Selective elimination of alloreactivity in vitro and in vivo while sparing other T-cell-mediated immune responses.

Selective elimination of alloreactive cells was carried out in the set-up of T-cell-mediated immunotherapy in an effort to gain the benefits of hematopoietic allogeneic transplantation while reducing the risk of GVHD. Low MW chemical compounds were screened for their effect on T-cell-mediated immune responses of murine- and human-derived lymphocytes. Selected compounds were further tested in secondary MLR assays in which sensitization to alloantigens was carried out in vitro, in the presence or absence of a given compound, followed by exposure to related and unrelated alloantigens or T-cell mitogenic stimulation. At a low concentration of <1 µM, a quinazoline derivative named AO#349 [2-(3,4,5-trimethoxyphenyl)-N-p-tolylquinazolin-4-amine], was able to induce 78-90% inhibition of a selective allogeneic response while retaining >92% immune reactivity to unrelated alloantigens and mitogenic stimuli in vitro. Following allogeneic sensitization in the presence of AO#349, elimination of alloreactivity to the priming alloantigens was also proved in a murine model of GVHD: 10 out of 15 sub-lethally irradiated mice inoculated with these sensitized cells were GVHD-free for >200 days. AO#349 was efficient in induction of a selective elimination of alloreactivity and should be considered for clinical application in allogeneic cell-mediated immunotherapy.

Immune reconstitution following allogeneic stem cell transplantation in recipients conditioned by low intensity vs myeloablative regimen.

We have investigated the immune status of patients with hematologic malignancies treated with a low intensity conditioning in preparation for allogeneic stem cell transplantation. Conditioning consisted of fludarabine, anti-T lymphocyte globulin and low-dose busulfan, followed by infusion of allogeneic blood stem cells. This protocol resulted in rapid engraftment and complete replacement of host with donor hematopoietic cells. Immunological parameters of these patients were compared to those patients who were conditioned by an aggressive myeloablative regimen. Distribution of cell surface markers of lymphocyte subsets from both groups of patients was similar, but different from that of normal control cells. Reduced intensity or non-myeloablative conditioning prior to allogeneic stem cell transplantation (NST), hardly lowered the normal T cell-dependent mitogenic response even during the early period following transplant, while the myeloablative treatments resulted in a suppressed mitogenic reaction and in slow immune recovery. Reactivity of non-MHC restricted cytotoxic T cells was also at a normal level in patients who were treated with NST. We conclude that stem cell engraftment following reduced conditioning may result in early reconstitution of immune responses assessed in vitro. We hypothesize that clinical application of NST may lead to faster development of effective immune responses against residual host-type malignant and abnormal non-malignant hematopoietic cells, although the role of fludarabine on post-transplant infections remains to be investigated in a larger cohort of patients.

Cell therapy with preimmunized effector cells mismatched for minor histocompatible antigens in the treatment of a murine mammary carcinoma.

Cell therapy with allogeneic donor cells mismatched for minor histocompatible (MiHC) antigens was applied to a murine mammary carcinoma (4T1) model to test the feasibility of graft versus tumor (GVT) effect against metastatic epithelial tumor cells. BALB/c mice bearing a 4T1 tumor of BALB/c origin were given syngeneic or MiHC-mismatched splenocytes. GVT effects were determined in secondary recipients of adoptively transferred lung cells derived from primary hosts who had previously been inoculated intravenously with 4T1 cells, and injected with one of the following: 1) naive BALB/c splenocytes, 2) naive DBA/2 splenocytes, 3) 4T1-immune DBA/2 splenocytes, or 4) DBA/2 splenocytes immunized with host-derived BABL/c spleen cells. Naive DBA/2 splenocytes inhibited tumor growth only slightly and only slightly prolonged the survival of secondary recipients, in comparison with fully matched tumor/host BALB/c spleen cells. An efficient GVT reaction was demonstrated in vitro and in vivo with MiHC-mismatched DBA/2 splenocytes from mice presensitized by multiple injections of irradiated tumor or BALB/c-derived spleen cells. All 30 mice adoptively inoculated with lung cells from primary hosts that had previously been treated with these presensitized effector cells were tumor free for >250 days. Secondary recipients inoculated with lung cells from mice given naive BALB/c or DBA/2 spleen cells died of metastatic tumors within 33 to 46 days. These results suggest that preimmunized donor cells represent an effective tool against metastatic disease; hence, the next goal should be to control graft-versus-host disease while exploiting the GVT potential.

