Antigen-binding Cells Research Articles

Detection of antigen-binding cells by combined rosette formation and autoradiography.

ANTIGEN-BINDING cells (ABC) are generally detected by autoradiography using radioiodinated soluble antigen1 or by rosette formation using either a particulate antigen2 or an erythrocyte or other particle coated with soluble antigen3. Whether the same cell is detected by these two techniques was unknown. Using a soluble antigen, chicken globulin (CG), we have now demonstrated that the ABC detected by these methods is not invariably the same, which suggests that there is a receptor density difference between the thymus-derived (T) and the non-thymus-derived (B) rosette forming cell (RFC).

Antigen binding lymphocytes in congenitally athymic (nude) mice.

THE autoradiographic detection of the binding of various radiolabelled antigens to a proportion of lymphocytes from animals not exposed to those antigens (“nonprimed” lymphocytes) is well documented1–4. Such lymphocytes are thought to have patches of surface immunoglobulin, primarily IgM, which act as specific receptors for antigen5,6. A proportion at least of these unprimed lymphocytes are immunologically competent as shown in vivo7,8 and hence are true antigen reactive cells. Most assays have used peripheral lymphocyte suspensions from tissues of man, mouse, rat and chicken, not enriched or fractionated in any way for the two distinct lines of lymphocytes, thymic derived (T) and non-thymic derived (B)9. It is not clear whether antigen-binding cells (ABC), detected in routine assays where autoradiographs are exposed for 1–2 weeks, are of both T and B cell type or are predominantly of only one type. Experiments using unlabelled and radiolabelled immunoglobulin antisera with isolated T and B cells have inferred specific antigen binding on both populations although T cells seem to have far fewer antigen binding receptors than B cells10.

Specific Detection of Antigen-Binding Cells by Localized Growth of Bacteria

A new method for the enumeration of lymphoid cells with specific surface-receptors for antigen is described, based on the use of beta-D-galactosidase (EC 3.2.1.23), either directly as an antigen or as a conjugated antigen. Binding of beta-D-galactosidase is revealed by its activity in releasing riboflavin from a synthetic substrate, riboflavin-beta-D-galactopyranoside. The riboflavin, inactive as a vitamin in the galactosidic form, becomes active when released by the enzyme, and can be detected by bioassay. Hence, lymphoid cells with receptors for beta-D-galactosidase on their surface can be detected after they have been exposed to the enzyme, washed, and then plated in agar containing riboflavin-beta-D-galactopyranoside, streptomycin, riboflavin-deficient medium, and a streptomycin-resistant strain of Streptococcus faecalis that requires riboflavin. Release of riboflavin is signalled by the growth of characteristic bacterial colonies over the cell that bound beta-D-galactosidase.

Studies on immunological paralysis. IV. The relative contributions of continuous antibody neutralization and central inhibition to paralysis with type 3 pneumococcal polysaccharide.

High-zone tolerance to type III pneumococcal polysaccharide (SIll) in CBA mice is demonstrable by complete inhibition of the direct plaque-forming cell (PFC) response in the spleen. The serum antibody which protects mice against pneumococcal infection and agglutinates SIll-sensitized mouse erythrocytes is not detectable following injection of amounts of antigen one order of magnitude lower. Evidence is presented that this is attributable to continuous peripheral neutralization of antibody by recirculating, non-degradable antigen. This zone of 'pseudo-paralysis’ masks an immune response which, at the cellular level, is as intense as that induced by sheep erythrocytes. Tolerance can be lost spontaneously after many weeks and be succeeded by a strong immune response, but secreted antibody can again be neutralized by the presence of antigen in serum. Raised numbers of antigen-binding cells (rosettes) are detectable in fully tolerant (PFC-suppressed) animals. Serum anti-SIII activity can be detected in 'pseudo-paralysed’ mice by haemagglutination, or preferably by haemolysis, of heavily-sensitizedsheeperythrocytes. Evidence is presented that this represents the same IgM as is synthesized in immunized, protected mice, but which is here partially neutralized by co-existence with serum antigen. Only direct PFC’s were detectable in response to purified SIll and the characteristics of their specific neutralization implied that IgM of essentially similar avidity was formed in response to all antigen doses. Partial neutralization of IgM by SIll is discussed in relation to differences in availability or affinity of combining sites on macroglobulin.

