Treatment Of Spleen Cells Research Articles

Role of self carriers in the immune response and tolerance. VI. Induction and reversal of TNP-modified self unresponsiveness in vitro.

The involvement of T-suppressor cells in hapten-modified self unresponsiveness induced in vivo is well established. However, TNP-modified syngeneic spleen cells (TNP-SC) can block the anti-TNP PFC response of normal or nude spleen cells to TNP-immunogen in vitro, an effect that is apparently T cell-independent. In this study, we have used a 2-stage culture system in which both the induction of tolerance by irradiated syngeneic TNP-SC and the subsequent challenge with TNP-immunogen take place in vitro to dissect the cellular events that lead to unresponsiveness. Our results show that not only are spleen cells precultured with TNP-SC tolerant to challenge, but that these cells are able to specifically suppress the response of freshly prepared normal spleen cells to the T-independent antigens TNP-POL or TNP-LPS. Irradiation of the precultured cells abrogates the suppressor activity and rules out hapten carryover as the mechanism of suppression. Suppressor cells were able to inhibit the response of anti-Ba theta and C-treated spleen cells, suggesting that suppressor function is directed toward the B cell and/or accessory cells. Anti-Ba theta and C treatment of spleen cells before preculture with TNP-SC prevented the induction of both tolerance and suppression. Nylon wool-enriched T cells, on the other hand, could generate suppressor cells in response to TNP-SC. These results demonstrate that, like the in vivo situation, T cells are involved in the generation of this form of B cell tolerance in vitro. Implications relevant to the mechanisms of tolerance induction are discussed.

Mode of regulation of natural killer cell activity by interferon.

Whereas xenogeneic tumors such as baby hamster kidney or HeLa cells grow in nude mice, the same cells persistently infected with a variety of viruses are rejected. Spleen cells from normal nude mice were found to be induced to produce interferon and to exert natural killer (NK) activity on virus persistently infected (PI) tumor cells, and not on uninfected parental cells in vitro. The phenotype of the interferon-producing cells and the NK effector cells was found to be the same namely, Qa 5(+), Ly 5(+), ganglio-N- tetraosylceramide, with 35 percent of the NK cells also expressing Thy 1.2. NK activity against virus PI tumor cell lines could be nonspecifically augmented both in vivo and in vitro by prior contact with virus PI tumor cells. It was unambiguously demonstrated with chemically homogeneous mouse interferon that interferon, and not a contaminant, was responsible for the augmentation of NK activity in vitro. Studies on the mode of interferon action in augmenting NK activity revealed that the target cell for interferon action was serologically distinct from the NK effector cell. Anti-Ly 5 + complement (C)-treated spleen cells were depleted of NK activity and the ability to produce interferon, but, upon incubation with interferon for 1-3 h, regained both NK activity and susceptibility to anti-Ly 5 + C. Treatment with anti-Qa 5 + C eliminated NK activity, which could not be restored by the addition of interferon. We conclude that interferon produced by Ly 5(+) cells in response to virus PI tumor cells acts on Ly 5(-) precursor cells and induces their differentiation into functional Ly 5(+) NK effector cells.

Inhibition of in vitro antibody synthesis by cyclophosphamide-induced suppressor cells

A population of suppressor lymphocytes appears in the spleens of mice 5 to 14 days after treatment with a high dose of cyclophosphamide (100–200 mg/kg body wt). Removal of carbonyl iron adherent cells or Ig − cells from cyclophosphamide (CP)-treated spleen cells does not abolish suppressive activity. These suppressors are, however, sensitive to removal by treatment with anti-Thy-1.2 and rabbit complement. CP-treated spleen cells can suppress the in vitro primary response of normal spleen cells to the soluble hapten-protein conjugate DNP-MON or the particulate antigen HRBC when added at time of culture initiation or up to the second day of culture. CP-treated spleen cells can themselves respond in vitro to DNP-MON, as well as to HRBC, but with altered kinetics from that of normal spleen cells. Collectively, the data suggest that the CP-induced suppressors act late in the in vitro antibody response, possibly by prematurely shutting off antibody synthesis by B cells.

Prostaglandin (PG) release in the mixed lymphocyte culture; effect of presensitization by a skin allograft; nature of the PG-producing cell.

