scholarly journals A CTLA-4 blocking strategy based on Nanobody in dendritic cell-stimulated cytokine-induced killer cells enhances their anti-tumor effects

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wu Wang ◽  
Xi Wang ◽  
Wenli Yang ◽  
Kai Zhong ◽  
Na He ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Tumor Antigens ◽  
Malignant Tumors ◽  
Scid Mice ◽  
Adoptive Therapy ◽  
Killer Cells ◽  
Cik Cells ◽  
In Vivo Experiments

Abstract Background Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, it sefficacy is restricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells, we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36. Methods Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, we tested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity, or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy. Results After stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice. Conclusions Our findings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential to treat malignant tumors through in vivo adoptive therapy.

scholarly journals A CTLA-4 Blocking Strategy Based on Nanobaby in Dendritic Cell-stimulated Cytokine-induced Killer Cells Enhances Their Anti-tumor Effects

2021 ◽  
Author(s):  
Wu Wang ◽  
Kai Zhong ◽  
Xi Wang ◽  
Wenli Yang ◽  
Na He ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Tumor Antigens ◽  
Malignant Tumors ◽  
Scid Mice ◽  
Adoptive Therapy ◽  
Killer Cells ◽  
Cik Cells ◽  
In Vivo Experiments

Abstract Background: Cytokine-induced killer cells which were induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, the efficacy was restricted by the immunosuppression of tumor microenvironment mediated by the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) pathway. We, therefore, screened a nanobody which is targeted for CTLA-4 (Nb36), and blocked the CTLA-4 signaling with Nb36 to overcome the negative co-stimulation of effector T cells. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors and used to induce CIK cells in vitro, then co-cultured with DC cells that have received tumor antigens. We tested whether blocking CTLA-4 signaling with Nb36 could promote DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity in vitro. In vivo experiments, The NOD/SCID mice were injected subcutaneously with HepG2 cells to induce solid tumor. We observe whether this therapy can more effectively inhibit tumor growth in mice. Results: After being stimulated with Nb36, DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, thereby strengthening the killing effect on tumor cells. For in vivo experiments, Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted tumors of liver cancer, reduced tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice. Conclusions: These findings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited superior anti-tumor efficacy. Our findings suggested that DC-CIK cells + Nb36 has potential to treat malignant tumors through in vivo adoptive therapy.

Quantification of the Active Decitabine-Triphosphate (DAC-TP) Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia (AML)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3578-3578
Author(s):  
Hongyan Wang ◽  
Ping Chen ◽  
Jiang Wang ◽  
Ramasamy Santhanam ◽  
Josephine Aimiuwu ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Dna Methyltransferases ◽  
Analytical Methodology ◽  
In Vivo Experiments ◽  
Accuracy And Precision ◽  
Coefficients Of Variation ◽  
The Mean

