Constant Region Determinants Research Articles

Reconstitution of antigen-specific suppressor activity with translation products of mRNA.

It has been well established that antigen-specific suppression of antibody responses can be mediated by soluble factors derived from suppressor T cells1. However the chemical and functional properties of antigen-specific suppressor factors (TsF) are still controversial. We have recently described an antigen-specific suppressor T-cell factor composed of two distinct molecules. One is encoded by the gene located in the I–J subregion of the mouse H–2 complex on the 17th chromosome2,3, and the other which possesses the antigen-binding moiety2,3 and the constant region determinant (Ct), is the product of the Ct gene linked to the immunoglobulin heavy chain (Igh) gene cluster on the 12th chromosome4,5. The association of these two molecules is essential for the expression of the TsF activity2,3. However, Fresno et al.6 have shown that the TsF specific for sheep red blood cells (SRBC) is composed of a single polypeptide chain carrying the antigen-binding moiety. Kapp et al.7 have described another TsF with the antigen (GAT)-binding moiety and the I–J determinants on the same molecule. We describe here the identification of messenger RNAs (mRNAs) coding for the Ct-bearing molecules with antigen-binding moiety and the I–J encoded molecules in mRNA fractions derived from the T suppressor cell hybridoma by translation in Xenopus laevis oocytes. Furthermore, the mixture of the translation products of these two separate mRNA fractions was shown to reconstitute the antigen-specific TsF activity.

Two distinct allotypic determinants on the antigen-specific suppressor and enhancing T cell factors that are encoded by genes linked to the immunoglobulin heavy chain locus.

The alloantiserum was raised in BALB/c (H-2d, Igh-1a) mice hyperimmunized with spleen cells of Igh allotype congenic mice, CB-20 (H-2d, Igh-1b). It was found to define the new allotypic determinants (distinct from B cell Igh constant region determinants: Igh allotype) expressed only on a small population of T cells belonging to the Thy-1 dull-stained Lyt-2- or Lyt-2+ population of Igh-1b mice. Genes coding for the determinants were shown to be accommodated somewhere in the right side of the Igh variable region gene (Igh-V) cluster, as the antibody activity was completely absorbed with BAB-14 thymocytes. It was also demonstrated that the products detected by the antiserum represent the allotypic determinants (probably constant region determinants) on the antigen-binding moiety of the antigen-specific augmenting (TaF) and suppressor (TsF) T cell factors. Moreover, determinants on TsF were found to be distinct from those on TaF. Therefore, it can be suggested that the two genes coding for the T cell allodeterminants (distinct from those of the B cell Igh) are located in the right side of the B cell Igh-V on the 12th chromosome, and that both encode the antigen-recognition units of the functionally distinct T cell factors.

Isolation and partial characterization of an antigen-specific T-cell factor associated with the suppression of delayed type hypersensitivity.

Antigen-specific factors associated with immunosuppressive activity, released by cultured T cells from mice tolerant to the haptens trinitrophenyl, dinitrophenyl and oxazolone, were purified by hapten affinity chromatography. Their binding specificity for antigens paralleled their immunoregulatory activity. Like some immunoglobulin molecules, these factors had blocked NH2 termini and could be bound to Fc-like receptors on macrophages. However, neither immunoglobulin constant region determinants (isotypes) nor antigens encoded by the major histocompatibility complex were detected on the suppressive factors. The purified factors occurred as 68,000-dalton proteins and non-covalently linked dimers. No associated immunoglobulin light chain molecules were detected. The factors showed a marked propensity toward degradation with major breakdown products of 45,000-50,000 and 25,000-30,000 daltons. These results suggest that these molecules are the T-cell products analogous to B-cell immunoglobulin (equivalent to heavy chains) and that they may be the antigen-specific components which act in conjunction with major histocompatibility-controlled gene products to perform antigen-specific suppression.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XI. Pseudogenetic restrictions of hybridoma suppressor factors.

