Antigen-specific suppression in genetic responder mice to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Characterization of conventional and hybridoma-derived factors produced by suppressor T cells from mice injected as neonates with syngeneic GAT macrophages.

Abstract

Spleen cells from C57BL/10 mice injected with syngeneic B10 L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-pulsed macrophages (GAT-M phi) within 18 h of birth were unable to respond to soluble GAT, GAT-methylated bovine serum albumin, or B10 GAT-M phi as adults. Spleen cells from these neonatally treated mice responded at control levels to GAT presented in allogeneic M phi and to sheep erythrocytes. Partially purified T cells from these neonatally treated mice suppressed responses by syngeneic virgin, but not primed, spleen cells in an antigen-specific manner and acted during the early phases of the response. These responder GAT-specific suppressor T cells (GAT-TSR) were sensitive to anti-Thy-1 + C and 500-rad irradiation and have the phenotype Ly-1-2+, I-J+; GAT-TSR cells can only suppress responses by spleen cells syngeneic with the GAT-TSR cells at the I-J subregion of H-2. Restimulation of these Ts cells with syngeneic GAT-M phi induces an antigen-specific suppressor factor within the supernatant fluid. The factor, GAT-TsFR, is a glycoprotein with a molecular weight between 48,000 and 63,000, as determined by gel filtration chromatography using isotonic buffers; it bears serologically detectable determinants encoded by the I-J subregion of the H-2 complex, has an antigen-binding site for GAT and L-glutamic acid50-L-tyrosine50, and shares idiotypic determinants with anti-GAT antibodies. The presence of GAT-TsFR in the first 36 h of in vitro culture is required for significant suppression. Furthermore, only responses by spleen cell syngeneic with the cells producing GAT-TsFR at the I-J subregion are suppressed. The fusion of GAT-TsFR-producing cells with BW5147 resulted in generation of two hybridomas with properties and characteristics identical to those of the conventional GAT-TsFR with one exception: conventional and hybridoma 372.D6.5 GAT-TsFR only suppress responses by spleen cells of the I-Jb haplotype, whereas suppression mediated by the second hybridoma GAT-TsFR (372.B3.5) is genetically unrestricted. These hybridoma GAT-TsFR are compared with nonresponder GAT-Ts factor (GAT-TsF) and these responder and nonresponder GAT-TsF are considered in the context of suppressor pathways.

Purification and characterization of an L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factor from genetic responder mice.

“…The smallest biologically functional unit is an 18,000 mol wt protein (glycoprotein) whose precise relationship to the predominant 28,000 mol wt and larger forms is unclear. One interpretation is that the 18,000 mol wt form is actually a breakdown product of the major 28,000 mol wt form that has retained all of the function and serological determinants detected on the larger molecule (6). At this time we cannot rule out this possibility.…”

“…Spleen cells from C57BL/10 mice (5 X 10 6 /well) injected as neonates with syngeneic GAT-Mo were restimulated with GAT in vitro and fused with the AKR thymoma BW5147, as previously described (10)(11)(12)(13) . Individual clones were screened for GAT-specific suppressive activity and the biological activity of supernatant fluids from the hybridoma to be discussed, 372B3 .5, has been described in detail (6) . Supernatant fluids from BW5147 and other wells from the same fusion were consistently negative for both GAT-specific and nonspecific suppressive activity .…”

“…Peanut agglutinin (PNA) agarose was purchased from E-Y Laboratories, San Mateo, CA . Immunoadsorbents of monoclonal anti-1-J', anti-I J", and an antiidiotype (guinea pig anti-mouse anti-GAT antibody), anti-cGAT, were prepared as described previously (6) . Purified GAT-TsF k was applied to these immunoadsorbents ; the effluent and material eluted with 2.0 M KCI were tested for biological activity .…”

“…GAT (45,000 mol wt, Vega Biochemicals, Tucson, AZ) was prepared for use as antigen in culture (7), preparation of GAT-Md (8,9) and coupling to sheep erythrocytes (SRBC) for use as indicator cells in the hemolytic plaque assay (7), as described . Neonatal mice were injected with 2 X 10 6 syngeneic GAT-Mo for the induction of GAT-TSR, as previously described (6) . Spleens were removed from these animals at 10-20 wk of age and used for the generation of T cell hybridomas .…”

The Biochemistry of Antigen-Specific T-Cell Factors

“…In many cases these cells have been shown to release suppressor factors that block the appearance of antibody-forming cells (18,20,34,48). For example, several different synthetic polypeptides, the immune response to which is known to be under Ir gene control, induce suppressor cells.…”