Proliferation Of PBMCs Research Articles

Abstract 4016: mRNA encoding a CAR specific stimulating protein (CSSP) mediates tumor metabolism regulated (TMR) CAR signaling for CAR-T expansion beyond the tumor microenvironment

Abstract Background: Unlike hematologic targets, TMR Chimeric Antigen Receptors with binding restricted to antigen in the tumor microenvironment lack CAR dependent stimulatory signals for proliferation outside the tumor. Increased exposure to CAR-T is associated with improved clinical outcomes and therefore tools to safely and selectively increase proliferation represent potential adjunctive CAR-T therapeutics. Therefore, we developed a surrogate CAR Specific Stimulating Protein (CSSP) approach encoded by synthetic mRNA for expression in antigen presenting cells to boost CAR-T proliferation in vitro and in vivo. Methods: NFAT-Luc reporter lymphocytes were transduced with lentivectors encoding TMR CARs with or without epitopes reactive to CSSP’s in the CAR. Subsequently, PBMCs were transfected with LNPs containing synthetic mRNAs for either GFP or candidate membrane bound CSSPs. Transfection efficiency was confirmed by microscopy and flow cytometry. The activity of CSSP’s expressed in monocytes cultured with T cells expressing CAR variants with NFAT-luc reporter was measured by luciferase expression and compared to CD3 stimulation. Additionally, the kinetics of CAR stimulation with monocytes expressing CSSPs were determined by serial luciferase measurements. Finally, the proliferation of PBMCs transduced with a TMR-HER2 CAR co-cultured with CSSP treated purified monocytes were evaluated. Results: Following exposure of PBMCs expressing CSSPs to TMR-CAR-expressing NFAT T cells, CAR specific luciferase signaling was readily detectable compared to non-specific CD3 stimulation, indicating that the CSSPs were capable of CAR specific stimulation through the CD3ζ chain of the CAR. Two of the CSSP variants demonstrated stimulation in a CAR specific fashion at levels similar to or greater than non-specific CD3 stimulation. In kinetic experiments, stimulation of NFAT could be detected over a period of 8 days, indicating persistence of the CSSP similar to GFP in antigen presenting cell controls. Finally, co-culture of CSSP mRNA expressed in purified monocytes were capable of TMR-HER2-CAR T cells stimulation, which do not proliferate when cultured with HER2 antigen expressing cells under physiologic conditions. Conclusion: TMR-CAR-T cells can achieve proliferative capacity outside the tumor microenvironment using synthetic mRNA encoding CAR Specific Stimulatory Proteins. This novel mRNA approach may provide additional tools to promote the proliferation and/or persistence of solid tumor targeted CAR-T cells outside the tumor environment. Citation Format: Frederic Vigant, Anirban Kundu, Michael Betts, Sid P. Kerkar, Gregory I. Frost. mRNA encoding a CAR specific stimulating protein (CSSP) mediates tumor metabolism regulated (TMR) CAR signaling for CAR-T expansion beyond the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4016.

Assessing the functional potential of conditioned media derived from amniotic epithelial stem cells engineered on 3D biomimetic scaffolds: An in vitro model for tendon regeneration

Tendon diseases pose a significant challenge in regenerative medicine due to the limited healing capacity of this tissue. Successful tendon regeneration requires a combination of angiogenesis, immune response, and tenogenesis processes. An effective tendon engineering (TE) strategy must finely tune this systems’ interplay toward homeostasis.This study explores in vitro the paracrine influence of amniotic epithelial stem cells (AECs) engineered on a validated 3D electrospun PLGA scaffolds on HUVECs (angiogenesis), PBMCs/Jurkat (immune response), and AECs (tenogenic stem cell activation).The results revealed the role of scaffold's topology and topography in significantly modulating the paracrine profile of the cells. In detail, AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p < 0.0001 vs. CMCTR). Moreover, biological tests demonstrated 3D scaffolds' proactive role in potentiating AECs' paracrine inhibition on PBMCs proliferation (CM3Dvs. CTR, p < 0.001) and LPS-mediated Jurkat activation with respect to controls (CM3D and CM2Dvs. CTR, p < 0.01 and p < 0.05, respectively), without exerting any in vitro pro-angiogenic role in promoting HUVECs proliferation and tubule formation. Teno-inductive paracrine ability of AECs engineered on 3D scaffolds was assessed on co-cultured ones, which formed tendon-like structures. These latter demonstrated an upregulation of tendon-related genes (SCX, THBS4, COL1, and TNMD) and the expression TNMD and COL1 proteins.Overall, this research underscores the pivotal role of the 3D topology and topography of PLGA tendon mimetic scaffolds in orchestrating effective tendon regeneration through modulating cell behavior and crosstalk between engineered stem cells and different subpopulations in the damaged tendon.

Multi-omics analysis to reveal the synergistic mechanism underlying the multiple ingredients of Stephania tetrandra extract on rheumatoid arthritis through the PI3K/Akt signaling pathway.

