A new mouse immunoglobulin: IgG3.

A new subclass of mouse IgG for which we propose the name IgG3 has been shown to have a mol wt of 150,000 consistent with an L(2)H(2) structure, and is present in normal mouse serum at a concentration of 0.1-0.2 mg/ml. Its molecular weight, low carbohydrate content, glycopeptide analysis, and C-terminal analysis are all typical of the IgG class. The intact protein had a strong tendency to form noncovalent aggregates with itself which were dissociable in acid. Upon papain digestion an Fab fragment of 47,000 mole wt was generated along with an Fc fragment which was insoluble at neutral pH. As for its biology, the protein did not fix complement, was not cytophilic for gammaG2 receptor sites on macrophages, and did not show passive cutaneous anaphylaxis. It was very efficiently transported across the placenta so that its concentration in the newborn was twice that in the serum of the mother, compared to the concentration of IgG1 and IgG2 proteins which were only present at one-third the concentration of that found in the serum of the mother. The Fc fragment of this protein reacted with and was solubilized by the staphylococcal A protein which also precipitated the intact immunoglobulin. In addition, the myeloma protein which was the prototype for this gammaG subclass exhibited binding activity for levan which was localized to the Fab fragment.

Receptors for aggregated IgG on mouse lymphocytes: their presence on thymocytes, thymus-derived, and bone marrow-derived lymphocytes.

An autoradiographic binding assay employing (125)I-labeled heat-aggregated mouse IgG2b myeloma protein (MOPC 141) was used to demonstrate receptors for IgG on 20-45% of Balb/c thymocytes and on 70-80% of splenocytes. Binding could also be shown with heat or BDB aggregates of another IgG2b (MOPC 195), with IgG1 and with human gamma-globulin, but not with aggregated chicken gamma-globulin, IgA, BSA, nor with aggregated Fab fragments of IgG2b. Optimum binding was obtained at 37 degrees C. Detection of binding was dependent upon aggregate size with complexes of more than 100 IgG molecules being optimal, aggregates of 6-25 detecting splenocytes but not thymocytes, and aggregates of less than 6 binding to a negligible extent. Comparison of grain counts on various cell types showed mastocytoma cells (P815) and macrophages averaging 40-50 grains/cell/day, allogeneically activated thymocytes 20-30, splenocytes 2-3, L5178 lymphoma cells 1, and positive thymocytes 0.6 grains/cell/day. Double labeling experiments for surface Ig, theta-antigen, and agg IgG receptor on mouse spleen cells indicated that a relatively high density of receptor was present on about 80% of B cells, 30% of T cells, and 60% of SIg(-), theta(-), null cells.

Thymus-derived (T) cell immunoglobulins. Presence of a receptor site for IgG and absence of large amounts of "buried" Ig determinants on T cells.

Quantitation of surface and total cell Ig obtained after lysis by detergent, urea-acid treatment, and freeze-thawing were determined on spleen cells, thymus cells, and spleen cells specifically depleted of B cells. A two- to four-fold increase in measurable Ig was found after cell lysis. All cell populations showed a similar increase in measurable Ig indicating that no discordantly large amounts of buried Ig determinants were associated with the surface of T cells. The lack of appreciable amounts of T cell Ig was confirmed by immunoprecipitation of radioiodinated cells. A theta-positive lymphoma was described which, when grown in culture, lacked detectable surface Ig but contained a receptor site for IgG. This resulted in appreciable amounts of surface IgG being associated with the tumor line when isolated from ascitic fluid of tumor-bearing mice or after preincubation of cultured cells with either heat-aggregated IgG or normal mouse serum.

Recombination of heavy and light chains of human gamma-a-myeloma proteins: formation of hybrid molecules and configurational specificity.

