Biomedical subjects

T H Hansen

Publications and source records attributed to T H Hansen.

At least 55 records · Page 3Linked to original sources

PubMed1994

Exogenous peptide ligand influences the expression and half-life of free HLA class I heavy chains ubiquitously detected at the cell surface.

A pool of free HLA class I chains has been detected at the plasma membrane of all cells concomitantly expressing folded and assembled class I molecules. To determine the origin of these free HLA heavy chains, we have examined the biosynthesis of a single HLA class I molecule, HLA-B27, expressed by a murine cell line (L-B27). In L-B27 cells, as previously shown in Epstein-Barr virus-transformed lymphoblastoid cell lines, a precursor/product relationship exists, early in biosynthesis, between free (HC10-reactive) and beta-2-microglobulin (beta 2m)-associated (W6/32-reactive) class I heavy chains as demonstrated by pulse/chase experiments. At later stages in class I biosynthesis, both HC10- and W6/32-reactive heavy chains display complex oligosaccharides and accumulate at the cell surface. HC10- and W6/32-reactive molecules are both very stable at the cell surface, with half-lifes (t1/2) of > 7 h and approximately 4 h, respectively. Interestingly, cell surface expression and turnover of HC10- and W6/32-reactive molecules were affected by the addition of peptide ligands to the culture media. Culturing cells in the presence of HLA-B27 ligands resulted in the increased expression of W6/32-reactive molecules and the decreased expression of HC10-reactive molecules. Moreover, addition of exogenous peptide extended the t1/2 of W6/32-reactive molecules to > 7 h and reduced that of HC10-reactive molecules to 4 h. These results indicate that surface HC10-reactive molecules result largely from W6/32-reactive molecules following peptide and beta 2m dissociation. Therefore, HC10-reactive species are not only the precursors but also the end products in class I biosynthesis.

Adult↗

PubMed1994

Bcl-2 is upregulated at the CD4+ CD8+ stage during positive selection and promotes thymocyte differentiation at several control points.

In vivo thymocyte maturation models were used to investigate the differentiation role of Bcl-2. In alpha/beta T cell receptor (TCR) class II-restricted transgenic mice, Bcl-2 was upregulated at the CD4+ CD8+ stage during positive selection. The lckpr-bcl2 transgene was bred onto MHC classes I-I- and II-I-, MHC-I-, and alpha/beta TCR backgrounds to determine whether Bcl-2 promoted thymocyte maturation in the absence of coreceptor-MHC interaction. Bcl-2 rescued CD8+ thymocytes in class I-I- and alpha/beta TCR in mice; however, they were not exported to the periphery. Bcl-2 had no effect on CD4 lineage maturation in class II-I- mice. No single-positive thymocytes accumulate in MHC-I- mice despite overexpressed Bcl-2. Thus, Bcl-2 enables selection of certain TCRs on class II molecules and their differentiation along the CD8 pathway; however, Bcl-2 did not substitute for positive selection. In RAG-1-I- mice, Bcl-2 promoted differentiation to the CD4+ CD8+ stage. Bcl-2 can promote thymocyte maturation at several control points.

Animals↗

PubMed1994

Definition of TCR recognition sites on Ld-tum- complexes.

The P911 variant of the P815 mastocytoma was shown by Lurquin et al. (Cell 58:293, 1989) to elicit rapid tumor rejection in a syngeneic host. This rejection was mediated by Ld-restricted cytotoxic T lymphocytes (CTL) for which targets could be sensitized by the synthetic peptide designated tum- (P91A-.12-24). In a previous study, T cell clones specific for Ld-tum- complexes displayed very restricted TCR usage and a characteristic TCR motif in the V alpha CDR3 region, predicted to interact with peptide. However, in contrast to the majority of Ld peptide ligands that are nonamers, the tum- peptide is a 13-mer and its sequence does not fit the Ld binding motif. Thus, to define shorter versions of the tum- 13-mer and residues involved in TCR recognition, nonamer derivatives were synthesized and compared in several different binding and functional assays. From these comparisons, the peptide TQNHRALDL was found to be the optimal nonamer. CTL recognition of Ala-substituted analogues of this peptide indicated that the His and Arg residues at positions 4 and 5 are important for TCR contact. We propose that these basic residues of the tum- peptide interact with the previously defined acidic residues in the CDR3 region of several TCR known to recognize Ld-tum- complexes.

