Cellular events in tolerance. V. Detection, isolation and fate of lymphoid cells which bind fluoresccinated antigen in vivo.

The binding of tolerogen to specific receptors of lymphocytes and the subsequent fate of such cells was directly studied in Lewis rats injected with fluorescein-labeled sheep gamma globulin (F-SGG). This tolerogen produced unresponsiveness both in SGG-specific T cells (carrier tolerance) and F-specific antibody-forming cell precursors. The former (T-cell tolerance) was still significant more than 60 days after tolerogen whereas tolerance in the latter (B-cell tolerance) had waned by that time.Cells which have bound the tolerogen (antigen-binding cells, ABC) in vivo were detectable by direct immunofluorescence of washed spleen cell suspensions from rats injected with F-SGG up to 7 days previously. These cells were isolated using antifluorescein affinity columns, and shown to contain immunocompetent precursors for F- and SGG specific responses.The frequency of such ABC was between 30 and 80 per 10⁵ spleen, lymph node or bone marrow cells; no ABC were detected in the thymus. Both Ig positive and Ig negative cells were found to be ABC; Ig negative ABC usually showed a “capped” fluorescent pattern whereas Ig positive ABC generally were “spotted.”By 10 days after injection, ABC were not detectable in the spleen, lymph nodes, thymus or bone marrow of tolerant rats. Furthermore, reinjection of F-SGG after this time did not label any cells. This suggests that antigen-binding cells are not present at this time or that such cells, if available, lack receptors. In contrast, rats previously injected with a lower non-tolerogenic dose of F-SGG or an immunogenic form (F-SGG on bentonite) possessed cells at these later times which could be labeled with F-SGG. Thus, ABC remain detectable following immunogen or a subtolerogeic dose of F-SGG, but disappear in tolerant rats.By approximately 40 days after initial high dose tolerogen injection (when B cell tolerance has started to wane), cells capable of binding a second dose of F-SGG again became detectable. It is suggested that high doses of F-SGG are bound by specific lymphocytes (identifiable as ABC) and that these cells either fail to regenerate new receptors or die. As tolerance begins to wane, either new receptors or new cells are generated.     

Antigen binding to lymphoid cells from unimmunized mice: high frequency of beta-galactosidase binding cells at optimal conditions

The median frequency of antigen-binding cells for beta-galactosidase in mouse organs is: thymus—0.1%; lymph nodes—0.35%; spleen 2.0%; bone marrow—4.8%. We propose that these frequencies closely approximate the actual numbers of cells bearing receptors with beta-galactosidase specificity.We attribute these relatively high frequencies to (a) incubation of cells with saturating concentrations of antigen, (b) the multimeric nature of beta-galactosidase, and (c) retention of receptors by fixation.Several types of artifactual binding of beta-galactosidase were excluded from major consideration: “sticky cells;” GZ substrates on cell surfaces; spreading of histochemical reaction product; nonimmunoglobulin-related binding.We conclude that the high frequency of antigen binding cells found in this and other current studies is inconsistent with a model of unispecificity in precursor cells of either the T or B cell lines.     

Avian antigen-binding cells. I. Characterization of antigen-binding T cells from bursectiomized chickens by autoradiography.

Antigen-binding cells (ABC) from spleens of HGG-immunized, bursectomized agammaglobulinemic (Bx) chickens were detected by direct autoradiography with 125I-HGG and by sandwich autoradiography with HGG plus 125I-goat-anti-HGG. The specificity of antigen binding was demonstrated by 1) inhibition of binding of 125I-HGG by preincubation with unlabeled HGG and 2) a specific increase in ABC after immunization. The ABC from Bx chickens were not B cells, as shown by the virtual absence of immunoglobulin-bearing cells in this population and by the lack of inhibition of antigen binding by anti-immunoglobulin sera. The ABC were not macrophages and did not bind HGG via Fc receptors because their frequency was unchanged after passage over nylon wool or incubation with antigen-antibody complexes. The temperature dependence and azide stabilization of the ABC were characteristic of antigen-binding T cells. Therefore, T cells capable of binding soluble antigen were demonstrated in Bx chicken spleen, which is free of contamination by B cells and passively adsorbed antibody.     

Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface. Interactions with concanavalin A and with Cytoplasmic structures

The effect of concanavalin A (Con A) on the capping of mouse lymphocyte surface immunoglobulin (surface Ig), cross-linked by rabbit anti-mouse Ig antibody, and on the capping of mouse thymocyte theta antigen, cross- linked by anti-theta alloantibody and rabbit anti-mouse Ig antibody, has been studied by immunofluorescence, using fluorescein conjugated Con A and rhodamine-conjugated anti-mouse Ig antibody, and by electron microscopy, using native or fluorescein-conjugated Con A and ferritin- conjugated anti-mouse Ig antibody. Prior incubation of the cells with Con A inhibited only partially capping os surface Ig, whereas it blocked almost completely capping of theta antigens. Both on cells with rings and on cells with caps the staining for surface Ig or theta antigen was superimposed to the staining for Con A. When Con A receptors on spleen cells were capped by Con A at concentrations of 10 mug/ml or higher, and the distribution of surface Ig was examined under noncapping conditions, all detectable surface Ig were found in the caps. As shown by electron microscopy, surface Ig remained dispersed in a layer of Con A. The ability of Con A to cap surface Ig was not altered by the presence of cohchicine or vinblastine. These results suggest that surface Ig are cross-linked by Con A to other Con A receptors. In these conditions surface Ig behave essentially as Con A receptors, as for example, in their sensitivity to cytochalasin B during inhibition or reversal of capping induced by this drug. The behavior of surface Ig parallels that of Con A receptors also in the presence of vinblastine. It is concluded that in the presence of Con A, antimitotic drugs do not modify directly the interaction between Con A receptors and surface Ig, but probably influence the capping ability of the Con A receptors or, more in general, affect the ability to elicit movements over the cell surface. The role in capping of cytochalasin- sensitive and vinblastine-sensitive structures is discussed. Both types of structures appear to play an active role in the formation of a cap, although the former probably corresponds to the main mechanical system responsible for the active displacement of cytoplasmic and surface material.     

A comparison of ligand-induced redistribution of surface immunoglobulins, alloantigens, and concanavalin A receptors on mouse lymphoid cells

Redistribution of surface immunoglobulins (Ig), H-2^b, Thy-1.2 and TL. 1,2,3 alloantigens, and concanavalin A (Con A) receptors on mouse thymus, lymph node and spleen cells into “caps” induced by bivalent antibodies or ligands was compared by immunofluorescence. Surface Ig was capped rapidly following attachment of anti-Ig antibody at 37°. Capping of alloantigens and Con A receptors occurred very slowly following attachment of alloantibody or Con A, but much more rapidly after addition of a secondary bivalent antibody. An inverse relationship between the number of surface component sites per cell and the extent of capping of that component was observed. Capping of alloantigens sparsely represented on the cell surface was not inhibited by high concentrations of alloantibody, in contrast to capping of alloantigens present in greater quantities. These results suggest that factors in addition to molecular cross-linking may be involved in ligand-induced redistribution of cell surface components.     

"Stop and go" kinetics of receptor movements induced by anti-Ig or antigen on individual lymphocytes.

Serial observations were conducted on the time course of surface immunoglobulin (Ig) redistribution (capping) on individual mouse spleen lymphocytes. Capping of surface Ig by anti-Ig fluorescein and also antigen-induced capping of receptors on specific sheep erythrocyte antigen-binding cells were observed and the times required for individual cells to clear 90 and 180 ° of their circumference were recorded. There were striking differences between individual cells in both the onset and duration of receptor movements. Although the number of cells achieving complete clearing of first and second quadrants in successive time intervals declined, there was no correlation between the time required by a cell to clear the first quadrant and the time required by the same cell to clear the second quadrant. Thus, instead of observing gradual progressive migration of marker toward one pole of each individual cell at a rate resembling that of the whole population, we observed grossly discontinuous receptor movements, characterized by brief major shifts in receptors followed by a period of relative stability. Capping is thus viewed as a series of discrete contractile events related to the activity of membrane-associated cytoskeletal elements rather than a manifestation of “membrane flow”.     

Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.     

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 documented^1–4. Such lymphocytes are thought to have patches of surface immunoglobulin, primarily IgM, which act as specific receptors for antigen^5,6. A proportion at least of these unprimed lymphocytes are immunologically competent as shown in vivo^7,8 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.     