The present studies demonstrate that the conditions necessary for reductive cleavage, isolation, and recombination of L and H polypeptide chains of human gammaA-myeloma globulins parallel those required for similar manipulation of the component chains of gammaG-globulin. Specificity of recombination was shown for chains derived from the same protein. In contrast, no intradass preferential recombination was demonstrable. Hybrid molecules, formed by reassociation of noncovalent bonds, could be synthesized from isolated chains of two immunoglobulin classes resulting in the formation of molecules of the type gammaA-H-gammaG-L and gammaG-H-gammaA-L. Several sera containing both gammaA- and gammaG-"monoclonal" peaks were studied, one of which demonstrated the L chains associated with both peaks to be identical both by electrophoretic mobility in acid-urea gel and antigenic analysis. The possibility is considered that this case represents a naturally occurring analogue of the artificially produced hybrid molecules described in this study. Configurational antigenic specificity of gammaA-myeloma proteins, imposed by the presence of kappa L chains in native and appropriately recombined molecules, provides a further indication of the importance of noncovalent bonds in the establishment of the quaternary structure of these proteins.

Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line.

To evaluate subclass specificity and aggregate size requirements of IgG receptors on mouse cells, we measured binding of radiolabeled monomeric and BDB-aggregated mouse myeloma proteins fractionated into various sizes by means of gel filtration. Monomers, tetramers, and high molecular weight (approximately 10(7) daltons) aggregates were used. The various cells and cell lines studied could be segregated into three patterns of reactivity: (a) Macrophage and macrophage-like cell lines bound monomer IgG2a preferentially; high molecular weight IgG aggregates bound as follows: IgG1 = IgG2b = IgG2a. (b) Lymphoid lines D2N and S49 showed no capacity to bind monomer IgG2a; high molecular weight aggregates bound as follows: IgG1 = IgG2b less than IgG2a. (c) Other Thy-1-positive lymphoid cell lines (EL4 and L5178) and normal T and B cells showed no capacity to bind monomer IgG; high molecular weight IgG aggregates bound to a lesser extent than to cells of the first two categories in the following manner: IgG1 less than IgG2b greater than or equal to IgG2a. The variable pattern of reactivity of the macrophage-like cell lines with monomer and aggregated IgG suggested that two distinct receptors for IgG were present: one capable of binding IgG2a and another capable of binding all aggregates. Further evidence for this hypothesis was obtained by analysis of the inhibitory capacity of different IgG subclasses on the binding of aggregated IgG and monomer IgG2a to P388 cells. Inhibition of monomer IgG2a binding was effected only by monomer or aggregated IgG2a, whereas inhibition of binding of aggregated IgG1 or IgG2b was noted with aggregates of all three subclasses with some preferential inhibition by monomer IgG2b being observed. Furthermore, monomer IgG2b binding was preferentially inhibitable by monomer IgG2b. It is postulated from these data that two receptor sites are present on this macrophage-like cell line, one reactive with aggregates of all three subclasses as well as monomer IgG2b, and another receptor specific for monomer IgG2a which also binds aggregated IgG2a. Support of this concept was obtained by trypsinization experiments in which the binding of monomer IgG2a was markedly decreased by trypsin treatment of cells, whereas the binding of aggregated IgG2b was unaffected by this treatment.

Immunoglobulins on the surface of lymphocytes. I. Distribution and quantitation.

The distribution, and quantity of immunoglobulins on the surface of lymphocytes has been studied by means of immunofluorescence and a quantitative radio-immunoassay. Surface immunoglobulins were found on approximately 45% of spleen and marrow lymphocytes and 7-14% of lymphocytes from lymph nodes, peripheral blood, and peritoneal exudate. Thymic lymphocytes contained undetectable amounts of immunoglobulin. In the spleen the different immunoglobulins were present in the following order: gammaG2 > gammaG1 > M > gammaA > gammaG3. The surface immunoglobulin was largely removable by brief treatment with trypsin. Quantitative analysis indicated that 50,000-150,000 molecules of immunoglobulin were present on an individual cell. A variety of observations make it likely that this lymphocyte-associated immunoglobulin. is a product of the cell to which it is attached rather than a form of cytophilic antibody.

Immunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin.

