A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens.

A first or dominant immunization with one antigen markedly inhibited specific cytolytic T lymphocyte (CTL) responses to a second unrelated alloantigen without suppressing antibody responses to other antigens. Suppression was induced rapidly, became systemic, and could be transferred passively with only serum. Suppression did not result from elimination of cells capable of responding to the second antigen. The mechanisms responsible for this "priority of the first response" may be the same that help protect the fetus during pregnancy, promote renal allograft survival after multiple blood transfusions, and prevent effective CTL-mediated immunity to variants of tumor cells or infectious agents that arise during tumor progression or chronic infections.

A first or dominant immunization. II. Induced immunoglobulin carries transforming growth factor beta and suppresses cytolytic T cell responses to unrelated alloantigens.

Fresh sera from mice immunized by bearing an immunogenic tumor or by repeated injections of allogeneic spleen cells or xenogeneic erythrocytes powerfully suppress cytolytic T cell responses in one-way mixed lymphocyte cultures. Suppression is not antigen specific, though is mediated by immunoglobulin (Ig)G specific for the immunizing antigen. Suppression caused by IgG mimics that caused by active transforming growth factor beta (TGF-beta). IgG associates with or carries latent TGF-beta; however, suppression caused by the complex of IgG-TGF-beta requires macrophages (M phi), whereas active TGF-beta alone does not. Also, IgG dissociated from TGF-beta does not cause suppression, suggesting that M phi may take up Ig-TGF-beta, process the complex, and deliver active TGF-beta to lymphocytes. Indeed, suppression by immune serum was prevented by antibody to Fc receptors, by saturating Fc receptors with heterologous IgGs, and by antibodies against TGF-beta. The overall findings reveal a previously unrecognized regulatory circuit whereby IgG produced in response to one antigen nonspecifically downregulates cytolytic T lymphocyte responses to unrelated antigens. The findings introduce the intriguing possibility that TGF-beta delivered by IgG and processed by M phi may mediate important biological effects in processes such as wound healing, tumor growth, and some autoimmune diseases.

Regulation by complementary idiotypes. Ig protects the clone producing it.

A/He mice actively producing complementary or anti-idiotypic antibody directed against a combining site structure for phosphorylcholine (PC) have profound and long-lasting suppression of their response to PC. B cells from unresponsive mice are unresponsive in vitro, and attempts to demonstrate suppressor cells in unresponsive mice were unsuccessful. Although the process ultimately responsible for suppression has not been defined, suppression can be initiated by anti-idiotypic antibody alone and prevented by complementary Ig; i.e., by anti-PC antibody. Furthermore, a suppressed anti-PC response can be rescued by sublethal irradiation and anti-PC antibody given passively. The recovery of the suppressed response is slow and presumably results from maturation from "stem" cells, which are protected from tolerization by the passively given antibody. Thus, by extrapolation, one of the functions of secreted Ig may be to protect the clone that produces it.

Simultaneous complementary idiotypic responses: absence of reciprocal regulation.

Complementary antibodies, i.e. antibodies having combining site structures which are at least partially directed against each other, were induced in A/He mice by immunization with phosphorylcholine (Pc) coupled to keyhole limpet hemocyanin or with the Pc-binding IgA myeloma protein, HOPC-8 (H8). Both responses were monitored by enumerating plaque-forming cells and assaying serum antibody levels to Pc and H8. Prior immunization with H8 markedly suppressed subsequent immunization with Pc and vice versa; neither plaque-forming cell response was diminished, however, when mice were immunized simultaneously with Pc and H8. Experiments were designed to determine if the absence of reciprocal regulation was due to change in idiotypes. This was determined by measuring inhibition of plaque formation using complementary antibody. Plaque formation by cells was equally inhibited by high dilutions of the appropriate complementary antibody whether cells were from mice immunized with one, the other, or both antigens. Thus, the absence of regulation could not be accounted for by emergence of different idiotypes. Interestingly, sera from mice immunized to have high responses to both antigens were relatively ineffective in inhibiting plaque formation or suppressing immunization to Pc. However, such sera contained complexes of the complementary antibodies; apparently antibody to Pc in such sera quenches or neutralizes the activity of anti-H8 antibody. But the formation of complexes, at least measurable levels of circulating complexes, must be a result rather than the cause for the absence of reciprocal regulation, since regulation was also absent when immunization to Pc was manipulated so that responses were too low to result in detectable levels of circulating antibody to Pc. It is proposed that simultaneous complementary responses may occur in nature to other antigens and antibodies, and that such simultaneous responses may cause pathologic changes.

