The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.     

Receptor-mediated B cell antigen processing. Increased antigenicity of a globular protein covalently coupled to antibodies specific for B cell surface structures

Helper T cell recognition of globular protein antigens requires the intracellular processing of the native molecule by an antigen-presenting cell and subsequent presentation of a peptide fragment, containing the antigenic determinant, on the cell surface where it is recognized by the specific T cell in conjunction with Ia. B lymphocytes can function as antigen-presenting cells and, when antigen is bound by their surface Ig, are greatly enhanced in this capacity. In this report it is demonstrated that pigeon cytochrome c covalently coupled to antibodies directed toward either B cell surface immunoglobulin, class I or class II are effectively processed and presented by B cells to cytochrome c-specific T cells, requiring up to 1000-fold less cytochrome c as compared with cytochrome c alone or cytochrome c coupled to nonspecific immunoglobulin. The potent activity of the cytochrome c-antibody conjugates appears to be due to the ability of B cells to concentrate the antigen when the process becomes receptor mediated rather than to a signal provided to the B cell by the conjugate binding, because cytochrome c was not more effectively presented in the presence of unconjugated antibodies as compared with cytochrome c alone. Furthermore, the binding of the native antigen to B cell surfaces is not alone sufficient for T cell activation, in that the cytochrome c-antibody conjugates require processing and are major histocompatibility complex restricted. The results presented here indicate that surface immunoglobulin is not unique in its ability to facilitate antigen processing and/or presentation and that Ig, class I and class II are capable of transporting the cytochrome c to a cytoplasmic vesicle where proteolysis occurs yielding the required peptide, minimally of 10 amino acids. Cytochrome c coupled to monovalent fragments of anti-Ig-antibodies was nearly as effectively presented as cytochrome c coupled to bivalent antibodies, indicating that phenomena mediated by bivalent binding, such as patching and capping of the surface Ig, were not required for effective antigen presentation. The cytochrome c-antibody conjugates, which allow antigen processing to be initiated by receptor-mediated endocytosis, may provide the necessary tools to unravel the intracellular processes by which protein antigens are processed and presented by B lymphocytes.     

Characterization of the physical interaction between antigen-specific B and T cells

It has been assumed that physical interaction between B cells and helper T cells in the presence of specific antigen is an early and essential step in the physiologic antibody response to thymus-dependent antigens. The present studies were designed to examine this physical interaction by employing carrier-specific T hybridoma cells that can provide help to highly enriched hapten-binding B cells. Direct conjugation of the B and T cells can be visualized at both the light and electron microscopic level and the number of conjugates can be directly quantified. Before their effective conjugation with T cells, the B cells must be incubated with specific antigen for 4 to 6 hr. After this time, the T cells form conjugates with the B cells within 5 min. Conjugate formation requires hapten specificity, carrier specificity, covalent linkage between hapten and carrier, and is MHC restricted. Two types of T-B conjugates were observed by electron microscopy: an antigen-independent attachment of B cell microvilli to small portions of the T cell surface and an antigen-dependent, intimate apposition of large areas of the plasma membranes of the T and B cells. The kinetics of development of the two modes of interaction suggest that the second type may be important for signal transduction, since the number of T and B cells showing intimate interactions increases with time. Monoclonal antibodies directed against Thy-1.2, LFA-1 alpha, L3T4, and I-A partially block conjugation of the two cell types, suggesting that these surface molecules are involved in T-B interaction.     

Antigen-specific clones of proliferating T lymphocytes. I. Methodology, specificity, and MHC restriction

In this report we describe in detail a new method for cloning antigen-specific, proliferating T lymphocytes directly from primed murine lymph nodes after 3 days of activation in vitro. After expansion in liquid culture the cells from the colonies were shown to be antigen specific and to require I-A histocompatible, irradiated spleen cells for stimulation. For hapten-carrier-type antigens, the T cells were shown to be carrier specific in their recognition but they were also capable of distinguishing the presence of the hapten. Recloning of small numbers of these cells in soft agar under conditions of high plating efficiency yielded true clones (i.e., populations derived from a single cell) whose antigen specificity was identical to that of cells from the original colony. The fact that a clone of T cells was I-A restricted in its antigen recognition demonstrates that suppressor T cell function cannot account for the phenomenon of major histocompatibility complex restriction.     

