Cells In Peripheral Blood Mononuclear Cells Research Articles

The Impact of the Immunophenotyping Characteristics of Patients' Peripheral Blood on the Manufacturing and Clinical Outcome of CD7-Targeted Chimeric Antigen Receptor T Cells

BackgroundT cell acute lymphoblastic leukemia and T cell acute lymphoblastic lymphoma (T-ALL/LBL) is a highly aggressive hematological tumor characterized by immature lymphoblasts invading the bone marrow. Treatment options for patients with T-ALL/ LBL are currently limited. Chimeric antigen receptor (CAR)- T cell therapy has opened an era in the treatment of B-cell malignancies. However, the development of CAR-T therapy for T-ALL/LBL faces many challenges. One of them is that therapeutic targets are usually expressed on both tumor and normal T cells, which causes the potential risk of “cell fratricide”. Therefore, the difficulty of manufacturing CAR T cells for T-ALL/LBL is dramatically increased. CD7, is 40kD membrane-bound glycoprotein majorly expressed on peripheral T-cells and NK cells and their precursors. CD7 is highly expressed in almost all T-ALL/LBL and considered to be one of the most promising targets for T-ALL/LBL treatment.Patients and MethodsThis study is based on a phase I clinical trial (NCT04004637) for patients with relapse/refractory CD7 + NK/T cell lymphoma and T-ALL/LBL. To manufacture CAR-T cells, the peripheral blood mononuclear cells (PBMC) were collected from the patients who met the enrollment criteria. The proportion of viability and the ratio of the T cell markers were analyzed. Subsequently, the isolated T cells were co-transduced with CD7 protein expression blocker (PEBL) and CD7-CAR lentiviruses to obtain CD7-CAR-T cells, which can avoid the fratricide of CD7-CAR-T cells. Before the infusion, the phenotypic characteristics and cytotoxicity of CD7-CAR-T products were analyzed. Then peripheral blood (PB) of patients was collected regularly after receiving treatment to analyze the immunophenotyping of T cells.ResultsFrom August 2019 to June 2021, 24 leukopaks from patients with CD7-positive T-ALL/LBL were collected, and a total of 32 batches of CD7-CAR T cells were manufactured, with a 78.13% (25/32) successful rate. Among the 7 batches of failure cases, one patient had undergone blood collection twice and CAR-T preparation for three times, but all of three attempts failed (brown icon). Another four patients failed to prepare once. Eight patents were recruited for CD7-CAR-T treatment and 87.5% of complete remission (CR) rate was achieved (7/8), of which a patient (P4, blue icon) has been maintaining CR for more than 15 months. Two other patients, P7 (red icon) and P8 (light red icon), had CD7 - relapse at the time of 6th month and 3rd month after CR, respectively.We divided all samples into successful preparation group (GS), infusion group (GI) and preparation failure group (GF). As shown in Fig. 1A, all three groups exhibited good viability of PBMC. There was no significant difference between GS and GF, but GI was higher than that of GF. The proportion of CD3 + cells in PBMC of GS was significantly lower than that of GF, and GI also showed this feature. Meanwhile, GS and GI both have a higher CD4 +/CD8 + ratio compared with GF.The immunophenotyping results showed CD7-CAR-T products had a majority of the central memory subsets (T CM; 69.41 ± 10.71%) and effect memory subsets (T EM; 28.56 ± 10.19%), with limited number of effector T cell (T E) and naive T cells (T N) (Fig. 1B). The percentage of CAR +CD8 +CD27 + and CD4 +CD25 +CD127 - subsets associated with T cells activation and proliferation, as well as CD223 + and CD279 + subsets related to T cells suppression and exhaustion were lower, except for CD366 + subgroup that also indicated depletion signal (Fig. 1B). In addition, CD7-CAR-T cells showed strong cytotoxicity against CEM (CD7 +) tumor cells accompanied by the release of cytokines, in which the level of IL-2 is extremely low (Fig. 1C).Subsequently, we performed statistics on the proportion of CD3 + and CD4 +/CD8 + cells in the PB of patients after infusion. The proportion of CD3 + cells in the PB of the P4 has been maintained at a high level, and the ratio of CD4 +/CD8 + keeps low (Fig. 1D). P7 showed a significant decrease in the amount of T cells on the 60th day after CAR T infusion, while the ratio of CD4 +/CD8 + showed an upward trend.ConclusionThe results indicate that the success rate of CD7-CAR-T manufacturing is positively correlated with higher viability, lower CD3 + and higher CD4 + of PBMC. There was no significant difference among P4 (CR more than 15 months), P7 (CD7 - relapse at 6 th month after CR) and P8 (CD7 - relapse at 3rd month after CR). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Orelabrutinib, a Selective Bruton's Tyrosine Kinase (BTK) Inhibitor in the Treatment of Primary Immune Thrombocytopenia (ITP)

