Dissection of cross-reactivities using a panel of H-2Ld alloreactive T cell hybridomas.

In order to explore the role which class I structure plays in alloreactivity, we have generated Ld-reactive T cell hybridomas by fusion of a dm2 anti-BALB/cJ MLR with the BW5147 cell line and examined their stimulation by the following class I molecules (alpha 1/alpha 2/alpha 3): Lq, Dq, dm1, Ld/Ld/Dd, Lq/Dq/Ld, and Q10/Q10/Ld. We found that their specificities differed in their patterns of cross-reactions and were reasonably representative of those present in the bulk population of MLR-generated CTLs. Ld/Ld/Dd and Q10/Q10/Ld stimulated the majority of the hybridomas, Lq and dm1 were recognized by over half of the panel, and Lq/Dq/Ld stimulated only modestly, while Dq was not recognized by any hybridoma. Correlation of these observed reactivities with class I structure suggests that putative TCR contact residues may play a significant role in recognition when compared to the polymorphic amino acid residues which control pocket specificity and peptide binding. Specifically, Lq and Dq possess very similar or identical pockets, in contrast to those of dm1 and Q10. However, Q10 has identical TCR contact residues to Ld, both on the alpha 1 and alpha 2 alpha helices, unlike Dq which is mismatched on both helices. Lq and dm1 are mismatched compared to Ld on only one helix. Thus, a molecular rationale for the cross-reactions observed in this study involves the direct participation of residues of class I molecules in allorecognition.

Immunogenicity and tolerogenicity of self-major histocompatibility complex peptides.

Mechanisms involved in self-antigen processing and presentation are crucial in understanding the induction of self-tolerance in the thymus. We examined the immunogenicity of determinants from major histocompatibility complex (MHC) molecules that are expressed in the thymus and have tested peptides derived from the polymorphic regions of class I and class II molecules. We found that two peptides corresponding to NH2 termini of the class II alpha and beta chains (Ak alpha 1-18 and Ak beta 1-16) could bind to self-Ak molecules with high affinity and, surprisingly, were immunogenic in that they could elicit strong proliferative T cell responses in B10.A mice (Ak, Ek). Neonatal injection of peptide Ak beta 1-16 resulted in complete unresponsiveness to this peptide at 8 wk of age showing that these T cells were susceptible to tolerance induction. We have also tested certain class I MHC peptides and showed that some can interact efficiently with class II MHC peptides to induce an autoreactive T cell proliferative response. Among these class I peptides is one (Dd 61-85) that has the capacity to bind to self-Ia without being immunogenic, and therefore represents an MHC determinant that had induced thymic self-tolerance. We conclude that some self-MHC molecules can be processed into peptides that can be presented in the context of intact class II molecules at the surface of antigen-presenting cells. Autoreactive T cells recognizing optimally processed self-peptide/MHC complexes are eliminated during development, whereas other potentially autoreactive T cells escape clonal inactivation or deletion. Incomplete tolerance to self-antigens enriches the T cell repertoire despite the fact that such T cells may eventually become involved in autoimmune disease.

Origin of the fourth component of complement related Chido and Rodgers blood group antigens.

We have reviewed the relationship between C4 and its related blood group and discussed the mechanisms whereby a fragment of C4 could become attached to erythrocytes (E). We hypothesize that there is chronic fluid-phase activation of C4 by either C1 to form C4b or spontaneous cleavage of the thioester to form iC4. These activated molecules bind to E. Proteolytic degradation of the bound C4b or iC4 would leave a covalently attached fragment of C4 on E and thereby give rise to the Ch and Rg blood group antigens. This system is of further immunopathologic interest since this 'normal' activation or turnover of C4 is closely regulated. In patients deficient in regulatory proteins, this spontaneous or normal turnover of C4 and C3 may initiate a pathologic condition.

The C4 and Slp genes of the complement region of the murine H-2 major histocompatibility complex.

Recent analyses, at the protein and DNA levels of structure, of the murine complement components C4 and the closely related sex-limited protein, Slp have led to new insights into the H-2/S region-linked C4 and Slp genes and their products. The primary products are 200 000 Da precursors which are cleaved, intracellularly and extracellularly, into the the mature alpha-beta-gamma-subunit molecules of plasma. Precursor order of subunits is beta-alpha-gamma; a complementary DNA clone spanning the alpha-gamma junction has been extensively analysed. The C-terminal of the alpha-chain is of particular interest because of post-secretion processing which differentiates 'secreted' and 'plasma' forms of C4, both apparently functional, and because allelic variants of C4 and the Slp protein, which differ substantially in molecular masses, owe their differences principally to different levels of glycosylation of the alpha-chain. Allelic variations in rate of C4 synthesis (C4-high compared with C4-low) have been analysed in cultures of hepatocytes and macrophages. Three distinct modes of genetic regulation of the expression of the Slp protein have been identified.

Purification of Fc gamma receptor from rabbit alveolar macrophages that retains ligand-binding activity.

The Fc gamma receptor of rabbit alveolar macrophages was purified by affinity chromatography by using rabbit gamma-globulin (Rab gamma G) coupled to Sepharose. Macrophage preparations were efficiently labeled with 125I by using a modified lactoperoxidase method. After incubation of NP-40 cell lysates with Rab gamma G-Sepharose, elution at 4 degrees C with 0.5 N acetic acid containing 1% NP-40 and rapid neutralization allowed recovery of active Fc gamma receptor. Purified Fc gamma receptor retained its ligand-binding activity, since approximately 41 to 72% of labeled material specifically rebound to Rab gamma G-Sepharose. Active receptor also rebound to human IgG- and rat IgG-sepharose. Active Fc gamma receptor did not bind to Sepharose coupled to rabbit Fab, rabbit F(ab)'2 or human F(ab)'2 fragments, nor to Sepharose coupled to chicken IgG. Analysis of Fc gamma receptor by SDS polyacrylamide gels demonstrated a broad peak of radioactivity in the apparent m.w. range of 50,000 to 70,000 in 5.6% acrylamide gels and 35,000 to 55,000 in 9% gels. Labeled receptor with similar structural characteristics and ligand-binding activity was also obtained from highly purified adherent cell populations and from macrophages biosynthetically-labeled with [14C]glucosamine in culture.

Improved cell surface radioiodination of macrophages.

Rabbit alveolar macrophages at high cell concentrations are inefficiently radiolabeled by the usual lactoperoxidase-catalyzed iodination method. Soluble macromolecules secreted by macrophages were found to inhibit the radioiodination of macrophages and other cells. A modified procedure is described which minimizes the presence of such inhibitory material and thereby considerably improves the radioiodination efficiency for both alveolar macrophages and the macrophage-like P388D1 cell line.