The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

The necessity for T cell help for human tonsil B cell responses to pokeweed mitogens: induction of DNA synthesis, immunoglobulin, and specific antibody production with a T cell helper factor produced with pokeweed mitogen.

Human B lymphocytes obtained from tonsils do not proliferate when stimulated with pokeweed mitogen. A soluble factor produced from T cells cultured with pokeweed mitogen stimulates B cells to synthesize DNA and differentiate into immunoglobulin producing cells. This PWM produced supernatant induced a PFC response to SRBC. The T cell supernatant activity is produced within 12 hr of stimulation in the presence of serum and without a requirement for T cell division. Optimal stimulation of B cells occurred at 7 to 9 days of culture. This helper factor activity eluted postalbumin from a column of Sephadex G-200. Insolubilized pokeweed mitogen was not mitogenic for B cells. The continuous presence of the lectin in culture was not required for B cell proliferation or for immunoglobulin synthesis.     

Synthesis of Gm allotypes by human tonsillar lymphocytes in culture: concordance with serum phenotype.

Isolated human tonsillar lymphocytes were cultured with pokeweed mitogen, phytohemagglutinin, and without mitogen for 9 to 28 days. IgK, Gm(a) and Gm(f) were then quantitated in the cell suspensions. In cultures of cells derived from persons whose blood was heterozygous for IgGl allotype antigens Gm(a+f+), approximately equal amounts of Gm(a) and Gm(f) were found. In cultures of cells of Gma or Gmf homozygotes, there was complete concordance between the Gm allotype antigens produced by the cultures and the donor's serum phenotype-with no instance, either at zero time or at culture termination in which a Gm antigen was detected which was absent from the donor's serum. It was concluded that in vitro genetic allotype synthesis in tonsillar lymphocytes during short-term culture mirrored accurately in vivo Gm expression. IgK and Gm antigen synthesis was highest in the flasks containing pokeweed mitogen although both phytohemagglutinin and no-mitogen control flasks showed, in certain experiments, proliferation and an increase in the Ig per viable cell. It was observed that no-mitogen flasks contained twice as much allotype antigen as did phytohemagglutinin flasks suggesting an inhibition of Ig synthesis associated with the mitogen. The tonsillar lymphocytes, under the experimental conditions employed, were shown by a radio-incorporation and immunoprecipitation technique to be synthesizing polyclonal Ig de novo, at the termination of the cultures.     

Synergistic interaction of a cytokine produced by embryonic fibroblasts and a lymphokine contained in Con A-stimulated spleen cell culture for murine macrophage differentiation: a model experiment using cell line cells, M1

The modulating activity of the culture supernatant of Con A-stimulated murine spleen cells for macrophages was investigated by using M-1 cells, which could differentiate into macrophage-like cells (referred to as M+1 hereafter), cocultured in a conditioned medium (CM) containing a differentiation factor (DF) obtained from the secondary culture of murine embryonic fibroblasts. DF induced Ia antigens on M-1 cells at a high rate in parallel with the appearance of Fc-receptor (FcR)-dependent phagocytic activity for erythrocytes coated with an antibody (EA). In contrast, Con A-sup alone had no modulating effect on M-1 cells. However, the Con A-sup stimulates synergistically M-1 cells with DF. Thus, coculture of M-1 cells with Con A-sup and DF generates M++1 cells which possess higher phagocytic activity than M+1 cells. These cells also exhibited stronger accessory cell activity than M+1 cells when tested for their promoting effect on IL-2 production by Sephadex G-10-passed spleen cells. The accessory cell activity of M++1 cells was further enhanced by culture with lymphocytes in the presence of indomethacin while that of M+1 cells did not change. These findings suggest that M-1 cells probably acquire potentiating, as well as inhibitory activity at the same time when cultured with DF and Con A-sup. The functional maturation caused by Con A-sup seemed to be associated with the expression of a receptor for a lymphokine, termed phagocytosis-augmenting factor (PAF) which is present in the Con A-sup. Such a receptor appeared to be common to macrophage lineage, since PAF in Con A-sup was absorbed out with splenic adherent cells and peritoneal exudate cells (PEC) in addition to M+1 cells, but not with nonadherent splenic lymphocytes or lymphoid cell line cells, such as EL-4 and L-1210. This fact suggests that PAF is different from interferon-gamma (IFN) which is known to modulate the function of lymphocytes. Inability of PAF to bind Cibacron Blue-Sepharose, unlike IFN, supports this notion. The molecular weight of PAF is approximately 2-3 X 10(4). Thus, the present studies suggest the requirement of at least two signals for the full maturation of macrophages, a cytokine represented by DF and a lymphokine, by PAF.     

