Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Thymus dependency of cells involved in transfer of delayed hypersensitivity to listeria monocytogenes in mice

Delayed-type hypersensitivity (DH) to Listeria antigens was induced in inbred C3Hf/Umc mice by intravenous injection of a sublethal dose of viable Listeria monocytogenes. Bone marrow, spleen, and lymph node cells from the immune mice were capable of passive transfer of DH to syngeneic neonatally thymectomized or lethally (900 R) irradiated recipients. Immune thymus cells as well as immune serum were ineffective in transferring DH to irradiated animals. In vitro treatment with antitheta isoantibody (anti-θ) and complement abolished the capacity of spleen and bone marrow cells from immune donors to transfer DH to irradiated hosts, indicating the thymus dependency of this cell population. The results with bone marrow indicate the existence of a small, but biologically significant, thymus-dependent population in this tissue.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Different effects of cortisone on the humoral immune response to T-dependent and T-independent antigens.

The effect of the administration of cortisone on the murine humoral immune response to either thymus-dependent (TD) or -independent (TI) antigens was studied in vivo. Whereas the thymus-dependent immune response was markedly suppressed, the thymus-independent immune response was preserved. The opposing effect of steroids on these two types of immune responses appears to be due to the relative independence of thymus-independent antigens of a radioresistant cortisone-sensitive accessory cell.     

Cellular events in tolerance IX: Maintenance of immunological tolerance in the presence of normal B-cell precursors and in the absence of demonstrable suppression

The cellular events involved in immunological tolerance to fluoresceinated sheep gammaglobulin (FL-SGG) were analyzed at the level of hapten-specific B cells. One single iv injection of FL-SGG induced tolerance as measured by challenge with thymus-dependent (FL-KLH) or thymus-independent (FL-Ficoll) antigens in vivo or thymus-independent (FL-LPS) antigen in vitro. As noted earlier, unresponsiveness was maintained until 6–8 weeks after tolerance induction. Limiting-dilution precursor analysis demonstrated a reduction in B-cell precursors on Day 7 after tolerogen treatment; precursor frequencies returned to control levels by 3–4 weeks. This recovery of precursors in the presence of stable tolerance was not due to suppressor activity. Rather, results show that tolerant hapten-specific B cells are clonally anergic and display a reduced burst size in response to antigen. Hence, unresponsiveness is maintained in the presence of apparently normal precursor levels by an intrinsic defect in antigen-specific B cells.     

Role of cyclic AMP on differentiation of T- and B-lymphocytes during the immune induction.

Spleen cell cultures from genetically thymus-deficient nude mice were restored with a T-cell replacing factor obtained from normal spleen cells of Balb/c-Ig^b mice stimulated with concanavalin A. Treatment of these cultures with an inhibitory dose of cyclic AMP did not result in reduction of the number of specific antibody-forming cells after stimulation by antigen, whereas the same treatment led to inhibition in cultures restored with normal hydrocortisone-resistant thymus lymphocytes. Further experiments lead to the conclusion that the early effect of cAMP on the immune induction seen in vitro reflects inhibition of the production or secretion of a T-cell factor which is a prerequisite for triggering B-cells with a thymus-dependent antigen.     

Reciprocal changes in interferon production and immune responses of mouse spleen cells fractionated over columns of insolubilized conjugates of histamine.

Cultures of spleen cells from unimmunized and immunized BALB/c mice can support both direct and indirect anti-sheep erythrocyte plaque-forming responses. The responsiveness of spleen cells to this thymus-dependent sheep cell antigen can be altered by fractionation of the cells over insolubilized conjugates of histamines (H-R-S). Cells that do not adhere to H-R-S (i.e., those that pass through the columns) produce a significantly greater plaque-forming cell response than do non-chromatographed cells or cells that have passed through a control column of rabbit serum albumin-Sepharose (R-S). In contrast, the direct plaque-forming cell response of the same culture to Salmonella typhimurium lipopolysaccharide (a T-lymphocyte-independent antigen) was not significantly different in any of the groups of cells tested. In addition, spleen cell filtration over H-R-S also resulted in a significant increase in the blastogenic response to phytohemagglutinin as well as a significantly lower production of interferon in response to phytohemagglutinin. The possibility that suppressor cells (which were shown to adhere to H-R-S) produce their inhibitory effect via interferon is discussed.     

