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In JoVE (1)
- Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Other Publications (43)
- Molecular Cell
- Immunological Reviews
- Proceedings of the National Academy of Sciences of the United States of America
- Molecular & Cellular Proteomics : MCP
- The Journal of Biological Chemistry
- European Journal of Immunology
- The Journal of Experimental Medicine
- Immunology Letters
- Trends in Immunology
- EMBO Reports
- Science's STKE : Signal Transduction Knowledge Environment
- International Immunology
- Science's STKE : Signal Transduction Knowledge Environment
- Journal of Immunological Methods
- The Journal of Experimental Medicine
- Proceedings of the National Academy of Sciences of the United States of America
- Trends in Biochemical Sciences
- Immunology Letters
- Immunology Letters
- PloS One
- Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et Al.]
- International Immunology
- Advances in Experimental Medicine and Biology
- Advances in Experimental Medicine and Biology
- Advances in Experimental Medicine and Biology
- Analytical Biochemistry
- Journal of Immunotherapy (Hagerstown, Md. : 1997)
- Current Pharmaceutical Design
- Methods in Molecular Biology (Clifton, N.J.)
- Immunology Letters
- Immunology Letters
- International Immunology
- European Journal of Immunology
- FEBS Letters
- PloS One
- The Journal of Clinical Investigation
Articles by Wolfgang W. A. Schamel in JoVE
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Gina J. Fiala1,2,3, Wolfgang W. A. Schamel*2,3, Britta Blumenthal*3
1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics
In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.
Other articles by Wolfgang W. A. Schamel on PubMed
Recruitment of Nck by CD3 Epsilon Reveals a Ligand-induced Conformational Change Essential for T Cell Receptor Signaling and Synapse Formation
Cell. Jun, 2002 | Pubmed ID: 12110186
How membrane receptors initiate signal transduction upon ligand binding is a matter of intense scrutiny. The T cell receptor complex (TCR-CD3) is composed of TCR alpha/beta ligand binding subunits bound to the CD3 subunits responsible for signal transduction. Although it has long been speculated that TCR-CD3 may undergo a conformational change, confirmation is still lacking. We present strong evidence that ligand engagement of TCR-CD3 induces a conformational change that exposes a proline-rich sequence in CD3 epsilon and results in recruitment of the adaptor protein Nck. This occurs earlier than and independently of tyrosine kinase activation. Finally, by interfering with Nck-CD3 epsilon association in vivo, we demonstrate that TCR-CD3 recruitment of Nck is critical for maturation of the immune synapse and for T cell activation.
Molecular Cell. Nov, 2002 | Pubmed ID: 12453414
We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.
Immunological Reviews. Feb, 2003 | Pubmed ID: 12614350
The number of possible T cell activation outcomes resulting from T cell receptor (TCR) engagement suggests that the TCR is able to differentially activate a myriad of signaling pathways depending on the nature of the stimulus. The complex structural organization of the TCR itself could underlie this diversity of responses. Assembly and stoichiometric studies have helped us to shed some light on the initiation of TCR signaling. The TCR is composed of TCR and CD3 dimers. Changes in the interaction between CD3 subunits within the CD3 dimers and in the interaction of these dimers with the TCR heterodimer could be the triggering mechanism that initiates the first activation events. One of the hallmarks of these early changes in TCR conformation is the induced recruitment of the adapter protein Nck to a proline-rich sequence of the cytoplasmic tail of CD3epsilon, but there may be others. According to our most recent observations, the TCR is organized in pre-existing clusters within plasma membrane microdomains, exhibiting a complexity above and beyond that of dimer composition complexity. How the presence of TCR in clusters influences TCR avidity and propagation of TCR signals is something that has yet to be investigated.
