T lymphocyte-mediated suppression of myeloma function in vitro. IV. Generation of effector suppressor cells specific for myeloma idiotypes

BALB/c mice immunized intravenously with syngeneic splenocytes, to which affinity-purified IgA produced by the MOPC 315 myeloma is covalently coupled, develop suppressor T cells (Ts1) that inhibit IgA secretion by MOPC 315 cells after 3-4 d of co-culture. Immunization with M315-coupled splenocytes subcutaneously, followed by administration of a soluble extract of Ts1 cells, leads to the generation of effector Ts that are also idiotype specific and inhibit myeloma function within 1 d. Moreover, effector Ts are Lyt-1-2+, whereas Ts1 are either Lyt-1+2+ or require Lyt-1+ and Lyt-2+ cells to mature into effector Ts in vitro. Such a protocol should be useful for analyzing the interactions that result in the maturation of Ts and in defining the mechanisms of action of Ts, whose effect can be measured on a homogeneous target population and that are specific for a well-characterized myeloma idiotype.     

T lymphocyte-mediated suppression of myeloma function in vitro. II. Evidence for regulation of hapten-binding myelomas by syngeneic hapten- specific cytolytic T lymphocytes

BALB/c splenocytes stimulated in vitro with trinitrophenyl (TNP)-modified syngeneic cells inhibit the secretion of antibody by the TNP-binding BALB/c myeloma MOPC 315 in the presence of soluble TNP-Keyhole limpet hemocyanin (KLH). The effector cells are hapten-specific, H-2-restricted, Thy-1.2-bearing, Ly-2-positive T lymphocytes whose precursors are resistant to pretreatment with cyclophosphamide. These phenotypic properties are typical of hapten-specific cytolytic T lymphocytes (CTL). The TNP-reactive CTL that inhibit MOPC 315 cells fail to suppress H-2d myelomas that do not bear TNP-specific surface receptors, and this is not attributable to differences in total binding of TNP-KLH to the different myeloma cells. Moreover, azobenzene arsonate (ABA)-specific CTL inhibit MOPC 315 cells in the presence of the double conjugate TNP-ABA-KLH, but not in the presence of soluble TNP-KLH or ABA-KLH. These results show that H-2-restricted, hapten-specific lymphocytes regulate the function of myeloma cells that bind the hapten only to specific surface receptors, and provide a model for associative recognition of surface H-2 determinants and receptor-bound antigen. The results are discussed with reference to the mechanisms of T lymphocyte-target cell interactions, and the possible physiologic role of hapten-reactive CTL in specifically regulating anti-hapten antibody responses.     

Regulation of reaginic antibody production in mice. I. Suppression by antigen of IgE antibody production in vitro

Regulation of IgE production by antigen in a primed murine splenic lymphocyte culture system was described. Maximum IgE antibody production was found to occur when cells were cultured in the absence of exogenously added antigen. A cells and T lymphocytes did not affect the production of anti-DNP IgE antibody. By using a hapten-carrier antigen system (DNP-EA) for priming mice in vivo, it was found that the production of anti-DNP IgE by spleen cells in vitro was inhibited by hapten when coupled to homologous (EA) or heterologous (BGG) carrier, and was not enhanced or inhibited by homologous carrier. Anti-DNP IgE antibody production by cultures depleted of macrophages or T lymphocytes was found to be as sensitive to the suppressive effects of hapten as was the IgE production by whole spleen cell cultures. Both IgM and IgG secondary anti-DNP PFC responses in vitro were enhanced by the presence of the homologous hapten-carrier or carrier alone. DNP-BGG had no effect on the anti-DNP IgM or IgG PFC responses of the cultures. These data suggest that endogenous production of antibody (IgM or IgG) was not responsible for the observed suppression of the IgE response in vitrol The experimental results presented indicate that the regulation of the IgE production by antigen in the primed mouse splenic lymphocyte cultures was a consequence of the direct interaction of hapten with IgE B cells.     