AbstractA release of prostaglandin (PG) has been demonstrated in lymphocyte culture supernatants. The amount of PG produced is strikingly increased in mixed cultures between allograft donor and recipient. This phenomenon is not yet detectable after 6 h of culture, appears at 24 h and reaches its maximum at 48 h. Little or no increase in PG production is found in the supernatant of primary mixed lymphocyte cultures (i.e. without previous allograft) at least in the first two days of culture. The difference between allografted and control animals begins at day 6 after grafting, reaches a maximum at day 12, remains high until day 20 and decreases thereafter.This increased amount of PG in mixed cultures between donor and recipient spleen cells is probably, at least to a large extent, produced by macrophages. Evidence for this interpretation includes the following observations: (a) cells responsible for the increase are adherent cells and (b) the phenomenon is no longer found following a treatment of spleen cells with silica.When mouse peritoneal macrophages (or adherent spleen cells) are incubated in vitro with supernatants of mixed cultures between allograft donor and recipient, their PG production is considerably increased. Hence, it is suggested that the above‐described phenomenon could result from the stimulation of macrophages by lymphokines released in mixed cultures following allografts.If such a release of PG takes place within the graft, it may play a regulatory role in the control of the magnitude of the anti‐allograft immune response.

Selection of cytotoxic T-cell precursors specific for minor histocompatibility determinants. I. Negative selection across H-2 barriers induced with disrupted cells but not with glutaraldehyde-treated cells: evidence for antigen processing.

Intravenous injection of CBA mice with H-2-compatible irradiated B10.BR spleen cells led to a sequence of negative and positive selection of the host T-cell response against the multiple foreign minor histocompatibility antigens (HA) on the injected cells. By 1 d posttransfer, thoracic duct lymphocytes (TDL) of the host had lost the capacity to differentiate in vitro into cytotoxic cells specific for the injected minor HA; spleen and lymph node cells, by contrast, gave normal or enriched responses at this time. By 5 d posttransfer, TDL were hyperresponsive to the injected antigens. Selection with disrupted (sonicated) cells gave similar findings. With injection of either irradiated of disrupted spleen cells, the H-2 haplotype of the minor HA-bearing cells had no apparent effect on the magnitude of selection. By contrast, treatment of spleen cells with glutaraldehyde before injection led to H-2 restriction of selection, i.e., negative selection of the CBA response to B10.BR was marked with injection of glutaraldehyde-treated H-2-compatible B10.BR cells but was minimal with H-2-different B10 or B10.D2 cells. These data are taken to imply that, at least in H-2-incompatible situations, the minor HA-bearing cells must be processed by host cells, i.e., to allow the antigens to become associated with self H-2 determinants. Circumstantial evidence from studies on the specificity of selection induced with glutaraldehyde-treated cells from mice of the B10 recombinant strains suggested that I region-restricted T cells may control the induction of H2K, D-restricted cytotoxic precursor cells.

Induction of Antibody Responses In Vivo by Antigen Specific Helper Factor

T helper cell factors (HF) have been preparated against protein and synthetic antigens by restimulating in vitro induced helper cells with small amounts of antigen. HF when tested in vitro are antigen specific and capable of replacing T cells, initiating a thymus dependent IgM response. In the in vivo adoptive transfer assay HF is capable of replacing T helper cells and is active at very high dilution, inducing both IgM and IgG responses. When tested on unirradiated DNP primed animals the HF was able to initiate a thymus dependent anti-hapten response, comparable to that seen with helper T cells, of both IgM and IgG class. It was also shown that HF acts on anti-Thy 1 treated spleen cells. The results indicate that antigen specific helper factors may be one of the physiologic mediators of T -B interactions in intact animals.

C-reactive protein (CRP)-mediated inhibition of the induction of in vitro antibody formation : I. T-cell dependence of the inhibition

Purified human C-reactive protein (CRP) inhibited the in vitro anti-hapten antibody plaque-forming cells (PFC) response of both carrier keyhole limpet hemocyanin (KLH)-primed and unimmunized Balb/c spleen cells to TNP-KLH. The inhibitory effect was neutralized by the CRP-substrate, C-polysaccharide. The response to the T-independent antigens, TNP-T4 and DNP-lys-Ficoll, was not inhibited by CRP. A cell population that was suppressive for the in vitro PFC response was generated by incubating normal spleen cells with CRP. These cells suppressed the PFC response of syngeneic KLH-primed cells to TNP-KLH in proportion to the number of added lymphoid cells with bound CRP. Selective depletion of B cells, T cells or macrophages before incubation with CRP revealed that T cells were required for the induction of suppressive cells. Treatment of spleen cells after incubation with CRP, with T cell-specific antisera and C abolished suppressor-cell activity. Mitomycin-C treatment of the CRP-binding cells did not alter their suppressive activity. These results indicated that CRP mediates suppression of antibody induction to T-dependent antigens by interacting with T cells and generating a suppressive T-cell population.