Amino Acid Sequence↗

PubMed1994

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Aged↗

PubMed1993

Model for the in vivo assembly of nascent Ld class I molecules and for the expression of unfolded Ld molecules at the cell surface.

To characterize the process of class I assembly and maturation, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of intracellular and surface Ld molecules can be detected in an alternative conformation designated Ldalt1. Nascent Ldalt molecules are non-peptide ligand associated and are weakly associated with beta 2-microglobulin (beta 2m). Unexpectedly, when monoclonal antibodies were added directly to the lysis buffer, significant amounts of Ldalt/beta 2m heterodimer were detected, suggesting that beta 2m association is not necessarily sufficient to induce Ld conformation. By contrast, addition of peptide to cell lysates rapidly induced the folding of beta 2m-associated Ldalt to conformed Ld. Furthermore, the time course and dynamics of this conversion correlated precisely with peptide binding to Ld. The precursor-product relationship of Ldalt and conformed Ld was also visualized in vivo by pulse-chase analysis of BALB/c splenocytes. To investigate the factors that regulate intracellular transport of class I molecules, expression of Ld was studied in the peptide transport-deficient cell line, RMA.S-Ld, and in beta 2m-/- splenocytes. In contrast to wild-type cell lines, both Ldalt and conformed Ld are poorly expressed at the cell surface of RMA.S-Ld and beta 2m-/- splenocytes. Therefore, surface expression of Ldalt is dependent upon the concomitant expression of conformed Ld molecules. To determine whether surface Ldalt molecules can result from melting of conformed Ld molecules, surface Ld molecules were loaded with several different known Ld peptide ligands. Complexes of Ld with different ligands were found to have dramatically disparate surface half-lives. Importantly, the Ld peptide complexes that turned over the most rapidly resulted in the most gain in surface Ldalt, implying that peptide dissociation can induce the accumulation of nonconformed Ld heavy chains at the cell surface.

Amino Acid Sequence↗

PubMed1993

Binding of peptides lacking consensus anchor residue alters H-2Ld serologic recognition.

CTL recognize class I MHC/peptide complexes on the surface of target cells. Crystallographic and serologic data have indicated that peptide ligands can influence the conformation of class I molecules and hence T cell recognition. How the binding of peptides with disparate sequence motifs affects the conformation of distinct regions within a class I molecule remains unknown. A series of site-directed mutants of the murine class I molecule H-2Ld was studied to address this question. These mutants were generated by in vitro mutagenesis and used to map the serologic epitopes recognized by a panel of Ld-reactive mAb. The influence of six different ligands on serologic recognition by these mAb was then examined. Of 12 mAb tested, only one, B22/249, was found to be significantly influenced by the bound peptide. Peptide discrimination by B22/249 was observed at the cell surface and in immunoprecipitates of Ld after incubation with two of the six ligands. The two peptides that caused suboptimal B22/249 recognition of Ld/peptide lack a proline at position 2, which is present in the other four peptides and has previously been defined as an anchor residue for Ld ligands. The epitope on Ld detected by mAb B22/249 includes residues 63 to 70 on the alpha 1 domain helix. Two of these residues are in pocket B, which computer modeling predicts to be in contact with the second residue of Ld-binding peptides. Therefore, these data imply that a mAb to a class I molecule can distinguish peptides with different motifs, possibly reflecting peptide-dependent conformational changes in the class I molecule.

Amino Acid Sequence↗

PubMed1993

Specific prolongation of allograft survival by a T-cell-receptor-derived peptide.