Abstract Abstract 3578 Decitabine (DAC) is successfully used for treatment of patients (pts) with myelodysplastic syndromes and AML. Following cellular uptake, DAC is thought to be activated to DAC-TP and incorporated into DNA. The DAC-TP/DNA complex binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately anti-leukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated pts has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a sensitive and specific LC-MS/MS method for quantification of DAC-TP. The assay exhibited excellent accuracy and precision. The accuracy values were 83.7–109.4%, as determined by calculating the percentage of measured DAC-TP relative to the respective nominal concentrations (50, 500 and 5,000 nM) of the quality control samples. The within-day coefficients of variation (CVs) were 19.9 % (n=6) at 50 nM and 4.7–7.0 % between 500–5,000 nM; the between-day CVs (n=3) were 15.2 % at 50 nM and 7.5–10.2 % between 500–5,000 nM. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant MV4–11, and in THP-1 and FDC-P1/Kitmut cells (in vitro); and in bone marrow (BM) and spleen of normal and FDC-P1/Kitmut-driven AML mice (in vivo). DAC-TP reached peak levels (0.8, 1.4 and 0.5 pmol/106 cells) in 1–4 hours and declined to 20 % of its peak concentration after 24 hours incubation with 2.5 μM DAC in MV4–11, THP-1 and FDC-P1/Kitmut cells, respectively. Inhibition of hENT1 that mediates DAC transport into the cells and dCK that phosphorylates DAC into DAC-TP by NBTI and 2-thio-2′-deoxycytidine, respectively, significantly inhibited DAC-TP accumulation in AML cells. DAC-TP decay was instead blocked by tetrahydrouridine (THU)-induced inhibition of CDA, the catabolizing enzyme for cytidine and deoxycytidine and analogs. Consistent with these results, low dCK and hENTs but not CDA expression were detected in DAC-resistant MV4–11 cells, which showed 60 % decrease in DAC-TP levels as compared to their parental counterparts. DAC/DAC-TP-mediated downregulation of DNMT proteins (preferentially DNMT1 and DNMT3a) was also demonstrated in the AML cells even at DAC-TP concentrations as low as 0.1–1.3 pmol/106 cells in vitro after 4 hours DAC incubation. In the in vivo experiments, DAC-TP levels in leukemic mice were comparable to that in normal C57BL/6 mice, 0.3 pmol/106 cells in BM and 199.2 pmol/g tissue in spleen at 4-hours and 0.2 pmol/106 cells in BM and 165.3 pmol/g tissue in spleen at 24-hours following an i.v. bolus of 6.5 mg/kg DAC. In BM of leukemic mice, not only DNMT1 and DNMT3a but also DNMT3b protein expression reduced 80 % (DNMT3a) or diminished (DNMT1 and DNMT3b). The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells (PBMC) from AML pts treated with a 10-day regimen of DAC given 20 mg/m2/day i.v. over 1 hour. In BM samples, the mean DAC-TP levels were 0.8 ± 0.6 (Day 1) and 0.9 ± 0.5 pmol/106 cells (Day∼5) in complete responsive (CR) pts (n=4); and 0.4 ± 0.3 (Day 1) and 0.12 ± 0.02 pmol/106 cells (Day∼5) in non-responsive (NR) pts (n=3). In PBMC samples, the mean DAC-TP levels were 0.5 ± 0.2 (Day 1) and 1.2 ± 0.4 pmol/106 cells (Day∼5) in CR pts (n=3); and 0.02 ± 0.02 (Day 1) and 0.21 ± 0.04 pmol/106 cells (Day∼5) in NR pts (n=3). These data suggested that higher levels are seemingly associated with clinical response, but a larger number of pts need to be tested. In conclusion, monitoring the intracellular concentration of DAC-TP is feasible, and DAC-TP levels correlate with DNMT downregulation and may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Low-dose chidamide restores immune tolerance in ITP in mice and humans

Blood ◽  
2019 ◽  
Vol 133 (7) ◽  
pp. 730-742 ◽  
Author(s):  
Hong-yu Zhao ◽  
Ya-hui Ma ◽  
Da-qi Li ◽  
Tao Sun ◽  
Li-zhen Li ◽  
...  
Keyword(s):  
Immune Tolerance ◽  
Histone Deacetylase Inhibitors ◽  
Low Dose ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Peripheral Blood Mononuclear ◽  
In Vivo Experiments ◽  
Macrophage Phagocytosis

Abstract Increased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi’s) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi’s attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi’s could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

scholarly journals The priming role of dendritic cells on the cancer cytotoxic effects of cytokine-induced killer cells

2019 ◽  
Vol 22 (2) ◽  
pp. 196-212
Author(s):  
Binh Thanh Vu ◽  
Nguyet Thi-Anh Tran ◽  
Tuyet Thi Nguyen ◽  
Quyen Thanh-Ngoc Duong ◽  
Phong Minh Le ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mononuclear Cells ◽  
Interferon Γ ◽  
Interleukin 4 ◽  
In Vitro Cultivation ◽  
Killer Cells ◽  
Flow Cytometry Data ◽  
Cik Cells ◽  
Ifn Γ

Introduction: In vitro cultivation of DCs and cytokine-induced killer cells (CIK cells) - a special phenotype of T lymphocyte populations — for cancer treatment has gained significant research interest. The goal of this study is to understand whether the priming from DCs helps CIK cells to exert their toxic function and kill the cancer cells. Methods: In this research, DCs were differentiated from mononuclear cells in culture medium supplemented with Granulocyte-macrophage colony-stimulating factor (GM-CSF), and Interleukin-4 (IL-4), and were induced to mature with cancer cell antigens. Umbilical cord blood mononuclear cells were induced into CIK cells by Interferon-γ (IFN-γ), anti-CD3 antibody and IL-2. After 4-day exposure (with DC:CIK = 1:10), DCs and CIK cells interacted with each other. Results: Indeed, DCs interacted with and secreted cytokines that stimulated CIK cells to proliferate up to 133.7%. In addition, DC-CIK co-culture also stimulated strong expression of IFN-γ. The analysis of flow cytometry data indicated that DC-CIK co-culture highly expressed Granzyme B (70.47% ± 1.53, 4 times higher than MNCs, twice higher than CIK cells) and CD3+CD56+ markers (13.27% ± 2.73, 13 times higher than MNCs, twice higher than CIK cells). Particularly, DC-CIK co-culture had the most specific lethal effects on cancer cells after 72 hours. Conclusion: In conclusion, co-culture of DCs and CIK cells is capable of increasing the expression of CIK-specific characteristics and CIK toxicity on cancer cells.    

