Development and applications of surface-linked single chain antibodies against T-cell antigens.

In this report the use of surface-linkage to expand the potential experimental and therapeutic applications of single chain antibody (scFv) constructs is reviewed. A strategy for the generation and functional characterization of surface-linked scFvs that bind selectively to the T-cell proteins CD3epsilon, CD28, and CD152 (CTLA-4) is described in detail. Experimental examples are provided of the use of these constructs to study the positive and negative regulation of T-cell activation and to manipulate the in vivo immunogenicity of tumor cells. In addition, a novel system for Simultaneous T-cell Activation and Retroviral Transduction (START) is described in which retroviral packaging cells are rendered mitogenic for T lymphocytes by combined expression of surface-linked scFvs. Finally, the use of random mutagenesis and yeast surface display to increase the affinity and functional efficacy of scFv constructs is demonstrated.

Anergic T lymphocytes selectively express an integrin regulatory protein of the cytohesin family.

It has been proposed that the maintenance of T cell anergy depends on the induction of negative regulatory factors. Differential display of reverse transcribed RNA was used to identify novel genes that might mediate this function in anergic Th1 clones. We report that anergic Th1 clones do indeed express a genetic program different from that of responsive T cells. Moreover, one gene, the general receptor of phosphoinositides 1 (GRP1), was selectively induced in anergic T cells. The GRP1, located in the plasma membrane, regulated integrin-mediated adhesion and was invariably associated with unresponsiveness in multiple models of anergy. T cells expressing retrovirally transduced GRP1 exhibited normal proliferation and cytokine production. However, GRP1-transduced T cells were not stable and rapidly lost GRP1 expression. Thus, although GRP1 may not directly mediate T cell anergy, it regulates cell expansion and survival, perhaps through its integrin-associated activities.

RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation.

A novel T cell-specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon gamma, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.

Cloning of ATAC, an activation-induced, chemokine-related molecule exclusively expressed in CD8+ T lymphocytes.

A cDNA clone, designated ATAC, was isolated from a collection of human T cell activation genes. Analysis of tissue distribution determined that ATAC mRNA of approximately 0.9 kb is exclusively expressed in activated CD8+ T cells. Induction of the ATAC gene requires stimulation by both phorbol 12-myristate 13-acetate and Ca2+ ionophore A23187 ("two-signal gene") and is fully abrogated by the immunosuppressive agent cyclosporin A. Upon stimulation, ATAC mRNA is detectable within 30 min, maximal expression is seen after 4 h. The polypeptide encoded by the open reading frame of ATAC mRNA is 114 amino acids long with a calculated M(r) of 12.52 kDa. The structural features predict the cleavage and secretion of a mature ATAC protein of approximately 10 kDa from the 12.52-kDa precursor. ATAC is highly similar to a very recently identified murine molecule designated lymphotactin both at the cDNA (73.8% identity) and the protein (61.4% identity) levels, and related to members of the C-C and C-X-C chemokine families. Two variants of the ATAC protein were expressed and tested for chemotaxis and Ca2+ release on a variety of target cells. The ATAC gene was located to chromosome 1q23.

A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation.

TRAP is a tumor necrosis factor (TNF)-related, 33-kDa type II transmembrane protein almost exclusively expressed on the surface of activated CD4+ T lymphocytes. Interaction of TRAP with CD40 on B cells is of paramount importance for immunoglobulin class switching and subsequent synthesis of IgG, IgA or IgE in vivo. We now provide evidence that activated T cells not only express cell membrane-associated TRAP but also a soluble form of TRAP (sTRAP). After generating monoclonal antibodies against TRAP and establishing a TRAP-specific enzyme-linked immunosorbent assay we were able to detect substantial amounts of sTRAP in the supernatants of activated T cells. The onset and rate of sTRAP release was found to parallel the expression of TRAP on the cell surface. sTRAP, an 18-kDa protein, is generated by proteolytic processing of full-length TRAP in an intracellular compartment. Starting with methionine 113 of full-length TRAP, sTRAP lacks the transmembrane region and a part of the extracellular domain but contains the entire TNF-alpha homology region and can, therefore, bind to CD40. Like other members of the TNF superfamily (e.g. TNF-alpha, Fas/APO-1 ligand), TRAP thus has the potential to be biologically active not only in a transmembrane form but also as a soluble molecule.

