Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease

Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Adoptive cellular immunotherapy: NK cells and bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) has been increasingly used for the treatment of both neoplastic and non-neoplastic disorders. However, serious obstacles currently limit the efficacy and thus more extensive use of BMT. These obstacles include: graft-versus-host disease (GVHD), relapse from the original tumor, and susceptibility of patients to opportunistic infections due to the immunosuppressive effects of the conditioning regimen.Overcoming these obstacles is complicated by dual outcome of existing regimens; attempts to reduce GVHD by depleting T cells from the graft, result in increased rates of tumor relapse and failure of engraftment. On the other hand, efforts to increase graft-versus-tumor (GVT) effects of the transplant also promote GVHD. In this review, the use of natural killer (NK) cells to overcome some of these obstacles of allogeneic BMT is evaluated. Adoptive immunotherapy using NK cells after allogeneic BMT has several potential advantages. First, NK cells can promote hematopoiesis and therefore engraftment by production of hematopoietic growth factors. Second, NK cells have been shown to prevent the incidence and severity of GVHD. This has been shown to be at least partially due to TGF-beta, an immunosuppressive cytokine. Third, NK cells have been shown to augment numerous anti-tumor effects in animals after BMT suggesting a vital role of NK cells in mediating GVT effects. Finally, NK cells have been demonstrated to affect B cell recovery and function in mice. Therefore, understanding the mechanisms of beneficial effects of NK cells after BMT may lead to significant increases in the efficacy of this procedure.     

Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine.

The absence of surface costimulatory molecules explains in part the lack of an effective anti-tumor immune response in tumor-bearing animals, even though unique tumor antigens may be presented by class I MHC. We determined that the immunogenicity of a murine neuroblastoma, Neuro-2a, which lacks surface costimulatory molecules, could be increased by electrically induced fusion with dendritic cells. Electrofusion induced a higher level of cell fusion than polyethylene glycol, and tumor/dendritic cell heterokaryons expressed high levels of costimulatory molecules. While Neuro-2a was unable to induce the proliferation of syngeneic or allogeneic T cells in vitro, fused cells were able to induce T cell responses both in vitro and in vivo. When fused dendritic tumor cells were used as a cancer vaccine, immunized mice were significantly protected from challenge with Neuro-2a. We propose that electrofusion with patient-derived tumor and dendritic cells may provide a rapid means to produce patient-specific tumor vaccines.     

Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia

 The feasibility of inducing graft versus leukemia (GVL) effects with allogeneic T cells in recipients of autologous bone marrow transplantation (BMT) was studied in a murine model (BCL 1) of human B cell leukemia/lymphoma. Allogeneic cell therapy, induced by infusion with peripheral blood lymphocytes, a mixture of allogeneic spleen and lymph node cells and allogeneic activated cell therapy, induced by in vitro recombinant-interleukin-2(rIL-2)-activated allogeneic bone marrow cells in tumor-bearing mice, prevented disease development in adoptive BALB/c recipients. Concomitant in vivo activation of allogeneic lymphocytes with rIL-2 suppressed even more effectively the development of leukemia in secondary adoptive recipients of spleen cells obtained from treated mice. In contrast, in vivo administration of rIL-2 after syngeneic BMT, with or without equal numbers of syngeneic lymphocytes, led to disease development in secondary recipients. Our data suggest that effective cell therapy can be achieved after SBMT by allogeneic but not syngeneic lymphocytes and that anti-leukemic effects induced by allogeneic lymphocytes can be further enhanced by in vitro or in vivo activation of allogeneic effector cells with rIL-2. Therefore, cell therapy by allogeneic lymphocytes following autologous BMT could become an effective method for inducing GVL-like effects on minimal residual disease provided that graft versus host disease can be prevented or adequately controlled. Received: 14 May 1996 / Accepted: 6 August 1996     

Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells

Posttransplant infusion of donor bone marrow cells (BMC) induces tolerance to allografts in adult mice, dogs, nonhuman primates, and probably humans. Here we used a mouse skin allograft model and an allogeneic radiation chimera model to examine the role of MHC Ags in tolerance induction. Infusion of MHC class II Ag-deficient (CIID) BMC failed to prolong C57BL/6 (B6) skin grafts in ALS- and rapamycin-treated B10.A mice, whereas wild-type B6 or MHC class I Ag-deficient BMC induced prolongation. Removal of class II Ag-bearing cells from donor BMC markedly reduced the tolerogenic effect compared with untreated BMC, although graft survival was significantly longer in mice given depleted BMC than that in control mice given no BMC. Infusion of CIID BMC into irradiated syngeneic B6 or allogeneic B10.A mice produced normal lymphoid cell reconstitution including CD4+ T cells except for the absence of class II Ag-positive cells. However, irradiated B10.A mice reconstituted with CIID BMC rejected all B6 and a majority of CIID skin grafts despite continued maintenance of high degree chimerism. B10.A mice reconstituted with B6 BMC maintained chimerism and accepted both B6 and CIID skin grafts. Thus, expression of MHC class II Ag on BMC is essential for allograft tolerance induction and peripheral chimerism with cells deficient in class II Ag does not guarantee allograft acceptance.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Generation of lymphokine-activated killer (LAK) cells from tumor-infiltrating lymphocytes

Culture of tumor-infiltrating lymphocytes (TIL) containing about 20% BMC2 tumor cells with recombinant human interleukin 2 (rIL-2) resulted in the diminish of tumor cells and the growth of lymphocytes. These IL-2-activated lymphocytes showed a strong cytotoxic activity against not only syngeneic tumor cells but also allogeneic tumor cells. Such broad-reactive killer cells, termed lymphokine-activated killer (LAK) cells, are also inducible from spleen cells by in vitro activation with IL-2. However, LAK cells generated from TIL (TIL-LAK) showed higher cytotoxic activity against BMC2 than LAK cells generated from spleen cells (S-LAK). Furthermore, it was demonstrated that TIL-LAK cells revealed marginal cytotoxic activity against normal Con A blasts and YAC-1 cells as opposed to S-LAK. Flow cytometric analysis of TIL-LAK indicated that TIL-LAK cells mainly consisted of Thy 1.2+, Ly 2+, asialo GM1+ cells. TIL-LAK cells displayed not only in vitro cytotoxicity but also in vivo anti-tumor activity. Furthermore, it was also confirmed that TIL-LAK cells could be induced in autochthonous mouse tumor systems and human gastric tumor systems.     

The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2^b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2^b) (B6) and allogeneic C3H/Hej (H-2^k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.     

Donor-type activated natural killer cells promote marrow engraftment and B cell development during allogeneic bone marrow transplantation.

Purified NK cells were obtained from mice with severe combined immune deficiency and were activated with human IL-2 (hrIL-2) in vitro to determine if, once activated, these cells could be transferred with compatible bone marrow cells (BMC) and promote marrow engraftment in irradiated allogeneic recipients. After culture with hrIL-2, these cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. These activated NK cells were then adoptively transferred with the donor BMC and rhIL-2 into lethally irradiated allogeneic hosts. The addition of NK cells with the BMC allowed for more rapid hematopoietic engraftment as determined through short term studies, and greater donor-derived chimerism with accelerated reconstitution of the B cell population as determined with long term analysis. No evidence of graft-vs-host disease was detected in the recipients receiving the activated NK cells with allogeneic T cell replete BMC and hrIL-2. The mechanism by which the transferred NK cells improved BMC engraftment was at least partly through the abrogation of the host effector cell's ability to mediate resistance to the marrow graft. Thus, the administration of donor-type activated NK cells with BMC and hrIL-2 may significantly augment hematopoietic engraftment and immune reconstitution in the clinical setting of allogeneic BMT without giving rise to graft-vs-host disease.     

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.     

Resistance to 402AX teratocarcinoma involves immunity to minor histocompatibility antigens

The 402AX teratocarcinoma is a 12/J-derived mouse major histocompatibility complex (MHC) antigen negative tumor that is induced to express H-2^b class I antigens during rejection. Resistance to 402AX by MHC allogeneic and syngeneic mice is immunologically mediated and involves the recognition of tumor-associated antigens (TAA) in the context of induced MHC class I antigens. The current studies were undertaken to define the 402AX TAAs. Reconstitution of irradiated susceptible hosts (129/J) with 402AX-primed resistant spleen cells (C57BL/6) results in acute graft-versus-host disease, suggesting that tumor-primed C57BL/6 splenocytes are reactive to tumor genotype (129/J) minor histocompatibility (Hm) antigens. C57BL/6 anti-129/J effector cells, although not directly cytotoxic for 402AX cells, are specifically cold target inhibited by 402AX cells. Genetically susceptible hosts (C3H.SW) immunized to 129/J Hm antigens by skin grafting become resistant to an i.p. challenge of 402AX cells. These results suggest that 129/J Hm antigens may be the TAAs recognized during genetically controlled rejection of the 402AX teratocarcinoma.     