Background:Primary immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disorder characterized by decreased platelet count. increased risk of bleeding, and poor quality of life. Only about 70% patients response to first-line treatments, some patients are still refractory or relapsed after combined therapies, therefore it is necessary to explore new therapeutic targets. Bruton's tytosine kinase (BTK) is a non-receptor tyrosine kinase of Tec family, which is widely expressed in hematopoietic cells including B cells, monocytes/macrophages, and others. BTK participates in a variety of signaling pathways of innate and adaptive immunity, and plays an important role in cell survival and maturation. Platelet destruction mediated by anti-platelet glycoprotein antibodies is considered to be the main cause of ITP. B cells differentiate into plasma cells and produce autoantibodies due to the intolerance to autoantigens, which are important effectors in the pathogenesis of ITP. We speculated that inhibition of BTK may reduce platelet destruction by inhibiting B cell activation and autoantibody production. Orelabrutinib is a new generation of BTK inhibitor which has been used in hematological malignancies, this is the first study to explore the mechanisms of BTK inhibitor in the treatment of ITP.Methods:The concentrations of orelabrutinib were set at 1 nM, 10 nM, 100 nM and 1 μM in the in vitro study. Peripheral blood mononuclear cells (PBMCs) were isolated from active ITP patients and healthy controls and cultured for 72 hours, the apoptosis rate of PBMCs in each group was measured by Annexin V/PI double staining. CD19 + B cells of ITP patients were sorted by magnetic beads and stimulated with anti-human IgM to evaluate the activation of B-cell receptor (BCR) pathway and differentiation of plasma cells, respectively. Further, we transfused the splenocytes of immunized CD61-KO mice (C57BL/6) into the severe combined immunodeficient (SCID) mice to establish the active ITP murine models. Orelabrutinib was administered intragastrically at 10mg/kg, once a day. The control group was treated with 0.5% CMC at the same volume and frequency. Platelet count was measured weekly, the peripheral blood was collected and the B cell subsets in spleen were detected by flow cytometry at days 28 after splenocyte transfusion.Results:The proportion of early apoptotic cells (Annexin V +PI -) in PBMCs from both ITP patients and healthy controls was increased by orelabrutinib at 1μM,but there was no statistical difference. Orelabrutinib significantly inhibited the expression of CD69 in a dose-dependent manner at the concentrations of 10nM, 100nM and 1μM. Another early activation marker of BCR signaling pathway, CD86, was also found to be inhibited by orelabrutinib at 100nM and 1μM. The number of CD138 + plasma cells was decreased after treated with orelabrutinib at 10nM, 100nM and 1μM without dose-dependent manner. In the murine models, mice administered with orelabrutinib had significantly higher platelet count than the control mice at days 7, 14, 28 after splenocyte transfusion. The frequency of total B cells in peripheral blood leukocytes, the proportion of GL-7 + germinal center B cells and plasma cells in splenocytes were all determined to be lower in mice treated with orelabrutinib than the control group, though did not reach the statistical significance.Conculsion:Orelabrutinib could effectively suppress the activation and differentiation of B cells in vitro and in vivo, thus alleviate the thrombocytopenia in active ITP murine models. It could be the new treatment strategy for refractory/relapsed ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Peripheral neutrophils and naive CD4 T cells predict the development of heart failure following acute myocardial infarction: A bioinformatic study.

Heart failure (HF) secondary to acute myocardial infarction (AMI) is still a worldwide problem with a high mortality rate. The current study aimed to explore early and reliable predictive biomarkers of HF following AMI. The gene expression profile GSE59867 was downloaded from GEO. Array data from peripheral blood mononuclear cells (PBMCs) was used from 46 control patients and 111 patients with AMI at four time points: (i) first day of AMI; (ii) 4-6 days after AMI; (iii) one month after AMI; and (iv) six months after AMI. Among the 111 AMI patients, nine with HF and eight without HF were studied. CIBERSORT was used to analyze the relative proportions of immune cells in PBMCs. The proportions of immune cells in different groups were compared. Differentially expressed genes (DEGs) were analyzed with R language packages. The percentages of monocytes and neutrophils increased significantly on the first day of AMI, and then decreased gradually. The percentage of regulatory T cells increased significantly 4-6 days after AMI, while the percentage of resting memory CD4 cells, CD8 T cells, and resting natural killer cells decreased significantly on the first day of AMI, and then increased gradually. Patients who developed HF had a significantly higher proportion of neutrophils in PBMCs on the first day of AMI, but had a significantly lower proportion of naive CD4 T cells. Two shared genes, interleukin-1 receptor 2 (IL1R2) and leucine-rich repeat neuronal protein 3 (LRRN3), were found to have potentially important roles in predicting the development of HF following AMI. A higher proportion of neutrophils and a lower proportion of naive CD4 T cells in PBMCs on the first day of AMI may be correlated with the development of HF following AMI. IL1R2 and LRRN3 may exert functions in the development of HF following AMI.

