Tax and M1 peptide/HLA-A2-specific Fabs and T cell receptors recognize nonidentical structural features on peptide/HLA-A2 complexes

Both TCRs and Ab molecules are capable of MHC-restricted recognition of peptide/MHC complexes. However, such MHC restriction is the predominant mode of recognition by T cells, but is extremely rare for B cells. The present study asks whether the dichotomy in Ag recognition modes of T and B cells could be due to fundamental differences in the methods by which TCRs and Abs recognize peptide/MHC complexes. We have compared MHC and peptide recognition by panels of CTL lines specific for the Tax and M1 peptides presented by HLA-A2 plus Tax and M1 peptide/HLA-A2-specific human Fabs that were selected from a naive phage display library. Collectively, the results indicate both striking similarities and important differences between Fab and TCR recognition of MHC and peptide components of the Tax and M1/HLA-A2 complexes. These findings suggest that these two classes of immunoreceptors have solved the problem of specific recognition of peptide/MHC complexes by nonidentical mechanisms. This conclusion is important in part because it indicates that Ab engineering approaches could produce second-generation Ab molecules that more closely mimic TCR fine specificity. Such efforts may produce more efficacious diagnostic and therapeutic agents.     

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.     

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.     

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes （CTLs） by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and aUogeneic HLA-A2. 1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.     

The MHC class II molecule I-Ag7 exists in alternate conformations that are peptide dependent

Insulin-dependent diabetes mellitus is an autoimmune disease that is genetically linked to the HLA class II molecule DQ in humans and to MHC I-Ag7 in nonobese diabetic mice. The I-Ag7 beta-chain is unique and contains multiple polymorphisms, at least one of which is shared with DQ alleles linked to insulin-dependent diabetes mellitus. This polymorphism occurs at position 57 in the beta-chain, in which aspartic acid is mutated to a serine, a change that results in the loss of an interchain salt bridge between alphaArg76 and betaAsp57 at the periphery of the peptide binding groove. Using mAbs we have identified alternative conformations of I-Ag7 class II molecules. By using an invariant chain construct with various peptides engineered into the class II-associated invariant chain peptide (CLIP) region we have found that formation of these conformations is dependent on the peptide occupying the binding groove. Blocking studies with these Abs indicate that these conformations are present at the cell surface and are capable of interactions with TCRs that result in T cell activation.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

Cross-immunizing potential of tumor MAGE-A epitopes recognized by HLA-A*02:01-restricted cytotoxic T lymphocytes

Almost all melanoma cells express at least one member of the MAGE-A antigen family, making the cytotoxic T cells (CTLs) epitopes with cross-immunizing potential in this family attractive candidates for the broad spectrum of anti-melanoma immunotherapy. In this study, four highly homologous peptides (P264: FLWGPRALA, P264I9: FLWGPRALI, P264V9: FLWGPRALV, and P264H8: FLWGPRAHA) from the MAGE-A antigens were selected by homologous alignment. All four peptides showed high binding affinity and stability to HLA-A*02:01 molecules, and could prime CTL immune responses in human PBMCs and in HLA-A*02:01/K(b) transgenic mice. CTLs elicited by the four epitope peptides could cross-lyse tumor cells expressing the mutual target antigens, except MAGE-A11 which was not tested. However, CTLs induced by P264V9 and P264I9 showed the strongest target cell lysis capabilities, suggesting both peptides may represent the common CTL epitopes shared by the eight MAGE-A antigens, which could induce more potent and broad-spectrum antitumor responses in immunotherapy.     

Mechanisms for generating cell membrane antigens that are recognized by cytotoxic T lymphocytes

Studies with many viruses have revealed that viral specific protein synthesis is an obligatory step in generating antigens on target cells for antiviral cytotoxic T lymphocytes. This has been most clearly demonstrated with DI particles, virions that are structurally complete but lack infectious RNA. Adsorption of such particles onto target cell membranes does not render these cells susceptible to lytic attack by antiviral effector cells, unless some viral protein synthesis transpires. However, some viruses, such as Sendai virus, circumvent the requirement for viral protein synthesis via fusion of the viral envelope with the target cell membrane, a process mediated by a specialized fusion protein. Once inserted into the lipid bilayer, it is likely that viral components and self H-2 noncovalently associate so that the complex can be recognized by antiviral cytotoxic T cells. This idea is supported by the demonstration that viral proteins and H-2 containing membrane proteins, incorporated into reconstituted membrane vesicles or liposomes are recognized by cytotoxic T cells. These data further show that native rather than altered viral and H-2 molecules are the moieties recognized. Associations between antigen and H-2 have been detected by a variety of techniques and in some cases are not random but selective; that is, viral antigens perferentially associate with some H-2 alleles and not others. In summary, these findings indicate that although viral antigens are present in the mature virions, these components are not recognized by antiviral killer cells until integrated into the plasma membrane. This may be achieved either through direct fusion of the viral envelope with the target cell or following viral protein synthesis and insertion of viral antigens into the plasma membrane.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Peptide recognition by two HLA-A2/Tax11-19-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11-19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11-19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.     

