Regulatory cells in human bone marrow: Suppression of an in vitro primary antibody response

Human bone marrow (BMC) contains regulatory cells that can suppress the in vitro primary PFC response of normal allogeneic spleen or tonsillar cells and autologous peripheral blood cells. Suppression is dependent upon the dose of BMC added, but is not due to cell crowding nor to excessive cytotoxicity, and requires the presence of viable, metabolically active BMC. BMC are maximally inhibitory when added during the first 24 hr of culture and do not cause an induced shift in the kinetics of the response. Thus, suppression reflects inhibition of early inductive events in the antibody response. The target of suppression is the non-T cell, with either polyclonal activator or Ag being required for maximal suppression. DNA synthesis of normal tonsillar cells is not inhibited by BMC. Characterization of the human bone marrow-suppressor cell has shown it to be radiosensitive, E-rosette negative, Fc receptor positive, and to reside in the large, weakly adherent cell population after velocity sedimentation and in the lymphocyte-depleted fraction after sucrose density gradient separation. Pretreatment of the bone marrow-suppressor cell with anti-human thymocyte serum does not abrogate suppression. We speculate on a possible physiologic role for this cell.     

Ultrastructural aspects of the primary in vitro antibody response of mononuclear cells in human blood

Investigations on the morphology of cells that participate in immune responses in vitro have been limited because the recovery and identification of immunocompetent cells growing dispersely in conventional liquid cultures are technically difficult and allow only the observation of individual antibody-forming cells. Here we used a system in which focal proliferation of antisheep erythrocyte antibody-secreting cells has been induced in semisolid cultures of peripheral blood mononuclear cells. By this method, intact colonies can be observed by light microscopy at the center of each hemolytic area and then processed for electron microscopy without disrupting the connections existing among the cells. Two types of colonies develop: type I colonies which grow deeply into the agar and contain cells that undergo a complete process of differentiation from blast to mature plasma cell, and type II colonies which grow more superficially and do not seem to be directly involved in antibody production.     

Human C3a-mediated suppression of the immune response. I. Suppression of murine in vitro antibody responses occurs through the generation of nonspecific Lyt-2+ suppressor T cell

C3a derived from the third component of human complement was found to suppress in vitro murine anti-SRBC responses. C3a-mediated suppression occurs through the generation of nonspecific Lyt-2+ suppressor T cells. The generation of suppressor cells occurs at an early phase in the response because incubation of naive T cells with C3a for as little as 30 min results in suppression of the anti-SRBC response. The generation of suppressor T cells requires the interaction of T cells, C3a, and a Sephadex G-10-adherent cell, presumably a macrophage. Although the mechanism of action of these suppressor cells has not been elucidated, several possibilities have been eliminated. C3a-suppressor T cells do not apparently release inhibitory lymphokines, nor is helper cell activity inhibited by a 2-day co-culture with these suppressor cells. The observation that interleukin 2 (IL 2)-containing lymphokine preparations could overcome C3a-induced suppression led us to investigate the interaction of the suppressors with IL 2 producer cells. However, neither C3a nor C3a-generated suppressor T cells can block the synthesis of IL 2.     

Regulation of the in vitro secondary antibody response: I. Suppression by relatively high,but physiological levels of calcium ion

Secondary antibody responses generated in vitro with spleen cells from mice primed and boosted with SRBC or TNP-KLH antigen were found to be influenced by the amount of Ca²⁺ in the culture medium. Relatively low levels of Ca²⁺ (0.1 mM) were optimally supportive for the generation of PFC in vitro, with higher, more physiological levels of Ca²⁺ (1.0–1.7 mM) suppressing the generation of PFC by as much as 100%. Suppression by high levels of Ca²⁺ was most pronounced when the amount of antigen used to elicit the in vitro antibody response was high, whereas responses generated by lesser amounts of antigen were minimally affected by Ca²⁺ level. Ca²⁺-mediated suppression was localized to an intermediate phase (24–48 hr) of the response. Mitogenic and polyclonal antibody responses were not affected by high levels of Ca²⁺. The effect of Ca²⁺ concentration on the secondary, IgG-producing antibody response may be significant in terms of understanding the various control mechanisms interacting in regulation of IgG synthesis.     

In vitro antibody response to influenza virus. I. T cell dependence of secondary response to hemagglutinin.

A good secondary IgG response to the hemagglutinin (HA) of influenza virus has been obtained in vitro in Marbrook-type cultures of influenza-primed mouse spleen cell suspensions stimulated with inactivated influenza virus. Anti-HA antibody was quantitated by a solid phase radioimmunoassay (RIA) by using purified HA as substrate. The T dependence of this secondary response was shown by depletion of T cells and reconstitution with a source of primed or unprimed T cells. The help given by T cells primed to the homologous virus was many times greater than that given by unprimed T cells, although the latter was significant. The system described will allow investigation of the specificity requirements of helper T cells engaged in the anti-HA response.     

T cell tolerization in vitro: modulation of helper and suppressor cell activities

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.     

The in vitro antibody response to cell surface antigens. I. The xenogeneic response to human leukemia cells.

An in vitro spleen fragment culture system has been developed for the production and analysis of xenogeneic antibody responses to cell surface antigens. Depending on the methods of immunization and in vitro stimulation employed, mouse spleen fragments can produce antibody of both IgG and IgM classes directed against human cell surface antigens for more than 30 days in culture. A saturation binding analysis of the antibody products indicates that their range of specificities was more restricted than that of serum antibody. Approximately 5% of the in vitro antibody products raised against a homogeneous population of human leukemia cells could distinguish between the antigens present on the leukemia cells and those present on normal human lymphocytes. Methods previously employed to influence the range of serum antibodies expressed against complex immunogens, such as suppression of certain responses by passive administration of antibody at the time of immunization, were tested in the in vitro spleen culture system and resulted in successful modulation of the antibody response patterns observed.     