Peripheral neutrophils and naive CD4 T cells predict the development of heart failure following acute myocardial infarction: A bioinformatic study

IntroductionHeart failure (HF) secondary to acute myocardial infarction (AMI) is still a worldwide problem with a high mortality rate. The current study aimed to explore early and reliable predictive biomarkers of HF following AMI. MethodsThe gene expression profile GSE59867 was downloaded from GEO. Array data from peripheral blood mononuclear cells (PBMCs) was used from 46 control patients and 111 patients with AMI at four time points: (i) first day of AMI; (ii) 4-6 days after AMI; (iii) one month after AMI; and (iv) six months after AMI. Among the 111 AMI patients, nine with HF and eight without HF were studied. CIBERSORT was used to analyze the relative proportions of immune cells in PBMCs. The proportions of immune cells in different groups were compared. Differentially expressed genes (DEGs) were analyzed with R language packages. ResultsThe percentages of monocytes and neutrophils increased significantly on the first day of AMI, and then decreased gradually. The percentage of regulatory T cells increased significantly 4-6 days after AMI, while the percentage of resting memory CD4 cells, CD8 T cells, and resting natural killer cells decreased significantly on the first day of AMI, and then increased gradually. Patients who developed HF had a significantly higher proportion of neutrophils in PBMCs on the first day of AMI, but had a significantly lower proportion of naive CD4 T cells. Two shared genes, interleukin-1 receptor 2 (IL1R2) and leucine-rich repeat neuronal protein 3 (LRRN3), were found to have potentially important roles in predicting the development of HF following AMI. ConclusionA higher proportion of neutrophils and a lower proportion of naive CD4 T cells in PBMCs on the first day of AMI may be correlated with the development of HF following AMI. IL1R2 and LRRN3 may exert functions in the development of HF following AMI.

Gram-Negative Bacterial Lipopolysaccharide Promotes Tumor Cell Proliferation in Breast Implant-Associated Anaplastic Large-Cell Lymphoma.

Simple SummaryThe development of a rare cancer of the immune system (lymphoma) associated with breast implants has been increasingly reported around the world. It has been hypothesized that the cancer is triggered by inflammation from bacteria residing within the textured surface of these implants, transforming the lymphocytes of some genetically prone patients over many years. This study shows that bacteria rather than the implant itself can trigger activation and multiplication of these cancer cells in the laboratory, lending support that bacteria and their products play an important role in causation. The unique response of these cancer cells to bacterial antigen was dampened significantly in the presence of a Toll-like receptor 4 inhibitor peptide. This finding has significance for both cancer prevention and treatment.Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is a distinct malignancy associated with textured breast implants. We investigated whether bacteria could trigger the activation and multiplication of BIA-ALCL cells in vitro. BIA-ALCL patient-derived BIA-ALCL tumor cells, BIA-ALCL cell lines, cutaneous ALCL cell lines, an immortal T-cell line (MT-4), and peripheral blood mononuclear cells (PBMC) from BIA-ALCL, capsular contracture, and primary augmentation patients were studied. Cells were subjected to various mitogenic stimulation assays including plant phytohemagglutinin (PHA), Gram-negative bacterial lipopolysaccharide (LPS), Staphylococcal superantigens enterotoxin A (SEA), toxic shock syndrome toxin-1 (TSST-1), or sterilized implant shells. Patient-derived BIA-ALCL tumor cells and BIA-ALCL cell lines showed a unique response to LPS stimulation. This response was dampened significantly in the presence of a Toll-like receptor 4 (TLR4) inhibitor peptide. In contrast, cutaneous ALCL cells, MT-4, and PBMC cells from all patients responded significantly more to PHA, SEA, and TSST-1 than to LPS. Breast implant shells of all surface grades alone did not produce a proliferative response of BIA-ALCL cells, indicating the breast implant does not act as a pro-inflammatory stimulant. These findings indicate a possible novel pathway for LPS to promote BIA-ALCL cell proliferation via a TLR4 receptor-mediated bacterial transformation of T-cells into malignancy.

Labial gland-derived mesenchymal stem cells and their exosomes ameliorate murine Sj\xf6gren's syndrome by modulating the balance of Treg and Th17 cells

