T-cell receptor-CD3 Complex Research Articles

Structures of human γδ T cell receptor–CD3 complex

Gamma delta (γδ) T cells, a unique T cell subgroup, are crucial in various immune responses and immunopathology1–3. The γδ T cell receptor (TCR), which is generated by γδ T cells, recognizes a diverse range of antigens independently of the major histocompatibility complex2. The γδ TCR associates with CD3 subunits, initiating T cell activation and holding great potential in immunotherapy4. Here we report the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR–CD3 complexes5,6, revealing two distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR–CD3 complex is monomeric, with considerable conformational flexibility in the TCRγ–TCRδ extracellular domain and connecting peptides. The length of the connecting peptides regulates the ligand association and T cell activation. A cholesterol-like molecule wedges into the transmembrane region, exerting an inhibitory role in TCR signalling. The Vγ5Vδ1 TCR–CD3 complex displays a dimeric architecture, whereby two protomers nestle back to back through the Vγ5 domains of the TCR extracellular domains. Our biochemical and biophysical assays further corroborate the dimeric structure. Importantly, the dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. These findings reveal organizing principles of the γδ TCR–CD3 complex, providing insights into the unique properties of γδ TCR and facilitating immunotherapeutic interventions.

The structural basis of T-cell receptor (TCR) activation: An enduring enigma.

T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)-CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR-CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR-CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR-CD3 complex.

I-107 Long-range allosteric effects induced by Tax–HLA-A2 binding to an anti-HTLV-1 TCR: Implications for early T cell signaling

The mechanism whereby αβ T cell receptor (TCR) engagement by peptide–MHC (pMHC) is first communicated to the CD3 signaling apparatus of the TCR–CD3 complex, a process termed early T cell activation, is not well understood. To address the possibility that pMHC binding induces allosteric changes in TCR conformation and/or dynamics that are relayed to CD3, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and β chains of a human antiviral TCR (A6) that recognizes the Tax antigen from HTLV-1 bound to HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR V domains that propagate to 3 distinct sites in the C domains: (1) the Cβ FG loop that projects from the Vβ/Cβ interface, (2) a cluster of Cβ residues near the Cβ αA helix, a region involved in interactions with CD3, and (3) the Cα AB loop located at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. In addition, the pattern of long-range ligand-induced changes in TCR A6 seen by NMR is broadly similar to that predicted by MD simulations. We propose that the unique structure of the TCR β chain enables allosteric communication between the TCR binding sites for pMHC and CD3.

Instructional signals for nascent T cells

Thymic Selection Signals relayed through the T cell receptor (TCR)–CD3 complex are critical to determining whether immature T cells in the thymus undergo positive or negative selection. Positive selection enables T cell survival and subsequent export to the periphery, whereas negative selection leads to T cell death. Neier et al. developed a proteomics-based approach to profile proteins bound to the TCR–CD3 complex and compared differences between signaling associated with thymocyte survival and signaling associated with death. The full range of cellular outcomes after thymic selection could be attributed to quantitative differences in a shared core set of biochemical signals. Thus, differences in the affinity of TCRs for peptide–major histocompatibility complex complexes are transduced into downstream signals that are quantitatively variable, but qualitatively similar. Sci. Immunol. 4 , eaal2201 (2019).

Introducing Membrane Charge and Membrane Potential to T Cell Signaling.

While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR) signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Proteomics and Network Analyses Reveal Inhibition of Akt-mTOR Signaling in CD4+ T Cells by Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan.

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose‐capped lipoarabinomannan (ManLAM) inhibits CD4+ T‐cell activation by inhibiting proximal T‐cell receptor (TCR) signaling when activated by anti‐CD3. To understand the impact of ManLAM on CD4+ T‐cell function when both the TCR–CD3 complex and major costimulator CD28 are engaged, we performed label‐free quantitative MS and network analysis. Mixed‐effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti‐CD3‐ and anti‐CD28‐activated CD4+ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF‐κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x‐Akt‐mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T‐cell proteome by affecting Akt‐mTOR signaling, resulting in broad functional impairment of CD4+ T‐cell activation beyond inhibition of proximal TCR–CD3 signaling.

