H-2Ld Molecule Research Articles

Determination of a Key Antigen for Immunological Intervention To Target the Latent Stage of Toxoplasma gondii.

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a chronic infection by forming cysts preferentially in the brain. Up to one third of the human population worldwide is estimated to be chronically infected with this parasite. However, there is currently no drug effective against the cyst form of the parasite. In addition, the protective immunity against the cysts remains largely unknown. We analyzed the molecular mechanisms by which the immune system detects host cells harboring the cysts to eliminate the latent stage of the parasite using mice with the H-2d haplotype, which are genetically resistant to the infection. Our study revealed that CD8+ immune T cells bearing TCR Vβ8.1, 8.2 chain have a potent activity to remove T. gondii cysts from the brain. Our studies also uncovered that H-2Ld is the major Ag-presenting molecule to CD8+ T cells for initiating cyst elimination, and that CD8+Vβ8.1, 8.2+ immune T cells recognize the N-terminal region (aa 41-152) of dense granule protein 6 (GRA6Nt) of the parasite presented by the H-2Ld molecule. Furthermore, CD8+ immune T cells induced by immunization with recombinant GRA6Nt were eventually capable of removing the cysts from the brain when transferred to infected immunodeficient mice lacking T cells. Thus, GRA6Nt is a novel and potent Ag to activate CD8+ T cells capable of removing T. gondii cysts. These observations offer a basis for immunological intervention to combat chronic infection with T. gondii by targeting the persistent cysts of the parasite.

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

AbstractRecent clinical trials have shown that evasion of CD8+ T-cell responses against viral capsid is critical for successful liver-directed gene therapy with adeno-associated viral (AAV) vectors for hemophilia. Preclinical models to test whether use of alternate serotypes or capsid variants could avoid this deleterious response have been lacking. Here, the ability of CD8+ T cells (“cap-CD8,” specific for a capsid epitope presented by human B*0702 or murine H2-Ld molecules) to target AAV-infected hepatocytes was investigated. In a murine model based on adoptive transfer of ex vivo expanded cap-CD8, AAV2-transduced livers showed CD8+ T-cell infiltrates, transaminitis, significant reduction in factor IX transgene expression, and loss of transduced hepatocytes. AAV8 gene transfer resulted in prolonged susceptibility to cap-CD8, consistent with recent clinical findings. In contrast, using an AAV2(Y-F) mutant capsid, which is known to be less degraded by proteasomes, preserved transgene expression and largely avoided hepatotoxicity. In vitro assays confirmed reduced major histocompatibility complex class I presentation of this capsid and killing of human or murine hepatocytes compared with AAV2. In conclusion, AAV capsids can be engineered to substantially reduce the risk of destruction by cytotoxic T lymphocytes, whereas use of alternative serotypes per se does not circumvent this obstacle.

Oral ingestion of Lentinula edodes mycelia extract can restore the antitumor T cell response of mice inoculated with colon-26 cells into the subserosal space of the cecum

We previously reported that oral ingestion of Lentinula edodes mycelia (L.E.M.) extract can inhibit the growth of a subcutaneously established melanoma in a Tcell-dependent manner via mitigation of regulatory T cell (Treg)-mediated immunosuppression. In this study, we tested the antitumor effect and mechanism of oral ingestion of L.E.M. extract following inoculation of murine colon carcinoma colon-26 (C26) cells into the subserosal space of the cecum (i.c.) of syngeneic mice. In this model, the primary site of the immune response was gut-associated lymphoid tissue (GALT), which is known to be an immunological tolerance-inducing site for numerous dietary antigens. Oral ingestion of the L.E.M. extract suppressed the growth of i.c.-inoculated C26 cells in a T cell-dependent manner and restored the Tcell response of the mesenteric lymph nodes and the spleen, not only to a tumor antigen-derived peptide, presented on H-2Ld molecules, but also to C26 cells. I.c. inoculation of C26 cells increased the potential of CD4+ T cells of the mesenteric lymph nodes to produce transforming growth factor (TGF)-β, but ingestion of the L.E.M. extract decreased the ability of both CD4+ and CD8+ T cells in the mesenteric lymph nodes to produce this immunosuppressive cytokine. Although ingestion of L.E.M. showed only a marginal effect on Tregs in this model, this treatment significantly reduced the plasma levels of TGF-β and IL-6, both of which were increased in the i.c. C26-inoculated mice. In summary, our results indicate that oral ingestion of L.E.M. extract can restore antitumor T cell responses of mice even when the primary antitumor immune response is elicited in GALT, and provide important implications for anticancer immunotherapy of human colon cancer.