Suppression of the in vitro secondary antibody response of rabbit lymphoid cells by Concanavalin A.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 mug/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 mug/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 mug/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 mug/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance(s).     

Suppression of the primary cell-mediated immune response by human alpha1-fetoprotein in vitro.

A possible immunoregulatory role of human α₁-fetoprotein (HAFP) was investigated. HAFP-enriched fractions as well as pure HAFP were obtained by means of two different procedures, as follows. After passage of HAFP-containing ascites of patients with primary liver carcinoma (PLC) on an anti-HAFP immunosorbent column, the retained proteins were eluted first by a glycine-NaOH buffer, pH 10.0 (resulting in HAFP I), and second by NaSCN (HAFP II). HAFP was further purified by passage of the HAFP-containing fractions an on anti-human whole serum (anti-HWS) immunosorbent column. This resulted in semipurified HAFP I and II. HAFP, pure by means of SDS-disc gel electrophoresis, Ouchterlony gel diffusion, and immunoelectrophoresis, was obtained by recycling on the anti-HWS immunosorbent column, as well as by a final Sephadex G-100 gel filtration. A possible immunoregulatory activity was assessed by testing the influence of semipurified as well as pure HAFP I and II on the uptake of tritiated thymidine by human lymphocytes stimulated by allogeneic lymphocytes in vitro. Only HAFP I, semipurified as well as pure, consistently exerted a profound suppressive effect on this primary cell-mediated immune response at concentrations of 150–200 μg/ml. In contrast, HAFP II did not show a comparable immunoregulatory effect either because there are two biologically different HAFPs or because of a loss of biological activity from HAFP II due to the use of the sodium thiocyanate elution technique.     

Suppressor cell regulation of the spontaneously induced in vitro secondary antibody response.

The cytotoxic effect of peripheral blood lymphocytes from patients with chronic active hepatitis (CAH) on Chang liver cells was studied using a chromium-51 release assay. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of glass-adherent mononuclear cells, indicating that the major effector cell is not a classical monocyte. Lymphocytes from 12 of 18 patients with CAH were cytotoxic, and the cytotoxic cells were contained within T-cell-depleted fractions. Further studies revealed that these effector cells were K cells, not B cells. K cells were identified as Fs- and complement receptor-bearing cells without detectable surface immunoglobulin. Serial studies in CAH showed continuous cytotoxicity in contrast to transient cytotoxicity in acute viral hepatitis, suggesting that the cytotoxic activity of lymphocytes is responsible for persistence of the disease.     

Mouse mononuclear cell chemotaxis. I. Differential response of monocytes and macrophages.

The chemotactic responses of peripheral blood monocytes and peritoneal macrophages to lymphocyte- and complement-derived stimuli were studied in vitro. Monocyte and macrophage chemotactic responses to these stimuli were different in both dose-response and time course. These differences provide a useful functional marker between these two cell populations.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Elicitation of antigen-induced primary and secondary murine IgE antibody responses in vitro

Described herein are methods for eliciting and quantitating primary and secondary murine IgE antibody responses in vitro, and the important role of antigen concentration in determining the level of IgE produced during an immune response. The methods for quantitating IgE antibody levels in culture supernatant fluids and in serum by ELISA are presented in detail. The specificity of such methods was confirmed in that (1) no other isotype of antibody registered in the IgE-ELISA, and (2) parallel determinations of IgE antibody concentrations could be obtained by independent analysis using Fc epsilon RI-dependent basophil degranulation. We examined various parameters of cell donor immunization and lymphoid cell culture which allow for optimal in vitro primary and secondary IgE responses. High relative antigen doses result in diminished IgE antibody responses in experimental animals, a finding confirmed herein. High antigen concentrations in vitro also result in relative suppression of IgE antibody synthesis. This was also true for in vitro production of IgG1 and IgA antibodies. Conversely, IgM and IgG2a responses were elicited at both low and high antigen concentrations; IgG2b and IgG3 were not produced under the conditions of priming and culture used herein. Finally, production of IgE in vitro depended on the presence of carrier-primed CD4+ T cells and hapten-specific B cells. Generation of maximal IgE antibody secretion, and hence elicitation of an allergic reaction, is thus dependent on the amount of antigen acting as stimulant for the immune response.     

Suppression of in vitro antibody response by ribosome-associated factor(s) from interferon-treated cells.

Initiation factor preparation (eIF-IF) from mouse L cells treated with virus-type interferon suppressed the in vitro plaque-forming cell (PFC) response to sheep red blood cells. The eIF-IF preparation had previously been shown to block formation of the ternary complex Met-tRNA_f-eIF-GTP. The formation of this complex is a necessary step in initiation of protein synthesis. Initiation factor preparation (eIF) from untreated L cells affected neither the PFC response nor the participation of eIF in the formation of ternary complex. The induced factor was shown not to be Interferon by antibody neutralization experiments with anti-interferon. The factor must be present in the PFC cultures during the early stages of antigen induction in order to suppress the immune response. Speculatively, eIF-IF may act at the level of the macrophage, perhaps entering the cell by pinocytosis. This may account for its inability to inhibit virus replication in L cells. The production of the inhibitory factor is blocked or partially blocked by actinomycin D. It is possible that this factor is a mediator of the immunosuppressive effects of virus-type interferon. This is the first report of biological activity on cells, which is associated with a ribosome-associated factor induced by interferon.