Identification and characterization of a T cell-inducing epitope of bovine ribonuclease that can be restricted by multiple class II molecules.

An immunodominant epitope of bovine RNase restricted by I-Ek molecules was identified using a T cell hybridoma recognizing RNase. This epitope was localized to the peptide RNase(90-105). Single conservative amino acid substitutions were made at each of the positions 94 through 105. It was found that only at one position, Asn-103, were conservative substitutions not allowed. This residue was shown to be the critical residue in determining T cell specificity. The ability of RNase(90-105) and the well-defined T cell epitope, HEL(46-61) to stimulate mouse strains expressing different independent H-2 haplotypes was examined using a T cell proliferation assay. The response to HEL(46-61) was completely restricted to mice expressing an I-Ak molecule. In striking contrast, 6 of 10 different mouse strains, H-2b,f,k,q,s,u, mounted vigorous T cell responses to RNase(90-105). The response was restricted to both I-A and I-E molecules, including I-Ab, I-Af, I-Ek, I-Aq, and I-As. H-2d mice were nonresponders to RNase(90-105), which was shown to be due to the failure of RNase(90-105) to bind to I-Ad molecules. A variant RNase(90-105) peptide was generated, containing an I-Ad binding motif, that could bind to I-Ad molecules. Despite its ability to bind, this variant peptide was not able to stimulate a response in H-2d mice. This result demonstrates that the ability of a peptide to bind to an Ia molecule is necessary but not always sufficient for a response to occur. Thus, in contrast to the highly restricted HEL(46-61) determinant, the RNase(90-105) determinant is permissive in its binding to Ia molecules. These results show that in the universe of T cell inducing epitopes contains both highly restricted and broadly restricted epitopes are found.     

Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.     

Analysis of the interaction of peptide hen egg white lysozyme (34-45) with the I-Ak molecule

We examined the structural characteristics of a peptide Ag that determine its ability to interact with class II-MHC molecules and TCR. The studies reported here focused on recognition of the hen egg white lysozyme (HEL) tryptic fragment HEL(34-45) by two I-Ak-restricted T cell hybridomas. HEL(34-45) bound to I-Ak created more than one antigenic specificity. Experiments with truncated peptides and alanine-substituted peptides indicated that two T cell hybrids either recognized distinct regions of the HEL(34-45) peptide, or different determinants generated by interaction of the peptide with I-Ak. Although we identified residues of HEL(34-45) that were critical to T cell recognition, no positions in the peptide were identified as I-Ak contact sites using single alanine substitutions. This suggests that more than one site or region of the peptide contributes to the binding to I-Ak. Finally, the murine lysozyme equivalent of 34-45 did not bind to I-Ak. Substitution of the corresponding murine lysozyme (self) residue at position 41 of HEL(34-45) abrogated I-Ak binding of the peptide.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

Conformational restriction of the Tyr53 side-chain in the decapeptide HE.

A series of conformationally restricted analogs of the hen egg lysozyme (HEL) decapeptide 52-61 in which the conformationally flexible Tyr53 residue was replaced by several more constrained tyrosine and phenylalanine analogs was prepared. Among these tyrosine and phenylalanine analogs were 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (Htc), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 4-amino- 1,2,4,5-tetrahydro-8-hydroxy-2-benzazepine-3-one (Hba), 4-amino-1,2,4,5-tetrahydro-2-benzazepine-3-one (Aba), 2-amino-6-hydroxytetralin-2-carboxylic acid (Hat) and 2-amino-5-hydroxyindan-2-carboxylic acid (Hai) in which the rotations around Calpha-Cbeta and Cbeta-Cgamma were restricted because of cyclization of the side-chain to the backbone. Synthesis of Pht-Hba-Gly-OH using a modification of the Flynn and de Laszlo procedure is described. Analogs of beta-methyltyrosine (beta-MeTyr) in which the side-chains were biased to particular side-chain torsional angles because of substitution at the beta-hydrogens were also prepared. These analogs of HEL[52-61] peptide were tested for their ability to bind to the major histocompatibility complex class II I-Ak molecule and to be recognized in this context by two T-cell hybridomas, specific for the parent peptide HEL[52-61]. The data showed that the conformation and also the configuration of the Tyr53 residue influenced both the binding of the peptide to I-Ak and the recognition of the peptide/I-Ak complex by a T-cell receptor.     