Rapid and Massive Expansion of Cord Blood Derived Cytokine Induced Killer (CIK) Cells: An Innovative Proposal for the Treatment of Leukemia Relapse after Cord Blood Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 723-723
Author(s):  
Martino Introna ◽  
Marta Franceschetti ◽  
Alice Ciocca ◽  
Gianmaria Borleri ◽  
Elena Conti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Cord Blood Transplantation ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Cik Cells ◽  
Blood Transplantation

Abstract Cytokine induced killer cells (CIK) are CD3+/CD56+ T/NK cells with cytotoxic potential against leukemic and other tumor cells but not normal bone marrow in vitro and in vivo. They are expanded in vitro with rhIL-2 after stimulation of peripheral blood mononuclear cells with OKT3 and IFN-γ. We have shown in a recent phase I study that 107/kg allogeneic CIK cells can be safely given to patients relapsing after allogeneic bone marrow transplantation and show evidence of anti-leukemic activity in vivo with very little GVHD. Cord blood (CB) transplantation is progressively becoming an extensively used treatment for patients with malignant disorders. One major limitation of this procedure is the lack of donor derived cells to perform donor lymphocyte infusions in case of relapse. In order to be able therefore to extend the use of CIK cells to the CB transplantation setting, we have standardised a 21 days expansion protocol to produce CIK cells starting from very small amounts of nucleated cells isolated from cord blood. Using this protocol, 15x106 mononuclear cells (MNC) from CB containing a mean 0.3x106 CD3+/CD56+ yielded on average 805 x 106 MNC (50 fold expansion) containing 630 x106 CD3+/CD56+ cells (corresponding to a fold expansion of 1860 for CIK). In order to transfer the method to a clinical setting, we explored the possibility of expanding the residual cells recovered from the empty bags after CB transplantion. Three used CB bags were returned to the laboratory after transplantation and repeatedly washed. An average of 22 x106 nucleated cells could be recovered, yielding a mean 473 x106 CD3/CD56+ cells at the end of the culture period (1485 fold expansion of CIK cells). CIK cells generated from CB showed strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias (42–72% killing at a 30:1 E:T ratio). More importantly, they were cytotoxic against AML blasts isolated from 2 patients (41% lysis). During expansion CB derived CIK cells upregulated the NKG2D marker from a mean fluorescence intensity of 49 to 209. Furthermore they expressed perforin and granzyme molecules in >90% of cells. These observations open up the possibility of a future clinical application of this protocol, performed in GMP conditions. Patients relapsing following cord blood transplantation may be treated with CIK cells expanded from the same cord blood unit, where donors would not be anymore available for cell mediated immunotherapy.

