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PubMed1992

Human-mouse lymphoid chimeras: host-vs.-graft and graft-vs.-host reactions.

Human peripheral blood lymphocytes (huPBL) were injected into mice with severe combined immune deficiency (SCID). It was ascertained that murine natural killer (NK) cells were capable of affecting engraftment of human lymphocytes in SCID mice. The presence of host NK cells resulted in the clearance of the human lymphocytes. Human T lymphocytes were the primary cell to engraft in the SCID recipients, with human T cells being detected in the spleen, lymph nodes, bone marrow and peritoneal cavity of the mice up to several months after transfer. No human T cells were detected in the murine thymus and the level of engraftment in the periphery could be highly variable. Additionally, there appeared to be significant reactions between the human lymphocytes and the murine host resulting in a xenogeneic graft-vs.-host reaction (XGVHR). The predominant manifestation involved splenomegaly resulting from an expansion of murine hematopoietic cells in the spleens of these xenogeneic chimeras. The severity of the XGVHR could be correlated with the extent of human T cell engraftment and the recovered human T cells were found to be in a proliferative state. Thus, there appear to be significant host-vs.-graft and graft-vs.-host interactions occurring in human/mouse lymphocyte chimeras.

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PubMed1992

Peritoneal sanctuary for human lymphopoiesis in SCID mice injected with human peripheral blood lymphocytes from Epstein-Barr virus-negative donors.

The successful engraftment in SCID mice of intraperitoneally (i.p.) injected human lymphocytes (hu-PBL-SCID) and the failure of intravenously injected peripheral blood lymphocytes (PBL) directed the present study to investigate the early events of donor cell proliferation in the peritoneal cavity. We found focal lymphocyte engraftment together with histio-monocytic interleukin (IL)-6+ cell phenotypes which must have been transferred with the human cell inoculum, which could explain certain immune functions observed in hu-PBL-SCID chimeras. Following i.p. injection of 10(8) PBL, human cells suspended in peritoneal fluid as well as those adherent to the serosal peritoneum and abdominal organs were investigated by immunocytology and immunohistology. Human cells were found to form foci consisting predominantly of proliferating human lymphoblastoid CD3+ cells, which were mostly activated HLA-DR+ CD8+ lymphocytes. Among the lymphoid cells larger epithelioid-like cells were found to belong to the monocytic series and to stain strongly with anti-HLA-DR and anti-CD11c antibodies. Some of these cells were also positive with anti-ICAM and anti-IL-6. Congenic as well as allogeneic mouse PBL, injected i.p. into SCID mice, temporarily produced analogous foci, which shifted later on to foci similar in appearance to milky spots. However, the human cell foci appeared less compact, more closely resembling in vitro-culture soft agar colonies. It is possible that cytokines in the human histio-monocytic cells of the foci may have a feeder effect on the human lymphocytes and be a prerequisite for proliferation of human PBL in SCID mice. The observed early HLA-DR activation of human lymphocytes in the peritoneal foci could reflect triggering of immune reactions like xenogeneic graft-versus-host reactions in the peritoneal site, where the human CD11c+ HLA-DR+ histio-monocytic cells may act as antigen-presenting cells.

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PubMed1993

The fate of human peripheral blood lymphocytes after transplantation into SCID mice.

Human peripheral blood lymphocytes (hu-PBL) can be adoptively transferred by intraperitoneal injection into mice with severe combined immunodeficiency (SCID). The transplanted lymphocytes can produce immunoglobulin (Ig), respond to antigens, and survive for months in this chimeric model (hu-PBL SCID). However, whether the lymphocytes actually repopulate and reconstitute lymphoid structures and organs has been subject of some debate. To address this question and to characterize the hu-PBL SCID model better, we employed a novel technique for the identification of human cells in xenogeneic mice. We used fluorescence in situ hybridization (FISH) with a biotinylated DNA probe to all human centromeres. We demonstrated that FISH could be used to detect human cells when they accounted for less than 1% of human/mouse cell mixtures; it could also be employed for the identification and localization of individual human cells in tissue sections. By using FISH, we studied 31 SCID mice injected with 1.5 x 10(7)-4 x 10(7) hu-PBL via intravenous (i.v.) or intraperitoneal (i.p.) routes. In the 6 i.v.-injected mice, we found that the human cells were removed from the circulation into the lung within 1 h. In 22 of 25 i.p.-injected animals, 90-3716 micrograms/ml of human IgG was found in the sera at 3 to 13 weeks after transplantation (a.t.). Human cells colonized the peritoneal cavity and persisted for up to 13 weeks a.t. and, in the 12 mice studied, accounted for 4% to 57% of the cells in the peritoneal fluid. However, only rare, isolated human cells were found in the spleen, blood, bone marrow, lung or Peyer's patches. In 7 of 19 mice that received hu-PBL i.p. from Epstein-Barr virus-seropositive donors, we found masses of human cells usually beneath the peritoneal lining but sometimes infiltrating normal tissue. We conclude that FISH offers a simple means for accurate identification of human cells in the xenogeneic mouse. Although there is colonization of the peritoneal cavity in most mice, and development of lymphoid masses in some, there is no reconstitution of lymphoid structures and only minimal engraftment of lymphoid organs by human cells in conventionally-prepared hu-PBL SCID constructs.