Immunofluorescent studies using live cells from antibody-forming organs and anti-immunoglobulin antibodies demonstrate two populations of small lymphocytes which are differentiated by the presence or absence of Ig on their surface membranes. Most of the lymphocytes with detectable surface Ig appear to derive from cells of the bone marrow, while most of the Ig-negative lymphocytes derive from the thymus. Thus, adult mice thymectomized, lethally irradiated, and transplanted with bone marrow cells showed a normal number of lymphocytes with surface Ig but were depleted of the Ig-negative lymphocytes. Injection of thymocytes into these mice did not result in an increase in the number of lymphocytes with surface Ig in spleen and lymph nodes. Most of the injected thymocytes could be identified by means of histocompatibility markers. Also, the spleen and lymph nodes of neonatally thymectomized mice contained lymphocytes with surface Ig but were depleted of the Ig-negative lymphocytes. Attempts were made to identify light chains on thymocytes by a sensitive radioimmunoassay. In some experiments no light chains were detected and in others a small amount, i.e. no more than 2.6% of the amount present on spleen lymphocytes, could be detected. Whether these low figures are significant or represent a small amount of serum contamination is not clear as yet.

Two different complement receptors on human lymphocytes. One specific for C3b and one specific for C3b inactivator-cleaved C3b.

IN THE PRESENT STUDY IT WAS SHOWN THAT NORMAL PERIPHERAL LYMPHOCYTES HAVE TWO DIFFERENT COMPLEMENT RECEPTORS: one for C3b (the immune adherence receptor) and one for C3b subsequent to its cleavage by C3b inactivator. The two receptors are not cross-reactive and were shown by tests with various antisera to be antigenically distinct. Both the immune adherence receptor and the receptor for C3b inactivator-cleaved C3b were found on normal peripheral lymphocytes and on cultured lymphoblastoid cells. In 15 out of 18 chronic lymphatic leukemia patients, the immune adherence receptor was either partially or completely missing from the peripheral lymphocytes, while the lymphocyte receptor for C3b inactivator-cleaved C3b was retained. Normal erythrocytes, on the other hand, were found to have only the immune adherence receptor. Granulocytes from normal peripheral blood appeared to have only a receptor for C3b and did not have a receptor for C3b inactivator-cleaved C3b.

A subclass of human gamma-A-globulins (gamma-A2) which lacks the disulfied bonds linking heavy and light chains.

The gammaA2-subgroup of gammaA-globulins, previously delineated by antigenic studies, was found to differ strikingly from other immunoglobulins in the manner in which the polypeptide chains are bound together. The heavy and light chains were not linked to each other by disulfide bonds. Instead the light chains were disulfide linked to one another, and were present in the gammaA2-molecule as disulfide bridged L-L dimers. Antisera specific for gammaA2-proteins indicated the occurrence of two different antigenic types in all normal sera as well as saliva and colostrum. Both of these showed the unique interchain disulfide linkage. Quantitative analyses indicated higher levels of gammaA2-proteins in external secretions.

The stimulation of low-affinity, nontolerized clones by heteroclitic antigen analogues causes the breaking of tolerance established to an immunodominant T cell epitope.

H-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-E(k)-restricted epitope from moth cytochrome c (MCC) 88-103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88-103 analogues that are heteroclitic antigens as assessed on representative MCC 88-103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88-103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vbeta3(+)/Valpha11(+), none of the hybridomas derived from tolerance-broken animals expressed Vbeta3, although they were all Valpha11(+). Also, the Vbeta complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88-103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vbeta3(+)/Valpha11(+) transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.

The minimal number of class II MHC-antigen complexes needed for T cell activation.

Major histocompatibility complex (MHC) molecules are exposed to large quantities of self and nonself antigens. It is not known what fraction of MHC molecules needs to be occupied by antigen to induce a T cell response. A quantitative study of naturally processed antigen indicated that T cells could be activated when only 0.03 percent of the total I-Ed purified from antigen-presenting cells (APCs) was occupied with antigen. B cells and macrophages processed hen egg lysozyme (HEL) with different efficiencies, but similar degrees of occupancy were required for T cell stimulation. Higher occupancy was needed for I-Ed-transfected L cells, possibly reflecting the requirement for other accessory molecules for efficient APC-T cell interaction.

