Adoptive Transfer System Research Articles

Immunity to transplantable nitrosourea-induced neurogenic tumors. III. Systemic adoptive transfer of immunity.

The effect of intravenously injected tumor immune spleen cells on growth of 3 X 10(5) gliosarcoma T9 cells injected intradermally (ID) or intracerebrally (IC) into sublethally irradiated CDF rats was evaluated. Spleen cells from donor rats with sufficient immunity to reject 5 X 10(5) T9 cells inhibited the growth of T9 cells mixed with spleen cells in a ratio of 1:25 and injected ID, but could not act after intravenous transfer. However, donor rats which had rejected increasing T9 challenge doses up to 1 X 10(7) cells produced immune spleen cells which, upon IV transfer, could inhibit growth of ID T9 challenge but not of EB-679, an unrelated glioma, in recipient rats. Rejection of IC T9 challenge was also obtained after IV transfer, in recipients of such "hyperimmune" spleen cells, but was less (60% maximum) than that noted after ID T9 challenge (100% maximum). The removal of B cells from the transferred spleen cells did not affect the results, suggesting that the specific immunity was mediated by T cells. We conclude that the special immunological circumstances of tumors growing in the brain renders them less accessible to rejection by systemically transferred immune cells, but it is nevertheless possible to effect a significant incidence of rejection of syngeneic tumor growth in the brain by the intravenous transfer of hyperimmune spleen cells.

Suppressor T cell memory. Induction and recall of HGG-specific memory suppressor T cells and their role in regulation of antibody production.

Regulation of the antibody response to the hapten, dinitrophenyl (DNP), was studied using human gammaglobulin (HGG) as the carrier in an adoptive transfer system. CBA mice immunized at least 4 weeks previously to HGG or DNP served as the source of T helper cells and hapten-primed B cells, respectively. The addition of spleen cells from donors recently primed to HGG in immunogenic form (aHGG) suppressed the collaborative anti-DNP PFC response as effectively as cells from tolerant donors. The suppressive effect was antigen-specific and was mediated by a T cell with the same phenotype (Ly-1-, Ly-23+, Ia+) and induction kinetics as those previously identified in HGG-tolerant animals. During the primary response to HGG an early burst of helper activity was detected initially, followed by a wave of suppression which peaked at day 7 and subsequently waned, allowing adoptive helper function to reappear during the third week. When previously immunized animals were boosted with soluble HGG after primary suppression had waned, the sequential appearance of helper and suppressor activity was accelerated, and the secondary suppressive effect was more profound and of longer duration than that observed during a primary response. Although helper activity was not apparent during the peak of secondary suppression, helper T cells (Th) had not been functionally deleted since treatment of the donor spleen cells with anti-Ia and complement to deplete suppressor T cells (Ts) before adoptive transfer resulted in significant augmentation of the anti-DNP response. The secondary suppressive effect was formally shown to be mediated by activated Ts effector cells bearing the same surface markers as the cells responsible for primary suppression. The results suggest that the Ts population, like other lymphocyte subsets, contain memory cells capable of rapid reexpression of effector function upon secondary exposure to antigen. These cells are considered to be of a major importance in maintenance of immune homeostasis.

Opposite effects of total lymphoid irradiation on T cell-dependent and T cell-independent antibody responses.

The effect of total lymphoid irradiation (TLI) on the primary antibody response to the dinitrophenylated heterologous protein, keyhole limpet hemocyanin (DNP-KLH), in complete Freund's adjuvant (CFA), and to the trinitrophenylated polysaccharide antigen, Brucella abortus (TNP-BA), was studied in BALB/c mice. The antibody response to both antigens was diminished in comparison with nonirradiated mice when antigens were injected within 3 days after TLI. When the mice were immunized 30 days after completion of TLI the antibody response to DNP-KLH in CFA was still diminished, but the antibody response to TNP-BA was enhanced 5- to 10-fold as compared with that of control animals. The opposite effect of TLI on the two antibody responses was also observed in a syngeneic primary adoptive transfer system.