A High-molecular-weight Complex of Membrane Proteins BAP29/BAP31 is Involved in the Retention of Membrane-bound IgD in the Endoplasmic Reticulum
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2003 | Pubmed ID: 12886015
B cell antigen receptors (BCRs) are multimeric transmembrane protein complexes comprising membrane-bound immunoglobulins (mIgs) and Ig-alpha/Ig-beta heterodimers. In most cases, transport of mIgs from the endoplasmic reticulum (ER) to the cell surface requires assembly with the Ig-alpha/Ig-beta subunits. In addition to Ig-alpha/Ig-beta, mIg molecules also bind two ER-resident membrane proteins, BAP29 and BAP31, and the chaperone heavy chain binding protein (BiP). In this article, we show that neither Ig-alpha/Ig-beta nor BAP29/BAP31 nor BiP bind simultaneously to the same mIgD molecule. Blue native PAGE revealed that only a minor fraction of intracellular mIgD is associated with high-molecular-weight BAP29/BAP31 complexes. BAP-binding to mIgs was found to correlate with ER retention of chimeric mIgD molecules. On high-level expression in Drosophila melanogaster S2 cells, mIgD molecules were detected on the cell surface in the absence of Ig-alpha/Ig-beta. This aberrant transport was prevented by coexpression of BAP29 and BAP31. Thus, BAP complexes contribute to ER retention of mIg complexes that are not bound to Ig-alpha/Ig-beta. Furthermore, the mechanism of ER retention of both BAP31 and mIgD is not through retrieval from a post-ER compartment, but true ER retention. In conclusion, BAP29 and BAP31 might be the long sought after retention proteins and/or chaperones that act on transmembrane regions of various proteins.
Two-dimensional Blue Native/SDS Gel Electrophoresis of Multi-protein Complexes from Whole Cellular Lysates: a Proteomics Approach
Molecular & Cellular Proteomics : MCP. Feb, 2004 | Pubmed ID: 14665681
Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after gamma interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.
Biochemical Differences in the Alphabeta T Cell Receptor.CD3 Surface Complex Between CD8+ and CD4+ Human Mature T Lymphocytes
The Journal of Biological Chemistry. Jun, 2004 | Pubmed ID: 15060077
We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.
European Journal of Immunology. Jan, 2005 | Pubmed ID: 15580656
Anergic B lymphocytes exert compromised signal transduction towards the activation of NF-kappa B in response to B cell antigen receptor (BCR) triggering, whereas activation of the ERK pathway appears normal. How this differential down-regulation of the NF-kappa B pathway is regulated remains still elusive. Here, we demonstrate that stimuli known to enhance 3',5'-cyclic adenosine monophosphate (cAMP) are capable of selectively suppressing the activation both of NF-kappa B downstream of the BCR and Toll-like receptor 4 in splenic B lymphocytes and of the high-affinity receptor for IgE in BM-derived mast cells. This suppression is accomplished by blocking phosphorylation and subsequent degradation of the inhibitor of NF-kappa B. A cAMP-dependent protein kinase (PKA) inhibitor reverses this suppressive effect, indicating that PKA is a downstream effector of cAMP in this process. Importantly, not only drugs that artificially elevate intracellular cAMP levels, but also the nucleoside adenosine, which is known to be a mediator of cellular distress, inhibit the NF-kappa B pathway. This suggests that adenosine-mediated signals represent an important step in the molecular decision process controlling inflammation versus anergic immune responses.
The Journal of Experimental Medicine. Aug, 2005 | Pubmed ID: 16087711
A long-standing paradox in the study of T cell antigen recognition is that of the high specificity-low affinity T cell receptor (TCR)-major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native-polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (alphabetagammaepsilondeltaepsilonzetazeta) and multivalent complexes with two or more ligand-binding TCRalpha/beta subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity.
Proteomics. Sep, 2005 | Pubmed ID: 16097032
Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.
Differences in Pairing and Cluster Formation of T Cell Receptor Alpha- and Beta-chains in T Cell Clones and Fusion Hybridomas
Immunobiology. 2005 | Pubmed ID: 16323705
The questions of T cell receptor (TCR) clustering and preferential pairing of TCR alpha- and beta-chains are discussed controversially. We here describe the rare case of a non-pairing TCR alpha-TCR beta combination detected in the murine T cell hybridoma Hy-E6. Of its two TCR alpha-chains (Valpha3.2, Vbeta17) and one Vbeta16-chain only the Valpha17/Vbeta16 TCR is exposed on the surface, despite intracellular expression of Valpha3.2 protein. The lack of Valpha3.2/Vbeta16 pairing was confirmed by TCR transfections. Surprisingly, however, the parental T cell clone CTL-E6 expressed both alpha-chains on its plasma membrane. Different size distribution of TCR clusters in CTL-E6 versus Hy-E6 and transfectants as determined by Blue-Native gel electrophoresis indicated differences in the supra-molecular TCR assembly as one possible reason for this phenomenon. Our data further reveal that the nominal specificity of CTL-E6 for the fully agonistic trinitrophenyl (TNP) modified peptide M4L-TNP was specifically mediated by the trimeric Valpha3.2/Valpha17/Vbeta16 TCR of CTL-E6. In contrast, the Valpha17/Vbeta16 combination in Hy-E6 only conferred specificity for the cross-reactive partial agonist O4-TNP. Both specificities are H-2Kb-restricted and, hence, appear to be positively selected. The differences in TCR clustering in CTL and hybridoma might indicate differences in the reception and transmission of TCR-signals between these two cell types.
