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Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.

CD94/NKG2 receptors on mouse NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by fetal NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas mouse L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the alpha 1 and alpha 2 domains of Qa-1b and the alpha 3 domain of H-2Db. This allowed the use of a monoclonal antibody directed against H-2Db whilst retaining the peptide-binding groove of Qa-1b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with beta 2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with beta 2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1b-presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1b/Db molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2Dk, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.

Single amino acid disparities in MHC class I molecules can elicit transplantation responses. Since beta 2 microglobulin (beta 2m) is noncovalently associated with class I antigens on the cell membrane we investigated whether the single amino acid polymorphism at position 85 (Asp-->Ala) in the mouse beta 2m molecule can cause skin graft rejection. A B2mb transgene was introduced into CBA(B2ma) mice which subsequently expressed both forms of beta 2m. Skin from these CBA beta 2mb transgenic mice was not rejected by the parental CBA strain. Previous studies showed that cytotoxic T lymphocyte (CTL) responses directed against beta 2mb use H2Kb as a restriction element. We therefore produced mice expressing H2Kb and H2Ab as well as beta 2mb by crossing CBA.beta 2mb mice with either CBA.Kb (CBK) transgenic mice or C3H.SW mice and used these as skin graft donors for beta 2mb negative littermates. In both cases rejection of transgenic skin only occurred when mice had received both a beta 2mb graft and an H2-disparate allograft lying adjacent in the same site. Introduction of the male specific antigen, H-Y, as a helper determinant did not result in rejection of beta 2mb skin. Neither did two CTL determinants (P91A and beta 2mb) on the same graft complement one another to elicit a transplantation response. Prior immunisation with tissues expressing the beta 2m disparity alone did not generate in vivo or in vitro beta 2mb-specific CTL responses, suggesting that this single amino acid difference is not sufficient to elicit a CTL or helper T cell response.

Participation of transmembrane (TM) and glycosyl-phosphatidylinositol (GPI) anchored H-2Db molecules in antigen presentation and thymic selection events was investigated using transgenic mice. Both GPI-Db and TM-Db can efficiently present H-Y antigen, influenza and lymphocytic choriomeningitis virus (LCMV) peptides to primed cytotoxic, H-2Db-restricted T cells. Transgenic mice expressing GPI-Db, although unable to reject TM-Db skin grafts, nevertheless generate secondary CTL responses which can lyse TM-Db-bearing targets, indicating that GPI-Db mice fail to delete all TM-Db-reactive T cells. Furthermore, double-transgenic mice bearing GPI-Db and a T-cell receptor (TcR) for H-2Db+LCMV do not positively select receptor positive, CD8+CD4- T cells. This paradoxical behaviour of GPI-Db molecules suggests that the structural requirements for antigen presentation and thymic selection by class I molecules are different and may explain why GPI-linked class I molecules, such as Qa-2, do not appear to function as restriction elements in vivo.

KT16, like other anti-Thy-1 antibodies, induces T cell aggregation. Protein A-gold labelling shows the antibody to be concentrated along areas of intercellular contacts. Electron micrographs of KT16 treated T cells reveal a consistent type of junction between the cells. We demonstrate that this type of cell junction is Thy-1 specific, is predominantly the property of antibodies directed against a particular epitope, and is distinct from cellular aggregation caused by concanavalin A or anti-CD3 antibodies. The degree of adhesiveness induced by different anti-Thy-1 antibodies is related to their mitogenic capacity.