Activation of human B lymphocytes by a particulate antigen

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.     

Antigen presentation by hapten-specific B lymphocytes. IV. Comparative ability of B cells to present specific antigen and anti-immunoglobulin antibody

The ability of trinitrophenyl (TNP)-binding murine B lymphocytes to present native rabbit IgG (RGG), TNP-modified RGG, and rabbit anti-mouse Ig (RAMG) to an Ia-restricted, RGG-specific helper/inducer T cell clone was compared. By three independent assays (lymphokine secretion, T cell proliferation, and B cell differentiation), TNP-RGG was presented at 10(2)- to 10(3)-fold lower concentrations than RGG, and RAMG at 10(2)- to 10(3)-fold lower concentrations than TNP-RGG. The available data suggest that the efficiency of antigen presentation is dependent primarily on the avidity of binding of a ligand to B cell surface Ig and/or the extent of subsequent endocytosis (modulation). Despite the observed quantitative differences between anti-Ig (RAMG) and specific antigen (TNP-RGG), these results demonstrate that qualitatively both are essentially similar in their ability to mediate specific T-B interactions. Thus, anti-Ig antibodies are valid models for analyzing cognate interactions between antigen-specific B and helper T lymphocytes.     

Antigen presentation by hapten-specific B lymphocytes. V. Requirements for activation of antigen-presenting B cells

Splenic B cells specific for the haptens, 2,4,6-trinitrophenyl (TNP) or fluorescein isothiocyanate (FITC) were cultured with a range of concentrations of unmodified or TNP- or FITC-conjugated conalbumin and the conalbumin + I-Ak-specific, interleukin (IL) 1-dependent helper T cell clone, D10 . G4, in the presence and absence of IL-1. Lymphokine secretion, T cell proliferation, and antibody secretion by B cells all exhibited identical antigen dose responses. Thus, hapten-binding B cells presented low concentrations of haptenated conalbumin for activation of both the T and the antigen-presenting B cells. Whereas proliferation of D10 . G4 required the addition of IL-1, both lymphokine production and stimulation of B cells to antibody secretion occurred without exogenous IL-1. These results demonstrate that when B lymphocytes function as presenting cells for antigens that bind to their immunoglobulin receptors, activation of the responding T cells and the B cells themselves occur at similar concentrations of antigen. Moreover, for functional T-B interactions, antigen-presenting B and responding T lymphocytes constitute a complete system that requires no other accessory stimuli, whereas clonal expansion of T cells is dependent on accessory factors such as IL-1. Finally, since D10 . G4 secretes IL-4 but neither IL-2 nor interferon-gamma, our results demonstrate that differentiation of B cells as a consequence of direct ("cognate") interactions with helper T cells as well as of bystander B cells can occur in the absence of IL-1, IL-2, and interferon-gamma.     

Characterization of antigen processing and presentation by resting B lymphocytes

The production of antibody to a thymus-dependent Ag requires cooperation between the B cell and an Ag-specific Th cell. MHC restriction of this interaction implies that the Th cell recognizes Ag on the B cell surface in the context of MHC molecules and that the Ag-specific B cell gets help by acting as an APC for the Th cell. However, a number of studies have suggested that normal resting B cells are ineffective as APC, implying that the B cell must leave the resting state before it can interact specifically with a Th cell. Other studies, including our own with rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possible explanation for the above contradiction is that our B cells have become activated before presentation. Here we show that presentation by size-selected small B cells is not the result of nonspecific activation signals generated by the T cells or components of the medium. Also, although LPS activation does increase the efficiency of presentation by small B cells, use of large cells in place of small cells or preincubation of resting B cells with mitogenic doses of anti-Ig does not. Another possibility that we considered was that small B cells are unable to process Ag and that we had selected T cell lines that were capable of recognizing native Ag on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that Ag presentation requires Ag processing, a sequence of events that includes internalization of Ag into an acid compartment, denaturation or digestion of Ag into fragments, and its return to the cell surface in the context of class II MHC molecules. The experiments reported here show that our T cell lines require an Ag processing step and that small resting B cells, like other APC, process Ag before presenting it to T cells. Specifically, we show that an incubation of 2 to 4 h is required after the Ag pulse before Ag presentation becomes resistant to irradiation. Shortly after the pulse, the Ag enters a pronase-resistant compartment. Although efficient Ag presentation requires initial binding to membrane Ig, Ag is no longer associated with membrane Ig at the time of presentation and is not presented in its intact form, because removal of membrane Ig by goat anti-Ig blocks presentation before but not after the Ag pulse.     