BackgroundSjögren's syndrome (SS) is a chronic, systemic autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The disease severely affects the health and life of patients, and the treatment of SS has always been a clinical challenge and essentially palliative. Mesenchymal stem cells (MSCs) have been reported to exert immunomodulatory effects and as a potential novel therapeutic strategy for SS. Labial gland-derived MSCs (LGMSCs) are a population of resident stem cells in the labial gland, first isolated by our group. Exosomes released by MSCs contain a large variety of bioactive molecules and considered to function as an extension of MSCs.MethodsLGMSCs were isolated from patients who were needed surgery to remove the lip mucocele and LGMSCs derived exosomes (LGMSC-Exos) were isolated by ultracentrifugation. The non-obese diabetic (NOD) mice were treated with LGMSCs or LGMSC-Exos by tail vein injection. The saliva flow rate of mice was determined and salivary glands were dissected and stained with hematoxylin and eosin. In vitro, peripheral blood mononuclear cells (PBMCs) from SS patients were cocultured with LGMSCs or LGMSC-Exos. Percentage of T helper 17 (Th17) cells and regulatory T (Treg) cells were determined by flow cytometry. The serum levels of cytokines in NOD mice and in the supernatant of the co-culture system by ELISA.ResultsTreatment with LGMSCs or LGMSC-Exos reduced inflammatory infiltration in the salivary glands, and restored salivary gland secretory function in NOD mice. Importantly, LGMSCs or LGMSC-Exos were demonstrated to inhibit the differentiation of Th17 cells but promote the induction of Treg cells in NOD mice and PBMCs from SS patients in vitro, accompanied by reduced interleukin 17 (IL-17), interferon gamma, and IL-6 levels and enhanced transforming growth factor beta and IL-10 secretion by T cells.ConclusionsLGMSCs are potential candidates for MSCs-based therapy and LGMSC-Exos might be utilized for establishing a new cell-free therapy against SS.

A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer.

IntroductionCD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cells, and promote anti-PD-1 mAb therapy-induced immune escape. Consequently, developing a CD73 inhibitor as monotherapy and a potential beneficial combination partner with immune-checkpoint inhibitors needs investigation.MethodsCD73 inhibitors were evaluated in vitro with soluble and membrane-bound CD73 enzymes, as well as its PD biomarker responses in human peripheral blood mononuclear cells (PBMC) by flow cytometry and ELISA. The binding modes of the molecules were analyzed via molecular modeling. The anti-tumor activity and synergistic effect of SHR170008 in combination with anti-PD-1 mAb were evaluated in a syngeneic mouse breast cancer model.ResultsSHR170008 was discovered during the initial structural modifications on the link between the ribose and the α-phosphate of AMPCP, which significantly improved the stability of the compound confirmed by the metabolite identification study. Further modifications on the adenine base of AMPCP improved the potency due to forming stronger interactions with CD73 protein. It exhibited potent inhibitory activities on soluble and endogenous membrane-bound CD73 enzymes, and induced IFNγ production, reversed AMP-suppressed CD25+ and CD8+/CD25+ expression, and enhanced granzyme B production on CD8+ T cells in human PBMC. SHR170008 showed dose-dependent anti-tumor efficacy with suppression of adenosine in the tumors in EMT6 mouse breast tumor model. The increase of adenosine in tumor tissue by anti-PD-1 mAb alone was suppressed by SHR170008 in the combination groups. Simultaneous inhibition of CD73 and PD-1 neutralization synergistically enhanced antitumor immunity and biomarkers in response, and exposures of SHR170008 were correlated with the efficacy readouts.ConclusionOur findings suggest that CD73 may serve as an immune checkpoint by generating adenosine, which suppresses the antitumor activity of anti-PD-1 mAb, and inhibition of CD73 may be a potential beneficial combination partner with immune-checkpoint inhibitors to improve their therapeutic outcomes in general.

The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs.

SummaryThe generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.

SIGIRR and TNFAIP3 Are Differentially Expressed in Both PBMC and TNF-α Secreting Cells of Patients With Major Depressive Disorder.

Background: Major depressive disorder (MDD) is associated with the activation of the immune/inflammatory system. TNF-α is associated with MDD and poor treatment response. Toll-like receptors (TLR) are responsible in innate immune response, and is associated with MDD and antidepressant response. Some negative regulators of TLR pathway such as SOCS1, TOLLIP, SIGIRR, TNFAIP3, and MyD88s, are reported to be differentially expressed in the peripheral blood samples of patients of MDD.Methods: We recruited patients with MDD and healthy controls, collect their demographic data, and measured their mRNA levels of negative TLR regulators, using peripheral blood mononuclear cells (PBMC) and isolated TNF-α secreting cells. Clinical symptoms were evaluated using Halmiton Depression Rating Scale (Ham-D). Some patients were evaluated again after 4 weeks of antidepressant treatment.Results: Forty-seven patients with MDD and 52 healthy controls were recruited. Between the PBMC samples of 37 MDD patients and 42 controls, mRNA levels of SOCS1, SIGIRR, TNFAIP3, and MyD88s were significantly different. Between TNF-α secreting cells of 10 MDD patients and 10 controls, mRNA levels of SIGIRR and TNFAIP3 were significantly different. Change of Ham-D score only correlated significantly with TOLLIP mRNA level after treatment.Conclusion: SIGIRR and TNFAIP3, two negative regulators of TLR immune response pathways, were differentially expressed in both PBMC and TNF-α secreting cells of patients with MDD as compared to healthy controls. The negative regulations of innate immune response could contribute to the underlying mechanism of MDD.

Impact of high-dose rate radiotherapy on B and natural killer (NK) cell polarization in peripheral blood mononuclear cells (PBMCs) via inducing non-small cell lung cancer (NSCLC)-derived exosomes.