Background: Stephania tetrandra has been used for treating rheumatic diseases for thousands of years in rural areas of China. Several studies have found that tetrandrine and fangchinoline can inactivate the PI3K/Akt signaling pathway by reducing the expression and phosphorylation of AKT. However, the mechanism underlying the therapeutic actions of S. tetrandra on RA is not well known. Methods: In this study, we determined the molecular mechanism of the therapeutic effects of the multiple ingredients of S. tetrandra extract (STE) on collagen-induced arthritic (CIA) rats by integrating pharmacometabolomics, proteomics, and PTMomics. Results: In the multi-omics joint analysis, first, the expression signatures of proteins, PTMs, metabolites, and STE ingredients were profiled in CIA rats PBMCs that underwent STE treatment. Bioinformatics analysis were subsequently probed that STE mainly regulated tryptophan metabolism, inflammatory response, and cell adhesion pathways in CIA rats. The interrelated pathways were further constructed, and the findings revealed that STE attenuated the inflammatory response and proliferation of PBMCs in CIA rats by mediating the key targets of the PI3K/Akt pathway, including Hint1, ACP1, FGR, HSP90@157W + dioxidation, and Prkca@220N + 845.4540Da. The rheumatic functions of Hint1 and ACP1 were further confirmed by applying a transcriptomic data of RA patients who clinically received abatacept therapy. Furthermore, a cross-ome correlation analysis was performed and major in vivo ingredients of STE, including coclaurine-N-glucuronide, Me,coclaurine-O-glc, N-gluA-schefferine, corydamine, corypamine, tetrandrine, and fangchiniline, were found to act on these targerts to inactivate the PI3K/Akt pathway. Conclusion: These results elucidated the molecular mechanism by which the ingredients of STE mediate the expression of the key targets in the PI3K/Akt pathway, leading to anti-rheumatic functions. The findings of this study provided new insights into the synergistic effect of STE against arthritis in rats.

Preclinical immunological characterization of rademikibart (CBP-201), a next-generation human monoclonal antibody targeting IL-4Rα, for the treatment of Th2 inflammatory diseases

Rademikibart (CBP-201) is a next-generation human monoclonal antibody targeting IL-4Rα, undergoing evaluation in Phase 2 clinical trials for the treatment of moderate-to-severe Th2 inflammatory diseases. We report the immunological characterization of rademikibart. Rademikibart and dupilumab were associated with KD of 20.7 pM and 45.8 pM, respectively, when binding to distinct human IL-4Rα epitopes. Rademikibart did not bind to IL-4Rα from other species. Rademikibart inhibited IL-4 and IL-13-mediated STAT6 signaling (mean ± SD IC50: 7.0 ± 2.5 and 6.6 ± 1.5 ng/mL, respectively), TF-1 cell proliferation (IC50: 8.0 ± 1.6 and 9.7 ± 0.8 ng/mL, respectively) and TARC production in PBMCs (IC50: 59.2 ± 3.9 and 13.5 ± 0.2 ng/mL, respectively). Rademikibart versus dupilumab was more potent in the STAT6 assays (IL-4, p < 0.01; IL-13, p = 0.03), with non-significant trends towards greater potency in the TF-1 cell assays (IL-4, p = 0.09; IL-13, p = 0.20), and similar potency in the TARC assays. In experiments with mice expressing human IL-4Rα and IL-4, rademikibart and dupilumab demonstrated similar potency; both monoclonal antibodies eliminated IL-4 (p < 0.0001) and IL-13 (p < 0.05) mediated B cell activation in vitro and ovalbumin-induced IgE (p < 0.01) and eosinophilic lung infiltration (p < 0.0001) in vivo. In Th2-stimulated human skin explants, rademikibart rapidly downregulated IL-4, IL-13, and TARC gene expression, with greater effectiveness than dupilumab for IL-4 (p < 0.01) and a non-significant trend towards superiority for IL-13. In summary, rademikibart bound to a distinct IL-4Rα epitope with high affinity and demonstrated reductions in Th2 inflammatory biomarkers with at least similar and potentially superior potency to dupilumab.

Comparative Analysis of the Therapeutic Effects of Fresh and Cryopreserved Human Umbilical Cord Derived Mesenchymal Stem Cells in the Treatment of Psoriasis.

Psoriasis, an inflammatory autoimmune skin disease, is characterized by scaly white or erythematous plaques, which severely influence patients' quality of life and social activities. Mesenchymal stem cells derived from the human umbilical cord (UCMSCs) represent a promising therapeutic approach for psoriasis because of its unique superiority in ethical agreeableness, abundant source, high proliferation capacity, and immunosuppression. Although cryopreservation provided multiple benefits to the cell therapy, it also greatly compromised clinical benefits of MSCs due to impaired cell functions. The current study aims to evaluate the therapeutic efficacy of cryopreserved UCMSCs in a mouse model of psoriasis as well as in patients with psoriasis. Our results showed that cryopreserved and fresh UCMSCs have comparable effects on the suppression of psoriasis-like symptoms such as thickening, erythema, and scaling, and serum IL-17A secretion in mice model of psoriasis. Moreover, psoriatic patients injected with cryopreserved UCMSCs had a significant improvement in the Psoriasis Area and Severity Index (PASI), Physician Global Assessment (PGA), and Patient Global Assessments (PtGAs) scores compared to baseline values. Mechanically, cryopreserved UCMSCs markedly inhibit the proliferation of PHA-activated PBMCs, type 1 T helper (Th1) and type 17 T helper (Th17) cell differentiation and secretion of inflammatory cytokines including IFN-γ, TNF-a and IL-17A in PBMCs stimulated by anti-CD3/CD28 beads. Taken together, these data indicated that cryopreserved UCMSCs exhibited great beneficial effect on psoriasis. Thus, cryopreserved UCMSCs can be systemically administered as ''off-the-shelf'' cell product for psoriasis therapy.Trial Registration ChiCTR1800019509. Registered on November 15, 2018-Retrospectively registered, http://www.chictr.org.cn/ .