Regulation of the antibody response to type III pneumococcal polysaccharide by contrasuppressor T cells

A soluble membrane component of type III pneumococcal polysaccharide-coupled spleen cells (S3-SCSM) induces S3-specific suppressor T cells (Ts) in mice. These Ts can be detected only if mice are pretreated with cyclophosphamide (Cy) or if cells adherent to the lectin Vicia villosa are removed from the spleen cell population prior to transfer. The V. villosa-adherent spleen cells from mice injected with S3-SCSM could abrogate suppression mediated by Ts induced by S3-SCSM in Cy-treated mice. The V. villosa-adherent contrasuppressor cells were shown to be T cells that were I-J+ and of the Lyt-1 phenotype. Contrasuppressor T cells (Tcs) were not present in V. villosa-adherent spleen cell fractions obtained from normal mice, from mice injected with polyvinylpyrrolidone-coupled spleen cells, or from Cy-treated mice injected with S3-SCSM, i.e., mice in which Ts activity is dominant. The V. villosa-adherent cells that abrogated the activity of Ts induced by S3-SCSM in Cy-treated mice did not abrogate suppression mediated by a different subset of S3-specific Ts, suggesting that the Tcs described here do not have activity against all Ts subsets. The ability of S3-SCSM to activate Tcs in normal mice provides an explanation for the inability to detect S3-specific Ts in several previous studies.     

Haplotype-specific suppression of antibody responses in vitro. I. Generation of genetically restricted suppressor T cells by neonatal treatment with semiallogeneic spleen cells

C57BL/10 mice were injected with semiallogeneic (B10.D2 X C57BL/10)F(1) spleen cells via the anterior facial vein within 24 h of birth to induce tolerance to B10.D2 (H-2(d)) alloantigens. Spleen cells from these mice as adults developed reduced, but significant, mixed lymphocyte and cytotoxic lymphocyte responses in vitro to H-2(d) stimulator cells and these treated mice rejected first-set B10.D2 skin grafts within a normal time-course, indicating that at best only a state of partial tolerance had been induced. Spleen cells from these mice failed to develop antibody responses to a variety of antigens in vitro when H-2(d) macrophages were in the cultures. Partially purified T cells from these neonatally treated mice suppressed primary antibody responses by normal syngeneic spleen cells in the presence of H-2(d) but not other allogeneic macrophages. These radiosensitive, haplotype-specific suppressor T (Ts) cells inhibited primary antibody responses by blocking initiation of the response, but failed to suppress secondary antibody responses and mixed lymphocyte or cytotoxic lymphocyte responses by appropriate responding spleen cells. To activate H-2(d) haplotype-specific Ts cells, stimulation with IA(d) subregion antigen(s) was necessary and sufficient; syngenicity at the I-A subregion of H-2 between the activated Ts cells and target responding spleen cell populations was also necessary and sufficient to achieve suppression. Comparable results have been obtained with spleen cells from BALB/c mice injected as neonates with (B10.D2 × C57BL/10)F(1) spleen cells where IA(b) antigens activate the haplotype-specific Ts cells. Implications for the significance of this population of haplotype-specific Ts cells in immune regulation are discussed and the properties of these Ts cells are compared and contrasted with other antigen-specific and nonspecific Ts cells whose activity is restricted by I- region products.     

T lymphocytes specific for immunoglobulin allotype. I. Igh- 1(b)-specific T cells demonstrated by suppression in vivo and cytotoxicity in vitro

We show that determinants of IgG(2a) of C57BL/6 mice (Igh-1(b)) stimulate allotypespecific T cells in BALB/c mice. Such cells are detected in two different functional assays; chronic allotype suppression and T cell-mediated cytotoxicity. A population of suppressor T cells capable of inducing chronic Igh-1(b) suppression was demonstrated by rosetting procedures to possess Igh-1(b)-specific receptors, a result interpreted as indicating that suppressor T cells may act directly upon allotype-bearing B cells. From similar populations we were also able to demonstrate Igh-1(b)-specific cytotoxic T cells. Such cells were lytic for target myeloma cells expressing the Igh-1(b) allotype of IgG28, and were ineffective against a variant cell line failing to express Igh-1(b), and other target cell lines expressing different allotypes or isotypes. The similar specificity of suppressor T cells and cytotoxic T lymphocytes for Igh-1(b) allotype raises the possibility that the target in allotype suppression is a B cell, and that allotype-specific cytotoxic T cells may play some role in regulation of allotype expression in the suppressed state.     