Of swords and ploughshares: immunosurveillance and inflammation in melanoma

Of swords and ploughshares: immunosurveillance and inflammation in melanoma

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2Kd, H-2Dd and H-2Ld molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2Ld molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NFκB. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism.Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2′-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.

Inactivation of a defined active site in the mouse 20S proteasome complex enhances major histocompatibility complex class I antigen presentation of a murine cytomegalovirus protein.

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the delta/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the delta/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

Direct Binding of the MHC Class I Molecule H-2Ld to CD8: Interaction with the Amino Terminus of a Mature Cell Surface Protein

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing alphabeta TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 x 10(-7) M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8alpha and beta chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.

Double-cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region.

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens, and the gamma-interferon-inducible proteasome activator PA28 has been thought to play an important role in the generation of immunodominant MHC ligands by proteasomes. Several attempts have been made to show that proteasomes have the ability to yield cytotoxic T lymphocyte (CTL) epitopes effectively from model polypeptides derived from viral and intracellular proteins in vitro, but their antigen processing mechanism is poorly understood. Proteasomes produce the tumour rejection antigen precursor peptide pRL1b (SIIPGLPLSL), but not pRL1a (IPGLPLSL), bound to the H-2Ld molecule, from synthetic peptides covering the CTL epitope. This double cleavage production of pRL1b by proteasomes seemed to depend on the length of the flanking regions adjacent to either end of the CTL epitope, in which their successive deletions caused the almost complete prevention of pRL1b excision. The newly identified PA28 collaborates with proteasomes for efficient production of pRL1b, by promoting not only single cleavage of all susceptible peptides, but also dual cleavage in some peptides harboring certain characteristic lengths. The flanking regions outside pRL1b of suitable length appear to be essential for the correct CTL epitope production, possibly functioning as anchors to trap target peptides for proteasomal degradation. We propose a novel mechanism for dual-cleavage excision of immunodominant epitopes by proteasomes and PA28.

Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H-2Ld and is recognized by cytotoxic T lymphocytes against mouse tumor P815.

Mouse mastocytoma P815 expresses tumor antigens P815A and P815B encoded by a single gene called P1A and carried by a single peptide named P1A 35-43 (NH2-Leu-Pro-Tyr-Leu-Gly-Trp-Leu-Val-Phe-COOH). P1A 35-43 is presented to anti-P815A and anti-P815B cytotoxic T lymphocytes (CTL) by major histocompatibility complex (MHC) H-2Ld molecules. In order to determine the individual role played by each amino acid residue of P1A 35-43 in binding to H-2Ld and in recognition by anti-A and anti-B T cell receptors (TcR), a series of P1A35-43 peptides substituted by alanine at single positions was synthesized and tested for binding to H-2Ld and for CTL recognition. Binding to H-2Ld was estimated by measuring the ability of the peptide to up-regulate cell surface expression of H-2Ld. We found that three residues were important for interaction of P1A 35-43 with H-2Ld. Two of them, Pro at position 2 and Phe at position 9 were consistent with the described H-2Ld binding motif. A third residue, Trp at position 6, was also required for effective MHC binding of the tumor antigen. CTL sensitization assays showed that alanine substitution at position 7 (Leu) or at position 8 (Val) dramatically affected peptide recognition by anti-A CTL while positions 3 (Tyr) and 4 (Leu) were critical for recognition by anti-B CTL. We conclude that Pro2, Trp6 and Phe9 constitute the anchor residues of P1A 35-43 to H-2Ld, whereas the dipeptidyl sequences Tyr3-Leu4 and Leu7-Val8 form the core epitopes recognized by the TcR of anti-P815B and anti-P815A CTL, respectively.

Ab initio association with beta 2-microglobulin during biosynthesis of the H-2Ld class I major histocompatibility complex heavy chain promotes proper disulfide bond formation and stable peptide binding.