High throughput sequencing revealed enhanced cell cycle signaling in SLE patients

The multi-system involvement and high heterogeneity of systemic lupus erythematosus (SLE) pose great challenges to its diagnosis and treatment. The purpose of the current study is to identify genes and pathways involved in the pathogenesis of SLE. High throughput sequencing was performed on the PBMCs from SLE patients. We conducted differential gene analysis, gene ontology (GO) analysis, kyoto encyclopedia of genes and genomes (KEGG) analysis, and quantitative real-time PCR (qRT-PCR) verification. Protein–protein interaction (PPI) analysis, alternative splicing analysis, and disease correlation analysis were conducted on some key pathogenic genes as well. Furthermore, si-CDC6 was used for transfection and cell proliferation was monitored using a cell counting kit-8 (CCK-8) assay. We identified 2495 differential genes (1494 upregulated and 1001 downregulated) in SLE patients compared with healthy controls. The significantly upregulated genes were enriched in the biological process-related GO terms of the cell cycle, response to stress, and chromosome organization. KEGG enrichment analysis revealed 7 significantly upregulated pathways including SLE, alcoholism, viral carcinogenesis, cell cycle, proteasome, malaria, and transcriptional misregulation in cancer. We successfully verified some differential genes on the SLE pathway and the cell cycle pathway. CDC6, a key gene in the cell cycle pathway, had remarkably higher MXE alternative splicing events in SLE patients than that in controls, which may explain its significant upregulation in SLE patients. We found that CDC6 participates in the pathogenesis of many proliferation-related diseases and its levels are positively correlated with the severity of SLE. Knockdown of CDC6 suppressed the proliferation of Hela cells and PBMCs from SLE patients in vitro. We identified SLE-related genes and their alternative splicing events. The cell cycle pathway and the cell cycle-related biological processes are over-activated in SLE patients. We revealed a higher incidence of MXE events of CDC6, which may lead to its high expression in SLE patients. Upregulated cell cycle signaling and CDC6 may be related to the hyperproliferation and pathogenesis of SLE.

A small molecule inhibitor of VSIG-8 prevents its binding to VISTA.

The V-region immunoglobulin-containing suppressor of T cell activation (VISTA), a unique B7 family member, is an attractive immunotherapeutic target for cancer, autoimmune and inflammatory diseases. In 2016, a patent demonstrated V-Set and Immunoglobulin domain containing 8 (VSIG-8) was the putative VISTA receptor. Antagonistic or agonistic agents can conceivably modulate VISTA and its interacting partners, which will greatly benefit the treatment of many diseases. The interaction of VISTA and VSIG-8 were measured by Enzyme-linked Immuno Sorbent Assay (ELISA), Microscale Thermophoresis (MST) and coimmunoprecipitation (Co-IP) experiments. The bioactivity of VSIG-8 inhibitor L557-0155 was evaluated in vitro and in vivo. Kd value of human VISTA binding to human VSIG-8 was 1.58 ± 0.44μM by MST. When the related amino acid binding site of VISTA was mutated to alanine, the interaction between VISTA and VSIG-8 disappeared. VSIG-8 protein induced an decrease in the level of IL-2 in VISTA-overexpressing cells but a increase in VISTA-/- cells. Furthermore, VSIG-8 inhibitor L557-0155 promoted cytokine production and cell proliferation in PBMCs and suppressed melanoma growth. VSIG-8/VISTA coinhibitory pathway may provide a novel strategy for the treatment of human cancers, and VSIG-8 blockade may increase antitumor immunity. This study was the first time to report that VSIG-8 interacts with VISTA, and inhibited T cell function.

Efficiency of bis-amidate phosphonate prodrugs

Bis-amidate derivatives have been viewed as attractive phosphonate prodrug forms because of their straightforward synthesis, lack of phosphorus stereochemistry, plasma stability and nontoxic amino acid metabolites. However, the efficiency of bis-amidate prodrug forms is unclear, as prior studies on this class of prodrugs have not evaluated their activation kinetics. Here, we synthetized a small panel of bis-amidate prodrugs of butyrophilin ligands as potential immunotherapy agents. These compounds were examined relative to other prodrug forms delivering the same payload for their stability in plasma and cell lysate, their ability to stimulate T cell proliferation in human PBMCs, and their activation kinetics in a leukemia co-culture model of T cell cytokine production. The bis-amidate prodrugs demonstrate high plasma stability and improved cellular phosphoantigen activity relative to the free phosphonic acid. However, the efficiency of bis-amidate activation is low relative to other prodrugs that contain at least one ester such as aryl-amidate, aryl-acyloxyalkyl ester, and bis-acyloxyalkyl ester forms. Therefore, bis-amidate prodrugs do not drive rapid cellular payload accumulation and they would be more useful for payloads in which slower, sustained-release kinetics are preferred.