Function and behavior of surface immunoglobulin receptors in antigen-specific T cell-B cell interaction

The expression and function of surface immunoglobulin (sIg) receptors was analyzed using antigen-specific monoclonal B cells and T cells. When B cells were incubated with antigen (hapten carrier), hapten-specific sIg receptor expression was greatly reduced. The specific antigen-presentation activity of the B cells was also markedly reduced. Following the removal of antigen, expression of the sIg receptors was restored to original levels within 3-4 hr. Specific antigen-presentation activity of these B cells was concomitantly restored. Reduction of sIg receptor expression and specific antigen presentation were inhibited by the addition of competing free hapten, suggesting that crosslinking of sIg receptors is required for the observed changes in sIg receptor expression and that specific antigen presentation is hapten specific. Using oligopeptide-specific Th cells, we could show the requirement for processing of antigen in the interaction between hapten-specific B cells and carrier-specific T cells. These results indicated that the expression of sIg receptors for a specific antigen and the ability to present the antigen are interrelated.     

Anergy in immature B lymphocytes. Differential responses to receptor-mediated stimulation and T helper cells

We have compared the responses of purified neonatal and adult B lymphocytes to stimulation by anti-Ig antibodies, which are functional analogues of Ag, and by Th cells. Neonatal B cells are markedly deficient in proliferative responses to anti-Ig antibodies + IL-4 or to anti-Ig conjugated to dextran, both of which induce strong proliferation of adult B cells in the absence of T lymphocytes. Anti-Ig antibodies actually inhibit the functional responses of neonatal B cells, even to polyclonal stimuli such as LPS. However, Th cells induce both proliferation and Ig secretion by neonatal B cells in the presence of Ag that bind to B cell Ig and are subsequently presented by the B cells. Thus, in neonatal B lymphocytes, cross-linking of membrane Ig in the absence of Th cells has a net inhibitory effect, and this inhibition is overcome by T cell help. These results also suggest that unresponsiveness or tolerance to thymus-independent Ag is induced in the B cells themselves, but tolerance to thymus-dependent proteins resides primarily in the T cell compartment.     

Regulation of helper cell activity by specifically adsorbable T lymphocytes.

Mice immunized with soluble proteins such as human serum albumin (HSA) or ovalbumin (OA) develop in their spleens antigen-specific T and B lymphocytes. These populations of lymphocytes can be separated from each other by different means; e.g. treatment with anti-theta-antiserum and complement removes selectively T lymphocytes, whereas passage through glass bead columns coated with mouse immunoglobulin (Ig): anti-Ig complexes creates a relatively pure population of T lymphocytes. During the course of such separation studies it was observed that the helper capacity of HSA (or OA) immune mouse spleen cells after Ig:anti-Ig column passage frequently was higher than expected from the enrichment in theta-positive cells. In addition, after adsorption onto antigen coated Bio-Gel beads this effect was even more pronounced, i.e., and increase in the relative helper capacity of about 3 or 4 times compared with an increase in the content of theta-positive cells from about 30% to 40 to 50% after adsorption. The present results will demonstrate that the increased helper capacity was a specific phenomenon which was regulated by theta-positive cells. The regulatory cells specifically adsorbed onto antigen-coated Bio-Gel beads have not been successfully eluted by EDTA or excess-free antigen so far, and they were still adsorbed after pre-incubation with anti-Ig antibodies under conditions where specific B lymphocyte adsorption was almost prevented.     