Ublituximab (TGTX-1101), A Novel Anti-CD20 Monoclonal Antibody (mAb), Demonstrates Activity in Rituximab-Sensitive and Rituximab–resistant B Non-Hodgkin Lymphoma (B-NHL) Pre-Clinical In vitro and in Vivo models.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2756-2756 ◽  
Author(s):  
John Miller ◽  
Matthew J. Barth ◽  
Cory Mavis ◽  
Ping-Chiao Tsai ◽  
Pavel Klener ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Primary Tumor ◽  
Scid Mice ◽  
In Vivo Experiments ◽  
Non Hodgkin Lymphoma ◽  
Tail Vein Injection ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Abstract 2756 The addition of rituximab to chemotherapy regimens utilized in treating B-cell non-Hodgkin lymphoma (B-NHL) has resulted in significant improvement in treatment response and clinical outcomes. On the other hand, the use of rituximab is changing the biology and response to second-line therapy in patients with relapsed/refractory disease. Novel anti-CD20 mAbs continue to be developed that may offer additional treatment options for relapsed/refractory rituximab-pre-treated patients. Ublituximab (TGTX-1101) is a novel, chimeric mAb targeting a unique epitope on the CD20 antigen. Ublituximab has been glycoengineered to enhance affinity for all variants of FcγRIIIa receptors. To further characterize the activity of ublituximab, we evaluated its anti-tumor activity in a panel of rituximab-sensitive (RSCL), rituximab–resistant (RRCL) cell lines, primary tumor cells isolated from patients with B-NHL by negative selection using magnetic beads, and in lymphoma SCID mice xenograft models. RSCL (Raji, RL, U2932, Granta, HBL-2, Jeko-1, Mino, Rec1 and Z-138), RRCL (Raji-2R, Raji 4RH, RL-4RH, and U2932-4RH); and cytarabine-resistant (AraCR) mantle cell lymphoma cell (MCL) lines (Granta-AraCR, HBL-2-AraCR, Jeko-AraCR, Mino-AraCR and Rec1-AraCR) were labeled with 51Cr. Subsequently, cells were exposed to ublituximab, rituximab or isotope control and human serum (25%) for complement dependent cytotoxicity (CDC) assays or to effector cells isolated from healthy volunteers (effector:target ratio 40:1) for antibody dependent cellular cytotoxicity (ADCC) assays, respectively. Antibody-induced direct anti-proliferative effects and induction of apoptosis were determined by alamar blue reduction assay and Annexin-V and propidium iodide staining, respectively. Primary tumor cells (n=11) were exposed to ublituximab, rituximab or isotype control +/− pooled human serum for 48 hr. Changes in ATP content were determined using the CellTiterGlo assay. For in vivo studies, 6–8 week old SCID mice were inoculated via tail vein injection with 1×106 Raji cells on day 0 and assigned to rituximab (10mg/kg/dose), ublituximab (10mg/kg/dose) or control group. MAb was given via tail vein injection on days +3, +7, +10 and +14. Differences in survival were analyzed by Kaplan-Meier curves and p values calculated using log rank test. Ublituximab induced significantly higher ADCC when compared to rituximab in 13 out of 17 cell lines tested (including all RRCL and cytarabine resistant MCL cells): (Raji 44.4% vs. 19.8%; Raji 4RH 17.5% vs. 8.3%; Raji 2R 28.2% vs. 12%; RL 40.9% vs. 17.8%; RL-4RH 33.5% vs. 17.2%; U2932 46.9% vs. 28.8%; U2932-4RH 40.2% vs. 22.1%; HBL-2AraCR 30.7% vs. 16.6%; Jeko 34.8% vs. 18.4; Jeko-AraCR 23.8% vs. 9.6; Mino 47.4% vs. 11.6%; Mino-AraCR 32.5% vs. 15.5; Rec1 30.9% vs. 0%; p-values <0.05). There was no significant difference between ublituximab and rituximab in terms of CMC (including studies performed in primary tumor cells) or direct signaling (i.e. apoptosis or cell proliferation). While ublituximab therapy prolonged the survival of lymphoma-bearing SCID mice when compared to controls, the anti-tumor activity in vivo was similar to rituximab. Our results suggest that ublituximab exhibits higher ADCC than rituximab in vitro, including in RRCL and elicits similar CDC and direct anti-tumor effects. Despite this enhanced ADCC activity, initial in vivo experiments did not result in improved survival compared to rituximab, however additional in vivo experiments investigating the activity of ublituximab in RRCL and MCL mouse models, testing alternative dose/schedule regimens and/or in combination with other anti-lymphoma agents are planned. Updated research results will be presented at the annual meeting. A Phase I/II trial of ublituximab in patients with relapsed/refractory NHL is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ALPK2 acts as tumor promotor in development of bladder cancer through targeting DEPDC1A

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Yuchen Wang ◽  
Jie Wu ◽  
Wenjie Luo ◽  
Hailiang Zhang ◽  
Guohai Shi ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Malignant Tumors ◽  
Direct Interaction ◽  
In Vivo Experiments ◽  
Tumor Promotor ◽  
Promising Target ◽  
Bladder Cancer Cells

