T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.     

B and T lymphocyte colony formation in agar: 2-mercaptoethanol-activated albumin can substitute for 2-mercaptoethanol.

The effect of 2-mercaptoethanol (2-ME) activated albumin (MaSF) on mouse B lymphocyte colony (BLC) formation and on human phytohemagglutinin (PHA)-induced T lymphocyte colony formation (TLC) formation in semisolid agar medium was studied. MaSF was found to stimulate colony formation comparable to the stimulation obtained with 2-ME. MaSF could not substitute for serum in any of the agar culture systems. BLC formation stimulated by MaSF was obtained with spleen cells from athymic nude mice and nonadherent spleen and lymph node cells of normal mice suggesting a direct effect of MaSF on the colony-forming B cell without interference with T cells or macrophages. The results suggest that the stimulatory effect of 2-ME in the BLC and TLC agar systems is mediated by 2-ME-activated albumin present in the culture medium.     

In vitro induction of polyclonal killer T cells with 2-mercaptoethanol and the essential role of macrophages in this process.

Killer T cells against allogeneic and syngeneic tumor cells were generated in vitro by the addition of 2-mercaptoethanol (2-ME) to the murine spleen cell culture in the absence of any antigenic stimulation. The maximum activity of T cell-mediated cytotoxicity (CMC) induced with 2-ME was observed on day 4 of culture and the induction of CMC was completely inhibited by the addition of inhibitor of DNA synthesis, hydroxyurea, or cytosin arabinoside. CMC induced with 2-ME was specifically inhibited by the addition of unlabeled target cells to the 51Cr-release assay system. These results indicated that killer T cells were generated in the presence of 2-ME as a result of nonspecific polyclonal activation of precursors into cytotoxic effector cells and that they recognized target cells with antigen-specific recognition receptors. Spleen cells deprived of adherent cells showed impaired induction of CMC with 2-ME. The addition of peritoneal exudate macrophages to splenic T cells restored this response. The result indicated that macrophages were essential for the induction of CMC with 2-ME. The possibility that the function of macrophages was mediated by soluble factor(s) released from macrophages was demonstrated by the separate culture of splenic T cells and macrophages in double-chambered, Marbrook-type vessels and by the addition of supernatants from macrophage cultures to splenic T cells. 2-ME and soluble factor(s) released from macrophages seemed to be required for the activation of precursors into killer cells.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

Effect of a synthetic adjuvant on the induction of primary immune responses in T cell-depleted spleen cultures.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) stimulates in vitro primary immune responses to SRBC in T cell-depleted (nude) spleen cultures. The stimulation of immune responses by muramyl peptide was antigen dependent. A microculture system was used to compare the T cell-replacing activities of several structural analogues of muramyl dipeptide and to compare the activity of muramyl dipeptide to helper T cells. In a limiting dilution analysis with excess helper T cells or muramyl dipeptide, the frequency of B cell precursors that respond to SRBC was similar, ranging from 1.5 to 5 X 10(-5). Decreasing the cell density in microcultures did not affect the efficiency of B cell precursor responses in the presence of muramyl dipeptide. Muramyl dipeptide was examined for mitogenic activity in spleen cell cultures. In serum-free medium, muramyl dipeptide stimulates slight (3-fold) increases in DNA synthetic activity. In medium supplemented with 5 to 20% fetal calf serum, muramyl dipeptide showed no significant mitogenic activity. There are a number of possible explanations for the T cell-replacing activity of muramyl dipeptide. The most likely is that muramyl dipeptide interacts directly with B cells to mimic the helper T cell signal in the inductive stimulus.     

Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.     

Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Mitogenic Activity of the Cell Walls of Mycobacteria,Nocardia, Corynebacteria and Anaerobic Coryneforms

The mitogenic activity of the cell walls prepared from Mycobacterium bovis BCG, Nocardia rubra, Corynebacterium diphtheriae PW8, and four species of Propionibacterium, Corynebacterium parvum ATCC 11829, Propionibacterium acnes C7, Propionibacterium granulosum ATCC 25564 and Propionibacterium avidum ATCC 25577, were investigated. These cell walls were active as mitogens on normal spleen cells, anti-θ sera-treated spleen cells, macrophage-depleted spleen cells of C57BL/6J mice and cortisone-treated thymocytes of C57BL/6J mice. It was also shown that these cell walls were mitogenic on spleen cells and macrophage-depleted spleen cells of congenitally athymic (nude) mice. The above results suggest that the cell walls investigated in this study act as mitogens on both thymus-derived lymphocytes (T-cells) and bone marrow-derived lymphocytes (B-cells).     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.     

Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells

Purified, recombinant-derived murine granulocyte-monocyte colony-stimulating factor was found to enhance the primary in vitro immune response to SRBC by murine spleen cells. In determining the mechanism of this augmentation, it was found that only splenic adherent cells and neither resting nor activated T cells nor B cells expressed specific receptors for GM-CSF. When splenic adherent cells were pulsed briefly with GM-CSF before addition to macrophage-depleted cultures, they reconstituted the PFC response to a significantly greater degree than did control macrophages. Splenic adherent cells incubated overnight with SRBC plus GM-CSF were also more efficient antigen-presenting cells than splenic adherent cells incubated with antigen alone. The mechanism of this enhanced antigen presentation was found to be due to a GM-CSF-dependent increase in the level of IL 1 secretion and Ia antigen expression. Consistent with these data was the finding that GM-CSF augmented IL 2 production by splenic T cells in response to suboptimal concentrations of Con A. Finally, the day 5 in vivo antibody response (as measured by serum titers) of mice immunized with a low dose of SRBC was enhanced by two daily inoculations of GM-CSF. Thus, the role that GM-CSF plays in augmenting immune responses may not be solely accounted for by its ability to cause the proliferation or differentiation of macrophages, but more than likely includes its ability to enhance the function of antigen-presenting macrophages.     

The effects of sodium metaperiodate on T and B lymphocytes.

The differential mitogenic response of T and B lymphocytes to sodium metaperiodate has been investigated. It was found that periodate treatment leads to lymphocyte stimulation in spleen cells from Balb/c mice but not in spleen cells from the congenitally athymic nu/nu mice. In addition, treatment of Balb/c spleen cells with anti-θ serum plus complement lowers the mitogenic response to periodate and to concanavalin A without affecting the response to lipopolysaccharide. These results suggest a requirement for the presence of T lymphocytes in the initiation of a response to periodate. Spleen cells from nude mice also react with periodate, and their ability to respond to B cell mitogens is impaired after treatment with the chemical reagent.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Immune dysfunction associated with graft-vs-host reaction in mice transplanted across minor histocompatibility barriers. II. Reversible defect in T-dependent antibody responses

B cell and Th cell functions were assessed in mice undergoing a graft-vs-host reaction (GvHR) in response to minor histocompatibility Ag by using the plaque-forming cell (PFC) response to the T-independent Ag TNP-Brucella abortus and the T-dependent Ag TNP-SRBC. Bone marrow plus spleen cells from B10.D2 mice were transplanted into lethally irradiated B10.D2 (syngeneic recipient) or H-2d-compatible BALB/c (allogeneic recipient) to produce a chronic form of GvHR. BALB/c recipients of an allogeneic transplant demonstrated a marked and proportional lymphoid depletion of the spleen with normal percentages of B cells, T cells, and CD4+ and CD8+ T cell subsets. Mice with GvHR made normal numbers of PFC/10(5) spleen cells in response to the T-independent Ag, but a significantly depressed number of PFC/10(5) spleen cells to the T-dependent Ag compared with normal B10.D2 mice and with irradiated B10.D2 recipients of syngeneic B10.D2 marrow plus spleen cells. Mice undergoing the minor Ag GvHR made significantly larger numbers of PFC/10(5) spleen cells after secondary immunization with TNP-SRBC compared with controls. In vitro assays demonstrated that B cells from mice with GvHR responded to T help from normal B10.D2 mice and that T cells from mice with GvHR provided help to normal B cells after in vivo immunization. These data demonstrate that radiation chimeras with GvHR in response to minor histocompatibility Ag have relatively normal B cell function and an apparent defect in T helper cell function that is reversible by immunization with appropriate Ag.     

Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice.