Restoration of T-cell function in aged mice with long-term administration of levamisole

The in vivo effect of levamisole on antibody production against sheep red blood cells (SRBC) in aged mice was examined. Aged C57BL/6 mice were treated with levamisole, 2.5 mg/kg per os, for 5 months. The long-term administration of levamisole restored the ability of immune response to SRBC. The effect of levamisole on B cells was examined by an in vitro coculture technique. There was no change in the number of plaque-forming cells (PFC), indicating that levamisole did not act on B cells. Suppressor function of T cells after long-term administration of levamisole was also examined. Concanavalin A (Con A)-treated spleen cells from aged mice previously treated with levamisole suppressed the number of PFC of cocultured spleen cells from young mice, but Con A-treated spleen cells from aged control mice (no levamisole treatment) failed to suppress the number of PFC. The in vitro effect of levamisole was assessed by the number of PFC. The number of PFC was not altered significantly when levamisole was added to the culture medium. These results indicate that long-term administration of levamisole to aged mice can restore helper as well as suppressor T-cell function, and suggest that levamisole itself is not active in vitro but exerts its effect through metabolites in vivo.

Regulation of Antibody Response in Different Immunoglobulin Classes

Abstract Establishment of a mouse T hybrid cell line secreting suppressor factor(s) specific for the IgE antibody response is described. Fusion was made with polyethyleneglycol between AKR-derived T lymphoma cells (BW5147) and T cells from mice sensitized with DNP-Mycobacterium. Treatment of spleen cells with nondialyzable factor(s) in the culture supernatants of the T cell hybrid clone, 26-M10, showed a suppressive effect on IgE formation but not on IgG formation in adoptive transfer experiments. The suppressive effect was exerted through inactivation of normal or antigen-primed B cells responsible for IgE formation. It was also shown by direct cytotoxic test that the hybrid cells expressed H-2 and Thy-1 antigens derived from both parental cells on their surface. Karyotype analysis of the hybrid cells revealed that the number of chromosomes was less than the sum of the two parental cells' and the average was 50 (45 to 55). Although the 26-M10 hybrid cells lost the ability to secrete active suppressive factor(s) into culture medium 21 weeks after hybridization when the number of chromosomes in most of the cells was less than 41, recloning of the 26-M10 cells successfully recovered active suppressive clones.

Thymopoietin Enhances the Allogeneic Response and Cyclic GMP Levels of Mouse Peripheral, Thymus-Derived Lymphocytes

The action of the purified thymic factor, thymopoietin, on populations of post-thymic lymphocytes has been studied. Thymopoietin, at concentrations as low as 1.5 ng/ml, uniquely enhanced the proliferative response of peripheral T cells from lymph node and spleen to allogeneic stimulation. Enhancement of the allogeneic response (MLR) was not produced by several polypeptide hormones, including insulin, ACTH, HCG, or Ubiquitin. Treatment of spleen cells with anti-Thy-1 antiserum almost completely abolished the MLR. Thymopoietin's stimulatory effects could not reverse this. Thymopoietin treatment of Thy-1+-enriched spleen cell populations enhanced the MLR even when thymopoietin was removed as early as 2 min after incubation with responding cells. The interaction of thymopoietin with peripheral Thy-1+ cell populations produced a rapid and transient rise in cyclic GMP levels and slightly decreased cyclic AMP levels. These results suggest that thymopoietin interacts with one or more Thy-1+ subpopulations and that this interaction involves early changes in cyclic nucleotide metabolism.

Specific and non-specific activation of human and mouse lymphocytes in vitro by pathogenic and apathogenic Neisseria strains.