Idiotypes of anti-major histocompatibility complex antibodies.

Our studies to date indicate that treatment with anti-idiotype to monoclonal anti-MHC antibodies can markedly influence the repertoire of anti-MHC antibodies expressed. The antibodies discussed here appear to represent two classes, one of which is public, probably representing expression of a germ line gene, and the second of which probably represents either a somatic variant of a germ line gene or one of a very large number of germ line genes devoted to the same specificity. In either case, this class of idiotype arises only rarely following antigen, but is readily induced by anti-idiotype treatment. There may indeed exist a third class of anti-MHC monoclonal antibodies representing distant somatic diversification from a germ line gene. Our only indication of this so far is that certain idiotypes are only induced after multiple boosts with anti-idiotypes rather than a single treatment. This finding, however, may reflect again the enormous number of different ways in which anti-MHC antibodies to the same nominal specificity can be produced. Finally, our results in adoptive transfer systems indicate that manipulation of idiotype expression by anti-idiotype treatment probably involves a complex pathway of cellular interactions. If, as we expect, these intercellular interactions involve idiotype and/or anti-idiotypic receptors, they should provide a model for mechanistic studies of the in vivo immune network.

Prolongation of rat kidney allografts by pretransplant administration of donor antigen extract or whole blood transfusion combined with a short course of cyclosporine.

The immunosuppressive effect of the combination of a three day course of cyclosporine with one i.v. injection of 3M KCL-extracted donor antigen or donor blood transfusion was tested across the strong histocompatibility barrier causing rejection within 8 days of kidney transplants from Buffalo (Buf, RT1b) to Wistar-Furth (WFu, RT1u) inbred rats. Administration of 10 mg/kg/day cyclosporine alone for three days (-1, 0, and 1) slightly prolonged graft survival time from 7 to 11 days. The combination of cyclosporine with donor Buf 3M KCl antigen or with a Buf blood transfurion administered one day prior to transplantation caused greater prolongation of graft survival--19 and 25 days, respectively. Neither third-party BN soluble antigen nor BN blood transfustions acted synergistically with cyclosporine to prolong Buf graft survival. Increasing doses of donor-soluble antigen up to an optimal dose of 5 mg proportionately prolonged graft survival; however, administration of 10 mg antigen was less effective than 5 mg. On the other hand, administration of 1 ml of donor blood achieved the maximal effect. Lymphocytes harvested ten days after transplantation from recipients that had received combined therapy with cyclosporine and donor 3M KCl antigen not only displayed specific unresponsiveness to donor stimulator cells in mixed lymphocyte culture, but also specifically suppressed the proliferative response of syngeneic, virgin WFu responder cells to allogeneic donor Buf but not to third-party BN cells. Furthermore, suppressor cell activity was suggested by diminished responses in an in vivo local adoptive mixed lymphocyte culture assay and by prolongation of Buf kidney survival following systemic adoptive transfer. These findings suggest that immunosuppression with cyclosporine permits induction of specific suppressor cells by 3M KCl donor antigen, resulting in specific unresponsiveness to allografts.

Resistance of T suppressor cells to cyclophosphamide administration in total-lymphoid-irradiated mice.

The effect of administration of cyclophosphamide (Cy) on the susceptibility of mice to tolerance induction and its influence on the rate of induced T suppressor cells in antigen-specific and nonspecific systems was assessed using mice that had undergone total lymphoid irradiation (TLI). Tolerance to bovine serum albumin (BSA) was induced in adult mice conditioned by a short course of fractionated total lymphoid irradiation with an injection of nondeaggregated BSA; Cy was administered upon termination of TLI, two days prior to administration of the tolerizing antigen. Susceptibility to tolerance induction and rate of induced T suppressor cells were assessed one month later. Cy-treated TLI mice and mice treated with TLI alone did not respond to a challenge with DNP-BSA in complete Freund's adjuvant, whereas a good response was obtained to an unrelated protein antigen. The induced T suppressor cell rate was measured by inhibition of a secondary anti DNP-BSA response in an adoptive transfer system. The rate of non-antigen-specific T suppressor cells was measured by inhibition of the mixed leukocyte reaction with Concanavalin A-induced T suppressor cells obtained from spleen cells of TLI-treated, or Cy-treated mice, or those given both treatments. The results reveal that Cy treatment of TLI-conditioned mice does not reduce the rate of induced of T suppressor cells in antigen-specific and antigen-nonspecific systems.