Induction and characterization of splenic suppressor cells directed to natural killer cells in Japanese quails

Intravenous inoculation of chicken amniotic fluid (ChAmF) markedly reduced natural killer (NK) cell activity of spleen cells from Japanese quails. The reduction of NK activity was mediated by non-adherent thymus-dependent lymphoid cells which were resistant to treatment with anti-immunoglobulin serum and sensitive to treatment with anti-thymocyte serum in the presence of complement. The suppressing activity was selectively directed to NK cells, since Rous sarcoma virus-specific cytotoxicity or hemagglutinating antibody production against sheep erythrocytes was not suppressed in ChAmF-treated quails. Spleen cells from normal 1-week-old quails had similar characteristics to those from ChAmF-treated 4-week-old quails, lacking NK activity and exhibiting suppressive effect on NK activity, and were also shown to be thymus-dependent. Biological significance of the presence of NK cells and their suppressor cells is discussed in relation to embryonic development and tumor-surveillance mechanism.     

An immunostimulatory substance in the ascites fluid of patients with cancer metastatic to peritoneum.

The ascitic fluids from patients with cancer metastatic to the peritoneum contain a factor(s) which stimulates the primary antibody response to sheep red blood cells (SRBC) in vitro. This enhancement is manifested by an increase in the number of plaque-forming cells per culture and a slight increase in plaque size. This factor has a molecular weight in the range 30,000–100,000 as determined by Sephadex gel filtration. The factor, which we have called “stimulatory factor” (SF), will completely replace the requirement for fetal calf serum in the Mishell-Dutton type of assay. Enhancement of the antibody response is most apparent at suboptimal culture conditions. SF does not increase the number of plaque-forming cells to the T-independent antigen Escherichia coli but there is a marked increase in the size of the plaques produced to the lipopolysaccharide using coated SRBC as targets. The stimulation induced by this factor is not due to endotoxin contamination since endotoxin is heat stable and the SF is heat inactivated at 80 °C for 10 min. In addition endotoxin does not act in a manner similar to SF. Thus, the SF appears to influence both T and B cells. With thymus-dependent antigen the factor results in increased numbers of antibody cells being generated; with thymus-independent antigen the factor results in increased quantity of antibody being produced.     

An evaluation of antigen-driven expansion and differentiation of hapten-specific B lymphocytes purified from aged mice

Hapten-specific, trinitrophenyl antigen-binding B cells (TNP-ABC) were purified from inbred strains of mice representative of short-, intermediate-, and long-lived animals. Such populations of B cells were stimulated by either thymus-independent or thymus-dependent antigens in vitro and evaluated for both proliferation and differentiation into antibody-secreting cells. In the thymus-dependent system, the three strains of mice were selected on the basis of all being able to interact appropriately with the same T helper cell line. The results indicate that TNP-ABC purified from aged animals of all three strains responded to both forms of antigenic stimulation similar to TNP-ABC selected from young, littermate control animals. These results are discussed in terms of concepts of intrinsic B cell defects during the aging process.     

