No abstract

Although rarely elicited during natural human infection, the most broadly neutralizing antibodies (BNAbs) against diverse human immunodeficiency virus (HIV)-1 strains target the membrane-proximal ectodomain region (MPER) of viral gp41. To gain insight into MPER antigenicity, immunogenicity, and viral function, we studied its structure in the lipid environment by a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and surface plasmon resonance (SPR) techniques. The analyses revealed a tilted N-terminal alpha helix (aa 664-672) connected via a short hinge to a flat C-terminal helical segment (675-683). This metastable L-shaped structure is immersed in viral membrane and, therefore, less accessible to immune attack. Nonetheless, the 4E10 BNAb extracts buried W672 and F673 after initial encounter with the surface-embedded MPER. The data suggest how BNAbs may perturb tryptophan residue-associated viral fusion involving the mobile N-terminal MPER segment and, given conservation of MPER sequences in HIV-1, HIV-2, and SIV, have important implications for structure-guided vaccine design.

Thymus-derived lymphocytes protect mammalian hosts against virus-or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric ␣␤, CD3⑀␥, CD3⑀␦, and CD3 subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3⑀ lobe that they approach diagonally, adjacent to the lever-like C␤ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3⑀ and CD3␥ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (ϳ50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands. The T cell receptor (TCR)3 is a multimeric transmembrane complex composed of a disulfide-linked antigen binding clonotypic heterodimer (␣␤ or ␥␦) in non-covalent association with the signal-transducing CD3 subunits (CD3⑀␥, CD3⑀␦, and CD3) (reviewed in Ref. 1). TCR signaling via CD3 dimers evokes T cell lineage commitment and repertoire selection during development, maintains the peripheral T cell pool, and further differentiates naïve T cells into effector or memory cell populations upon immune stimulation (2-5). The interaction between an Fab-like ␣␤ TCR heterodimer and an antigenic peptide bound to a major histocompatibility complex molecule (pMHC) initiates a cascade of downstream signaling events via the immunoreceptor tyrosine-based activation motif elements in the cytoplasmic tails of the associated CD3 subunits (6 -9). The length of these CD3 cytoplasmic tails is substantial, relative to those of the TCR ␣ and ␤ chains (6, 7).How recognition of pMHC by a weakly interacting (ϳ1-100 M K d ) clonotypic heterodimer on the T cell surface evokes intracellular signaling via the adjacent CD3 components remains undefined (1). Solution structures of CD3⑀␥ and CD3⑀␦ heterodimers reveal a unique side-to-side hydrophobic interface with conjoined ␤-sheets involving the G-strands of the two Ig-like ectodomains of the pair (10, 11). The squat and rigid CD3 connecting segments contrast sharply with the long and flexible TCR ␣ and ␤ connecting peptides linking their respective constant domains to the transmembrane segments.To investigate the basis of signal transduction involving the ectodomain components within the TCR membrane complex,...

A series of monoclonal antibodies was used to define three discrete stages of human intrathymic T-cell differentiation. The earliest stage was confined to <10% of thymocytes, which were.reactive with both OKT9 and OKT1O. Subsequently, approximately 70% of human thymocytes acquired a thymocyte-restricted antigen, OKT6, lost OKT9 antigen, and expressed reactivity with OKT4 and OKT5. These last two monoclonal antibodies were previously shown to define inducer (helper) and cytotoxic/suppressor populations, respectively, in peripheral blood. The OKT4+, OKT5+, OKT6+ "common" thymocyte population represents the majority of thymocytes and accounts for more than 70% of thymocytes. With further maturation, thymocytes lose OKT6 reactivity, segregate into OKT4+ and OKT5+ subsets, and acquire reactivity with OKT3 (and OKT1). This latter stage corresponds to the more functionally mature subset. The possible relationship of acute lymphoblastic leukemia of T-cell lineage to these proposed stages of intrathymic differentiation was determined. Analysis of25 tumor populations showed that 21 could be related to one or another differentiative stage. The majority (15/21) were derived from an early thymocyte or prothymocyte subpopulation, 5/25 were derived from a common thymocyte subpopulation, and 1/25 was derived from a mature (OKT3+) subpopulation. These data suggest that is it now possible to define stages of T-cell differentiation that can be related to T-cell malignancies in humans.The importance of a thymic microenvironment in the differentiation and functional maturation of T cells has been demonstrated in several species. Moreover, profound changes in cell-surface antigens mark the various stages of T-cell ontogeny (1-7). For (12). These thymcytes were the most functionally mature and probably ready for peripheral exportation. Human peripheral T cells also consist of functionally distinct subsets (13)(14)(15)(16)(17)(18)(19).In the present study, we used a series of monoclonal antibodies reactive selectively with subpopulations of human thymocytes and inducer and suppressor T-cell subsets in order to further define stages of thymic differentiation (18, 19). We show that three major stages of intrathymic differentiation exist and that the majority of tumor populations from patients with acute lymphoblastic leukemia of T-cell lineage (T-ALL) can be shown to