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T H Hansen

Publications and source records attributed to T H Hansen.

At least 91 records · Page 5Linked to original sources
PubMed1986

Responses of B6.C-H-2bm12 to heterologous insulins show no correlation with the putative gene conversion but define Iabm12 as functionally unique.

The IA mutant mouse strain, B6.C-H-2bm12 (bm12) has been used to address several important questions for the role of Ia molecules in immune responses to foreign antigens. Numerous publications using bm12 mice have led to conclusions concerning (1) the number and relative importance of functional sites on Ia molecules; (2) the effects of qualitative versus quantitative differences in Ia; (3) whether T cells recognize Ia sequence or conformation; and (4) if gene conversion events, such as the one that putatively occurred in bm12, transfer functional Ir gene epitopes. Because of the importance of these conclusions, as well as their controversial nature, we have undertaken a comprehensive and systematic analysis of the aberrant immune response of bm12 mice to heterologous insulin. Responses to beef, horse, and sheep insulin were compared in B6 and bm12 mice by T cell proliferation, enumeration of plaque-forming cells, and quantitation of serum antibody levels. Various doses of antigen were administered and the kinetics of each response was monitored at various times. The findings of these studies suggest (1) B6 and bm12 mice both mount comparably high levels of response to sheep and horse insulins; (2) in contrast to the good response of B6 mice to beef insulin, bm12 mice showed dramatically impaired responses, as evident from both the lower magnitude of the response in all three assays as well as the difference in the kinetics of the response in B6 and bm12 mice; and (3) the response to sheep insulin is controlled by IA and IE encoded genes. These new findings differ from and extend previously published reports using bm12 mice, and therefore have substantive implications on the above stated conclusions regarding recognition of Ia. One such implication is that the bm12 gene conversion did not result in the transfer of a functional epitope for sheep insulin, but rather resulted in the creation of a functionally unique Ia molecule. Furthermore, this critical definition of the Ir gene lesion in bm12 permits us to address mechanistic questions regarding the nature of its Ir gene defect to beef insulin.

Animals↗
PubMed1985

Characterization of a dominant anti-Ia idiotype using the IA mutant mouse strain B6.C-H-2bm12.

Initial studies of antibody recognition of Ia molecules using the IA mutant mouse strain bm12 suggested that two anti-Ia monoclonal antibodies (mAbs), 25-9-17 and 34-5-3, share several features: (1) indistinguishable serologic specificity including a lack of reactivity with Iabm12, (2) binding of the same spatial epitope (cluster), and (3) definition of a cross-reactive idiotype (CRI) as defined by xenogeneic antisera. In the present study we characterize a rabbit anti-idiotype (anti-Id) to 25-9-17 by affinity chromatography, and demonstrate that it detects at least two distinct idiotopes, one shared by 25-9-17 and 34-5-3 designated CRI (25-9-17) and one unique for 25-9-17 molecules. Experiments were also undertaken to determine whether CRI (25-9-17) represents a measurable component of allogeneic humoral responses to Iab antigens. By both absorption analyses of a polyspecific antiserum and production of antigenically-restricted antisera using bm12 mice, CRI (25-9-17) was found to represent a significant proportion of the antibodies to Iab. By several criteria it was shown that the CRI (25-9-17)+ molecules were among the antibodies defining the serologic lesion of bm12 mice. In preparation for future studies to alter in vivo T-cell responses involving recognition of Ia (e.g. graft vs host disease and allogeneic transplant rejection), various immunization protocols and mouse strains were tested for induction of Id (25-9-17) following in vivo administration of various anti-idiotypic reagents. Rabbit anti-Id (25-9-17) successfully induced CRI (25-9-17) positive molecules in all strains tested regardless of IA or Ig genotype. Moreover, some of these treated mice produced antibodies to an Ia determinant missing on bm12 cells, suggesting that they recognize the same serologic determinant as mAb 25-9-17.

Animals↗
PubMed1985

The murine bm12 gene conversion provides evidence that T cells recognize predominantly Ia conformation.