Intra-lymphatic administration of GAD-alum in type 1 diabetes: long-term follow-up and effect of a late booster dose (the DIAGNODE Extension trial)

AimTo evaluate the long-term effect of intra-lymphatic administration of GAD-alum and a booster dose 2.5 years after the first intervention (DIAGNODE Extension study) in patients with recent-onset type 1 diabetes.MethodsDIAGNODE-1: Samples were collected from 12 patients after 30 months who had received 3 injections of 4 μg GAD-alum into a lymph node with one-month interval. DIAGNODE Extension study: First in human, a fourth booster dose of autoantigen (GAD-alum) was given to 3 patients at 31.5 months, who were followed for another 12 months. C-peptide was measured during mixed meal tolerance tests (MMTTs). GADA, IA-2A, GADA subclasses, GAD65-induced cytokines, PBMCs proliferation and T cells markers were analyzed.ResultsAfter 30-month treatment, efficacy was still seen in 8/12 patients (good responders, GR). Partial remission (IDAA1c < 9) had decreased compared to 15 months, but did not differ from baseline, and HbA1c remained stable. GAD65-specific immune responses induced by the treatment started to wane after 30 months, and most changes observed at 15 months were undetectable. GADA subclasses IgG2, IgG3 and IgG4 were predominant in the GR along with IgG1. A fourth intra-lymphatic GAD-alum dose to three patients after 31.5 months gave no adverse events. In all three patients, C-peptide seemed to increase the first 6 months, and thereafter, C-peptide, HbA1c, insulin requirement and IDAA1c remained stable.ConclusionThe effect of intra-lymphatic injections of GAD-alum had decreased after 30 months. Good responders showed a specific immune response. Administration of a fourth booster dose after 31.5 months was safe, and there was no decline in C-peptide observed during the 12-month follow-up.

English

Bovine respiratory diseases are widespread and too costly disease impacting contributes to economically essential diseases like mastitis and pneumonia worldwide. The present study aimed to explore the influence of M. bovis field strain 07801 on the status of PBMCs in the challenged cattle group and the immunized one. For this aim, the PBMCs proliferation, apoptosis, and cytokine profile changes were determined. In this study, M. bovis strain Mb 07801 and reference strain PG45 were used; ten calves (2-3 months old) were arranged into two groups (5 calve each): 1) PBS-challenged group (with M. bovis 07801 at about 1010 CFU/ml), and 2) immunized group (by Inactivated M. bovis 07801); both groups were treated nasally and intra-tracheal. Blood samples were obtained from both groups and examined for PBMCs proliferation and apoptosis, as well as serum cytokine profile, before infection and at day zero and days 7, 14, 21, and 36 post bacterial treatment. The results revealed that M. bovis strain (07801and PG45) antigen increased the proliferative response of the stimulated PBMC compared with the unstimulated cells and ConA-alone stimulated. The PBMC apoptosis showed a non-significant increase in both challenged and immunized groups compared to the negative and positive control (treated with apoptosis inducer) groups. Besides, the levels of cytokines profile showed a significant up-regulation in IFN-γ, IL-4, IL-2, IL-18, TNF-α, IFN-α, IL-6, IL-10 IL-1β, and IL-13, in both groups, except IL-4 and IL-18 those recorded a down-regulation in the immunized group at days 7,14, 21, and 36 post-M. bovis infection. In conclusion, immunization markedly ameliorated the immune deterioration induced by M. bovis strain 07801.

Abstract 3176: Inhibitory activity of Globo-H and SSEA-4 on activated T-cells

Abstract Background/Aim: Globo-H (GH) and stage-specific embryonic antigen-4 (SSEA-4) are globo-series glycosphingolipid (GSL) antigens highly expressed on a variety of epithelial cancers. GH and SSEA-4 are reported to play an important role in cancer cell development and survival and thus are promising targets for developing immunotherapeutic agents for cancer treatment. Shedding of GSLs by tumor cells and uptake by neighboring cells may generate a preferable environment for tumor progression and survival within the tumor microenvironment. GH shed from tumor cells was shown to promote angiogenesis and suppress immune responses in vitro. In addition, exogenous Globo-H ceramide (GHCer) inhibits proliferation of human PBMCs and murine CD4+ T-cells. Given the important role of sialic acid in regulating the immune system, SSEA-4, a polysaccharide with sialic acid at the terminal, may play an important role in the regulation of T-cell responses. This study aimed to investigate the GHCer and SSEA-4 ceramide (SSEA-4Cer) effects on T-cell activation. Methods: C57BL/6 mice bearing murine B16F10 melanoma were treated with GHCer by peritumoral injection and monitored for 26 days. T-cell tumor subpopulations were evaluated by FACS and immunohistochemical analysis. Presence of GHCer and SSEA-4Cer expression on human T cells after the treatment with GHCer and SSEA-4Cer was monitored by FACS analysis. Cell proliferation and apoptosis of T cells after treatment with GHCer, SSEA-4Cer, and glucoceramide (GlcCer) were assessed using ATP assay and annexin V staining, respectively. After treatment with GHCer/SSEA-4Cer/GlcCer, expression levels of CD25, cytokine IFN-γ, and Ki-67 in human PBMCs stimulated with anti-CD3/CD28 were measured by flow cytometry. Results: B16F10 tumor growth rate increased 2.54-fold (P&amp;lt;0.05, n=6) in GHCer-treated mice with a lower number of tumor-infiltrating CD3+ cells (0.45-fold; P&amp;lt;0.01, n=6) compared with control mice. Not surprisingly, addition of GHCer and SSEA-4Cer to human T cells was shown to increase GH and SSEA-4 levels on the surface of T cells. Furthermore, addition of GHCer and SSEA-4Cer to the activated T cells showed a 26.4% and 36.9% decrease in cell proliferation, respectively, while no induction of apoptosis was observed. In contrast, GHCer had no impact on cell viability and proliferation of resting T cells. Exogenous GHCer and SSEA-4Cer significantly reduced CD3+CD25+ and CD3+IFNγ+ T cells in anti-CD3/CD28-stimulated PBMCs. SSEA-4Cer exhibited higher activity in the suppression of CD8+CD25+ T cells in PBMCs than GHCer did. In addition, suppression of the proliferation of activated T-cells by SSEA-4Cer was abolished in the presence of NF-κB inhibitor. Conclusion: GHCer inhibits T-cell infiltration into the tumor in vivo and SSEA-4 exhibits immunosuppressive activity on activated T cells, suggesting that both GHCer and SSEA-4Cer may play a role in regulating immunologic responses in the tumor microenvironment. Citation Format: Tzer-Min Kuo, Chin-Chan Lee, Jiann-Shiun Lai, Chung-Chen Su, Ming-Tain Lai. Inhibitory activity of Globo-H and SSEA-4 on activated T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3176.