Characterization of subpopulations of T lymphocytes: I. Separation and functional studies of peripheral T-cells binding different amounts of fluorescent anti-Thy 1.2 (theta) antibody using a fluorescence-activated cell sorter (FACS)

Peripheral T lymphocytes in mice can be distinguished by the presence of the Thy 1.2 (theta) cell surface antigen. The fluorescence-activated cell sorter (FACS) was used to analyze and separate T-cells from peripheral lymphoid cell suspensions after incubation with fluorescein-labeled anti-Thy 1.2 (F anti-Thy 1.2). Stained cells were markedly reduced in nu/nu mice, in mice carrying the Thy 1.1 allele (theta-AKR), and were not seen after incubation with anti-Thy 1.2 that had been absorbed with CBA brain. According to these criteria, the stained cells were termed “T lymphocytes.”Among the T lymphocytes, there was considerable heterogeneity of fluorescent staining. The FACS was used to separate T-cells from other cells and further to separate T-cells with high intensity F anti-Thy 1.2 fluorescence (bright T-cells) from those with less F anti-Thy 1.2 fluorescence (dull T-cells). Separated bright T and dull T lymphocytes were shown to have several different functional properties. Dull T-cells appeared more sensitive to small doses of ALS in vivo, homed to lymph node in higher proportions than did bright T-cells, and were not affected by the short-term effects of thymectomy in adult life. Bright T lymphocytes, by contrast, were resistant to the in vivo effects of ALS, homed preferentially to spleen rather than lymph node in irradiated hosts, and were reduced shortly after adult thymectomy. Separated populations of bright and dull T-cells showed reduced ability to produce cytotoxic activity after in vitro sensitization, while mixtures of these two subpopulations of T-cells produced synergistic cytototoxic responses. The ontogenic and functional implications of these findings are discussed.     

Physiology of receptor capping induced by erythrocyte surface antigens

The capping of antigen-binding cell receptors by bound sheep erythrocytes (SRC) demonstrates that antigen mounted on a cell surface can generate a signal leading to the capping reaction. SRC-induced capping of ABC is: (a) highly dependent on both aerobic and anaerobic glycolysis, (b) unaffected by agents altering intracellular cyclic nucleotide concentrations, (c) slightly more vigorous in strain A than in CBA mice, (d) inhibited by calcium ionophore, (e) inhibited by the local anesthetic dibucaine and the tranquillizer chlorpromazine, (f) dependent on cytoskeletal activity (i.e., inhibited by the simultaneous presence of colchicine and cytochalasin B), (g) not dependent on the membrane ATPases inhibited by ouabain, (h) not dependent on motility, in that agents which inhibit motility (cytochalasin B alone) or stimulate motility (carbachol) do not alter capping behavior. These properties represent similarities between the capping of surface Ig by the cellular antigens on SRC and by proteins such as anti-Ig. SRC-induced capping is much slower than anti-Ig-induced capping, and only engages 30–40% of ABC, indicating that the nature of the crosslinking agent can influence the kinetics and extent of capping. But SRC cap with the rapid kinetics typical of anti-Ig-induced capping if the surface membrane of the ABC is first cleared of other glycoproteins with trypsin. The removal of negatively charged sialic acid residues by neuraminidase has no such effect. It is probable that the compression of bound SRC into a small area of the membrane requires more energy than does the capping of protein ligands, and that some cells cannot muster enough energy to achieve it.     

Membrane defects in the tolerant B cell. II. Mechanism of capping failure and reversal by antigen exposure.

The demonstration that TNP-binding B lymphocytes from animals whose B cells have been rendered tolerant to TNP by trinitrobenzene sulfonic acid cannot undergo antigen-induced capping of their TNP receptors for at least a year despite recovery of immune responsiveness has led to a search for the mechanism of the capping failure. Microtubule-dependent membrane “locking” analogous to that induced by concanavalin A appears to afflict the tolerant B cells, in that capping TNP receptors is restored after exposure to 10^?4M colchicine or overnight incubation at 4 °C. Assignment of the defect to the cytoskeleton rather than the receptors themselves is also supported by the observations that enzymatic stripping and regrowth of receptors does not unlock the cell and that non-Ig membrane molecules recognized by antilymphocyte serum also cannot be capped on the tolerant cells. Cells which have remained locked for 4 days to 8 months after a single tolerogen exposure become unlocked 4 days after immunogen is given. Four days after immunogen, tolerogen fails to lock the membranes of TNP-binding cells. These results suggest that tolerogen contact interferes in a much broader range of functions in the TNP-binding cell than those which affect the immune response. Among these effects is a remarkably stable “locked” configuration of the cytoskeleton which is independent of immune responsiveness or receptor turnover, but which can be reversed by exposure to immunogen whether or not an immune response ensues.     