Allograft rejection results from the specific recognition by host CD8+ T cells of allogeneic major histocompatibility complex (MHC) molecules on the tissue graft. The specificity of this cellular response is determined by the molecular interaction of the T-cell receptor (TCR) on host T cells with the MHC molecule and its bound ligand on the grafted tissue. To better understand the precise manner by which the TCR interacts with the MHC-peptide complex and how to therapeutically intervene, we have studied the allogeneic response to the mouse class I MHC molecule Ld. In this report, the therapeutic potential of a synthetic peptide derived from the TCR V beta 8 variable region that predominates in responses to Ld was tested. This V beta 8-derived peptide was found to dramatically and specifically block the in vivo and in vitro allogeneic response to Ld. Furthermore, this specific blocking is not dependent upon the presence of V beta 8+ effector cells nor does the V beta 8 peptide bind to the Ld ligand binding cleft. We propose that this peptide functions as an antagonist, competing with the native TCR for recognition of the Ld molecule.

Amino Acid Sequence↗

PubMed1993

Alloreactive cytotoxic T lymphocytes generated in the presence of viral-derived peptides show exquisite peptide and MHC specificity.

The nature of alloreactivity to MHC molecules has been enigmatic, primarily because of the observation that allogeneic responses are considerably stronger than syngeneic responses. To better determine the specificity potential of allogeneic responses, we have generated alloreactive CTL specific for exogenous, viral-derived peptide ligands. This approach allowed us to critically evaluate both the peptide- and MHC-specificity of these alloreactive T cells. Exploiting the accessibility of the H-2Ld class I molecule for exogenous peptide ligands, alloreactive CTL were generated that are specific for either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis virus (LCMV) peptides bound by Ld alloantigens. Peptide specificity was initially observed in bulk cultures of alloreactive CTL only when tested on peptide-sensitized T2.Ld target cells that have defective presentation of endogenous peptides. Subsequent cloning of bulk alloreactive CTL lines generated to MCMV yielded CTL clones that had exquisitely specific MCMV peptide recognition requirement. All of the MCMV/Ld alloreactive CTL clones were also exquisitely MHC-specific in that none of the CTL clones lysed targets expressing MCMV/Lq complexes, even though Lq differs from Ld by only six amino acid residues and Lq also binds the MCMV peptide. This observation clearly demonstrates that alloreactive CTL are capable of the same degree of specificity for target cell recognition as are syngeneic CTL in MHC-restricted responses.

Amino Acid Sequence↗

PubMed1993

A highly polymorphic microsatellite in the class II Eb gene allows tracing of major histocompatibility complex evolution in mouse.

A hallmark of major histocompatibility complex (MHC) genes is their extraordinarily high level of polymorphism. Polymorphic residues on MHC molecules determine which peptide ligands they bind and present to effector T lymphocytes. Although the genetic mechanisms responsible for MHC polymorphism have been delineated, the timetable and the pathway of their diversification remain unclear. To trace MHC evolution, we have characterized a highly polymorphic microsatellite containing tandem repeats (TRs) of two tetranucleotide units, TGGA and GGCA, located at the 3' end of the second intron in the class II Eb gene of mouse. On the basis of length as well as sequence variations, 11 TR alleles were defined in 55 inbred mouse strains, which included MHC recombinant haplotypes and haplotypes derived from different subspecies of mouse. In this extensive sampling, a striking concordance was observed between the serologically identified class II proteins and the associated TR alleles. Examination of several strains carrying the same MHC haplotypes as well as strains carrying recombinant MHC haplotypes indicates that TR alleles are extremely stable. These observations suggest that TR polymorphism predates the separation of various subspecies of mouse. On the basis of sequence divergence, a genealogical tree has been constructed to depict evolution of the different TR alleles. Finally, evidence is presented that suggests this microsatellite polymorphism is generated by slipped-strand mispairing during DNA replication.

Alleles↗

PubMed1993

Major histocompatibility complex-specific prolongation of murine skin and cardiac allograft survival after in vivo depletion of V beta+ T cells.

