A non-conserved amino acid variant regulates differential signalling between human and mouse CD28

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside

Aurora A drives early signalling and vesicle dynamics during T-cell activation

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3z-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.We thank S. Bartlett for English editing and critical reading of the manuscript, Dr A. Akhmanova for providing reagents, Maria Navarro for the her critical reading of the manuscript and scientific recommendations, Miguel Vicente-Manzanares for his critical reading of the manuscript, and Aitana Sanguino and Maria Jose Lopez for the technical support. We also thank the Confocal Microscopy \& Dynamic Imaging Unit (CNIC), Madrid, Spain. This study was supported by grants SAF2011-25834, SAF2014-55579-R and BIO2012-37926 from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid ERC-2011-AdG 294340-GENTRIS and ERC-2013-AdG 334763-NOVARIPP. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation, and the Pro-CNIC Foundation.S

SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron–microglia–astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.This work was supported by grants from the Spanish AEI (BFU2013-43213-P; BFU2016-75412-R with FEDER support and PID2019-104186RB-I00), Fundacion Tatiana Perez de Guzman el Bueno and CIBERER to PB. JRC (BES-2011-047189), GP (BES-2017- 080318) and MIM (BES-2014-068797) were supported by FPI fellowships from the AEI. We also acknowledge a CBM Institutional Grant from the Fundacion Ramon Areces.Peer reviewe

Identificacion de los dominios de CD3-'eta' implicados en endocitosis y ensamblaje

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

A non-conserved amino acid variant regulates differential signalling between human and mouse CD28

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P in human vs. A in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this YAPP sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside.Progetto Ateneo” (Sapienza University of Rome, Italy) and Multiple Sclerosis Italian Foundation (FISM 2016/R/29)Peer Reviewe

Conformational changes in the T cell receptor differentially determine T cell subset development in mice.

International audienceIn the thymus, immature T cells differentiate from common precursors to become T cells expressing either the αβ or γδ T cell receptor (TCR) complex. The CD3ε subunit of the TCR complex is thought to transduce ligand-induced conformational changes in the TCR by recruiting the cytosolic adaptor protein Nck. To investigate the role of conformational changes in the TCR in T cell development, we generated mice with a germline mutation (C80G) in the extracellular domain of CD3ε, which prevents the outside-in transmission of conformational changes in the TCR. The development of αβ T cells in the C80G mice was blocked at an early stage that depends on signaling by a precursor form of the TCR. In contrast, the C80G mutation did not impair the development of some subsets of γδ T cells, including Vγ1.1(+) cells; however, development of other γδ T cell subsets was blocked. A similar phenotype was observed in mice with a mutation in the cytoplasmic proline-rich sequence (PRS) of CD3ε, the binding site for Nck. In a genetic complementation test, the PRS CD3ε mutant failed to rescue the wild-type phenotype when expressed in heterozygosity with the C80G mutant. These data suggest that Nck may function as an effector of TCR conformational changes during T cell development. Additional experiments showed differential effects of the C80G mutation on the activation of TCR-dependent signaling pathways, which suggests that there are pathways that are either dependent on or independent of the transmission of conformational change in the receptor

Conformational changes in the T cell receptor differentially determine T cell subset development in mice.

International audienceIn the thymus, immature T cells differentiate from common precursors to become T cells expressing either the αβ or γδ T cell receptor (TCR) complex. The CD3ε subunit of the TCR complex is thought to transduce ligand-induced conformational changes in the TCR by recruiting the cytosolic adaptor protein Nck. To investigate the role of conformational changes in the TCR in T cell development, we generated mice with a germline mutation (C80G) in the extracellular domain of CD3ε, which prevents the outside-in transmission of conformational changes in the TCR. The development of αβ T cells in the C80G mice was blocked at an early stage that depends on signaling by a precursor form of the TCR. In contrast, the C80G mutation did not impair the development of some subsets of γδ T cells, including Vγ1.1(+) cells; however, development of other γδ T cell subsets was blocked. A similar phenotype was observed in mice with a mutation in the cytoplasmic proline-rich sequence (PRS) of CD3ε, the binding site for Nck. In a genetic complementation test, the PRS CD3ε mutant failed to rescue the wild-type phenotype when expressed in heterozygosity with the C80G mutant. These data suggest that Nck may function as an effector of TCR conformational changes during T cell development. Additional experiments showed differential effects of the C80G mutation on the activation of TCR-dependent signaling pathways, which suggests that there are pathways that are either dependent on or independent of the transmission of conformational change in the receptor

1H-NMR analysis of CD3-ε reveals the presence of turnhelix structures around the itam motif in an otherwise random coil cytoplasmic tail

The conformation adopted in solution by the cytoplasmic tail of CD3-B has been analyzed by 'H-nmr. Tlie cytoplasmic tail is mostly random coil except for the amino acids conforming the imimmoreceptor tyrosine-based activation motif (ITAM), YxxL/IxxxxxxxYxxL. Although the N-terminal YxxL sequence of the motif is poorly folded, adopting 6-residue turn-like conformations with the Tyr side chain in t\vo different orientations, the C-tenninal YxxL sequence is placed in a more complex structure involving a set of nonclassical a-helix turns and -tums that comprises 11 amino acids. Tills st met lire is not modified by phosphorylation of the tyrosine residue. Tlie differences in the conformation adopted around the t\vo tyrosines of the ITAM motif suggest that they may play different roles pertaining to either binding signal transducing proteins or, alternatively, proteins involved in other processes such as endoplasmic reticulum location. ©1997 John Wiley & Sons, Inc.This work was supported by grants from CICYT PM95-0005, Comunidad de Madrid (AE13/95) , the European Union Biotech Program (BIOCT920164) , the Fundación Rodríguez Pascual, and Fundación Ramón Areces.Peer Reviewe