Soluble erythrocyte antigens preferentially enhance cross-reactive specificities in human antibody responses

Stimulation of human tonsillar lymphocytes in culture with pokeweed mitogen (PWM) results in the production of a large number of antibody-forming cells which can be measured in the sheep erythrocyte (SE)-specific, direct haemolytic plaque-forming cell (PFC) assay. For optimal responsiveness in this system the human AB serum supplementing the culture medium must have been preabsorbed with SE. This effect is due, at least in part, to release during the absorption step of soluble immunogenic material from SE which acts synergistically with PWM to produce an enhanced response but which is unable to stimulate a response in the absence of the mitogen. In contrast to SE, absorption of AB serum with ox or horse erythrocytes failed to increase erythrocyte-specific responses. However, absorption with goat erythrocytes (GE) enhanced both SE- and GE-specific responses. An analysis of PFC specificities in cultures containing absorbed or unabsorbed serum revealed that enhanced responsiveness in the presence of absorbed serum was due to an increase in the proportion of PFC which cross-reacted with both SE and GE. The significance of this observation is discussed in terms of the antigenic requirements of interacting T- and B-cell subpopulations.     

Activation of human B lymphocytes: XVI. Cellular requirements,interactions, and immunoregulation of pokeweed mitogen-induced total-immunoglobulin producing plaque-forming cells in peripheral blood

The optimal culture and assay conditions for the detection of spontaneously occurring and pokeweed mitogen (PWM)-induced polyvalent Ig (IgG + IgM + IgA) and individual Ig class-specific plaque-forming cells (PFC) in human peripheral blood have been described in detail. Culture conditions are critical, particularly with regard to cell density and batches of supplemental serum. Fetal calf serum is a much more supportive serum supplement for PWM-induced PFC than is human serum. The assay system is a modified reverse hemolytic PFC assay using staphylococcal protein A coupled to sheep red blood cells by the chromic chloride method. PFC are developed by rabbit anti-human polyvalent Ig or anti-human individual Ig class antisera. Human peripheral blood contains 468 (±78) spontaneously occurring Ig secreting PFC per 10⁶ lymphocytes at Day 0 and 20,500(± 1971) PWM-induced Ig secreting PFC after 6 days in culture. The response is T-cell dependent; however, T cells can be replaced by a soluble T-cell factor prepared from a 48-hr allogeneic mixed lymphocyte reaction supernatant. The relative dependence on monocytes is a reflection of the culture conditions employed. Under the conditions of round-bottom tubes which promote cell-to-cell contact, depletion of monocytes to 0 to 2% does not result in a diminution of PFC responses. In fact, under such conditions, in certain individuals monocytes are markedly suppressive such that removal of monocytes results in a substantial enhancement of PFC responses. This system is simple and reproducible and should prove extremely useful in the delineation of the mechanisms of B-cell triggering and immunoregulation in normals and in disease states.     

CD4+ suppressor cells differentially affect the production of IFN-gamma by effector cells of experimental autoimmune encephalomyelitis