In vitro translation studies indicate that the beta 2-microglobulin (beta 2-m) light chain influences the formation of intrachain disulfide bonds in class I major histocompatibility complex (MHC) molecules during their biosynthesis. We now have examined the influence of beta 2-m on class I MHC intrachain disulfide bond formation in vivo. Using beta 2-m+ and beta 2-m- derivatives of a cell line transfected with the mouse H-2Ld gene, we show that all of the H-2Ld molecules from beta 2-m+ cells have both the alpha 2 and alpha 3 intrachain disulfide bonds, whereas about 50% of the H-2Ld molecules from beta 2-m- cells have only one of these bonds. All of the free H-2Ld heavy chains from beta 2-m+ cells can undergo a peptide-induced conformational change and can bind exogenous peptide and beta 2-m stably in vitro. Only those H-2Ld molecules from beta 2-m- cells, which have both intrachain disulfide bonds, undergo a peptide- and beta 2-m-induced conformational change in vitro. These H-2Ld molecules do not bind beta 2-m and peptide stably in vitro. From these results emerges a greater understanding of the role of beta 2-m at the time of class I MHC molecule synthesis: beta 2-m promotes intrachain disulfide bond formation in the class I MHC molecule and additionally affects class I MHC structure to render it competent to form stable trimolecular complexes with peptide and beta 2-m.

MHC class I and non-MHC-linked capacity for generating an anti-viral CTL response determines susceptibility to CTL exhaustion and establishment of virus persistence in mice.

The influence of the murine MHC gene complex H-2 class I alleles or of the genetic background and the role of virus variants on establishment of a persistent infection with lymphocytic choriomeningitis virus (LCMV) was analyzed in immunocompetent mice of H-2d haplotype by evaluation of cytotoxic T cell responses. Susceptibility to establishment of a virus carrier state increased in various H-2d mice with lower CTL response and slower CTL kinetics. Low responder BALB/c-H-2dm2, lacking H-2Ld molecules to present the major T cell epitope amino acids 118-126 of the LCMV nucleoprotein or DBA/2 mice possessing relatively few CD8+ T cells, were more susceptible than high responder BALB/c mice expressing H-2Ld. Additional critical factors were LCMV isolate and dose of infection. The rapidly replicating LCMV-DOCILE and Cl 13 Armstrong induced viral persistence readily, whereas the slowly replicating parental virus strains WE or Armstrong did not. The presented findings illustrate a model of MHC-linked or MHC-unlinked susceptibility for virus persistence and may help to explain pathogeneses of chronic virus infections in humans that are often associated with slowly progressing immunopathology.

Class I histocompatibility molecule association with phosphorylated calnexin. Implications for rates of intracellular transport

Recent studies have shown that the endoplasmic reticulum (ER)-resident protein, calnexin, associates with class I major histocompatibility complex (MHC) molecules early in their biosynthesis. It has been suggested that calnexin participates in the assembly of class I MHC molecules or in the retention within the ER of unassembled class I molecules. We have examined the role of phosphorylation of calnexin in its association with mouse class I MHC molecules. We show that phosphocalnexin associates with H-2Ld and H-2Db molecules but not with H-2Kb and H-2Dd molecules, although calnexin-H-2Kb association can be demonstrated. These observations are interesting in light of the fact that H-2Kb and H-2Dd molecules are transported out of the ER more rapidly than are H-2Ld and H-2Db molecules. H-2Ld and H-2Db molecules differ in amino acid sequence only in their membrane-distal alpha 1 and alpha 2 domains. Nevertheless, the affinity of phosphocalnexin for H-2Ld is greater than its affinity for H-2Db. Furthermore, H-2Db becomes endoglycosidase H-resistant more slowly in cells in which it associates with phosphocalnexin than in cells in which it does not. Ca2+ ionophore A23187 prevents association of phosphocalnexin with H-2Ld molecules in vivo but does not cause the disruption of phosphocalnexin-H-2Ld complexes after they have formed. A23187 does not prevent assembly of H-2Ld-beta 2-microglobulin (beta 2-m) heterodimers. Furthermore, phosphocalnexin is found associated with H-2Ld molecules regardless of their state of assembly with beta 2-m and antigenic peptide. These results suggest that phosphocalnexin association with class I MHC molecules does not play a role in assembly of the class I MHC-beta 2-m-peptide complex nor in preventing release of unassembled class I molecules from the ER but may otherwise influence their rate of transport through the ER.

Natural killer cells recognize common antigenic motifs shared by H-2Dd, H-2Ld and possibly H-2Dr molecules expressed on bone marrow cells.