Dendritic cells that have interacted with antigen are targets for natural killer cells.

Natural killer (NK) cells (poly I:C induced, x-ray resistant, nonadherent, Thy-1-, Ly-1.1-, Ly-2.1-, anti-asialo GM1-positive, and cytotoxic for YAC-1) suppressed T lymphocyte proliferation in mixed lymphocyte reaction (MLR) and autologous MLR cultures. Dendritic cells (DC) were required for proliferation of lymphocytes in both responses. The question whether lymphocytes or DC were the targets for NK cells was resolved by taking advantage of the fact that NK cells, but not DC, lose activity after 24 h in culture. Three findings indicate that DC, not lymphocytes, are targets for NK cells. First, responses suppressed by NK cells were fully restored by adding small numbers of DC to cultures 24 h after NK cells had been added. Second, DC incubated alone with NK cells and antigen for 24 h did not stimulate proliferation of lymphocytes. Third, lymphocytes incubated alone with NK cells for 24 h proliferated normally when DC were added. Additional experiments showed that DC became targets only after interaction with antigen. Thus, we suggest that NK cells may regulate lymphocyte proliferation by monitoring antigen presentation by DC.

Autoantibodies produced spontaneously by young 1pr mice carry transforming growth factor beta and suppress cytotoxic T lymphocyte responses.

Young MRL/MPJ-lpr (lpr) mice 8-12 wk old challenged with alloantigen had significantly lower specific cytolytic T lymphocyte (CTL) responses than control MRL/MPJ +/+ mice. Serum from lpr mice compared with serum from ++ or normal C3H mice powerfully suppressed CTL responses in mixed lymphocyte cultures (MLC); absorbing lpr serum on protein G, adding antibody against transforming growth factor beta (TGF-beta) to cultures or dissociating immunoglobulin G (IgG) and TGF-beta before additions to cultures prevented suppression. Apparently autoantibody, similar to IgG produced by normal mice in response to immunization, carries TGF-beta which suppresses CTL responses in vivo and in vitro.

Thy-1+ and Thy-1- natural killer cells. Only Thy-1- natural killer cells suppress dendritic cells.

Cells enriched for NK activity (poly I:C induced, x-ray resistant, and nonadherent), include two phenotypically and functionally different populations. Both populations of NK cells are AGM1+, Ly-1.1-, Ly-2.1-, Ia-, and have the morphology of large granular lymphocytes. One population, however, is Thy-1+ while the second population is Thy-1-. Thy-1+ NK cells lyse YAC-1 and P815 target cells; Thy-1- NK cells lyse YAC-1 but not P815 target cells. The FACS was used to obtain homogeneous populations of Thy-1+ and Thy-1- NK cells, which retain high cytotoxicity. While Thy-1- NK cells suppress the antibody response in vitro by suppressing or eliminating DC, Thy-1+ NK cells do not suppress antibody responses in vitro.

The specific selection of recirculating lymphocytes by antigen in normal and preimmunized rats.

Thoracic duct lymphocytes (TDL) from normal rats will restore a primary antibody response to sheep erythrocytes (SRBC) in irradiated recipients and cause a graft-versus-host reaction in F(1) hybrid rats; lymphocytes from rats immunized with either tetanus toxoid or dinitrophenylated bovine gamma globulin (DNP BGG) will generate specific antibody after cell transfer and challenge. The ability of TDL to mediate each of these responses is severely depressed by giving a single intravenous dose of the specific antigen shortly before cannulation of the thoracic duct, although the lymphocyte donors themselves respond normally. The injection of antigen does not decrease the output of lymphocytes in the thoracic duct and the effect is specific for the antigen injected. The findings are most readily accounted for by assuming that small subpopulations of specific lymphocytes are selected from the recirculating pool by antigen which has localized in lymphoid tissue. The observation that passive antibody abolishes selection by SRBC supports this interpretation. The strong selection exerted by a subcutaneous injection of SRBC in Freund's complete adjuvant, which induces delayed hypersensitivity but little early antibody, suggests that a common cell type may be involved in the induction of both delayed hypersensitivity and antibody formation. The anti-DNP antibody response generated by TDL from rats immunized with DNP BGG was abolished by a selecting injection of the homologous conjugate. The response was depressed to a smaller degree by injections of either BGG or dinitrophenylated human serum albumin, suggesting that carrier-specific (T) and hapten-specific (B) lymphocytes could be separately selected from the recirculating pool. The regional selection of recirculating lymphocytes by antigen may explain a number of phenomena in which the prior injection of antigen has been found to inhibit a subsequent immune response.