T cell recognition of bovine ribonuclease. Self/non-self discrimination at the level of binding to the I-Ak molecule

Bovine RNase A specific T-cell hybridomas were generated to study the recognition of foreign Ag by T lymphocytes. One hybrid, TS12, was shown to recognize RNase in association with I-Ak. This hybridoma required bovine RNase to be processed before recognition. The immunogenic determinant on the RNase molecule recognized by TS12 was localized to the tryptic fragment RNase(40-61). All of the stimulatory ability of this determinant was shown to be contained within the synthetic 14mer RNase(43-56). When this segment of bovine RNase was compared with the self murine sequence, only one amino acid difference was found, a substitution of a proline residue at position 50 for a serine residue. This substitution completely abolishes binding to the I-Ak molecule, as shown by both functional and direct binding assays. This finding shows that self/non-self discrimination not only occurs at the level of the T cell, but also can be caused by an inability of the self peptide to associate with a class II molecule.     

Fine specificity of murine class II-restricted T cell clones for synthetic peptides of influenza virus hemagglutinin. Heterogeneity of antigen interaction with the T cell and the Ia molecule

We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule.     

The sites in the I-Ak histocompatibility molecule photoaffinity labeled by an immunogenic lysozyme peptide

The class II histocompatibilty molecule I-Ak was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled alpha and beta chains were digested with protease from Staphylococcus aureus strain V-8 (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a major labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)-iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-Ak. The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicating that the photoaffinity labeling was site-specific. Conversely, the NH2-terminal conjugate iodo-4-azidosalicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-Ak. The labeling was also site-specific because reverse-phase HPLC of primary digests with protease V-8 and secondary digests with trypsin showed single major labeled components. The labeling of I-Ak by IASA-(46-61) was fully inhibitible by HEL-(46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment. The site of the I-Ak molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was determined. IASA-(46-61) labeled selectively at Pro-118 of a primary alpha chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary beta chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46-61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the sequence 52-61 and also competed for the binding of this smaller peptide to I-Ak. Thus, peptides that bind to the allele-specific binding site and are long enough to extend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both alpha and beta chains.     

IA mutant functional antigen-presenting cell lines

We describe a protocol for the selection of mutant cells with an altered pattern of Ia antigenic determinants and antigen-presenting properties from a homogeneous population of functional antigen-presenting cells (APC). The APC line used in this work was obtained by fusing lipopolysaccharide-stimulated B cells from (BALB/c x A/J)F1 donors with cells from the M12.4.1 BALB/c B lymphoma cell line. The resulting hybridomas, including TA3, retained the potent antigen-presenting activity of the parental B lymphoma line and expressed Ia antigens and immune response gene-determined antigen-presenting properties of the A/J type. Mutants of TA3 were obtained by subjecting the cells to negative immunoselection with one monoclonal anti-(alpha) 1-Ak antibody and complement followed by positive immunoselection via electronic cell sorting with a second monoclonal alpha I-Ak or alpha I-Ek antibody. Two types of mutants were obtained. One, A8, appeared to have undergone a fairly limited alteration, since it lost only some of the I-Ak antigenic determinants; the second type appeared to have lost the entire I-Ak molecule but to have retained the I-E molecule. Functional studies with the A8 mutant demonstrated that the loss of a limited number of I-Ak determinants correlated with the loss of a specific I-Ak-encoded restriction element, since A8 failed to present a specific antigen, hen egg lysozyme (HEL), to a HEL-specific I-Ak-restricted T cell hybridoma but retained some capacity to present a second antigen, poly(Glu60Ala30Tyr10) (GAT), to a GAT-specific I-Ak-restricted T cell hybridoma. These results indicate that Ia antigens are the products of immune response gene loci. The availability of such mutants should allow an examination of the relationship between the structure of an Ia molecule and the antigens with which it is co-recognized by T cells.     

