PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain.
Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.
Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.
Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein-Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.
Genetic alterations in tumor cells provide promising targets for antitumor therapy. Recently, loss of methylthioadenosine phosphorylase (MTAP), a deletion frequently occurring in cancer, has been shown to create vulnerability to the inhibition of the protein arginine methyltransferase 5 (PRMT5). MTAP deficiency leads to accumulation of methylthioadenosine (MTA), which reduces PRMT5 activity, and thus, sensitizes the tumor cells to selective PRMT5 inhibitors (PRMT5i). PRMT5i are investigated as a new strategy to selectively kill MTAP-deficient tumor cells by blocking residual PRMT5 activity, but also to treat PRMT5-overexpressing tumors. Although many studies investigated the role of PRMT5 in cancer, only little data exist about the effect of PRMT5 inhibition on immune cells. As we could show that the tumor metabolite MTA suppresses T cells, we asked whether selective PRMT5 inhibition is detrimental for T-cell immune responses. Therefore, we examined the effect of the synthetic PRMT5 inhibitor EPZ015666 on human CD8 þ T cells in direct comparison with the naturally occurring PRMT5-inhibiting molecule MTA. Both compounds reduced T-cell proliferation, viability, and functionality. In addition, T-cell metabolism was impaired upon PRMT5 inhibition. These effects coincided with the induction of p53 expression and reduced AKT/mTOR signaling. Our data clearly demonstrate that PRMT5 activity is involved in various cellular processes of human CD8 þ T cells associated with essential T-cell functions. Therefore, not only tumor cells, but also antitumor immune responses, are compromised by PRMT5 inhibitors. This emphasizes the importance of considering side effects on the immune system when developing new strategies to specifically target not only MTAP-deficient tumors.
BackgroundA major complication after allogeneic hematopoietic stem cell transplantation (aSCT) is the reactivation of herpesviruses such as cytomegalovirus (CMV) and Epstein–Barr virus (EBV). Both viruses cause significant mortality and compromise quality of life after aSCT. Preventive transfer of virus-specific T cells can suppress reactivation by re-establishing functional antiviral immune responses in immunocompromised hosts.MethodsWe have developed a good manufacturing practice protocol to generate CMV/EBV-peptide-stimulated T cells from leukapheresis products of G-CSF mobilized and non-mobilized donors. Our procedure selectively expands virus-specific CD8+ und CD4+ T cells over 9 days using a generic pool of 34 CMV and EBV peptides that represent well-defined dominant T-cell epitopes with various HLA restrictions. For HLA class I, this set of peptides covers at least 80% of the European population.ResultsCMV/EBV-specific T cells were successfully expanded from leukapheresis material of both G-CSF mobilized and non-mobilized donors. The protocol allows administration shortly after stem cell transplantation (d30+), storage over liquid nitrogen for iterated applications, and protection of the stem cell donor by avoiding a second leukapheresis.ConclusionOur protocol allows for rapid and cost-efficient production of T cells for early transfusion after aSCT as a preventive approach. It is currently evaluated in a phase I/IIa clinical trial.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1498-3) contains supplementary material, which is available to authorized users.
Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.
Reconstitution of T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by peptide stimulation and adoptively transferred them to a patient with angioimmunoblastic T-cell lymphoma (AITL), who had developed persisting high titers of EBV concomitant to relapse after transplantation. T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.Author summaryA characteristic feature of all herpesviruses is their persistence in the host’s body after primary infection. Hence, the host’s immune system is confronted with the problem to control these viruses life-long. Well-known representative of the herpesvirus group are the classic Herpes-Simplex Virus (HSV-1) and Varicella Zoster Virus (VZV, causing chicken pox); a less known representative is Epstein Barr Virus (EBV, causing mononucleosis). When the immune system is severely compromised, for example after stem cell transplantation from a foreign (allogeneic) donor, these viruses can reappear, as they are already in the host’s body. Especially EBV cause life-threatening complications after stem cell transplantation and only reinforcement of the host’s immune system can reestablish viral control. Here we show that ex vivo manufactured EBV-specific T cells can reestablish long-term control of EBV and that these cells persist in the host’s body over months. These results give us a better understanding of viral immune reconstitution post-transplant and of clinically-relevant T cell populations against EBV.
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