Victor Tybulewicz: Projects

B and T cell migration and adhesion

Migration and adhesion are critical for the physiology of B and T lymphocytes. Using an RNAi screen, we identified a novel pathway involving the WNK1 kinase that regulates both adhesion and migration. We showed that WNK1 is a negative regulator of TCR- and chemokine-induced adhesion through the LFA1 integrin in T cells. Our work showed that WNK1 negatively regulates LFA1 via the RAP1 GTPase. In addition, WNK1 is a positive regulator of chemokine-induced T cell migration. In this process it acts via the OXSR1 (OSR1) and STK39 (SPAK) kinases to phosphorylate SLC12A2 (NKCC1) thereby allowing Na+, K+ and Cl- ions to enter the cell.

More recently we have shown the WNK1 is also a negative regulator of adhesion and a positive regulator of migration in B cells. Current work is aimed at understanding how WNK1 regulates adhesion and migration at the molecular level and at identifying further novel pathways using CRISPR/Cas9 based screens. Furthermore, we are also investigating how the WNK1 pathway acting in B or in T cells impacts on immune responses.

The WNK1 pathway

The WNK1 pathway. WNK1 transduces TCR and CCR7 signals leading to negative regulation of the RAP1 GTPase and LFA1-mediated adhesion. WNK1 also transduces CCR7 signals leading to migration via OSR1, SPAK and NKCC1. (Click to view larger image)

Selected Publication

Köchl, R, Thelen, F, Vanes, L, Brazão, TF, Fountain, K, Xie, J, Huang, C-L, Lyck, R, Stein, JV, Tybulewicz, VLJ (2016). WNK1 kinase balances T cell adhesion and migration in vivo. Nat Immunol, 17, 1075-1083

 

B cell survival

The number of B cells is strictly controlled. This homeostasis is achieved by signalling through at least two receptors: the B cell antigen receptor (BCR) and the BAFF receptor (BAFFR). We have shown that these two receptors act co-operatively, with BAFFR transducing signals via the BCR leading to the activation of the SYK tyrosine kinase, and subsequent activation of the ERK MAPkinase and PI3 kinase pathways, which contribute to B cell survival.

Current studies are aimed at understanding the molecular mechanisms by which the BAFFR transduces survival signals, in particular how BAFFR signals to the BCR. We are also investigating novel pathways that may contribute to B cell survival. We are addressing these research areas using a combination of proteomics, transcriptomics, metabolomics and CRISPR/Cas9-based screens.

BAFFR transduces survival signals via the BCR.

BAFFR transduces survival signals via the BCR. BAFFR transduces survival signals, via Src-family kinases (SFK) leading to the phosphorylation of the ITAM motif on BCR-associated CD79A and subsequent recruitment and activation of the SYK tyrosine kinase. SYK in turn transduces signals leading to activation of ERK MAPkinase and PI3 kinase pathways. BAFFR also transduces signals via NIK and IKK1 to the non-canonical NF-kB pathway. Other possible pathways supporting B cell survival are illustrated above. For more details see Schweighoffer et al Curr Opin Cell Biol (2018). (Click to view larger image)

Selected Publications

Schweighoffer, E, Tybulewicz, VL (2018). Signalling for B cell survivalCurr Opin Cell Biol, 51, 8-14

Schweighoffer, E, Nys, J, Vanes, L, Smithers, N, Tybulewicz, VL (2017). TLR4 signals in B lymphocytes are transduced via the B cell antigen receptor and SYKJ Exp Med, 214, 1269-1280

Schweighoffer, E, Vanes, L, Nys, J, Cantrell, D, McCleary, S, Smithers, N, and Tybulewicz, VLJ (2013). The BAFF receptor transduces survival signals by co-opting the B cell antigen receptor signaling pathway. Immunity, 38, 475-488

Mócsai, A, Ruland, J and Tybulewicz, VLJ (2010). The Syk tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol, 10, 387-402

Schweighoffer E, Vanes L, Mathiot A, Nakamura T, Tybulewicz VL (2003) Unexpected requirement for ZAP-70 in pre-B cell development and allelic exclusion. Immunity  18, 523-33

Turner, M, Gulbranson-Judge, A, Quinn, ME, Walters, AE, MacLennan, ICM, and Tybulewicz, VLJ (1997) Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. J Exp Med,186, 2013-2021

Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, Furlong MT, Geahlen RL, Tybulewicz VL. (1995) Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 378, 298-302

 

Memory B cells

Memory B cells (Bmem) form a crucial part of immunological memory. They are long-lived and are rapidly re-activated following re-challenge with previously encountered pathogens. We are interested in how Bmem survival is regulated. We have previously shown that the SYK tyrosine kinase is essential for the survival of Bmem. By analogy with our studies on the survival of naïve B cells, this suggests that the BCR may also be important in Bmem survival, possibly by transducing BAFFR signals (see above). Thus, we are investigating the roles of BCR and BAFFR in Bmem survival along with other potential signalling proteins.