Functional analysis of the antigenic structure of a minor T cell determinant from pigeon cytochrome C. Evidence against an alpha-helical conformation

A minor T cell determinant from pigeon cytochrome c, composed of residues 43 to 58 (p43-58), was synthesized along with a series of 48 analogs containing amino or carboxyl-terminal deletions or single amino acid substitutions. These peptides were analyzed functionally for their ability to elicit unique T cell populations on immunization of C57BL/10 mice and to stimulate a degenerate T cell clone capable of recognizing p43-58 in association with two different Ia molecules, A beta b:A alpha b and A beta d:A alpha d. These experiments allowed us to identify the residues in the determinant that are critical for T cell activation. Residues 50 and 52 had the dominant influence on T cell specificity, and residues 47, 48, 49, 51, and 53 had weak effects. Residues 46 and 54 were hardly recognized by the TCR at all, but appeared to influence the potency of the determinant by interacting with the Ia molecule. Finally, substitutions at positions 55 to 58 had no effect, but removal of these residues reduced the potency of the peptide, suggesting a contribution from the peptide backbone of this part of the molecule during T cell activation. An analysis of the spatial relationship of these dominant epitopic and agretopic residues suggests that this determinant does not assume a pure alpha-helical secondary structure when bound to the Ia molecule.     

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.     

Specificity of the T cell receptor: two different determinants are generated by the same peptide and the I-Ak molecule

The determinants recognized by two I-Ak-restricted hen egg-white lysozyme-specific T cell hybridomas were differentiated with a series of truncated or substituted peptides. The 10mer 52-61 was the smallest peptide that was immunogenic for both T cells. This peptide differed by a single residue, Leu56, from the corresponding autologous lysozyme peptide, which was nonimmunogenic. The addition of amino acids to the amino terminus of 52-61 increased the immunogenicity of the peptides for 3A9 T cells and decreased the immunogenicity for 2A11 T cells. By deleting or diiodinating Tyr53, the resulting peptides were rendered totally nonimmunogenic. In contrast, the 3-NO2-Tyr derivative was fully immunogenic for the 3A9 cells but completely nonimmunogenic for the 2A11 cells. Thus, two different, but very similar, determinants were generated by the same HEL peptide and the I-Ak molecule.     

T cell-mediated recognition of foreign antigen and the Ia molecule observed by stopped-flow fluorometry

Th cell-mediated rapid recognition of foreign Ag and the Ia molecule was studied using azobenzenearsonate-L-tyrosine (ABA-L-tyrosine)-specific Th cells (I-Ak restricted), foreign Ag (ABA-L-tyrosine), and APC (H-2k). Initial transmembrane signals in Th cell hybridomas (2-45-12) and in Th cell lines (A24-17 or A33-7) were monitored by stopped-flow fluorometry with fluorescent probes. It was found that Th cells recognized foreign Ag within 1 s at 25 degrees C on the APC (B10.BR spleen cells or L cells into which I-Ak genes were transferred). Recognition of foreign Ag and the Ia molecule was shown to deliver the initial signals to Th cell hybridomas and T cell lines. First, Th cells had membrane fluidity increased and then calcium was transported from the external medium into the T cells. The initial transmembrane signals to Th cell hybridomas were inhibited by the addition of an anti-I-Ak mAb. None of the initial signals were observed in the absence of either specific foreign Ag or APC.     

Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide antigen

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.     

Processing and reactivity of T cell epitopes containing two cysteine residues from hen egg-white lysozyme (HEL74-90)

The Ag processing and structural requirements involved in the generation of a major T cell epitope from the hen egg-white lysozyme protein (HEL74-88), containing two cysteine residues at positions 76 and 80, were investigated. Several T cell hybridomas derived from both low responder (I-Ab) and high responder (I-Ak) mice recognize this region. These hybridomas are strongly responsive to native HEL, but unresponsive to the reduced and carboxymethylated protein. Air-oxidized HEL74-88 peptide was unable to bind I-Ak molecules and failed to stimulate T cells in the absence of intracellular Ag processing. Further functional competition assays showed that alkylation of cysteine residues with bulky methyl groups interferes with the contacts for the MHC class II molecules (I-Ak) of high responder mice and the I-Ab-restricted TCR of low responder mice. Serine substitutions of the cysteine residues of HEL74-88 either enhanced or abrogated T cell stimulation by the peptides without significant alterations in the class II binding. These results suggest that the cysteine residues of peptides must be free from disulfide bonding for efficient stimulation of T cells and yet frequently used modifications of cysteine residues may not be suitable for peptide-based vaccine development.     

The antigen-processing mutant T2 suggests a role for MHC-linked genes in class II antigen presentation.