Suppressor factor derived from three different murine T cell hybridomas were characterized . They specifically inhibited 4-hydroxy-3-nitrophenyl acetyl cutaneous sensitivity responses. The factors bind antigen and bear I-J and idiotypic determinants, but lack conventional immunoglobulin constant-region determinants. The factors function during the induction phase of the immune response, by inducing a second population of suppressor cells (Ts(e)). Suppressor factor can inhibit both cellular and plaque-forming cell responses in appropriate strains of mice. These hybridoma suppressor factors directly suppress strains of mice that are Igh-V homologous with the strain producing the factor. Thus, there is an apparent Igh-V restriction in the activity of these factors. However, this is a pseudogenetic restriction because these factors generate second order suppressor cells (Ts(e)) in Igh-incompatible mice, but in order to express the suppressive activity, the cells must be adoptively transferred into recipients that are Igh compatible with the strain producing the suppressor factor. Finally, it was shown that the factor-induced Ts(e) population is under an apparent dual genetic restriction. Thus, Igh and H-2 homology is required in order for the Ts(e) population to express its suppressive activity.

Haplotype-specific suppression of antibody responses in vitro. II. Suppressor factor produced by T cells and T cell hybridomas from mice treated as neonates with semiallogeneic spleen cells.

Culture supernatant fluids from spleen cells from C57BL/10 or BALB/c mice neonatally treated with semiallogeneic (B 10.D2 x B10)F1 cells to induce haplotype-specific suppressor T cells and restimulated with macrophages syngeneic at I-A with the allogeneic haplotype encountered as neonates contain a soluble factor capable of suppressing primary in vitro antibody responses of normal syngeneic spleen cells in a non-antigen-specific manner. This haplotype-specific suppressor factor, TsF-H, has also been recovered in culture fluids of a T cell hybridoma produced by fusion of the AKR thymoma BW5147 and the haplotype-specific suppressor T cells. TsF-H is inactivated by low pH (3.5) trypsin, for 30 min at 50 degrees C, and has a molecular weight in the range of 45,000 to 68,000. Studies with specific immunoabsorbents demonstrate the presence of determinants encoded by the I-A subregion of the haplotype of the T cell producing TsF-H but not I-J subregion or immunoglobulin constant-region determinants on the TsF-H. Suppression is restricted to primary in vitro antibody responses, and not secondary antibody, mixed lymphocyte, or cytotoxic lymphocyte responses by spleen cells syngeneic at the I-A subregion of H-2 with the T cell producing the factor. The properties and activities of TsF-H and the haplotype-specific suppressor T cell are compared and contrasted with antigen-specific and genetically restricted suppressor T cells and their factors.

A marmoset T-lymphocyte protein related to defined human serum immunoglobulin and fragments.

Current concepts of the antigen-specific receptor of thymus-derived (T) lymphocytes suggest that this molecule bears immunoglobulin (Ig) variable (V) regions, but does not represent a classical serum antibody. We immunized rabbits with a globulin fraction obtained from an in vitro cultured marmoset T cell lymphoma line which expressed surface components related to immunoglobulins. Quantitative immunoprecipitation and competition radioimmunoassay studies carried out using characterized monoclonal human Igs and their chains and fragments show that this rabbit antiserum, raised against a T cell product, reacts with a particular, conformational VH-determinant which is formed by interaction with lambda light chains and with two mu chain (IgM) related determinants. Neither mu chain determinant is a major isotype specific marker, and each occurs only on certain distinct subsets of serum mu chains. One is located in the Fc fragment; the location of the other mu related determinant is not known. These results show that this rabbit antiserum recognizes both variable and constant region determinants on the T cell receptor heavy chain. Rabbit antibody to the T cell product which cross-reacts with serum mu chain was isolated by immune affinity chromatography on Sepharose-derivatized covalently with one particular reactive IgM protein, the Waldenstrom macroglobulin Can (kappa mu). This antibody precipitated a biosynthetically-labelled component of approximate M.W. 70,000 as assessed by polyacrylamide gel electrophoresis in Na Dod SO4-containing buffers. These data support the conclusion that certain T cells express and synthesize a molecule related to Ig VH-regions which also has a constant region sharing antigenic markers with IgM subpopulations.