In vitro lymphocyte responses to gonococcal antigen (T2), apathogenic Neisseria pharyngis (APN), PPD and PHA were studied in patients with uroarthritis and in healthy controls. Only the T2 antigen induced a significantly higher DNA synthetic response in cells from patients than in controls, whereas no difference was observed with the other substances. Peak responses occurred on day 6 of culture with a concentration of 4 × 107 bacteria/ml. In order to test whether lymphocyte responses were the result of specific sensitization, experiments were carried out with cord blood cells. These cells reponded to both T2 and APN with maximum activity on day 6 of culture. Since specific sensitization was ruled out in these experiments, we concluded that T2 and APN were capable of inducing a non‐specific mitogenic response in cord blood cells. The mitogenic activity of T2 and APN was further elucidated in experiments with mouse lymphocytes. Both substances induced increased DNA synthesis in mouse spleen cells, with peak activity on days 2–3 of culture. Stimulation occurred in spleen cells depleted of macrophages, in anti‐Thy 1.2 treated spleen cells and in Nude spleen cells, but not in thymus cell cultures. It was concluded that T2 and APN contained polyclonal B cell activating (PBA) substances, which was subsequently proved by their ability to induce a polyclonal antibody response in cell culture. The B cell subpopulation that responds to the mitogenic properties of these bacteria is normally absent from blood, present as a minor proportion of cord blood lymphocytes and present as a major cell population in spleen. Therefore, in spite of the mitogenic properties of these bacterial strains, they are useful tools for the elucidation of a specific reactivity in patients with uroarthritis.

Generation of T Helper Cells in Vitro

Abstract A sequential culture technique for the in vitro induction and subsequent assay of T helper cells is employed to examine the histocompatibility requirements for antigen recognition by murine T helper cells. F1 T cells are primed in vitro with antigen-pulsed parental strain macrophages and tested for antigen-specific helper activity in cultures containing anti-Thy 1.2 serum and C treated spleen cells from hapten-primed parental or F1 mice. A semiallogeneic system is used and appropriate controls are included to avoid possible complicating effects of allogeneic interactions. The results indicate that F1 T helper cells preferentially stimulate carrier-specific anti-hapten plaque-forming cell responses in spleen cells which are H-2 identical with the macrophage used initially to prime the T cells. Parental spleen cell cultures do not respond to F1 T helper cells which were primed with the other parental strain macrophage. Supplementing this culture with macrophages which are histocompatible with those used to prime the F1 T cells is sufficient to restore T helper cell activity. Thus, the genetic restriction described here is between the primed T cell and the macrophage used to elicit secondary responses and not between the T cell and B cell. The results in this semiallogeneic system, however, do not rule out the possibility of additional allogeneic genetic restrictions in the subsequent interaction of T cells with B cells.

Activation of Specific Cellular Immunity toward Murine Leukemia in Mice Rejecting Syngeneic Somatic Hybrid Cells

Abstract ASL-1 × LM(TK)- somatic hybrid cells form both H-2a and H-2k antigen complexes. After forming a localized tumor in syngeneic (A/J × C3H/HeJ)F1 mice, they are rejected. Such mice are resistant to otherwise invariably lethal injections of ASL-1 cells, surviving for prolonged and, in some instances, indefinite periods. To examine the basis of immunity, the capacity of spleen cells from mice rejecting hybrid cells to stimulate the release of 51Cr from labeled ASL-1 cells was investigated. Cells from the spleens of mice rejecting ASL-1 × LM(TK)- cells stimulated the release of 51Cr from labeled ASL-1 cells, but not from Ehrlich ascites or P815 cells. Cells from mice injected with mitomycin-C-treated ASL-1 cells led to the release of 51Cr from labeled ASL-1 cells as well, but the extent of 51Cr release was approximately one-third as occurred in the presence of cells from hybrid cell-injected mice. Cells from noninjected mice or from mice injected with LM(TK)- cells failed to lead to the specific release of 51Cr from ASL-1 cells. The presence of unlabeled ASL-1 cells, but not Ehrlich ascites cells, competitively inhibited the spleen cell-stimulated release of 51Cr from labeled ASL-1 cells. Sera from A/J mice injected with mitomycin-C-treated ASL-1 cells contained antibodies specific for the tumor-associated antigen of ASL-1 cells. The specific release of 51Cr from ASL-1 cells stimulated by spleen cells from hybrid-cell injected F1 mice was inhibited by such antisera, but not by H-2a antisera. Treatment of spleen cells from mice injected with hybrid cells with Thy 1.2 antiserum before incubation with 51Cr-labeled leukemic cells reduced the proportion of 51Cr released by approximately one-half. Spleen cells from animals injected with hybrid cells, as well as to a lesser extent from noninjected mice, which are capable of adhering to plastic Petri dishes were active in the cytotoxic reactions as well. Results similar to these were obtained with 51Cr-labeled RADA-1 cells, a radiation-induced leukemia of A/J mice and spleen cells from (A/J × C3H/HeJ)F1 animals injected with RADA-1 × LM(TK)- hybrid cells.