BackgroundNon-small cell lung cancer (NSCLC) is the most commonly diagnosed solid tumor. While it has been established that stereotactic body radiotherapy for NSCLC plays an important role in antitumor immune response, the possible effects of the dose rate on this response has not been fully clarified.MethodsIn vitro, A549 cells were irradiated on a Varian TrueBeam® Linear Accelerator with dose and dose rate escalation using the flattening filter-free (FFF) technique, which was followed by coculturing with peripheral blood mononuclear cells (PBMCs). The exosomes from irradiated A549 cells were isolated and then cocultured with PBMCs. Flow cytometry was performed to analyze the proportion of lymph cell clusters in PBMCs.ResultsThe proportion of CD3− immune cell clusters in PBMCs was significantly higher in the 10 Gy treatment group than in the nonirradiated group and other lower-dose (2, 6 Gy) treatment groups at the dose rate of 1,000 MU/min. However, no influence was observed on the proportion of CD3+ T cell subsets. Further results showed that both natural killer (NK) and B cell proportions reached peaks in the 14 Gy treatment group when a dose rate of 1,200 MU/min was used. Notably, the peak values of these two cell proportions were reached at a lower radiation dose of 10 Gy when a greater dose rate, ranging from 1,600 to 2,400 MU/min, was used. We further found that a single, high dose of irradiation (10 Gy), as compared with a single, low dose of irradiation (2 Gy), could markedly stimulate the A549-related exosome secretion in a radiation dose rate-dependent manner. The ultrahigh dose rate radiation–derived exosomes contributed to the polarization of B and NK cell subsets in PBMCs.ConclusionsThe optimized radiation regime, which depends on the appropriate radiation dose and dose rate, results in the production of exosomes derived from NSCLC cells and eventually the redistribution of immune cells in PBMCs.

Abstract 1524: Peripheral blood mononuclear cells engineered via microfluidic Cell Squeeze® technology to generate APCs that drive mutant-KRas-specific CD8+ T cell responses

Abstract KRAS mutations are shared across multiple cancer types and are most commonly found in cancers of the lung, pancreas, and colon. While recently developed drugs blocking signaling driven by KRAS G12C mutations have shown promise in the clinic, there is a great need for therapies targeting the more common G12V and G12D mutations. We utilize our microfluidic Cell Squeeze® technology to deliver antigens directly into the cytosol of peripheral blood mononuclear cells (PBMCs) for the generation of cancer antigen-targeted cellular vaccines. This method bypasses the need for cross-presentation and directly enables efficient antigen presentation on MHC-I in multiple cell types. Murine and human in vitro studies have demonstrated that this allows SQZ PBMCs to drive CD8+ T cell activation. Here, we demonstrate the delivery of various antigens, including KRas-derived peptides containing the most common mutations (G12D and G12V) to PBMC cell subsets. In addition, we find that human PBMCs squeezed with either KRasG12V or KRasG12D peptides can specifically stimulate KRasG12V- and KRasG12D- reactive CD8+ T cell responders. When KRasG12V and KRasG12D peptides are co-delivered via microfluidic squeezing, the human PBMCs enable activation of CD8+ T cells of both specificities. This work with KRasmutant-presenting human PBMCs lays a strong foundation for developing a potentially potent cell therapy to treat KRASmutant associated cancers. Citation Format: Defne Yarar, Carolyne Smith, Amritha Ramakrishnan, Scott Loughhead, Armon Sharei, Katherine Seidl, Howard Bernstein. Peripheral blood mononuclear cells engineered via microfluidic Cell Squeeze® technology to generate APCs that drive mutant-KRas-specific CD8+ T cell responses [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 1524.

CD8+ T cells actively penetrate hepatocytes via the CD44/p-ERM/F-actin pathway in autoimmune hepatitis.

Emperipolesis is a pathological feature for the diagnosis of autoimmune hepatitis (AIH). We have previously found that CD8+ T cells participated in the emperipolesis in AIH. In this study we aimed to clarify the characteristics and molecular mechanisms of emperipolesis in patients with AIH in vitro and in mice with α-Galactosylceramide (α-GalCer)-induced acute hepatitis. The peripheral blood mononuclear cells (PBMC) of patients with various chronic liver diseases and healthy controls were co-cultured with hepatic cell lines to induce emperipolesis in vitro. Confocal staining was performed to illustrate the cellular types and potential mechanisms of emperipolesis in AIH. In addition, a murine model of α-GalCer-induced acute hepatitis that mimics human AIH was used to confirm the role of CD44/p-ERM/F-actin in the emperipolesis process in vivo. In the co-cultured system of PBMC and hepatic cell line, emperipolesis was observed most commonly in patients with AIH. The main cells participating in emperipolesis were CD8+ T cells, and they penetrated hepatic cells actively via the CD44/p-ERM/F-actin pathway. As a result, most CD8+ T cells engulfed by hepatic cells underwent apoptosis. In the α-GalCer-induced acute hepatitis model, emperipolesis was observed around the inflammatory foci and was inhibited by the ezrin phosphorylation inhibitor NSC668394. Similarly, activated murine CD8+ T cells penetrated primary hepatocytes via the CD44/p-ERM/F-actin pathway in vitro. CD8+ T cells penetrate hepatic cells actively via the CD44/p-ERM/F-actin signaling pathway and undergo apoptosis. This may be a compensatory mechanism to attenuate the overwhelming immune attack in AIH.