Carboxy-terminal amino acids of gamma-A and gamma-M heavy chains.

The carboxy-terminal amino acids of alpha-and micro-chains from human immunoglobulins and alpha-chains from mouse immunoglobulins have been determined by carboxypeptidase digestion and hydrazinolysis. The data suggest the following carboxy-terminal sequences: human micro: Ala-Gly-Thr-Cys-TyrCOOH; human alpha: Thr-Cys-TyrCOOH; murine alpha: (Ileu, Cys)-TyrCOOH.

Autologous peptides constitutively occupy the antigen binding site on Ia.

Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype was efficient in inhibiting the binding of 125I-labeled I-Ad-specific peptide to I-Ad, but did not significantly inhibit the binding of an I-Ed-specific peptide to I-Ed; the reciprocal isotype-specific inhibition was demonstrated with low molecular weight material derived from I-Ed. The inhibitory material was predominantly peptide in nature, as shown by its susceptibility to protease digestion. It was heterogeneous as measured by gel filtration (mean molecular weight approximately 3000), and when characterized by high-performance liquid chromatography, it eluted over a wide concentration of solvent. Such self peptide-MHC complexes may have broad significance in the biology of T cell responses, including generation of the T cell repertoire, the specificity of mixed lymphocyte responses, and the immune surveillance of self and nonself antigens in peripheral lymphoid tissues.

Low-molecular-weight proteins related to Bence Jones proteins in multiple myeloma.

Urinary proteins distinct from Bence Jones proteins, but sharing antigenic determinants, were found in the urine of a number of patients with multiple myeloma. These components were smaller in size and antigenically deficient compared with Bence Jones proteins. They were best detected with antiserums to the homologous Bence Jones proteins and, in some cases, were related to the variable portion of the Bence Jones protein molecule.

Elevated salivary and synovial fluid beta2-microglobulin in Sjogren's syndrome and rheumatoid arthritis.

Beta2-Microglobulin is normally present in low concentrations in serum and other bodily fluids. By use of a radioimmunoassay, elevated concentrations of beta2--microglobulin were found in saliva and synovial fluid from patients with Sjogren's syndrome and rheumatoid arthritis, autoimmune inflammatory diseases that attack and destroy the salivary glands and articular tissues, respectively. Elevated beta2-microglobulin concentrations decreased in the saliva of two patients who simultaneously showed a clinical response to systemic treatment. Measurement of beta2-microglobulin in inflammatory fluids may offer a simple method of quantifying local activity in autoimmune states.

The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides.

The capacity of purified I-Ad, I-Ed, I-Ak, and I-Ek to bind to protein derived peptides that have been previously reported to be T cell immunogens has been examined. For each of the 12 peptides studied strong binding to the relevant Ia restriction element was observed. All the peptides bound more than one Ia molecule; however, for 11 of 12 peptides, the dominant binding was to the restriction element, whereas in one instance the dominant binding was to a nonrestriction element. When the peptides were used to inhibit the presentation of antigen by prefixed accessory cells to T cells, an excellent correlation was found between the capacity of a peptide to inhibit the binding of an antigen to purified Ia and the capacity of the peptide to inhibit accessory cell presentation of the antigen. Thus, the binding of peptide to purified Ia is immunologically relevant, and Ia seems to be the only saturable molecule on the surface of the accessory cell involved in antigen presentation. Inhibition analysis also indicated that all peptides restricted to a particular Ia molecule competitively inhibited one another, suggesting that each Ia restriction element has a single binding site for antigen. Cross-linking of labeled peptides to Ia followed by electrophoretic analysis and autoradiography suggested that this single binding site is made up of portions of both alpha and beta chains of Ia.

Immunological self, nonself discrimination.

