Effect of macrophages and antibodies on in vivo growth of Moloney sarcoma in the rat.

Brown Norway and Lewis rats were challenged with a Brown Norway Moloney sarcoma tumor, MST-1, admixed with nonimmune peritoneal exudate macrophages syngeneic to the host; or admixed with nonimmune peritoneal exudate macrophages and hyperimmune anti-MST-1 antibodies. In vivo growth of MST-1 in BN and Lewis rats was inhibited by admixing Brown Norway or Lewis macrophages, respectively, with BN anti-MST-1 antibodies. The inhibiting BN antibodies were of the IgG2 class, lacking IgG2a antibodies. Brown Norway anti-MST-1 of IgG2 class without macrophages did not affect growth of MST-1. Brown Norway and Lewis anti-MST-1 antibodies of IgG2a class enhanced tumor growth, whether admixed with macrophages or not. Anti-MST-1 antibodies of IgM and IgG1 classes did not influence tumor growth. Peritoneal exudate macrophages removed from Lewis donors 8 to 10 days after inoculation of MST-1 inhibited completely growth of the challenge tumor; macrophages of Brown Norway origin were inhibitory only when harvested from hyperimmune donors, that is, 40 or more days after inoculation of MST-1. Macrophages from hyperimmune donors were specifically cytotoxic to MST-1 and did not inhibit an unrelated syngeneic BN tumor of chemical origin.     

Tumor-associated antigens of rat moloney sarcoma cells. II. Cytosol antigens.

Rat Moloney sarcoma cells (MST) were pulsed with 35S-L-methionine for 10 and 60 min and lysed by vortexing in 0.5% deoxycholate, 0.5% NP40, 0.02 M Tris, 0.05 M NaCl, pH 7.5, for 30 sec. The lysate was centrifuged at 16,300 X G for 10 min and the supernatant was co-precipitated with Ig fractions of normal BN serum, normal Lewis serum, BN antiserum to Moloney sarcoma cells (BNaMST), BN antiserum to tumor-associated antigens (BNaTAA), BN antiserum to murine leukemia virus (BNaMuLV), BN antiserum to p30 (BNap30), BN antiserum to gp70 (BNagp70), Lewis antiserum to BN (LeaBN), and BN antiserum to BC5 tumor (BNaBC5). With BNaTAA and BNaMST, a cytoplasmic TAA with m.w. 85,000 was detected. In addition, BNaTAA detected three other species of cytoplasmic TAA with m.w. 220,000, 170,000 and 39,000.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Lack of mast cell reactivity during leukemic infiltration of the rat mesentery under syngeneic and allogeneic conditions. A study by transmission electron microscopy (TEM)

The ultrastructural morphology of mast cells localized in rat mesenteries was studied after intraperitoneal implantation of L5222 rat leukemia cells in syngeneic and allogenic hosts. It became evident that the mast cells in the syngeneic (BDIX rat) as well as the allogeneic system (BN rat) showed nor morphological alterations. Degranulation was never observed. This is in contrast to the behavior of macrophages which displayed a strong phagocytotic activity in allogeneic hosts. Thus, it seems that mast cells, under the present experimental conditions, remained inactive during a phase of intense tumor rejection.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

W3/25+ T cells mediate the induction of immunologic unresponsiveness in enhanced rat recipients of cardiac allografts

