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Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo.

ABSTRACT: Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene (F8). Treatment with recombinant or plasma-derived FVIII replacement therapy is standard therapy. A major problem in treating hemophilia A patients with therapeutic FVIII is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies (inhibitors) because they are not immunologically tolerant to this human protein. Hence, there is a need to establish tolerogenic protocols to FVIII epitopes. To specifically target FVIII-specific B cells, we engineered immunodominant FVIII domains (A2 and C2) as a chimeric antigen receptor expressed by both human and murine cytotoxic T cells. This FVIII domain engineered B-cell antibody receptor (BAR) that expresses T cells was capable of killing FVIII-reactive B-cell hybridomas in vitro and in vivo. Moreover, FVIII BAR CD8 T cells blocked the development of specific antibody from unimmunized spleen cells stimulated polyclonally with lipopolysaccharide in vitro. In addition, adoptive transfer of FVIII A2- and C2-BAR CD8 T cells significantly reduced the anti-FVIII antibody formation in hemophilic mice. These data suggest that BAR-engineered T cells are a promising approach for future prophylactic treatment for patients with severe hemophilia A who are at high risk of developing inhibitors.

SUBMITTER: Parvathaneni K

PROVIDER: S-EPMC6156881 | biostudies-other | 2018 Sep

REPOSITORIES: biostudies-other

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Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo.

Parvathaneni Kalpana K Scott David W DW

Blood advances 20180901 18

Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene (<i>F8</i>). Treatment with recombinant or plasma-derived FVIII replacement therapy is standard therapy. A major problem in treating hemophilia A patients with therapeutic FVIII is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies (inhibitors) because they are not immunologically tolerant to this human protein. Hence, there is a need to establish tolerogenic protocols to F ...[more]

PMID: 30232086

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Project description:BACKGROUND:Mesenchymal/stromal stem cells (MSCs) are favorably regarded in anti-cancer cytotherapies for their spontaneous chemotaxis toward inflammatory and tumor environments associated with an intrinsic cytotoxicity against tumor cells. Placenta-derived or TRAIL-engineered adipose MSCs have been shown to exert anti-tumor activity in both in-vitro and in-vivo models of multiple myeloma (MM) while TRAIL-transduced umbilical cord (UC)-MSCs appear efficient inducers of apoptosis in a few solid tumors. However, apoptosis is not selective for cancer cells since specific TRAIL receptors are also expressed by a number of normal cells. To overcome this drawback, we propose to transduce UC-MSCs with a bicistronic vector including the TRAIL sequence under the control of IL-6 promoter (pIL6) whose transcriptional activation is promoted by the MM milieu. METHODS:UC-MSCs were transduced with a bicistronic retroviral vector (pMIGR1) encoding for green fluorescent protein (GFP) and modified to include the pIL6 sequence upstream of the full-length human TRAIL cDNA. TRAIL expression after stimulation with U-266 cell conditioned medium, or IL-1?/IL-1?, was evaluated by flow cytometry, confocal microscopy, real-time PCR, western blot analysis, and ELISA. Apoptosis in MM cells was assayed by Annexin V staining and by caspase-8 activation. The cytotoxic effect of pIL6-TRAIL + -GFP + -UC-MSCs on MM growth was evaluated in SCID mice by bioluminescence and ex vivo by caspase-3 activation and X-ray imaging. Statistical analyses were performed by Student's t test, ANOVA, and logrank test for survival curves. RESULTS:pIL6-TRAIL + -GFP + -UC-MSCs significantly expressed TRAIL after stimulation by either conditioned medium or by IL-1?/IL-1?, and induced apoptosis in U-266 cells. Moreover, when systemically injected in SCID mice intratibially xenografted with U-266, those cells underwent within MM tibia lesions and significantly reduced the tumor burden by specific induction of apoptosis in MM cells as revealed by caspase-3 activation. CONCLUSIONS:Our tumor microenvironment-sensitive model of anti-MM cytotherapy is regulated by the axis pIL6/IL-1?/IL-1? and appears suitable for further preclinical investigation not only in myeloma bone disease in which UC-MSCs would even participate to bone healing as described, but also in other osteotropic tumors whose milieu is enriched of cytokines triggering the pIL6.

Project description:BackgroundSmall metal nanoparticles obtained from animal blood were observed to be toxic to cultured cancer cells, whereas noncancerous cells were much less affected. In this work, engineered zinc and copper metal nanoparticles were produced from bulk metal rods by an underwater high-voltage discharge method. The metal nanoparticles were characterized by atomic force microscopy and X-ray photoelectron spectroscopy. The metal nanoparticles, with estimated diameters of 1 nm-2 nm, were determined to be more than 85% nonoxidized. A cell viability assay and high-resolution light microscopy showed that exposure of RG2, cultured rat brain glioma cancer cells, to the zinc and copper nanoparticles resulted in cell morphological changes, including decreased cell adherence, shrinking/rounding, nuclear condensation, and budding from cell bodies. The metal-induced cell injuries were similar to the effects of staurosporine, an active apoptotic reagent. The viability experiments conducted for zinc and copper yielded values of dissociation constants of 0.22 ± 0.08 nmol/L (standard error [SE]) and 0.12 ± 0.02 nmol/L (SE), respectively. The noncancerous astrocytes were not affected at the same conditions. Because metal nanoparticles were lethal to the cancer cells at sub-nanomolar concentrations, they are potentially important as nanomedicine.PurposeLethal concentrations of synthetic metal nanoparticles reported in the literature are a few orders of magnitude higher than the natural, blood-isolated metal nanoparticles; therefore, in this work, engineered metal nanoparticles were examined to mimic the properties of endogenous metal nanoparticles.Materials and methodsRG2, rat brain glioma cells CTX TNA2 brain rat astrocytes, obtained from the American Type Culture Collection, high-voltage discharge, atomic force microscope, X-ray photoelectron spectroscopy, high-resolution light microscopy, zeta potential measurements, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay were used in this work.ResultsEngineered zinc and copper metal nanoparticles of size 1 nm-2 nm were lethal to cultured RG2 glioma cancer cells. Cell death was confirmed by MTT assay, showing that the relative viability of RG2 glioma cells is reduced in a dose-dependent manner at sub-nanomolar concentrations of the nanoparticles. The noncancerous astrocytes were not affected at the same conditions.ConclusionThe engineered and characterized zinc and copper nanoparticles are potentially significant as biomedicine.

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Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo.

Publications

Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo.

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