It is unclear if the interaction between CD8 and the TCR-CD3 complex is constitutive or antigeninduced. FRET microscopy between fluorescent chimeras of CD3ζ and CD8β showed that this interaction was induced by antigen recognition, within the immunological synapse. Non-stimulatory endogenous or exogenous peptides, presented simultaneously with antigenic peptides, increased the CD8-TCR interaction. This indicates that the interaction between the intracellular regions of a TCR-CD3 complex recognizing its cognate peptide-major histocompatibility complex (pMHC) antigen, and CD8 (plus Lck), is enhanced by a non-cognate CD8-MHC interaction. Thus the CD8 interaction with non-stimulatory pMHC helps mediate T cell recognition of antigen, improving the coreceptor function of CD8.The αβ T cell receptor (TCR) is responsible for the affinity and specificity of antigen recognition 1,2 , whereas the coreceptors, CD8 or CD4, enhance the sensitivity of TCR recognition 3 . Disruption of coreceptor-pMHC interactions inhibit or change the quality of the T cell response 3-5 . Coreceptors act in two main ways. Firstly, they bind to non-polymorphic regions of MHC 4 . This can aid in adhesion, but the main role is generally believed to be increasing the sensitivity of T cell activation via the entropic facilitation of TCR-pMHC binding, rather than through energetic stabilization of the tri-molecular complex 6-9 ). Secondly, CD4 and CD8 are associated with the kinase Lck. Coreceptor binding to pMHC recruits Lck close to the TCR, enabling it to phosphorylate components of the TCR's signaling complex (CD3), thus enhancing signal transduction 3 .There are conflicting data on the interaction between the TCR-CD3 complex and coreceptors, and in particular whether any interaction between these molecules is constitutive or induced by antigen recognition. Various co-immunoprecipitation and flow cytometry fluorescence resonance energy transfer (FRET) experiments suggest a constitutive interaction between some TCR-CD3 and coreceptors in unstimulated T cells 7,10-13 . Others show interaction only after T cell activation 14-17 . Co-capping and co-modulation experiments also support an interaction of coreceptor with TCR 18-21 . CD8αβ and CD4 reside on glycolipid-enriched microdomains or rafts, whereas TCR association with rafts is greatly increased upon stimulation 22,23 . It is therefore questionable whether all of these assays measure a direct interaction or simply colocalization of the molecules on the same rafts.Whether the interaction between CD8 and TCR is constitutive or antigen-induced has important consequences for the role of CD8. Constitutive association implies that CD8 acts as a universal amplifier in antigen recognition. Inducible interaction suggests an extra level of fine-tuning, where CD8 could sharpen and amplify sensitivity and specificity of recognition. In order to address definitively whether CD8 and TCR interact, and to what extent the interaction is induced upon antigen recognition, we have used microscopy to measure FR...

Themis (Thymocyte expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis is expressed in high amounts in thymocytes between the pre-T cell receptor (TCR) and positive selection checkpoints, and in low amounts in mature T cells. Themis-deficient thymocytes exhibit defective positive selection, which results in reduced numbers of mature thymocytes. Negative selection is also impaired in Themis-deficient mice. A higher percentage of Themis-deficient T cells exhibit CD4+CD25+Foxp3+ regulatory and CD62LloCD44hi memory phenotypes than in wild-type mice. Supporting a role for Themis in TCR signaling, this protein is phosphorylated quickly after TCR stimulation, and is needed for optimal TCR-driven Ca2+ mobilization and Erk activation.

How T cells translate T cell receptor (TCR) recognition of almost identical pMHC ligands into distinct biological responses has remained enigmatic. Although differences in affinity or off rate are important, they offer at best an incomplete explanation. By using Förster resonance energy transfer (FRET), we have visualized the ligand-induced interaction between OT-I TCR and CD8. We found that both recruitment of TCR to the immunological synapse and the TCR-CD8 interaction induced by weak agonists (positive-selecting ligands) was delayed but not necessarily weaker than strong agonists (negative selectors). A delayed and perhaps longer lasting CD8-TCR interaction results in delayed phospho-ERK recruitment to the synapse. The kinetics of the TCR-CD8 interaction can reconcile previously anomalous data, where biological activity did not correlate with TCR-pMHC binding kinetics for certain ligands. Our findings indicate that the T cell translates antigen recognition into T cell responses by differential recruitment of CD8 to the TCR.

Aims/hypothesis: We evaluated diabetes-related pregnancy outcomes in a cohort of Spanish women in relation to their glucose tolerance status, prepregnancy BMI and other predictive variables. Methods: The present paper is part of a prospective study to evaluate the impact of American Diabetes Association (2000) criteria in the Spanish population. A total of 9,270 pregnant women were studied and categorised as follows according to prepregnancy BMI quartiles and glucose tolerance status: (1) negative screenees; (2) false-positive screenees; (3) gestational diabetes mellitus (GDM) according to American Diabetes Association criteria only; and (4) GDM according to National Diabetes Data Group criteria (NDDG). We evaluated fetal macrosomia, Caesarean section and seven secondary outcomes as diabetes-related pregnancy outcomes. The population-attributable and population-prevented fractions of predictor variables were calculated after binary logistic regression analysis with multiple predictors. Results: Both prepregnancy BMI and abnormal glucose tolerance categories were independent predictors of pregnancy outcomes. The upper quartile of BMI accounted for 23% of macrosomia, 9.4% of Caesarean section, 50% of pregnancy-induced hypertension and 17.6% of large-for-gestational-age newborns. In contrast, NDDG GDM accounted for 3.8% of macrosomia, 9.1% of pregnancy-induced hypertension and 3.4% of preterm births. Conclusions/ interpretation: In terms of population impact, prepregnancy maternal BMI exhibits a much stronger influence than abnormal blood glucose tolerance on macrosomia, Caesarean section, pregnancy-induced hypertension and large-for-gestational-age newborns.

The earliest molecular events in T cell recognition have not yet been fully described, and the initial T cell receptor (TCR) triggering mechanism remains a subject of controversy. Here, using TIRF/FRET microscopy, we observe a two-stage interaction between TCR, CD8, and MHCp. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR triggering event is induced by free Lck.