B cells from C3H/HeJ mice fail to respond to an endotoxin (LPS K235) which is mitogenic for normal mice including the closely related C3H/HeN strain. The cellular basis for this unresponsive state has been investigated. The C3H/HeJ mice have normal numbers of B cells, which are capable of normal responses to other B cell mitogens, such as polyinosinic acid (Poly I). Addition of normal macrophages or spleen cells fails to reconstitute the normal response. Furthermore, neither macrophages nor spleen cells from the C3H/HeJ strain suppress the normal C3H/HeN spleen cells. Finally, spleen cells enriched for B cells by the removal of macrophages or T cells demonstrate the same differences in responsiveness to LPS. These results indicate that LPS unresponsiveness is a defect of the B cell itself and not due to suppressor cells or the absence of helper cells. When LPS is added to Poly I-stimulated cultures, there is additional enhancement of the response of normal C3H/HeN spleen cells. However, LPS causes a dose-dependent suppression of the Poly I response of C3H/HeJ spleen cells. This suppression is dependent on the time of addition of LPS to the Poly I-stimulated cultures. These data are interpreted as indicating that the binding of LPS to the membrane of C3H/HeJ B cells results in their inactivation or suppression, and that this is the basis of LPS unresponsiveness in this mouse strain.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

The mitogenic properties of rabbit anti-mouse brain serum

The mitogenic activity of heterologous rabbit anti-mouse brain sera (RAMB) was investigated. By complement-dependent cytotoxicity and indirect immunofluorescence, RAMB was T-cell specific. Mitogenic activity was assessed by determination of [³H]thymidine incorporation into DNA. RAMB was mitogenic for spleen cells for Thy 1.1- and Thy 1.2-positive mouse strains. Maximal mitogenic responsiveness to RAMB occurred on Day 3 of culture. The incorporation of [³H]uridine into RNA and [³H]leucine into protein and percentage of blast cells in culture were also significantly increased following RAMB stimulation. The mitogenic activity of RAMB was abrogated by adsorption of the sera with BALB/c or AKR thymocytes or brains or with RL♂ 1.3+, a Thy 1.2-bearing T-cell lymphoma of BALB/c origin. In contrast, the mitogenic activity was not removed when RAMB sera were absorbed with RL♂ 1.4?, a variant of RL♂ 1 which appears to specifically lack cell surface Thy 1 determinants. These data suggest that the mitogenic activity of RAMB is Thy 1 directed. RAMB mitogenicity is T-cell dependent. Spleen cells from normal and heterologous nu/+ mice respond to RAMB, while spleen cells from nu/nu mice do not respond. Normal thymocytes and cortisone-resistant thymocytes do not respond mitogenically to RAMB. The response of unseparated spleen cells to RAMB is also macrophage dependent. Nylon-wool column-purified splenic T cells respond to high concentrations of RAMB in the absence of exogenous macrophages but do not respond to lower concentrations of RAMB unless exogenous macrophages are added to the cultures. Nylon-wool-adherent cells, which are B-cell enriched and relatively T-cell depleted, also respond to RAMB, suggesting that in the presence of even small numbers of T cells, B cells can be recruited into the response.     

Spontaneously Induced Suppressor Cells In Vitro: Nonspecific Suppression of In Vitro Antibody Formation

Nonspecific suppressor cells were induced during in vitro culture of normal mouse spleen cells (SPC) using the Marbrook culture system. The suppressor cells inhibited both the primary and secondary antibody-formation responses antigen nonspecifically in vitro, and both IgM- and IgG-responses were inhibited. The supernatants from suppressive precultured cells were not suppressive. The suppressor cells also inhibited the response of allogeneic SPC beyond H-2 compatibility. The induction of the suppressor cells did not require the presence of antigen but required fetal calf serum (FCS) or both FCS and 2-mercaptoethanol (2-ME). The suppressor cells were generated from the nylon-wool adherent, radiation-sensitive T cell population. On the other hand, the suppressor cells were nylon-wool nonadherent, relatively radiation-sensitive T cells. Actively antibody-producing cells were not affected by the suppressor cells. The suppressor cells inhibited the mitogenic responses of normal SPC to phytohemagglutinin-P (PHA), bacterial lipopolysaccharide (LPS) and concanavalin A (Con A). The suppressor cells themselves inhibited the growth of EL4 cells (T-cell leukemia of C57BL/6 mouse origin) and MOPCll cells (B cells, plasmacytoma of BALB/c mouse origin) even at a low effector-to-target cell ratio (E:T ratio = 1:1), but did not kill these tumor cells. These results indicate that the target cells of the suppressor cells are both T and B cells, and that the mechanism of action of the suppression is either inhibition of proliferation or inhibition of early events in the course of the immune response.