The structure of the highly polymorphic Ia dimer is the genetically determined factor that controls the immune response to foreign antigens, albeit the mechanism remains unresolved. However, it is clear that, in diverse immune responses, effector T lymphocytes require recognition of self-Ia and foreign antigenic determinants on the surface of an antigen-presenting cell or an antibody-secreting B cell. Furthermore, a single Ia molecule has been found to possess several independently acting functional domains. In this report T-cell recognition of Ia was limited to a single, defined structure by using the Ia mutant mouse strain B6.C-H-2bm12 (bm12). The Ia determinant being recognized is the site of the mutation that represents a difference in three of five amino acid residues in a hypervariable region of its beta chain. This mutation has been proposed to have resulted from a gene conversion-like event and is known to have functional importance. Recognition of the bm12 mutation site was studied here in in vitro cultures of T cells generated against Iabm12 antigens. The specificity of these alloreactive T cells was tested by using stimulator cells expressing various Ia alloantigens of known structure. Our findings provide direct genetic evidence that T cells recognize predominantly conformational determinants on Ia molecules and not their primary structure. The implications of these findings on our understanding of the genetic control of the immune response and the potential to modulate these responses in an antigen-specific way are discussed.

Animals↗
PubMed1985

Long-term passive enhancement of allogeneic skin grafts with monoclonal antibodies.

Monoclonal antibodies (mAb) to graft-specific class I or class II major histocompatibility antigens were tested for their ability to enhance the survival of allogeneic skin transplants. Mutant mouse strains were grafted with wild type tissue to restrict the antigenic differences being recognized. For allogeneic recognition of the class I antigen Ld, mutant BALB/c-H-2dm2 (dm2) mice were grafted with wild type BALB/cKh skin, and two dm2 anti-BALB/cKh mAb, 23-10-1 and 30-5-7, were tested for their ability to enhance. The anti-Ld antibody 23-10-1 (IgM) was found not to enhance the survival of BALB/c skin on dm2 mice. 30-5-7, however, an IgG2a antibody of indistinguishable specificity from 23-10-1, prolonged graft survival for approximately 5 days. For recognition of selected Iab determinants, mutant B6.C-H-2bm12 (bm12) mice were grafted with wild type B6/Kh skin, and mAb specific for the serological change(s) in bm12 were tested for their ability to enhance. The anti-Iab antibody 25-9-17 (IgG2a) was found not to enhance B6 grafts on bm12 mice. However, the enhancement seen with 25-9-17 using (C3H X bm12)F1 recipients was extraordinary, such that treated mice had a mean survival time three times that of the controls. Since 25-9-17 is of C3H origin, these results suggest that allotype (or possibly idiotype) compatibility is important in antibody enhancement. Another anti-Iab antibody 28-16-8 (IgM), also of C3H origin, failed to enhance a B6 graft on (C3H X bm 12)F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Structural definition of a family of Ld-like molecules distributed among four of seven haplotypes compared.

Comparative tryptic peptide analyses were performed on 12 different D region molecules representing seven different haplotypes. The Dd, Dq, and Dw16 regions were shown to encode multiple, antigenically distinct molecules (Dd Ld, Dq Lq Rq, and Dw16 Lw16, respectively). In addition, each of these molecules was found to have a unique primary structure, implying that they are the products of separate genes. However the previously described Rd molecule, which was identified by sequential immuno-precipitation and 2-D gel analyses, was indistinguishable from Ld by tryptic peptide mapping, implying that these two molecules may be products of the same gene. The Db, Ddx, Dk, and Dp regions were found to determine a single molecule with the reagents tested. Intra- and/or inter-haplotype comparisons of the peptide maps of each of these D region molecules revealed widely disparate structural relationships. For example, the Db, Dq, Lq, Rq, Dw16, and Lw16 molecules all showed striking homology with the Ld molecule. Members of this family share between 43 to 55% peptide homology with Ld, indicating a high conservation of primary structure (greater than 90%). However, because Dq and Dw16 region-encoded molecules show no exceptional relationship to each other, the portion of the conserved sequence is not the same for each of these Ld-like molecules. By contrast, comparisons of the Dk, Dd, Ddx, and Dp molecules with Ld or with each other revealed tryptic peptide homologies ranging from 22 to 38%, suggesting a sequence homology of 70 to 85%. When compared with the Kb molecule, each of the D region molecules showed between 21 to 36% peptide map homology (70 to 85% sequence homology). These studies indicate, therefore, that there is a family of Ld-like molecules representing several distinct haplotypes. This definition of a highly homologous family of D region molecules suggests that many D-region molecules have evolved from an Ld-like primordial gene and that in different haplotypes different portions of this prototypic structure have been maintained.

Amino Acid Sequence↗
PubMed1984

Primary structural evidence that the H-2Dq region encodes at least three distinct gene products: Dq, Lq, and Rq.

Class I gene products D, L, and R encoded by the D region of the H-2q haplotype were isolated by sequential immunoprecipitation from Nonidet P-40 extracts of B10.AKM murine spleen cells. Primary structural comparisons of these molecules were made by tryptic peptide map analyses and radiochemical amino acid sequence determinations. Partial NH2-terminal radiosequence analyses of the peptides generated by CNBr cleavage of these three molecules defined the Rq molecule as a gene product that is distinct from the Dq and Lq molecules. Comparative tryptic peptide map analyses of [3H]arginine-, [3H]lysine-, or [3H]mannose-labeled molecules were performed to determine if the Dq and Lq molecules could be distinguished. The two molecules were identical in their [3H]lysine-labeled peptides (17 in common) and [3H]mannose-labeled glycopeptides but could be differentiated by their [3H]arginine-labeled peptides (18 of 21 in common). These results suggest an amino acid sequence homology of 99% or greater between the Dq and Lq molecules. These data therefore provide structural evidence for three D region gene products and suggest that their antigenic differences are not due to post-translational glycosylation differences. Thus, the D region of the H-2q haplotype may provide an example of a relatively recent class I gene duplication, indicating that these genes are in a dynamic state of evolution through expansion or contraction of the gene pool.

Amino Acid Sequence↗
PubMed1984

Delineation of Ia:nominal antigen complementary determinants recognized by T cells in studies of gene complementation in response to insulin.

The immune response to beef insulin in mice is controlled by genes in the IA subregion. We have previously shown that B6.C-H-2bm12 (bm12) mice, an A beta gene mutation of B6, have a selective loss of responsiveness to beef insulin, whereas other IAb controlled responses such as (TG)AL and collagen are unchanged. F1 hybrid mice between two nonresponder genotypes Ik and Ibm12 were found to be good responders to beef insulin suggesting functional complementation. In this report, we define the cellular and molecular basis of this complementation by investigating the determinants on Ia molecules and nominal antigen that are recognized by (B10.A X bm12)F1 proliferating T cells. Genetic analyses demonstrated that the Ik region was the only nonresponder genotype that complemented Ibm12, thus restoring responsiveness to beef insulin. More precisely an IAk and not an IEk gene product was found to be responsible for this complementation. Antibody blocking studies furthermore showed that the A alpha b:A beta k hybrid Ia mediated the response to beef insulin in (B10.A X bm12)F1 mice. Clonal analyses of the response to beef insulin in these F1 mice confirmed these conclusions, because the insulin-specific response in all 21 F1-T cell clones studied thus far was found to be dependent upon presentation via the A alpha b:A beta k hybrid Ia molecule. Dissection of the antigenic specificity of the F1-T cell clones demonstrated recognition of at least two insulin determinants, one A-loop (A8-A10) associated and the other non-loop (A4 or B chain) associated. Therefore these studies identify the molecular and antigenic basis of the Ir gene complementation seen in the response to beef insulin of (B10.A X bm12)F1 hybrids.

Animals↗
PubMed1984

Assessment of antigen-specific restriction sites on Ia molecules as defined by the bm12 mutation.

To further delineate the functional defects of bm12 mice in antigen presentation, we analyzed antigen-specific T lymphocyte clones derived from B6 or bm12 for their ability to recognize antigens presented on either B6 or bm12 APC. Both complex antigens, such as PPD and GAT, and restricted antigens, such as beef insulin, were used. Our results indicate that for complex antigens, more than 50% of the B6 T lymphocyte clones recognized antigens only if presented by B6 APC, whereas the rest could not discriminate B6 from bm12 APC. Similarly, bm12 T lymphocyte clones responding to complex antigens could be divided into two groups depending on the sources of the APC. We have also isolated B6 T lymphocyte clones specific for the more restricted antigen, beef insulin, to which bm12 failed to respond. All B6 T lymphocyte clones could be stimulated only with B6 APC and not with bm12 APC. These data are consistent with the notion that there are antigen-specific association sites on the Ia molecule, and that complex antigens have more than one such association site. Furthermore, these studies demonstrate that both the gain and loss determinants associated with the bm12 mutation are recognized by a significant number of bm12 and B6 antigen-specific T lymphocyte clones, respectively, thus defining the importance of this region of the A beta polypeptide chain in antigen presentation.

Animals↗
PubMed1984

Chemical and serologic definition of two unique D region-encoded molecules in the wild-derived mouse strain B10.GAA37.

Detailed serologic and biochemical characterization of D region products of the wild-derived mouse strain B10.GAA37 (Dw16) were performed and compared with previous studies of the D region products of the H-2d,b, and q haplotypes. Serologic analysis revealed that the antigens encoded by the Dw16 region express a unique combination of specificities defined by monoclonal antibodies (mAb) with established activity for the Ld and Dd molecules. Two out of five anti-Ld-reactive mAb reacted with B10.GAA37 cells, whereas one of three anti-Dd mAb showed B10.GAA37 reactivity. Sequential immunoprecipitation of B10.GAA37 antigens demonstrated the existence of at least two antigenically distinct molecules (designated Dw16 and Lw16) encoded by genes associated with the Dw16 region. Peptide map comparisons of the Dw16 and Lw16 molecules defined multiple differences in their primary protein structure, suggesting they are products of separate genes. Structural comparisons of the Lw16 and Dw16 molecules with the Ld and Dd molecules implied a) that the Dw16 and Dd regions did not result from a recent evolutionary divergence of a common primordial haplotype, and b) that the Lw16 and Dw16 molecules are more structurally homologous to each other than the Ld and Dd molecules are. Comparison of these findings with our previous studies of antigens encoded by the D regions suggest that each of these haplotypes has unique properties in terms of the number of gene products expressed and/or the structural relatedness of products of the same region.

Animal Population Groups↗
PubMed1983

Idiotypic and fluorometric analysis of the antibodies that distinguish the lesion of the I-A mutant B6.C-H-2bm12.

The serologic lesion of the I-A mutant mouse strain, bm12, was investigated with the use of monoclonal anti-Iab antibodies and anti-idiotypic (Id) reagents produced against these antibodies. In a fluorometric analysis, three different monoclonal anti-Iab antibodies (25-9-17, 34-5-3, 28-16-8) failed to bind bm12 cells, whereas two anti-Iab antibodies (25-5-16 and 17/227), which bound bm12 cells, showed about one-half the fluorescence intensity that they showed in binding to Iab antigens. Of the three monoclonal antibodies that failed to react with bm12 cells, two antibodies (25-9-17 and 34-5-3) were found to bind the same steric site on Iab molecules (cluster I). In contrast, the antibodies (25-5-16 and 17/227) that reacted with both Iab and Iabm12 antigens were found to bind a second distinct site (cluster II). The binding of antibody 28-16-8 to Iab antigens inhibited reciprocally the binding of cluster I and II anti-Iab antibodies, suggesting a possible third site, sterically located intermediate between the other two sites. To assess the relatedness of the antibodies defining the serologic lesion of bm12 mice, xenogeneic and syngeneic anti-Id reagents were produced against antibodies 25-9-17 and 28-16-8. By using these anti-Ids in a binding site-related inhibition assay, a cross-reactive idiotype was detected that is shared by 25-9-17 and 34-5-3 antibodies; thus these two monoclonal antibodies share several features, including 1) idiotypic determinants, 2) failure to bind bm12 cells, 3) binding the same spatial Iab site, and 4) having indistinguishable serologic fine specificity that corresponds with a previously defined predominant alloantigenic determinant recognized in the bm12 anti-Iab humoral response. Therefore, several parameters of antibody recognition of Ia can now be correlated with structural changes in Ia molecules. These findings will potentiate future studies of the T cell recognition of these same Ia epitopes.

Animals↗
PubMed1983

Expression of a bone marrow-associated Ly-6 determinant on the T cell population expanding in the lymph nodes of the autoimmune mouse strain MRL/Mp-lpr/lpr.

In this report we describe the reactivity patterns of two monoclonal anti-Ly-6.2 antibodies toward lymph node (LN) cells of the autoimmune MRL/Mp-lpr/lpr (lpr) and the normal congenic MRL/Mp-+/+ (+/+) mouse strains. The monoclonal antibody 34-11-3, which has previously been found to detect a LN-associated Ly-6.2 antigen, reacted with both lpr and +/+ LN cells. Another monoclonal antibody, 34-10-7, which has been demonstrated to detect a bone marrow- (BM) associated determinant, was found to react with a small proportion of +/+ LN cells and unexpectedly with a high percentage of lpr LN cells. The expression of this BM determinant found in the LN of lpr mice was age dependent, and its presence on Thy-1.2+ cells increased with the expansion of a subset of Lyt-1+2- cells. In contrast, dual labeling experiments showed that in +/+ and young lpr mice a small subset of Lyt-1+2+ cells express this BM-associated Ly6.2 determinant. Therefore these findings demonstrate that the lpr gene-dependent T cell expansion in the LN results in both aberrant expression and association of lymphocyte cell surface markers.

Animals↗
PubMed1983

Two-dimensional gel comparisons of murine H-2D region-associated antigens of different H-2 haplotypes.

The heterogeneity of histocompatibility antigens encoded within the H-2D region of murine chromosome 17 was investigated in several mouse strains using monoclonal antibodies. Sequential immunoprecipitation and two dimensional gel electrophoresis (2D) were used to determine the number and structural relatedness of antigenically distinct products of the same H-2D region. Strikingly different, haplotype-specific patterns were observed. The three antigenically distinct molecules, Dd, Ld and Rd, all encoded within the Dd region, each showed a unique constellation of spots by 2D analysis, although the Ld and Rd molecules showed more structural similarity to each other than either molecule showed to the Dd molecule. Comparisons of the Ld and Rd molecules using sequential precipitations and a cloned Ld gene suggested that they are not conformation or glycosylation variants of the same polypeptide, implying they have different primary amino acid sequences. Using an anti-Ld antibody, the Ldml molecule was isolated from cells of the Dd region mutant mouse strain B10.D2-H-2dml and was found to have a 2D gel profile clearly distinguishable from the profile of either Ld or Dd molecules isolated from the wild-type strain B10.D2. Of the three antigenically-distinct molecules determined by the Dq region, the 2D gel profile of the Rq molecule was different from that of the Dq and Lq, the latter two molecules being indistinguishable. In contrast to these findings, analysis of the gene products of the Db region failed to resolve either antigenic or molecular heterogeneity and the multiple, antigenically distinct gene products of the Dw16 region were indistinguishable by 2D comparisons. These haplotype specific variations in the structural relatedness and number of antigenically distinct molecules determined by D region genes support the hypothesis that class I MHC genes are in a dynamic evolutionary state of gene expansion and contraction.

Animals↗
PubMed1982

Ia.W39 expression correlates with a specific Ir function.

The xid- defective mouse (N X B6)F1 male and the I-Ab mutant of the B6 mouse bm12 have mutations on different chromosomes, and therefore in separate genes. However, both variant strains have a trait in common; namely, they are unresponsive to the A chain loop determinant(s) of beef insulin to which mice of the H-2b haplotype respond. We show here that in both mutant strains, this unresponsiveness is concurrent with the absence of the Ia.W39 specificity from the M phi and B cell surface. The concordance is specific for Ia specificity W39 and no other Ia determinant, suggesting that Ia.W39 is identical with the Ir epitope necessary for generating an immune response to a particular amino acid sequence of beef insulin. In the xid-defective mouse, the molecule carrying this antigen is synthesized but not expressed on the plasma membrane, presumably because a mature M phi and B cell subset is missing. By comparison, the bm12 mouse has mature B lymphocytes, but is unable to synthesize Ia.W39. Thus, bm12 has a structural mutation in the gene coding for Ia.W39, whereas xid is a mutant regulatory or maturation gene controlling the membrane expression of Ia.W39.

Animals↗
PubMed1982

Characterization of H-2Db antigens implies haplotype differences in the number of H-2 molecules expressed.

Investigation of the molecular heterogeneity of H-2Db region antigens by using monoclonal antibodies and complex alloantisera has yielded evidence for the existence of only one gene product in this region. Sequential immunoprecipitation and isoelectric focusing analysis suggest that the private H-2.m2 and public H-2.m64 specificities defined by monoclonal or alloantibodies are present on the same molecule. Blocking studies with these reagents indicate these specificities represent two determinants spatially separated on the H-2Db molecule. Functional studies suggest that effector T cells may be generated against both of these determinants or closely associated epitopes, but the majority of the T cells recognize an epitope associated with or identical to the private specificity H-2.m2. Studies of the D region-encoded products of haplotypes besides H-2b have shown two or more distinct molecules can be detected by anti-H-2.m64 in both the H-2d and H-2q haplotypes. The presence of H-2.m64 on a single Db molecule is unique and gives further evidence for the lack of other gene products encoded in this region. Speculations based on the molecular heterogeneity of the D region gene products are made concerning the evolution and genetic organization of H-2 genes.

Animals↗
PubMed1982

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12.

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.

Animals↗