Spleen cells from rats that have recovered from experimental autoimmune encephalomyelitis (EAE) suppress the production of IFN-gamma by effector T cells of EAE in an Ag-specific manner. These postrecovery suppressor cells also inhibit EAE in vivo. Fractionation of the postrecovery suppressor spleen cells on nylon wool and OX-8 coated plates yields a nylon wool-adherent CD4+ suppressor cell population that, when cocultured with effector T cells, suppresses IFN-gamma production by these effector cells. In contrast, the nylon wool-adherent, CD4+ postrecovery suppressor cell population fails to inhibit the production of IL-2 by the effector T cells. In further experiments, the effector T cell population was depleted of CD8+ cells and cocultured with the nylon wool-adherent, CD4+ postrecovery suppressor cells, and the supernatants were assayed for IFN-gamma and IL-2. IFN-gamma production was inhibited in these cultures but IL-2 production was not inhibited. Irradiated effector T cells were cocultured with CD4+ postrecovery suppressor cells, without myelin basic protein, in an effort to determine whether the mechanism of differential lymphokine suppression involved an anti-idiotypic response against effector T cells. No IL-2 was produced, indicating that there was no CD4+ suppressor cell mediated anti-idiotypic response against effector T cells. These studies suggest that the suppressor cell is a nylon wool adherent, CD4+ T cell that functions to down-regulate EAE effector T cells by differential inhibition of lymphokine production.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Antibody responses of human lymphocytes in vitro. Specificity and physical properties of plaque-forming cell precursors.

When human tonsillar lymphocytes are cultured with pokeweed mitogen they generate a larger number of antibody-secreting cells. A substantial number of the plaque-forming cells generated have specificity for sheep erythrocytes but very few are specific for either bovine or horse erythrocytes. We have investigated the physical properties of sheep erythrocyte-specific precursors and have found that they have the characteristics of larger lymphocytes and are heterogeneous with respect to density. Our results support the concept that the percursors which can be induced by pokeweed mitogen to develop into plaque-forming cells belong to a pre-activated sub-population of lymphocytes.     

Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice.

Spleen cells from mice bearing a progressively growing syngeneic tumor failed to respond to stimulation with mitogens in vitro. This lack of reactivity was due to the presence of nylon wool-adherent cells in the population that could inhibit the mitogen response of normal lymphocytes. Paradoxically, at times when strong suppressor cell activity could be detected in tumor-bearing mice, the animals responded normally to in vivo immunization with sheep erythrocytes and allogeneic tumors, and to in vitro sensitization with allogeneic tumor cells. Regression of a highly antigenic syngeneic tumor also was unaffected by the presence of these suppressor cells. Thus, the occurrence of nonspecific suppressor cells in the spleens of tumor-bearing mice did not influence the overall immunologic competence of these animals.     

Kinetics of long-term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin-activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen-stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H-thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.     

A mutant human T-cell line producing immunosuppressive factor(s)

6T-CEM-20, a subclone of a 6-thioguanine-resistant mutant derived from the human-T-cell line CEM, secreted into the medium, a high titered immunosuppressive factor specific for T cells. The cell-free supernatant was very potent in suppressing, via a noncytotoxic mechanism, mitogen-activated T-cell proliferation, cytotoxic T-cell functions, and pokeweed mitogen (PWM)-stimulated plaque-forming cells (PFC). Lower dilutions of the supernatant (10(-1)-10(-2] abrogated T-cell functions within 24 hr whereas higher dilutions (10(-3)-10(-7] required a culture period of up to 4 days with lymphocytes to arrest T-cell activities. The suppressive activity was most pronounced when the factor was added in the early part of the culture period. The factor was sensitive to heat treatment and both low and high pH (most stable at physiological pH). Preliminary purification with column chromatography indicates that the active moiety was contained in the high-molecular-weight fraction (MW greater than 200,000). Data from coculture experiments suggested that T lymphocytes, which were exposed for 5-12 hr to the active supernatant or the partially purified material, suppressed allogeneic T-cell proliferation in a dose-related manner. This suppressor factor which we called suppressor-activating factor (SAF) might have activated a suppressor population or induced the production of a suppressor factor which in turn mediated the observed suppression. Both the molecular structure and the detailed mechanism of action are under investigation.     