Antigen processing requirements for T cell activation: differential requirements for presentation of soluble conventional antigen vs cell surface MHC determinants

The present studies were undertaken to characterize the antigen-processing requirements involved in the responses to T cells to soluble antigen (antigen specific), to allogeneic cell surface MHC determinants (alloreactive), and to syngeneic MHC determinants (autoreactive). T cell clones were used that have dual cross-reactive specificities either 1) for self MHC plus soluble antigen and for allogeneic MHC products or 2) for syngeneic MHC and for allogeneic MHC, in order to permit comparison of the processing requirements for responses of the same T cell to distinct antigenic stimuli. The proliferative responses of antigen-specific, Ia-restricted T cell clones to soluble antigens were sensitive to treatment of antigen-presenting cells (APC) with 125 to 250 microM chloroquine, a lysosomotropic agent previously shown to inhibit the processing of soluble antigens. In contrast, the same T cell clones were only minimally affected in their ability to respond to similarly chloroquine-treated APC expressing allogeneic MHC products. The responses of autoreactive T cell clones to syngeneic stimulating cells and their cross-reactive responses to allogeneic cells were both resistant to chloroquine treatment of stimulating cells. The failure of chloroquine to inhibit antigen presentation to autoreactive T cell clones suggests that these clones are specific for self Ia not associated with in vitro processed foreign antigen. Thus, chloroquine sensitivity distinguishes the in vitro antigen-processing requirements for presentation of the soluble antigens tested from the requirements for presentation of syngeneic or allogeneic cell surface MHC determinants to the same T cells.     

Tumor-associated neural differentiation antigens detected on C 1300 neuroblastoma cells by hybridoma monoclonal autoantibodies

Immune isoantisera and hybridoma monoclonal autoantibodies against syngeneic C1300 neuroblastoma (NB) cells were produced from BALB/c mice. Isoantisera were obtained (i) from mice immunized with membrane preparations from cloned NB cells and (ii) from mice bearing NB tumors. After repetitive absorptions on several different syngeneic or allogeneic tumor cell lines and syngeneic normal kidney, liver, spleen, bone marrow, and brain mouse tissue powders, these sera still retained antibodies reacting with tissue-differentiation antigens present on both NB cells and normal nerve sympathetic cells on cryostat whole body sections of neonatal mice. Monoclonal autoantibodies against NB cells were the products of the fusion between plasmacytoma cells and spleen cells from mice bearing syngeneic NB tumors. These anti-NB monoclonal antibodies revealed a restricted spectrum of distinct alloantigenic specificities against syngeneic bone marrow, fetal and adult brain cells, and nerve sympathetic cells present on neonatal rather than adult mice. A mixture of four monoclonal antibodies, recognizing, respectively, an epitope of the Ia complex and three distinctive neuronal-restricted antigens, proved to be a powerful and specific probe for histological immunodiagnosis of neuroblastoma, on cryostat sections of NB tumors, metastases, and tumor-draining lymph nodes.     

Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

Activation of natural killer (NK) cells in vivo with H-2 and non-H-2 alloantigens

Intraperitoneal inoculation of allogeneic lymphoid cells rapidly activates cytotoxic cells in the peritoneum which are nonadherent and express the NK-1, asialo-GM₁, and Thy-1 antigens. Allogeneic spleen cells were very efficient at activating these natural killer (NK) cells, while allogeneic thymocytes were much less effective. Heat-killed allogeneic cells or sonicates also could augment NK activity. — Incompatibility atH-2K, H-2I-A, orH- 2D readily evoked NK cell activity, whileH-2S- andH-2I-E/C-associated disparities did not. Non-H- 2 differences also stimulated NK activity and augmentation was particularly evident inMls-disparate combinations. Thus, the same alloantigens which efficiently activate T cells also activate NK cells.     

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.     

