Comparison of structural requirements for interaction of the same peptide with I-Ek and I-Ed molecules in the activation of MHC class II-restricted T cells.

We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107-116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules.     

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.     

Identification of distinct predominant epitopes recognized by myoglobin-specific T cells under the control of different Ir genes and characterization of representative T cell clones

We characterized the fine specificity of antigen recognition of myoglobin-immune T cells from B10.D2, B10.GD, and B10.A(5R) mice. Polyclonal H-2d T cells show a predominant response to an epitope centering on Glu 109 and His 116. These residues localize a dominant epitope to one segment of the myoglobin surface, but probably are not the only amino acid residues involved. This response pattern maps genetically to I-Ad (or Kd) based on the B10.GD recombinant strain. A different epitope specificity was detected in B10.A(5R) T cells mapping to I-E beta b E alpha k or I-Cd, but no difference was detected between strains differing at the Igh locus. Thus, epitope specificity varied with Ia haplotype but not Igh allotype. Myoglobin-specific B10.D2 T cell lines were established, and five clones specific for the Glu 109, His 116 epitope were isolated; these were all restricted to I-Ad antigen-presenting cells. These clones represent the dominant specificity in the polyclonal T cell response to myoglobin and will be useful in characterizing the structure and function of T cell receptors for antigen and Ia. The differences in number and nature of T cell epitopes compared to B cell epitopes of myoglobin are discussed.     

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.     

Binding to Ia protects an immunogenic peptide from proteolytic degradation

A 34 amino acid hen egg-white lysozyme (HEL) peptide was designed and synthesized to investigate if an immunogenic peptide once bound to an Ia molecule becomes proteolytically inaccessible. The determinant recognized by T cells, HEL(52-61) was composed of L-amino acids whereas the 12 amino acid extension on each side of this core were composed of D-epimers. This peptide, HEL(40-73) was resistant to proteolysis, except in the core region, where any cleavage would destroy the determinant. Initially HEL(40-73) was shown to be able to stimulate the HEL specific T cell, 3A9, indicating that an I-Ak molecule can bind and present large peptides that extend beyond the theoretical binding groove. HEL(40-73) was then used to examine the proteolytic sensitivity of determinants recognized by T cells. If HEL(40-73) was treated with chymotrypsin before binding to I-Ak, the determinant was totally destroyed; however, if HEL(40-73) was allowed to first bind to I-Ak, then the determinant became resistant to chymotrypsin cleavage. Thus an Ia molecule can protect a determinant from proteolytic degradation, a finding that has important implications for proposed pathways of Ag processing.     

T cell clones to two major T cell epitopes of myoglobin: effect of I-A/I-E restriction on epitope dominance

The murine T cell response to sperm whale myoglobin was analyzed for polyclonal and monoclonal T cells. For polyclonal T cells, the immunodominant epitope included residue Glu 109 when the antigen-presenting cells expressed I-Ad, whereas a Lys 140-containing epitope was immunodominant when the antigen-presenting cells expressed I-Ed only. Monoclonal T cells specific for each epitope were derived from a polyclonal line. T cell clones specific for the Glu 109 epitope were restricted to I-Ad, whereas the clones specific for the Lys 140 epitope were restricted to I-Ed. Thus, for an antigen that can be presented in association with either I-Ad or I-Ed, the immunodominance of particular epitopes depends strongly on the restriction element used. The immunodominance of each epitope-Ia combination may be due to a limited repertoire of T cells or selective presentation of epitope and Ia by accessory cells.     