Specific in vitro antimannan-rich antigen of Candida albicans antibody production by sensitized human blood lymphocytes.

We have developed a new antigenic system for the induction of specific in vitro antibody response in man. The antigen used was purified from the cell wall of Candida albicans strain A and contained greater than 96% polysaccharide mannan. Peripheral blood mononuclear cells from Candida-sensitized donors produced specific antimannan antibodies during a 7-d culture in the presence of mannan absorbed with methylated bovine serum albumin. Two methods were used to detect antimannan antibody responses. Antimannan antibody-producing cells were identified by radioautography with tritiated mannan. Antibody concentration in culture supernatants was measured by an enzyme-linked immunosorbent assay. In both methods, specific IgM and IgG (but not IgA) antibodies were detected. The antibody production to mannan was specific, since an antigenically unrelated polysaccharide (pneumococcal antigen S III) did not bind to methylated bovine serum albumin-mannan-induced blast cells and did not induce antimannan antibody-containing cells. Furthermore, a pulse with an excess of unlabeled mannan abolished [3H]mannan binding, whereas an excess of unlabeled S III did not. Similarly, no antimannan antibody was obtained in influenza virus-stimulated cultures and mannan-stimulated cultures were not inducing antiinfluenza antibodies. The antimannan antibody production was shown to be a T cell-dependent phenomenon. The T helper effect appeared to be radiosensitive. It was under a genetic restriction as it occurred only in autologous or semi-identical but not in allogeneic situations. This system is relatively simple, reproducible, and well suited for the study of specific secondary in vitro antibody responses to polysaccharide antigens in humans.     

Haplotype-specific suppression of antibody responses in vitro. II. Suppressor factor produced by T cells and T cell hybridomas from mice treated as neonates with semiallogeneic spleen cells

Culture supernatant fluids from spleen cells from C57BL/10 or BALB/c mice neonatally treated with semiallogeneic (B 10.D2 x B10)F1 cells to induce haplotype-specific suppressor T cells and restimulated with macrophages syngeneic at I-A with the allogeneic haplotype encountered as neonates contain a soluble factor capable of suppressing primary in vitro antibody responses of normal syngeneic spleen cells in a non- antigen-specific manner. This haplotype-specific suppressor factor, TsF- H, has also been recovered in culture fluids of a T cell hybridoma produced by fusion of the AKR thymoma BW5147 and the haplotype-specific suppressor T cells. TsF-H is inactivated by low pH (3.5) trypsin, for 30 min at 50 degrees C, and has a molecular weight in the range of 45,000 to 68,000. Studies with specific immunoabsorbents demonstrate the presence of determinants encoded by the I-A subregion of the haplotype of the T cell producing TsF-H but not I-J subregion or immunoglobulin constant-region determinants on the TsF-H. Suppression is restricted to primary in vitro antibody responses, and not secondary antibody, mixed lymphocyte, or cytotoxic lymphocyte responses by spleen cells syngeneic at the I-A subregion of H-2 with the T cell producing the factor. The properties and activities of TsF-H and the haplotype- specific suppressor T cell are compared and contrasted with antigen- specific and genetically restricted suppressor T cells and their factors.     

Viruses disrupt functions of human lymphocytes. Effects of measles virus and influenza virus on lymphocyte-mediated killing and antibody production

We present experimental data that offer, in part, a better understanding of the immunosuppression that accompanies measles virus infection. We note that measles virus "silently" infects human lymphocytes and that the infection does not alter lymphocyte survival in vitro. Yet such infected lymphocytes fail to generate natural killer (NK) cell activity or synthesize immunoglobulins (Ig). Thus, the presence of virus within lymphocytes impairs their specific immune functions in the absence of cytolysis. Influenza virus also infects human lymphocytes. In contrast to measles virus infection of resting lymphocytes in which viral antigen is rarely expressed, influenza virus infection of these cells yields viral antigens expressed in the cytoplasm and on the cell surface. Influenza virus-infected lymphocytes have normal NK cell activity but fail to synthesize IgG or IgM.     