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor.

T-cell receptor-CD3 complex (TCR) is a versatile signaling machine that can initiate antigen-specific immune responses based on various biochemical changes of CD3 cytoplasmic domains, but the underlying structural basis remains elusive. Here we developed biophysical approaches to study the conformational dynamics of CD3ε cytoplasmic domain (CD3εCD). At the single-molecule level, we found that CD3εCD could have multiple conformational states with different openness of three functional motifs, i.e., ITAM, BRS and PRS. These conformations were generated because different regions of CD3εCD had heterogeneous lipid-binding properties and therefore had heterogeneous dynamics. Live-cell imaging experiments demonstrated that different antigen stimulations could stabilize CD3εCD at different conformations. Lipid-dependent conformational dynamics thus provide structural basis for the versatile signaling property of TCR.

Molecular architecture of the αβ T cell receptor-CD3 complex.

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

AB0093 Association of cd247 polymorphisms with rheumatoid arthritis: a replication study and a meta-analysis

BackgroundThe T-cell receptor T3 zeta chain (CD3ζ), also called CD247, is essential for assembly, surface expression and signaling cascade of the T-cell receptor-CD3 complex (TCR/CD3 complex). Abnormalities in this pathway...

Lipid Membrane Association of the T Cell Antigen Receptor ζ Subunit: Affinities and Structure

The T cell antigen receptor (TCR) is a cell surface protein complex that binds peptide antigen fragments presented by the Major Histocompatibility Complex (MHC). The interaction of TCR with MHC leads to T cell activation and initiation of an immune response,1 but the molecular details of the transmembrane signaling process remain unknown. TCRζ is the 115 amino acid long cytosolic signaling domain of the CD3-T cell receptor complex that plays a central role in this process. TCRζ is predominantly unstructured2 and carries several copies of the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs become phosphorylated upon receptor engagement, constituting an early and obligatory event in the signaling cascade that blocks lipid association and lipid-associated conformational rearrangements3 of TCRζ. We present a characterization of the affinity of TCRζ to tethered lipid bilayers by SPR and a structural study of its association with such membranes using neutron reflectometry (NR). We studied the binding of unphosphorylated TCRζ to highly charged stBLMs rich in anionic phosphatidylserine (PS) or phosphatidylglycerol (PG) and observed affinity constants of Kd ≈ 10 μM. Minute amounts of phosphoinositides (PI(4,5)P2) increase the membrane affinity of the protein by more than 10-fold. An NR characterization of TCRζ in the membrane-associated state shows the major portion of the protein interfacially associated with the bilayer surface and a minor protein penetrating the bilayer deeply. This is consistent with a model in which an α-helical structure is aligned on the membrane surface that may be connected via a hinge with another α-helical segment inserted into the bilayer.[1] Call, M.E., and Wucherpfennig, K.W., Annu. Rev. Immunol. 23:101 (2005).[2] Sigalov, A.B., et al., Biochemistry 45:15731 (2006).[3] Aivazian, D.A., et al., Nat. Struct. Biol. 7:1023 (2000).

Change in expression pattern of TCR–CD3 complex in patients with multiple myeloma