Immunology Letters. Apr, 2006 | Pubmed ID: 16356554
SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.
Trends in Immunology. Apr, 2006 | Pubmed ID: 16527543
During antigen recognition, T cells show high sensitivity and specificity, and a wide dynamic range. Paradoxically, these characteristics are based on low-affinity receptor-ligand interactions [between the T-cell antigen receptor (TCR-CD3) complex and the antigen peptide bound to MHC]. Recent evidence indicates that the TCR-CD3 is expressed as multivalent complexes in the membrane of non-stimulated T cells and that conformational changes in the TCR-CD3 can be induced by strong but not weak agonists. Here, we propose a thermodynamic model whereby the specificity of the TCR-CD3-pMHC interaction is explained by its multivalent nature. We also propose that the free energy barriers involved in the change in conformation of the receptor impose a response threshold and determine the kinetic properties of recognition. Finally, we suggest that multivalent TCR-CD3s can amplify signals by spreading them from pMHC-engaged TCR-CD3s to unengaged complexes as a consequence of the cooperativity in the system.
EMBO Reports. May, 2006 | Pubmed ID: 16670682
The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range.
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes
Science's STKE : Signal Transduction Knowledge Environment. Jul, 2006 | Pubmed ID: 16868305
Multiprotein complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCs--for example, multisubunit receptors or transcription factors; and (ii) signal-induced, transient, low copy number MPCs--for example, complexes that form upon binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a separation method with a higher resolution than gel filtration or sucrose density ultracentrifugation that can be used to analyze abundant, stable MPCs from 10 kD to 10 MD. In contrast to immunoprecipitation and two-hybrid approaches, it allows the determination of the size, the relative abundance, and the subunit composition of an MPC. In addition, it shows how many different complexes exist that share a common subunit, whether free monomeric forms of individual subunits exist, and whether these parameters change upon cell stimulation. Here, we give a detailed protocol for the separation of MPCs from total cellular lysates or of prepurified MPCs by one-dimensional BN-PAGE or by two-dimensional BN-PAGE and SDS-PAGE.
Identification of Disulfide Bonds in the Ig-alpha/Ig-beta Component of the B Cell Antigen Receptor Using the Drosophila S2 Cell Reconstitution System
International Immunology. Sep, 2006 | Pubmed ID: 16877534
Structural information about immune receptor complexes is important for understanding signal transduction mechanisms. We have used the Drosophila S2 cell reconstitution system for identification of disulfide bonds within and between CD79a (Ig-alpha) and CD79b (Ig-beta), the heterodimeric signal transducing element of the B cell antigen receptor (BCR). Cysteines 113 and 135 of Ig-alpha and Ig-beta, respectively, form the intermolecular disulfide bridge stabilizing the Ig-alpha/Ig-beta heterodimer in both S2 cells and the B cell line J558L. Furthermore, using transfected S2 cells, two putative intramolecular disulfide bonds in the Ig-like domain of Ig-beta were identified. Ig-betaC65 and Ig-betaC120 form the canonical Ig fold disulfide bond. In addition, Ig-betaC43 and Ig-betaC124 also bind covalently. Individual cysteine to serine mutations in Ig-alpha had no influence on membrane-bound Ig (mIg)-M expression on the surface of S2 cells. In contrast, mIgM expression on the surface of B cells expressing Ig-alphaC113S was reduced, indicating that this intermolecular bond is prerequisite for efficient IgM-BCR formation. Our data also suggest that the Ig-alpha/Ig-beta heterodimer can assemble into oligomers.
Overlapping Functions of Human CD3delta and Mouse CD3gamma in Alphabeta T-cell Development Revealed in a Humanized CD3gamma-mouse
Blood. Nov, 2006 | Pubmed ID: 16888097
Humans lacking the CD3gamma subunit of the pre-TCR and TCR complexes exhibit a mild alphabeta T lymphopenia, but have normal T cells. By contrast, CD3gamma-deficient mice are almost devoid of mature alphabeta T cells due to an early block of intrathymic development at the CD4(-)CD8(-) double-negative (DN) stage. This suggests that in humans but not in mice, the highly related CD3delta chain replaces CD3gamma during alphabeta T-cell development. To determine whether human CD3delta (hCD3delta) functions in a similar manner in the mouse in the absence of CD3gamma, we introduced an hCD3delta transgene in mice that were deficient for both CD3delta and CD3gamma, in which thymocyte development is completely arrested at the DN stage. Expression of hCD3delta efficiently supported pre-TCR-mediated progression from the DN to the CD4(+)CD8(+) double-positive (DP) stage. However, alphabetaTCR-mediated positive and negative thymocyte selection was less efficient than in wild-type mice, which correlated with a marked attenuation of TCR-mediated signaling. Of note, murine CD3gamma-deficient TCR complexes that had incorporated hCD3delta displayed abnormalities in structural stability resembling those of T cells from CD3gamma-deficient humans. Taken together, these data demonstrate that CD3delta and CD3gamma play a different role in humans and mice in pre-TCR and TCR function during alphabeta T-cell development.
