A Novel CAR Expressing NK Cell Targeting CD25 With the Prospect of Overcoming Immune Escape Mechanism in Cancers.

For many years, high-affinity subunit of IL-2 receptor (CD25) has been considered as a promising therapeutic target for different pathologic conditions like allograft rejection, autoimmunity, and cancers. Although CD25 is transiently expressed by newly-activated T cells, it is the hallmark of regulatory T (Treg) cells which are the most important immunosuppressive elements in tumor microenvironment. Thus, Tregs can be considered as a potential target for chimeric antigen receptor (CAR)-based therapeutic approaches. On the other hand, due to some profound adverse effects pertaining to the use of CAR T cells, CAR NK cells have caught researchers' attention as a safer choice. Based on these, the aim of this study was to design and develop a CAR NK cell against CD25 as the most prominent biomarker of Tregs with the prospect of overcoming immune escape mechanism in solid and liquid cancers. In the current study, an anti-CD25 CAR was designed and evaluated by comprehensive in silico analyses. Then, using lentiviral transduction system, NK-92 cell line was engineered to express this anti-CD25 CAR construct. In vitro functional analyses of anti-CD25 CAR for its reactivity against CD25 antigen as well as for cytotoxicity and cytokine production assays against CD25 bearing Jurkat cell line were done. In silico analyses demonstrated that the anti-CD25 CAR transcript and scFv protein structures were stable and had proper interaction with the target. Also, in vitro analyses showed that the anti-CD25 CAR-engineered NK-92 cells were able to specifically detect and lyse target cells with an appropriate cytokine production and cytotoxic activity. To conclude, the results showed that this novel CAR NK cell is functional and warrant further investigations.

Computational study for suppression of CD25/IL-2 interaction.

Cancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

Integral membrane protein expression of human CD25 on the cell surface of HEK293 cell line: the available cellular model of CD25 positive to facilitate in vitro developing assays.

Typically, CD25 is expressed on the cellular surface of regulatory T (Treg) cells. These cells are significant in regulating the self-tolerance and also preventing the immune system from attacking a person's own tissues and cells. They promote the cancer progression by playing an important role in evading the immune system. Thus, the experimental procedures was aimed to clone and express human CD25 in HEK293 cell line, as the available cellular model, for the purpose of developing assays to facilitate and enhance the studies on an available CD25 positive cell. The secondary RNA structure of CD25 was evaluated by in silico analysis. Then, cDNA of human CD25 were synthesized from isolated total mRNA of cultured and stimulated PBMCs from blood donors. After cloning the cDNA of CD25 into a pcDNA3.1(+) plasmid, using the effective transfection of the recombinant pcDNA3.1(+) in HEK293, qRT-PCR and flow cytometry methods were used to quantitatively evaluate CD25 transcripts and protein level. There was a 4.8 fold increase in transcripts and a 76.2% increase in protein levels of CD25 when comparing the transfected and control cell lines. The genetically engineered HEK293 cell line expressing Treg cell surface marker of CD25 was introduced in this study for the first time. This cell line can be used to overcome the problematic issues for studying Treg cells including low population of Tregs in peripheral blood, low recovery methods for Treg isolation, time-consuming and non-cost benefit methods in the conditions of in vitro cell culture experiments for the studies focused on the binding of IL-2 to CD25.

Expansion of alpha-galactosylceramide-stimulated Valpha24+ NKT cells cultured in the absence of animal materials.

Valpha24+ NKT is an innate lymphocyte with potential antitumor activity. Clinical applications of Valpha24+ natural killer (NK) T cells, which are innate lymphocytes with potential antitumor activity, require their in vitro expansion. To avoid the potential dangers posed to patients by fetal bovine serum (FBS), the authors evaluated non-FBS culture conditions for the selective and efficient expansion of human Valpha24+ NKT cells. Mononuclear cells (MNCs) and plasma from the peripheral blood of normal healthy donors were used before and after G-CSF mobilization. MNCs and plasma separated from apheresis products were also used. MNCs were cultured for 12 days in AIM-V medium containing alpha-galactosylceramide (alpha-GalCer) (100 ng/mL) and IL-2 (100 U/mL) supplemented with FBS, autologous plasma, or autologous serum. The cultured cells were collected and their surface markers, intracellular cytokines, and cytotoxicity were evaluated. The highest expansion ratio for Valpha24+ NKT cells was obtained from G-CSF-mobilized MNCs cultured in medium containing 5% autologous plasma. Cultures containing MNCs and autologous plasma obtained before and after G-CSF mobilization had approximately 350-fold and 2,000-fold expansion ratios, respectively. These results suggest that G-CSF mobilization conferred a proliferative advantage to Valpha24+ NKT cells by modifying the biology of cells and plasma factors. Expanded Valpha24+ NKT cells retained their surface antigen expression and production of IFN-gamma and exhibited CD1d-independent cytotoxicity against tumor cells. Valpha24+ NKT cells can be efficiently expanded from G-CSF-mobilized peripheral blood MNCs in non-FBS culture conditions with alpha-GalCer and IL-2.

