Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9

Human histocompatibility leukocyte antigen E (HLA-E) and mouse major histocompatibility complex (MHC) class Ib antigen, Qa-1, share the same substitutions at two normally conserved positions 143 and 147, which are likely to affect binding of the C terminus of peptides. Qa-1 is able to bind a peptide derived from the leader sequence of H-2 D and H-2 L molecules. We developed a peptide binding assay in vitro to compare the binding specificity of HLA-E with the mouse MHC class Ib molecule Qa-1. We demonstrate that HLA-E binds, although poorly, the peptide which binds to Qa-1 and that it also binds nonamer signal sequence-derived peptides from human MHC class I molecules. Using alanine and glycine substitutions, we could define primary anchor residues at positions 2 and 9 and secondary anchor residues at position 7 and possibly 3.     

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.     

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2^b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2K^b and H-2D^b MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2K^b and H-2D^b and the predictive value of these motifs is shortly discussed. The high-affinity H-2D^b-binding peptide and putative CTL epitope E₇ 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E₇ 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.     

Qa-1, a nonclassical class I histocompatibility molecule with roles in innate and adaptive immunity

Qa-1, a nonclassical class I histocompatibility molecule expressed in mice, predominantly assembles with a single nonameric peptide, Qdm, derived from the signal sequence of certain class Ia molecules. The Qa-1/Qdm complex is the primary ligand for CD94/NKG2A inhibitory receptors expressed on a major fraction of natural killer (NK) cells. Cells become susceptible to killing by NK cells under conditions where surface expression of the Qa-1/Qdm inhibitory ligand is reduced. The CD94/NKG2 "missing-self" recognition system serves as mechanism for removing cells that have abnormalities in the intracellular machinery required for assembly and expression of class I-peptides complexes, as a consequence of viral infection, for example. Despite its highly focused peptide-binding specificity, Qa-1 also has a capacity to act as an antigen-presentation molecule for CD8+ T cells. It appears that a small subpopulation of these T cells undergoes positive selection by interaction with Qa-1 in the thymus, and they maintain their specificity for Qa-1 after maturation. The role of these unusual T cells in adaptive immune responses remains to be defined.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Major histocompatibility complex class I-restricted presentation of influenza virus nucleoprotein peptide by B lymphoma cells harboring an antibody gene antigenized with the virus peptide

We analyzed the capacity of B cells to process and present a peptide from the variable region of an endogenous immunoglobulin heavy (H) chain to a major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) clone. The H-chain gene was engineered to express 14-amino acid peptide from the sequence of the influenza virus nucleoprotein (NP) antigen in the third complementarity-determining region (CDR3). This NP peptide is presented in association with the D^b allele in H?2^b mice. We demonstrate that B lymphoma cells (H-2^b) harboring the antigenized H-chain gene process and present the NP peptide in association with the D^b molecule and are lysed by a CTL clone specific for that peptide in an MHC-restricted way. In contrast, the soluble antigenized antibody failed to mediate lysis of H?2^b target cells. The endogenously processed immunoglobulin CDR3 peptide could be eluted from surface D^b molecules in transfected cells. This study formally demonstrates that peptides from the hypervariable loops of endogenous immunoglobulin are processed through the endogenous degradative pathway and are presented to CD8⁺ T cells in the context of MHC class I molecules. The implication of these findings for processing and presentation of endogenous immunoglobulin peptides in B cells and network regulation by idiopeptides is discussed.     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2D^b using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind D^b. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to D^b. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the D^b pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited D^b-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all D^b-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, D^b-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to D^b, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.     

Glycosylphosphatidylinositol-linked Db does not induce an influenza-specific cytotoxic T lymphocyte response or recycle membrane-bound peptides

Major histocompatibility complex (MHC) class I molecules, as well as MHC class I-bound peptides, are known to recycle between the cell surface and an undefined, endosomal-like compartment. Little is known about the functional significance of this process. We have explored this using two different forms of the H-2D^b molecule expressed in transgenic mice, either transmembranous (D^b-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (D^b-GPI). The recycling capacity of peptides bound to D^b-tm and D^b-GPI was investigated using glycosylated D^b-binding glycopeptides, which were detected by flow cytometry. Only the tm form of D^b was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus (PR8) strain and tested for cytotoxic T lymphocyte (CTL) responses against an immunedominant nucleoprotein epitope (366–374, ASNENMETM), onyl D^b-tm mice were found to generate specific CTL responses. The results support the idea that membrane recycling of MHC class I-bound peptides on antigen-presenting cells may be important for the generation of certain CTL responses.     

Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries

MHC class I molecules bind short peptides for presentation to CD8⁺ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class I a molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1^c, which carries unusual key residues at both ends of the groove, and RT1.A^a which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7 – 15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-K^b, 8 – 13-mer and H2-D^b, 9 – 15-mer peptides). The rat MHC class Ia molecule, RT1-A^a, revealed a preference for 9 – 15-mer peptides, whereas RT1-A1^c showed a more stringent preference for 9 – 12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2K^d to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2K^d. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2K^d-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2K^d; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2K^d. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2K^d to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2K^d class I molecule, and only a fraction of these serve as CTL epitopes.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

MHC Class I Phenotype and Function of Human β2-Microglobulin Transgenic Murine Lymphocytes

Lymphoid cells from β₂-microglobulin (β₂m) knockout mice transgenic for human (h) β₂m (C57BL/10 mβ₂m^?/hβ₂m⁺) were compared with normal mice for their binding to exogenously added hβ₂m, binding to a H-2D^b peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous hβ₂m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/hβ₂m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous hβ₂m to MHC-I, in particular, to the H-2D^b molecule through an exchange with endogenous mouse β₂m. In contrast to normal H-2D^b molecules, hybrid H-2D^b expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added hβ₂m suggesting that a stable fraction of hybrid H-2D^b molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/hβ₂m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

Carboxy-terminal residues of major histocompatibility complex class II-associated peptides control the presentation of the bacterial superantigen toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II-associated peptides. For example, presentation of the toxic shock syndrome toxin-1 (TSST-1) superantigen by antigen-processing-defective T2-I-A^b cells (which expresses I-A^b that is either empty or associated with invariant chain-derived peptides) can be strongly enhanced by some, but not other, I-A^b-binding peptides. Here we investigate the contribution of I-A^b-associated peptides in the presentation of TSST-1 to T cells. The data show that overlapping peptides expressing the same core I-A^b-restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST-1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C-terminal end of the peptide have a dramatic effect on TSST-1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST-1 molecule, but appears to depend on the length of the C-terminal region. These data are consistent with crystallographic studies suggesting that TSST-1 may interact with the C-terminal residues of MHC class II-associated peptides. We also examined the capacity of naturally processed peptides to promote TSST-1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST-1 binding to I-A^b. Taken together, these data suggest that TSST-1 binding to MHC class II molecules is controlled by the C-terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST-1 to T cells. Thus, the peptide dependence of TSST-1 binding to class II molecules may significantly reduce the capacity of TSST-1 to stimulate T cells.     

Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor

Recent studies involving T cells that express gamma delta T-cell receptor (gamma delta TcR) have raised the possibility that Qa-1/Tla region class I major histocompatibility complex (MHC)-like molecules are antigen-presenting molecules for gamma delta TcR. In this report, cytotoxic T lymphocyte (CTL) clones specific for a Qa-1/Tla region gene product were isolated from a bulk B10. QBR (Kb, Ib, Dq Qa-1/Tlab) anti-B10.MBR (Kb, Ik, Dq, Qa/Tlaa) CTL line. These CTL lysed blasts from all Qa-1a strains regardless of the H-2 haplotype, indicating that the recognition of the Qa-1 antigen by these CTL is not restricted by other class I molecules. In bulk populations, CTL activity of this specificity was found only in the CD8+CD4- subpopulation. Accordingly, all established CTL clones were phenotyped as Thy-1+, CD8+CD4-. Furthermore, these clones were shown to express alpha beta TcR rather than gamma delta TcR. Thus, the results indicate that Qa-1 antigen can be recognized by alpha beta TcR T cells in a manner similar to recognition of classical class I molecules.     

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft

The MHC class I molecule H-2D^d (D^d) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered D^d molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the D^b molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (D^dS73WD156Y) impaired the protective effect of the D^d molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A⁺ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the α helix of the α2 domain, facing the solvent and without direct contact with the peptide (D^dA150S) had no effect. D^d recognition by Ly-49A⁺ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the D^d molecule that make the trimolecular complex less permissive for inhibition of Ly-49A⁺ NK cells.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

The ability of H-2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld

F₁ hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non-class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the “self/non-self” property of class I molecules and the molecular domains responsible for this function. H-2^b/Hh-1^b lymphoma cells were transfected with class 1 H-2D^d or L^d gene, and its effect on the Hh-1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H-2^b/Hh-1^b bone marrow grafts by irradiated F₁ hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the D^d gene, but not of L^d, renders the Hh-1^b-positive cells incapable of inhibiting HR in F₁ mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H-2^b/d F₁ mice for which D^d and L^d are self, but also in H-2^b/k F₁ mice for which both D^d and L^d are non-self. Thus, this function of the D^d molecule is an intrinsic property, not necessarily related to its self/non-self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a “target interference” model as the underlying mechanism of the D^d effect. To locate the relevant domain(s) of the D^d molecule, mutant D^dm1 gene was tested and found to have the same effect as the non-mutant D^d. D^dm1 is a hybrid molecule between D^d and L^d, sharing with D^d only the α1 domain and a portion of the α2 domain. The two N-terminal domains of D^dm1 differ from those of D^d by three amino acid substitutions, two of which affect the molecules' peptide-binding properties.