2’Fucosyllactose and Bifidobacterium longum subsp. infantis Supplementation Modulates Immune Development of Piglets

ObjectivesProbiotics and 2’fucosyllactose (2’FL) have been shown to affect immune development in infants, but less is known about their synbiotic administration. Herein, the effects of 2’FL and Bifidobacterium longum subsp. infantis (Bi-26) on immune development was investigated in the young pig. MethodsMale piglets (N = 53 were provided ad libitum access to milk replacer without (CON) or with 1g/L 2’FL (FL) from postnatal day 2 to 34/35. Pigs were then stratified to receive Bi-26 prepared in glycerol stock (109 CFU) or glycerol stock alone (BI and FLBI). Blood, mesenteric lymph nodes (MLN), and ascending colon (AC) and rectal (RC) contents were collected. MLN and peripheral blood mononuclear cells (PBMC) immune cells were quantified by flow cytometry. Immunoglobulins (Igs) were measured by ELISA. PBMC and MLN cells were stimulated ex vivo with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) for 72 h. Cytokines in serum and ex vivo cell supernatants were measured by multiplex assay. Cytokine data were analyzed by multivariate LCA-model. All other data were analyzed by a 2-way ANOVA with fixed effects of prebiotic and probiotic. ResultssIgA tended (P = 0.07) to be higher in RC than AC, with no treatment effect. Serum IgG and IgM and MLN and PBMC immune cells were unaffected by treatment. Serum, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-10, IL-12 and IL-18 were all higher in FL than CON (p < 0.05). In unstimulated PBMC, IL-18 was lower in FL, BI and FLBI than CON (p < 0.05). In LPS-stimulated PBMCs, IFNγ was higher in FL and IL1-RA was higher in FLBI than CON (p < 0.05). LPS had no effect on MLN cytokines; but, PHA increased IL-17 in FL and FLBI versus CON (p < 0.05). ConclusionsCompared to CON, serum cytokines involved in Th1 T-cell differentiation, IL-2, IL-12 and IL-18 were higher in FL piglets. This was countered by the increased production of IL-4 and IL-10. IL-1RA also increased, potentially balancing higher IL-1α and IL-1β in FL piglets. These differences were not observed in FLBI. LPS stimulated IFNγ secretion by PBMC from FL, whereas cells from FLBI secreted more IL-1RA. These findings suggest that dietary 2’FL primes T-cells for proinflammatory cytokine secretion, which is modulated by co-administration of Bi-26. Funding SourcesDuPont Nutrition and Biosciences.

Structure-Based Design and Development of Chemical Probes Targeting Putative MOR-CCR5 Heterodimers to Inhibit Opioid Exacerbated HIV-1 Infectivity.

Crystal structures of ligand-bound G-protein-coupled receptors provide tangible templates for rationally designing molecular probes. Herein, we report the structure-based design, chemical synthesis, and biological investigations of bivalent ligands targeting putative mu opioid receptor C-C motif chemokine ligand 5 (MOR-CCR5) heterodimers. The bivalent ligand VZMC013 possessed nanomolar level binding affinities for both the MOR and CCR5, inhibited CCL5-stimulated calcium mobilization, and remarkably improved anti-HIV-1BaL activity over previously reported bivalent ligands. VZMC013 inhibited viral infection in TZM-bl cells coexpressing CCR5 and MOR to a greater degree than cells expressing CCR5 alone. Furthermore, VZMC013 blocked human immunodeficiency virus (HIV)-1 entry in peripheral blood mononuclear cells (PBMC) cells in a concentration-dependent manner and inhibited opioid-accelerated HIV-1 entry more effectively in phytohemagglutinin-stimulated PBMC cells than in the absence of opioids. A three-dimensional molecular model of VZMC013 binding to the MOR-CCR5 heterodimer complex is constructed to elucidate its mechanism of action. VZMC013 is a potent chemical probe targeting MOR-CCR5 heterodimers and may serve as a pharmacological agent to inhibit opioid-exacerbated HIV-1 entry.

Supraphysiological estradiol promotes human T follicular helper cell differentiation and favours humoural immunity during in vitro fertilization.