Immunity. Jan, 2007 | Pubmed ID: 17188005
T cell receptor (TCR-CD3) triggering involves both receptor clustering and conformational changes at the cytoplasmic tails of the CD3 subunits. The mechanism by which TCRalphabeta ligand binding confers conformational changes to CD3 is unknown. By using well-defined ligands, we showed that induction of the conformational change requires both multivalent engagement and the mobility restriction of the TCR-CD3 imposed by the plasma membrane. The conformational change is elicited by cooperative rearrangements of two TCR-CD3 complexes and does not require accompanying changes in the structure of the TCRalphabeta ectodomains. This conformational change at CD3 reverts upon ligand dissociation and is required for T cell activation. Thus, our permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.
High-sensitivity Detection and Quantitative Analysis of Native Protein-protein Interactions and Multiprotein Complexes by Flow Cytometry
Science's STKE : Signal Transduction Knowledge Environment. Jun, 2007 | Pubmed ID: 17551170
Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase-fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.
A Native Antibody-based Mobility-shift Technique (NAMOS-assay) to Determine the Stoichiometry of Multiprotein Complexes
Journal of Immunological Methods. Jul, 2007 | Pubmed ID: 17568608
Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view of protein interaction networks and prerequisite for a molecular understanding of how a certain MPC functions. Here, we present a technique utilizing monoclonal subunit-specific antibodies for an electrophoretic immunoshift assay in Blue Native-gels (NAMOS-assay), which allows the determination of the stoichiometry of MPCs. First, we use the B cell antigen receptor as a model MPC whose stoichiometry is known, confirming the HC(2)LC(2)Igalpha/beta(1) stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of alphabetaepsilon(2)gammadeltazeta(2). We then show that the NAMOS-assay does not require purified MPCs, since it can determine the stoichiometry of an MPC in cell lysates. The NAMOS-assay is also compatible with use of epitope tags appended to the protein of interest, as e.g. the widely used HA-tag, and anti-epitope antibodies for the assay. Given its general applicability, this method has a wide potential for MPC research.
Different Composition of the Human and the Mouse Gammadelta T Cell Receptor Explains Different Phenotypes of CD3gamma and CD3delta Immunodeficiencies
The Journal of Experimental Medicine. Oct, 2007 | Pubmed ID: 17923503
The gammadelta T cell receptor for antigen (TCR) comprises the clonotypic TCRgammadelta, the CD3 (CD3gammaepsilon and/or CD3deltaepsilon), and the zetazeta dimers. gammadelta T cells do not develop in CD3gamma-deficient mice, whereas human patients lacking CD3gamma have abundant peripheral blood gammadelta T cells expressing high gammadelta TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human gammadelta TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR-specific antibodies. The gammadelta TCR isolated in digitonin from primary and cultured human gammadelta T cells includes CD3delta, with a TCRgammadeltaCD3epsilon(2)deltagammazeta(2) stoichiometry. In CD3gamma-deficient patients, this may allow substitution of CD3gamma by the CD3delta chain and thereby support gammadelta T cell development. In contrast, the mouse gammadelta TCR does not incorporate CD3delta and has a TCRgammadeltaCD3epsilon(2)gamma(2)zeta(2) stoichiometry. CD3gamma-deficient mice exhibit a block in gammadelta T cell development. A human, but not a mouse, CD3delta transgene rescues gammadelta T cell development in mice lacking both mouse CD3delta and CD3gamma chains. This suggests important structural and/or functional differences between human and mouse CD3delta chains during gammadelta T cell development. Collectively, our results indicate that the different gammadelta T cell phenotypes between CD3gamma-deficient humans and mice can be explained by differences in their gammadelta TCR composition.