Multiple cancers. Tumor burden permits the outgrowth of other cancers.

We demonstrate that tumor-bearing hosts permit the outgrowth of "potentially malignant" cells that are located at a different site. These second cancers continued to grow and kill their hosts even though they retain the "premalignant" phenotype, even after removal of the original malignancy. The potentially malignant cells used in these experiments were ultraviolet light- or methylcholanthrene-induced regressor tumor cells that are rejected regularly by normal mice at any testable dose, and only form progressive tumors in immunosuppressed individuals. The immunological rejection of these highly immunogenic, potentially malignant cells was suppressed by Thy-1+, Ly-2-, nonadherent, radio-sensitive suppressor cells in the tumor-bearing mice. These suppressor cells were absent in nude tumor-bearing mice. Unlike helper and cytolytic T cell-mediated responses, which are exquisitely tumor specific, the suppression caused by a progressively growing tumor was crossreactive among many syngeneic, independently derived tumors induced by different carcinogens. However, T cell-mediated immune responses to alloantigens, allogeneic tumors, certain syngeneic tumors, and humoral responses to xenogeneic red blood cells were normal in these mice. The immune suppression in the tumor-bearing animals closely simulated that induced by ultraviolet light irradiation, and both types of suppression might therefore share common mechanisms. Our findings may contribute to understanding the growth, development, and possible control of multicentric malignancies and add a precaution to the potential use of strongly immunogenic tumor variants for active immunotherapy in hosts bearing less immunogenic tumors.

Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells.

Inoculated immunogenic cancer cells after initial growth are potentially rejected by specific host immunity; however, the outcome of the interaction between host and inoculated cancer cells is a function of multiple factors including the route of inoculation, the number of cells, the density of antigens on the injected cancer cells, and the state of the immune system of the host. In the present study, we have examined a different kind of variable: the stroma that inoculated tumor cells initially reside in. The impetus to examine this factor arises from observations that cancer cells from several lines inoculated as fragments of solid tumors often grow progressively, whereas the same number or more than 10-fold larger numbers of identical type cells injected as a suspension are rejected, even though fragments or suspended cells are both tumorigenic at the same doses in nude mice. In the present studies, we found that: (a) indeed, cancer cells inoculated as fragments were more tumorigenic than cancer cells in suspension; (b) the tumorigenicity of suspended cancer cells was increased by injection of the cells into polyurethane sponge implants; (c) cancer cells were more tumorigenic embedded in syngeneic stroma than in transgenic antigenic stroma expressing the K216 major histocompatibility complex class I antigen; and (d) antigenic, bone marrow-derived, stromal components (presumably passenger leukocytes) were sufficient to cause rejection of immunogenic but antigenically unrelated cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression by autogenous complementary idiotypes: the priority of the first response.

Complementary idiotypes or antibodies are considered to have combining site structures which are at least partly directed against each other. Complementary antibodies were induced in A/He mice by immunization with phosphorylcholine (PC)-containing antigens and by immunization with the PC-binding IgA myeloma protein TEPC-15 (T15). Both responses were monitored by enumerating plaque-forming cells (PFC) and assaying serum antibody levels against the corresponding antigens. Mice immunized at least three times with T15 in adjuvants had markedly suppressed responses to subsequent immunization with PC; similarly, mice preimmunized multiple times with PC had suppressed responses to immunizations with T15. In contrast, mice immunized with T15 in the interval between "primary" and "secondary" immunizations with PC had undiminished PFC responses to both antigens but significantly decreased antibody titers to PC. Simultaneous responses were also induced by immunizations with T15 superimposed on weekly immunizations with PC; with this regime, immunization with T15 actually enhanced the PFC response to PC, but serum antibody to PC was significantly lower than for mice immunized with PC only. Levels of serum antibody to PC were probably lower, either because anti-PC antibody was complexed with the complementary antibody directed against T15, or because the antibody directed against T15 prevented synthesis and/or release of anti-PC antibody by cells in vivo. Thus, an established prior autogenous immune response can dramatically suppress a subsequent primary complementary response, but the effects of complementary responses on each other are more complex with different sequences of immunization. Also, the effects of variables such as the amounts and ratios of the classes of antibodies on regulation of complementary responses remain to be defined.