Antigen-Specific T-Cell Lines in DNCB-Contact Sensitivity in Guinea Pigs

Expanded DNP-specific guinea pig T cells exhibit enhanced in vitro and in vivo activity as demonstrated by DNA synthesis and systemic adoptive transfer of contact sensitivity. Moreover, expanded lymphocytes are capable of accomplishing local passive transfer of contact sensitivity and producing interleukin 2, vascular permeability-increasing and macrophage migration-inhibiting factors. Expansion of hapten-specific lymphocytes offers a suitable model for studying eliciting mechanism of allergic contact dermatitis in humans.

Mechanisms of protection against virulentYersinia pestis infection without participation of humoral antibody: H-2 restriction in athymic mouse model

Athymic mice, deficient in mature T-lymphocytes, were killed when subcutaneously challenged with 100 organisms of a highly virulent strain ofYersinia pestis. None of the methods for activating the macrophages protected the animals from death; however, the survival time of the mice primed withYersinia pseudotuberculosis containing VW antigen 14 days before the challenge was significantly prolonged. Neither IgM nor IgG specific forY. pestis was detectable in these mice 14 days after priming. The primed athymic mice given the spleen cells of the twice-primed thymus-bearing syngeneic or semiallogeneic mice resisted the challenge with virulentY. pestis. On the basis of these results, the protective activity of the immune T-cells againstY. pestis infection assayed in an adoptive transfer system resides in cell-mediated immunity with H-2 restriction.

Modulation of B and T cell subsets in mice treated with fractionated total lymphoid irradiation. II. Tolerance susceptibility of B cell subsets.

Total lymphoid irradiation (TLI) results in long-lasting changes in the characteristics of both T and B cells suggestive of arrested maturation. A characteristic feature of immature B cells is their high susceptibility to tolerance induction. This study examines the susceptibility to tolerance to bovine serum albumin (BSA) of TLI-treated mice. Two experimental protocols were designed. In the first, tolerance to BSA was induced in TLI-treated adult (BALB/c X C57BL/6)F1 mice, and the ability of B cells of those mice to respond to BSA was assessed in an adoptive transfer system. In the second experimental protocol, tolerance was induced in adoptive hosts reconstituted with purified B cells originating from TLI-treated mice and with splenic T cells of normal, untreated mice. Results obtained in these two systems clearly demonstrated that splenic B cells of TLI-treated mice are highly susceptible to tolerance induction. This high susceptibility of B cells is linked neither with the elevation of immature T cells nor with induced T suppressor cells which arise due to the long-term malfunction of the thymus. Tolerance could be induced in TLI cells even 4 months after termination of the treatment. Thus, maturation processes of B cells in TLI-treated mice are arrested for long periods of time.

A new experimental approach to the specific immunotherapy for malignant brain tumor. Immunobiological characterization of T-cell growth factor

