Harley Y. Tse

Characterization of a dominant anti-Ia idiotype using the Ia mutant mouse strain B6.C-H-2bm12

Initial studies of antibody recognition of Ia molecules using the IA mutant mouse strain bm12 suggested that two anti-Ia monoclonal antibodies (mAbs), 25-9-17 and 34-5-3, share several features: (1) indistinguishable serologic specificity including a lack of reactivity with Iabm12, (2) binding of the same spatial epitope (cluster), and (3) definition of a cross-reactive idiotype (CRI) as defined by xenogeneic antisera. In the present study we characterize a rabbit anti-idiotype (anti-Id) to 25-9-17 by affinity chromatography, and demonstrate that it detects at least two distinct idiotopes, one shared by 25-9-17 and 34-5-3 designated CRI (25-9-17) and one unique for 25-9-17 molecules. Experiments were also undertaken to determine whether CRI (25-9-17) represents a measurable component of allogeneic humoral responses to Iab antigens. By both absorption analyses of a polyspecific antiserum and production of antigenically-restricted antisera using bm12 mice, CRI (25-9-17) was found to represent a significant proportion of the antibodies to Iab. By several criteria it was shown that the CRI (25-9-17)+ molecules were among the antibodies defining the serologic lesion of bm12 mice. In preparation for future studies to alter in vivo T-cell responses involving recognition of Ia (e.g. graft vs host disease and allogeneic transplant rejection), various immunization protocols and mouse strains were tested for induction of Id (25-9-17) following in vivo administration of various anti-idiotypic reagents. Rabbit anti-Id (25-9-17) successfully induced CRI (25-9-17) positive molecules in all strains tested regardless of IA or Ig genotype. Moreover, some of these treated mice produced antibodies to an Ia determinant missing on bm12 cells, suggesting that they recognize the same serologic determinant as mAb 25-9-17.

T-Cells Recognize IA Conformation in the Interaction with Antigen Presenting Cells

The presentation of soluble antigens by antigen presenting cell (APC) to T-cells requires that the antigen be recognized in the context of an Ia determinant (Shevach and Rosenthal, 1973). Although the T cell antigen receptor has been identified and sequenced (Hedrick et al., 1984; Fink et al., 1986), the nature of the ligand that triggers the T cells remains unclear. The early experiments of Lin et al. (1981) and Michaelides et al. (1983) using the IA mutant strain of mouse B6.C-H-2 bml 2 (bm1 2) demonstrated that limited alteration of the Ia molecule concomitantly led to changes in the response pattern of T-cells to soluble antigens. Specifically, it was shown that bml 2 lost immune responsiveness to the antigens beef insulin and H-Y while their responses to other antigens remained normal when compared to the wild type C57BL/6. Besides providing convincing evidence equating Ia to the Ir gene product, these experiments also suggest that there are multiple funcitonal domains (Beck et al., 1983) on the Ia molecule that by some unknown mechanisms determine responsiveness to certain antigens. Indeed, this conclusion is consistent with other studies using a variety of approaches such as monoclonal antibody blocking (Frelinger et al., 1984), site-directed mutagenesis (Cohen et al., 1986), in vitro selection of variant APC lines (Glimcher et al., 1983b) and inhibition of specific antigen response with structurally related co-polymers (Benacerraf and Rock, 1984). More recently, Babbitt and coworkers (1985) using biochemical methods demonstrated a weak but measurable physical association between a hen egg lysozyme antigenic fragment and purified Ia molecules. This association was demonstrated for Ia derived from responder strains but not that from non-responder strains. In another analysis, Ashwell and Schwartz (1986) also provided arguments that antigen and Ia physically interacted to form a ternary complex recognizable by the T-cell antigen receptor.

Do T Cells Recognize the Same Ia Epitopes on Antigen Presenting Cells and on B Cells

While it is generally believed that antigen presenting cells exhibit Ir gene functions, there has been a long debate as to whether Ir genes are also expressed at the level of B cells (1,2). Since both antigen presenting cells and B cells possess surface Ia molecules, this argument is partially resolved by the recent demonstration in several systems, that Ia molecules are indeed products of Ir genes (3–5). The question remains, however, whether T cells “see” the same Ia epitopes on both antigen presenting cells and on B cells. A direct approach to this question would require knowledge about the nature of the putative “T cell receptors”. In the absence of that information, we attempted to combine the use of the T cell cloning technique and the Ia mutant strain C57BL/6-C-H-2 bm12 (BM12) to examine the Ia specificity by which T cells interact with antigen presenting cells and B cells.

Genetic Restriction of Polyclonal Stimulation of B Cell Proliferation by Antigen-Specific T Cell Clones

Since the demonstration of immune response (Ir) gene effects in animal models (1), much interest has been centered on the identification of Ir gene products and the elucidation of the mechanisms of Ir gene functions.Studies using serological reagents, chimeras, lymphocyte functional assays and mutants (2–4) have all demonstrated that Ia is probably the Ir gene product itself. This is not surprising since Ia has been implicated in a number of cellular interaction processes, notably antigen presentation (5) and T-B cell collaboration (6). It is believed that Ia serves as self-recognition molecules in antigen responses and failure of foreign antigens to associate with a given set in Ia specificities results in unresponsiveness (4,7). Other mechanism that involve the generation of suppressor T cells have also been proposed (8).