Abstract 1712: Neospora caninum: An immunotherapeutic protozoan against cancer

Abstract Background: Immunotherapy induces, provides, and/or reactivates anti-tumor immune responses. Some microorganisms also can initiate response that lyzes infected tumor and/or stimulates systemic immunity. Attenuated viruses or bacteria are well studied as oncotherapeutics, but no protozoa except Toxoplasma gondii. We assessed the effect on tumors of other protozoa that were naturally non-pathogenic to humans. Thus, we discovered the ability to use Neospora caninum (Nc) in a manner and form that demonstrated a synergistic array of pertinent immunotherapeutic characteristics against solid cancers. We report on the most recent data notably from Nc engineered to secrete human IL-15 within the tumor. Methods: In vitro, the immunostimulatory properties of Nc strains wildtype and engineered to secrete human IL-15 were studied. In vivo experiments of treatment with of Nc tachyzoites administered locally (intra and peri tumoral) or remotely (subcutaneous) in a murine thymoma EG7 tumor and in human Merkel cell carcinoma (MCC). Results: We demonstrated that the treatment of thymoma EG7 by Nc strongly inhibited tumor development. Analysis of immune responses and interactions between Nc and tumor cells showed that Nc had the ability to lyze infected cancer cells, reactivated immune competence within the tumor microenvironment (TME), and activated the systemic immune system by promoting the recruitment of immune cells to the site of tumor. We also established in a NOD/SCID mouse model that Nc was able to induce a strong regression of human MCC. Recently, to further enhance oncotherapeutic effect, we engineered an Nc strain to secrete human IL-15 (cross reactive with mouse cells), associated with alpha subunit of IL-15 receptor, increasing its stability. This strain induced proliferation of human PBMCs and their secretion of IFN-γ. In the EG7 model, human IL-15 secreting Nc showed greater protection against tumor development, confirming enhancement of immunotherapy by engineering Nc to deliver/secrete IL-15. Conclusion: These results highlight Neospora caninum as a potentially extremely efficient, and non-toxic anti-cancer agent, capable of being engineered to express at its surface or to secrete bio-drugs, like human IL-15 cytokine. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine capable of vectoring molecular therapy, overcoming TME defenses. Citation Format: Louis Lantier, Agathe Poupée-Beaugé, Anne Di Tommaso, Céline Ducournau, Stéphanie Germon, Gordon S. Lee, Antoine Touze, Nathalie moiré, Isabelle Dimier-Poisson. Neospora caninum: An immunotherapeutic protozoan against cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1712.

Blockade of IL-2 and IL-15 Gamma Chain Receptor Signaling Decreases Leukemic Cell Viability in T-Cell Large Granular Lymphocyte Leukemia and Adult T-Cell Leukemia