Cellular suppressor mechanisms developing during the induction of immunologic enhancement were studied in LEW rats immunized actively with BN spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving a (LEW X BN)F1 cardiac allograft, respectively. With this regimen, graft survival is prolonged from 7.4 +/- 0.5 days in unmodified, acutely rejecting hosts to 25 +/- 12 days in enhanced recipients, with one-third of the grafts surviving indefinitely. To test for suppressor capacities, 60 X 10(6) splenic T helper/inducer (W3/25+) and T suppressor/cytotoxic (OX8+) cells were adoptively transferred 7 and 14 days after transplantation either into unmodified syngeneic LEW animals that received (LEW X BN)F1 test grafts 24 hr later or into T cell-deprived B rats with indefinitely functioning heart transplants that were reconstituted with sensitized lymph node cells (100 X 10(6). W3/25+ T cells harvested on days 7 and 14 from enhanced recipients prolonged test graft survival in unmodified hosts (13.1 +/- 2.3 and 13.3 +/- 1.3 days, respectively, p less than 0.001) and delayed rejection in reconstituted B rats from 6.7 +/- 0.5 to 18.2 +/- 6.5 and 23.3 +/- 5.8 days, respectively (p less than 0.001). OX8+ and B lymphocytes had no suppressor activity. However, enzymatic stripping of the surfaces of W3/25+ cells abrogated suppressor function. Similarly, after i.p. treatment with cyclophosphamide, W3/25+ T cells lost their suppressor properties. Lack of donor specific but not third party alloaggressiveness was demonstrated by the profoundly diminished ability of W3/25+ lymphocytes from enhanced hosts to recreate rejection in nonreconstituted B rats, even when exogenous interleukin 2 was administered. After pronase treatment, however, W3/25+ T cells were capable of inducing immunoresponsiveness at a tempo similar to naive T helper cells (31.5 +/- 12.5 vs 32.8 +/- 7.9). Thus, the present studies provide evidence for the development of a specific W3/25+ suppressor cell in the induction of active and passive enhancement. Coincident abrogation of specific T effector alloaggressiveness is apparently mediated by surface-blocking factors.     

Tumor progression in murine leukemia virus-induced T-cell lymphomas: monitoring clonal selections with viral and cellular probes.

Clonal selections occurring during the progression of Moloney murine leukemia virus (MuLV)-induced T-cell lymphomas in mice were examined in primary and transplanted tumors by monitoring various molecular markers: proviral integration patterns, MuLV insertions near c-myc and pim-1, and rearrangements of the immunoglobulin heavy chain and beta-chain T-cell receptor genes. The results were as follows. Moloney MuLV frequently induced oligoclonal tumors with proviral insertions near c-myc or pim-1 in the independent clones. Moloney MuLV acted as a highly efficient insertional mutagen, able to activate different (putative) oncogenes in one cell lineage. Clonal selections during tumor progression were frequently marked by the acquisition of new proviral integrations. Independent tumor cell clones exhibited a homing preference upon transplantation in syngeneic hosts and were differently affected by the route of transplantation.     

Ultraviolet light induces tumors with both unique and host-associated antigenic specificities

Murine tumors induced by ultraviolet light (UV) are immunogenic in syngeneic and semi-syngeneic hosts, evoking antibody of several different specificities. Cytotoxic antibody specific for the immunizing syngeneic tumor (tumor-specific antigen) comprises the early response and a minor portion of the later response of C3H and C3H.SW mice. It is the primary specificity to which C57BL/6 and C57BL/10 hosts respond. The major portion of the antibody produced by C3H and C3H.SW against syngeneic tumors cross-reacts strongly with other tumors, both UV and chemically induced, arising in C3H and C3H.SW but not in B6.H2k, C57BL/6, C57BL/10, or (C3H X B6)F1. Normal adult cells or embryonic fibroblasts do not cross-react with the antisera. These results are interpreted as evidence for the involvement of a host component (non-MHC) in this tumor-associated antigen (TAA). (C3H X B6)F1 and (C3H X BALB/c)F1 hosts respond to C3H tumors with antibody with cross-reactive specificities identical to those of the C3H and C3H.SW hosts. thus detecting the TAA(C3H) specificity. (C3H X B6)F1 hosts respond to syngeneic F1 tumors, however, with a totally cross-reactive antibody that is interpreted as evidence for the existence of a common antigen in addition to the evident immune response control. An undetected TAA (B6) specificity in the (C3H X B6)F1 tumors is speculatively proposed.     

Therapeutic efficacy of human recombinant interleukin-2 (TGP-3) alone or in combination with cyclophosphamide and immunocompetent cells in allogeneic,semi-syngeneic,and syngeneic murine tumors