Regulation of ige antibody production by serum molecules. III. Induction of suppressive activity by allogeneic lymphoid cell interactions and suppression of IgE synthesis by the allogeneic effect

Antibody responses of the IgE class are, like other immunoglobulin classes, regulated by a finely-tuned network of complex cellular and molecular interactions (1). Previous studies conducted in our laboratory (2, 3) have provided new insights into the differences in control mechanisms that result in individuals manifesting either the high (allergic) or low (nonallergic) IgE responder phenotype. These experiments have shown that certain manipulations (i.e. low dose X-irradiation) convert normally low responder mice to high IgE responders, apparently by diminishing a suppressor T-cell mechanism which normally dampens, rather selectively, IgE antibody production in such individuals. Similar findings have been made by Watanabe et al. (4). Recently, we have been studying the types of manipulations that could reverse the high IgE responsive state back to a low one. These studies (2, 3, 5, 6) have demonstrated that the high IgE responses induced in low responder mice can be substantially diminished, and even abolished, by passively transfusing serum or ascetic fluid from donor mice previously inoculated with mycobacterial-containing complete Freund''s adjuvant (CFA). Because the suppressive activity of CFA-immune serum or ascitic fluid is so highly selective for IgE antibody production, we have recently termed these serum substances suppressive factors of allergy (SFA) (2, 3). The present study was undertaken to determine whether alternative means, particularly those that avoid administration of CFA, could be devised for the induction of SFA. Herein, we report the effectiveness of allogeneic lymphoid cell interactions in inducing SFA, both in vivo and in vitro, as well as the potent suppressive effects of an in vivo allogeneic effect on irradiation enhanced IgE antibody production in low responder mice.     

Specific anti-influenza virus antibody production in vitro by lymphocytes from a subset of patients with hypogammaglobulinemia.

Specific anti-influenza virus antibody production in vitro was studied in peripheral blood mononuclear cells from 17 patients with hypogammaglobulinemia. Cells obtained from 6 of 12 patients with common variable hypogammaglobulinemia produced anti-influenza virus antibody, predominantly of the IgM isotype, when cultured in vitro with type A influenza virus. No antibody was produced in vitro, however, by cells from either of two patients with Bruton's type X-linked hypogammaglobulinemia or by cells from any of three patients with X-linked hypogammaglobulinemia and isolated growth hormone deficiency. These studies demonstrate that peripheral blood mononuclear cells from a subset of patients with common variable hypogammaglobulinemia retain the potential to produce specific antibody in response to antigenic stimulation.     

Antigen receptors on murine T lymphocytes in contact sensitivity. III. Mechanism of negative feedback regulation by auto-anti-idiotypic antibody

Contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) is maximal 6 d after sensitization but declines rapidly. Previous studies have shown that this rapid decline is due to auto-anti-idiotypic (anti-Id) antibodies produced by the host. The present study was done to investigate the mechanism(s) involved in his down-regulation of the effector phase of the CS reaction. Using transfer of CS to mimic the natural effector phase, we found that the inhibition of transfer by treating DNFB-sensitized lymph node (LN) cells with either auto-anti-Id or syngeneic anti-Id serum is complement (C) independent. This inhibition requires Ia+ T cells in the immune population. Depleting immune LN cells of Ia+ T cells rendered them insensitive to inhibition by anti-Id alone, although the same population is inhibited by anti-Id plus C. This cell population is rendered sensitive to inhibited by anti-Id alone by addition of untreated DNFB-sensitized LN cells, but not by addition of normal LN cells. Further studies showed that the suppression of immunity by anti-Id-activated Ia+ T cells is not systemic, but rather occurs locally at the skin test site and is antigen nonspecific. We interpret these data to indicate that the natural regulation of CS to DNFB by auto-anti-Id antibodies is an active process that involves a negative feedback regulatory loop.     

