Biology subjects

Zoete, V.

Publications and source records attributed to Zoete, V..

4 recordsLinked to original sources

bioRxiv2022

Rapid BCMA downmodulation on myeloma cells upon CAR T cell contact is mediated by trogocytosis and BCMA internalization

BackgroundChimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here, we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs, and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL, single-chain-variable-fragment, heavy-chain-only). MethodsThree new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo, for their polyfunctionality, immune synapse formation, memory, exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms, we analyzed BCMA levels and cellular localization and quantified CAR T cell - target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. ResultsAPRIL-BB{zeta} CAR T cells in a trimeric ligand binding conformation conferred the best polyfunctionality, immune synapse formation and fast anti-tumor function in vivo in two different mouse xenograft models. Upon CAR T cell - myeloma cell contact, we found rapid BCMA downmodulation on target cells with all three evaluated binding moieties. CAR T cells acquired BCMA on their cell surface by trogocytosis, and BCMA on MM cells was rapidly internalized. Since trogocytosis can lead to CAR T cell exhaustion and presence of CAR T cells does not protect patients from relapse, we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell - MM cell interactions activated BCMA pathway, we did not find enhanced tumor growth in vitro or in vivo. ConclusionWe designed and characterized distinct APRIL-CAR T cells for dual-antigen targeting of MM. Rapid BCMA downmodulation occurred upon CAR T cell - tumor cell encounter independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect, but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM and can inform the development of improved treatment strategies.

immunology↗

bioRxiv2022

Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes

CD4+ T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on MHC-II molecules. The high polymorphism of MHC-II genes represents an important hurdle towards accurate prediction and identification of CD4+ T-cell epitopes in different individuals and different species. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across human, mouse, cattle and chicken. Analysis of these binding specificities combined with X-ray crystallography refined our understanding of the molecular determinants of MHC-II motifs and revealed a widespread reverse binding mode in MHC-II ligands. We then developed a machine learning framework to accurately predict binding specificities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T-cell epitopes, and enabled us to discover and characterize several viral and bacterial epitopes following the aforementioned reverse binding mode.

immunology↗

bioRxiv2022

The actin assembly requirements of the formin Fus1 to build the fusion focus

Formins form the largest family of actin filament nucleators and elongators, involved in the assembly of diverse actin structures. Actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, common to all formins. However, the rate of these reactions varies between formins by at least 20-fold. Typically, each cell expresses several distinct formins, each contributing to the assembly of one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in the fission yeast Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates for function in vivo. Fus1 assembles the actin fusion focus, an aster-like structure of actin filaments at the contact site between gametes, necessary for the process of cell fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on the architecture of the fusion focus, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in the Fus1 FH2 domain that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional property of the FH2 domain. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

cell biology↗

bioRxiv2020

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germline mosaicism in one of the parents; the mosaicism however could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling, and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant which is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism related gene. Our results show that PAK2 induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

genetics↗