Biased T cell receptor V gene usage in rheumatoid arthritis. Oligoclonal expansion of T cells expressing V alpha 2 genes in synovial fluid but not in peripheral blood.

OBJECTIVE: To analyze the T cell receptor (TCR) variable (V) region gene usage in the rheumatoid joint. METHODS: Monoclonal antibodies (MAb) were used to determine the prevalence of selected V elements on T cells in synovial fluid (SF) from rheumatoid arthritis (RA) patients and in peripheral blood (PB) from RA patients and normal controls. V alpha 2-positive PB and SF T cells from 1 patient were cloned by immediate limiting-dilution and analyzed by restriction mapping. RESULTS: In 9 of 14 RA patients, SF was enriched in at least 1 of the selected V elements, compared with PB. TCR genes of the V alpha 2 family were the most frequently overrepresented in the SF (4 patients). The expanded V alpha 2-positive cells were oligoclonal in SF but heterogeneic in PB. CONCLUSION: Our data showing biased and clonally restricted TCR elements in the rheumatoid joint indicate major histocompatibility complex-restricted antigen recognition, rather than a "superantigen," in the pathogenesis of RA.

Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM.

X chromosome-linked immunodeficiency with hyper-IgM (HIGM1, MIM number 308230) is a rare disorder characterized by recurrent bacterial infections, very low or absent IgG, IgA and IgE, and normal to increased IgM and IgD serum levels. HIGM1 has been suggested to result from ineffective T-cell help for B cells. We and others have identified a novel, TNF-related activation protein (TRAP) that is exclusively expressed on the surface of stimulated T cells. TRAP, a type II transmembrane protein of M(r) 33,000, is the physiological ligand for CD40 (refs 5-8). Crosslinking of CD40 on B cells induces, in the presence of lymphokines, immunoglobulin class switching from IgM to IgG, IgA or IgE. Mapping of the TRAP gene to the X-chromosomal location q26.3-q27.1 (ref. 6) suggested a causal relationship to HIGM1, which had previously been assigned to Xq26 (refs 12-14). Here we present evidence that point mutations in the TRAP gene give rise to nonfunctional or defective expression of TRAP on the surface of T cells in patients with HIGM1. The resultant failure of TRAP to interact with CD40 on functionally intact B cells is responsible for the observed immunoglobulin isotype defect in HIGM1.

Cloning of TRAP, a ligand for CD40 on human T cells.

A cDNA clone, designated TRAP (TNF-related activation protein) was isolated from a collection of T cell activation genes. The polypeptide encoded by a mRNA of approx. 2.3 kb is 261 amino acids long with a calculated M(r) of 29.3 kDa. The structural features predict a type II transmembrane protein, but are also compatible with a secreted form. TRAP is highly similar to an identified murine CD40 ligand both at the cDNA (82.8% identity) and the protein (77.4% identity) levels, and related to tumor necrosis factor/lymphotoxin. Expressed in a murine myeloma, TRAP was identified as a ligand for CD40 by binding to a soluble CD40 construct. TRAP mRNA is expressed in a T cell-specific fashion with a maximum at 8 h after stimulation. The TRAP gene is located in the q26.3-q27.1 region of the X chromosome.

Oligoclonal T cells in rheumatoid arthritis: identification strategy and molecular characterization of a clonal T-cell receptor.

Immunodominant antigens in rheumatoid arthritis (RA) should induce an expansion of T cells bearing a corresponding T-cell receptor (TCR). We therefore analysed the TCR repertoire at the site of inflammation using two fundamentally different strategies. The total TCR repertoire was examined by generating 'representative' T-cell clone panels, which were subsequently tested for clonality by restriction mapping of the TCR beta gene locus. No clonality was detected in large T-cell clone panels generated with cells from three patients. However, when we selectively analysed the TCR repertoire of in vivo pre-activated, interleukin-2 (IL-2)-responsive T cells, significant T-cell/TCR clonality was found in 2 out of 4 patients. The clonal T cells represented a minority of the total T-cell population with an estimated frequency of 1 in 300 to 1 in 1000 cells. Molecular characterization of a clonal TCR and the use of a specific TCR V beta MoAb ruled out an over-representation of T cells bearing the same V beta element in the total T-cell population, rendering the involvement of super-antigens in the induction of T-cell clonality in this case unlikely.