Freeze-thawing procedures have no influence on the phenotypic and functional development of dendritic cells generated from peripheral blood CD14+ monocytes.

Little is known about the potential influence of cryopreservation on the biologic activities of dendritic cells (DCs). In this study, we examined the effects of freeze-thawing on the phenotypic and functional development of human DCs obtained from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood CD14+ cells. CD14+ cells were cultured, immediately or after freeze-thawing, with granulocyte-macrophage CSF and interleukin-4 for 9 days, and then with added tumor necrosis factor-alpha for another 3 days. For both fresh and freeze-thawed monocytes, immature DCs harvested on day 6 and mature DCs harvested on day 9 of culture were examined under the same conditions. Cells were compared with regard to their 1) capacities for antigen endocytosis and chemotactic migration (immature DCs), and 2) allogeneic mixed lymphocyte reaction and antigen-specific cytotoxic T lymphocyte responses (mature DCs). Freeze-thawing did not affect the viability or subsequent maturation of DCs at any stage of development. Furthermore, essentially no difference was observed in phenotype or function between cells generated from fresh or cryopreserved/thawed cells. Although this study design was limited with the use of fetal bovine serum, the observation still suggests that freeze-thawing does not affect viability, phenotype, subsequent maturation, or functions of DCs at any stage of maturation.

Role of NKT cells and alpha-galactosyl ceramide.

Alfa-Galactosyl Ceramide was isolated from Ocean sponge which has antitumor effect against several tumors in in vivo animal model with no cytotoxicity. KRN7000(KRN) is the most potent alpha-Galactosyl Ceramide modified from the one isolated from Ocean sponge. KRN is also active against metastatic tumors through the activation ofanimal immune system. Research efforts in learning the mechanism of action, we found the important role of dendritic cells(DC) and NKT cells. NKT cells was first characterized in 1988 which is overlap some part with NK cells and T-Cells and majority is different from NK and T. KRN is active through the activation of DC and NKT in giving antigen specific immune stimulation in animal. This antigen specific stimulation is memorized by immune system and can reject second tumor challenge. KRN is not active in nude mice and NKT deficient animal. NKT cells level in blood is lower in patients with autoimmune disease, cancer, HIV positive or aplastic anemia. NKT rapidly releases IL-4 and IFN-gamma at high level when activated. NKT is CD1d and TCR restricted. NKT plays important role in autoimmune disease such as Type 1 Diabetes, Scleroderma and Systemic Lupus Erythematosus, infections such as Mycobacteria, Listeria and Malaria, GVHD control and tumor rejection. NKT acts as double edge sword, aggressive and suppressive ways. KRN can prevent the onset of Type 1 Diabetes, inhibit replication of hepatitis virus B in liver and suppress malaria replication in activating NKT cells. KRN can activate NKT through DC and activated NKT activates NK, T and macrophage. KRN also expands NKT cells and expanded NKT has full function. Although the exact role of DC and NKT is not clear, KRN clinical study results in conjunction with DC and NKT cell activation are expected.

The role of PGE(2) in the differentiation of dendritic cells: how do dendritic cells influence T-cell polarization and chemokine receptor expression?