Proceedings of the National Academy of Sciences of the United States of America. Oct, 2007 | Pubmed ID: 17925433
Trends in Biochemical Sciences. Feb, 2008 | Pubmed ID: 18201888
The T cell antigen receptor (TCR-CD3) is the most complex receptor known to date, consisting of eight transmembrane subunits. Its activation by an antigen is the initial step in an immune response. Here, we present the permissive geometry model explaining how antigen binding initiates intracellular signalling cascades. We propose that a dimeric antigen imposes its geometry on two TCR-CD3 receptors by simultaneously binding to both. This causes the TCRalphabeta subunits to rotate with respect to each other leading to displacement of the ectodomains of the associated CD3 dimers. This results in a scissor-like movement of the CD3 dimers that opens the cytoplasmic tails for interaction with signalling proteins, thus initiating signalling cascades.
The Short Length of the Extracellular Domain of Zeta is Crucial for T Cell Antigen Receptor Function
Immunology Letters. Mar, 2008 | Pubmed ID: 18207249
The T cell antigen receptor (TCR-CD3) consists of the pMHC-binding TCRalphabeta heterodimer and the signalling dimers CD3deltaepsilon, CD3gammaepsilon and zetazeta. The very short length of the extracellular domain (EC) of the zeta chain is preserved through evolution, however a rational explanation for this observation has not been elucidated. Here, we show that TCR-CD3 assembly is clearly defective when the murine zeta EC domain is artificially enlarged. Under these conditions, the TCR-CD3 complex is super-competent in transducing activation signals upon engagement. Furthermore, the TCR-CD3 complexes containing enlarged zeta EC domains underwent ligand-induced conformation changes with higher efficiency than TCR-CD3 complexes with an unmodified zeta EC domain. Together these data suggest that a short zeta EC domain is needed to correctly assemble the TCR-CD3 complex. When this domain is enlarged, the resulting TCR-CD3 complex is distorted leading to a hyperactive phenotype and enhanced T cell activation.
Immunology Letters. Mar, 2008 | Pubmed ID: 18215426
The zeta chain is a key component of the T cell antigen receptor (TCR-CD3) complex, required for the expression of the receptor on the cell surface. It contains an extremely small extracellular (EC) part of nine amino acids. Interestingly, the length, but not the sequence, of the zeta EC has been highly conserved through evolution. Here, we examined the effect of increasing the length of human zeta EC on TCR-CD3 assembly and surface expression. Appending a 30 kDa polypeptide to the N-terminus of zeta completely abolished assembly and transport of the TCR-CD3 to the cell surface. Addition of only 17 amino acids, including the HA-tag (HAzeta), strongly reduced the efficiency of TCR-CD3 assembly and led to reduced expression on the surface, suggesting that the short zeta EC region is located within the receptor complex. In Blue Native gels (BN-PAGE) these receptors had a normal size, indicating that they have a stoichiometry of alphabetagammaepsilondeltaepsilonzetazeta. In resting TCR-CD3s the HA-tag, and thus the zeta EC region, was not accessible for anti-HA antibody binding, demonstrating that it was indeed buried in a cavity within the receptor complex. However, prolonged stimulation with antigen permitted the access of the anti-HA antibody, thus suggesting that stimulation led to architectural changes in the TCR-CD3.
T Cell Receptor Engagement Triggers Its CD3epsilon and CD3zeta Subunits to Adopt a Compact, Locked Conformation
PloS One. 2008 | Pubmed ID: 18320063
How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3epsilon subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3epsilon and CD3zeta subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3epsilon and CD3zeta, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.
Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et Al.]. Mar, 2008 | Pubmed ID: 18360818
Multiprotein complexes play crucial roles in nearly all cell biological processes. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a powerful method to study these complexes. It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa. If a second-dimension SDS-PAGE is applied (two-dimensional BN/SDS-PAGE), the size, subunit composition, and relative abundance of the different multiprotein complexes can be studied. In recent years, there has been a large increase in the number of publications where BN-PAGE was used to study protein-protein interactions. Here, we give detailed protocols for the separation of multiprotein complexes by two-dimensional BN/SDS-PAGE and for a related technique to determine the stoichiometry of these complexes.