Summary The potential for a recombinant human interleukin-2 (rIL-2, TGP-3) alone, in combination with cyclophosphamide, and in combination with cyclophosphamide and normal immunocompetent cells to manifest biological activity in vivo was tested using allogeneic, semi-syngeneic, and syngeneic tumor-host systems in mice. The biological activity of rIL-2 was evaluated by the inhibition of the growth of tumors and the inhibition of metastases in short-term assays and, in long-term assays, the prolongation of the survival time of mice bearing subcutaneously (s.c.) or intradermally transplanted tumors. rIL-2 was injected s. c. daily continuously for up to 40 days or intermittently two to four times into mice bearing established tumors. In the short-term assays, the dose and schedule dependence of activity of rIL-2 alone was significantly manifested against sarcoma 180 in ICR mice (allogeneic) by the regression of the tumor, and was confirmed against Meth-A fibrosarcoma in BALB/c mice (syngeneic) by retarding the growth of the tumor. When assessed using these tumors, it was found that the antitumor activity of rIL-2 was scheduledependent: the growth of tumors was more significantly suppressed when rIL-2 was injected every day for 10 days, starting on the 7th day after tumor transplantation, than when rIL-2 was injected five times every other day or twice every 5th day, even if the total amounts of rIL-2 injected were same. The continuous injection for 10 days was considered to be a standard regimen and the daily effective doses of rIL-2 were 5, 10, and 25 µg/mouse. Using the standard regimen and the effective doses, the activity of rIL-2 alone was also observed against two other syngeneic tumors: Colon carcinoma 26 in BALB/c mice, by retarding the growth of the tumor, and Lewis lung carcinoma in C57BL/6 mice by reducing the formation of lung metastases. When assessed using M5076 reticulum cell sarcoma, in a long-term assay, the activity of rIL-2 alone was not manifested in C57BL/6 mice (syngeneic) even when rIL-2 was injected for a long period (20 days) but it was observed in BDF1 (semi-syngeneic) mice. On the other hand, it was found that rIL-2 was effective in combination with cyclophosphamide in prolonging the survival time of C57BL/6 mice bearing the tumor. After cyclophosphamide (2.0 mg) had been administered orally to mice on the 6th day after tumor transplantation, the tumor regressed temporarily but regrew; however, when rIL-2 at a dose of 10 µg was also injected daily for a long period (40 days), the regrowth was retarded and the survival time of the mice was significantly prolonged. Moreover, when normal immunocompetent cells were transferred at the tumor sites, the regrowth of the tumors was retarded more significantly even at a daily dose of 1 µg or 3 µg rIL-2, and mice were observed to be cured by daily doses over 3 µg. The results obtained in the syngeneic tumor-host systems indicate that the continuous injection of rIL-2 is necessary and important for its activity to be manifest in vivo, and that, when combined with cytotoxic drugs and/or with immunocompetent cells, the potential efficacy of rIL-2 is valuable in cancer therapy.     

Neonatal induction of tolerance to skeletal tissue allografts without immunosuppression

Vascularized allogeneic skeletal tissue transplantation without the need for host immunosuppression would increase reconstructive options for treating congenital and acquired defects. Because the immune system of a fetus or neonate is immature, it may be possible to induce tolerance to allogeneic skeletal tissues by alloantigen injection during this permissive period. Within 12 hours after birth, 17 neonatal Lewis rats were injected through the superficial temporal vein with 3.5 to 5 million Brown Norway bone marrow cells in 0.1 ml normal saline. Ten weeks after the injection, peripheral blood from the Lewis rats was analyzed for the presence of Brown Norway cells to determine hemopoietic chimerism. The Lewis rats then received a heterotopic, vascularized limb tissue transplant (consisting of the knee, the distal femur, the proximal tibia, and the surrounding muscle on a femoral vascular pedicle) from Brown Norway rat donors to determine their tolerance to the allogeneic tissue. A positive control group (n = 6) consisted of syngeneic transplants from Lewis rats into naive Lewis rats to demonstrate survival of transplants. A negative control group (n = 6) consisted of Brown Norway transplants into naive Lewis rats not receiving bone marrow or other immunosuppressive treatment. The animals were assessed for transplant viability 30 days after transplantation using histologic and bone fluorochrome analysis. All the syngeneic controls (Lewis to Lewis) remained viable throughout the experiment, whereas all the Brown Norway to Lewis controls had rejected. Ten of the 17 allografts transplanted into bone marrow recipients were viable at 30 days, with profuse bleeding from the ends of the bone graft and the surrounding graft muscle. The percent of chimerism correlated with survival, with 3.31 percent (SD = 1.9) of peripheral blood, Brown Norway chimerism present in the prolonged survival groups and 0.75 percent (SD = 0.5) of Brown Norway chimerism in the rejected graft group. This study demonstrated prolonged survival of allogeneic skeletal tissue without immunosuppression after early neonatal injection of allogeneic bone marrow in a rat model.     

3H-deoxythymidine incorporation in graft-versus-host disease in the Norway rat. I. Liquid scintillation studies.

