A B S T R A C T Although the association between human histocompatibility leukocyte antigen (HLA) B27 and ankylosing spondylitis is the prototype of HLAdisease association, the mechanism underlying these associations has not been determined. We have investigated the possibility that the B27 molecules from patients with ankylosing spondylitis are different from those of normals, and only the "different" molecules predispose the individual to disease. Biosynthetically radiolabeled HLA-B27 molecules from patients with ankylosing spondylitis and normal individuals were compared by two-dimensional gel electrophoresis and tryptic peptide mapping with high pressure liquid chromatography. Extensive charge heterogeneity in the 45,000-dalton heavy chain was detected when B27 molecules were analyzed by two-dimensional gel electrophoresis; the charge heterogeneity was reduced, but not eliminated, when the B27 molecules were treated with neuraminidase to remove sialic acid residues before analysis. No structural difference in the B27 molecules from an ankylosing spondylitis patient and a normal individual were detected by two-dimensional gel electrophoresis. Analysis of [3H]leucine-labeled and[3H]arginine-labeled tryptic peptides and chymotryptic peptides of the trypsin insoluble material by reverse-phase high pressure liquid chromatography revealed identity of the B27 molecules from ankylosing spondylitis patients and normal individuals. These studies indicate that development of akylosing spondylitis in only some B27 positive individuals is not at-
In this communication we examined the functional activity of rabbit antisera produced against guinea pig T lymphocytes that had been stimulated in vitro by culture with macrophage-associated antigen. One antiserum (96-2) was raised against strain 13 T cells that were primed by culture for a week with trinitrophenyl-(TNP) modified syngeneic macrophages. Addition of 96-2 serum to TNP-primed strain 13 T cells or strain 13 T cells primed in a mixed leukocyte reaction (MLR) stimulated a dramatic increase in DNA synthesis in vitro. In contrast, strain 13 T cells primed in vitro with several protein antigens failed to be stimulated by the 96-2 serum. In addition, 96-2 serum produced little stimulation of TNP or MLR-primed strain 2 cells, or of strain 13 T cells taken directly from the animal. Another antiserum (144-2) was raised against glutamic acid-lysine copolymer (GL)-immune strain 2 T cells selected by culture with GL-pulsed strain 2 macrophages. Addition of 144-2 serum to GL-selected or MLR-primed strain 2 T cells also produced a dramatic proliferative response. However, 144-2 serum did not stimulate TNP-primed strain 2 or strain 13 T cells, or strain 2 T cells selected with unrelated protein antigens. By several techniques we showed that contaminating B cells in the primed T cell preparations were not responsible for the observed proliferation and that the antisera directly stimulated highly purified T cells. The proliferative activity of 96-2 and 144-2 could be removed by absorption with nonimmune spleen cells. Physicochemical analysis of the cellular antigens detected by the antisera showed molecular species of 98,000, 70,000, 33,000 and 25,000 daltons. By sequential immunoprecipitation analyses, the latter two peaks were determined to be Ia antigens, the 70,000 peak of in vitro primed cells contained some immunoglobulin-like molecules and another nonimmunoglobulin-like antigenic species, and the 98,000 species appeared unrelated to immunoglobulin or Ia antigens.
The guinea pig MHC, the GPLA complex, codes for the B and S classic histocompatibility antigens. Four alleles have been postulated at the B locus and two alleles at the S locus. Strain 2 and strain 13 guinea pigs have serologically identical B.1 and S antigens but differ by the I region. However, since these two strains do not have common parentage, it was unknown whether their B.1 molecules were structurally identical. This is an important consideration since the large body of immunogenetic data attributed solely to I region differences is based on this premise and therefore would be subject to reinterpretation if it should prove untrue. We have demonstrated that the primary structures of these two B.1 molecules are identical by performing isoelectric focusing and comparative tryptic-peptide mapping analysis. The DHCB (PCA) guinea pig expresses the same I region as that of the strain 13 guinea pig, but has a different B alloantigen (i.e., B.3). Isoelectric focusing and comparative tryptic-peptide mapping analysis of this B.3 alloantigen have substantiated the serologic finding of nonidentity of the B.1 and B.3 molecules by showing that the two molecules do not share identical primary structure. However, the mapping data showed that there was 40 to 50% homology between the B.1 and B.3 molecules. This result is consistent with the findings in the mouse system for alleles of the same locus. Therefore, our study has demonstrated that the B.1 molecules from the two noncongenic strain 2 and strain 13 guinea pigs are structurally identical and that the B.3 molecule of the DHCBA guinea pig is most likely an allelic product of the B.1 molecule.
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