Differential Antibody Binding to the Surface AlphabetaTCR.CD3 Complex of CD4+ and CD8+ T Lymphocytes is Conserved in Mammals and Associated with Differential Glycosylation
International Immunology. Oct, 2008 | Pubmed ID: 18653700
We have previously shown that the surface alphabeta T cell antigen receptor (TCR).CD3 complex borne by human CD4(+) and CD8(+) T lymphocytes can be distinguished using mAbs. Using two unrelated sets of antibodies, we have now extended this finding to the surface alphabetaTCR.CD3 of seven additional mammalian species (six non-human primates and the mouse). We have also produced data supporting that differential glycosylation of the two main T cell subsets is involved in the observed TCR.CD3 antibody-binding differences in humans. First, we show differential lectin binding to human CD4(+) versus CD8(+) T lymphocytes, particularly with galectin 7. Second, we show that certain lectins can compete differentially with CD3 mAb binding to human primary CD4(+) and CD8(+) T lymphocytes. Third, N-glycan disruption using swainsonine was shown to increase mAb binding to the alphabetaTCR.CD3. We conclude that the differential antibody binding to the surface alphabetaTCR.CD3 complex of primary CD4(+) and CD8(+) T lymphocytes is phylogenetically conserved and associated with differential glycosylation. The differences may be exploited for therapeutic purposes, such as T cell lineage-specific immunosuppression of graft rejection. Also, the impact of glycosylation on CD3 antibody binding requires a cautious interpretation of CD3 expression levels and T cell numbers in clinical diagnosis.
Advances in Experimental Medicine and Biology. 2008 | Pubmed ID: 19065784
Ligand binding to the multichain immune recognition receptors (MIRRs) leads to receptor triggering and subsequent lymphocyte activation. MIRR signal transduction pathways have been extensively studied, but it is still not clear how binding of the ligand to the receptor is initially communicated across the plasma membrane to the cells interior. Models proposed for MIRR triggering can be grouped into three categories. Firstly, ligand binding invokes receptor clustering, resulting in the approximation of kinases to the MIRR and receptor phosphorylation. Secondly, ligand binding induces a conformational change of the receptor. Thirdly, upon ligand-binding, receptors and kinases are segregated from phosphatases, leading to a net phosphorylation of the receptor. In this review, we focus on the homodclustering induced by multivalent ligands, the heterodustering induced by simultaneous binding of the ligand to the MIRR and a coreceptor and the pseudodimer model.
Advances in Experimental Medicine and Biology. 2008 | Pubmed ID: 19065785
Many antigen receptors of the immune system belong to the family of multichain immune recognition receptors (MIRRs). Binding of ligand (antigen) to MIRR results in receptor phosphorylation, triggering downstream signaling pathways and cellular activation. How ligand binding induces this phosphorylation is not yet understood. In this Chapter, we discuss two models exploring the possibility that kinases and phosphatases are intermingled on the cell surface. Thus, in resting state, MIRR phosphorylation is counteracted by dephosphorylation. Upon ligand binding, phosphatases are removed from the vicinity of the MIRR and kinases, such that phosphorylated MIRRs can accumulate (segregation models). In the first model, clustering of MIRRs by multivalent ligand leads to their concentration in lipid rafts where kinases, but not phosphatases, are localized. The second model takes into account that the MIRR-ligandpair needs dose apposition of the two cell membranes, in cases where ligand is presented by an antigen-presenting cell. The intermembrane distance is too small to accommodate transmembrane phosphatases, which possess large ectodomains. Thus, phosphatases become spatially separated from the MIRRs and kinases (kinetic-segregation model).
Advances in Experimental Medicine and Biology. 2008 | Pubmed ID: 19065789
Ligand binding to the T-cell antigen receptor (TCR) evokes receptor triggering and subsequent T-lymphocyte activation. Although TCR signal transduction pathways have been extensively studied, a satisfactory mechanism that rationalizes how the information of ligand binding to the receptor is transmitted into the cell remains elusive. Models proposed for TCR triggering can be grouped into two main conceptual categories: receptor clustering by ligand binding and induction of conformational changes within the TCR. None of these models or their variations (see Chapter 6 for details) can satisfactorily account for the diverse experimental observations regarding TCR triggering. Clustering models are not compatible with the presence of preformed oligomeric receptors on the surface of resting cells. Models based on conformational changes induced as a direct effect of ligand binding, are not consistent with the requirement for multivalent ligand to initiate TCR signaling. In this chapter, we discuss the permissive geometry model. This model integrates receptor clustering and conformational change models, together with the existence of preformed oligomeric receptors, providing a mechanism to explain TCR signal initiation.
Analytical Biochemistry. Sep, 2009 | Pubmed ID: 19482002
We describe the Blue Native (BN)-PAGE retardation assay for the detection of interactions of biomolecules with protein complexes. Potential interactors of proteins are included in the BN gel matrix, resulting in retardation of proteins that interact with the added molecule. After validation using the T-cell antigen receptor, we applied the assay for a general identification of dextran interactors in combination with mass spectroscopy. The proteomic screen revealed triosephosphate isomerase oligomer as a dextran-binding, high M(R) complex.