An improved method for preparing anti-B lymphocyte serum.

THE preparation of a heterologous antiserum specific to the bursa equivalent (B) lymphoid cells of the mouse requires extensive absorption with mouse tissues, especially thymocytes, to obtain specificity. The cell surface antigen(s) against which the serum reacts has been termed mouse specific bone marrow derived lymphocyte antigen(s) (MBLA)1. The specificity of anti-MBLA for the B lymphoid population has been demonstrated by cytotoxicity tests, by its ability to inhibit a portion of antigen binding cells and by adoptive immune responses of antiserum purified lymphoid cells (refs. 1 and 2 and unpublished results of E. Moller and myself).

Receptors on immunocompetent cells. 3. Specificity and nature of receptors on dinitrophenylated guinea pig albumin- 125 I-binding cells of immunized guinea pigs.

Guinea pigs immunized with 2,4-dinitrophenyl-guinea pig albumin (DNP-GPA) possess lymphocytes which specifically bind sufficient DNP-GPA-(125)I to their surface to be detected by radioautography. These lymphocytes are present in the draining lymph nodes in a frequency of approximately 50/1000 lymphocytes in animals immunized 2-4 wk earlier with DNP-GPA in complete Freund's adjuvant. Nonimmunized animals have approximately 0.4 DNP-GPA antigen-binding cells (ABC) per 1000 lymphocytes. An increase in the frequency of DNP-GPA ABC in peripheral blood is detectable by 5 days after immunization, which is before the time that serum anti-DNP antibody is measurable. The receptors of these ABC are hapten specific in that free epsilon-DNP-L-lysine, at low concentration, inhibits the binding of DNP-GPA-(125)I; DNP bovine serum alumbin (DNP-BSA) is equivalent to DNP-GPA in the inhibition of binding of DNP-GPA-(125)I to ABC; and both DNP-GPA agarose beads and DNP-BSA agarose beads specifically adsorb DNP-GPA-(125)I ABC. Anti-immunoglobulin antisera, particularly anti-gamma(2) sera, inhibit the binding of DNP-GPA-(125)I to these cells implying that the receptors are immunoglobulin, primarily of the gamma(2) heavy chain class. DNP-GPA-(125)I ABC appear to represent precursors of antibody-secreting cells and have specificity characteristics which are very different from cells, of similarly immunized guinea pigs, which mediate a cellular immune response to DNP-GPA.

Receptors on immunocompetent cells. II. Specificity and nature of receptors on dinitrophenylated guinea pig albumin- 125 I-binding lymphocytes of normal guinea pigs.

Nonimmunized guinea pigs possess rare lymphocytes which bind sufficient 2,4-dinitrophenyl-guinea pig albumin-(125)I (DNP-GPA) to their surface to be detected by short-term radioautography. The cells occur in the lymph nodes, spleen, peripheral blood, and bone marrow with a frequency of approximately 40/100,000 lymphocytes, but are absent from the thymus. The receptors of these cells are largely specific for the haptenic group (epsilon-DNP-L-lysine) as shown by inhibition of DNP-GPA-(125)I binding with epsilon-DNP-L-lysine and with DNP bovine serum albumin (DNP-BSA). Furthermore, these cells specifically adsorb to agarose beads to which either DNP-GPA, DNP-BSA, or DNP-keyhole limpet hemocyanin (KLH) has been covalently linked. This hapten specific depletion of DNP-GPA-(125)I antigen-binding cells (ABC) correlates with a similar diminution in the capacity of adsorbed populations to transfer primary responsiveness to DNP-KLH to irradiated syngeneic recipients. Fluoresceinated anti-immunoglobulin binds to the surface of some guinea pig lymphocytes, and all DNP-GPA-(125)I ABC, as shown by a double-label technique. The great majority of DNP-GPA ABC and human gamma-globulin ABC possess surface Ig molecules of the gamma(2) heavy chain class. Preincubation of cell suspensions with anti-gamma(2) antibody markedly diminishes the number of DNP-GPA-(125)I ABC which are detected, strongly suggesting that the receptors of these cells are immunoglobulin molecules, most of which possess gamma(2) heavy chains. The specificity characteristics of DNP-GPA-(125)I ABC are strikingly different from those of cells mediating a cellular immune response to DNP-GPA, indicating major differences in the specificity and nature of the receptors of these cell types.