In haematological malignancy, cell-mediated immunity has been shown to be suppressed in advanced disease. This immune dysfunction may be due, in part, to altered expression of the T cell receptor (TCR)–CD3 complex. The distribution and clonality of the TCR Vbeta repertoire and the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in T cells from patients with multiple myeloma (MM) were investigated. Specific Vbeta subfamily primers, reverse transcription polymerase chain reaction, and the GeneScan® technique were used to analyse the expression of the TCR Vbeta subfamily and the clonality of Vbeta T cells in 11 patients with MM. Real-time reverse transcription polymerase chain reaction was used to detect the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in peripheral blood mononuclear cells from 19 patients with MM. The beta2-microglobulin gene was used as an endogenous reference. A total of 5–22 Vbeta subfamily T cells were detected in different patients (mean value of expressed Vbeta subfamilies was 12·55±6·11), whereas all 24 Vbeta genes were identified in all control samples. The most frequently expressed Vbeta subfamilies were Vbeta1 (100%), Vbeta2, Vbeta3, Vbeta9, Vbeta13, and Vbeta16 (81·8%), while the expression of Vbeta20 was undetectable in all MM samples. Oligoclonal expansion of one or more Vbeta subfamily of T cells was detected in all patients. Such expansions involved different MM stages, and the numbers of expanded clonal Vbeta subfamilies seemed higher in stage I/II groups than in stage III; however, there was no significant difference. Among MM samples, of the Vbeta subfamily members, Vbeta13, Vbeta1, and Vbeta21, were expanded most frequently. A significant decrease in the expression level of the CD3gamma gene was observed in MM samples; in contrast, a higher expression of CD3epsilon was found in the MM group than in the healthy group. The expression pattern of the four CD3 chains was epsilon>zeta>delta>gamma in peripheral blood mononuclear cells from MM, while a gamma>epsilon>zeta>delta expression pattern was found in healthy controls. In conclusions, the present study presents precise data on changes in the variability of Vbeta patterns and expression of TCR signal transduction molecules in MM patients compared to controls, which may be associated with immune dysfunction. This study contributes to a better understanding of the cellular immune features in MM patients.

Alternative Splicing Factor/Splicing Factor 2 Regulates the Expression of the ζ Subunit of the Human T Cell Receptor-associated CD3 Complex

T cells from patients with systemic lupus erythematosus express decreased levels of the T cell receptor-associated CD3 zeta chain, a feature directly linked to their aberrant function. The decrease in CD3zeta protein expression is in part due to decreased levels of functional wild type isoform of the 3'-untranslated region (UTR) of CD3zeta mRNA with concomitant increased levels of an unstable alternatively spliced isoform. In order to identify factors involved in the post-transcriptional regulation of CD3zeta, we performed mass spectrometric analysis of Jurkat T cell nuclear proteins "pulled down" by a CD3zeta 3'-UTR oligonucleotide, which identified the splicing protein alternative splicing factor/splicing factor 2 (ASF/SF2). We show for the first time that ASF/SF2 binds specifically to the 3'-UTR of CD3zeta and regulates expression of CD3zeta protein by limiting the production of the alternatively spliced isoform. During activation of human T cells, an increase in the wild type CD3zeta mRNA is associated with increased expression of ASF/SF2. Finally, we show a significant correlation between ASF/SF2 and CD3zeta protein levels in T cells from systemic lupus erythematosus patients. Thus, our results identify ASF/SF2 as a novel factor in the regulation of alternative splicing of the 3'-UTR of CD3zeta and protein expression in human T cells.

Effect of TNF-α on CD3-ζ and MHC-I in Postnatal Rat Hippocampus

The zeta subunit of the CD3 T-cell receptor complex and the major histocompatibility complex class 1 (MHC-I) are important not only for the immune response to antigens, they also function as signal molecules in the brain, where they play a role in the postnatal maturation process. The expression of these molecules can be regulated by cytokines. In situations associated with increased cytokine production, such as neonatal hypoxia, the hippocampus is particularly susceptible to permanent damage. This has prompted us to examine the MHC-I and CD3-zeta expression in hippocampus from early postnatal, weanling and adolescent rats and to record the effects of TNF-alpha and IL-1beta, cytokines commonly increased in neonatal hypoxia, on MHC-I and CD3-zeta expression in the hippocampus. We show that there is a robust postnatal up-regulation of CD3-zeta and MHC-I protein as well as of MHC-I mRNA and that TNF-alpha down-regulates the expression of CD3-zeta protein and MHC-I mRNA in early postnatal but not in weanling nor in adolescent rats. These results may offer a molecular explanation to the adverse effects of increased circulating levels of cytokines on brain in neonatal hypoxia.