Target-dependent T-cell Activation by Coligation with a PSMA X CD3 Diabody Induces Lysis of Prostate Cancer Cells
Journal of Immunotherapy (Hagerstown, Md. : 1997). Jul-Aug, 2009 | Pubmed ID: 19483653
Recently, we have described a bispecific PSMA x CD3 diabody with one binding site for the T-cell antigen receptor (TCR-CD3) and another for the Prostate Specific Membrane Antigen (PSMA). It effectively eliminates human prostate cancer cells by redirecting T-lymphocytes in vitro and in vivo. Here, we show that activation of the T-cells and killing of the tumor cells, only occurred when the T-cells were coincubated with PSMA-positive tumor cells and the PSMA x CD3 diabody. Both CD4+ and CD8+ human T-lymphocytes were activated. Surprisingly, they were equally potent in their cytotoxic activity, proliferation, and up-regulation of activation markers. Both, CD4+, and CD8+ T-cells mainly used the perforin-granzyme- based pathway and to a somewhat lesser extent the FasL pathway to lyse tumor cells. When Jurkat T-cells were stimulated with the diabody alone, the TCR-CD3 was not triggered. In contrast, when the diabody was clustered with a secondary antibody the TCR-CD3 was stimulated as detected by Ca(2+)-influx and Erk, IkappaB, and linker of activated T-cell phosphorylation. Clustering of the diabody could also be achieved by the dimeric PSMA antigen expressed on tumor cells. Thus, although the diabody binds to all T-cells, only those in contact with PSMA-expressing cancer cells are activated. In conclusion, the PSMA x CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer.
Current Pharmaceutical Design. 2009 | Pubmed ID: 19860673
Vaccination techniques have developed rapidly over the last several decades from the immunization with live attenuated pathogens to the use of peptide and DNA subunit vaccines, from the use of classical adjuvants to cell-directed delivery. Vaccination techiques are also under investigation for the treatment of tumors and autoimmune diseases. However, profound knowledge of activation mechanisms of the immune cells on a molecular level is prerequisite for a better understanding of the immune response, and for the development of effective immunomodulatory tools. In this review we discuss the models of BCR and TCR activation, and using the example of some vacciantion technologies, we show, how the understanding of these models could help in the design of a new generation of vaccines.
Methods in Molecular Biology (Clifton, N.J.). 2009 | Pubmed ID: 19048218
The T cell antigen receptor (TCR) is a multi-protein complex composed of six different transmembrane subunits, which form complexes of various sizes on the surface of resting T cells. The stoichiometry of the smallest form was recently determined to be alphabetagammaepsilondeltaepsilonzetazeta, whereas that of the larger forms is unknown. The roles of the different forms and their ratios are poorly defined. Biochemical analyses to address these questions must focus on the detergent and the best native conditions to maintain the integrity of the complexes. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a high-resolution native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. Using this powerful approach, the size, subunit composition and the relative abundance of the different TCR forms can be studied. We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE.
Analysis of Novel Phospho-ITAM Specific Antibodies in a S2 Reconstitution System for TCR-CD3 Signalling
Immunology Letters. May, 2010 | Pubmed ID: 20005895
The T cell antigen receptor (TCR-CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR-CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR-CD3 subunit zeta. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of zeta (phospho-zetaY1) and antisera raised against the first and the second phospho-tyrosine of CD3epsilon (phospho-epsilonY1 and phospho-epsilonY2).
Detection of Phosphorylated T and B Cell Antigen Receptor Species by Phos-tag SDS- and Blue Native-PAGE
Immunology Letters. May, 2010 | Pubmed ID: 20005898
Detection of phospho-proteins and differently phosphorylated forms of the same protein are important in understanding cell behaviour. One novel method is Phos-tag SDS-PAGE. A dinuclear Mn(2+) complex that binds to phosphate groups (the Phos-tag) is covalently attached to the polyacrylamide gel matrix. Thus, phosphorylated proteins are retarded in their migration and can be distinguished from their non-phosphorylated counterparts. We applied Phos-tag SDS-PAGE to the analysis of the zeta, CD3epsilon and CD3delta subunits of the T cell antigen receptor (TCR-CD3). Pervanadate stimulation generated six different phospho-zeta and each two different CD3epsilon and CD3delta forms. This corresponds to the phosphorylatable tyrosines on their cytoplasmic tails. The phosphorylation pattern was compatible with random phosphorylation events. Further, we showed that the Phos-tag technology can be applied to Blue Native (BN)-PAGE. This extends the applicability to the analysis of native protein complexes. Upon pervanadate stimulation the TCR-CD3 complex was predominantly detected as two distinct phospho-complexes. In contrast, the B cell antigen receptor (BCR) appeared as one phospho-form. Thus, Phos-tag BN-PAGE is useful for the analysis of different phosphorylation states of multiprotein complexes.