More recently, using RNAseq we have identified a number of genes whose expression differs between Bmem and naïve B cells. We are using this to construct a novel reporter and deleter mouse strain that will permit genetic manipulations to be limited to Bmem cells. This strain will be used to investigate the roles of specific signalling molecules and transcription factors in the biology Bmem cells. Finally, we are interested in the differentiation pathways that are induced following B cell activation, leading to their differentiation into germinal centre B cells, memory B cells and plasma cells. 

 

Differentiation of B cells following activation.

Differentiation of B cells following activation. Activation of naïve B cells by binding of antigen to the BCR, in combination with T cell help, leads to differentiation of the B cells into germinal centre B cells, memory B cells and plasma cells. (Click to view larger image)

Selected Publication

Ackermann, JA, Nys, J, Schweighoffer, E, McCleary, S, Smithers, N, Tybulewicz, VLJ (2015). Syk tyrosine kinase is critical for B cell antibody responses and memory B cell survival. J Immunol, 194, 4650-4656

 

Genetics of Down syndrome

Trisomy of human chromosome 21 (Hsa21) occurs in ∼1 in 750 live births, and the resulting gene dosage imbalance gives rise to Down syndrome (DS), the most common known genetic form of mental retardation. DS is a constellation of different phenotypes: while all people with DS have hypotonia, cognitive impairment, neurodegeneration and craniofacial alterations, most also present with a spectrum of other disorders such as heart defects and leukaemia.

In collaboration with Professor Elizabeth Fisher (Institute of Neurology, UCL) we are interested in identifying dosage-sensitive genes on Hsa21 which, when present in three copies, cause the many different phenotypes seen in DS. We are addressing this using mouse models. We have created a novel mouse strain, termed Tc1, which carries a freely segregating copy of Hsa21. Tc1 mice show defects in memory, synaptic plasticity, locomotor function, heart development and in the craniofacial skeleton (O'Doherty et al, 2005; Morice et al, 2008; Galante et al 2009; Witton et al 2015).

People with DS have increased rates of mekaryoblastic leukaemia, but lower rates of solid tumours. We have shown that Tc1 mice have perturbed megakaryopoiesis, a pathology that may contribute to the increased rates of leukaemia in human DS (Alford et al, 2010). In contrast, studies of Tc1 mice have shown that the reduction in solid tumours may be due to defective angiogenesis (Reynolds et al, 2010).

Currently our main aim is to identify 'dosage-sensitive' genes, which, when present in three copies cause DS phenotypes. To do this we have constructed a series of novel mouse strains with duplications and deletions of regions mouse chromosomes orthologous to Hsa21 (Lana-Elola et al 2016). Analysis of these mouse strains has allowed us to narrow down the region containing the genes that cause congenital heart defects. We are currently working to identify the specific dosage-sensitive genes that cause congenital heart defects, and to understand how increased copy number of these genes leads to pathology.

More broadly, we are also using the same approaches to identify the dosage-sensitive genes that cause cognitive impairment, neurodegeneration and craniofacial abnormalities.

Generation of the transchromosomic Tc1 mice

Generation of the transchromosomic Tc1 mice. Using gene targeting we inserted a neomycin resistance gene into human chromosome 21 (Hsa21) in a human cell line. These cells carrying a tagged Hsa21 were arrested in metaphase and then centrifuged to isolate microcells carrying one or just a few chromosomes. The microcells were fused to murine embryonic stem (ES) cells, which were then selected with G418 for uptake of the neomycin resistance gene, and screened for lines carrying Hsa21. These ES lines were injected into mouse blastocysts to generate chimeric mice, which were bred to establish the Tc1 mouse strain carrying a freely segregating Hsa21. (Click to view larger image)

 

A genetic mapping panel of 9 mouse strains to find dosage-sensitive genes contributing to DS phenotypes.