Graft-versus-host-disease was produced in newborn Brown Norway (BN) rats with an intravenous (iv) injection of adult allogeneic Lewis (L) lymph node cells (experimental) and the response was compared to littermates injected with adult syngeneic BN cells (control). By 4 days the reaction in the spleen of experimental animals was such that the spleen index was 1.70 and 2.58 on day 7, and continued to increase until death. A one hour iv pulse of tritiated deoxythymidine (3HdT) administered to experimental and control animals revealed a whole organ peak incorporation of 3HdT on day 6 in experimental spleens. A second larger peak occurred on day 10 in the experimental spleen as compared to a single peak at days 6 or 7, respectively, in the experimental mesenteric and combined superficial lymph nodes. However, analysis of the incorporation of 3HdT with respect to organ weight revealed a peak incorporation in animals receiving L cells on day 4--6 with a second smaller peak on day 10 in the experimental spleen and again a single peak on day 5 or 6 in the lymph nodes. Total 3HdT incorporation within both experimental lymph node compartments became less than controls by day 15 even though experimental nodes had a larger mass. 3HdT incorporation per milligram tissue weight decreased in all tissue compartments of experimental animals by day 13--14. The contribution of donor and host cell proliferation to the various peaks observed is discussed.     

Studies of H-2 restriction in cell-mediated cytotoxicity and transplantation immunity to Leukemia-associated antigens.

H-2 dependency of T cell-mediated cytotoxicity and transplantation immunity to leukemia-associated antigens has been investigated. Through the use of a 20-hr 125IUdR release assay, it was found that the induction of T cell-mediated cytotoxicity against Friend virus-induced leukemias of different H-2 haplotype orgins could be produced by immunization with both syngeneic and allogeneic tumor cells; the effector cells that were generated by syngeneic immunization could also provide effective killing of allogeneic tumor cells, although the killing of allogeneic targets might require a longer incubation time (20 to 40 hr). Furthermore, in vivo transplantation immunity against Friend virus-induced leukemias also was induced by immunization with both syngeneic and allogeneic tumors and syngeneic immunization could induce specific protection against the challenge with a-logeneic tumor in x-irradiated hosts. These findings clearly indicate that, both at the sensitizing phase and effector phase of the immune response, there is no strict H-2 dependency for T cell-mediated cytotoxicity or in in vivo transplantation imunity to leukemia-associated antigens.     

Graft versus neuroblastoma reaction is efficiently elicited by allogeneic bone marrow transplantation through cytolytic activity in the absence of GVHD

Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.     

Effect of tumor cell culture media and sera from tumor hosts on spreading,phagocytosis, and antitumor cytotoxicity of C. parvum-activated murine macrophages

Summary We investigated whether the media of tumor cell cultures and sera from tumor-bearing hosts exert inhibitory effects upon macrophage spreading, phagocytosis, and cytotoxicity. Peritoneal macrophages from normal and Corynebacterium parvum-treated C3Hf/Bu mice were incubated in media from a syngeneic mammary carcinoma, allogeneic Ehrlich ascites carcinoma, and human malignant melanoma or cervical carcinoma cell cultures, or in the serum of hosts bearing these tumors. Such media and sera inhibited the ability of both normal and C. parvum-activated macrophages to spread on glass surfaces and to ingest latex particles. In contrast, they did not interfere with the in vitro destruction of tumorigenic L929 cells by C. parvum-activated macrophages. Media of murine embryo fibroblasts and a human benign tumor and sera from healthy mice or humans did not, however, inhibit either of the macrophage functions tested.     

Autoaggressive T lymphocyte lines recognizing the encephalitogenic region of myelin basic protein: in vitro selection from unprimed rat T lymphocyte populations

Autoimmune T lymphocyte lines have been established from unprimed normal rat lymph node cell populations. In a first, negative-selection round, spontaneously proliferating (SMLR) T cells were eliminated by a pulse of BUdR followed by short wave light irradiation. In a second, positive-selection round, the SMLR-depleted populations were confronted with MBP presented by syngeneic spleen adherent cells. Reactive T cells were propagated until stable, permanent T lines were established. All lines were exclusively specific for the selecting antigen, MBP, and were restricted in recognition by determinants of the own MHC. All lines expressed the differentiation marker W3/25, but not OX8. Line vLe, which was derived from Lewis (LEW) rat lymphocytes, and which recognized the encephalitogenic sequence 48-88 of MBP, was extremely efficient in mediating EAE to normal untreated LEW rats. Doses of 1 X 10(6) and greater transferred lethal EAE, whereas transient although definite disease was caused by a minimum of 1 X 10(4) cells. Rats recovering from disease were resistant against subsequent active induction of EAE. In contrast, BN rat-derived line vBN was completely incapable of transferring EAE to syngeneic rats. This lack of encephalitogenicity was a property of the T line, because vLe cells transferred severe EAE to (LEW X BN)F1 hybrid rats, whereas none of hybrid rats injected with vBN cells showed any sign of disease. The data provide strong evidence in favor of the presence of potentially autoaggressive T clones in the normal immune system, and they might suggest that the actual proportion of these clones within the natural T cell repertoire is genetically determined.     

Prevention of transplantable tumors by adoptive transfer of spleen cells from immunized rats.

Fischer 344 rats were specifically hyperimmunized with allogeneic, nonvirus-producing [Kirsten murine sarcoma virus (KiMSV)] or syngeneic, virus-producing [KiMSV (Rasheed)] rat tumors. Spleen cells taken from these rats adoptively transferred protection against a 100 to 1,000 X rat tumor dose50 cell challenge with several different transplantable rat tumors. Protection was obtained with spleen cells after removal of adherent cells and macrophages but not peritoneal cells. The spleen cells were not directly cytotoxic but required more than 3 days residence in the recipient before protecting the recipient against challenge. No protection against tumor cell challenge was observed when spleen cells were lethally x-ray irradiated before injection into nontreated rats. Spleen cells taken from rats immunized with normal histocompatibility antigens did not protect in this test system.     

Selective localization of radiolabeled immune lymphocytes into syngeneic tumors.

Newly formed long-lived small lymphocytes (LLSL) generated during the immunization of mice to tumor specific transplantation antigens (TSTA) of syngeneic MCA-induced sarcomas were labeled. We then studied the localization of these labeled cells into tumor implants. "Crisis-cross" experiments were performed in which MCA sarcomas with individually distinct TSTA were studied in parallel. A selective localization of LLSL into tumors to which the lymphocytes were immune was found when small tumor pieces were implanted into immune mice whose LLSL had been labeled. Selective localization was also detected upon adoptive transfer of immune LLSL to tumor-bearing mice, but only when these mice had, before transfer, received a sublethal dose of whole body irradiation.     

Augmentation of in vivo cytotoxic T lymphocyte activity and reduction of tumor growth by large multivalent immunogen.

Class I alloantigen incorporated into cell-size supported membranes provides an effective stimulus for in vitro stimulation of CTL responses. When alloantigen-bearing cell-size (5 microns) microspheres, termed large multivalent immunogen (LMI), were administered in vivo, no primary cytotoxic response to the Ag could be detected. However, coadministration of LMI and allogeneic tumor stimulator cells resulted in substantial augmentation of the resulting CTL response, compared with that obtained from mice that received just stimulator cells. Responses were augmented only when the same alloantigen was present on the LMI and on the stimulator cells, and the effector cells remained specific for the cognate alloantigen-bearing targets. The physical form of the alloantigen was critical for augmentation; alloantigen in liposomes had no effect on response levels. Tumor cell Ag in the form of purified plasma membrane vesicles can also be incorporated onto the surface of cell-size microspheres. As with allogeneic responses, tumor Ag on LMI specifically augmented the in vivo CTL activity generated in response to irradiated tumor cells in syngeneic mice. Administration of Ag-bearing LMI to mice inoculated i.p. with live P815, EL4, or RDM4 tumor cells resulted in a significant reduction in growth of the tumors in their syngeneic hosts. Similarly, LMI treatment significantly reduced growth of P815 as a solid s.c. tumor. LMI-mediated growth reduction occurred only when plasma membrane Ag from the cognate tumor was used to prepare the LMI, and Ag in the form of free plasma membrane vesicles was not effective. Although Ag has been used to manipulate in vivo humoral and Th responses, this has proven to be much more difficult for CTL responses. The ability of Ag-bearing LMI to affect significantly the in vivo levels of cytolytic response and to reduce syngeneic tumor growth has potential for application to tumor immunotherapy and, possibly, treatment of other diseases in which CTL can provide a protective effect.