T Cell-Mediated Cytotoxicity against Trinitrophenyl-Modified Cells: Effect of Glutaraldehyde Treatment on the Immungenicity and Antigenicity of Trinitrophenyl-Modified Cells

Abstract Cells treated with low concentrations of glutaraldehyde for a 10-sec interval were unable to incorporate 3H-leucine into TCA precipitable protein, respond to H-2 allogeneic cells in mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays, or display capping of cell surface immunoglobulin (Ig) with a fluoresceinated anti-Ig reagent. Such cells could stimulate and specifically block H-2 allogeneic CML activity but could not stimulate an H-2 allogeneic MLR response. Trinitrobenzenesulfonic acid (TNBS) treated spleen cells were used to sensitize syngeneic splenocytes into displaying a cytotoxic effect against trinitrophenyl (TNP)-modified target cells. Treatment of the stimulator cells with glutaraldehyde immediately after modification with TNBS did not impair their immunogenic activity. Similar treatment of TNP-modified concanavalin A-stimulated lymphoblasts that were used as inhibitors in a CML cold target competition assay allowed such cells to retain their antigenicity. Cells treated with glutaraldehyde before TNP-modification, however, were not antigenic in the cold target competition assay. These data are compatible with TNBS acting on plasma membrane molecules directly to cause cells to be antigenic and immunogenic in the CML assay rather than affecting internal cellular components.

Specific suppression of the immune response in the adoptive transfer system

Injection of spleen cells (SC) of syngeneic animals immunized with large doses of sheep's red cells (SRBC) into intact mice led to marked specific suppression of the immune response of the recipients. The highest suppressive activity was shown by SC taken from donors on the 14th day after intraperitoneal injection of SRBC. The SC of intact animals and of mice receiving a preliminary injection of rat red cells did not affect the immune response of the intact recipients on immunization with SRBC. Treatment of the immune SC with anti-T serum (ATS) or anti-B globulin (ABG) and complement considerably reduced or completely abolished the suppressive activity. Injection of a mixture of two suspensions of immune SC, one treated with ATS and the other with ABG, into intact recipients did not lead to suppression of the immune response. It is postulated that the suppressor cells in this particular model are T lymphocytes, expressing common antigens or antigens cross-reacting with B cells.

Nonspecific activation of murine lymphocytes. I. Proliferation and polyclonal activation induced by 2-mercaptoethanol and alpha-thioglycerol.

The effect of 2-mercaptoethanol (2-ME) and alpha-thioglycerol (alpha TG) on proliferation and polyclonal activation of lymphocytes was studied in cultures of spleen cells from C3H mice. Inclusion in serum-free or serum-containing medium of the optimal concentration (5 x 10(-5) M) of either 2-ME or alpha TG resulted in highly significant uptake and incorporation of tritiated thymidine ([3H]TdR) into DNA and in morphological blast transformation. These phenomena were dose-dependent, with both lower and higher doses causing less marked effects. The kinetic peak of these responses was found to occur at day 3 of culture. Improved cellular viability could not explain these results, because by day 3 there was no significant difference in viability between cells cultured in the presence or absence of 2-ME. 2-ME evoked a proliferative response in cultures of congenitally athymic (nu/nu) spleen cells that exhibited a similar but lower dose-response profile compared with that of heterozygous (nu/+) littermates. Cultures of bone marrow-derived (B) lymphocytes, generated by treatment of spleen cells with rabbit antithymocyte serum and complement, incorporated [3H]TdR to a degree at least equal to that of normal spleen cell cultures. Thymus-dependent (T) cells did not support significant 2-ME, alpha TG, or Concanavalin A responses in the absence of serum. However, when cultured in 5% fetal calf serum, definite T-cell responses occurred, though always of a lower magnitude than B-cell responses in this system. When the enriched B-cell and T-cell preparations were co-cultured, a synergistic response was noted. Macrophage dependency of the 2-ME and alpha TG effect was shown to be minimal. It is likely that the greater effectiveness of alpha TG relative to 2-ME is due to differences in the chemical structure of these two thiol compounds. The advantages of utilizing 2-ME and alpha TG as probes in the study of lymphocyte activation are evaluated and their possible mechanisms of action are discussed.

Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. III. Alterations of splenic B and T cells in Friend virus-infected mice.

Lymphoid tissues of mice infected with murine leukemia virus (Friend) (MuLV-F) were examined for the presence of cellular markers of MuLV-F infection. The Friend virus-associated cell membrane antigen (FVMA) and the virus group-specific antigen (GSA) were detectable on cells from the spleen and, to a lesser degree, on cells from the bone-marrow. In contrast, neither FVMA nor GSA was found in cells from the thymus. Alterations in the B-cell and T-cell spleen populations of MuLV-F-infected mice were then studied. The proportion of Ig-positive cells declined from the initial 45% (in non-infected controls) to about 10% after 2 weeks of infection. A similar decline of theta-positive cells was noted. However, complement-bearing cells (EAC rosettes) declined even more rapidly and became undetectable in the second week after infection. The treatment of spleen cells from MuLV-F-infected mice with anti-FVMA serum plus complement in vitro reduced the number of detectable Ig-positive cells, specifically, whereas the number of theta-positive cells remained unchanged. Furthermore, B and T cells from spleens of infected mice were separated on an affinity column with anti-Ig antibody-coated beads. The initial cell suspension contained about 45% FVMA-positive cells, about 40% Ig-positive cells and about 40% theta-positive cells. Ig+ cells were retained on the column. The theta-positive cell fraction was collected in the eluate and contained very few FVMA-positive cells with some "null" cells. Most of the FVMA-positive cells were retained on the column, which strongly suggested that they were B cells. These results confirm the previous experiments which showed the selective infections of purified splenic B cells by MuLV-F in cultures.

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice: I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.

Failure of ovalbumin associated with allogeneic spleen ceels to stimulate proliferation of lymph node cells of immune mice.

Ovalbumin-pulsed spleen cells were found to stimulate thymidine uptake of lymph node cells of syngeneic mice immunized with ovalbumin in complete Freund's adjuvant after treatment of spleen cells with Mitomycin C but not after heating the spleen cells at 56degrees for 30 min. Ovalbumin-pulsed spleen cells of allogeneic mice failed to stimulate the immune lymph node cells more than unpulsed cells, although a net increase in the thymidine uptake above the allogeneic stimulation was observed when free ovalbumin was added to the mixed culture. To eliminate the high background of the mixed lymphocyte reaction, F1 mice were made chimeric with bone marrow of one of the parental strains. Using lymph node cells of the immunized chimeras, the stimulation by pulsed spleen cells was much greater when antigen was presented on cells of the parental strain used for bone marrow injection than when presented on cells of the other parental strain.

In Vitro Generation of Suppressor Cell Activity: Suppression of in vitro Induction of Cell-Mediated Cytotoxicity

Abstract It was observed that when normal mouse spleen cells were cultured alone in vitro (precultured) for 3 to 7 days, these cells lost the ability to generate cell-mediated cytotoxicity (CML) during subsequent in vitro sensitization with allogeneic spleen cells, trinitrophenyl (TNP)-modified syngeneic spleen cells, or syngeneic tumor cells. These precultured cells, which were themselves unable to generate CML, were also shown in mixing experiments to suppress, actively, the generation of CML by freshly explanted spleen cells. Suppression occurred at the sensitization phase of CML, and not at the effector level; supernatants from suppressive precultured cells were not suppressive. Suppression was totally abrogated by the treatment of spleen cells with a T cell-specific rabbit anti-mouse brain serum and complement (RαMB + C) either before or after preculturing, suggesting that a T cell was essential both to the generation of suppressor activity and to its expression. Suppressor activity was entirely absent in precultured nylon wool column-nonadherent spleen cells, a T cell-enriched population containing most of the RαMB + C-sensitive cells in the spleen. Precultured nylon column-adherent cells (T cell-depleted) did have suppressive activity, and a mixture of nylon-adherent and nylon-nonadherent cells was as suppressive after preculture as the precultured unseparated spleen. Moreover, the ability of nylon-adherent spleen cells to generate suppressive activity during preculturing was abrogated by treatment with RαMB + C. Thus, the “spontaneous” generation of CML-suppressive activity was dependent upon a limited subpopulation of splenic T cells isolated in the nylon columnadherent fraction. The relationship of these data to a previously described synergy between subpopulations of normal mouse spleen in the generation of CML is discussed, and the findings related to other suppressor systems described in the literature.