Carrier function in anti-hapten antibody responses. IV. Experimental conditions for the induction of hapten-specific tolerance or for the stimulation of anti-hapten anamnestic responses by "nonimmunogenic" hapten-polypeptide conjugates.

Administration of nonimmunogenic 2,4-dinitrophenyl (DNP) conjugates of copolymers of D or L-glutamic acid and lysine (GL) induces hapten-specific tolerance in nonimmune and DNP-ovalbumin-primed strain 13 guinea pigs. This tolerant state is evidenced by depressed anti-DNP antibody synthesis in response to challenge with DNP-ovalbumin and by a diminished frequency of DNP-specific antigen-binding cells and of anti-DNP antibody-secreting cells. Such a nonimmunogenic compound (DNP-D-GL) will nevertheless elicit a DNP-specific anamnestic antibody response when administered at an appropriate time to DNP-ovalbumin-primed guinea pigs undergoing a graft-versus-host reaction. These experiments are discussed in terms of a two-cell theory of stimulation of antibody responses.

Antigen-binding cells in mice immune or tolerant to Escherichia coli polysaccharide.

The number of PFC and of RFC was studied in mice which were unimmunized, immunized, or tolerant against lipopolysaccharide of E. coli 055:B5 origin. The number of PFC/10(6) spleen cells increased from 0.5 in normal to 209 in immunized mice. The corresponding figures for RFC were 93 and 513 RFC/10(6) spleen cells. In tolerant animals, which contained few or no PFC, the number of RFC was increased as compared to that found in unimmunized mice. The formation of rosettes was specific, since their formation was inhibited by soluble coli polysaccharide and by rabbit antisera against mouse immunoglobulins. The antigen-binding cells were not derived from thymus, neither in immune or tolerant mice, because they did not carry the theta antigen. It is suggested that the majority of antigen-binding cells present in tolerant animals are cells having receptors for the antigen of rather low affinity. The relevance of these findings for the induction of high and low zone tolerance is discussed.

Immunoglobulin synthesis and secretion. VI. Synthesis and intracellular transport of immunoglobulin in nonsecretory lymphoma cells.

Cells from an established line of Burkitt's lymphoma (Daudi) and a mouse myeloma (P(3)K) were pulse-labeled in vitro with (3)H-leucine, and immunoglobulin was immunologically precipitated from cell lysates and secretions. In contrast to P(3)K cells, Daudi cells synthesize a small amount of Ig which is not secreted. Subcellular fractionation experiments indicated that Ig of Daudi cells is synthesized on membrane-bound polyribosomes and enters the cisternae of the microsomes. Ig in the microsomes could be labeled with either (3)H-galactose or (3)H-fucose suggesting that transport proceeds to the Golgi complex. Additional evidence indicates that Ig molecules are transported to the plasma membrane but are not cleaved from the cell surface. These results together with other studies of Burkitt lymphoma cells suggest that the Daudi line may represent a clone of neoplastic cells derived from normal lymphocytes which synthesize but do not secrete Ig. Similarities between lymphoma cells and antigen-binding cells are discussed.

Antigen binding cells in embryonic chicken bursa and thymus.

THE autoradiographic detection of the binding of 125I-labelled antigen to a proportion of normal lymphocytes is now well established and characterized1–4. Such cells are thought to possess patches of immunoglobulin surface receptors which combine specifically with an appropriate antigen5. Evidence for the specificity of the antigen binding includes the capacity of specific anti-L and anti-µ chain antisera to block the receptor sites6, and the demonstration that at least the more heavily labelled antigen binding lymphocytes in mouse spleen are immunologically competent7.

Antigen-binding cells in normal mouse thymus.

The thymus of a normal adult mouse contains lymphocytic cells with a large number of antigen-binding receptors. Binding of the antigen by these cells is specific and can be inhibited by cross-reactive materials. It is possible that the interaction of thymocytes with antibody-forming precursor cells, required in the primary immune response to certain antigens, is mediated by these specific antigen-binding cells of the thymus.

Antigen binding cells in tolerant animals.