Leu-3+ T cells produce gamma-interferon in patients with recurrent herpes labialis

Interferon-gamma (IFN-gamma) was spontaneously secreted by peripheral blood mononuclear cells cultured from patients soon after recurrent herpes labialis (RHL) or was induced from macrophage-T lymphocyte cultures in vitro with HSV antigen. Circulating Leu-3+/Leu-2- cells produced the spontaneous IFN almost exclusively. In the HSV antigen-stimulated culture system the same subset was the predominant producer of IFN-gamma. The IFN-gamma producing leu-3+ lymphocytes were plastic nonaderent but nylon wool adherent, and may be analagous to the murine Th 2 helper cell. In contrast to one lymphocyte subset being the major IFN-gamma producer in this viral disease, mitogen stimulation induced IFN-gamma from all (Leu-2+/2- and Leu-3+/3-) subsets, with panning as the separation technique. As mitogens circumvent the normal processing and presentation of antigen, the RHL system described above may provide a more accurate picture of the relative contributions of helper (Leu-3+) and cytotoxic/suppressor (Leu-2+) T cells to IFN-gamma production in herpes viral disease.     

Differentiation from precursors in normal bone marrow of spontaneously cytotoxic cells showing anti-self-MHC specificity

Colonies containing spontaneously cytotoxic effector cells with specificity for target cells carrying self-MHC can be grown from normal mouse bone marrow (BM). BM was first depleted of nylon wool-adherent cells and was then cultured at low cell number (1 to 300 cells/culture) in multiple replicate microcultures in liquid culture medium containing supernatant from EL4 thymoma cells stimulated with PMA. Frequency of colony growth followed one-hit limiting dilution kinetics. Colonies contained lymphoid, myeloid, or both kinds of cells. About 5% of colonies contained self-specific cytotoxic effector cells. Analysis using the X chromosome-linked isoenzyme PGK-1 confirmed that colonies containing autoreactivity could be clonal. A factor other than IL 2, IL 3, or PMA appears to be required for the growth of autoreactive colonies. Similar colonies, both with and without autoreactive effector cells, could also be grown from the BM of athymic nude mice with frequencies and cytotoxic activities directly comparable to those found for normal BM. C.B-17 scid mice lack both B and T cells, apparently due to a block in the development of lymphoid stem cells. Colonies could be grown with comparable frequency from their BM, but these colonies lacked both lymphoid cells and spontaneous cytotoxic activity. Evidence is presented against the self-reactive effector cells being NK cells, macrophages, or mature T cells. It is speculated that they represent an early stage of the T cell differentiation pathway.     

Functional differentiation of B lymphocytes in agammaglobulinemia. III. Characterization of spontaneous suppressor cell activity.

The addition of fresh peripheral blood mononuclear cells (PBL) from four of seven patients with agammaglobulinemia to generated hemolytic plaque-forming cells (PFC) resulted in a dose-dependent suppression of PFC. This spontaneous suppressor cell activity (SSA) was restricted to the four patients who could generate a PFC response in vitro. SSA was mediated by a small subset of E-rosetting T lymphocytes characterized by theophylline-sensitive E-receptors and surface receptors for Fc-IgG. The effects of SSA were temperature dependent and reversible, and pokeweed mitogen could prevent the rapid decline of SSA observed during culture. Augmentation of SSA was achieved by agents known to increase intracellular levels of cyclic AMP, whereas lithium chloride abrogated SSA, including the drug-induced effects. Cells mediating SSA may play a role in preventing the normal transition of pre-B cells to B cells in patients with agammaglobulinemia without B lymphocytes.     

Regulation of human in vitro anti-hapten responses: demonstration of a carrier effect

Human peripheral blood mononuclear cells, tonsillar lymphocytes, or mixtures of allogeneic or autologous B and T cells from these tissues were stimulated in vitro with the soluble hapten:carrier complexes TNP-OA, TNP-KLH, TNP-Myo, or TNP-Lac. These complexes were able to induce TNP-specific, direct PFC during 5–6 days in culture. The response involved proliferating PFC precursor B cells, nonproliferating T helper cells, and radiosensitive T-suppressor cell precursors. Exposure to high concentrations of free or carrier-bound hapten resulted in the inactivation of PFC precursor B cells. Carrier specific suppressor T cells could be induced by priming with nonhaptenated carrier protein and were able to block the PFC response when added to fresh target cultures. The use of hapten:carrier complexes permits the characterization of distinct T-cell functions independently of the assay system for B-cell activation.     