The ability of immunodominant peptides derived from several antigen systems to compete with each other for T cell activation was studied. Only peptides restricted by a given transplantation antigen are mutually competitive. There is a correlation between haplotype restriction, ability to bind to the appropriate transplantation antigen, and ability to inhibit activation of other T cells restricted by the same transplantation antigen. An exception was noted in that a peptide derived from an antigen, bacteriophage lambda cI repressor, binds to the I-Ed molecule in a specific way, yet is not I-Ed-restricted. Comparison of the sequence of the repressor peptide with that of other peptides able to bind to (and be restricted by) I-Ed and a polymorphic region of the I-Ed molecule itself revealed a significant degree of homology. Thus, peptides restricted by a given class II molecule appear to be homologous to a portion of the class II molecule itself. The repressor-derived peptide is identical at several polymorphic residues at this site, and this may account for the failure of I-Ed to act as a restriction element. Comparison of antigenic peptide sequences with transplantation antigen sequences suggests a model that provides a basis for explaining self, nonself discrimination as well as alloreactivity.

Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad.

Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule I-Ad. Sequences for nine of these were obtained by a combination of automated Edman degradation and tandem mass spectrometry. All of the peptides are derived from secretory or integral membrane proteins that are synthesized by the antigen-presenting cell itself. Peptides were 16 to 18 residues long, had ragged NH2-and COOH-termini, and contained a six-residue binding motif that was variably placed within the peptide chain. Binding data on truncated peptides suggest that the peptide binding groove on class II molecules can be open at both ends.

Immunoglobulins on the surface of lymphocytes. IV. Distribution in hypogammaglobulinemia, cellular immune deficiency, and chronic lymphatic leukemia.

The distribution of peripheral blood lymphocytes that contain surface Ig has been studied by means of immunofluorescence in humans. Normal individuals, individuals with sex-linked and acquired agammaglobulinemia, selective IgA deficiency, cellular immune deficiencies, and individuals with chronic lymphatic leukemia (CLL) were studied. Approximately 28% of the peripheral blood lymphocytes from normal individuals contained surface Ig. On an average 15% contained IgG, 6%, IgA, and 8%, IgM; and the kappa: lambda ratio was 2:1. Lymphocytes from patients with CLL appeared to be "monoclonal" in that the cells from a given individual had a single Ig associated with them (e.g., kappa IgM). In three-quarters of the cases the H chain class was IgM; in the remaining one-quarter no H chain could be detected on the cell surface. The L chain class was kappa in 12 cases and lambda in 8. Four patients with sex-linked agammaglobulinemia and one with "acquired" agammaglobulinemia had markedly decreased numbers of cells with surface Ig (0-4%). In contrast, the three patients with selective IgA deficiency and no detectable serum IgA contained normal numbers of cells (6-8%) with surface IgA. Five patients with cellular deficiency states, including two with Wiskott-Aldrich syndrome, contained a normal or low percentage of cells with surface Ig.

Catabolism of human gammaG-immunoglobulins of different heavy chain subclasses. I. Catabolism of gammaG-myeloma proteins in man.

The rates of catabolism of human gammaG-immunoglobulins of subclasses gammaG(1), gammaG(2), gammaG(3), and gammaG(4) were studied by determining the rates of elimination from the circulation of pairs of (131)I-and (125)I-labeled gammaG-myeloma proteins in 57 patients suffering from cancer other than multiple myeloma. On the average, gammaG(1)-, gammaG(2)-, and gammaG(4)-myeloma proteins were catabolized at a rate similar to that of normal gammaG-immunoglobulin, whereas gammaG(3)-myeloma proteins were catabolized more rapidly than normal gammaG-immunoglobulin. The average half-lives for the myeloma proteins were 12.3 days for normal gammaG, 11.6 days for gammaG(1), 12.4 days for gammaG(2), 8.2 days for gammaG(3), and 11.3 days for gammaG(4). However, significant differences in catabolic rates were observed when individual myeloma proteins of a single subclass were compared. These individual variations were present within all four heavy chain subclasses. The extent of differences ranged from 10 to 47%. The catabolic rate of normal gammaG was in an intermediate range when compared with myeloma proteins of relatively long and short half-lives. The rate of catabolism of an individual myeloma protein did not correlate with its light chain type, Gm factor, carbohydrate content, or electrophoretic mobility. These findings indicate that the structure(s) related to the catabolism of gammaG-immunoglobulins are complex and differ from one immunoglobulin molecule to another.