Immunotherapy of relapsed resistant chronic myelogenous leukemia post allogeneic bone marrow transplantation with alloantigen pulsed donor lymphocytes.

Allogeneic cell-mediated immunotherapy with donor lymphocyte infusion (DLI) can successfully reverse chemoradiotherapy-resistant relapse in patients with chronic myeloid leukemia treated by allogeneic bone marrow transplantation (BMT). We describe the first successful attempt in 1992 to treat DLI-resistant relapse in a patient with CML in full hematologic relapse, using immunized donor lymphocytes. Donor lymphocytes were pulsed in vitro with a mixture of irradiated peripheral blood lymphocytes (PBL) obtained from both parents, in order to trigger alloactivation of donor lymphocytes against host alloantigens presented by parental cells, using as stimulating cells maternal PBL expressing the shared maternal haplotype and paternal PBL expressing the shared paternal haplotype of the patient. Full hematologic, cytogenetic and molecular remission was induced for the first time, independently of GVH, and has persisted for more than 9 years. To the best of our knowledge, this report represents the first successful immunotherapy with donor lymphocytes activated against host-type antigens. We suggest that immune donor PBL may be superior to DLI, possibly effective even when all other modalities fail, perhaps even independently of GVHD.

Induction of antitumor immunity by indomethacin.

Irradiated tumor cells given, together with indomethacin, to syngeneic mice induced an antitumor response and conferred protection against a challenge of a lethal dose of murine mammary (4T1) and lung (3LL) carcinoma cells. Continuous administration of indomethacin was crucial throughout the entire period of immunization and challenge, as no protection was achieved when the drug was given during only one of these procedures. Antitumor immunity was long-lasting and, when tested in the 4T1 model, 48% of mice were resistant to a second challenge of lethal tumor cells. Tumor-free immune mice that were given indomethacin for more than 300 days remained healthy with normal white blood cell counts and normal spleen size. Cells isolated from immune mice were able to kill tumor cells in culture after in vitro activation by interleukin-2, in a manner similar to cells from naive normal control mice. In addition, the mitogenic response of their T cells was as high as that of the control naive mice. While indomethacin was able to induce antitumor immunity to 4T1 and 3LL murine carcinoma cells, both of which contain a high concentration of endogenic prostaglandin E(2) (PGE2), no such immunity was achieved to murine tumor cells with a low concentration of endogenic PGE2. These results suggest a correlation between PGE2 concentration and the ability of indomethacin to induce antitumor immunity. We therefore suggest that an immunotherapy protocol with long-term dispensation of a tolerable dose of an immunomodulator, given together with irradiated autologous tumor cells, may stimulate antitumor responses to tumors containing high concentrations of endogenic PGE2.

The use of soybean agglutinin (SBA) for bone marrow (BM) purging and hematopoietic progenitor cell enrichment in clinical bone-marrow transplantation.

Soybean agglutin (SBA) is a plant lectin that has been used to fractionate bone marrow cells. It binds bone marrow mononuclear cells, including mature myeloid, erythroid, and lymphoid cells, but has very low binding affinity and no toxic effect to the human hematopoietic cells. In this article we describe the possibilities of enriching bone-marrow-derived CD34+ hematopoietic progenitor cells using SBA. As the method is simple and elegant. SBA is of vast importance to the field of clinical bone marrow transplantation.

Allogeneic cell therapy for a murine mammary carcinoma.

