Rebecca E. Hussey

An alternative pathway of T-cell activation: A functional role for the 50 kd T11 sheep erythrocyte receptor protein

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11(1), the SRBC binding site expressed on all T lymphocytes and thymocytes; T11(2), an epitope unrelated to the SRBC binding site but with a similar distribution; and T11(3), a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11(2) and T11(3) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.

Antigen recognition by human T lymphocytes is linked to surface expression of the T3 molecular complex

Four distinct surface molecules on human T cells are defined by the monoclonal antibodies anti-T1, anti-T3 (anti-T3A), anti-T11 and anti-T12. Following cell binding, anti-T3 (anti-T3A) and anti-T1 induce independent modulation of their respective ligands, whereas anti-T11 and anti-T12 do not. To explore the biological consequences of this modulation, we used cloned populations of T4 and T8 cytotoxic T lymphocytes. Anti-T3 (anti-T3A), but not anti-T1, inhibits cytotoxic T lymphocyte effector function by T4 and T8 clones as well as antigen-specific T cell recognition. The latter is not secondary to a generalized inhibitory effect since responsiveness to interleukin 2 is maintained. Moreover, after modulation, cytotoxic T lymphocytes recover cytolytic function in parallel with reexpression of surface T3 molecules. We provide evidence for a direct linkage between antigen recognition by T lymphocytes and surface expression of the T3 molecular complex.

The human T cell receptor: appearance in ontogeny and biochemical relationship of α and β subunits on IL-2 dependent clones and T cell tumors

Abstract The human T cell receptor for antigen (Ti) has recently been identified on IL-2 dependent T cell clones as a 90 kd disulfide-linked heterodimer comprised of one 49–51 kd alpha (α) and one 43 kd beta (β) chain. These subunits are noncovalently associated with a monomorphic 20–25 kd T3 molecule. Here, we produce monoclonal antibodies to a human tumor (REX) derived from an earlier stage of thymic differentiation in order to determine whether clonotypic structures are expressed and to define the ontogeny of Ti. The results of SDS-PAGE and peptide map analyses indicate that an homologous T3-associated heterodimer is synthesized and expressed by REX. This glycoprotein shares several peptides in common with clonotypic structures on an IL-2 dependent T cell clone. In addition, similar Ti related molecules appear during intrathymic ontogeny in parallel with surface T3 expression. The latter findings provide the structural basis for the immunological competence observed exclusively within the T3+ thymocyte compartment.

Structural Basis of CD8 Coreceptor Function Revealed by Crystallographic Analysis of a Murine CD8αα Ectodomain Fragment in Complex with H-2Kb

Abstract The crystal structure of the two immunoglobulin variable–like domains of the murine CD8αα homodimer complexed to the class I MHC H-2K b molecule at 2.8 A resolution shows that CD8αα binds to the protruding MHC α3 domain loop in an antibody-like manner. Comparison of mouse CD8αα/H-2K b and human CD8αα/HLA-A2 complexes reveals shared as well as species-specific recognition features. In both species, coreceptor function apparently involves the participation of CD8 dimer in a bidentate attachment to an MHC class I molecule in conjunction with a T cell receptor without discernable conformational alteration of the peptide or MHC antigen-presenting platform.

Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic αβ heterodimeric structure (Ti) non‐covalently associated with the monomorphic CD3 signaling components. A crystal structure of an αβ TCR‐anti‐TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cβ domain, situated beneath the Vβ domain. This loop, along with the partially exposed ABED β sheet of Cβ, and glycans attached to both Cβ and Cα domains, forms a cavity of sufficient size to accommodate a single non‐glycosylated Ig domain such as the CD3ϵ ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre‐TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module.

Identification of a common docking topology with substantial variation among different TCR–peptide–MHC complexes

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.

A cdc15‐like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2‐triggered adhesion

A human CD2 cytoplasmic tail‐binding protein, termed CD2BP1, was identified by an interaction trap cloning method. Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing. Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C‐terminal SH3 domain. Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300–309), its association is augmented markedly by the CD2BP1 N‐terminal segment. Upon ligand‐induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co‐localizing with CD2. In turn, CD2‐stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)‐PEST to CD2. These findings offer the first molecular view into the control processes for T cell adhesion.

Peptide-Induced Negative Selection of Thymocytes Activates Transcription of an NF-ΚB Inhibitor

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

Expression, Purification, and Functional Analysis of Murine Ectodomain Fragments of CD8αα and CD8αβ Dimers

Soluble mouse CD8αα and CD8αβ dimers corresponding to the paired ectodomains (CD8f) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8ααf for H-2Kb as measured by BIAcore revealed a ∼65 μm K d , similar to that of CD8αβf. Consistent with this result, CD8ααf as well as CD8αβf blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8αβ versus CD8αα co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8αα, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.

A rapid method for separating functionally intact human T lymphocytes with monoclonal antibodies

Abstract In the present study, we exploited a simple adherence technique utilizing monoclonal antibodies to separate highly purified human T-cell subpopulations. Unfractionated T cells were reacted with one of two hybridoma antibodies, termed anti-T4 and anti-T8. The anti-T4 reactive (T4+) subset was previously shown to define the human inducer T-cell subset, whereas the T8+ subset contained suppressor T cells. The monoclonal antibody-coated T cells were separated into adherent and nonadherent fractions based on binding to plastic dishes coated with affinity-purified goat antimouse immunoglobulin. This fractionation resulted in separation of lymphocytes into T4+ and T8+ T-cell subsets by either positive or negative selection. Reanalysis by indirect immunofluorescence indicated that they were ≧95% pure. Moreover, in vitro studies showed that the separated populations were functionally intact with regard to proliferative responses and regulatory functions. This procedure should be useful for efficiently and inexpensively separating lymphocytes based on monoclonal antibody binding.