Separation of the immune response genes for LDH-B and MOPC-173. II. Description of an immune response defect in B10.ASR7

By using the intra-I region recombinant mouse strain B10.ASR7 (H-2as3), the immune response (Ir) genes for LDH-B and MOPC-173 were genetically and serologically separated, as assayed by T cell proliferation. Previous work demonstrated that H-2s and H-2b strains respond to LDH-B and MOPC-173 whereas H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak molecule-activated T helper cells by I-Ek molecule-activated T suppressor cells. In the experiments reported here, B10.ASR7 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to LDH-B but not to MOPC-173. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A beta sA alpha sE beta sE alpha k) macrophages with A.TL anti-B10.HTT serum (anti-As beta Es beta Js) adsorbed with B10.ASR7 spleen cells blocked the MOPC-173 response but not the LDH-B response. Unadsorbed serum blocked both antigen responses. The B10.ASR7 E beta allele was determined to be s due to the ability of (A.TL X B10.ASR7)F1 hybrids to mount a T cell proliferative response to the terpolymer GLPhe. Monoclonal antibody blocking of the B10.ASR7 T cell proliferative response to LDH-B demonstrated that the Ia.2 and Ia.17, and not the Ia.15 epitopes are spatially related to the Ia epitopes involved in the restriction of the B10.ASR7 LDH-B T cell proliferative response. In addition, B10.ASR7 helper T cells generated in response to LDH-B were suppressed in a haplotype-specific manner by I-Ek molecule-restricted suppressor T cells in that (A.TL X B10.ASR7)F1 hybrids failed to respond to LDH-B. This nonresponsiveness was eliminated by treatment with monoclonal antibodies directed against the I-Ek molecule. These results suggest the possibility that the immune response defect in B10.ASR7 could be related to the site of recombination.     

Activation of type B T cells after protein immunization reveals novel pathways of in vivo presentation of peptides

Type B T cells recognize a peptide-MHC conformer generated in recycling endosomes and eliminated by H2-DM in late endosomes; as a result, they recognize exogenous peptide, but fail to respond to the identical epitope generated from the native protein. To investigate the behavior of these cells in vivo, we generated mice transgenic for a type B TCR recognizing the 48-62 epitope of hen egg white lysozyme (HEL) presented by I-A(k). Type B T cells responded only to peptide ex vivo, but responded in vivo to immunization with either protein or peptide in the presence of Freund's adjuvant or LPS. Presentation of the type B conformer was MyD88-independent, evident within 24 h after HEL immunization, and restricted to the CD11b/c(+) APC subset. Immunization with listeriolysin O, a potent inducer of cell death, also primed type B T cells in vivo, and transfer of HEL-bearing allogeneic dendritic cells activated type B T cells. We conclude that a number of conditions in vivo, some of which induce inflammation and cell death, lead to peptide presentation through mechanisms distinct from the classical pathways involving H-2DM molecules.     

Immunoregulation of lysozyme-specific suppression. III. Epitope-specific amplification of immunosuppression induced by monoclonal suppressor-T-cell products

The hen egg-white lysozyme (HEL)-specific suppression induced by soluble molecules produced by a monoclonal T-cell lymphoma line (LH8-105) obtained from HEL-specific suppressor T lymphocytes has been examined. Injection of I-J+ molecules from LH8-105 cell culture supernatant (TsFa) in HEL-primed mice during the afferent phase of the response induced Lyt-2+ second order suppressor T (Ts) cells which, upon transfer into HEL-CFA-primed syngeneic recipients, inhibit the delayed-type hypersensitivity (DTH) response to HEL. Transfer of spleen cells from TsFa-injected mice primed with HEL or human lysozyme suppresses the DTH response to HEL in recipient mice whereas this response is not affected by cell transfer from ring-necked pheasant egg-white lysozyme (REL)-primed and TsFa-injected mice, indicating that induction of second order Ts by TsFa is specific for a lysozyme epitope including phenylalanine at position 3. Fine antigenic specificity of second order Ts-cell induction is confirmed by similar results obtained upon injection of TsFa in mice primed with HEL N-terminal synthetic peptide or with an analog in which, as in REL, phenylalanine has been substituted by tyrosine at position 3. The same fine antigenic specificity observed in the induction of second order Ts cells is also present in the expression of TsFe suppressive activity. The similar antigenic specificity of Tsa and Tse suggests that Tse cells could result from amplification of the Tsa cell population or these two cell subsets could reflect different maturation stages of the same cell type rather than distinct T-cell populations activated in cascade.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Murine T cell responses to melittin and its analogs.