The molecular machinery for cAMP-dependent immunomodulation in T-cells

cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I-Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR-CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP-PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, beta-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.

The Impact of Single Amino Acid Substitutions in CD3γ on the CD3ϵγ Interaction and T-Cell Receptor–CD3 Complex Formation

The human T-cell receptor–CD3 complex consists of at least eight polypeptide chains; CD3γϵ- and δϵ-dimers associate with the disulfide linked αβ- and ζζ-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3γ and CD3ϵ in human T-cells. For this purpose, we have generated site-directed mutants of CD3γ that were introduced in human T-cells defective in CD3γ expression. Cell-surface and intracellular expression of the introduced CD3γ chains was determined, as was the association with CD3δ, CD3ϵ, and the T-cell receptor. Although the introduction of wild type CD3γ and CD3γ (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3γ (82C-S), CD3γ (85C-S), and CD3γ (76Q-E) all resulted in an impaired association between CD3γ and CD3ϵ and a lack of cell-surface expressed CD3γ. Finally, the introduction of CD3γ (76Q-L) and CD3γ (78Y-A) restored the expression of TCR-CD3δϵγϵζ 2 complexes, although the association between CD3γ and CD3ϵ was impaired. These results indicate that several amino acids in CD3γ are essential for an optimal association between CD3γ and CD3ϵ and the assembly of a cell-surface expressed TCR-CD3δϵγϵζ 2 complex.

Inherited and SomaticCD3ζ Mutations in a Patient with T-Cell Deficiency

A four-month-old boy with primary immunodeficiency was found to have a homozygous germ-line mutation of the gene encoding the CD3zeta subunit of the T-cell receptor-CD3 complex. CD3zeta is necessary for the development and function of T cells. Some of the patient's T cells had low levels of the T-cell receptor-CD3 complex and carried the Q70X mutation in both alleles of CD3zeta, whereas other T cells had normal levels of the complex and bore the Q70X mutation on only one allele of CD3zeta, plus one of three heterozygous somatic mutations of CD3zeta on the other allele, allowing expression of poorly functional T-cell receptor-CD3 complexes.

Differential desensitization of homozygous haplotypes of the beta2-adrenergic receptor in lymphocytes.

Single-nucleotide polymorphisms of the beta(2)-adrenergic receptor gene and its 5' promoter have been associated with differences in receptor function and desensitization. Linkage disequilibrium may account for inconsistencies in reported effects of isolated polymorphisms. Therefore, we have investigated the three most common homozygous haplotypes of the beta(2)-adrenergic receptor (position 19 [Cys/Arg] of the 5' leader cistron and positions 16 [Arg/Gly] and 27 [Gln/Glu] of the receptor) for putative differences in agonist-induced desensitization. Lymphocytes of well defined nonasthmatic, nonallergic subjects homozygous for the haplotype CysGlyGln, ArgGlyGlu, or CysArgGln were isolated. Desensitization of (-)-isoproterenol-induced cyclic adenosine monophosphate (cAMP) accumulation and beta(2)-adrenergic receptor sequestration and downregulation were measured in relation to beta(2)-adrenergic receptor-mediated inhibition of IFN-gamma and interleukin-5 production. We observed that lymphocytes of individuals bearing the CysGlyGln haplotype were more susceptible to desensitization of the beta-agonist-induced cAMP response than those of individuals with the ArgGlyGlu or CysArgGln haplotype. The haplotype-dependent desensitization of beta-agonist-induced cAMP response was not associated with haplotype-dependent beta(2)-adrenergic receptor sequestration or downregulation. In addition, our data suggest reduced inhibition, in lymphocytes of subjects with the CysGlyGln haplotype, of interleukin-5 production induced by treatment with antibodies to the T-cell receptor-CD3 complex and to costimulatory molecule CD28 (alphaCD3/alphaCD28). This is the first study demonstrating haplotype-related differences in agonist-induced beta(2)-adrenergic receptor desensitization in primary human cells. This haplotype-related desensitization of the beta(2)-adrenergic receptor in lymphocytes might have consequences regarding the regulation of helper T-cell type 2 inflammatory responses.