During pregnancy, humoural immunity is essential for protection against many extracellular pathogens; however, autoimmune diseases may be induced or aggravated. T follicular helper (Tfh) cells contribute to humoural immunity. The aim of this study was to test whether Tfh cell function can be manipulated via hormones. Seventy‐four women who underwent in vitro fertilization were recruited and divided into four groups: menstrual period (MP), controlled ovarian hyperstimulation (COH), embryo transfer (ET) and pregnant after embryo transfer (P). A flow cytometry analysis was performed to identify Tfh cells in peripheral blood mononuclear cells (PBMCs). Bioinformatics analysis revealed a possible pathway between Tfh and B cells. Enzyme‐linked immunosorbent assays were used to detect interleukin (IL)‐21 and IL‐6. The quantitative polymerase chain reaction was performed to quantify BCL‐6, BACH2, XBP‐1, IRF‐4 and G protein‐coupled (GP)ER‐1 mRNA expression. Compared with the MP group, the COH, ET and P groups showed more Tfh and B cells, as well as higher IL‐21, IL‐6, BCL‐6 and BACH2 expression. Furthermore, Tfh cell frequency in PBMCs, as well as serum IL‐21 and IL‐6 levels, were all positively correlated with serum estradiol (E2) levels; the B cell percentage also correlated positively with Tfh cells in PBMCs. Combined with the bioinformatics analysis, XBP‐1, IRF‐4 and GPER‐1 expression was related to E2 levels, both in vivo and in vitro. We speculate that E2 augments Tfh cells and favours humoural immunity. This study indicates that Tfh cell regulation may be a novel target in maintaining the maternal‐foetal immune balance.

Expression profile of PU.1 in CD4+T cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. PU.1 is an important member of the ETS transcription factors family which has diverse functions such as regulating the proliferation, differentiation of immune cells and multiple inflammatory cytokines. Previous studies preliminary explored the relation between PU.1 and SLE. To further explain the potential role of PU.1 in the pathogenesis of SLE, 40 SLE patients and 20 age-sex matched healthy controls (HC) were recruited in this study. Flow cytometry was used to test the percentages of CD4+PU.1+T cells in peripheral blood mononuclear cells (PBMCs) from patients with SLE and HC. Expression levels of PU.1 mRNA in CD4+T cells from SLE patients and HC were analyzed by real-time transcription-polymerase chain reaction. Expression levels of plasma IL-1β, IL-9, IL-18, IL-6, IFN-α, TNF-α, IL-10 and TGF-β1 were measured by enzyme-linked immunosorbent assay. The percentage of CD4+PU.1+T cells in PBMCs from patients with SLE was significantly higher than that from HC (P < 0.001). In addition, the PU.1 mRNA expression in CD4+T cells from SLE patients was increased than that from HC (P = 0.002). In SLE patients, no significant correlation was found between the percentage of CD4+PU.1+T cells and the expression of PU.1 mRNA in CD4+T cells (P > 0.05). Associations of PU.1 mRNA expression in CD4+T cells with major clinical and laboratory parameters of SLE patients were also analyzed, but no significant correlations were found. Consistent with previous studies, SLE patients had increased IL-1β, IL-18, IL-6, IFN-α, TNF-α and IL-10 plasma concentrations than HC (P < 0.01). The expression level of plasma TGF-β1 was significantly decreased in SLE patients than in HC (P < 0.001). In SLE patients, the expression level of IL-1β was positive correlated with PU.1 mRNA expression in CD4+T cells (P = 0.001). Our study first time evaluated the expression profile of PU.1 in CD4+T cells from SLE patients confirming that PU.1 may participate in the pathogenesis of SLE.

An orally available PD-1/PD-L1 blocking peptide OPBP-1-loaded trimethyl chitosan hydrogel for cancer immunotherapy

Blockade of the immune checkpoint PD-1/PD-L1 with monoclonal antibodies demonstrated unprecedented clinical efficacies in many cancers. But the orally available low molecular weight inhibitors remain infancy. Compared to small molecules, peptide exhibits better selectivity and fewer side effects, but poor half-life and a big challenge to be orally administrated. Here, we developed a proteolysis-resistant D peptide OPBP-1 (Oral PD-L1 Binding Peptide 1) which could selectively bind PD-L1, significantly block PD-1/PD-L1 interaction and enhance IFN-γ (interferon γ) secretion from CD8+ T cells in human PBMCs (Peripheral blood mononuclear cells). OPBP-1 could significantly inhibit tumor growth in murine colorectal CT26 and melanoma B16-OVA models at a relatively low dose of 0.5 mg/kg, with enhancing the infiltration and function of CD8+ T cells. More interestingly, oral delivery of OPBP-1 loaded TMC (N, N, N-trimethyl chitosan) hydrogel (OPBP-1@TMC) showed promising OPBP-1 oral bioavailability (52.8%) and prolonged half-life (14.55 h) in rats, and also significantly inhibited tumor growth in CT26 model. In conclusion, we discovered and optimized a PD-1/PD-L1 blocking peptide OPBP-1, and subsequently loaded into a TMC based hydrogel oral delivery system, in order to maximally elevate the oral bioavailability of the peptide drug and effectively inhibit tumor growth. These results opened up a new prospect for oral drug development in cancer immunotherapy.