Cellular requirements for production and release of the lymphocyte costimulator

The lymphocyte costimulator (CoS) is a lymphokine required for the activation of T cell responses to H-2 alloantigens or mitogen, CoS activity is found in the supernatant medium of Concanavalin A (Con A) stimulated spleen cells, In this paper we investigate the cellular requirements for CoS production by Con A-activated mouse spleen cells. Maximal lymphokine production in response to Con A depends on a co-operative interaction between T cells and a nylon wool-adherent cell present in the spleen of nude mice. T cells appear to be the major producers of CoS activity, doing so only in response to an initial inductive stimulus supplied by nude spleen cells. The inductive stimulus is found as a soluble factor in the supernatant of Con A-activated spleen cells, and can also be provided by stimulatory (S+), but not by non-stimulatory (S-), tumour cells H-2 identical with the responding T cells. The activation of lymphokine-producing T cells is thus a two-signal process, requiring both mitogen and an additional inductive signal. Once activated, homogeneous populations of T cells will release lymphokine in response to mitogen alone.     

Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages.

Human monocytes, after in vitro activation by mixed lymphocyte culture (MLC) supernatants produce a monokine (MK) that enhances the plaque-forming cell (PFC) response of pokeweed mitogen (PWM)-stimulated human B lymphocytes. Technical conditions and kinetics of MK production were established. Irradiation of monocytes (5000 rads) does not abolish MK production but heat-killed cells are unable to release the factor. Highly T cell-depleted monocyte populations still produced the PFC-enhancing factor. The same MK has an inconsistent enhancing effect on the PFC responses of lipopolysaccharide (LPS) and nocardia water-soluble mitogen (NWSM)-stimulated B cells. Other macrophage activators such as LPS, phytohemagglutinin (PHA), and latex particles failed to induce consistently the liberation of the PFC-enhancing MK. The target cell for the MK activity on PWM-stimulated B cells appears to be the B lymphocyte itself. These studies demonstrate that soluble monocyte products can have substantial modulatory effects on human B cell function.     

A series of murine interleukin molecules which stimulate both murine and human lymphocytes: I. Production by Phytolacca americana (pokeweed) lectin 2 (Pa-2)-stimulated thymus and thymus-derived cells

Cell-free supernatant fluid, from cultures of Phytolacca americana (pokeweed) lectin 2 (Pa-2)-pulsed murine spleen or thymus cells, contains factors which induce cultured lymphocytes to differentiate into IgM-secreting cells (assayed by a reverse plaque technique) and to proliferate (measured by the incorporation of tritiated thymidine) without the addition of mitogen. The factors in this supernatant fluid responsible for these activities have been designated as lymphocyte stimulating factors (LSF). LSF showed no genetic restrictions related to the major histocompatability complex; LSF made in one strain of mice worked in other strains. Indeed, LSF is not restricted by species barriers; human peripheral blood mononuclear cells were also stimulated by murine LSF to proliferate and differentiate into immunoglobulin-secreting cells without further addition of antigen or mitogen. Maximum production of LSF was achieved within 12 hr of culture and was independent of cell division. In contrast to TRF, no further production of LSF was detectable after 24 hr of culture. Unlike T-cell growth factor, this material stimulated increased mitosis of thymic, T, and B lymphocytes without the addition of mitogen or antigen. LSF also stimulated polyclonal B-cell differentiation into IgM-secreting cells. Maximal numbers of immunoglobulin-secreting cells were generated when LSF was added at the initiation of the culture. Indeed, unlike TRF, LSF needed to be present only during the first 6 hr of culture to achieve maximum stimulation, and did not require the presence of antigen. The production of LSF by a T-cell population in the spleen was shown by two independent methods. Spleen cells treated with anti-Thy 1 plus complement failed to produce detectable levels of LSF. On the other hand, purified populations of surface immunoglobulin-negative spleen cells produced LSF. Furthermore, the subset of thymocytes responsible for LSF production was the small population (approximately 10%) of cells in the thymus, which are not agglutinated by peanut agglutinin.