Murine natural killer (NK) cells can mediate specific rejection of bone marrow cell (BMC) allografts. Whereas positive recognition of allogeneic MHC antigens forms the basis for T cell alloreactivity, it has been postulated that NK cells are reactive against targets that do not express certain self-encoded MHC class I antigens. Here, we study the immunogenicity of BMC grafts from two class I transgenic mice, D8 (B6 mice with an H-2Dd transgene) and C3H.Ld (C3H mice with an H-2Ld transgene). D8 BMC grafts are acutely rejected by B6 but not D8 recipients. This suggests that antigenic motifs associated with the H-2Dd molecule are recognized. B6 mice depleted of their CD3+ but not NK1.1+ cells can still reject D8 BMC grafts. These data suggest that NK1.1+/CD3- cells recognize the H-2Dd derived antigenic motifs. Similarly, C3H.Ld BMC grafts are rejected by B6 x C3H F1 but not B6 x C3H.Ld F1 recipients. Thus, antigenic motifs associated with the H-2Ld molecule can also be recognized. Furthermore, expression of either H-2Dd or H-2Ld by the recipients renders them unable to reject D8 or C3H.Ld BMC grafts. Therefore, H-2Dd and H-2Ld molecules appear to express common antigenic motifs recognized by NK cells. Additional studies with B6.R4 (KbIbSbDr), an intra-H-2 recombinant mouse, indicated that a third class I molecule, possibly H-2Dr, also shared the common antigenic motifs with both H-2Dd and H-2Ld molecules. Thus, positive recognition of class I antigens by NK cells can occur. However, expression of some of these antigenic motifs appear to be negatively controlled by certain H-2r genes as suggested by rejection of D8 and B6.R4 BMC grafts by D8 x B10.RIII F1 and B6.R4 x B10.RIII F1 hybrids respectively.

The cytoplasmic domain of the H-2Ld class I major histocompatibility complex molecule is differentially accessible to immunological and biochemical probes during transport to the cell surface.

An antiserum was generated against a synthetic peptide corresponding to a portion of the cytoplasmic domains of the H-2Ld and H-2Db class I major histocompatibility complex molecules of the mouse. This antibody preparation, R4, binds exclusively to endoglycosidase H-resistant H-2Ld/Db molecules which are not associated with beta 2-microglobulin. Interestingly, acquisition of resistance to endoglycosidase H precedes acquisition of R4 reactivity by 30 min. R4-reactive H-2Ld and H-2Db molecules occur on the cell surface and are phosphorylated in vivo. Other studies show that the tyrosine in the cytoplasmic domain is accessible to radioiodination on only a subset of H-2Ld molecules, and that the two-dimensional electrophoretic profiles of phosphorylated H-2L/Db molecules, of R4-reactive molecules, and of H-2Ld molecules radiolabeled on this cytoplasmic domain tyrosine are virtually identical. R4-reactive H-2Ld molecules do not undergo the peptide- and beta 2-microglobulin-induced conformational changes characteristic of free class I major histocompatibility complex heavy chains. The accessibility of the H-2Ld cytoplasmic domain to R4 and to radioiodination late in biosynthesis and its biological significance are discussed.

Biochemical and functional characteristics of soluble MHC molecules derived from H-2Ld/Q10d chimeric gene.

We have constructed a chimeric class I gene in which the 5' half of the H-2Ld gene is linked to the 3' half of Q10d. The resulting H-2Ld/Q10d protein is homologous to the native H-2Ld heavy chain for the three external domains except for an Arg to His substitution at position 260. The transmembrane and intracytoplasmic domains of the H-2Ld chain are replaced by the short low hydrophobic transmembrane-like domain of the Q10d chain. Following DNA-mediated gene transfer into mouse L cells, transformants were selected for the presence of specific mRNA. Radiolabelling and immunoprecipitation analysis revealed secretion of a 48-46 kd chain weakly associated with beta 2-microglobulin. This molecule reacts with H-2Ld-specific mAb that identify determinants on the first and second domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum, but is not recognized by a mAb specific for a determinant of H-2Ld third domain. The integrity of antibody reactivity of the first and second domains together with beta 2-microglobulin association suggest that our molecule may be considered a good soluble counterpart of the native membrane H-2Ld molecule with which to perform functional studies. In order to analyze the immunogenic capacities and T-cell recognition of the soluble H-2Ld molecules, T-cell lines were produced from mice of various inbred strains immunized with supernatant from H-2Ld/Q10d-transfected fibroblasts. Characterization of these T cells revealed that they expressed a CD4+CD8- phenotype, and recognized H-2Ld/Q10d products in a class II-restricted manner.