AbstractBladder cancer is one of the most common malignant tumors in the urinary system. The development and improvement of treatment efficiency require the deepening of the understanding of its molecular mechanism. This study investigated the role of ALPK2, which is rarely studied in malignant tumors, in the development of bladder cancer. Our results showed the upregulation of ALPK2 in bladder cancer, and data mining of TCGA database showed the association between ALPK2 and pathological parameters of patients with bladder cancer. In vitro and in vivo experiments demonstrated that knockdown of ALPK2 could inhibit bladder cancer development through regulating cell proliferation, cell apoptosis, and cell migration. Additionally, DEPDC1A is identified as a potential downstream of ALPK2 with direct interaction, whose overexpression/downregulation can inhibit/promote the malignant behavioral of bladder cancer cells. Moreover, the overexpression of DEPDC1A can rescue the inhibitory effects of ALPK2 knockdown on bladder cancer. In conclusion, ALPK2 exerts a cancer-promoting role in the development of bladder cancer by regulating DEPDC1A, which may become a promising target to improve the treatment strategy of bladder cancer.

Anti-PR1/HLA-A2 Antibody (8F4, A TCR-Mimic) Mediates Specific Lysis of AML Stem Cells but Not Normal Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 15-15
Author(s):  
Anna Sergeeva ◽  
Gheath Alatrash ◽  
Karen Clise-Dwyer ◽  
Kathryn E Quintanilla ◽  
Sijie Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Healthy Donor ◽  
Mononuclear Cells ◽  
Scid Mice ◽  
Specific Lysis ◽  
Isotype Control

Abstract Abstract 15 PR1 (VLQELNVTV) is an HLA-A2-restricted peptide derived from endogenous myeloid leukemia-associated antigens (LAA) proteinase 3 and neutrophil elastase. PR1/HLA-A2 is targeted by PR1-specific cytotoxic T lymphocytes (PR1-CTL) that contribute to cytogenetic remission in patients with CML after allogeneic stem cell transplantation or interferon therapy, and PR1 peptide vaccination induces specific CD8 immunity in AML, MDS, and CML patients. Because clinical effects after PR1 vaccination are associated with a low amount of leukemia and because the effects on leukemia stem cells (LSCs) are unknown, we sought to develop a PR1/HLA-A2-specific MAB that could be more effective in high disease burden cases and that might also target LSC. We previously reported a mouse IgG2A antibody (8F4) with PR1/HLA-A2 specificity. We now used 8F4 to study PR1 expression by FACS and confocal mircroscopy on leukemia subsets and normal peripheral blood mononuclear cells (PBMC). Circulating blasts from peripheral blood (PB) or leukapheresis products (LP) from AML patients (5 HLA-A2+, 2 control HLA-A2-,) were studied (see Table 1). PR1 expression (MFI±SEM) was higher in AML (23.7±5.18) compared to control (4.0±2.42) and normal PBMC (13.6±0.23; p = 0.046). Confocal microscopy confirmed bright heterogeneous surface expression of PR1 on AML, but was nearly absent on healthy donor PB. Interestingly, PR1 expression was higher on LSC (CD34+CD38-lin-) (76±44) compared to healthy donor HSC (CD34+CD38-lin-) (13.4), which suggests that only differentiated myeloid cells might be more susceptible to 8F4 toxicity. To study whether 8F4 mediated specific lysis of PR1+ cells, we determined cell-mediated cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of 8F4 compared to isotype control. 8F4 induced 51% specific lysis by CDC and 15% specific lysis by ADCC of PR1-pulsed T2 cells, which was dose-dependent on both 8F4 and PR1, with no lysis of target cells by isotype control. Moreover, 8F4 induced CDC-mediated lysis of blasts from 5/5 HLA-A2+ AML patients, but not blasts from an HLA-A2- AML patient (see Table 1), or mononuclear cells from HLA-A2+ healthy donors. To study the in vivo effects of 8F4 on AML, we used an established xenogeneic model of human AML in NOD/SCID mice. 1 × 106 blasts from 4 HLA-A2+ AML patients (including the HLA-A2+ patients susceptible to 8F4-mediated lysis in vitro) and 1 HLA-A2- patient were mixed for one hour under serum-free conditions with 8F4 or isotype control antibody and adoptively transferred to NOD/SCID mice. Engraftment of AML was compared on day 14 and AML growth was confirmed on day 28 by comparing FACS analysis and histopathology of BM from sacrificed animals. 8F4 treatment of AML cells, but not isotype treatment, abrogated engraftment of 4/4 HLA-A2+ AML patients, but not of 1 HLA-A2- AML patient on d14. By day 28, AML expanded by 150% in one surviving isotype-treated animal. Thus, 8F4-mediated specific lysis of AML and LSC by CDC and ADCC mechanisms in vitro were confirmed by in vivo results of AML engraftment. Taken together, these results show that 8F4, a novel TCR-mimic antibody that targets only PR1/HLA-A2+ cells, mediates lysis of AML blasts and LSC, but not total normal BM cells or HSC. Therefore, 8F4 may be useful as an immunotherapeutic agent in the treatment of HLA-A2+ myeloid leukemia, and as the first antibody therapy that may target and eliminate LSC. Disclosures: No relevant conflicts of interest to declare.

Immunoglobulin-Like Transcript 5 Inhibits Macrophage-Mediated Bacterial Killing and Antigen Presentation During Sepsis

2019 ◽  
Vol 220 (10) ◽  
pp. 1688-1699
Author(s):  
Siqi Ming ◽  
Musheng Li ◽  
Minhao Wu ◽  
Jianhui Zhang ◽  
Haibo Zhong ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Underlying Mechanism ◽  
Peripheral Blood Mononuclear ◽  
Bacterial Killing ◽  
In Vivo Experiments ◽  
Healthy Donors ◽  
Antigen Presenting ◽  
Species Production

Abstract Background Immunosuppression contributes to the mortality of sepsis. However, the underlying mechanism remains unclear. Methods In the present study, we investigated the role of inhibitory receptor immunoglobulin-like transcript 5 (ILT5) in sepsis. We first screened the expression of ILT family members, and we found that ILT5 was dramatically up-regulated in the peripheral blood mononuclear cells from sepsis patients versus healthy donors. Results Knockdown of ILT5 by small interfering ribonucleic acid increased bacterial killing and reactive oxygen species production in THP-1 and RAW264.7 cells. Moreover, ILT5-expressing monocytes/macrophages exhibited lower expression of antigen-presenting molecules including major histocompatibility complex-II and CD80. In the in vitro coculture system with monocytes/macrophages, blockage of ILT5 facilitated Th1 proliferation and differentiation of CD4+ T cells. Furthermore, in vivo experiments demonstrated that pretreatment with ILT5 blocking peptide improved the survival and pulmonary pathology of septic mice. Conclusions Together, our study identified ILT5 as an immunosuppressive regulator during sepsis, which may provide potential therapeutic strategy for sepsis.

scholarly journals miR-181a Ameliorates the Progression of Myasthenia Gravis by Regulating TRIM9

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Qiang Wang ◽  
Yunquan Liu ◽  
Shixiang Kuang ◽  
Ruozhao Li ◽  
Ning Weng ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Target Genes ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Inflammatory Factors ◽  
Peripheral Blood Mononuclear ◽  
Inflammatory Microenvironment ◽  
In Vivo Experiments

Abnormally activated CD4+ T cells are considered to be an important factor in the pathogenesis of myasthenia gravis (MG). In the pathogenesis of MG, the imbalance of proinflammatory cytokines and immune cells maintains the imbalance of immune response and inflammatory microenvironment. Studies have shown that miRNA is involved in the pathogenesis of MG. In our experiment, we extracted peripheral blood mononuclear cells (PBMCs) from MG patients and detected the expression of miR-181a and TRIM9 in PBMCs by qRT-PCR. In vitro experiments were conducted to explore the regulatory mechanism of miR-181a on target genes and its influence on inflammatory factors related to MG disease. Experimental autoimmune myasthenia gravis (EAMG) model mice are established, and the effects of miR-181a on EAMG symptoms and inflammatory factors are explored through in vivo experiments. According to a total of 40 EAMG mice that were successfully modeled, all EAMG mice showed symptoms of muscle weakness; their diet was reduced; their weight gain was slow; and even weight loss occurred. In MG patients and EAMG mice, the expression of miR-181a was low and TRIM9 was highly expressed. Bioinformatics website and dual-luciferase report analysis of miR-181a had a targeting relationship with TRIM9, and miR-181a could target the expression of TRIM9. After upregulating miR-181a or interfering with TRIM9, serum miR-181a in EAMG mice was significantly upregulated; TRIM9 was significantly downregulated; its clinical symptoms were reduced; and the expression of inflammatory factors was reduced. The study finally learned that miR-181a can reduce the level of MG inflammatory factors by targeting the expression of TRIM9 and has the effect of improving the symptoms of MG.

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