The rate of division of antibody-forming cells during the early primary immune response.

Mitotic blocking agents, colchicine or Velban, were used to estimate cycle times of spleen cells which release hemolysin for sheep erythrocytes (plaque-forming cells). The cells were obtained either from rats immunized with sheep erythrocytes or from cultures of mouse spleen cells immunized in vitro with the same antigen. 2, 3, or 4 days after immunization, animals or cell cultures were treated with mitotic blocking agents for periods of time ranging from 2.5 to 7 hr; plaque-forming cells were then enumerated. Decreased numbers of plaque-forming cells were found after such treatment. The extent of reduction was a function of duration of the drug treatment and the method of immunization, but was independent of the time after immunization. The evidence presented is consistent with premises that: (a) plaque-forming cells in mitosis do not release sufficient antibody to be detected, (b) mitotic blocking agents, by arresting plaque-forming cells in metaphase, prevent not only detection of these cells but also the increase in number of plaque-forming cells which would have resulted from cell division, (c) mitotic blocking agents do not affect release of antibody by cells in interphase. Cell cycle times, based on the extent of reduction of plaque-forming cells per unit time of drug treatment, were estimated using a mathematical model appropriate for an exponentially increasing population of cells. Cell cycle times estimated using the mitotic blocking agents agreed well with cell doubling times calculated from the increase in plaque-forming cells occurring 1-4 days after immunization. Increased responses produced by higher antigen doses or treatment of immunized animals with an adjuvant resulted from an increased rate of division of responding cells and their progeny. The results are consistent with a cell selection theory of antibody formation. Antigenic stimulation causes relatively few cells to proliferate and to synthesize antibody; apparently the magnitude of the response is dependent primarily on the rate of division of responding cells. It is suggested on the basis of observations of in vitro-immunized cell cultures that the rate of division of responding cells may be dependent on the rate of interaction between two cell types, both of which are essential for the in vitro plaque-forming cell response.

Enhanced growth of primary tumors in cancer-prone mice after immunization against the mutant region of an inherited oncoprotein.

One major objective of tumor immunologists is to prevent cancer development in individuals at high risk. (TG.AC x C57BL/6)F1 mice serve as a model for testing the feasibility of this objective. The mice carry in the germline a mutant ras oncogene that has an arginine at codon 12 instead of glycine present in the wild-type, and after physical (wounding) or chemical promotion, these mice have a high probability for developing papillomas that progress to cancer. Furthermore, F1 mice immunized with Arg(12) mutant ras peptide in complete Freund's adjuvant (CFA) develop T cells within 10 d that proliferate in vitro on stimulation with the Arg(12) mutant ras peptide. Within 14 d, these mice have delayed-type hypersensitivity to the peptide. Immunization with CFA alone or with a different Arg(12) mutant ras peptide in CFA induced neither response. To determine the effect of immunization on development of tumors, mice immunized 3 wk earlier were painted on the back with phorbol 12-myristate 13-acetate every 3 d for 8 wk. The time of appearance and the number of papillomas were about the same in immunized and control mice, but the tumors grew faster and became much larger in the mice immunized with the Arg(12) mutant ras peptide. Thus, the immunization failed to protect against growth of papillomas. The peptide-induced CD4(+) T cells preferentially recognized the peptide but not the native mutant ras protein. On the other hand, mice immunized with Arg(12) mutant ras peptide and bearing papillomas had serum antibodies that did bind native mutant ras protein. Together, these studies indicate that active immunization of cancer-prone individuals may result in immune responses that fail to eradicate mutant oncogene-expressing tumor cells, but rather induce a remarkable enhancement of tumor growth.

Hypersensitivity: specific immunologic suppression of the delayed type.

Delayed-type cutaneous hypersensitivity to sheep erythrocytes was induced in rats by intradermal injection of the antigen mixed with Freund's adjuvant; hypersensitivity was sustained by weekly injections. Either passive immunization with rat antiserum to sheep erythrocytes or intravenous injection of sheep erythrocytes partially suppressed induction of hypersensitivity; these procedures used together specifically and completely suppressed induction of hypersensitivity. Complete suppression was sustained by antigen given intravenously before each weekly injection of the mixture of antigen and adjuvant. These findings provide the rational basis of a simple method for prolonging survival of allografts with only the biological agents, antigen and antibody, of the immunological response.

