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B cells surveil the body for foreign matter using their surface-expressed B cell antigen receptor (BCR), a tetrameric complex comprising a membrane-tethered antibody (mIg) that binds antigens and a signaling dimer (CD79AB) that conveys this interaction to the B cell. Recent cryogenic electron microscopy (cryo-EM) structures of IgM and IgG isotype BCRs provide the first complete views of their architecture, revealing that the largest interaction surfaces between the mIg and CD79AB are in their transmembrane domains (TMDs). These structures support decades of biochemical work interrogating the requirements for assembly of a functional BCR and provide the basis for explaining the effects of mutations. Here we report a focused saturating mutagenesis to comprehensively characterize the nature of the interactions in the mIg TMD that are required for BCR surface expression. We examined the effects of 600 single-amino-acid changes simultaneously in a pooled competition assay and quantified their effects by next-generation sequencing. Our deep mutational scanning results reflect a feature-rich TMD sequence, with some positions completely intolerant to mutation and others requiring specific biochemical properties such as charge, polarity or hydrophobicity, emphasizing the high value of saturating mutagenesis over, for example, alanine scanning. The data agree closely with published mutagenesis and the cryo-EM structures, while also highlighting several positions and surfaces that have not previously been characterized or have effects that are difficult to rationalize purely based on structure. This unbiased and complete mutagenesis dataset serves as a reference and framework for informed hypothesis testing, design of therapeutics to regulate BCR surface expression and to annotate patient mutations. [ABSTRACT FROM AUTHOR]

B cells from patients with common variable immunodeficiency (CVID) who are heterozygous for transmembrane activator and CAML interactor (TACI) mutation C104R, which abolishes ligand binding, fail to produce Igs in [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.08.044 Byline: Matthew E. Call (1)(2), Jason R. Schnell (3), Chenqi Xu (1), Regina A. Lutz (1), James J. Chou (3), Kai W. Wucherpfennig (1)(2) Abstract: The T cell receptor (TCR) [alpha][beta] heterodimer communicates ligand binding to the cell interior via noncovalently associated CD3[gamma]E, CD3[delta]E, and I[paragraph]I[paragraph] dimers. While structures of extracellular components of the TCR-CD3 complex are known, the transmembrane (TM) domains that mediate assembly have eluded structural characterization. Incorporation of the I[paragraph]I[paragraph] signaling module is known to require one basic TCR[alpha] and two I[paragraph]I[paragraph] aspartic acid TM residues. We report the NMR structure of the I[paragraph]I[paragraph].sub.TM dimer, a left-handed coiled coil with substantial polar contacts. Mutagenesis experiments demonstrate that three polar positions are critical for I[paragraph]I[paragraph] dimerization and assembly with TCR. The two aspartic acids create a single structural unit at the I[paragraph]I[paragraph] interface stabilized by extensive hydrogen bonding, and there is evidence for a structural water molecule (or molecules) within close proximity. This structural unit, representing only the second transmembrane dimer interface solved to date, serves as a paradigm for the assembly of all modules involved in TCR signaling. Author Affiliation: (1) Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA (2) Program in Immunology, Harvard Medical School, Boston, MA 02115, USA (3) Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 13 June 2006; Revised 23 July 2006; Accepted 11 August 2006 Article Note: (miscellaneous) Published: October 19, 2006

The atomic-resolution NMR structure of the membrane-embedded portion of a critical signaling component of the T cell receptor (TCR)-CD3 complex, the [zeta][zeta] homodimer is presented. This structural unit representing only the second trans-membrane dimer interface serves as a paradigm for the assembly of all modules involved in TCR signaling.

Research has been conducted on T cell receptors. The structure of these receptors has been investigated and the results demonstrate that the three-helix rearrangement represents the organizing principle for receptor structure formation.

The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligandinduced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eighthelix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δe:CD3γe:ζζ complexes. These findings demonstrate that the eight TCR-CD3 subunits form a compact and precisely organized structure within themembrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling. [ABSTRACT FROM AUTHOR]

Assembly of the I cell receptor (ICR) with its dimeric signaling modules, CD3δϵ, CD3γϵ, and ζζ is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3δϵ and CD3γϵ contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3δϵ and CD3γϵ demonstrated parallel β-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3δϵ or CD3γϵ dimerization. CD3δϵ assembled with a TCRα mutant that lacked both immunoglobulin domains, but shortening of the TCRα connecting peptide reduced assembly, consistent with membrane-proximal TCRα-CD3δϵ interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane- proximal domains of TCRα-CD3δϵ and TCRβ-CD3γϵ. [ABSTRACT FROM AUTHOR]

Simple Summary: The development and refinement chimeric antigen receptor (CAR)-T cell immunotherapy has significantly improved the prognosis of patients with B cell malignancies. Severe treatment related toxicities however remain a significant challenge for the field. This perspective reviews 17 clinical trials of the most widely used anti-CD19 (FMC63) CAR-T cell therapies, with the aim of dissecting the contribution of the structural and costimulatory domains of the CAR to clinical outcomes and toxicities. The CD28 structural hinge and transmembrane CAR domains are highlighted as strongly associated with both clinical efficacy and severe toxicity. This perspective supports further investigation into the structural CAR domains for improved CAR design and safer CAR-T cell therapies. Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of B cell malignancies, improving patient survival and long-term remission. Nonetheless, over 50% of patients experience severe treatment-related toxicities including cytokine release syndrome (CRS) and neurotoxicity. Differences in severity of toxic side-effects among anti-CD19 CARs suggest that the choice of costimulatory domain makes a significant contribution to toxicity, but comparisons are complicated by additional differences in the hinge and transmembrane (TM) domains of the most commonly used CARs in the clinic, segments that have long been considered to perform purely structural roles. In this perspective, we examine clinical and preclinical data for anti-CD19 CARs with identical antigen-binding (FMC63) and signalling (CD3ζ) domains to unravel the contributions of different hinge-TM and costimulatory domains. Analysis of clinical trials highlights an association of the CD28 hinge-TM with higher incidence of CRS and neurotoxicity than the corresponding sequences from CD8, regardless of whether the CD28 or the 4-1BB costimulatory domain is used. The few preclinical studies that have systematically varied these domains similarly support a strong and independent role for the CD28 hinge-TM sequence in high cytokine production. These observations highlight the value that a comprehensive and systematic interrogation of each of these structural domains could provide toward developing fundamental principles for rational design of safer CAR-T cell therapies. [ABSTRACT FROM AUTHOR]