Characteristics of TCR/CD3 complex CD3{varepsilon} chains of regulatory CD4+ T (Treg) lymphocytes: role in Treg differentiation in vitro and impact on Treg in vivo.

Tregs are anergic CD4(+)CD25(+)Foxp3(+) T lymphocytes exerting active suppression to control immune and autoimmune responses. However, the factors in TCR recognition underlying Treg differentiation are unclear. Based on our previous data, we hypothesized that Treg TCR/CD3 antigen receptor complexes might differ from those of CD4(+)CD25(-) Tconv. Expression levels of TCR/CD3, CD3ε,ζ chains, or other molecules involved in antigen signaling and the characteristics of CD3ε chains were analyzed in thymus or spleen Treg cells from normal mice. Tregs had quantitative and qualitatively distinct TCR/CD3 complexes and CD3ε chains. They expressed significantly lower levels of the TCR/CD3 antigen receptor, CD3ε chains, TCR-ζ chain, or the CD4 coreceptor than Tconv. Levels of kinases, adaptor molecules involved in TCR signaling, and early downstream activation pathways were also lower in Tregs than in Tconv. Furthermore, TCR/CD3 complexes in Tregs were enriched in CD3ε chains conserving their N-terminal, negatively charged amino acid residues; this trait is linked to a higher activation threshold. Transfection of mutant CD3ε chains lacking these residues inhibited the differentiation of mature CD4(+)Foxp3(-) T lymphocytes into CD4(+)Foxp3(+) Tregs, and differences in CD3ε chain recognition by antibodies could be used to enrich for Tregs in vivo. Our results show quantitative and qualitative differences in the TCR/CD3 complex, supporting the hyporesponsive phenotype of Tregs concerning TCR/CD3 signals. These differences might reconcile avidity and flexible threshold models of Treg differentiation and be used to implement therapeutic approaches involving Treg manipulation.

Dissociation of actin polymerization and lipid raftaccumulation by ligation of the Inducible Costimulator(ICOS, CD278

T lymphocyte antigen activation is facilitated by clustering of membrane glycosphingolipidenriched microdomains (GEMs, lipid "rafts") at the T cell/APC contact that is linked to changes in actin cytoskeleton and is one major mechanism of CD28 costimulation. Ligation of CD28 alone, or ligation of the CD28-like molecules CTLA-4 (CD152) and ICOS (CD278)induces act in polymerization with cell elongation and generation of lamellipodia and filopodia in T cells. These changes are dependent on Src, PI3-kinase, Vav, and Rho family GTPases. Whereas CD28 and CTLA-4 have been shown to be functional and physically associated with lipid rafts, the presence of ICOS in lipid rafts or its effect in raft clustering is not known. In this work, we have activated the T cell line D10 with anti-ICOS antibodies, alone or combined with anti-CD3 antibodies, bound or unbound to polystyrene microbeads or glass coverslips. The possible relationship of ICOS-induced changes in actin cytoskeleton to the ICOS localization in membrane rafts was then analyzed by fluorescence microscopy, or by immunoblot of detergent insoluble ("raft") or soluble ("non-raft") fractions of cell lysates. Our data show that ICOS promotes TCR/CD3 induction of raft clustering at the site of activation. However, ICOS, which, on its own, can induce accumulations of polymerized actin, is undetectable in membrane rafts, even when using CD3 or ICOS, ligands capable of inducing clear changes in the actin cytoskeleton (AU)

La activación de linfocitos T se facilita por la concentración, en el sitio de interacción con elligando, de microdominios de membrana enriquecidos en glicoesfingolípidos (GEM, o "balsas" lipídicas). Este fenómeno está unido a, y es dependiente de cambios en el citoesqueleto de actina, siendo uno de los principales mecanismos implicados en la coestimulación porCD28. El entrecruzamiento de CD28 aisladamente, o de moléculas de su familia como CTLA-4 (CD152) e ICOS (CD278) inducen en linfocitos T polimerización de actina acompañada de elongación celular y aparición de lamelipodia y filopodia. Estos cambios son dependientes de Src, PI3-cinasa, Vav, y GTPasas de la familia Rho. Se han descrito relaciones funcionales y físicas de CD28 y CTLA-4 con balsas lipídicas, pero se desconoce si ICOS se encuentra en estos dominios, o su efecto sobre la agrupación de balsas inducida porligandos. En este trabajo se han activado células T de la línea D10 con anticuerpos anti-ICOS, solos o combinados con anticuerpos anti-CD3, y unidos o no a microesferas de poliestireno oa cubreobjetos de vidrio. En estas células se ha determinado la posible relación entre los cambios en el citoesqueleto de actina y la localización de ICOS en las balsas lipídicasmediante microscopía de fluorescencia, o mediante "inmunoblot" de las fracciones de lisados insolubles ("balsas") o solubles ("no-balsas") en detergente. Nuestros datos muestran que ICOS incrementa el agrupamiento de balsas lipídicas inducida por anticuerpo antiCD3 en el sitio de contacto con el estímulo. Sin embargo, ICOS, que por sí solo induce acumulación d actina polimerizada, es indetectable en las balsas de membrana, incluso empleando ligandos (CD3 o ICOS) capaces de inducir cambios claros en el citoesqueleto deactina (AU)

Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278).

To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding to ICOS peptides phosphorylated at the Y(191)MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85α, p50-55α and p85ß regulatory subunits and p110α, p110δ and p110ß catalytic subunits. Intriguingly, T cells expressed high levels of both p110α or p110δ catalytic subunits, yet ICOS peptides, cell surface ICOS or PI3-kinase class IA regulatory subunits preferentially coprecipitated p110α catalytic subunits. Silencing p110α or p110δ partially inhibited Akt/PKB activation induced by anti-CD3 plus anti-ICOS antibodies. However, silencing p110α enhanced and silencing p110δ inhibited Erk activation. Both p110α- and p110δ-specific inhibitors blocked cytokine secretion induced by TCR/CD3 activation with or without ICOS costimulus, but only p110α inhibitors blocked ICOS-induced cell elongation. Thus, p110α and p110δ are essential to optimal T cell activation, but their abundance and activity differentially tune up distinct ICOS signaling pathways.