Homeostasis of the antibody response: immunoregulation by NK cells.

When injected into mice, the synthetic double-stranded polynucleotide poly(inosinic) X poly(cytidylic) acid induces high natural killer (NK) cell activity within 4 to 12 hours. Induction of NK activity in mice immunized 2 or 3 days previously, or the addition of NK cells to cultures immunized in vitro 2 or 3 days previously, promotes early termination of the ongoing primary immunoglobulin M antibody response. A target for NK cells is a population of accessory cells that has interacted with antigen and is necessary for sustaining the antibody response. The inference is strong that NK cells induced normally by immunization also terminate the usual antibody response in vivo by elimination of antigen-exposed accessory cells.

Specific suppression of immune responses.

The models we have discussed in detail demonstrate specific suppression of immune reactivity produced in normal adult animals by antibody and antigen. The mechanism of homeostasis of suppression in these models depends on continued exposure to antigen and on an active response by the host. The active response may include production of antibody directed against specific receptors as well as antibody directed against antigen. Thus, specific regulation of both antibody and cell mediated immunity to an antigen might be achieved by the use of only the biological agents of the response: antigen, antibody, and possibly antibody to receptors. The general implication is that these same biological agents are responsible for autoregulation of immune reactions occurring in nature. Presumably, these agents may be used to suppress or reverse immune responses for appropriate clinical objectives.

Neonatal tolerance induced by antibody against antigen-specific receptor.

Specific immunologic unresponsiveness is induced by injecting adult or neonatal mice with antibody against antigen-specific receptor (antireceptor antibody). Suppression in mice treated as adults lasts several weeks, and cells from these suppressed mice respond normally in culture. In contrast, unresponsiveness induced in neonatal mice is long-lasting; cells from these mice do not respond in culture and do not affect the response of normal cells. Evidently, antireceptor antibody reversibly blocks antigen receptors in adult animals, but induces unresponsiveness in neonatal mice by depleting the clone of receptor-bearing cells.

Cytokines and cancer: experimental systems.

The transfer of certain cytokine genes into cancer cells can provide very powerful suppression of tumor growth in the absence of any toxic side effects. Some of these cytokines, such as interleukin-4, granulocyte colony-stimulating factor and tumor necrosis factor, can mediate powerful immune suppression even in T-cell-deficient animals and appear to be effective for poorly or non-antigenic tumors. However, approaches must be found to induce or deliver cytokines locally at the tumor site.

Major histocompatibility complex class I and unique antigen expression by murine tumors that escaped from CD8+ T-cell-dependent surveillance.

The rejection of murine UV-induced skin cancers by normal mice is a striking example of powerful immune surveillance of the normal host against malignant cells. In this study, we show that UV-induced regressor tumors regularly grew progressively and killed mice that were depleted of CD8+ T-cells. Depletion of CD4+ T-cells had no effect, suggesting that CD8+ but not CD4+ T-cells were required for this immune surveillance. To determine whether change in major histocompatibility complex (MHC) class I expression was a frequent event that caused low immunogenicity of tumors or facilitated escape from immune destruction, recently isolated murine tumors of varying degrees of immunogenicity, including highly immunogenic UV-induced regressor, less immunogenic UV-induced progressor, and poorly immunogenic spontaneous progressor tumors, were compared. There was no correlation between the ability of a tumor to grow progressively in a normal immunocompetent host and the level of constitutive class I expression or the level of expression induced in vitro by gamma interferon. (Only 1 of more than 20 progressor tumors analyzed showed complete loss of a MHC class I molecule.) Some progressor variants showed loss of a unique tumor-specific cytotoxic T-lymphocyte-defined antigen, consistent with earlier evidence of antigen loss providing a mechanism for tumor escape. However, most of the host-selected progressor variants retained both MHC class I antigens and the unique tumor antigens that we could detect with cytotoxic T-lymphocyte clones, suggesting that mechanisms other than loss of MHC class I or of the unique target antigen may be involved in escape of some tumors from a highly effective CD8-dependent host surveillance.

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin.

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.