Escape of mouse mastocytoma P815 after nearly complete rejection is due to antigen-loss variants rather than immunosuppression.

Even though mastocytoma P815 often undergoes a nearly complete rejection in syngeneic mice, the tumor cells almost always escape to form progressive tumors. We found that this was not due to the establishment of an immunosuppressed state because genetically marked P815 cells, that were injected in mice where tumor escape was occurring, were readily rejected. An analysis of escaping tumor cell populations with anti-P815 cytolytic T lymphocyte (CTL) clones showed the presence of stable resistant variants. Using antigen-loss variants found in escaping populations or selected in vitro with CTL clones, we were able to define four different tumor-associated antigenic specificities, each recognized by a specific CTL clone. One of these specificities was absent from all escaping tumor cells and another had been lost by some of them.

H-2Kd-restricted antigenic peptides share a simple binding motif.

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

Synthetic peptides as antigens and competitors in recognition by H-2-restricted cytolytic T cells specific for HLA.

The specificity of peptide recognition by a number of Kd-restricted CTL clones specific for HLA-CW3 or HLA-A24 was investigated. The CTL clones were derived from DBA/2 (H-2d) mice immunized with syngeneic P815 mouse cells transfected with genes encoding HLA-CW3 or HLA-A24 class I molecules. We had previously shown that CTL clones that lysed P815-CW3 transfectant target cells could lyse P815 (HLA-) target cells incubated with synthetic CW3 peptides corresponding to the COOH-terminal end of the alpha 2 domain. In the present study, we found that Kd-restricted CTL clones that lysed P815-A24 transfectant target cells recognized a synthetic peptide from the same region (residues 170-182) of the A24 molecule. CW3 and A24 differ by only one amino acid within this region. Recognition of CW3 or A24 peptides corresponded exactly with lysis of P815-HLA transfectants both for clones that mutually exclusively lysed CW3 or A24 transfectant target cells and for CW3/A24 crossreactive CTL clones. The latter CTL clones that lysed both CW3 and A24 transfectant target cells showed a clear preference for the peptide corresponding to the immunizing HLA allele. The homologous CW3 and A24 peptides could compete with each other for recognition, in contrast to a peptide from the same region of HLA-B7. Peptides from the corresponding region of the endogenous Kd and Dd/Ld molecules could also inhibit recognition of CW3 and A24 peptides. Competition with peptides apparently occurred at the level of the target cell. These results are consistent with a model whereby MHC class I molecules position protein fragments or peptides for specific recognition by T cells.

Conversion of a self peptide sequence into a Kd-restricted neo-antigen by a Tyr substitution.

We have previously found that a Tyr residue was critical for the interaction of peptides with the Kd molecule, and therefore may be acting as an anchor residue. In the present report we show that it is possible to convert a self peptide sequence into a Kd-restricted neo-antigen by a single Tyr substitution at position 2 of the peptide. This supports the idea that Tyr is a critical element in the binding motif of Kd-restricted peptides and is a finding that could also prove useful for vaccine development.

Recognition of oligonucleotide-encoded T cell epitopes introduced into a gene unrelated to the original antigen.

We have previously demonstrated that H-2Kd-restricted CTL specific for HLA-CW3 or HLA-A24 can recognize synthetic peptides corresponding to residues 170-182 of the HLA molecules. Synthetic oligonucleotides encoding region 170-182 of CW3 or A24 were inserted into the influenza nucleoprotein (NP) gene. We demonstrate herein that P815 (H-2d) cells transfected with the NP-oligo recombinant genes are specifically lysed by HLA-specific Kd-restricted CTL clones. Our results imply that there must be a high degree of flexibility for the expression of T cell epitopes in different molecular contexts.

T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire.

We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage.

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.

Oligoclonal expansion of major histocompatibility complex class I-restricted cytolytic T lymphocytes during a primary immune response in vivo: direct monitoring by flow cytometry and polymerase chain reaction.