International Immunology. Mar, 2010 | Pubmed ID: 20145007
Antigen binding to the B-cell antigen receptor (BCR) leads to receptor triggering and B-lymphocyte activation. Here, we have probed the molecular requirements for BCR triggering in primary murine B cells using a set of defined soluble haptenated peptides. Bi- and trivalent haptens activated the BCR, as measured by protein phosphorylation, Ca(2+) influx, BCR down-modulation and CD69, CD86 and MHC class II up-regulation. In contrast, four distinct monovalent haptens were ineffective. Next, we used two different anti-idiotypic antibodies, which bind to the antigen-combining site of the BCR. Again, monovalent Fab fragments were ineffective, whereas bivalent antibodies could stimulate the BCR. These findings are compatible with ligand-induced clustering of monomeric BCRs or re-organization of BCR complexes within pre-formed BCR oligomers. Lastly, an increase in the valency of the haptenated peptides improved the activation potential, whereas variations in the distance between two haptens had no effect. This finding contributes to understand how the immune system can efficiently recognize structurally diverse antigens but still discriminate between foreign and self.
Innate, Antigen-independent Role for T Cells in the Activation of the Immune System by Propionibacterium Acnes
European Journal of Immunology. Sep, 2010 | Pubmed ID: 20690177
Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL-12-mediated IFN-gamma production. We show that P. acnes elicits IL-12p40 and p35 mRNA expression in macrophages, and IFN-gamma mRNA in liver CD4(+) T cells and NK cells. After priming with P. acnes, CD4(+) T cells serve as the major IFN-gamma mRNA source. In the absence of CD4(+) T cells, CD8(+) T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN-gamma to induce the P. acnes-driven immune effects. Moreover, in the absence of alpha beta T cells, gamma delta T cells also enable the development of strongly enhanced TNF-alpha and IFN-gamma responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T-cell types, independent of their antigen specificity, exert NK-cell-like functions, which contribute decisively to the activation of the innate immune system.
FEBS Letters. Dec, 2010 | Pubmed ID: 20828560
The T-cell antigen receptor (TCR) is a multisubunit transmembrane complex that mediates the antigen-specific activation of T cells. Using a variety of techniques, several research groups have shown that TCRs are at least partially pre-clustered before antigen binding. These new findings are contradictory to the "classical" view, according to which TCRs are randomly distributed on the cell surface and only associate upon antigen binding. In this review we try to answer the following questions: What are the experimental evidences for the existence of pre-clustered TCRs? How can the TCR pre-clusters be activated upon antigen binding? Which functional consequences for T-cell activation arise from the pre-clustering of TCRs.
Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-colour IP-FCM As an Input for Kinetic Modelling of Signalling Networks
PloS One. 2011 | Pubmed ID: 21829558
To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success.
Immunity. Aug, 2011 | Pubmed ID: 21867922
In the current issue of Immunity, Martínez-Martín et al. (2011) describe the central supramolecular activation cluster (cSMAC) as a site of clathrin-independent T cell receptor (TCR) internalization and trogocytosis. Further, they identify small Rho GTPases TC21 and RhoG as key mediators of these processes.
Increased Sensitivity of Antigen-experienced T Cells Through the Enrichment of Oligomeric T Cell Receptor Complexes
Immunity. Sep, 2011 | Pubmed ID: 21903423
Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.
A Leaky Mutation in CD3D Differentially Affects αβ and γδ T Cells and Leads to a Tαβ-Tγδ+B+NK+ Human SCID
The Journal of Clinical Investigation. Oct, 2011 | Pubmed ID: 21926461
T cells recognize antigens via their cell surface TCR and are classified as either αβ or γδ depending on the variable chains in their TCR, α and β or γ and δ, respectively. Both αβ and γδ TCRs also contain several invariant chains, including CD3δ, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3δ-deficient patients described to date showed a complete block in T cell development. However, CD3δ-KO mice have an αβ T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in αβ but not in γδ T cell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3δ proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant αβ T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of αβ T cells, surface TCR expression was more reduced in γδ than in αβ T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3δ requirements for αβ versus γδ T cell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.