Identification of natural antigenic peptides of a human gastric signet ring cell carcinoma recognized by HLA-A31-restricted cytotoxic T lymphocytes.

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.     

Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor

Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues.     

A new LAGE-1 peptide recognized by cytolytic T lymphocytes on HLA-A68 tumors

Antigens encoded by genes of the LAGE family, including LAGE-1 and NY-ESO-1, are of interest for cancer immunotherapy because they are tumor-specific and shared by tumors of different histological types. Several clinical trials are in progress with NY-ESO-1 peptides, protein, recombinant poxviruses, and dendritic cells pulsed with peptides. In this study, CD8 T lymphocytes from an individual without cancer were stimulated with dendritic cells infected with a recombinant avian poxvirus encoding a complete LAGE-1 protein. A CTL clone was isolated that recognized a new LAGE-1 peptide, ELVRRILSR, which corresponds to position 103–111 of the protein sequence. It is presented by HLA-A6801 molecules. When tumor cells expressing LAGE-1 were transfected with HLA-A68, they were lysed by the CTL clone, indicating that the peptide is processed in tumor cells. These results indicate that the LAGE-1.A68 peptide can be used for antitumoral vaccination. We observed also that specific T cells could be detected in a blood sample with a high sensitivity by using an A68/LAGE-1 fluorescent multimer.     

The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.     

The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

Two different T cell receptors use different thermodynamic strategies to recognize the same peptide/MHC ligand

A6 and B7 are two alphabeta T cell receptors (TCRs) that recognize the Tax peptide presented by the class I major histocompatibility molecule HLA-A2 (Tax/HLA-A2). Despite the fact that the two TCRs have different CDR loops and use different amino acid residues to contact their ligand, both receptors bind ligand with similar diagonal orientations. Here we show that they also bind with very similar binding affinities and kinetics (the DeltaDeltaG degrees for binding is approximately 0.3kcal/mol at 25 degrees C). The two receptors respond similarly to alterations in the MHC molecule, yet differ dramatically in their responses to ionic strength and temperature. The different responses to temperature indicate markedly different binding thermodynamics, which are not predictable from the surface area buried in the interfaces. A6 and B7 thus represent two TCRs that are both compatible with Tax/HLA-A2, although compatibility has been achieved through the use of different thermodynamic strategies. Finally, neither A6 nor B7 are predicted to undergo large conformational adaptations upon binding, distinguishing them from a number of other TCRs whose structure, thermodynamics, and kinetics have been characterized.     

Structural identity between HLA-A2 antigens differentially recognized by alloreactive cytotoxic T lymphocytes

Alloreactive CTL raised against HLA-A2 Ag often display heterogeneous recognition of HLA-A2+ target cells. This heterogeneity has been found to reflect structural polymorphism among the corresponding target Ag, thus defining HLA-A2 subtypes. A previous study (van der Poel et al. 1986. Human Immunol. 16:247) established the existence of a new HLA-A2.4 variant, A2-SCHU, that was distinguished from A*0206 (A2.4a) by HLA-A2-specific alloreactive CTL. The same CTL subdivided HLA-A2.1 Ag into two subgroups. In the present study, the molecular basis of this heterogeneity has been examined by double-label comparative peptide mapping analysis of differentially recognized A2.1 and A2.4 Ag. In addition, we have determined the complete sequence of polymerase chain reaction-amplified full length cDNA from A2-SCHU. The results show that: 1) A2-SCHU is indistinguishable from A*0206 by peptide mapping; 2) the cDNA sequence of A2-SCHU is identical to that of A*0206; and 3) two differentially recognized A2.1 Ag are both indistinguishable from A*0201 by comparative peptide mapping. These results indicate that differential recognition by alloreactive CTL can occur among structurally identical class I HLA Ag and suggest that allorecognition by such CTL may involve corecognition of endogenous peptides, presumably derived from polymorphic proteins.