Isolation of t cell membrane proteins (IgT) with antisera to non-isotypic determinants of immunoglobulins: Evidence thaT IgT ‘Light’ chains are not identical to B cell kappa chains

Radiolabelled cell membrane proteins obtained by detergent lysis of murine T cells labelled by lactoperoxidase-catalysed radioiodination were specifically immunoprecipitated with an antiserum to immunoglobulin Fab determinants. Absorption of the antiserum by myeloma proteins or normal immunoglobulins coupled to Sepharose and the use of antibody proteins eluted from these absorbents indicated that the antibody activity in the serum responsible for binding detergent-soluble T cell membrane proteins was directed towards variable region and not to the constant region determinants of kappa light chains. Immunoglobulin light chain sized polypeptides isolated from T cells with this antiserum were found to possess serological characteristics which differed from serum or B cell membrane immunoglobulin kappa chains. Evidence is presented which suggests that a portion of the light chain sized polypeptides isolated from T cells may be proteolytic fragments of a larger polypeptide chain. The results suggest that immunoglobulin-like detergent-soluble T cell membrane proteins isolated with antisera to immunoglobulins (IgT) possess neither immunoglobulin heavy nor light chain constant regions determinants but do carry determinants associated with immunoglobulin variable regions.

Are VH framework antigenic determinants expressed on both rabbit B and T lymphocytes?

AbstractThe extent to which VH framework determinants are represented on T cell receptors is unresolved and controversial. We have studied this question in the rabbit using antia (VH framework) allotype antisera and a goat anti‐rabbit VH antiserum. Flow micro‐ fluorometric analysis of spleen lymphocytes from hyperimmune animals (Freund's adjuvant plus picryl chloride) demonstrated closely similar frequencies of cells stained with the anti‐VH framework antisera compared to anti‐light chain antisera. To search for potentially hidden molecules, additional studies were performed with radioiodinated, detergent‐solubilized plasma membranes derived from immune spleen and lymph node cells. The membranes were analyzed by standard immunoprecipitation techniques. Absorption with anti‐Ig constant region immunoabsorbents removed all radioactivity precipitable by specific anti‐VH framework antisera compared to controls, whereas after absorption with control (normal goat globulin) immunosorbents, VH‐bearing surface IgM and putative surface IgD (presumably of B cell origin) were readily demonstrated by immunoprecipitation. Thus, no evidence for a population of cells or a class of molecule(s) bearing VH in the absence of light or heavy chain constant region determinants was obtained. Although these studies do not support the concept of similar VH framework determinants on T and B cell receptors, they do not exclude a much more limited sharing of determinants or a very minor T cell subpopulation which expresses VH framework that would not be detectable by the methods used in these studies.

Immunosuppressive factor(s) extracted from lymphoid cells of nonresponder mice primed with L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) III. Immunochemical properties of the GAT-specific suppressive factor.

The GAT-specific suppressor T-cell factor (GAT-TsF) extracted from lymphoid cells from GAT-primed, nonresponder DBA/1 mice has been partially characterized. It is a protein that has affinity for GAT and determinants encoded by the I region of the H-2 complex. On the basis of specificity and avidity, GAT-TsF resembles anti-GAT-MBSA antibodies produced by DBA/1 mice in spite of the fact that it is too small to be classical antibody and has no constant-region determinants of heavy or light chains. Further, GAT or a fragment of GAT is associated with the GAT-TsF. GAT-TsF has been partially purified from the crude extract by absorption to GAT-Sepharose and elution with 0.4 to 0.6 KCl. GAT-TsF purified on the basis of its affinity for GAT bears I-region determinants but not detectable GAT or GAT fragment.

Demonstration of an idiotypic antigen on a monoclonal cold agglutinin and on its isolated heavy and light chains.

A potent anti-idiotype serum produced in a rabbit immunized with the isolated heavy chains of an IgM cold agglutinin "Col" was rendered specific by solid-state adsorptions. The anit-Col idiotype was shown to bind specifically to both isolated Col heavy (mu) and light (kappa) chains as well as to intact Col IgM by three methods: (i) reversal of anti-idiotype inhibition of Col cold agglutinin in an automated hemagglutination-inhibition assay system; (ii) adsorption of the anti-idiotype by affinity gels consisting of Col IgM, mu, or kappa chains covalently coupled to Sepharose 2B; (iii) binding of Col IgM and its isolated chains by an anti-idiotype affinity gel. Fragments of Col light chain lacking constant region determinants but still capable of inhibiting anti-idiotype were produced by limited pepsin digestion of the light chains. The finding of shared idiotypic determinants on isolated heavy and light chains of a monoclonal antibody suggests that these chains share a common sequence in a hypervariable region. As an extension of the gene insertion theory of Wu and Kabat, we postulate that genes coding for hypervariable regions may be available for insertion into the DNA for both heavy and light chains.