In vitro and in vivo analysis of bone marrow-derived CD3+, CD4-, CD8-, NK1.1+ cell lines

The development of methods of avoiding graft-versus-host disease (GVHD) while retaining the alloengraftment-promoting and anti-leukemic effects of allogeneic T cells is a major goal of research in bone marrow transplantation (BMT). We have recently obtained evidence suggesting that natural suppressor (NS) cells derived from T cell-depleted (TCD) syngeneic marrow can protect against GVHD while permitting alloengraftment. We have now attempted to enrich and then propagate NS cells in vitro, with the goal of obtaining an enhanced anti-GVHD effect by adoptive transfer in vivo. Two long-term cell lines were generated culturing BMC depleted of Mac1-positive cells and of Mac1-positive plus Thy1-positive cells in high concentrations of IL-2. Both cell lines showed anti-GVHD effects when administered along with a GVHD-producing inoculum, while permitting complete allogeneic reconstitution. A clone derived from Mac1-depleted BMC protected completely against a more chronic pattern of GVHD. These cell lines demonstrated suppressive activity in vitro, cytolytic activity against a broad range of natural killer (NK)-sensitive and NK-resistant targets, and a novel cell surface phenotype, with characteristics of both alpha beta-TcR-bearing T cells and of NK cells. In some respects, these cells resemble LAK cells and differ from fresh NS cells, and from the cloned NS cells derived from spleens of total lymphoid irradiation (TLI)-treated mice and neonatal mice. To our knowledge, this is the first detailed phenotypic analysis of cell lines with in vivo anti-GVHD activity. If applicability can be demonstrated in large animal models, the ability to use bone marrow as a source of such protective cell lines might also have potential utility in clinical BMT.     

Stimulation of alloreactive cytotoxic T cells by paternal major histocompatibility antigens during murine pregnancy

In an effort to elucidate T cell reactivity toward paternal major histocompatibility (MHC) antigens during pregnancy, the ability of pregnant mice to develop alloreactive cytotoxic T lymphocytes (CTL) was studied in individual multiparous females mated with MHC congeneic strains of B10 background. Spleen cells obtained from B10.BR females mated to allogeneic males manifested strikingly higher CTL than those from animals mated to syngeneic males or from virgins; syngeneically mated animals were equivalent to virgin controls in CTL responses. The augmented CTL response in allogeneic pregnancy was detected not only by stimulation with the paternal MHC antigens but also by an unrelated MHC haplotype. However, this augmentation was found only during pregnancy in that 2-5 days after the delivery the CTL activity in allopregnant animals returned to a level comparable to that of virgin controls. No suppressor cells were detected at this stage. These observations suggest that maternal T cells recognize MHC disparity with the fetus in some way during pregnancy. Anti-MHC antibodies, immunoglobulin (Ig) M, and IgGs of all subclasses were not detected in these animals throughout multiple pregnancies.     

IFN-gamma- and cell-to-cell contact-dependent cytotoxicity of allograft-induced macrophages against syngeneic tumor cells and cell lines: an application of allografting to cancer treatment.

In allogeneic tumor or skin transplantation, the rejection process that destroys the allogeneic cells leaves syngeneic cells intact by discrimination between self and nonself. Here, we examined whether the cells infiltrating into the allografts could be cytotoxic against syngeneic immortal cells in vitro and in vivo. The leukocytes (i.e., macrophages (Mphi; 55-65% of bulk infiltrates), granulocytes (20-25%), and lymphocytes (15-20%)) infiltrating into allografts, but not into autografts, in C57BL/6 mice were cytotoxic against syngeneic tumor cells and cell lines, whereas the cytotoxic activity was hardly induced in allografted, IFN-gamma-/- C57BL/6 mice. Among the leukocytes, Mphi were the major population of cytotoxic cells; and the cytotoxic activity appeared to be cell-to-cell contact dependent. When syngeneic tumor cells were s.c. injected into normal C57BL/6 mice simultaneously with the Mphi-rich population or allogeneic, but not syngeneic, fibroblastic cells, tumor growth was suppressed in a cell number-dependent manner, and tumor cells were rejected either with a Mphi:tumor ratio of about 30 or with an allograft:tumor ratio of approximately 200. In the case of IFN-gamma-/- C57BL/6 mice, however, the s.c. injection of the allograft simultaneously with tumor cells had no effect on the tumor growth. These results suggest that allograft or allograft-induced Mphi may be applicable for use in cancer treatment and that IFN-gamma induction by the allograft may be crucial for the treatment.