Clustering of dendritic cells, helper T lymphocytes, and histocompatible B cells during primary antibody responses in vitro

Mouse spleen suspensions generate discrete cell clusters within 1-2 d of culture. We have isolated these clusters by velocity sedimentation to study their contribution to primary antibody responses. Clusters represent approximately 5% of the starting spleen cells and consist of 20-50% B cells, 20-50% T cells, and 10-20% dendritic cells (DC). When the cultures are stimulated with thymus-dependent antigens, like heterologous red cells or dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), the clusters are the principal site for the development of plaque-forming cells (PFC). Noncluster fractions form few PFC and only when supplemented with fresh DC. PFC responses in all cases are antigen specific. B cells cluster only in the presence of T cells and DC (1 DC/200 B-T cell mixtures) and only after encountering specific antigen. The elimination of either DC or Lyt-1+2- T cells, with monoclonal antibody and complement, ablates B cell development into PFC. PFC responses are restored with antigen-nonspecific helper factors formed in the syngeneic mixed leukocyte reaction between DC and T cells. Since PFC to DNP-KLH do not develop de novo when B cells are exposed to antigen and helper factors, anti-DNP PFC precursors must be stimulated within clusters to become responsive to helper factors. PFC development within clusters is restricted by the major histocompatibility complex (MHC). When DC and T cells are from strain P1, then P1 but not P2 B cells develop into PFC; when DC are from strain P2 and T cells from strain P1, strain P2 B cells are selected to become PFC in clusters. The entry of B cells into clusters is itself MHC restricted, since P1 DC/T cells aggregate six times as many B cells from strain P1 as strain P2. Thus, clusters are the site in which DC, B, and T cells interact to generate PFC. One can use clusters to retrieve B cells that have been selected in an antigen-dependent, MHC-restricted fashion and to show that clustering B cells become responsive to soluble, polyclonal helper factors.     

Kinetic study of T lymphocytes after sensitization against soluble antigen. III. Potentiation and suppression of the PHA response by antigen-activated lymphocytes of low density

Lymph node cells of ovalbumin-sensitized rats were separated on the basis of buoyant density into fractions reciprocally enriched in cells responsive to ovalbumin or phytohemagglutinin (PHA). Recombination of high density and low density fractions in varying proportions resulted in potentiation or suppression of the DNA synthetic response to PHA in culture. The response of cultures containing equal numbers of high and low density cells was markedly greater than the sum of the two populations stimulated separately. However, when decreasing numbers of low density cells were cultured with a constant number of high density cells, profound suppression was observed.     

Regulation of the T-cell response to ectromelia virus infection. I. Feedback suppression by effector T cells

Spleen cells and serum from mice immunized with ectromelia virus suppressed the immune response to infectious virus when transferred intravenously into recipient mice given an immunizing virus dose. The suppression was reflected in decreased cytotoxic T-cell activity directed against H-2 compatible virus-infected target cells in the spleens of recipients. Suppression was observed when immune cells or serum were transferred 1-2 h or 1 day after immunization of recipients, but not 2 days after, and was maximal when 6-day immune spleen cells were used as suppressor cells. H-2 compatibility between donor and recipient mice was necessary for suppression to be expressed. Use of recombinant mice showed that I-region compatibility was neither sufficient nor necessary, and that D-region compatibility was sufficient. Specificity of suppression was suggested by the finding that cells and serum from mice immunized with Listeria monocytogenes, a bacterium, had no suppressive activity on the antiviral response. Anti-theta treatment eliminated the ability of immune cells to suppress, and the suppressive effect was not markedly dose-dependent with respect to both cell dose and virus dose under the conditions employed. Virus levels in the spleens of recipients were significantly reduced after injection of immune cells. Adult thymectomy had no effect on the primary immune response to ectromelia virus infection, thus indicating no role for T1 cells in the suppressive mechanism. The results obtained therefore suggested that suppression in this system was due to effector T cells which triggered clearance of virus (and thus, of virus-induced antigens) necessary for the induction of precursors of effector T cells, and that this simple feed-back mechanism normally plays an important role in the regulation of the primary immune response to ectromelia infection at the level of precursor induction. The existence of other postinduction regulatory mechanisms, however, is unknown and under investigation.     

In vitro idiotypic suppression of chronic lymphocytic leukemia lymphocytes secreting monoclonal immunoglobulin M anti-sheep erythrocyte antibody.