Stoichiometry of the T-cell receptor-CD3 complex and key intermediates assembled in the endoplasmic reticulum.

The T-cell receptor (TCR)-CD3 complex is critical for T-cell development and function, and represents one of the most complex transmembrane receptors. Models of different stoichiometry and valency have been proposed based on cellular experiments and these have important implications for the mechanisms of receptor triggering. Since determination of receptor stoichiometry in T-cells is not possible due to the presence of previously synthesized, unlabeled receptor components with different half-lives, we examined the stoichiometry of the receptor assembled in endoplasmic reticulum (ER) microsomes of B-cell origin. The stoichiometric relationship among all subunits was directly determined using intact radiolabeled TCR-CD3 complexes that were isolated with a sequential, non-denaturing immunoprecipitation method, and identical results were obtained with two detergents belonging to different structural classes. The results firmly establish that the alphabeta TCR-CD3 complex assembled in the ER is monovalent and composed of one copy of the TCRalphabeta, CD3deltaepsilon, CD3gammaepsilon and zeta-zeta dimers.

A Brij to T-cell activation: rafts come out of the cold

Membrane rafts help coordinate protein trafficking patterns, signaling cascades and steps in viral pathogenesis. Stabilized by cholesterol and the long, saturated, acyl chains of sphingolipids, these liquid-ordered supramolecular domains of plasma membranes and other membranes segregate highly specific subsets of membrane proteins. The structural reality of rafts is no longer in doubt, but the precise roles they play remain an area of controversy. Although the field has incorporated new biophysical and cellular imaging approaches, biochemical isolation of rafts has relied on the now classic observation that raft membranes remain insoluble at 4°C in non-ionic detergents such as Triton X-100. Although instrumental in establishing raft properties, various problems compromise the utility of this method. These include artifactual modification of lipid-phase behavior at low temperatures, significant solubilization of the liquid-ordered domain and loss of proteins with low-affinity association with raft lipids.Now, Hai-Tao He and collaborators [1xTCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts. Drevot, P. et al. EMBO J. 2002; 21: 1–10Crossref | PubMed | Scopus (252)See all References][1] find that a polyoxyethylene ether detergent in the Brij series circumvents these problems and use this detergent to gain a new perspective on T-cell activation. They reasoned that the bulky headgroup and mono-unsaturated ether chain of Brij 98 would cause the detergent to solubilize the fluid phase of the membrane but leave the more tightly packed liquid-ordered phase intact. The hunch proved correct: membrane remaining insoluble at 37°C in the presence of 1% Brij 98 displays the characteristic properties of rafts, including enrichment in sphingomyelins, cholesterol, GPI-anchored, palmitoylated and myristoylated proteins and the absence of prenylated proteins.Previous work on T-cell activation pointed to rafts as dynamic centers that collect signaling components from the surrounding liquid-disordered phase, with the process initiated by T-cell receptors binding to cognate peptide-presenting MHC molecules and repartitioning into rafts. However, a different scenario emerges when rafts are isolated at 37°C. Signaling complexes are found fully primed in the liquid-ordered phase; rafts from cells that have not been activated contain essential signaling components, including the T-cell receptor–CD3 complex, the Lck and ZAP-70 kinases, and the CD4 co-receptor. Moreover, when such rafts are activated and supplied with ATP, appropriate initial phosphorylation events take place. The method also resolves a microheterogeneity among the rafts, with antibodies to Thy-1 precipitating a vesicle population distinct from that containing the signaling machinery. Preliminary data suggest that these populations interact upon T-cell activation. Perspectives opened by this new wrinkle on raft isolation extend within immunology (e.g. does CD8 also associate with the T-cell receptor before activation?) and well beyond.