B cell activation. II. Receptor cross-linking by thymus-independent and thymus-dependent antigens induces a rapid decrease in the plasma membrane potential of antigen-binding B lymphocytes

We report the specific induction of B cell plasma membrane depolarization with the use of thymus-dependent and -independent antigens. We have utilized various trinitrophenol-carrier conjugates for the stimulation of isolated trinitrophenol-binding mouse B cells. Membrane depolarization was assessed by flow cytometric analysis of 3-3'-pentyloxacarbocyanine iodide (DiOC5[3])-stained cells. Entry into the cell cycle was determined by flow cytometric analysis of acridine orange-stained cells. The results indicate that polyvalent antigens, but not free hapten, induce B cell membrane depolarization by a large proportion of antigen-binding cells within 2 hr of stimulation. Although all polyvalent antigens induce membrane depolarization, only thymus-independent antigens induce the subsequent G0 to G1 transition, suggesting that the membrane Ig cross-linking signal alone, although sufficient to induce membrane depolarization and subsequent increased IA expression, is insufficient to drive the entry of B cells into the cell cycle. The G0 to G1 transition appears to be dependent on a second signal, perhaps mediated by the thymus-independent carrier or antigen-specific, Ia-restricted T cell helper.     

Activation of rat B lymphocytes. I. Characterization of anti-immunoglobulin responses and isotype density of rat B cells

Spleen cells of two rat strains, Lewis and Brown Norway (BN), have been activated by lectins and by antibodies specific for immunoglobulin isotypes embedded in their cell membranes. Optimal concentrations of antibodies specific for mu, gamma, or delta-chains of rat augments in vitro incorporation of 3H-TdR 5 to 18-fold in Lewis B lymphocytes and 1.5 to 4-fold in BN B lymphocytes. In addition, F(ab')2 fragments of anti-Ig reagents induced Lewis splenic B cells but not BN B cells to incorporate 3H-TdR. Responses to LPS and dextran sulfate, B lymphocyte mitogens, measured by radioactive uptake, were five to 10 times greater in Lewis B cell populations than in BN B cell populations. Density of surface Ig isotypes and capping kinetics were similar in the two rat strains, although the percentage of T cells, T cell subsets, B cells, and Ia+ B cells differed in the spleens of these strains of rats. Both T lymphocytes and macrophages were needed in culture to effect an optimal response. IL-2 restored the response in B cell cultures depleted of T cells and macrophages, and enhanced 3H-TdR uptake in whole spleen cells of Lewis but not BN rats. The strain-dependent responsiveness of B cells to specific anti-Ig reagents or B cell mitogens appears to be associated with inherent (genetic) defects in T cells and B cells or defects in T cell to B cell cooperation in BN rats.     

Role of antigen-specific B cells in the induction of SRBC-specific T cell proliferation

In this study, we ask whether antigen presentation can be effected by antigen-activated B cells. Antigen-dependent in vitro proliferation of T cells from mice primed with SRBC or HoRBC occurs in the presence of B cells primed to the relevant antigen. B cells prepared from lymph nodes of mice primed with irrelevant antigens are not effective antigen-presenting cells for RBC-specific T cell proliferation over a wide range of SRBC doses. This is true even when both RBC and the antigen to which the B cells are primed are included in the culture. In contrast, B cells specific for a hapten determinant coupled to SRBC are able to support proliferation of T cells specific for SRBC determinants. We conclude from these data that antigen-specific B cells play a role in the induction of T cell proliferative responses to SRBC and HoRBC antigens. Two models are proposed: either B cells, upon antigen interaction with surface antibody, are able to act as accessory cells to induce Ia-dependent proliferation of immune T cells; or B cells augment the T cell proliferative response by secretion of antibody, leading to opsonization of the antigen for macrophage uptake and presentation.     

Biologic activity of antigen receptors artificially incorporated onto B lymphocytes

We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1.2 monoclonal antibody and complement causes only a slight reduction in the number of receptor-decorated B cells that respond to the relatively thymus-independent antigen DNP-POL. This type of phenomenon is also seen with natural antigen-specific B cells. Thus, polyclonal activation of receptor-decorated B cells exhibits the same gross helper cell requirements as antigenic activation of natural antigen-specific B cells. The results of this study are discussed in the context of the role of membrane-bound surface Ig in antigen-dependent B cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.     

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Interaction of gangliosides with B cells in splenic fragment cultures

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.