Single-chain VαVß T-cell receptors function without mispairing with endogenous TCR chains.

Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vß single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

Development of a novel strategy for engineering high-affinity proteins by yeast display.

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Psychosocial differences between male and female alcoholics.

This study was designed to further explore and compare psychological characteristics in male and female alcoholics at an inpatient treatment program of an urban general hospital. In addition to comparing male and female variables, the study also sets out to determine whether there is any correlation between depression and female alcoholics.