The effect of allogeneic cell therapy on tumor growth was studied in a murine model of mammary carcinoma (4T1) as an experimental model of solid tumors in humans. i.v. inoculation of 4T1 (H-2d) cells into syngeneic mice [BALB/c or (BALB/cXC57BL/6)F1] (F1) carrying the H-2d histocompatible antigens results in tumor colonies in the lungs that finally cause the death of all of the mice. Sublethally irradiated F1 mice were inoculated with 4T1 cells to simulate minimal residual disease and with immunocompetent splenocytes derived from naive donors of F1 (syngeneic), BALB/c (syngeneic to the tumor but semiallogeneic to the host), or C57BL/6 (allogeneic to the tumor and semiallogeneic to the host) mice. The survival of F1 tumor-bearing mice that were treated with allogeneic C57BL/6 splenocytes was significantly prolonged (P < 0.02) compared with hosts given F1 or BALB/c-derived splenocytes that are syngeneic to 4T1 tumor cells. Adoptive transfer of lung cells that were isolated from F1 primary mice inoculated with 4T1 cells and syngeneic BALB/c or F1 splenocytes led to local tumor growth and death in secondary recipients. In contrast, only 1 of 22 secondary recipients developed tumors when inoculated with lung cells derived from F1 mice given allogeneic C57BL/6 splenocytes. All of the 21 secondary hosts survived disease-free for a follow-up time of >200 days. These results indicate that immunocompetent cells allogeneic to the mammary carcinoma cells were able to inhibit tumor development in the primary hosts and to prevent tumor growth in the adoptive recipients, which suggests that allogeneic cell therapy may be an efficient antitumor tool to eradicate minimal residual disease in human solid tumors.

Tumorigenicity and immunogenicity in a murine model of B-cell leukemia/lymphoma (BCL1).

Multiple injections of intact irradiated BCL1 cells, a murine B-cell leukemia/lymphoma can trigger a dose-dependent anti-tumor immune response in naive syngeneic mice. The ability to induce anti-BCL1 immunity and the effect of various cell-modifications on BCL1 tumorigenicity and immunogenicity was evaluated. Newcastle disease virus (NDV) infection or transfer of cytokine genes by both retroviral and Adeno 5 vectors affect neither tumorigenicity nor immunogenicity of BCL1 cells given as a non-immunogenic cell-dose. New ways will have to be developed to elicit a reliable and reproducible anti-tumor effect in spontaneously arising and non-immunogenic hematological malignancies.

Cytokine gene transduction into non-immunogeneic murine tumor cells.

The effect of cytokine transduction on the tumorigenicity and immunogenicity of murine non-immunogeneic mammary carcinoma (4T1), acute myeloid leukemia (mAML) and partially immunogenic B-cell leukemia (BCL1) has been evaluated in syngeneic strains of mice. Transduction by retroviral vectors containing the genes for GM-CSF, IL-2 or IFN-gamma did not lead to a marked antitumor effect in 4T1 mammary tumor or BCL1. A reduced local tumor size was observed in mice inoculated with 4T1 cells transduced with both GM-CSF and IL-2 genes followed by an in vitro exposure to recombinant IFN-gamma, but survival was not prolonged. Tumorigenicity of mAML cells transduced with the gene coding for IFN-gamma was significantly reduced as manifested by prolonged survival of mice in comparison with animals inoculated with non-transduced mAML cells. Transduction by each of the aforementioned cytokines did not affect the immunogenicity of these tumor model cells. The results suggest that genetic modification of spontaneous and non-immunogenic experimental tumor models does not necessarily support direct utilization of cytokine gene therapy for clinical application. More effective methods have yet to be established in order to achieve an antitumor effect in spontaneous non-immunogenic malignancies.

Effect of indomethacin on tumorigenicity and immunity induction in a murine model of mammary carcinoma.

Indomethacin, an inhibitor of cyclo-oxygenase given orally, reduced the tumorigenicity of cancer cells in a non-immunogenic murine model of mammary adenocarcinoma (4T1). In the presence of indomethacin, a dose-dependent immune protection could be induced most effectively by immunizing mice with 1 to 3 doses of irradiated tumor cells inoculated at intervals of 7 days prior to challenge with a tumorigenic cell dose. Three immunizations given without indomethacin resulted in tumor growth in 88% of the recipients, and indomethacin treatment started 28 days prior to the challenge dose and given without immunizations led to tumor onset in 83% of mice. In contrast, tumor was documented only in 12% of mice vaccinated with 3 immunization doses and given concomitantly indomethacin. Moreover, 53% of disease-free survivors resisted a second challenge with a high tumorigenic dose. Induction of an anti-tumor immunity in indomethacin-treated mice was further studied as a therapy for tumor-bearing mice. Complete cure was induced in 50% of mice, and a significant reduction in tumor size as well as prolonged survival time were observed in the remaining animals. Immunostimulation by tumor cell vaccination given in the presence of a tolerable dose of indomethacin, therefore, may be incorporated into immunotherapy protocols to activate an anti-tumor response against residual tumor cells that escaped surgery and/or high-dose chemo/radiotherapy.

The Use of Soybean Agglutinin (SBA) for Bone Marrow (BM) Purging and Hematopoietic Progenitor Cell Enrichment in Clinical Bone Marrow Transplantation.

Soybean agglutinin (SBA) is a plant lectin, a glycoprotein of nonimmune origin that binds specifically to cell surface carbohydrates through noncovalent combinations and thus provokes agglutination of the bound cells. SBA has been used, therefore, to fractionate a variety of cell types. SBA binds approx 60-90% of bone marrow mononuclear cells, including mature myeloid, erythroid, and lymphoid cells, but has very low binding affinity and no toxic effect to the human hematopoietic progenitor cells. In addition, it binds very effectively to certain tumor cells, including neuroblastoma, breast cancer, and Burkitt's lymphoma cells. Based on these characteristics, SBA has multiple potential applications for clinical bone marrow, transplantation (BMT).

Activated long-term peripheral blood cultures as preparation for adoptive alloreactive cell therapy in cancer patients.

Different modes of in vitro activation of peripheral blood mononuclear cells (PBMC) were compared for their effect on long-term propagation. PBMC cultures were activated by short exposures to the mitogen phytohemagglutinin (PHA) and the CD3 complex, with or without secondary signals provided by ligands of CD28 costimulatory molecules. Activation and long-term cultures were carried out in the presence of recombinant interleukin-2 (rIL-2). Addition of supernatant derived from IL-2-activated PBMC improved culture cell yield. Cumulative fold expansions ranged between 10(3) and 10(5) within 21 days. The highest cell yield was found after PHA activation. Fewer cells were obtained after activation with a combination of CD3 and CD28, and even fewer were obtained after CD3 activation alone. An increase in CD8+ and CD56+ cells, without change in CD4+ cells, was found in activated cultures when compared with fresh PBMC. Non-MHC-restricted cytotoxic activity was documented in all activated cultures. Cytotoxic activity per culture was highest in PHA-activated PBMC because of the high cell yield on the day of harvest. Successful in vitro expansion of PBMC might be helpful for gene transfer into T lymphocytes, as well as for the induction of an antitumor response, particularly for prevention and treatment of relapse of hematologic malignancies following allogeneic or autologous bone marrow transplantation.

Induction of graft vs. tumor effect in a murine model of mammary adenocarcinoma.

We have attempted to induce immune-mediated graft-vs.-tumor (GVT) effects against solid tumors, using a murine model of mammary adenocarcinoma derived from BALB/c(H-2d) mice. A cell line (4Tl) isolated from this tumor model was highly tumorigenic in syngeneic (BALB/c) or haplo-identical (BALB/c x C57B1/6)F1 mice (F1), was only partially tumorigenic in an H-2d congenic strain of mice (DBA/2) and was non-tumorigenic in a major histocompatible (MHC)-unrelated (H-2b) strain of mice (C57B1/6). 4Tl cells express class I MHC antigens and adhesion molecules but do not express MHC class II antigens or B7-1 co-stimulatory molecules. Female BALB/c (H-2d) or F1 (H-2d/b) mice were reconstituted with male bone marrow (BM) cells derived from minor histocompatible (MiHC)-mismatched DBA (H-2d) donors or with MHC-mismatched C57B1/6 (H-2b) BM cells, respectively, 24 hr following lethal total body irradiation. Recipient mice carrying MiHC- or MHC-mismatched donor cells were inoculated with 4Tl cells 2-3 months following BM reconstitution. Chimeras reconstituted with allogeneic donor cells that were MiHC- or MHC-incompatible with tumor cells were able to down-regulate the development of the primary tumor expressing host-ype MHC alloantigens. Tumor size in BM chimeras across MiHC or MHC antigens was significantly smaller than tumor size observed in normal BALB/c or F1 controls. The GVT effect might be of help in improving immunotherapy for solid tumors in humans.

The effect of linomide, an immunoregulator in experimental autoimmune diseases, on humoral antibody responses in mice.

