J A Kapp

The development and characterization of encephalitogenic cloned T cells specific for myelin proteolipid protein

T-cell clones have been isolated from SJL/J mice after immunization with myelin proteolipid protein (PLP). The cloned cells responded strongly to PLP stimulation in vitro as well as to the synthetic PLP-related peptide 139-151. The response to PLP is Ia mediated, since it was inhibited by monoclonal antibodies to the matched I-As haplotype, but not with antibodies to other I-A haplotypes. Phenotypic analysis using immunofluorescence demonstrated the following characteristics of the clones: Thy-1+, CD4+, CD5+ and CD8-. Injection of 10-30 million PLP-activated cells from one clone induced severe experimental allergic encephalomyelitis in five mice, both clinically and histologically. This represents to our knowledge the first report of PLP-specific encephalitogenic cloned T cells.

Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells

Myelin proteolipid protein (PLP) contains one established antigenic epitope within the 139-151 amino acid sequence, with encephalitogenic activity for SJL mice (PLP139-151). In the current study, the processing and presentation of PLP by subsets of splenic antigen-presenting cells (APC) were examined by comparing their capacity to stimulate PLP-responsive T-cell clones, two of which are specific for PLP139-151, and one which is not specific for this peptide. In order to study whether PLP requires processing before its presentation by APC, PLP-pulsed and fixed APC were shown to stimulate PLP-specific T cells. However, the addition of PLP to unpulsed, fixed APC resulted in the absence of T-cell stimulation, while the ability of these fixed APC to bind antigenic peptide and efficiently present it to T cells, was demonstrated by their ability to use a synthetic peptide for the stimulation of the T cells. In order to study potentially different processing efficiencies among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC activities were studied separately. The non-adherent (NAd) APC were unable to stimulate PLP139-151 specific T-cell clones with PLP as antigen. In contrast, a T-cell clone specific for a separate, but unidentified epitope on PLP was stimulated by NAd APC efficiently. In addition, stimulation of PLP139-151-specific T-cell clones by NAd APC did occur when the synthetic peptide instead of intact PLP was used as antigen, indicating a defect in PLP processing by the NAd APC.(ABSTRACT TRUNCATED AT 250 WORDS)

H-2 GENE COMPLEX REGULATION OF MACROPHAGE-LYMPHOCYTE INTERACTIONS IN ANTIBODY RESPONSES IN VITRO

The genetic restrictions regulating the efficient interactions among antigen-bearing macrophages and syngeneic and allogeneic murine lymphoid cells in development of secondary antibody responses to a T cell-dependent antigen in vitro have been investigated. Immune spleen cells develop secondary antibody responses preferentially when stimulated in vitro with antigen on macrophages syngeneic to the macrophages used to immunize the spleen cells in vivo. These genetic restrictions governing efficient macrophage-lymphoid cell interactions in secondary antibody responses are antigen-specific, controlled by the I-A subregion of the H-2 complex and are operative at the level of the immune T cell. The implications of these genetic restrictions in regulation of antibody responses are briefly considered.

Regulatory mechanisms of the immune response to heterologous insulins: I. Development and regulation of plaque-forming cell responses in vitro☆

We, as well as many other investigators, have been studying the regulation of immune responses to insulin as a model system of H-2 linked immune response (Ir) gene control. Although antibody responses by mice to heterologous insulins are qualitatively controlled, antibodies that are generated to one species of heterologous insulin cross react extensively with other species. The exquisite control of responsiveness is regulated by T cells that appear to recognize differences in the amino acid sequences of the A-chain loop of insulin. Our previous studies of the mechanism(s) by which Ir genes regulate T cell activity to insulin have been confined to an adoptive transfer model because traditional cell culture techniques using normal or insulin-primed spleen cells have failed to generate insulin-specific plaque-forming cell responses in vitro. In this communication we demonstrate that more vigorous immunization protocols and the use of lymph node T cells as a source of helper T cells can circumvent this problem. More importantly, all of the major features of the regulation of responses to insulin that have been observed in vivo are reflected in this in vitro system. Thus, these experiments provide the essential foundation for future dissection of the mechanism of Ir gene control of responses to insulin.

Genetic analysis of immune suppression

We have analyzed the genetic control of susceptibility to suppression by 1-J+, suppressor-T-cell derived factors (TsF) specific for the synthetic polymer L-glutamic acid50-L-tyrosine50 (GT). GT-TsF activity was measured as specific inhibition of proliferative responses to GT developed in cultures of lymph-node T cells from mice primed with GT complexed to methylated bovine serum albumin (GT-MBSA). These experiments demonstrated that there is no MHC-encoded genetic restriction between donors and recipients of GT-TsF in suppression of proliferative responses. We have also confirmed the observations that mice of the H-2b, H-2d, and H-2khaplotypes can produce GT-TsF, whereas H-2amice do not, and that H-2b, H-2d, and H-2kmice are sensitive to GT-TsF from all producer strains, whereas H-2bmice are not sensitive to GT-TsF from any strain. Analysis of the effect of GT-TsF on responses by mice bearing recombinant haplotypes suggests that at least two genes are required for susceptibility to GT-TsF and that these genes show coupled complementation.