THE immune response involves the appearance of anti-body secreting cells as well as antigen binding cells. Antibody secreting cells can be counted in vitro using-Jerne and Nordin's local haemolysis in gel (LHG) assay1, and are then called plaque forming cells (PFC). Antigen binding cells can be recognized by their ability to bind particulate antigens such as sheep red cells (SRB) to their surfaces and have been named rosette forming cells (RFC)2,3. It is likely that RFC represent a mixture of antibody secreting cells and non-antibody secreting cells. Howard et al.4 studied the RFC response in the spleens of mice tolerant to pneumococcal polysaccharide. Although serum antibodies could not be seen in these animals a considerable number, larger, in fact, in “tolerant” than in optimally immunized animals, of RFC was found. They explained these findings on the basis that antibody secreting cells existed but that the humoral antibodies produced were neutralized extra-cellularly by persisting undegraded antigen.

Antigen binding and the immune response. II. The large number of antigen-binding cells in primed animals.

SummaryFour months after a primary injection of beta-galactosidase without adjuvant, washed spleen cells of A/Jax mice bind increased levels of the enzyme in vitro, at a time when no antibody activity can be demonstrated in the serum by other criteria. In such mice, a large proportion of spleen cells bear the antigen-binding activity. Hence, not all antigen-binding cells are necessarily antigen-sensitive or antibody-producing cells.This increased binding can be attributed to cells which have adsorbed cytophilic antibody in vivo during the period of antibody biosynthesis.

Studies on antigen-binding cells: I. The origin of reactive cells

The origin of rosette-forming cells of mice immunized with sheep red blood cells has been investigated. By the use of antigenic markers on thymus cells (H-2, θ) it was shown that a considerable proportion of RFC formed in the spleens in irradiated animals reconstituted with thymus and bonemarrow cells, as well as in intact normal and immunized animals were of thymus origin. The possible limitations of the test systems and the significance of the results have been discussed.

Studies on antigen-binding cells: II. Relationship to antigen-sensitive cells

The immunological significance of antigen-binding cells (“rosette”-forming cells) in mice injected with sheep erythrocytes has been investigated in different experimental situations by a comparative quantitative study of antibody-producing cells and antigen-binding cells as well as of antigen-sensitive cells utilizing a cell-transfer system. “Passive” antibody administration to animals immunized with sheep red cells caused a considerable depression of antibody-producing cells, whereas “rosette”-forming cells as well as antigen-sensitive cells were much less suppressed. The dose response of mice to sheep erythrocytes was studied and it was found that very low doses of antigen induced immunological priming or memory and a marked response with regard to rosette-forming cells in the absence of any significant development of antibody-producing cells. These results suggest that the antigen-binding lymphoid cells in the spleens of mice immunized against sheep erythrocytes include antigen-sensitive cells as well as cells actively engaged in antibody secretion. A considerable proportion of the former are thymus derived, possessing the thymus-specific antigen θ, and with immunoglobulin-like binding sites for antigen, as judged by inhibition of antigen binding by treatment of the cells with an anti-Fab and anti-light chain antibody.

Antigen binding cells in tolerance and immunity.

Antigen binding cells in tolerance and immunity.

ANTIGEN-BINDING LYMPHOCYTES IN HUMAN FETAL THYMUS

The thymus of the human fetus, already lymphoid at 20-22 weeks of gestation, contained antigen-binding lymphocytes detectable by autoradiography after exposing thymic lymphocytes to radioiodine labelled flagellin from Salmonella adelaideand S. waycross. Counts of antigen-binding lymphocytes were considerably higher for the fetal thymus than for the postnatal and adult thymus. There were relatively more antigen-binding lymphocytes in fetal thymus than in the corresponding fetal spleen and blood, but the reverse pertained for the adult thymus. " Grain-counts " over cells, representing " avidity " of lymphocytes for antigen, showed that the number of cells fell progressively as the grain-count increased. It is suggested that in man the fetal thymus is an important contributor of cells concerned in humoral antibody production, and is the site of generation or establishment of diversity of antibody pattern. The relatively large number of flagellin-reactive cells in the fetal thymus indicates that the development of antigen-binding cells within the fetal thymus does not happen entirely by a random process of somatic mutation. Our data favour genetic programming for antibody patterns so that the essential feature of the thymus may be to convert genomic potentiality to actual phenotypic expression.