BCG-Infected Dendritic Cells Induce TNF-alpha Dependent Cell Cluster Formation That Damage theBlood Brain Barrier

Abstract Central nervous system tuberculosis (CNSTB) is the most serious manifestation of extrapulmonary tuberculosis infections representing 5–10% of all cases. How Mycobacterium tuberculosis (Mtb) crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. We applied a novel in vitro model of BBB model that consists of primary brain astrocytes and microvessel endothelial cells to test the mechanisms how mycobacterium-infected DCs interact with peripheral blood mononuclear cells (PBMC) and affect the BBB. We show that mycobacterium hinders DC migration, and infected DCs adhere to brain microvessel endothelial cells and induce PBMC cell cluster formations. Infected DC-induced cell clusters contain 24% CD11c+, 57%CD11b+, and 16% CD4+ cells. PBMC and infected DC clusters strongly adhered to the in vitro BBB and over time induced cluster associated damage (CAD). CAD correlated with mitochondrial disorganization and the degradation of tight junction proteins ZO-1 and claudin-5, and upregulation of cellular adhesion molecules VCAM-1 and ICAM-1 on endothelial cells. Anti-TNF-a inhibition reduced cell cluster formation as well as ICAM-1 expression on brain microvessel endothelial cells and mitigated CAD. This research leads to better knowledge in understanding how mycobacteria infected cells affect the BBB that could lead to better therapeutic treatments for mycobacterial infection of the CNS.

Difference Expressions CD34 in Acute Myeloid Leukemia Cell Culture in the Administration of Cytarabine-Daunorubicine Dose Standards

The cure rate for patients with Acute Myeloid Leukemia (AML) is 20-75%. Standard-dose cytarabine + (SDAC)-daunorubicine gives a remission rate of ± 60%, and the case of relapse is frequently found. In-vivo CD34 expression is a reliable and straightforward test that must evaluate AML patients' response to predict the response of chemotherapy + induction phase accurately. Differences in in-vitro CD34 expression are expected to be able to predict chemosensitivity in AML patients. An experimental post-test-only control group study was conducted from May to December 2019, and 8 AML subjects were found. Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood samples of patients with AML collected in EDTA tubes. The PBMC isolated from peripheral blood were divided into two groups, and each group contained 106 PBMC cells in culture media. The control group (without treatment) and the SDAC-daunorubicine group were 0 + incubated for 4 hours at 37 C with a 5% CO2 atmosphere. The expression of CD34 was measured using FACSCaliburâ„¢, while + CD34+ percentage was calculated with CellQuestâ„¢ software. The percentage of CD34 in the control, SDAC + DNR, showed a significant difference with p &lt; 0.001. This study showed a significant difference between the control group and the group + administered with the standard dose of cytarabine-daunorubicine with p &lt; 0.001. The average CD34 expression in the + SDAC-DNR treatment group was higher than in the control group. CD34 markers cannot be used as predictors of chemosensitivity in the administration of chemotherapy.

Effects of vitamin K2 combined with methotrexate against mitogen-activated peripheral blood mononuclear cells of healthy subjects and rheumatoid arthritis patients.

Methotrexate (MTX) is used as anchor drug for patients with early and established rheumatoid arthritis (RA). Vitamin K2 administration was also reported to be associated with decreased disease activity in RA. Immunosuppressive pharmacodynamics of vitamin K2 combined with MTX was investigated. Mitogen-activated peripheral blood mononuclear cells (PBMCs) were used to evaluate immunosuppressive pharmacodynamics of drugs in vitro. Vitamin K2 alone dose-dependently suppressed T cell mitogen-activated proliferation of PBMCs of both healthy subjects and RA patients. 446.5 and 2232.5ng/mL vitamin K2 significantly decreased the IC50 values of MTX on the proliferation of PBMCs of RA patients, with little influences on the pharmacodynamics of MTX in the healthy PBMCs. 4465ng/mL vitamin K2 potentiated the pharmacodynamics of MTX in both RA patients and healthy PBMCs. The additional effects of vitamin K2 to potentiate the suppressive effects of MTX seemed not to be related to the regulation of CD4+ CD25+ T cells or CD4+ CD25+ Foxp3+ Treg cells. MTX alone at 100ng/mL significantly decreased the percentage of CD4+ T cells in PBMCs of healthy subjects (p<0.001) with a slight influence in that of RA patients (not significant) and the combination did not show synergistic inhibitory effect. Vitamin K2 alone tended to suppress the secretion of IL-17, IFN-γ, and TNF-α from the activated PBMCs of RA patients with smaller influences on the cytokine productions from healthy PBMCs. These additional effects of vitamin K2 were also observed in combination with MTX. The above information may partially elucidate the potentiation effects of vitamin K2 on the immunosuppressive efficacy of MTX.