IntroductionInterleukin-15 (IL-15) and IL-2 are important cytokines that drive the progression of many T-cell malignancies such as T-cell Large Granular Lymphocyte Leukemia (T-LGLL) and HTLV-1 derived Adult T-cell Leukemia (ATL).T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with a constitutively active JAK/STAT pathway controlled by cytokines including IL-15 and IL-2. A computational systems approach identified IL-15 as a master regulator of T-LGLL survival and patients have increased IL-15/IL-15Ra serum levels. Cytokine signaling leads to increased phosphorylation and activation of STAT proteins. T-LGLL cells exhibit STAT3 or STAT5 mutational activation in some cases and constitutive activation in all patients. T-LGLL treatments are not selective or curative, and the commonly utilized immunosuppressive agents are not effective in all patients.ATL is an aggressive T-cell disorder that also exhibits aberrant IL-2 and IL-15 cytokine signaling. Tax, identified as an essential oncogene in the disease, is associated with increased cytokine and cytokine receptor expression. ATL patients have high IL-2Ra gene expression and PBMCs from chronic/smoldering ATL patients can depend on autocrine production of cytokines for proliferation. Patients with ATL have poor prognosis and current cytokine-targeting therapies have not proven to be efficacious.The gc receptor is shared by the gc-family cytokines including IL-2, -4, -7, -9, -15 and -21. BNZ-1 (formerly known as BNZ132-1-40) is a pegylated peptide designed to specifically bind the gc receptor to block IL-2, IL-15 and, to a lesser extent, IL-9 cytokine signaling. Because these cytokines are vital to the survival of T cells in T-LGLL and ATL, these diseases represent ideal models to test the hypothesis that treatment of leukemic cells with BNZ-1 will result in reduced proliferation and viability.MethodsThese studies utilized a human T-LGL cell line (TL1), PBMCs from T-LGLL patients and ATL patients, and human IL-15 transgenic NSG mice injected with the ED40515(+) ATL cell line expressing luciferase. In vitro assays were performed after an overnight cytokine withdrawal and 20-minute pretreatment with BNZ-1 prior to addition of cytokine. Leukemic mice were divided into 3 treatment groups; BNZ-1 (40 mg/kg i.v. 2x per week for 4 weeks), JAK1/2 inhibitor Ruxolitinib (Ruxo) (50 mg/kg/day s.c. by pump for 28 days) and PBS control. Tumor burden was measured through bioluminescence imaging (Xenogen IVIS).ResultsOur results demonstrate that in vitro treatment of TL1 cells with BNZ-1 inhibits IL-2 and IL-15 mediated expansion and viability. BNZ-1 impaired the growth of these cytokine-dependent cells to the level observed in cells without exogenous cytokine supplementation. In addition, BNZ-1 was able to drastically block IL-15 and IL-2 induced phosphorylation events, specifically STAT1, STAT3, STAT5, ERK and Akt. The compound blocked IL-15 mediated viability and signaling more effectively than that mediated by IL-2.The BNZ-1 mediated decreased viability of PBMCs from T-LGLL patients was observed in the presence of either IL-2 or IL-15 stimulation and regardless of STAT3 mutational status. A potent reduction of IL-2 and IL-15 mediated STAT1, STAT3, STAT5 and ERK phosphorylation was also observed in T-LGLL PBMCs with BNZ-1 treatment. Proliferation of PBMCs from ATL patients was inhibited by 58 to 80 percent by the addition of either the antiIL-2Ra antibody daclizumab or and the combination treatment of daclizumab, anti-IL-15 and anti-IL-9. BNZ-1 at 10 µM inhibitedproliferation by 60 to 80 percent.Leukemic burden in the ATL tumor bearing mice was reduced by both BNZ-1 and Ruxo when compared to controls. The BNZ-1 treatment group showed comparable efficacy to that observed in the Ruxo group. Furthermore, BNZ-1 therapy was associated with lower levels of IL-2Ra, which reflects reduced tumor burden at 4 and 7 weeks when compared to that observed with the PBS group.In summary, BNZ-1 shows promise as a potential therapy for T-LGLL and ATL. Results from this study support its advancement in clinical trials in the treatment of T-LGLL and ATL patients. DisclosuresTagaya:Bioniz Therapeutics: Employment. Azimi:Bioniz Therapeutics: Employment.

Aberrant Level of Estrogen Correlates with Defective Thrombopoiesis during Pregnancy in Patients with Immune Thrombocytopenia

AbstractPregnancy seems to be a high-risk event in immune thrombocytopenia(ITP)because disease flares are frequently observed during gestation clinically, and the treatment with glucocorticoid is often ineffective. Sex hormones are changed dramatically during pregnancy to maintain a stable environment both for the mother and for the fetus. To determine the sex hormone levels during pregnancy in ITP, sex hormones were analyzed in patients with ITP and matched healthy women before, during, and after pregnancy. We demonstrated that there is a relationship between estrogen level and thrombopoiesis in ITP pregnancy. Moreover, we showed that estrogen plays a role in glucocorticoid resistance which is common in ITP patients who are pregnant.Thirty consecutive successful pregnancies in 30 patients with ITP, 21 pregnancies in 21 healthy controls, twenty patients with primary ITP and 19 healthy subjects were studied. Serum samples for sex hormone testing, including estrogen, progesterone, testosterone, prolactin, LH and FSH, were collected and analyzed. We confirmed that serum levels of all sex hormones varied significantly during pregnancy and postpartum, both in the patients with ITP and in healthy controls, as expected. However, the concentration of estrogen was significantly lower in pregnant ITP patients, compared with healthy pregnant subjects, leading to a completely different profile.To clarify whether the different estrogen concentration has a direct effect on megakaryocyte(MK) maturation and thrombopoiesis during pregnancy in ITP, BM-derived CD34+ cells from ITP patients and healthy controls in pregnancy, were treated with different doses of estrogen. We found that estrogen had the ability to promote MK polyploidization, maturation and proplatelet formation via estrogen receptor(ER) in healthy controls, but the concentration of estrogen, which was lower than that of the normal-pregnancy group, could not play the above role in CD34+ cells of pregnancy-ITP group. To determine the differences in MKs treated with estrogen of different doses in each group, we screened some essential transcription factors that may participate in the regulation of MK polyploidization and maturation. The expression levels of GATA1, FLI1, FOG-1, KLF-1, and NF-E2, were detected by RT-PCR and Western blot. We found that, in the pregnancy-ITP group treated with lower estrogen, the expression of GATA1 and NF-E2 was significantly reduced compared with the healthy control. We obtained similar results with the bone marrow samples, indicating that pregnant ITP patients showed more obvious detects in megakaryocyte maturation defect and lower expression of transcription factors. Further investigation is needed to evaluate the expression of ER and its nuclear translocation in MKs of the pregnancy-ITP group treated with the relatively lower level of estrogen.We also explored if the relatively reduced estrogen level plays a role in glucocorticoid resistance which is commonly observed in ITP patients during pregnancy. We evaluated the inhibitory effects of dexamethasone, with or without different concentrations of estrogen, on the proliferation of PBMCs from the pregnancy-ITP group induced by a mitogen. We found that estrogen impaired the inhibitory effects of dexamethasone. Further study with immunofluorescence demonstrated that estrogen upregulated the expression of protein phosphatase, which affected the glucocorticoid receptor activation.In conclusion, an unexpected lack of estrogen serum level increase occurs in patients with ITP during pregnancy. The relatively reduced level of estrogen cannot induce the expression of GATA1 and NF-E2 via ER in pregnant ITP patients, which is required for MK maturation and thrombopoiesis. Meanwhile, setrogen inhibits glucocorticoid action via protein phosphatase, which may account for the glucocorticoid resistance observed in pregnant ITP patients. DisclosuresNo relevant conflicts of interest to declare.