Previous T cell receptor (TCR) sequence analysis of a panel of 23 H-2Kd restricted cytotoxic T lymphocyte (CTL) clones recognizing the decapeptide HLA-CW3 170-179 revealed a striking conservation of TCR structure, in that all clones examined used V beta 10 and J alpha pHDS58 segments. We show here that the primary response in vivo after intraperitoneal injection of DBA/2 mice with HLA-CW3 expressing transfectants of syngeneic P815 (H-2d) tumor cells is characterized by a dramatic expansion of CD8+ V beta 10+ CTL in the peritoneal cavity and draining (mesenteric) lymph node, as well as in peripheral blood. Additional analysis of TCR on HLA-CW3 immune populations by flow cytometry and polymerase chain reaction further indicates that the vast majority of responding CD8+ cells express restricted V alpha domains, a dominant J alpha segment (pHDS58), and a conserved CDR3 length for both alpha and beta chains. This novel system provides a unique opportunity to directly monitor an oligoclonal primary antigen specific immune response in vivo at the single cell level independently of functional assays.

Structure of the gene of tum- transplantation antigen P198: a point mutation generates a new antigenic peptide.

Mutagen treatment of mouse tumor cell line P815 produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These tum- antigens are recognized by cytotoxic T lymphocytes (CTL) but induce no detectable antibody response. By transfecting P815 cell line P1.HTR with DNA of tum- variant P198, we obtained transfectants expressing tum- antigen P198 that could be identified on the basis of their ability to stimulate anti-P198 CTL. This was repeated with DNA of a cosmid library derived from variant P198, and a cosmid carrying the sequence encoding antigen P198 was recovered from a transfectant. Gene P198 is 3 kb long and contains eight exons. It shows no homology with previously identified tum- gene P91A, nor with any gene presently recorded in the data banks. The long open reading frame codes for a 23.5-kD protein. The antigenic allele of gene P198 differs from the normal allele by a point mutation located in exon 7. This mutation causes an Ala to Thr change, and was shown by site-directed mutagenesis to be responsible for the expression of the antigen. An 11-amino acid synthetic peptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P198 CTL. The homologous peptide corresponding to the normal sequence of the gene did not, but it was able to compete for binding to major histocompatibility complex molecule Kd. We conclude that tum- mutation P198 generates a new epitope recognized by syngeneic T cells. As observed with gene P91A, we found that a fragment of gene P198 that contained only exons 3-7, cloned in nonexpression vectors, transferred efficiently the expression of the antigen.

Cells expressing a major histocompatibility complex class I molecule with a single covalently bound peptide are highly immunogenic.

The 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, beta 2-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.

T cell receptor selection by and recognition of two class I major histocompatibility complex-restricted antigenic peptides that differ at a single position.

Peptides 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 class I major histocompatibility complex molecule H-2Kd for recognition by murine 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 beta junctional regions interact with the amino-terminal part of the HLA peptides.

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium 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 class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta 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-2Kd selects CTLs that bear TCRs 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.

A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution.

The development of T cell effector and memory responses against foreign antigens (Ags) involves the activation, differentiation and proliferation of naive T cells expressing distinct Ag-specific TCRs. Understanding the complexity of Ag-selected TCR repertoires in individual responders in terms of the sequences selected and their relative frequencies may provide indications about how a repertoire is established and suggest ways to influence the outcome of an immune response. Most methods of repertoire analysis are unsuitable for calculating the relative in vivo frequencies of Ag-specific clones (expressing distinct TCRs) selected during an immune response, whereas sequence data obtained by single-cell PCR analysis directly reflect cell frequencies if a sufficiently large number of cells is sampled. Using a CD8 T cell response in normal mice in which Ag-selected cells are identified by cell surface phenotype and rearranged TCRBV sequences are determined by PCR amplification of genomic DNA directly from single cells, we have analyzed a large number (>200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice. We found that each responder selects a unique Ag-specific TCR repertoire in which the various TCRBV sequences are present in a wide range of frequencies. However, the overall distribution of sequences is quite similar for different responder animals. Moreover, an individual's selected TCR repertoire is uniformly represented among Ag-specific CD8 cells circulating in the blood or localized in the spleen or liver. Relatively few sequences make up the bulk of the repertoire and account for the oligoclonality observed in earlier studies. We discuss various models that could account for this skewed distribution of an Ag-selected TCR repertoire.

Individuality of Ag-selected and preimmune TCR repertoires.

We have combined flow cytometry and single-cell PCR to characterize the TCRBV repertoires selected by individual mice in a model CD8 response against a defined peptide/MHC complex (CW3 170-1 79/Kd). Ourresults established thatdifferent mice select individually distinct yet structurally similar CW3-specific repertoires. Repertoire selection appears to be flexible depending on the immunizing cell dose. Using a single-donor, matched-pair-recipient adoptive transfer strategy, we demonstrated that the CW3-specific TCR repertoires of normal mice are already distinct at the preimmune level. We combine our data with computer simulations to test models for the composition of an Ag-specific preimmune repertoire and its selection during an immune response.

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides.

We have previously derived MHC class I (H-2Kd) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freund's adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.

A simple panning method for the selection of cell surface antigen transfectants.

Mouse Ltk- (H-2k) cells that became reactive with anti-H-2d monoclonal antibodies (McAb) after transfection with DNA from H-2d-positive cells were isolated by an indirect panning method. The cells were transfected with DNA from a plasmid containing the selective thymidine kinase (tk) gene in addition to total cell DNA, so that a first selection could be carried out in hypoxanthine/aminopterin/thymidine (HAT) medium. The HAT-selected tk+ transfectants were incubated with anti-H-2d McAb, washed and transferred to dishes coated with purified anti-immunoglobulin (anti-Ig). This 2-step method of panning has the advantage that only the anti-Ig reagent requires purification. Transfected cells clearly reactive with either anti-Ld/Rd or anti-Kd McAb were isolated after only 1 or 2 cycles of panning.

Isolation and characterization of protective cytolytic T cells in a rodent malaria model system.

Protective immunity against malaria is induced by immunization with irradiation-attenuated sporozoites. Here we report the isolation of cytolytic T-cell (CTL) clones from BALB/c (H-2d) mice immunized with either Plasmodium berghei or Plasmodium yoelii sporozoites. The epitopes recognized by these CTL can be mimicked by synthetic peptides corresponding to a homologous region in the CS proteins of both rodent malaria species. Both peptides are recognized by the CTL in the context of the same MHC class I molecule, H-2 Kd. In vivo adoptive transfer of the CTL clones into non-immune syngeneic mice protected them from a lethal challenge of infectious sporozoites.

The diversity of antigen-specific TCR repertoires reflects the relative complexity of epitopes recognized.

Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 Kd. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252-260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170-179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant. H-2 Kd molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/Kd complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.

Tumour cell variants with increased immunogenicity obtained by mutagen treatment.

By treating mouse tumour cells in vitro with mutagens it is possible to obtain at high frequency variants that are rejected by normal syngeneic mice. Such variants have been designated 'tum-' to distinguish them from the original tumorigenic (tum+) cell lines. Most tum- variants appear to express new, individual antigens that can be defined in vivo by cross-protection experiments or in vitro by cytolytic T lymphocytes. The failure of tum- variants to form progressive tumours is apparently the result of an immune rejection response. Mice that have rejected a tum- variant clone are often protected against a subsequent challenge with the original tum+ cells. This was even demonstrated for two spontaneous mouse tumours for which no immunogenicity could be demonstrated otherwise. An analysis of 21 variants derived from mastocytoma P815 with cytolytic T lymphocyte populations showed that the repertoire of tum- antigens probably exceeds 50 specificities. No two variants have been found to express the same new antigen. Variant-specific long-term cytolytic T lymphocyte clones were isolated and used for the immunoselection of secondary antigen-loss variants that could then be analysed for the presence of previously undefined residual variant antigenic determinants. Somatic cell hybrids were prepared between different P815 tum- variants or between tum- variants and the original P815 cells. An analysis of the hybrids with variant-specific cytolytic T lymphocyte clones showed that the expression of tum- antigens was dominant. The intriguing paradox of the high frequency of tum- variants in populations of mutagen-treated tumour cells may require new genetic and immunological approaches before being understood.