The role of prostaglandin E(2) (PGE(2)) in the function of dendritic cells (DCs), T-cell polarization, and expression of chemokine receptors was evaluated in human cells. Immature DCs were generated from peripheral blood CD14(+) cells using a combination of GM-CSF and interleukin-4 (IL-4) with or without PGE(2). On day 6, maturation of DCs was induced by the addition of tumor necrosis factor alpha with or without PGE(2). DCs harvested on day 6 (immature DCs) or day 9 (mature DCs) were examined using functional assays. In the presence of PGE(2), immature and mature DCs showed, phenotypically, a lower expression of CD1a and, functionally, a higher allostimulatory capacity at a high DC/T-cell ratio than control cells cultured in the absence of PGE(2). DCs cultured in the presence of PGE(2) induced the differentiation of naïve T cells toward a helper T-cell type 1 (Th1) response, which was independent of IL-12 secretion in the basal state despite a slightly lower interferon gamma secretion compared with control cells. However, the function of cytotoxicity-stimulating autologous T cells was not augmented by the addition of PGE(2). Immature DCs expressed the inflammatory chemokine receptors, CCR1 and CXCR4, but not CCR6, regardless of the presence or absence of PGE(2). Mature DCs expressed CCR7 equally, measured using a migration test and the measurement of calcium flux with macrophage inflammatory protein-3beta and reverse transcription-polymerase chain reaction assay in all of the groups. All of these findings suggest that PGE(2) affects the DC-promoted differentiation of naïve T cells to a Th1 response in the basal state, without affecting chemokine receptor expression on DCs.

The synergistic effect of thrombopoietin in erythropoiesis with erythropoietin and/or IL-3 and myelopoiesis with G-CSF or IL-3 from umbilical cord blood cells of premature neonates.

The authors sought to determine whether recombinant human thrombopoietin (TPO) acts synergistically with other cytokine(s) on burst-forming unit-erythroid (BFU-E)-derived and colony-forming unit-granulocyte/macrophage (CFU-GM)-derived colony formations from cord blood of premature neonates. Cord blood nonadherent mononuclear cells (MNC) from normal premature neonates were cultured in a methylcellulose system. When cultured with 1 x 10(4) MNC/mL, erythropoietin (EPO) 2 U/mL, interleukin-3 (IL-3) 50 ng/mL, and/or TPO 100 ng/mL, the addition of TPO to EPO gave rise to more BFU-E-derived colonies (p = .000). The addition of TPO to EPO + IL-3 gave rise to more BFU-E-derived colonies (p = .002) also. TPO synergizes erythropoiesis from cord blood of premature neonates. Likewise, the addition of TPO to G-CSF gave rise to more CFU-GM-derived colonies (p = .000) also. TPO synergizes myelopoiesis from cord blood of premature neonates. Thus, TPO has synergistic effects on both erythropoiesis and myelopoiesis from cord blood of premature neonates.

Human plasma thrombopoietin levels are regulated by binding to platelet thrombopoietin receptors in vivo.

BACKGROUND: Data from several studies support the hypothesis that thrombopoietin (TPO) plasma levels are regulated via circulating platelet (PLT) numbers by binding to PLT TPO receptors (TPO-Rs). In this study, PLT numbers and TPO plasma levels were measured following the transfusion of unmanipulated, sham-saturated, and TPO-R-saturated PLT preparations to provide additional in vivo evidence for this regulatory mechanism. STUDY DESIGN AND METHODS: Following in vitro experiments to characterize pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) binding characteristics, PLT numbers and TPO plasma levels were measured following the transfusion of unmanipulated, sham-saturated, and TPO-R-saturated PLT preparations in thrombocytopenic patients. Sham-saturated and TPO-R-saturated PLTs were prepared by a 1-hour incubation without and with 40 ng per mL of PEG-rHuMGDF, respectively, and subsequent washing and resuspension. RESULTS: In vitro, 2.72 +/- 0.8 ng of PEG-rHuMGDF per 1 x 10(8) PLTs was bound within 1 hour of incubation. No additional PEG-rHuMGDF was bound following a second incubation with PEG-rHuMGDF, and bound PEG-rHuMGDF was not released over time. In vivo, TPO plasma levels decreased significantly (p < 0.001), by 30.7 +/- 5.8 and 20.9 +/- 2.1 percent after transfusion of unmanipulated and sham-saturated PLT preparations, respectively. However, TPO plasma levels were unaffected after the transfusion of TPO-R-saturated PLTs despite comparable transfusion-induced PLT count increases. CONCLUSION: These data strongly support the concept that binding to PLT TPO-R is directly involved in human TPO plasma level regulation in vivo.