Vitamin K2 Supplementation for the Prevention and Treatment of Rheumatoid Arthritis

ObjectivesTo investigate the effects of vitamin K2 supplementation on the prevention and treatment of rheumatoid arthritis(RA). MethodsVitamin K2 and rheumatoid arthritis were searched in Pubmed from inception until December 2020. ResultsStudy 1: Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47 ± 4,910.02 ng/mL. There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p < 0.05), such as C-reactive protein (CRP), rheumatoid factor, anti-cyclic citrullinated peptide antibody (ACPA), matrix metalloproteinase-3, Pre-DAS-28 (CRP), and ∆DAS-28 (CRP). Methotrexate inhibited the proliferation of mitogen-activated PBMCs of RA patients with a IC50 value of 22.83 ± 12.47 ng/mL. IC50 values of methotrexate only showed significant correlation with ACPA (p = 0.0158, r = 0.6905); Study 2: A significant decrease in MK-7 treated group for the levels of undercarboxylated osteocalcin (ucOC), erythrocyte sedimentation rate (ESR), disease activity score assessing 28 joints with ESR (DAS28-ESR), C-reactive protein (CRP) and matrix metalloproteinase (MMP-3) was found. In MK-7 treated group, a marked decrease in RA clinical and biochemical markers for moderate and good response compared to non-responders was observed in ucOC, ESR and DAS28-ESR. A marked increase in the levels of MK-7 for the moderate and good responders compared to non-responders was observed; Study 3: as compared to the MK-4-naïve group, the MK-4-treated group showed lower serum CRP (1.7 ± 0.2 vs. 0.5 ± 0.1 mg/dl; P < 0.001), MMP-3 (220.4 ± 21.9 vs. 118.0 ± 14.4 ng/ml; P < 0.001), and DAS28-CRP (2.9 ± 0.1 vs. 2.4 ± 0.1; P < 0.05). The patients who were additionally treated with MK-4 showed significant decreases in serum CRP (1.1 ± 0.2 to 0.6 ± 0.2 mg/dl; P < 0.001), MMP-3 (160.1 ± 25.6 to 125.0 ± 17.8 ng/ml; P < 0.05), and DAS28-CRP (3.1 ± 0.2 to 2.4 ± 0.1; P < 0.001). ConclusionsVitamin K2 supplementation could provide a benefit for the prevention and treatment of RA patients via its immunosuppressive function and improving disease activity. Funding SourcesNo.

M351-0056 is a novel low MW compound modulating the actions of the immune-checkpoint protein VISTA.

Background and PurposeThe protein V‐domain immunoglobulin suppressor of T‐cell activation (VISTA) is a novel immune‐checkpoint molecule that belongs to the B7 family and regulates a broad spectrum of immune responses. So far, low MW compounds targeting VISTA for the treatment of autoimmune diseases or inflammation, have not been identified.Experimental ApproachWe developed a homology modelling for VISTA 3D structure and subsequent virtual screening for low MW ligands binding to VISTA. Visualization of the binding postures of docked ligands with protein VISTA indicated that compound M351‐0056 targeted VISTA. The biological activities of compound M351‐0056 targeting VISTA were investigated in vitro using monocytes and T cells and in vivo, using mice with imiquimod‐induced dermatitis.Key ResultsThe K D value of M351‐0056 for human VISTA‐extracellular domain was 12.60 ± 3.84 μM as assessed by microscale thermophoresis. M351‐0056 decreased cytokine secretion from PBMCs or human CD4+ T cells, suppressed proliferation of PBMCs and enhanced expression of Foxp3+ T cells. These effects of M351‐0056 modulating VISTA involved the JAK2–STAT2 pathway. Daily administration of M351‐0056 ameliorated imiquimod‐induced psoriasis‐like dermatitis. Expression of mRNA and protein of inflammatory cytokines in psoriatic lesions was decreased after M351‐0056 treatment.Conclusion and ImplicationsThe compound M351‐0056 showed high affinity for VISTA and may modulate its immune function in vitro and in vivo. Our finding provides a lead compound for therapeutically enhancing VISTA‐mediated pathways to benefit the treatment of autoimmune diseases or inflammation.