Level of mIa expression on mitogen-stimulated murine B lymphocytes is dependent on position in cell cycle

Using correlated flow cytometric analysis of cell surface Ia antigen expression (immunofluorescence) and cell cycle phase (pulse-width of axial light extinction), we have quantitated changes in expression of mIa antigen on murine B cells during progression through cell cycle. Our results indicate that density of mIa expressed on mitogen-stimulated B cells increases fourfold to fivefold during the transition from G0 to G1. By early S phase, mIa density has decreased by fourfold to fivefold relative to peak expression. This decrease becomes more evident by late S, G2, and M phases, when an eightfold decrease in mIa antigen density is observed relative to peak levels. This decrease results in mIa antigen expression lower than that of resting, unstimulated B cells. Therefore, maximum mIa antigen expression occurs during G0 to G1 transition and in early G1, when a requirement for I region-restricted, antigen-driven T cell help for thymus-dependent, antigen-driven B cell activation has been demonstrated.     

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.     

Immunosuppression by cobra factor: distribution, antigen-induced blast transformation and trapping of lymphocytes during in vivo complement depletion.

In vivo administration of cobra factor (CoF), the C3-activating protein of cobra venom, suppresses thymus-dependent antibody production. In a study of possible mechanisms for this effect binding of CoF to murine spleen cells in vitro was not detected, nor was there any effect on C3 or Fc receptors. The numbers of spleen cells bearing C3 receptors, Fc receptors, θ antigen or surface immunoglobulin were not altered by in vivo complement depletion of mice with CoF. The distribution and antigen-induced trapping of transferred ⁵¹Cr-labelled syngeneic spleen cells were unaffected by treatment of either donors or recipients with CoF. Furthermore, the antigen-induced generation, trapping and specific retention of immunospecific blast cells were normal in CoF-treated mice, despite profound suppression in these animals of IgG antibody production. The majority of these blast cells 3 days after immunisation were T cells, suggesting that complement depletion interferes with the process of T-dependent antibody production at a later stage than the activation of T cells by antigen.     

Restoration of the Immune Response to Sheep Erythrocytes by a Serum Factor

THE immune response of CBA mice to sheep erythrocytes (SRBC) is known to be thymus-dependent because strain members thymectomized during the first few hours of life exhibit a marked inability to respond to this antigen^1,2. Experiments with isoantisera suggested that a cell to cell interaction is involved in this response. Thymus cells per se do not develop into haemolytic plaque-forming cells, but in some, so far obscure, way they cause cells of bone marrow origin to become producers of haemolytic plaques^2,3. A study of spleen cells from neonatally thymectomized (NNT) mice in a tissue culture system indicated that the decreased responsiveness to SRBC is also expressed in vitro. In that case 15×10⁶ NNT spleen cells produced only 500 haemolytic plaques when assayed on day 4 of culture. But when 15×10⁶ thymus cells were added to identical cultures of NNT spleen cells at inception, the number of haemolytic plaque forming cells increased to 2,300 (ref. 4). When an equivalent number of thymus cells alone were incubated with SRBC there was no response.     

Interferon and the cellular immune response: separation of interferon-producing cells from DNA-synthetic cells

Spleen cells from mice were separated by albumin discontinuous density gradient centrifugation into six fractions. Cells from each fraction were cultured with a variety of general mitogens and a specific antigen PPD. The cultures then were assayed for mitogenesis and interferon production. The fraction of cells producing interferon were found to belong to a different population of cells from those undergoing a DNA synthetic response. Treatment of the interferon-producing fraction of cells with anti-theta serum and complement eliminated the interferon-producing capabilities of the remaining cells after exposure to PHA, Con A, and PPD but not after exposure to PWM. The results suggest at least a two-cell requirement for the production of interferon in response to PHA, Con A or PPD stimulation. They also suggest that the interferon-producing cells do not belong to the thymus-dependent lymphocyte subpopulation.     