The preferential usage of certain T cell receptor (TCR) V beta genes has been well established in several major histocompatibility complex (MHC)-restricted immune responses. However, V beta usage among allogeneic responses remains unclear. Because recent findings of ours and others indicate that V beta 8 predominates in certain Ld-restricted, peptide-specific responses, we examined the V beta 8 usage in allogeneic responses to Ld. To selectively recognize the Ld molecule, cells from BALB/c-H-2dm2 (dm2), the Ld-loss mutant mouse, were stimulated in vitro or in vivo with wild-type BALB/c cells. We report here that after the intraperitoneal administration of the anti-V beta 8 monoclonal antibody (mAb) F23.1, peripheral V beta 8 T cells were depleted from dm2 mice. This in vivo depletion abrogated the ability of dm2 splenocytes to mount a primary response to Ld molecules. This abrogation was specific, since the response of V beta 8-depleted dm2 cells to Kb/Db antigens was the same as that of control nondepleted dm2 cells. Furthermore, in vivo depletion of V beta 8 cells was found to cause a dramatic prolongation of Ld-disparate skin grafts (mean survival time [MST] 22.1 +/- 2.1 vs. 10.3 +/- 1.1 d for saline-treated controls, or 10.9 +/- 1.7 d for controls treated with mAb KJ23 to V beta 17). By contrast, V beta 8 depletion had no effect on recipients grafted with haplotype-mismatched skin or single Dk-locus-disparate skin. These findings demonstrate that V beta 8+ T cells predominate in allogeneic response to Ld but not other alloantigens. The effect of V beta 8 depletion was found to be even more dramatic on recipients grafted with Ld-disparate vascularized heart transplants (MST > 100 vs. 8.6 +/- 0.5 d for controls). In total, these findings establish the efficacy of using mAb to the V beta gene family to specifically and significantly enhance the survival of allografts. The implications of detecting V beta 8 usage in both alloreactive or MHC-restricted TCR responses to the same class I molecule are discussed.

Animals↗

PubMed1993

Residues in pockets B and F of HLA-B27 are critical in the presentation of an influenza A virus nucleoprotein peptide and influence the stability of peptide - MHC complexes.

Six pockets, designated A through F, which extend from the peptide binding site of class I HLA molecules, have been postulated to play an important role in determining peptide binding specificity. HLA-B27 mutant molecules with single amino acid substitutions at residues 9his-->phe, 24thr-->ser, 45glu-->thr, and 67cys-->ala in pocket B; 114his-->asn in pocket D; and 116asp-->phe in pocket F have been generated and characterized for their capacity to present an influenza A nucleoprotein peptide (NP 383-391) for cytotoxic T lymphocyte recognition. We report here that substitutions in residues 45, 67, and 116 affect presentation of NP 383-391 when peptide is processed and loaded during viral infection. Using 125I-labeled NP peptide, we demonstrate that substitutions in residues 67 and 116 alter the stability of NP-HLA-B27 complexes. A substitution at position 9 of the NP peptide complements the mutation introduced at residue 116, suggesting that the NP peptide binds with its carboxy terminal amino acid in pocket F. These findings indicate that polymorphic residues within pockets B and F of HLA-B27 play a crucial role in peptide binding and stability of peptide-MHC class I complexes. Furthermore, our results suggest that substitutions at allele-specific residues within pockets B and F alter the stability of NP-HLA-B27 complexes resulting in the diminution or abrogation of NP presentation during viral infection.

Adult↗

PubMed1992

Extensive peptide ligand exchange by surface class I major histocompatibility complex molecules independent of exogenous beta 2-microglobulin.

Certain class I major histocompatibility complex molecules expressed on live cells have been shown to bind exogenous peptide ligands. However, it remains controversial whether this binding occurs by peptide exchange or to empty surface class I molecules. In this report we compare the surface binding and dissociation of two virus-derived ligands of the Ld class I molecule of the mouse. The peptide ligands were previously identified in immune responses to cytomegalovirus or lymphochoriomeningitis virus as immunodominant, optimally sized, and Ld restricted. Ligand dissociation was monitored on live cells indirectly by measuring the surface turnover of Ld-peptide complexes or directly by using labeled peptides. The cytomegalovirus-derived and lymphochoriomeningitis virus-derived peptides appeared to dissociate relatively rapidly; however, the cytomegalovirus-derived peptide had a more rapid off-rate than the lymphochoriomeningitis-derived peptide. Furthermore, these rates of dissociation appear to span that seen with endogenous Ld-associated peptides expressed by cells at 37 degrees C. Exploiting the extraordinary accessibility of the surface Ld ligand binding site we developed an assay to quantitate peptide ligand exchange. Cells were precoated with saturating amounts of unlabeled peptide by overnight incubation and were then tested for secondary binding of labeled peptides in a 4-h assay. Our results unequivocally demonstrate the potential for surface class I molecules to undergo peptide exchange. Furthermore, peptide exchange was found to be largely independent of exogenous beta 2-microglobulin. This result implies that beta 2-microglobulin association and not beta 2-microglobulin exchange is the critical factor in peptide exchange by surface class I molecules. Because of the exquisite ability of T cells to discriminate different amounts of ligand bound to class I, the binding of exogenous peptides could play a critical role in normal or aberrant immune responses.

Animals↗

PubMed1992

[Fever of unknown origin (febris continua e causa ignota)].

Fever can be recognized as a higher set-point of the normal temperature regulation which is controlled by the center in the anterior part of hypothalamus. The change in this set-point is induced by interleukin-1 (IL-1) which is the common mediator of exogenic and endogenic pyrogenic factors. IL-1 is believed to act through an induction of a prostaglandin E cascade. The normal diurnal variation in temperature can often be recognized in infectious diseases but not always in non-infectious conditions. Four different fever curves can be defined but are without differential diagnostic importance, however, septic fever curves are more likely to occur in bacteremic patients. Comparison of the most important investigations about PUO since 1960 shows that the follow-up investigations revealed a high percentage of undiagnosed cases and that the mortality due to conditions related to PUO was 6-8%. Among the other investigations, a total of 83% were diagnosed: 23% had cancer, 33% had infections, 11% had collagenic diseases, 17% had other causes and 16% were undiagnosed. To establish the diagnosis in cases of PUO, liver biopsy can be of diagnostic value especially in patients with hepatomegaly. Abdominal CT-scan, ultrasonography and Gallium 67 scintigraphy are equal in sensitivity and specificity and can supplement each other with diagnostic information. Leucocyte scintigraphy can detect local inflammatory processes. Laparotomy or laparoscopy have high diagnostic values and can be considered in patients with signs of involvement of abdominal organs if no diagnosis has been established after noninvasive investigations. Lymphography gives only limited diagnostic information in cases of PUO.

Fever of Unknown Origin↗

PubMed1992

Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

Animals↗

PubMed1992

T cell receptor rearrangements in various S49 lymphoma sublines.

The S49 cell lines are a unique series of tumor sublines isolated from a single BALB/c thymoma. Several different sublines were previously isolated from non-mutagenized cells using pharmacologic agents that would select for different stages of thymic development. In this report we show that all seven of the sublines studied express TL class I Ag confirming their derivation from immature thymocytes. This uniform TL expression is in contrast to the previously characterized locus-specific shut off of Kd,Dd, and/or LdAg by various S49 sublines. Furthermore, S49 sublines were found to display disparate CD4/CD8 expression. Whereas the unselected subline is a CD4+/CD8+ double positive, each of the selected sublines is singly positive for either CD4 or CD8. All seven sublines were found to be CD3+ and express alpha beta TCR heterodimers. To establish whether the S49 sublines have a monoclonal or polyclonal origin, their TCR rearrangements were compared. Based on the detection of identical but unusual TCR gamma rearrangements and similarity of the alpha and beta rearrangements, we propose that the S49 sublines probably had a monoclonal origin. However, significant differences between the TCR alpha and beta gene rearrangement were observed, suggesting that these sublines have undergone further differentiation at TCR loci in addition to CD4/CD8 and MHC loci. Evidence is presented that much of this phenotypic diversity preceded their in vitro selection.

Animals↗

PubMed1991

[Interactions between oral contraceptives and other drugs].

Failures of oral contraceptives are possible when combined with rifampicin or antiepileptics, especially phenobarbitone and phenytoin. The mode of action is shown by clinical trials to be due to induction of hepatic enzymes thus increasing the steroid metabolism. Failure or oral contraceptives has occurred with the concomitant use of antibiotics, i.e. ampicillin and sulfonamides. Clinical trials have focused upon the changes in the intestinal flora induced by antibiotics. This might influence the enterohepatic circulation of estrogen and thereby decrease reabsorption of estrogen, but this has not been definitely proved. The failures may be caused by individual pharmacokinetics of oral contraceptives. Oral contraceptives are able to influence the pharmacodynamics of various other drugs metabolized by oxidation or conjugation but besides an increased pharmacological effect of prednisolone and increased toxicity of imipramine the clinical importance is uncertain.

Anti-Bacterial Agents↗

PubMed1991

Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes.

We have taken advantage of some unique properties of H-2Ld to investigate the determinant density requirements for cytotoxic T lymphocyte (CTL) priming versus effector function and to correlate the determinant density requirements with CD8 dependency. In a previous study (Lie, W.-R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, and T. H. Hansen. 1990. Nature [Lond.]. 344:439), we demonstrated that culturing normal cells with peptides known to be restricted by H-2Ld led to a two- to fourfold increase in surface Ld expression. In the present study, we demonstrate the generation of Ld-restricted, peptide-specific in vitro primary CTL by culturing spleen cells with murine cytomegalovirus or tum- peptide at concentrations previously shown to result in maximum induction of Ld expression. Target cells can be sensitized for recognition by these CTL with lower dose of peptide than are required for the primary sensitization. This demonstrates differences in the determinant density requirements for priming versus effector function. The in vitro primary CTL generated with peptide can weakly lyse target cells that express the determinant endogenously, and CTL lines and clones capable of strong lysis of endogenous expressors are easily obtained. In both cases, target cells treated with exogenous peptide are lysed better than target cells expressing antigen endogenously. This suggested that there are differences in the determinant density of peptide-fed versus endogenous targets. This interpretation was substantiated when it was observed that the level of lysis of target cells expressing endogenous determinants correlated inversely with the amount of peptide required to sensitize targets for recognition by various tum- -specific CTL clones. Furthermore, simultaneous titration of both the peptide used to treat target cells and the antibody to CD8 revealed that the various CTL clones analyzed displayed widely disparate CD8 dependencies. In each case, the CD8 dependency correlated inversely with the determinant density requirement. Therefore, CD8 dependency of CTL is relative, but shows an absolute and quantitative correlation with their dependency on determinant density. These findings suggest that under physiologic conditions, where only low determinant densities are likely to be encountered, all CTL clones will show at least partial CD8 dependency.

Amino Acid Sequence↗

PubMed1991

Mutation at amino acid position 133 of H-2Dd prevents beta 2m association and immune recognition but not surface expression.

The histocompatibility loss mutation H-2dm6 was derived from a mouse treated with the chemical mutagen ethylnitrosourea, and previous mapping studies implicated Dd as the affected locus. Southern blot analyses of DNA from H-2dm6 cells did not detect major deletions in the Ddm6 gene, suggesting that H-2dm6 was different from the previously characterized D region mutants H-2dm1 and H-2dm2. RNA blot analysis identified Ddm6 transcripts of appropriate size and a Ddm6 protein was immunoprecipitated from biosynthetically labeled H-2dm6 cells. Interestingly, the Ddm6 protein showed no beta 2m association and was only precipitated by a mAb to the alpha 3 domain. Furthermore, oligosaccharide maturation and low levels of surface expression of Ddm6 molecules were detected. However, the surface Ddm6 was nonfunctional as a target Ag in in vitro cytotoxicity assays, consistent with its original in vivo detection as a loss mutation. Sequence analyses of Ddm6 cDNA identified a single nucleotide base difference from wild-type, resulting in the substitution of a Trp to Arg at position 133. The significance of this substitution is discussed in the context of other class I expression variants.

Animals↗