The 26-residue peptide melittin from bee venom elicits high IgG1 and IgE responses in selected strains of mice. The antibody responses were shown previously to be specific mainly for the region of residue 20-26. The T cell epitope of melittin in H-2d-restricted mice is now found to be primarily in residue 11-19, corresponding to an alpha-helical amphiphilic segment of the molecule. Melittin-specific T cell lines have varying responses to different structural analogs of the melittin T cell epitope, and the results indicate that the antigenicity of T cell epitope peptides depend more on their primary structure than on their secondary structure. Melittin-specific T cell clones are found to be CD4+ and secrete IL-4, and are restricted to presentation on I-Ad or I-Ed. The I-Ad- or I-Ed-restricted clones differ in their responses to different analogs of melittin.     

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.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Role of L3T4 and Ia in the heteroclitic response of T cells to cytochrome c

The activation of helper T lymphocytes has been proposed to result from the sum of low-affinity interactions between the specific immune receptor, as well as nonpolymorphic receptors such as L3T4 on the T cell surface, and nominal antigen and Ia displayed in a multivalent array on the antigen-presenting cell surface. The present work takes advantage of a T cell hybridoma specific for pigeon cytochrome c in the context of I-Ek, which responds to tobacco hornworm moth cytochrome c at one hundredth the concentration of the homologous antigen, to determine if the T cell's requirement for L3T4 and Ia is directly related to its functional affinity for antigen. The results demonstrate that the T cell's activation by pigeon cytochrome c was blocked by antibodies directed to L3T4 and to I-Ek, even at antigen concentrations twofold to fourfold above those required for maximal responses. In contrast, the response to tobacco hornworm moth cytochrome c was not as affected by these antibodies under equivalent superoptimal conditions. The same phenomenon was observed for the T cell's activation by the carboxyl-terminal peptide fragments of the two cytochromes c, which do not require processing, indicating that the differences were not due to the relative efficiency of processing and/or presentation of the antigens. Although both I-Ek- and L3T4-specific antibodies blocked the T cell response to pigeon cytochrome, antibodies to I-Ak had no effect, even though I-Ak had been considered to be a ligand for L3T4. Thus, either Ia does not bind L3T4 or, if it does, I-Ek must be a sufficient ligand for L3T4 for T cells that recognize their antigen in the context of I-Ek. These studies provide more definitive evidence that the T cell's requirement for the functions of Ia and of L3T4 is dependent on the T cell's functional affinity for its antigenic determinant. This data is consistent with a model of T cell activation in which, given a high enough affinity of the T cell receptor for the processed antigen, the requirement for other components of a stimulatory complex, such as Ia and L3T4, may diminish to undetectable levels.     

Isolation and characterization of antigen-Ia complexes involved in T cell recognition

Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is herein demonstrated that the complexes, once formed, are very stable (kd approximately equal to 3 X 10(-6) s-1), but the rate of complex formation is very slow (ka approximately 1 M-1 s-1 explaining the overall low equilibrium constant of approximately 2 X 10(-6) M. Treating the complexes with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance of these complexes in antigen recognition.     

Unusually diverse T cell response to a repeating tripeptide epitope.

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA)5. This peptide represents one of the two T cell epitopes on the synthetic alpha-helical polypeptide antigen Poly 18, Poly EYK(EYA)5 in H-2d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA)5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA)5-specific and H-2d-restricted T cell hybridomas derived from H-2d, H-2bxd, and H-2b----H-2bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA)5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA)5 to the antigen-binding groove of I-Ad appears to generate a highly diversified T cell response. Therefore, (EYA)5-I-Ad complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.     

Functional mapping of MHC class II polymorphic residues. The alpha-chain controls the specificity for binding an Ad-versus an A k-restricted peptide and the beta-chain region 65-67 controls T cell recognition but not peptide binding.

MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.     

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.