The in vivo efficacy of T-cell growth factor (TCGF)-dependent tumor-specific cytotoxic T lymphocyte (CTL) clone against a syngeneic murine malignant glioma (a 20-methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin, 203-glioma) was investigated. Prior to this goal, the immunobiological properties of TCGF were first characterized in culture supernatants of concanavalin A (Con A)-activated murine (C57BL/6) or rat (Wistar) lymphocytes, respectively. 5×106/ml lymphocytes were cultured with 2 μg/ml Con A in RPMI-1640 medium supplemented with 2% fetal calf serum for 48 hours. The culture supernatants (Con A sup) were concentrated by ammonium sulphate precipitation and partially purified by successive diethylaminoethyl (DEAE)-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. TCGF was assayed for a quantitative analysis using a TCGF-dependent murine glioma-specific CTL clone (G-CTLL). The major portion of TCGF activity in murine and rat Con A sup was recovered in the 50 to 80%, and 40 to 80% (NH4)2SO4 fraction, respectively. These activities were found in proteins of (3.0 ?? 3.6) × 104, (1.2 ?? 1.8) × 104 daltons in size as estimated by gel filtration, respectively and eluted from ion exchange chromatography in a NaCl gradient on 150, and 50 mM, respectively. It was also found that rat Con A sup contained TCGF activity approximately 8 times as much as murine Con A sup and that the CTL activity of sensitized T lymphocytes was fully enhanced in a short-term culture with TCGF. The substantial in vivo anti-tumor activity of G-CTLL cell line was observed from the intracranial Winn-type neutralization assay and the systemic adoptive transfer assay. Accordingly, this new experimental approach to the specific immunotherapy with TCGF and the cloned CTL line such as G-CTLL may be particularly useful for malignant gliomas.

Mechanism of host defense suppression induced by viral infection: mode of action of inosiplex as an antiviral agent.

The mechanism of influenza virus (INFV)-induced immunosuppression and the mode of inosiplex action against INFV infection were studied. INFV suppressed both anti-lipopolysaccharide and anti-sheep erythrocyte antibody production in mice. INFV infection caused viral mRNA synthesis and increased total RNA synthesis in lymphocytes, but total mRNA synthesis was decreased. The translational ability of INFV-infected lymphocytes was also suppressed. Thus, INFV seemed to cause suppression of both mRNA synthesis and the translational ability of lymphocytes, resulting in suppression of lymphocyte functions. Inosiplex potentiated antibody production against sheep erythrocytes but not against lipopolysaccharide in normal and INFV-infected mice. Adamantanamine did not produce such a potentiating effect. The lymphocytes obtained from INFV-immunized and inosiplex-treated mice conferred resistance against INFV infection. This resistance was partially inhibited by anti-Thy 1.2 antibody treatment of the lymphocytes. In an adoptive cell transfer system, inosiplex treatment of T-cell donors potentiated antibody production when a non-immunosuppressive carrier (human serum albumin) was used. When an immunosuppressive carrier (INFV) was used, inosiplex treatment of either B-cell donors or T-cell donors increased antibody production. Direct introduction of inosiplex into lymphocytes by a cell fusion technique stimulated anti-sheep erythrocyte antibody production more effectively than the addition of inosiplex to cultures. Inosiplex increased total RNA and total mRNA syntheses in phytohemagglutinin-treated lymphocytes. In INFV-infected lymphocytes, inosiplex decreased syntheses of total RNA, total mRNA, and viral mRNA and restored translational ability. From these results, we concluded that inosiplex penetrates into lymphocytes and suppresses viral RNA synthesis and that it supports lymphocyte functions by promoting RNA synthesis and translational ability, both of which are necessary for hosts.

Encephalitogenic activity of guinea pig myelin basic protein in the SJL mouse.

GPBP was shown to be encephalitogenic in SJL mice by direct challenge and in experiments in which an adoptive transfer system was employed. The three fragments obtained by treating GPBP with pepsin were assessed in the same manner. The encephalitogenic activity resided in the C terminal half of the molecule (residues 89-169). LNC also proliferated to the same fragment in vitro. Fragments 1-37, and, to a lesser extent, 44-48 stimulated sensitized LNC to proliferate but did not induce disease.

The cellular basis for resistance to induction of tolerance in BXSB SLE male mice.

The autoimmune BXSB male mouse early in life displays a resistance to tolerance induction that is common with the other autoimmune strains (MRL/1, NZB x W, and NZB) whereas its female counterpart, which in early life is immunologically normal, is easily tolerized. The cellular basis for the BXSB male's resistance to tolerance induction with DHGG was investigated by using adoptive male-female cell transfer systems. We found that irradiated female recipients of male T-depleted bone marrow cells, plus either male or female thymus cells, resisted tolerance induction, had hyperactive splenic Ig-secreting cells, and developed early glomerulonephritis. Recipients of male thymic cells plus T-depleted female bone marrow cells could be tolerized, did not have enhanced Ig secretion, and did not develop glomerulonephritis. Furthermore, donor male T-depleted bone marrow cells--but not male thymic, female thymic, or female bone marrow cells--resisted induction of tolerance before their transfer to produce lymphoid chimeras.