Royal jelly alleviates the detrimental effects of aging on immune functions by enhancing the in vitro cellular proliferation, cytokines, and nitric oxide release in aged human PBMCS

Aging strongly delays the immunity. Our research aims to assess the in vitro effects of royal jelly (RJ) on the immune function of aged PBMCs. PBMCs were obtained from 10 healthy aged and young donors by the gradient density centrifugation method and further cultured in RPMI-1640 medium supplemented with or without RJ in the presence of Con A. Cell proliferation was assessed by MTT assay along with the measurement of interleukins, Nitric oxide (NO), Glutathione (GSH), and Malondialdehydes (MDA). Our results showed that RJ improved PBMCs proliferation significantly in the elderly subjects, accompanied by the increase in NO (p=.001) and the release of IL-2, IL-4, and IL-6 cytokines. RJ also increased the intracellular GSH (p= .001) and MDA (p=.001) levels in aged PBMCs. In young subjects, RJ enhanced PBMCs proliferation potency, IL-4, IL-6, GSH, and intracellular MDA levels but with a concomitant decrease in NO and IL-2 cytokine secretion as compared with non RJ-treated cells. In conclusion, RJ restored functions of the aged PBMCs as well as the young control subjects, indicating a beneficial effect on immune status during the aging process. PRACTICAL APPLICATIONS: Royal jelly is a well-known edible dietary compound, used traditionally to treat many diseases throughout the world. Since antiquity, it was shown to have medicinal importance. The immuno-enhancing potential of this food was largely and scientifically established by the lipid and protein fractions. The present study illustrates the anti-aging and stimulatory effects of the fresh RJ whole extract, from local Algerian honey bee: Apis mellifera intermissa, on the immunity of aged men. This study provides the experimental evidence supporting anti-immunosenesence effects of royal jelly. RJ supplementation can be used in the old age management and human age-related complications, especially, associated with the weaknesses of the immune response.

Umbilical cord mesenchymal stem cells as well as their released exosomes suppress proliferation of activated PBMCs in multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) degenerative disorder which is caused by a targeted autoimmune-mediated attack on myelin proteins. Previously, mesenchymal stem cells were considered as a novel and successful treatment of MS. One of the underlying mechanisms behind their immunomodulatory function is the release of extracellular vesicles, particularly exosomes. In this study, we aimed to evaluate the suppressive efficacy of MSCs and their exosomes on the proliferation of peripheral mononuclear blood cells (PBMC) in relapsing-remitting MS (RRMS) patients and healthy subjects. To do, mesenchymal stem cells were derived from human umbilical cord tissues and used for exosome isolation through ultracentrifugation. Suppressive function of MSCs and MSC-derived exosomes was examined in a coculture with CFSE-labelled PBMCs in vitro. PBMC proliferation of the patients and healthy individuals was measured using flow cytometry. We first demonstrated that proliferation of PBMCs decreased in the presence of MSCs and suppression was more efficient by MSC-derived exosomes, with a minimum alloreaction rate. However, suppression capacity of MSCs and their exosomes significantly decreased during extensive sub-culturing. The present study showed that MSC-derived exosomes as an effective cell-free therapy could prevent proliferation of PBMCs. However, further evaluations are need to move towards a functional approach that can be translated to the clinic.

3'UTR enhances hCD47 cell surface expression, self-signal function, and reduces ER stress in porcine fibroblasts.

Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3' untranslated region (3'UTR) of the hCD47 gene, which is missing from constructs previously used to make hCD47 transgenic pigs, is critical for efficient cell surface expression in human cells. The aim of this study was to investigate the impact of a modified form of the 3'UTR on the expression, localization, and function of hCD47 in transfected porcine cells. hCD47 constructs with and without the modified 3'UTR were knocked into the GGTA1 locus in porcine fetal fibroblasts using CRISPR. Flow cytometry of the transfected cells was used to analyze hCD47 localization. Endoplasmic reticulum (ER), mitochondrial, and oxidative stress were examined by gene expression analysis and confocal microscopy. Phagocytosis of transfected cells by human macrophages was measured by flow cytometry, and stimulation of human/non-human (NHP) primate lymphocytes by the cells was examined using a PBMCs proliferation assay. Cells transfected with the construct lacking the 3'UTR (hCD47(3'UTR-)) exhibited predominantly intracellular expression of hCD47, and showed evidence of ER stress, dysregulated mitochondrial biogenesis, oxidative stress, and autophagy. Inclusion of the 3'UTR (hCD47(3'UTR+)) decreased intracellular expression of hCD47 by 36% and increased cell surface expression by 53%. This was associated with a significant reduction in cellular stress markers and a higher level of protection from phagocytosis by human macrophages. Furthermore, hCD47(3'UTR+) porcine cells stimulated significantly less proliferation of human/NHP T cells than hCD47(3'UTR-) cells. Our results suggest the potential benefits of using hCD47 constructs containing the 3'UTR to generate genetically engineered hCD47-expressing donor pigs.

Designing peptide-based vaccine candidates for Plasmodium falciparum erythrocyte binding antigen 175

Plasmodium falciparum leads to a virulent form of malaria. Progress has been achieved in understanding the mechanisms involved in the malarial infection, still there is no effective vaccine to prevent severe infection. An effective vaccine against malaria should be one which can induce immune responses against multiple epitopes in the context of predominantly occurring HLA alleles. In this study, an integrated approach was employed to identify promiscuous peptides of a well-defined sequence of erythrocyte binding antigen-175 and promiscuous peptides for HLA alleles were designed using bioinformatics tools. A peptide with 15 amino acids (ILAIAIYESRILKRK) was selected based on its high binding affinity score and synthesized. This promiscuous peptide was used as stimulating antigen in lymphoproliferative responses to evaluate the cellular immune response. It was observed this peptide evokes lymphoproliferative and cytokine responses in individuals naturally exposed to the malaria parasite. The intensity of PBMCs proliferation was observed to be higher in sera obtained from P. falciparum exposed as compared to unexposed healthy individuals, suggesting earlier recognition of peptide of this region by T cells. Furthermore, the binding mode of HLA–peptide complex and their interaction may lead to a rational and selective peptide-based vaccine candidate design approach which can be used as a malaria prophylaxis.