.174xCEM.T2 (T2) is a human cell hybrid that has a large homozygous deletion within the MHC, including all of the functional class II genes. We have generated stable HLA-DR3 and H-2 I-Ak transfectants of T2 that express parental levels of class II molecules at the cell surface. T2.Ak transfectants fail to stimulate a hen egg lysozyme (HEL)-specific, I-Ak-restricted T cell when incubated with intact HEL. However, stimulation occurs if the appropriate HEL peptide is provided. The T2 cell line therefore has a defect in class II-restricted Ag processing. Biosynthetic studies demonstrate that the kinetics of I-Ak transport in T2.Ak are similar to the parental rates of transport, although the percentage of I-Ak molecules transported appears somewhat lower. I-Ak glycoproteins in T2.Ak associate normally with the I-chain, which appears to be proteolytically cleaved after transport through the Golgi apparatus in a similar fashion to that in the parent cell line, .174xCEM.T1 (T1). The DR alpha beta heterodimers in T2 differ from the parental phenotype in two ways. First, HLA-DR3 expressed in T2 does not have the epitope recognized by the DR3-specific mAb 16.23, although DR3 expressed in the parent does have the epitope. Second, the alpha beta subunits in the parent remain associated when exposed to SDS at room temperature, although those in T2 dissociate.     

Cutting edge: dueling TCRs: peptide antagonism of CD4+ T cells with dual antigen specificities.

T cells expressing two different TCRs were generated by interbreeding 3A9 and AND CD4+ TCR transgenic mice specific for the hen egg lysozyme (HEL) peptide 48-62:I-Ak and moth cytochrome c (MCC) peptide 88-103:I-Ek peptide:MHC ligands, respectively. Peripheral T cells in the offspring express two TCR V beta-chains and respond to HEL and MCC. We observed minimal or no additive effects upon simultaneous suboptimal stimulation with both agonist peptides; however, an antagonist peptide for the 3A9 TCR was able to inhibit the response of the dual receptor T cells to MCC, the AND TCR agonist. This HEL antagonist peptide did not affect AND single transgenic T cells, indicating that the antagonism observed in the dual TCR cells is dependent on the presence of the HEL-specific 3A9 TCR. In contrast, anti-TCR Abs mediate receptor-specific antagonism. These results demonstrate that peptide antagonism exerts a dominant effect.     

Specificity of peptide binding by the HLA-A2.1 molecule

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.     

A limited region within hen egg-white lysozyme serves as the focus for a diversity of T cell clones

The C57BL/6 (H-2b) mouse is a nonresponder to hen egg-white lysozyme (HEL) injected i.p., owing to a T suppressor cell-inducing determinant at the amino-terminal region. After immunization with a 93-amino acid fragment (a.a. 13-105) of HEL lacking this determinant, all clones from two independently derived C57BL/6 T cell lines were found to be specific for epitopes within a subregion of peptide 74-96. Three specificity patterns for the clones could be defined on the basis of cross-reactivities with only two other species variant lysozymes. Reactivities of all three specificity groups was consistent with the serine to threonine substitution at position 91, although reactivity of one of the groups could be affected by substitutions at position 84. The results confirm at the clonal level that even for distantly related antigens, only limited regions are recognized by T cells. They are consistent with the notion that specific sites on the antigen capable of interaction with Ia molecules lead to dominance of certain regions for T cell reactivity. Moreover, the diversity in specificity among clones suggests that the limiting feature of T cell responsiveness is not a lack of available T cells in the repertoire directed against a single antigenic site.     

Functional studies on the role of I-A molecules expressed by the antigen-specific T suppressor cell clone HF1

The antigen-specific T suppressor (Ts) cell clone HF1 (Thy-1.2+, I-Ak+, I-Ek+) was isolated from a CBA/J mouse tolerized to low doses of bovine serum albumin (BSA), and a subclone was adapted to grow in the absence of antigen-presenting cells. The functional role of the I-Ak molecules endogenously expressed by HF1 Ts cells was analyzed by using anti-I-Ak monoclonal antibodies (mAb) in attempts to block the antigen-induced proliferation of these cells. Among a panel of anti-I-Ak mAb tested, only the H118-49 mAb directed against the private determinant Ia.m1 blocked BSA-induced proliferation. The data presented here suggest a selective regulatory role of membrane-bound I-Ak molecules in the antigen-induced signal transmission.