Thymus-independent induction and antigen-dependent recovery of idiotype-specific suppression

BALB/c nude (nu/nu) mice and euthymic (nu/+) littermates were treated as neonates with anti-T15 antibody and challenged at various ages with either a thymus-independent, PC-Brucella abortus (PC-BA), or thymus-dependent, PC-keyhole limpet hemocyanin (PC-KLH), form of phosphorylcholine (PC). Nu/nu mice challenged with PC-KLH received KLH-primed splenic T cells prior to immunization. Neither neonatally anti-idiotype-treated nu/+ nor nu/nu mice responded with the production of T15-positive anti-PC antibodies after challenge with either form of PC antigen. It is concluded that neither induction nor maintenance of a state of T15-specific suppression requires thymus-matured T cells. Recovery of anti-PC responsiveness in suppressed nu/+ or nu/nu mice was similar and was found to be related to the form of antigen used to elicit the response. Immunization with PC-KLH revealed a long-lasting unresponsiveness (up to 16 weeks). In contrast, immunization with PC-BA elicited a full anti-PC response as early as at 6.5 weeks of age.     

An electron microscope autoradiographic study of DOC conversion to corticosterone in the rat adrenal cortex

Summary Seven thymuses from children between 1 and 12 years were examined by electron microscopy. Biopsies had been taken during surgical correction of congenital heart defects.In all cases we found interdigitating reticulum cells (IRC) in the medulla and inner cortex. These cells resembled the IRC which have been described previously in the thymus-dependent regions of the spleen and lymph node. They were characterized by an irregularly shaped nucleus, narrow cisterns of rough endoplasmic reticulum, and widespread interdigitation and invagination of the cell membrane. The surfaces of the IRC were in close contact with those of small lymphocytes, sometimes polysomal lymphatic cells, epithelial cells, and occasionally with those of lymphatic cells containing ergastoplasm.The IRC is apparently a specific cell of thymus-dependent regions. It may be that the IRC in the thymus, lymph node, and spleen contribute to the microenvironment needed for the differentiation of T-cells.Supported by the Deutsche Forschungsgemeinschaft, SFB 111/CII and III.—We wish to thank Miss M. Neubert and Mrs. R. Köpke for their technical assistance and Mrs. M. Soehring for her help with the translation.     

Effect of volatile excretions induced with thymus-dependent antigen in female mice on the behavioural responses of males

It was found that thymus-dependent antigen sheep red blood cells in the optimal immunogenic dose (1 x 10(8) cells/mouse) induced in female mice of CBA and B6 strain secretion of attractive urinary volatile components (VCs), and in the supraoptimal dose (1 x 10(9) cells/mouse)--aversive VCs for intact males CBA strain. In a direct comparison of the properties ofVCs-immunized mice of CBA and B6, a modification of the effect of constitutive chemosignalling: disturbance of ability of females VCs to attract allogeneic males, was observed. The role of thymus-dependent antigen dose and sex of animals in the mechanism of generation of antigen-induced chemosignals is discussed.     

Interdigitating cells in the lymphoid tissues of bovine fetuses and calves

Summary Electron-microscopic studies of lymphoid tissues from bovine fetuses and from calves disclosed a non-lymphoid cell type in the thymus-dependent zones of secondary lymphoid tissues and in the thymus that is distinguishable from reticulum cells, epithelial and endothelial cells, and macrophages. Based on morphological and topographical criteria, the cell is identified as the interdigitating cell. In addition, studies of the tissues of normal and virus-challenged fetuses, and of conventionally reared calves, indicated that the interdigitating cells originate from monocytoid cells, which undergo differentiation in the thymus-dependent zones during an immune response.     

Receptor activity of sorted lymphoid cells with a thymic regulatory factor.

A small molecular-weight regulatory factor from the thymus has been shown to enhance both cell-mediated and humoral immune responses. In this study, the relative binding capacity of the factor to thymus-dependent and bone marrow-dependent lymphocytes was measured directly. Spleen cells were separated using an automated, multiparameter, cell sorting instrument. Receptor activity for the iodinated thymic protein of each population was determined. Based on a number of criteria, thymus-dependent cells exhibited a greater affinity for the regulatory protein than did the bone marrow-dependent cells. These results indicate that the mechanism of action of regulatory control is via receptor-ligand interactions of specific subpopulations of lymphoid cells with a humoral factor.