Antibody-induced changes on rabbit sperm surface inhibit gamete interaction

Interaction of specific ligands with cell surface molecules may induce reorganization of surface components. A monoclonal antibody (B-12) against sperm surface antigens of 40kDa size induced molecules on the plasma membrane overlying the acrosome of rabbit sperm to cluster in small aggregates at 0°C (patching). At an elevated temperature of 37°C these clusters of antigen antibody complexes collected into a large aggregate on one pole of the cell forming a cap (capping). This cap-like structure showed a reduction in size over a period of time and eventually disappeared from the sperm surface. Inhibition of capping by sodium azide indicated that it is an energy-dependent process. Patching of antigens did not require energy. Involvement of sperm head cytoskeleton in the process of capping was evident from potentiation of cap formation by cytoskeleton disrupting agents like cytochalasin B and D. Patching of antigen antibody complexes was not affected by either of the agents. The loss of antigen antibody complexes from sperm surface was mainly due to shedding of the complexes in the surrounding media. Sperm with patches of antigen antibody complexes did not adhere to oocytes. Sperm from the group where a majority of the sperm were denuded of the antigen antibody complexes also did not bind to oocytes. © 1994 Wiley-Liss, Inc.     

Expansion and cell surface Ig isotype switching in the antigen-binding cell population of nu/nu mice and nu/+ littermates

Swiss-Webster nu/nu splenocytes placed in modified Mishell-Dutton culture containing sheep red blood cells (SRC) generated increased numbers of antigen-binding cells (ABC) compared with antigen-free cultures. In contrast Balb/c nu/nu cultures did not expand their ABC population in response to SRC, suggesting that strain background may influence the effect of the nu/nu gene on T-dependent immune responses. Cell surface Ig isotype analysis indicated that the SRC-induced expanded ABC population exhibited a significant decrease in cell surface IgD and a significant increase in ABC bearing both IgM and IgG. The Swiss-Webster nu/+ littermate cell surface Ig isotype patterns were generally similar to the nu/nu ABC patterns, but with different kinetics.     

Two distinct mechanisms of B-lymphocyte tolerance.

Based on the different characteristics of “pure” B-lymphocyte tolerance induction by polymeric and nonpolymeric antigens, it is proposed that there are two fundamentally distinct mechanisms by which mature B cells are tolerized: Polymeric antigens inhibit the expression of surface receptors irrespective of surface Ig isotype, whereas nonpolymeric antigens act by an unknown mechanism in which surface IgD is critically important in rendering cells “resistant” to tolerance induction. Further experiments to validate this hypothesis are highlighted.     

The effects of concanavalin A on the mobility of lymphocyte surface receptors

It has been found that concanavalin A (Con A) bound to the lymphocyte surface can either induce cap formation or inhibit cap formation of various receptors including those for Con A itself. The expression of these antagonistic activities is highly dependent on the conditions under which cells are incubated with Con A. Incubation with Con A at 37 °C resulted in cap formation in only a small percentage of the cells and inhibited patch and cap formation induced by other reagents such as anti-immunoglobulin. In contrast, incubation of cells with Con A at 4 °C, followed by removal of unbound Con A molecules and elevation of the temperature to 37 °C resulted in cap formation in more than 40 % of the cells. Quantitative analyses suggest that these effects involve cross-linkage of Con A receptors, which occur in two states, mobile and relatively immobile. A model is proposed to explain the various effects of Con A in terms of the association of these receptors with colchicine binding proteins.     