Linomide (quinoline-3-carboxamide), a well tolerated, orally administered compound was recently shown to be effective in the prevention and treatment of several autoimmune diseases in experimental animal models. We have investigated its effect on specific humoral immune responses directed to T-cell-dependent soluble or particulate antigens and to a T cell-independent antigen in several mouse strains. Linomide administered after antigen priming did not affect primary and secondary antibody responses directed to T-cell particulate antigens (SRBC) or soluble antigens given with or without complete Freund's Adjuvant (CFA). Linomide treatment given prior to antigen priming did not affect the antibody response to a soluble antigen (TNP-KLH) given with an adjuvant. In contrast, dose-dependent down regulation of primary antibody responses was observed when T cell-dependent (BSA-dextran) or T-cell-independent (TNP-Ficoll) antigens were administered in an immunogenic form without adjuvant after starting Linomide treatment. The primary anti-SRBC antibody response was also suppressed by high dose Linomide given prior to immunization although normal secondary responses were retained. It is worth noting that no immunosuppressive effects on antibody responses were found at low dose ranges which effectively reversed T cell dependent autoimmune manifestation.

Activated allogeneic cell therapy (allo-ACT) for relapsed chronic myelogenous leukemia (CML) refractory to buffy coat transfusions post-allogeneic bone marrow transplantation.

We describe a 17-year-old male patient with chronic myelogenous leukemia (CML) in hematologic and cytogenetic relapse 4 months post-non-T cell-depleted allogeneic bone marrow transplantation for accelerated CML. Two sequential buffy coat transfusion with donor peripheral blood cells (8.9 and 4.8 x 10(7) cells/kg), the second transfusion in combination with in vivo activation of donor cells by human recombinant interleukin-2 (rIL-2) 6 x 10(6) IU/m2 subcutaneously for 3 days, failed to induce remission . The patient responded to an infusion of donor peripheral blood lymphocytes (3.4 x 10(7) cells/kg) pre-activated in vivo with rIL-2 and additionally activated in vivo with rIL-2, 6 x 10(6) IU/m2/day subcutaneously for 3 days. Elimination of the Philadelphia (Ph) clone was confirmed by cytogenetic analysis showing a normal male karyotype and by disappearance of the bcr/abl transcript, using the polymerase chain reaction (PCR). At present, the patient is 26 months post-treatment with no evidence of disease, but with chronic graft-versus-host disease. Our data indicate that allogeneic activated cell therapy (allo-ACT) may provide antitumor effector cells that successfully induce graft-versus-leukemia (GVL) effects even when cell therapy with donor buffy coats was insufficient.

Tumor-cell vaccination induces tumor dormancy in a murine model of B-cell leukemia/lymphoma (BCL1).

Immunity to murine B-cell leukemia/lymphoma (BCL1) induced by multiple injections with irradiated tumor cells, prevented leukemia development in primary and adoptive transfer recipients despite long-lasting persistence of residual tumor cells. Detection of dormant BCL1 cells was carried out by PCR analysis using the VH-rearranged DNA sequence as a BCL1 clonal marker. Dormant tumor cells were detected > 250 days following immunity induction in 40% of spleens from healthy immune mice having no detectable symptoms of disease. Tumor dormancy was not abrogated by adoptive transfer of BCL1-containing splenocytes into syngeneic recipients, indicating that cell-mediated anti-tumor immunity contributes to maintenance of the tumor dormant state and prevents renewed tumor-cell growth. Splenocytes but not sera from immune mice conferred specific radiosensitive protection from a lethal dose of BCL1 cells included in cell mixtures transferred to secondary recipients. A therapeutic effect of transferred immune splenocytes was shown in BCL1-bearing mice, which remained disease-free for > 200 days after inoculation; nevertheless, dormant BCL1 cells were detected by PCR analysis in some of the surviving mice. Our results suggest that an efficient tumor-cell vaccine can lead to induction of tumor dormancy that can be maintained by a cell-derived mechanism for a long period of time.

Induction of tumor immunity by intact irradiated leukemic B cells (BCL1) bearing a tumor-associated cell-surface idiotype and the costimulatory B7 molecule.