Cells mediating graft rejection in the mouse. IV. The Ly-5, 6, and 8 effector cell phenotype.

Skin graft rejection was previously shown to be mediated by sensitized lymphocytes bearing the Thy-1+, Ly-1+2-3- cell surface phenotype and these cells were further characterized using antisera to the Ly-5, 6, and 7 specificities. By using an adoptive transfer system in which ATXBM mice (CBA/H) received sensitized cells treated with antiserum and complement, it was shown that the cells mediating skin graft rejection were of the Ly-5+6-7+ phenotype. Other studies have demonstrated that cytotoxic T cells are Ly-5+6+7-, whereas helper T cells are Ly-5+6-7+, so that the T cells-mediating graft rejection is again clearly distinguished from the cytotoxic T. cell. At this time, however, it is not possible to separate the T cells mediating helper T cell functions, delayed-type hypersensitivity (DTH), and allograft rejections.

Studies of defective tolerance induction in NZB mice. Evidence for a marrow pre-T cell defect.

NZB mice manifest a defect in tolerance induction by deaggregated heterologous gamma globulins. We have used an adoptive transfer system to study the defect. Thymectomized, intact, or thymectomized recipients given thymic epithelial grafts were studied after lethal irradiation and reconstitution with NZB, DBA/2, or (NZB x DBA(F1 marrow depleted of mature T cells. NZB thymocytes were responsible for the tolerance defect of NZB mice. The information for the defect was present in the NZB marrow prethymocyte. That defect could only be expressed when there was further maturation in association with a thymus. However, the normal DBA/2 thymic epithelium served as well as the abnormal NZB thymic epithelium. These studies resolve existing conflicts as to whether the NZB marrow or thymus is responsible for the loss of tolerance in association with autoimmunity.

Specific elimination of the T lineage cells: Effect of in vitro treatment with anti-Thy 1 serum without complement on the adoptive cell transfer system

Thymocytes from C57BL/6(B6) mice treated with anti-Thy 1 antiserum without complement in vitro were transferred to lethally irradiated AKR mice. Five days following transfer, the proportion of Thy 1.2(+) cells recovered from the recipient spleen was significantly lower (7%) than that from the control mice which had received untreated cells (64%). The B6 spleen cells were treated in the same manner and transferred with SRBC (T-dependent antigen) or DNP-Ficoll (T-independent antigen) to irradiated syngeneic recipients. The recipients developed a response to SRBC which was significantly lower than that observed in control mice, but showed the same number of plaque-forming cell (PFC) against TNP-SRBC as the control group of mice which had received untreated B6 spleen cells. These results clearly show that in vitro pretreatment of lymphocytes with anti-Thy 1 serum without complement specifically resulted in elimination or inactivation of the T lineage cells in the host environment. The mechanisms of the elimination are discussed in this study.

Selective killing of leukaemia cells by antibody-toxin conjugates: implications for autologous bone marrow transplantation.

The recent development of tumour-reactive monoclonal antibodies has stimulated interest in their use in targeting toxic agents to tumour cells. One such approach involves the covalent coupling of tumour-reactive antibodies to the toxic peptide of the plant toxin, ricin. Such conjugates are highly effective in killing various tumour cells in vitro1–6. Here we have used the murine B-cell tumour, BCL1, in an adoptive transfer system capable of detecting 1–10 tumour cells, to demonstrate the effectiveness and specificity of an anti-immunoglobulin(Ig)–ricin A chain conjugate in deleting tumour cells from infiltrated murine bone marrow. We report that the transferred cells were able to repopulate the haematopoietic system of lethally irradiated mice and that in most mice the tumour did not recur. We discuss the implications of this approach for autologous bone marrow transplantation following supralethal tumour therapy.

