effector cells.Therapeutic efficiency of CD73 mAb had been studied in C57BL/6 and BALB/C mice xenografted with several murine cancer cell lines. However, for human CD73 mAb with no binding to murine CD73, CD73 humanized mouse models are essential for medicinal development.We established CD73 humanized mouse model (hCD73) on C57BL/6 and BALB/c background in which the mCD73 was replaced with hCD73. Similar expression level of hCD73 in peripheral blood was observed compared with mCD73 in wildtype mice. Besides, the proportion of T, B and NK cells in spleen of hCD73 mice were also analogy to that in wildtype mice. After treating with anti-hCD73 antibody, tumor growth was inhibited in our CT-26-hCD73 tumor model.Furthermore, hPD1/hPDL1/hCD73 mouse models were also established on C57BL/6 and BALB/c backgrounds based on hCD73 mice. However, a better anti-tumor effect of anti-CD73 antibody was found on BALB/c-hPD1/hPDL1/hCD73 bearing CT26 than that on B6-hPD1/hPDL1/hCD73 bearing MC38. Besides, Combination of anti-CD73 and anti-PD1 significant elevated anti-tumor effect comparing with anti-CD73 and anti-PD1 mono therapy in BALB/c-hPD1/hPDL1/hCD73 mice bearing CT26-hPDL1hCD73.In conclusion, BALB/c-hPD1/hPDL1/hCD73 is a well efficient mouse model for therapeutic effect study of anti-CD73 and relevant combination therapy. Citation Format: Xing Liu, Cunxiang Ju, Hongyan Sun, Jing Zhao, Xiang Gao, Zhiying Li. Humanized mice for preclinical efficacy evaluation of drugs targeting CD73 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1665.

Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis

ObjectivesIn this report, we attempt to clarify the immune modulatory effects of Brazilian green propolis (BGP) and its major component, artepillin C, on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. We also estimate the physiological mechanism affecting artepillin C's upon the cells. MethodsMale C3H/HeN mouse spleen cells stimulated by antiCD3 monoclonal antibody were co-cultured with BGP, artepillin C, and HC030031, a transient receptor potential ankyrin 1 (TRPA1) Ca2+ channel antagonist. The synthesis of interferon (IFN)-γ, interleukin (IL)-6, IL-17, IL-4, IL-10, and IL-2 was assayed by enzyme-linked immunoassay. The expression of IL-2 mRNA and the protein product were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively. ResultsThe production of IL-2 was markedly enhanced, while that of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6, and IL-17 was significantly reduced in the antibody-stimulated spleen cells treated with BGP at a non-cytostatic concentration. These effects were reproduced in the cells treated with artepillin C. The expression of IL-2 mRNA was unaffected; however, that of the protein was significantly enhanced in the artepillin C-treated cells compared to untreated control cells. The enhancement of protein expression and the production of IL-2 by artepillin C was significantly alleviated by adding HC030031. ConclusionsArtepillin C is an important regulator of cytokine synthesis from activated spleen cells. The agent specifically augmented the expression of IL-2 via the Ca2+-permeable cation channel, TRPA1, at least in part, at the translational or secretion levels.