Discrepancy between in vitro measurable and in vivo virus neutralizing cytotoxic T cell reactivities. Low T cell receptor specificity and avidity sufficient for in vitro proliferation or cytotoxicity to peptide-coated target cells but not for in vivo protection.

The TCR-alpha beta of CTL recognize peptide Ag in association with MHC class I molecules. TCR binding should be highly specific to guarantee pathogen specificity and to avoid self-reactivity. Therefore, the in vivo relevance of T cells exhibiting cross-reactivities in vitro and the respective role of the TCR affinities involved are not clear. To analyze high and low avidity T cell activities both in vitro and in vivo, we investigated primary and clonal CTL responses specific for the lymphocytic choriomeningitis virus nucleoprotein 118-126 epitope in association with the two closely related H-2Ld or H-2Lq molecules. As expected, we found highly specific class I-allele-restricted CTL responses when antiviral protection or immunopathology in vivo and lysis of virus infected target cells in vitro were analyzed. In contrast, the CTL were MHC crossreactive and thus considerably less discriminatory against targets expressing high MHC-peptide densities and in proliferation assays. The data show that relatively high TCR avidities are required for virus neutralization in vivo, in contrast to in vitro analyses of peptide-coated target cells or proliferative T cell responses that may engage TCR of low avidity and broad specificity and therefore may not reflect biologically relevant TCR avidities.

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.

The association of H-2Ld with human beta-2 microglobulin induces localized conformational changes in the alpha-1 and- 2 superdomain

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2Ld molecule exhibited enhanced cross-reactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2Ld class I hybrid molecules were then used to establish the importance of the alpha-2 and -3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2Ld hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2Ld molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2Ld can affect the conformation of the alpha-1 and -2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.

An H-2Ld hybrid molecule with a Qa-2 alpha-3 domain and phosphatidyl-inositol anchor is not recognized by H-2Ld-specific cytotoxic T lymphocytes.

Ld/Q7d, a hybrid molecule consisting of alpha-1 and alpha-2 domains from H-2Ld and alpha-3 and carboxy-end components from Q7d, was expressed on the surface of CRL-3A rat liver cells. This molecule retained serologic H-2Ld epitopes. The Ag is attached to the cell membrane through a phosphatidyl-inositol linkage, characteristic of Qa-2 molecules. Both bulk cultured and cloned H-2Ld alloreactive CTL as well as H-2Ld restricted vesicular stomatitis virus-specific CTL lyse CRL-3A cells which express H-2Ld but show little or no lytic activity on cells which express the Ld/Q7d hybrid. These cells also fail to act as cold target competitors for alloreactive anti-H-2Ld CTL. However, cells expressing Ld/Q7d are not resistant to CTL mediated lysis because they can be killed in the presence of lectin. These data indicate that recognition of polymorphic class I CTL epitopes in the alpha-1 and alpha-2 domains are influenced by the structure of the carboxy-end of the molecule.

Species-specific structural differences in the alpha 1 + alpha 2 domains determine the frequency of murine cytotoxic T cell precursors stimulated by human and murine class I molecules.

The frequency of murine CTL precursors (CTLp) that recognize the human histocompatibility Ag HLA-A2 and HLA-B7 was measured and found to be approximately two orders of magnitude lower than the frequency of CTLp that recognize murine H-2 alloantigens. The possible contribution of other cell surface molecules to this difference in response was addressed by expression of the H-2Ld molecule on a human cell and the HLA-B7 molecule on a murine cell. It was found that both human and murine H-2Ld expressing cells elicited comparable levels of H-2Ld specific CTL. Although murine HLA-B7 positive cells stimulated a higher frequency of HLA-B7-specific CTLp than did human cells, this appeared to be largely due to stimulation of CTLp that recognized HLA-B7 in the context of H-2 molecules; consequently, it was concluded that the difference in the frequency of murine CTLp elicited by human and murine class I Ag is due to species specific structural differences in these molecules. The regions of the class I molecule that were responsible for this difference were mapped using chimeric class I molecules constructed to replace domains of the human molecule with their murine counterparts. It was found that the frequency of CTLp is controlled by structures within the alpha 1 and alpha 2 domains of the molecule. These results are discussed in the light of models for T cell recognition of class I Ag and the diversification of the T cell receptor repertoire.