A patient with chronic lymphocytic leukemia was found to have B cells with surface immunoglobulin (Ig)M manifesting anti-sheep erythrocyte (SRBC) specificity together with a high titer serum monoclonal anti-SRBC IgM antibody. By immunizing a sheep with the monoclonal IgM antibody, followed by multiple absorptions against normal human IgM, an anti-idiotype (Id) antibody was obtained. The serum IgM anti-SRBC antibody was then demonstrated to share the same idiotypic determinants with the surface IgM with anti-SRBC specificity on the patient's B cells. The anti-Id antibody suppressed the spontaneous secretion of anti-SRBC antibody as well as the pokeweed mitogen-induced anti-SRBC antibody production as measured by a hemolysis-in-gel plaque-forming cell assay. In contrast, pokeweed mitogen-induced anti-SRBC plaque-forming cell responses of normal individuals were not suppressed by the anti-Id antibody. Thus, this study demonstrates in vitro suppression of human B-cell function by anti-Id antibody.     

Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes

The relative functional maturity of neonatal mouse spleen T- and B-cell populations was assessed by comparing the ability to respond to the thymic-independent antigen, DNP-Ficoll, or thymic-dependent SRBC by producing antibody in vitro. Although mouse spleen cells responded to DNP-Ficoll at an earlier age than they responded to SRBC or TNP-SRBC, the reason for the lag in the T-dependent response was confounded by the finding of high numbers of suppressor T lymphocytes in the neonatal spleen. Thus, small numbers of neonatal spleen T cells or thymocytes significantly decreased the in vitro antibody response of adult spleen cells. Although B lymphocytes appear to be functionally mature soon after birth, their acitivity may be modulated by an excess of suppressor T cells; e.g., the reconstitution of helper cell function in the neonatal spleen required anti-theta treatment before addition of adult helper cells. Suppressive activity attributable to T cells seems to play a dominant role in determining the ability of the neonatal animal to react positively or negatively to antigenic stimulation.     

Mechanisms of genetic resistance to Friend virus leukemia. III. Susceptibility of mitogen-responsive lymphocytes mediated by T cells

Friend leukemia virus (FV) suppressed the proliferative responses of spleen, lymph node, marrow, and thymus cell populations to various T- and B-cell mitogens. Cells taken from mice, e.g. BALB/c genetically susceptible to leukemogenesis in vivo were much more susceptible to suppression of mitogenesis in vitro than similar cells from genetically resistant mice, e.g., C57BL/6. Nylon wool-purified splenic T cells from BALB/c and C3H mice lost susceptibility to FV-induced suppression of mitogenesis but became suppressible by addition of 10% unfiltered spleen cell. Thus, FV mediates in vitro suppression of lymphocyte proliferation indirectly by "activating" a suppressor cell. The suppressor cell adhered to nylon wool but not to glass wool or rayon wool columns. Pretreatment of spleen cells with carbonyl iron and a magnet did not abrogate the suppressor cell function. Suppressor cells were not eliminated by treatment with rabbit antimouse immunoglobulin (7S) and complement (C). However, high concentrations of anti-Thy-1 plus C destroyed suppressor cells of the spleen; thymic suppressor cells were much more susceptible to anti-Thy-1 serum. Nude athymic mice were devoid of suppressor cells and their B-cell proliferation was relatively resistant to FV-induced suppression in vitro. The suppressor cells in the thymus (but not in the spleen) were eliminated by treatment of mice with cortisol. Thus, FV appears to mediate its suppressive effect on mitogen-responsive lymphocytes by affecting "T-suppressor cells." Spleen cells from C57BL/6 mice treated with 89Sr to destroy marrow-dependent (M) cells were much more suppressible by FV in virto than normal C57BL/6 spleen cells. However, nylon-filtered spleen cells of 89Sr-treated C57BL/6 mice were resistant to FV-induced suppression in vitro, indicating that the susceptibility of spleen cells from 89Sr-treated B6 mice is also mediated by suppressor cells. Normal B6 splenic T cells were rendered susceptible to FV-induced suppression of mitogenesis by addition of 10% spleen cells from 89Sr-treated B6 mice. Thus, M cells appear to regulate the numbers and/or functions of T-suppressor cells which in turn mediate the immunosuppressive effects of FV in vitro. Neither mitogen-responsive lymphocytes nor T-suppressor cells are genetically resistant or susceptible to FV. The genetic resistance to FV is apparently a function of M cells, both in vitro as well as in vivo.