Mechanism responsible for the induction of I-J restriction on TS3 suppressor cells.

The mechanisms responsible for the induction of I-J restrictions on third-order suppressor T cells (TS3) were analyzed. The I-J phenotype of the antigen-coupled cells used for priming restricted the specificity of the TS3 population. Thus, TS3 cells were only generated after priming with antigen-coupled I-J homologous cells. Identity at the I-JM (and I-E) subregions was sufficient for TS3 induction. Furthermore, priming of H-2 heterozygous mice with antigen-coupled parental cells generated TS3 that were restricted to the parental haplotype used for priming. The splenic cell population responsible for antigen presentation and induction of TS3 cells was fractionated. The cells involved in antigen presentation were found in the splenic adherent population and were absent in the fraction containing splenic nonadherent T and B cells. The subsequent activation and interaction of TS3 cells is also restricted by genes in the H-2 complex. The results are discussed in terms of a general mechanism responsible for the induction of restrictions in T helper and TS3 cells.

The role of I-J and Igh determinants on F1-derived suppressor factor in controlling restriction specificity.

In the 4-hydroxy-3-nitrophenyl acetyl (NP) contact sensitivity system, the activity of third-order suppressor cells and their factors is restricted by H-2(I-J) and Igh linked genes. The present report analyzes the specificity of NP-specific Ts3 cells and factors derived from H-2 and Igh heterozygous (B6 X C3H)F1 mice. Two approaches were used. First, heterogeneous populations of F1 Ts3 cells were activated in vitro and then assayed in Ts3-depleted recipients which carried different combinations of H-2 and Igh alleles. The second approach was to hybridize the Ts3 cells and analyze the specificity of the F1-derived TsF3. The combined data demonstrated four functionally distinct populations of Ts3 cells. The activity of each population was restricted by a particular combination of H-2 and Igh haplotypes. Thus, Ts3 cells derived from F1 donors can demonstrate an apparent scrambling of H-2 and Igh restriction specificities. There was functional allelic exclusion of the H-2(I-J) and Igh determinants expressed on (B6 X C3H)F1 hybridoma-derived TsF3. Thus, TsF3 from each cloned hybridoma line expressed only one set of I-J and Igh determinants. Furthermore, there was a complete correlation between the I-J and Igh linked determinants expressed on TsF3 and the restriction specificity. In view of the recent findings on the molecular biology of the I-J region, an alternative interpretation of the role of I-J determinants on suppressor cells and factors is offered.

Analysis of the site on a TNF-alpha molecule which affects type II TNF receptor binding in human cells.

The epitope region on the TNF-alpha molecule recognized by monoclonal antibody (mAb) 3-D-6, which neutralizes the cytotoxic activity on murine LM cells, has been determined as Gly24-Gln-Leu-Gln-Trp-Leu-Asn-Arg31. To examine whether this region participates in TNF receptor binding in human cell lines, four kinds of TNF-alpha mutants (Gln25 --> Glu, Gln27 --> Glu, Leu29 --> Val, and Arg31 --> Ser) were prepared using site-directed mutagenesis. One mutant, mRS31, which has a nonconserative mutation at position 31 (Arg --> Ser), showed markedly reduced binding in U-937 cells and in HL-60 cells compared with the wild-type recombinant TNF-alpha (rTNF-alpha). These two cell lines have been reported to have both type I and type II TNF receptors. mRS31 also showed reduced cytotoxicity on U-937 cells. Another mutant, mLV29, which has a conservative mutation at position 29 (Leu --> Val), showed, to a lesser extent, reduced binding in U-937 cells and HL-60 cells and reduced cytotoxic activity in U-937 cells. However, all four TNF-alpha mutants showed a similar binding in HEp-2 cells and in HeLa cells, which have been reported to have only the type I TNF receptor. These results suggest that Leu29 may be involved in direct contact with the type II receptor and that the nonconservative mutation at position 31 may induce a local conformational change in the site involved in type II TNF receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)