Mouse mapping panel for Down Syndrome. Human chromosome 21 (Hsa21) is shown on the left in a diagram illustrating the centromere (oval) and the long and short arms of the chromosome. The long arm of Hsa21 is orthologous to regions of 3 different mouse chromosomes, Mmu16, Mmu17 and Mmu10 (grey bars). We have constructed a series of mouse strains (Dp1Tyb – Dp9Tyb) with duplications of different sections of the Hsa21-orthologous region on Mmu16 (black bars). Dp1Tyb is duplicated for a 23 Mb region from Lipi to Zbtb21, containing 148 coding genes and covering the entire orthologous region on Mmu16. This panel of mouse strains can be used to map the location of dosage-sensitive genes that are required in three copies to cause DS phenotypes (Lana-Elola et al 2016). (Click to view larger image)

Heart defects in Dp1Tyb mice.

Heart defects in Dp1Tyb mice. Using high resolution episcopic microscopy (HREM) to generate 3D images of Dp1Tyb hearts at embryonic day 14.5, we were able to identify different types of congenital heart defects (CHD) (red arrowheads): perimembranous and muscular ventricular septal defects (pVSD and mVSD) and atrio-ventricular septal defects (AVSD). Graph shows the percentage of CHD in Dp1Tyb embryonic hearts at E14.5 compared to wild-type (Wt) littermates. iAVC, inferior atrio-ventricular cushion; LV, left ventricle; MV, mitral valve; RV, right ventricle; sAVC, superior atrio-ventricular cushion; TV, tricuspid valve; VS, ventricular septum. (Click to view larger image)

Selected Publications

Lana-Elola, E, Watson-Scales, S, Slender, A, Gibbins, D, Martineau, A, Douglas, C, Mohun, T, Fisher, EMC, Tybulewicz, VLJ (2016). Genetic dissection of Down syndrome-associated congenital heart defects using a new mouse mapping panel. eLife, pii:e11614. doi:10.7554/eLife.11614.001

Witton, J., Padmashri, R., Zinyuk L.E., Popov V.I., Kraev, I., Line S. J., Jensen, T. P., Tedoldi, A., Cummings, D. M., Tybulewicz V.L.J., Fisher E.M.C., Bannerman, D. M., Randall A. D., Brown J. T., Edwards, F. A., Rusakov D., Stewart M. G., Jones M. W. (2015). Hippocampal circuit dysfunction in the Tc1 mouse model of Down syndrome. Nat Neurosci, 18, 1291-1298

Reynolds, LE, Watson, AR, Baker, M, Jones, TA, D'Amico, G, Robinson, SD, Joffre, C, Garrido-Urbani, S, Rodriguez-Manzaneque, JC, Martino-Echarri, E, Aurrand-Lions, M, Sheer, D, Dagna-Bricarelli, F, Nizetic, D, McCabe, CJ, Turnell, AS, Kermorgant, S, Imhof, BA, Adams, R, Fisher, EMC, Tybulewicz, VLJ, Hart, IR and Hodivala-Dilke, KM (2010). Tumour angiogenesis is reduced in the Tc1 mouse model of Down's Syndrome. Nature, 465, 813-817

Alford, K, Slender, A, Vanes, L, Li, Z, Fisher, EMC, Nizetic, D, Orkin, SH, Roberts, I and Tybulewicz, VLJ (2010) Perturbed hematopoiesis in the Tc1 mouse model of Down syndrome. Blood,  115, 2928-2937

Galante, M, Jani, H, Vanes, L, Daniel, H, Fisher, EMC, Tybulewicz, VLJ, Bliss, TVP and Morice, E (2009) Impairments in motor coordination without major changes in cerebellar plasticity in the Tc1 mouse model of Down syndrome. Human Molecular Genetics 18, 1449-1463

Morice, E, Andreae, LC, Cooke, SF, Vanes, L, Fisher, EMC, Tybulewicz, VLJ and Bliss, TVP (2008) Preservation of long-term memory and synaptic plasticity despite short-term impairments in the Tc1 mouse model of Down syndrome. Learning & Memory, 15, 492-500

O'Doherty, A, Ruf, S, Mulligan, C, Hildreth, V, Errington, ML, Cooke, S, Sesay, A, Modino, S, Vanes, L, Hernandez, D, Linehan, JM, Sharpe, PT, Brandner, S, Bliss, TVP, Henderson, DJ, Nizetic, D, Tybulewicz, VLJ and Fisher, EMC (2005) An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science 309, 2033-2037

Victor Tybulewicz portrait

Victor Tybulewicz

victor.t@crick.ac.uk
+44 (0)20 379 61612

  • Qualifications and history
  • 1984 PhD MRC Laboratory of Molecular Biology, Cambridge, UK
  • 1986-1991 Postdoctoral fellow, Whitehead Institute, MIT, Cambridge, MA, USA
  • 1991-2015 Group Leader, Medical Research Council National Institute for Medical Research, London, UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK