Dominant TCR V alpha usage by virus and tumor-reactive T cells with wide affinity ranges for their specific antigens

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA-A2 and derived from the Epstein-Barr virus proteins BMLF1 and BRLF1 and from the melanoma protein Melan-A/MART-1. Within each set, a majority of clones used a recurrent V alpha region, even though they expressed highly diverse TCR beta chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted V alpha usage was not associated with the affinity/avidity of the CTL clones. The V alpha dominance, which may be a frequent feature of antigen-specific T cells, likely reflects a restricted geometry of TCR/peptide/MHC complexes, primarily determined by V alpha CDR.     

Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells

Allospecific immune responses against the MHC of another individual are remarkably strong, due t a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H-2b-derived murine lymphoma mutant RMA-S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA-S. Up-regulation of the levels of H-2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.     

A viral peptide can mimic an endogenous peptide for allorecognition of a major histocompatibility complex class I product.

Alloreactive class I-restricted T cells may recognize the class I structure alone, in association with a specific peptide, or with any stabilizing peptide. We have tested the role of endogenous peptides in the recognition of H-2Kb molecules by two alloreactive cytolytic T lymphocyte (CTL) clones using the mutant tumor line RMA-S, which expresses its surface H-2b molecules devoid of peptides and is not lysed by these two CTL clones. Empty H-2b molecules on RMA-S cells can be stabilized by binding exogenously added peptides. H-2Kb-specific recognition of the RMA-S cells by one of the CTL clones was restored by endogenous peptide extracts which only minimally stabilized H-2Kb on the surface of RMA-S cells, indicating the requirement for a specific peptide on a limited number of H-2Kb molecules. In addition, one out of three peptides which greatly enhance the expression of H-2Kb, the nucleoprotein peptide 52-59 from vesicular stomatitis virus (VSV), was also able to restore the lysis of RMA-S cells by the clone. The recognition of a common motif by an alloreactive clone (H-2k anti-H-2Kb) and virus-specific Kb-restricted clones suggests that both H-2k and H-2b thymic environments allow selection of T cells capable of recognizing H-2Kb+VSV and that tolerance to self, as would be the case in the (H-2k x H-2b)F1 mice, would partially delete the repertoire of antiviral T cells.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity

Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8⁺ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8⁺ T cell population in TAP1 -/- mice. CD8⁺ T cells were polyclonal with regard to T cell receptor (TCR) Vβ expression. Overall, Vβ usage in TAP1 -/- mice appeared to be very similar to that in wild-type mice, with significantly reduced levels of Vβ5.1/5.2-expressing CD8⁺ T cells as the only clear exception. This polyclonal population of CD8⁺ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2^b class I. The present results demonstrate that a polyclonal CD8⁺ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8⁺ T cells.     

T cell receptor crossreactivity as a general property of T cell recognition

TCR recognition of MHC/peptide complexes directs many aspects of T cell biology, including thymic selection, survival of na?ve T cells and differentiation into effector and memory T cells. It was widely thought that TCR recognition is highly specific, with an individual T cell being capable of only recognizing a particular peptide and closely related sequence variants. By considering the structural requirements for peptide binding to MHC molecules and TCR recognition of MHC/peptide complexes, we demonstrated that T cell clones could recognize a number of peptides from different organisms that are remarkably distinct in their primary sequence. These peptides are particularly diverse at those sequence positions buried in pockets of the MHC binding site, while a higher degree of similarity is present at a limited number of peptide residues that create the interface with the TCR. Many examples have now been documented for human and murine T cells, indicating that TCR crossreactivity represents a general feature of TCR recognition.     

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.      

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.     

Identification of H-2K(b)-restricted T-cell epitopes within the nucleocapsid protein of Hantaan virus and establishment of cytotoxic T-cell clones

Although neutralizing antibodies against Hantaan virus (HTV) can protect hosts from viral infection, T-cell responses to HTV are also important in host defense against HTV. However, much less is known about cytotoxic T lymphocyte (CTL) responses to HTV. To identify CTL epitopes in the HTV nucleocapsid protein (NP), we selected 7 H-2K(b)-motif-fitting peptides. Of these peptides, 3 peptides (NP3, NP4, and NP7) were recognized by CTL responses derived from HTV-immunized mouse splenocytes. NP3 and NP4 peptides were also recognized by HTV-immunized splenocytes after secondary in vitro stimulation with the relevant peptide, but NP7 could not be recognized after in vitro stimulation. These results agree well with peptide immunization studies showing that peptide-specific CTL responses could be induced with NP3 and NP4 but not with NP7 peptide. Furthermore, CTL activity assay using targets, prepared to express the antigen (NP) endogenously, demonstrated that NP3 and NP4 peptides could be presented endogenously. CTL elicited with NP4 peptide retained some cross-reactivity and was difficult to long-term culture. However, NP3-elicited CTL was very specific for NP3 peptide and was stable enough to be cloned. Among many CTL lines elicited with HTV or HTV NP peptides, 6 NP3-specific CTL clones were established and have been maintained more than 2 years. All 6 CTL clones were characterized to be CD3+, CD4-, CD8+, CD25+, CD62L-, and NK1.1-, and to use TCR Vbeta6. This preferential usage of TCR Vbeta6 indicates that TCR Vbeta6 regions are important for recognition of the HTV NP3 epitope (NP221-228, SVIGFLAL) on H-2K(b) molecule. Our data demonstrate the definition of mouse CTL epitopes in HTV and the generation of HTV-specific mouse CTL clones.     

Comparison of the frequency of peptide-specific cytotoxic T lymphocytes restricted by self- and allo-MHC following in vitro T cell priming

T cell recognition of antigenic peptides is thought to occur preferentially in the context of self-MHC. Here, we have tested the ability of four different K(b)-peptide combinations to stimulate self- and allo-restricted CTL responses in three different mouse stains. Responder T cells were primed in vitro with peptide-loaded stimulator cells, followed by limiting dilution assays to measure the number of peptide-specific cytotoxic T lymphocytes (CTL). For three peptides the number of CTL restricted by self-MHC was higher than for allo-MHC-restricted responses, although the difference was surprisingly small (3- to 5-fold). For the fourth peptide there was no detectable difference in the number of self- and allo-restricted CTL. Peptide titration experiments revealed that high avidity CTL were present in both the self- and allo-restricted setting. These data showed that the bias for preferred peptide recognition in the context of self-MHC imposed by positive thymic selection seems marginal. This raised the possibility that the TCR repertoire is inherently biased towards MHC restriction, independent of MHC-guided thymic selection. This was supported by the analysis of mature T cells generated from the thymus of MHC-deficient mice by lectin stimulation. K(b)-restricted CTL were found amongst these T cells at numbers similar to those of allo-restricted CTL. In summary, the data suggest that MHC-restricted peptide recognition is an inherent feature of the TCR repertoire and does not require thymic selection by MHC molecules.     

Emergence in Cx knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant x light chain region

Allotype- or idiotype-specific CD4⁺ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide-specific CD8⁺ T cells. We have therefore investigated whether T-depleted spleen cells from Ig x light chain-expressing 129/Sv mice (129x^+/+) could induce, in Cx knockout mice (129 x^?/?), the generation of Ig constant x light chain region (Cx)-specific cytotoxic T lymphocytes (CTL). The determination of TCRβ chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole Cx sequence, show that the B cells from x^+/+ mice are able to elicit in Cx knockout mice, the emergence of a diverse CTL repertoire that recognizes one single Cx peptide presented by the H-2K^b class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own x light chains.     

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen

Murine MHC class I-restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL-defined epitopes of this viral envelope protein are the L^d -binding 12-mer S28 – 39 peptide IPQSLDSWWTSL in H-2 ^d mice, and the K^b -binding 8-mer S208 – 215 peptide ILSPFLPL in H-2^b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H-2 class I-binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the L^d -binding peptide S28 – 39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the K^b -binding peptide S208 – 215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I-binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.     

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.     

The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent

Thymus leukemia (TL) antigens belong to the family of MHC class Ib antigens. We have shown in our previous studies that they serve as transplantation antigens, and can be recognized by both TCR alpha beta and TCR gamma delta cytotoxic T lymphocytes (CTL) with TL but not H-2 restriction. Although TL are known to be expressed TAP independently, it is unclear whether peptide loading on TL molecules is necessary for the formation of CTL epitopes. In the present study, we first showed that TL expression is beta(2)-microglobulin (beta(2)m)-dependent but TAP1 independent by flow cytometric analysis of thymocytes from beta(2)m- or TAP1-deficient mice crossed with TL transgenic mice expressing Tla(a)-3-TL on their thymocytes. Subsequently, we investigated the epitope recognized by CTL derived from C3H mice immunized with skin from a transgenic mouse expressing T3(b)-TL ubiquitously. Bulk CTL lines against TL from primary mixed lymphocyte cultures showed comparable cytotoxicity against T3(b)-TL transfectants of TAP2-deficient murine RMA-S grown at 37 degrees C to that against those grown at 25 degrees C. Furthermore, TCR alpha beta and TCR gamma delta CTL clones against TL recognized TL expressed on T3(b)-TL transfectants of RMA-S and Drosophila melanogaster cells having broad defects in peptide loading of MHC, and lysed these target cells. These results together indicate that TL-specific CTL populations primarily recognize epitopes expressed TAP independently.     

Peptide presentation by class-I major histocompatibility complex molecules

MHC class-I molecules express distinct peptide-binding pockets within their antigen-binding groove. These are critically involved in the binding of antigenic peptides. The amino acid composition of a pocket dictates the structure of a peptide which can be bound in it. This is evident as a consensus amino acid motif which has to exist within a peptide in order for it to bind to a particular MHC allele. Perturbation of a MHC pocket by amino acid substitution can result in the abolition of peptide binding. Less drastic mutations of the peptide-binding groove, particularly the ones away from the critical pocket, can subtly alter the conformation of bound peptide. Both types of substitution exert an influence on the TCR recognition of antigenic peptide. Peptides are also critically involved in the positive selection of the class-I-restricted TCR repertoire in the thymus. These self peptides act by mimicking their foreign antigens. This mimicking involves the binding of self peptides and foreign antigenic peptides to the same pockets of the MHC class-I-antigen binding groove. Consequently, MHC class-I polymorphism in the antigen binding groove controls the intrathymic positive selection and peripheral antigen presentation by the same mechanisms. The majority of positively selecting self peptides could well originate from the extracellular processing of circulating self proteins. Using the diverse, extracellularly generated self peptides and the different determinant density requirements for positive versus negative selection, the immune system can ensure the repertoire diversity, avoiding both the massive clonal deletion of the selected repertoire and the autoreactivity of its T cells.     

A simplified procedure for the preparation of MHC/peptide tetramers: chemical biotinylation of an unpaired cysteine engineered at the C-terminus of MHC-I

Recently, a powerful approach for the detection of MHC/peptide-specific T cells has been made possible by the engineering of soluble-tetrameric MHC/peptide complexes, consisting of singly biotinylated MHC/peptide molecules bound to fluorescent-labeled streptavidin. These tetrameric molecules are thought to compensate for the low affinity and relative fast dissociation rate of the TCR/MHC-peptide interaction by increasing the avidity of this interaction, thus allowing the stable binding of MHC/peptide tetramers to TCR expressing cells. Here we describe a new more simplified procedure for obtaining MHC/peptide tetramers using the well-characterized H-2K(b)/VSV system. This procedure consists of the incorporation of an unpaired cysteine residue at the C-terminus of the H-2K(b) molecule, allowing site-specific biotinylation by a -SH-specific biotinylating reagent. The H-2K(b)/VSV tetramers bound only to hybridomas expressing H-2K(b)/VSV-specific TCRs. When coated on a plate, these tetramers were able to induce IL-2 release by those hybridomas. Furthermore, H-2K(b)/VSV tetramers bound to CTL populations obtained from mice immunized with VSV-peptide. The specificity of the binding was further refined by studying cross-recognition of VSV by CTL populations obtained from mice immunized with single amino acid substituted VSV peptide variants. H-2K(b)/VSV tetramers bound only to those CTL populations that cross-reacted with the wild-type VSV peptide. Our method provides a simple, efficient and inexpensive procedure for making MHC/peptide tetramers, a highly specific and very useful reagent with a number of important applications in basic and clinical T cell research.     

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes

CD8⁺ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide / TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-γ and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-γ and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be usefol for inducing T lymphocytes with qualitatively different effector functions.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.