EXPRESSION OF IR GENES IN T CELLS

An immunosuppressive factor has been identified in lymphoid cell extracts from nonresponder mice primed with the terpolymer L-glutamic acid 60 -L-alanine 30 -L-tyrosine 10 (GAT). This material suppresses the GAT-specific antibody response to GAT coupled to methylated bovine serum albumin (GAT-MBSA) or GAT coupled to pigeon erythrocytes by normal syngeneic mice in vivo and in vitro. Data presented below demonstrate that the suppressive moiety is a T cell product that binds to GAT and, to a lesser extent, the serologically cross-reactive polymers GA and GT. Despite its avidity for GAT, the active material does not bear μ, γ 1 , γ 2a , γ 2b or α heavy chain or κ light chain constant region determinants and is, therefore, not classical antibody. This factor is bound by immunoadsorbents containing allo-antibody to products of the I-region of the H-2 complex suggesting that it belongs to a new family of antigen-specific immunoregulatory molecules.

Cellular Requirements and Mechanisms of Action of Antigen-specific Suppressor T Cell Factors

The cellular requirements and mechanisms of action of antigen-specific suppressor T cell factors have been analyzed using partially purified T cell subsets or cloned T cells lines, B cells and monoclonal sources of antigen-specific suppressor T cell factors. Single chain suppressor factors (TsF1) specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) are unable to suppress antibody responses of helper T cells and B cells directly, but require an Lyt 2+ cell to suppress antibody responses to GAT. These GAT-TsF1 are unrestricted by MHC or IgCH locus genes. Their activity contrasts to two-chain suppressor factors (GAT-TsF2) which are suppressive in the absence of added Lyt 2+ cells, but are restricted by MHC locus genes. The target of the TsF2 is the helper T cell; the activity of cloned helper T cells is inhibited when the cells are pulsed with GAT-TsF2 in the presence of GAT. Moreover, these cloned cells adsorb syngeneic, but not allogeneic, GAT-TsF2 in the presence of GAT. Thus helper cells can serve as targets for MHC-restricted TsF2 and cloned helper T cells can be used as a homogeneous target population to analyze the molecular mechanism(s) of T cell-mediated suppression.

Antigen-Specific Suppressor T Cells as One Mode of Expression of Immune Response Genes

Two models to explain the cellular site and mechanism of expression of MHC linked immune response (Ir) genes are currently popular. One model holds that the immune response gene is expressed as a defect in the T cell receptor repertoire such that T cells from nonresponder animals are unable to recognize antigens under Ir gene control in the context of self MHC antigens and, thus, are not stimulated. However, the same antigen may be recognized in the context of allo-MHC components under conditions where alloreactivity has been abolished. Ample experimental evidence exists in the literature to support this model (1–4). The alternative model to explain Ir gene function, the determinant selection hypothesis, centers around the concept that antigen presenting cells from nonresponder animals fail to display foreign antigenic determinants in the proper array with self MHC determinants. Again, ample experimental evidence is available to support this model (5–8).

Purification and Analysis of Antigen-specific Suppressor Proteins derived from T-Cell

ABSTRACT The immune response in the mouse to the synthetic protein terpolymer L-G-A 30 -T 10 is controlled by genes that map within the I region of the major histocompatibility locus. Mice which are either responders or non-responders to GAT can be shown to contain GAT-specific suppressor cells. Recently we have prepared somatic T cell hybridomas from which can be isolated antigenspecific suppressor factors (TsF). Several of these suppressor factors have been subjected to purification and analysis. The suppressor factors come in two molecular weight classes, 29,000 and 60,000. The 29,000 dalton proteins bind specific antigen, contain I-J determinants and react with anti-idiotype antisera and possess suppressor activity which is not MHC restricted. The 60,000 dalton proteins consist of two chains, one which binds antigen and one which reacts with I-J antisera. These proteins show MHC restriction. The mRNA from hybridoma C4.4 (which makes 29,000 dalton TsF) has been partially purified. In vitro translation of this mRNA results in the production of a biologically active protein with all the properties of TsF obtained from cells except that it is slightly smaller. These proteins are now being analyzed by chemical and molecular cloning methodology.

L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT): A probe for regulatory mechanisms in antibody responses.

The synthetic random terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) has been used as a probe to investigate regulatory mechanisms in antibody responses in tissue culture systems. In this brief review, the mechanisms of H-2 linked Ir gene control of antibody responses to GAT and genetic restrictions governing Mphi-immune T cell interactions in antibody responses to GAT are summarized.