Modification of immune response by cinnarizine.

Effects of cinnarizine on immune response in mice were investigated. Mice were orally administered with cinnarizine and were immunized with sheep red blood cells (SRBC) intravenously. Numbers of plaque forming cells (PFC) to SRBC in spleen of these mice were assayed and delayed-type hypersensitivity (DTH) response to SRBC was measured. 1) PFC response in immunization with 5 X 10(6) cells/mouse of SRBC was enhanced by administration of 25 mg/kg of cinnarizine, while the response in immunization with 5 X 10(8) cells/mouse was suppressed by 25 to 200 mg/kg of cinnarizine. 2) From study on timing of administration, suppression of PFC response by 6.25 to 200 mg/kg of cinnarizine was observed at 24 hr. after the immunization. 3) 12.5 to 200 mg/kg of cinnarizine suppressed polyclonal B cell activation induced by lipopolysaccharide (LPS). 4) Colchicine induced suppressor T cell inactivation was prevented by administration of 50 mg/kg of cinnarizine and it was suggested that cinnarizine may induce suppressor T cells from the study of adoptive cell transfer system. 5) 50 mg/kg of cinnarizine showed the suppression of DTH response in expression phase, but not in induction phase. It was concluded that immune responses in mice were modified by cinnarizine.

The in vivo cytotoxic T cell response to alloantigen requires a Lyt-1+ helper T lymphocyte.

Previous evidence has shown that Lyt-1+ helper T cells are required for an in vitro cytotoxic T cell response. We now present evidence which demonstrates an in vivo requirement for Lyt-1+ helper T cells in the activation of a killer T cell response. An adoptive transfer system was designed in which alloantigen-primed parental helper cells were transferred to an F1 recipient. After 1 day, this recipient of primed helper cells was lethally irradiated and normal responder spleen cells syngeneic to the primed helper cells were injected into the F1 recipient. In these experiments the semiallogeneic cells of the irradiated F1 mouse serve as the source of stimulator cells. When responder cells and irradiated helper cells were both present in the F1 mouse, good cytotoxic T cell responses were obtained. When either responders alone or helpers alone were examined, cytotoxic T cell responses were much lower. The presence in vivo of suppressor cells which inhibit cytotoxic T cell responses made it necessary to culture spleen cells from these irradiated recipients for short periods in vitro to reveal the cytotoxic T cells which had been generated in vivo. Cell collaboration was shown to occur before this in vitro culture period commenced; the generation of cytotoxicity required that helper cells and killer cell precursors interact in vivo. Treatment of the primed helper cells with anti-Thy-1 serum plus complement or anti-Lyt-1+ serum and complement removed helper cell activity. This indicates that the primed helper cell in in vivo collaboration is a Lyt-1+ T lymphocyte.

Establishment of unresponsiveness in primed B lymphocytes in vivo.

As an approach to examine the influence of the state of cellular activation on the ability to tolerize B cells, the induction of unresponsiveness in human gamma-globulin-(HGG) primed B lymphocytes was studied in an adoptive transfer system. In contrast to transferred normal spleen cells, spleen cells from HGG-primed mice are not readily rendered unresponsive when exposed to the tolerogen, deaggregated HGG (DHGG), in irradiated recipients. A kinetic study showed that unfractionated primed spleen cells do not respond to an antigenic challenge given between 6 and 10 days after cell transfer and injection of DHGG, indicating that they are transiently depressed. In contrast, isolated primed B cells are tolerized when transferred to recipients and treated with DHGG in the absence of T cells. Furthermore, primed B cells exposed to tolerogen in the recipients do not recover the ability to respond to HGG either after a secondary challenge with AHGG given up to 14 days after transfer, or after 2 consecutive challenges given on days 14 and 24 after transfer. The presence of primed T cells at the time of tolerization interferes with the induction of unresponsiveness in these primed B cells. These studies suggest that the presence of primed T cells is responsible for the inability to tolerize unfractionated primed spleen cells populations and that primed B cells themselves are not intrinsically resistant to the induction of unresponsiveness.