The idioptypic (Id) determinant of immunoglobulin expressed on the cell surface of malignant B cells represents a prototypical tumor-associated antigen (TAA), which has been used in a purified soluble form for active immunization in experimental tumor models and human hematological malignancies. Using a spontaneous transplantable murine model of B cell leukemia/lymphoma (BCL1), we have demonstrated the expression of the B7 costimulatory molecules in addition to the previously described Id determinant and class II major histocompatibility antigens. Intact irradiated BCL1 cells bearing these distinct determinants induced long lasting antitumor immunity in naive syngeneic mice. Induction was dose-dependent and most effective when three doses of 30 x 10(6) intact irradiated BCL1 cells were given at intervals of 7-10 days. The induced immunity protected 96% of 28 mice inoculated with a lethal dose of 10(5)-10(6) nonirradiated BCL1 cells and 85% of 27 mice given a second challenge, whereas control mice died on day 20 after inoculation with 10(6) BCL1 cells. Adoptive transfer of splenocytes derived from immune mice did not induce leukemia in syngeneic recipients. Such splenocytes, harvested more than 365 days following immunization and administered together with fresh BCL1 cells to adoptive recipients, were able to confer protection for 90 days, even following a second challenge given 104 days after the first one. BCL1 immune splenocytes transferred into BCL1-bearing mice exerted a therapeutic effect, preventing leukemia onset for at least 180 days. Our results demonstrate the ability of tumor cells to trigger effective anti-tumor immunity. These findings could ultimately be applied to the prevention of tumor relapse in treatment of hematological and other malignancies expressing TAA, class II MHC antigen and costimulatory molecules.

Effect of various cytokine combinations on induction of non-MHC-restricted cytotoxicity.

Efforts were directed to achieve an increased lymphokine-activated non-MHC-restricted killing (LAK) activity greater than that induced by rIL-2 alone. Human peripheral blood (PB) and bone marrow (BM)-derived mononuclear cells (MC) were exposed in vitro to multiple cytokine combinations, including rIL-6, rIL-7, rIFN-alpha and rIFN-gamma in the presence of either suboptimal or optimal doses of rIL-2. Our results have shown that BMMC are a potential source for induction of increased LAK activity upon exposure to multiple cytokine combinations, whereas PBMC could not be successfully stimulated under the same conditions. Fifty-five to 62% of BM-derived samples stimulated with high dose rIL-2 + rIL-7 or rIL-2 + rIL-7 + rIL-6 + rIFN-gamma exhibited a higher degree of cytotoxicity than BM samples stimulated with rIL-2 alone. Exposure of PB-derived large granular lymphocytes (LGL) to various cytokine combinations led to increased proliferation after stimulation with suboptimal dose of rIL-2 in the presence of rIL-6 and rIL-7. This increase was not observed in induction of cytotoxicity. We suggest that BMMC activated by multiple cytokine combinations could play an active role in improving antitumor response in vivo by contributing to the control of minimal residual tumor cell growth, particularly post-BM transplantation.

Prevention and treatment of relapse by bone marrow transplantation.

High, myeloablative doses of chemoradiotherapy represent the treatment of choice for a large number of malignant hematological diseases that cannot be successfully treated with conventional chemotherapy. Residual tumor cells following high dose chemotherapy represent the most common treatment failure, resulting in frequent relapse following autologous bone marrow transplantation (ABMT) and even allogeneic bone marrow transplantation (BMT). Graft vs leukemia (GVL) effects mediated by immunocompetent donor lymphocytes represent a major therapeutic potential of allogeneic BMT which results in reduced rate of relapse, especially when immune interactions between immunocompetent donor's T lymphocytes and host allogantigens is apparent, a reaction which might result in graft vs host disease (GVHD). Recent experiments in animal models of murine leukemias suggest that post-transplant immunotherapy may be successfully accomplished by lymphokine-mediated immunotherapy (LMI) and cell-mediated immunotherapy (CMI). Following allogeneic BMT, provided GVHD can be prevented by T-cell depletion, CMI may be amplified by repeated administration of immunocompetent donor's lymphocytes in graded increments following successful induction of chimerism and sustained hematopoiesis. GVL effects induced by CMI may be further potentiated by in-vivo administration of a short course of recombinant human interleukin-2 (rhIL2). Taken together, our data suggest that post-transplant immunotherapy by cytokines and adoptive cell therapy may successfully prevent relapse in patients at high-risk and even result in complete elimination of tumor cells following overt relapse. Thus, immunotherapy may represent an optimal approach for prevention and treatment of minimal residual disease.