Membrane depolarization is induced in tolerant B lymphocytes by stimulation with antigen

Membrane depolarization is one of the earliest events in activation of cells by ligand receptor interaction. It is known that crosslinking of antigen-specific Ig receptors on B cells by antigen can induce membrane depolarization and subsequent Ia antigen expression on the cell surface. To determine whether a tolerance-inducing form of the antigen can also induce membrane depolarization after Ig receptor binding we used splenic B cells enriched for dinitrophenyl (DNP)-specific cells and determined relative membrane potential in these cells after binding of DNP-murine IgG2a (MGG) (tolerogen) or antigens (DNP-keyhole limpet hemocyanin (KLH) and DNP-Ficoll). Relative membrane potential was determined by loading the cells with the dye, 3.3-dipentyloxacarboxyanine (DiOC5(3)) after 2 hr incubation with ligand and determining relative fluorescence intensity on the fluorescence-activated cell sorter (FACS). Carriers alone did not depolarize these normal cell populations, but 100% of DNP-specific cells were depolarized by DNP-KLH and DNP-MGG while 85% were depolarized by DNP-Ficoll. To determine if tolerant B cells could be depolarized by antigen we induced tolerance in vitro or in vivo with DNP-MGG and measured the depolarization of DNP-specific B cells in response to antigens and tolerogen. DNP-specific B cells made tolerant by DNP-MGG underwent membrane depolarization when incubated with either DNP-KLH, DNP-MGG, or DNP-Ficoll but not with carriers alone. These data suggest that tolerogen induces membrane depolarization equally as well as antigen in normal cells. In addition, tolerant cells can be depolarized by Ig receptor crosslinking with either antigen or tolerogen. Thus, tolerance does not block the early membrane events induced by antigen in B cells.     

The isotype cycle: successive changes in surface immunoglobulin classes expressed by the antigen-binding B-cell population during the primary in vivo response

Using the antigen-binding inhibition method, capable of revealing any combination of three surface Ig (sIg) isotypes on a population of antigen-binding cells (ABC) (S. Kanowith-Klein, E. S. Vitetta E.L. Korn, and R.F. Ashman, J. Immunol.122, 2349, 1979) we have defined the sequence of antigen-induced changes in the expression of sIgM, sIgD, and sIgG on the sheep erythrocyte (SRC) antigen-binding B-cell population (SRC-ABC) throughout the in vivo primary immune response. The majority of nonimmune B-ABC simultaneously expressed M and D (M⁺D⁺G^?). By Day 3 sIgG had appeared, mainly on cells already bearing sIgM and sIgD. By Day 5, other G⁺ populations appeared: M⁺D^?G⁺ and M^?D^?G⁺. By Day 12, M⁺D^?G⁺ ABC declined, while M^?D^?G⁺ ABC remained predominant for another month. By 6 months, the sIg phenotypes on the ABC had returned to the original nonimmune pattern, mainly M⁺D⁺G^?; but the absolute number of 6-month immune ABC was four times greater than that of nonimmune ABC. This cyclical change in sIg expression was confined to the B-cell population expressing receptors specific for the immunizing antigen, and affected the large majority of such cells. Twelve days after immunization with SRC, ABC specific for a non-cross-reacting antigen still mainly expressed the nonimmune sIg phenotype, M⁺D⁺G^?.     

Crystal Structure of the Lamprey Variable Lymphocyte Receptor C Reveals an Unusual Feature in Its N-Terminal Capping Module

Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.     

Interaction and release of soulble immune complexes from mouse B luymphocytes

Soluble immune complexes (¹²⁵I BSA-anti-BSA-C) bind to B lymphocytes and accumulate at one pole of the cells (“caps”). The complexes remain on the membrane after incubation of the cells at 37 °C in tissue culture medium for several hours. The ¹²⁵I BSA can be quantitatively removed from the cell surface by incubation with excess BSA but not with excess antibody to BSA or preformed BSA-anti-BSA-C complexes. The release of ¹²⁵I BSA is probably due to the removal of the complexes from the cell membrane and not to an exchange between unlabeled BSA in the medium and the labeled BSA present in the membrane-bound complexes. Release of ¹²⁵I BSA by excess BSA is temperature dependent. The membrane-bound complexes can also be removed by incubating the cells with papain fragments of rabbit antibody to mouse Ig (anti-γ₁, γ₂, and k Ig chains). However, after exposure to divalent [F(ab′)² or 7S Ig] rabbit antibodies to mouse Ig, the complexes remain associated with the cells. In addition, after such treatment the complexes cannot be removed by excess BSA or by Fab anti-Ig.