SummaryWe previously showed that H-2Ka-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasraodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human dass I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2K a. We first sequenced the TCR o~ and/3 genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR c~ and/3 cDNA junctional regions of 23 independent H-2K drestricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of Vet, Jo~, V~ and J/3 segments, and in terms of length and sequence of the CDR3 cx and/3 loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2K d selects CTLs that bear TCILs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.

SummaryThe major histocompatibility complex (MHC) class I molecules expressed at the cell surface are associated with a large number of different peptides so that the density of a given MHC-peptide complex is relatively low. Here we describe the properties of MHC class I molecules genetically attached to a single antigenic peptide. Cells expressing these fusion proteins are recognized by T cells specific for the particular MHC-peptide complex. Coculture of naive splenocytes with cells expressing these MHC-peptide fusion proteins and the B7.1 antigen allows the induction of primary cytotoxic T lymphocytes (CTL) in vitro. Injection of these cells into naive mice enhances the frequency of specific CTL precursors and protects against a subsequent challenge with a tumor or a virus bearing the antigenic peptide. Soluble MHC-peptide fusions were also produced in which all three components, that is, the heavy chain, B2-microglobulin and the peptide, have fused into a single-chain protein. The availability of MHC class I molecules bound to a single peptide provides valuable tools for the manipulation of CTL responses and the analysis of the selection processes in the thymus.

SummaryPeptides derived from HLA-Cw3 and HLA-A24 within region 170-179 differ by a single substitution, at position 173, and are both presented by the dass I major histocompatibility complex molecule H-2K a for recognition by routine cytolytic T lymphocytes (CTLs). As a first approach to understand the way T cell receptors (TCRs) intact with the HLA peptides, we have analyzed the TCR selection by, and recognition of, the two HLA antigenic sites. First, we have compared the TCR repertoires selected by HLA-Cw3 and HLA-A24, not only by sequencing the TCRs carried by CTL clones isolated and grown in vitro, but also by analyzing the TCRs expressed in vivo by peritoneal exudate lymphocytes from immune animals. Second, we have compared the TCR crossrecognition of HLA-A24 by CTLs selected by HLA-Cw3 with that of HLA-Cw3 by CTLs selected by HLA-A24. The combined analysis of TCR selection by and recognition of these two related HLA antigenic sites provides evidence that the TCR/3 junctional regions interact with the amino-terminal part of the HLA peptides. CTLs recognize antigenic peptides presented by class I MHC molecules (1, 2). The specificity of this recognition is conferred by the TCR ot//~ (3, 4). Whereas the structure of several class I MHC molecules, and that of a class I MHC-peptide complex, were determined by crystallographic studies (5), such information is not available yet for the TCR. However, a model of the TCR ol/B tertiary structure was proposed, based on its homology with Igs (6). In any case, experimental evidence is lacking so far to support a topology of class I MHC-peptide recognition by TCRs.We previously reported that DBA/2 mice could mount a CTL response towards two related antigens, HLA-Cw3 and HLA-A24, in the context of the same murine class I MHC molecule, H-2K a, and that a fraction of the CTLs raised in response to either HLA antigen was not reactive to the second HLA antigen (7). The optimal synthetic peptides recognized by these specific CTLs, corresponding to the region 170-179, differ by a single nonconservative substitution at position 173 and bind their common restriction element H-2K d with a similar affinity (Table 1) (8--12). Regions 170-179 of the related HLA molecules A2 and A3 are identical to HLA-A24 and can also be recognized by H-2Ka-restricted CTLs (13).We recently showed that the TCRs carried by H-2K arestricted CTLs specific for the Cw3 170-179 peptide were very limited in primary structure (14). They were encoded by few germline gene segments: a single VB segment ORB10), a singleJo~ segment (Jc~pHDS58), few Vot segments (mainly Vc~3,4,8), and few J/~ segments (mainly J/31.2, 1.4, 2.3). Thdrjunctional CDR3 c~ and/3 loops also displayed limited diversity: a single length of nine and six amino adds, respectively, a conserved non-V-, non-J-encoded glydne amino acid at position 97 in the CDR3/~, and a high occurrence of non-J-encoded glycine or charged amino acids at positions 94 and 95 in the CDR3 c~. As a first approach to understand the topology of interaction of TCRs with...

No abstract

Transport of an immunogenic self-peptide from the second domain of the mouse major histocompatibility complex (MHC) H-2Kd class I molecule is blocked at the TAP1-TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1-TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self-peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies.