Biomedical subjects
Publications and source records attributed to C D Surh.
H-2O is a recently described non-polymorphic mouse MHC class II molecule. Here, Lars Karlsson and colleagues describe the discovery of the H-2Ob and H-2Oa loci and the pattern of expression of H-2O, and speculate on the possible function of this unusual molecule.
To test whether T and B cell differentiation can proceed across species barriers, rat fetal liver (FL) cells were used to reconstitute SCID mice. Provided that the hosts were conditioned with light irradiation, i.v. injection of FL cells caused near-complete repopulation with rat-derived lymphohematopoietic cells, including myeloid and erythroid cells, Ia+ cells of the macrophage/dendritic cell lineages, and mature T and B cells. In keeping with the known hypersensitivity of SCID cells to irradiation, host hematopoietic cells in the chimeras were almost undetectable, even with hosts exposed to as low as 250 rad. In the case of T cells, the distribution of immature and mature cells in the thymus of rat FL----SCID chimeras closely resembled the normal rat thymus in terms of architecture and expression of CD4, CD8, and alpha beta-TCR molecules. Thymopoiesis was followed by the appearance of large numbers of typical rat CD4+ and CD8+ cells in spleen and lymph nodes. These organs also contained substantial numbers of rat B (mu+) cells. The data thus indicate that the xenogeneic environment of SCID mice is fully capable of sustained de novo differentiation of rat T and B cells.
To seek information on the potential lifespan of normal B and T lymphocytes, lymph node (LN) cells from unprimed mice were transferred to H-2-identical severe combined immunodeficiency (SCID) hosts. At a population level, the donor B and T cells survived for at least 10 mo post-transfer with no reduction in their numbers. In terms of antibody production, LN-injected SCID mice remained responsive to several different antigens and contained unprimed precursors of memory cells for greater than or equal to 6 mo post-transfer. Most of the B and T cells recovered from LN-injected SCID mice expressed the typical virgin phenotype of mature lymphocytes from young mice. These findings suggest that many of the transferred lymphocytes might have remained in interphase as virgin cells from the time of injection. This did not apply to all of the transferred cells, however, because 20-40% of CD4+ cells from long-term SCID hosts displayed a memory phenotype, 7% incorporated 2-bromodeoxyuridine over 5 d, and total numbers of B and T cells increased gradually (twofold) over a 10-mo period. Collectively, the data favor the view that the pool of mature B and T cells in adult mice is largely self sufficient: some of the cells proliferate, presumably in response to environmental antigens, but many mature cells can remain quiescent for prolonged periods. Input of new cells from the primary lymphoid organs continues, but at a much reduced rate relative to young life.
Long-term H-2-heterozygous a----(a x b)F1 bone marrow (BM) chimeras prepared with supralethal irradiation (1,300 rad) are devoid of Ia+ host BM-derived antigen-presenting cells (APC), but show quite strong host Ia expression in germinal centers, probably on follicular dendritic cells (a class of nonhemopoietic stromal cells). To examine whether Ia expression on these non-BM-derived cells is capable of inducing post-thymic tolerance of T cells, thymectomized irradiated (a x b)F1 mice were reconstituted with parent alpha stem cells and then, 6 mo later, given parent alpha thymus grafts. As measured by primary mixed lymphocyte reactions and V beta expression, the CD4+ cells differentiating in the thymus-grafted mice showed no detectable tolerance to the H-2 (Ia) antigens of the host. To examine whether the thymus-grafted mice contained immunologically significant quantities of host Ia antigens, long-term alpha----(alpha x b)F1 chimeras were injected with normal strain alpha CD4+ cells; the donor cells were recovered from thoracic duct lymph of the chimeras and tested for host reactivity in vitro. The results showed that Ia expression in the chimeras was sufficient to cause selective trapping of a substantial proportion of host-Ia-reactive CD4+ cells soon after transfer and, at later stages, to induce strong priming. Tolerance was not seen. The data place constraints on the view that T cell recognition of antigen expressed on cells other than typical BM-derived APC leads to tolerance induction.
The repertoire of mature class II-restricted T cells is generated through a complex process of selection whereby early T cells confront class II molecules in the thymus, especially on epithelial cells. Expression of class II molecules on such cells is prominent both in the cortex and in the medulla. We have identified a novel class II molecule, H-20, which is expressed only in epithelial cells of the thymic medulla and in B cells. The unusual tissue distribution and the nonpolymorphic nature of H-20 suggest that its function is different from that of classical class II molecules.
To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.
Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease that includes the presence of lymphoid infiltrates in portal tracts, high titer autoantibodies against pyruvate dehydrogenase-E2 (PDH-E2) and branched chain ketoacid dehydrogenase-E2 (BCKD-E2), and biliary tract destruction. The mechanism by which the autoimmune response is induced, the specificity of damage to the biliary epithelium, and the role of T cells in PBC are still unknown. To address these issues, we have taken advantage of a mouse mAb, coined C355.1, and studied its reactivity against a panel of liver tissue from normal subjects as well as a panel of liver specimens from patients with PBC, progressive sclerosing cholangitis, and chronic active hepatitis (CAH). C355.1, much like human autoantibodies to PDH-E2, reacts exclusively by immunoblotting with PDH-E2, binds to the inner lipoyl domain of the protein, and inhibits PDH-E2 activity in vitro. In addition, we have also attempted to develop cloned T cell lines that react with PDH-E2 and/or BCKD-E2 using liver biopsies from patients with PBC, compared with CAH. Although monoclonal C355.1 produced typical mitochondrial fluorescence on sections of normal liver, pancreas, lung, heart, thyroid, and kidney, it produced a distinct and intense reactivity when used to stain the bile ducts of patients with PBC. Nine of 13 PBC liver biopsies studied herein contained bile ducts on light microscopy, all of which reacted intensely at a 1:100 culture supernatant dilution of monoclonal C355.1. In contrast, although bile ducts of liver specimens from normals, CAH, and progressive sclerosing cholangitis also reacted with C355.1, such reactivity was exclusively mitochondrial and readily detectable only at a dilution of 1:2. More importantly, we generated CD4+, CD8-, alpha beta TCR+ cloned T cell lines from patients with PBC, but not from CAH, that produced IL-2 specifically in response to PDH-E2 or BCKD-E2.
The E2 component (acetyltransferase) of the pyruvate dehydrogenase (PDH) complex is the major mitochondrial autoantigen recognized by autoantibodies in patients with primary biliary cirrhosis (PBC). Previous work, using only a partial length rat liver cDNA clone of PDH-E2, demonstrated that the immunodominant epitope was localized to the lipoic acid binding site. Human PDH-E2, in contrast to rat PDH-E2, has two lipoic acid binding sites. By using a full length human cDNA for PDH-E2, and by preparation of multiple overlapping recombinant fragments, we have determined that three autoreactive determinants are present on human PDH-E2: two cross-reactive lipoyl domains, and an area surrounding the E1/E3 binding region. The dominant epitope was localized to the inner lipoyl domain whereas the outer lipoyl domain only showed a weak cross-reactivity, and only 1/26 PBC sera reacted weakly to the E1/E3 binding region area. By probing recombinant fusion proteins expressed from small restriction fragments of the inner lipoyl domain, we have found that a minimum of 75 amino acids (residues 146-221) were required for detectable autoantibody binding, and that 93 amino acids (residues 128-221) were necessary for characteristically strong antimitochondrial autoantibody recognition. Such a requirement for a large region suggests the possibility that a conformational autoepitope may be recognized. In addition, we have found that absorption of PBC sera with the purified mammalian PDH complex does not remove reactivity against Escherichia coli Ag. The possible implications for such results are discussed.
Immunization with recombinant human pyruvate dehydrogenase (PDH)-E2, the major autoantigen of primary biliary cirrhosis, readily induces a vigorous murine antibody response but does not generate hepatic disease. To determine the fine specificity of this response, 18 mAb were generated from three strains of mice and the reactive epitopes mapped. An initial examination of mAb suggested that they behaved similarly to the antimitochondrial autoantibodies in primary biliary cirrhosis (PBC) because i) all polyclonal antisera and 2 of 18 mAb reacted with all species of mammalian PDH-E2 examined including mouse PDH-E2, ii) 15 of 18 mAb inhibited PDH enzyme function, and iii) the reactivity of mAb toward rPDH-E2 were blocked by PBC sera. However, fine examination of the reactive sequences of the PDH-E2 complex revealed that antibodies identical to those in PBC patients were not produced by experimental immunization. In contrast to PBC, none of the mAb or murine polyclonal sera were able to react with protein X, a lipoic acid-containing component of the PDH complex previously shown to cross-react with PDH-E2 when probed with PBC sera. Although the epitopes for 12 mAb were localized within the inner lipoyl domain, none reacted with mouse PDH-E2 or cross-reacted with the outer lipoyl domain as observed in PBC. In addition, the epitopes of the two mAb which did react with all mammalian species of mitochondria were not localized within the PBC epitope. These findings indicate the highly immunogenic nature of the inner lipoyl domain of PDH-E2. The inability to elicit antibodies of the same specificity in mice, considered together with the highly localized autoantibody response in humans, suggests that antimitochondrial autoantibodies are most likely the result of specific breakdown of tolerance to a unique autoepitope.
Primary biliary cirrhosis is characterized by the presence of autoantibodies to mitochondria with specific reactivity to proteins of 74 and 52 kilodaltons (kd). The 74-kd mitochondrial protein is the E2 component--dihydrolipoamide acetyltransferase--of the pyruvate dehydrogenase complex, and the 52-kd protein is the equivalent E2 component--dihydrolipoamide acyltransferase--of the branched-chain alpha-keto acid dehydrogenase complex. Current methods for the detection of antibodies to these proteins lack specificity or sensitivity, or they are time-consuming and not readily available. We therefore developed an enzyme-linked immunoassay to quantify specific antimitochondrial antibodies in patients with primary biliary cirrhosis. Recombinant polypeptides coding for both the 74-kd and the 52-kd mitochondrial autoantigens were used to analyze 217 coded serum samples, including samples from 93 patients with primary biliary cirrhosis and 124 controls, for reactivity by our immunoassay, immunoblotting, and immunofluorescence testing. Serum samples from 89 of the 93 patients with primary biliary cirrhosis reacted with either the pyruvate dehydrogenase-E2 or the branched-chain alpha-keto acid dehydrogenase protein. None of the 124 control samples from healthy volunteers (n = 86) or patients with primary sclerosing cholangitis (n = 38) had significant reactivity. Our results indicate that the use of recombinant, cloned autoantigens provides a simple, accurate, and rapid method of quantifying and monitoring the levels of specific mitochondrial autoantibodies in the serum of patients with primary biliary cirrhosis.
Antimitochondrial autoantibodies recognizing 68 to 74 and 50 to 52 kD inner membrane mitochondrial antigens are characteristically present in sera of patients with primary biliary cirrhosis. The biochemical identification of the antigens, however, has remained elusive. We report herein that the 52 kD antigen is the dihydrolipoamide acyltransferase of the branched-chain alpha-keto acid dehydrogenase complex. This was demonstrated by three experiments through the use of recombinant fusion protein expressed in Escherichia coli from a cDNA insert encoding the human autoantigen. First, 33 of 37 primary biliary cirrhosis patients exhibiting reactivity toward the 50 to 52 kD mitochondrial antigen by immunoblotting also showed reactivity toward the recombinant fusion protein. Second, absorption of primary biliary cirrhosis sera with recombinant fusion protein, but not with an irrelevant recombinant clone, the F-specific rat liver antigen, was effective in absorbing out reactivity against the 50 to 52 kD mitochondrial antigen but not the 68 to 74 kD antigen. Third, complete removal of reactivity toward all four different isoelectric point polypeptides at 50 to 52 kD was observed in two-dimensional gel analysis. Furthermore, primary biliary cirrhosis sera were analyzed with mitochondria from three sources, rat liver, human placenta and bovine heart, in order to compare reactivity patterns and to determine precisely the comparative molecular weights of the autoantigens in the three species. The availability of recombinant autoantigens will provide improved diagnostic tests and, more importantly, will allow definite issues in primary biliary cirrhosis to be studied, including identification of immunodominant epitopes, the significance of autoantigen recognition and the establishment of autoreactive T cell clones.
The availability of recombinant mitochondrial autoantigens may permit the experimental study of the pathophysiology of primary biliary cirrhosis. Previously, we demonstrated that high-titer antibodies to the 74 kD mitochondrial autoantigen dihydrolipoamide acetyltransferase could be generated when BALB/c mice were immunized with purified recombinant protein. Based on these data, we attempted an 8-month study to induce antibodies and liver dysfunction by immunizing AKR/J, C3H/J and CBA/HeJ mice as well as rats, guinea pigs, rabbits and rhesus monkeys with purified recombinant human dihydrolipoamide acetyltransferase. Antibodies to dihydrolipoamide acetyltransferase were readily induced and detected in all species of experimental animals with species and strain differences in the titer of the responses. Of particular interest, rabbits and guinea pigs produced antibodies which were specifically reactive with the functional site of dihydrolipoamide acetyltransferase, whereas the other strains and species produced antibodies to other epitopes on the molecule. Finally, similar to data on humans with primary biliary cirrhosis, the pyruvate dehydrogenase enzyme pathway was inhibited in the presence of immunized animal sera. These data imply that features other than simply an antibody response to mitochondrial enzymes are required for the development of primary biliary cirrhosis. Further studies will be necessary to determine the mechanisms by which mitochondrial proteins elicit an immune response.
Antimitochondrial autoantibodies are characteristically present in sera of patients with primary biliary cirrhosis. The antimitochondrial autoantibodies recognize four major antigens from beef heart mitochondria at relative molecular weights of 74, 56, 52 and 48 kD. In the present study, we report that the 56 kD antigen is the protein X of pyruvate dehydrogenase complex and that it possesses cross-reactive antimitochondrial autoantibody epitope(s) with the 74 kD antigen, the acetyltransferase (E2) of the pyruvate dehydrogenase complex. This was demonstrated by comparing the specificities of primary biliary cirrhosis sera with a protein X-specific rabbit antiserum and by absorbing primary biliary cirrhosis sera with recombinant pyruvate dehydrogenase-E2 fusion protein. In the two-dimensional gel analysis, primary biliary cirrhosis sera and protein X-specific rabbit antiserum reacted to the same two isoelectric point polypeptides at 56 kD molecular weight. The absorption of primary biliary cirrhosis sera with the human recombinant pyruvate dehydrogenase-E2 removed reactivity toward both the 74 and 56 kD antigens. Furthermore, analysis of 82 antimitochondrial autoantibody-positive primary biliary cirrhosis sera by immunoblotting did not reveal any sera which reacted solely against either the 74 or 56 kD antigen. Finally, primary biliary cirrhosis sera recognized protein X from human, bovine and porcine sources but not protein X from rat or mouse origin. The identification of protein X as another major target of the autoimmune response in primary biliary cirrhosis suggests that the pyruvate dehydrogenase complex may have a central role in the induction of this enigmatic disease.
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Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency (greater than 95%) of autoantibodies to a Mr 70,000 mitochondrial antigen, a component of the M2 antigen complex. We have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase (EC 2.3.1.12) of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.
Natural thymocytotoxic autoantibodies (NTA) have been suggested to contribute to the T cell mediated immune dysfunction prevalent in NZB mice. However, direct studies to confirm these suggestions have been difficult to perform due to the absence of large amounts of pure NTA. To overcome this problem, we generated a naturally occurring monoclonal antibody from an NZB mouse which possesses the serological characteristics of serum-NTA. Herein, we report the in vitro functional characteristics of this monoclonal antibody designated SAG-3. In vitro treatment of spleen and lymph node cells from BALB/c mice with SAG-3 and complement eliminated most of the proliferative response to Con-A and moderately reduced the response of these cells to PHA-P and PWM. Complement mediated cytolysis of SAG-3 reactive cells significantly reduced the capacity of unprimed lymph node cells to generate primary CTL responses in vitro against allogeneic cells. However, only a partial decrease in CTL activity was observed when primed secondary CTL effector cells were treated, due primarily to the depletion of CTL's specific towards MHC class I antigens. Furthermore, a moderate reduction in antigen-specific helper T cell function was observed. Pretreatment of responder cells with SAG-3 + C' did not appear to reduce the proliferative MLR response to a wide range of stimulator cells. In addition, contrary to previous reports on serum-NTA, SAG-3 appears not to be reactive against Con-A induced suppressor T cells. Finally, by FACS analysis SAG-3+ thymocytes constitute an overlapping population of Lyt-2+ and L3T4+ thymocytes. These findings are discussed in comparison to previous reports on NTA.
Naturally occurring thymocytotoxic autoantibodies (NTA) have been suggested to be the cause of thymic atrophy and T cell disorders in human and murine lupus. Definitive studies on NTA's role in the induction of SLE, however, have been lacking due to the lack of a pure source of NTA. Although it is clear that NTA are a heterogeneous group of antibodies, the nature of their antigens has remained obscure. We report the characteristics of a monoclonal NTA, designated SAG-3, which appears more reflective of the activities previously reported of serum NTA than other NTA-secreting clones. SAG-3 is an IgM autoantibody cytotoxic for 80 to 90% of thymocytes, 20 to 25% of splenic lymphocytes, 25 to 30% of lymph node cells, and less than 3% cortisol-resistant thymocytes, bone marrow, and fetal liver cells. SAG-3 is murine-specific without reactivity towards rat, hamster, or guinea pig, and appears very early in thymic development, on day 17 fetal thymocytes. SAG-3 is equally cytotoxic against several strains of mice, including both Thy-1.1 and Thy-1.2 allotypes, and the cytotoxicity is absorbed by brain but not liver cells. Reactive thymocytes occurred throughout the cortical regions of the thymus, indicating preferential affinity towards immature thymocytes. Although the serologic activities of SAG-3 suggest that Thy-1 alloantigen is its target, SAG-3 antigen is found to be distinct from Thy-1 and also from Lyt-1, Lyt-2, or L3T4 antigens. The binding of SAG-3 to thymocytes could be competitively inhibited by NTA-positive NZB sera.
A series of human-human hybridomas derived from a single fusion of UC 729-6 with lymph node lymphocytes were examined for the type and nature of macromolecules synthesized and secreted. One hybrid, VLN3G2, secreted fourfold higher IgG than that present in the cytoplasm over 4 days of growth, while the IgM distribution was opposite to that of IgG. VLN5C7, contrary to VLN3G2, contained several-fold more cytoplasmic IgG as well as IgM than the amounts secreted over the same period of time. Of the secreted IgG and IgM by both of these hybridomas, only the IgG showed immunoreactivity against target A431 cell surface antigen(s). Another hybridoma, termed VLN1H12, secreted immunoreactive IgM against target A431 cells, but no detectable IgG. Cytoplasmic proteins prepared by repeated freeze-thaw of the hybridoma cells, membrane proteins obtained by NP-40 extraction of the cell membrane, and secreted proteins present in the supernates of the various hybridomas were assayed by an enzyme-linked immunoassay (ELISA), to understand the discrepancy observed in the immunoglobulins of the cellular and extracellular compartments. The parental UC 729-6 cell line used in these cell fusions produced only trace amounts of immunologically inactive IgM and no detectable IgG. Molecular sieving column chromatography of these hybridoma supernates suggested the presence of intact IgG and IgM molecules and the absence of free heavy chains or hybrid antibodies containing both mu and gamma heavy chains. Intrinsic labeling of VLN3G2 hybridoma cells with 35S-methionine demonstrated the presence of not only a nonimmunoglobulin protein but also a small molecular weight protein-A-binding polypeptide in the culture supernatant. 35S-methionine-incorporated IgG and IgM antibodies, isolated from spent media, cytoplasm, and cell membranes of VLN3G2, also showed binding to protein-A-bearing bacteria. In conclusion, the differences observed in the amounts of secreted MAbs by the human-human hybridomas were not due to the decreased synthesis of these molecules.