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PubMed1994

The ability of H-2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld.

F1 hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non-class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the "self/non-self" property of class I molecules and the molecular domains responsible for this function. H-2b/Hh-1b lymphoma cells were transfected with class I H-2Dd or Ld gene, and its effect on the Hh-1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H-2b/Hh-1b bone marrow grafts by irradiated F1 hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the Dd gene, but not of Ld, renders the Hh-1b-positive cells incapable of inhibiting HR in F1 mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H-2b/d F1 mice for which Dd and Ld are self, but also in H-2b/k F1 mice for which both Dd and Ld are non-self. Thus, this function of the Dd molecule is an intrinsic property, not necessarily related to its self/non-self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a "target interference" model as the underlying mechanism of the Dd effect. To locate the relevant domain(s) of the Dd molecule, mutant Ddm1 gene was tested and found to have the same effect as the non-mutant Dd. Ddm1 is a hybrid molecule between Dd and Ld, sharing with Dd only the alpha 1 domain and a portion of the alpha 2 domain. The two N-terminal domains of Ddm1 differ from those of Dd by three amino acid substitutions, two of which affect the molecules' peptide-binding properties.

Animals↗
PubMed1975

Inhibition of syngeneic fibrosarcoma growth by lymphocytes sensitized on tumor-cell monolayers in the presence of the thymic humoral factor.

The present experiments were performed to investigate the possibility of inhibiting tumor growth in vivo with syngeneic lymphocytes sensitized in vitro on monolayers of the tumor under test, and to study the effect of a thymic humoral factor (THF) in this sensitization process. Monolayers of fibrosarcoma cells were used to sensitize spleen cells from syngeneic donors against the tumor. Such sensitized lymphocytes manifested cytotoxic activity against cells fo the fibrosarcoma in a microassay measuring tumor-cell detachment. However, when the sensitized lymphocytes were mixed with the fibrosarcoma cells and injected into syngeneic mice, enhanced tumor growth was observed in vivo. Addition of thymic humoral factor to the cultures during sensitization resulted in increased cytotoxic activity by the lymphocytes in vitro and a reduction in the tumor enhancement caused by these cells when injected in vivo. Enhanced tumor growth occured when activated lymphocytes of allogeneic as well as syngeneic origin were injected together with the fibrosarcoma cells. Enhancement, which was already apparent when the spleen cells had been sensitized for 24 h, could be circumvented by separate administration of lymphocytes and tumor cells. Syngeneic lymphocytes injected systemically after sensitization for 5 days exerted anti-tumor reactivity against the fibrosarcoma grafted in the foot-pad of syngeneic mice. Tumor growth was further inhibited by systemic injection of lymphocytes which had been sensitized in the presence of the thymic humoral factor.

Animals↗
PubMed1976

Immunogenic and immunosuppressive components in soluble preparations from Moloney murine leukemia virus-induced tumor cells.

Soluble extracts from Moloney virus-induced leukemia cells were shown to contain immunogenic and immunosuppressive components. Immunogenic components were divided into two classes based on their ability to :(1) combine with humoral antibody to inhibit cytotoxic activity; or (2) stimulate blastogenesis in normal syngeneic lymphocytes. Immunosuppressive components were demonstrated by the inhibition of DNA synthesis in normal syngeneic lymphocytes and suppression of a mixed lymphocyte reaction. Stimulating components in many soluble preparations were completely masked by immunosuppressive components. However, all three components could be resolved by Sephadex G200 chromatography.

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PubMed1977

Tumorigenicity of human hematopoietic cell lines in athymic nude mice.

Human hematopoietic cell lines, which had been classified on the basis of studies on clonality, and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation to nude mice and for capacity to grow in semi-solid medium in vitro. Recently established LCL failed to grow both in nude mice and in agarose. In contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity to form colonies in agarose. Most lymphoma lines formed colonies in agarose and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed to grow. Both myeloma lines, however, formed colonies in agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed to grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown to form tumors when inoculated subcutaneously in nude mice and had the capacity to grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic in nude mice. The capacity to form colonies in agarose is not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur in the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential to grow subcutaneously in nude mice and in agarose in vitro. (3) The inconsistency with regard to the capacity of come of the neoplastic cell lines to grow in nude mice or in agarose seems to underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines.

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PubMed1977

Depressed in vitro peripheral blood lymphocyte response to mitogens in cancer patients: the role of suppressor cells.

The reactivity of peripheral blood lymphocytes from patients with advanced malignancy was assessed by mitogen-induced stimulation of protein synthesis as measured by 3H-leucine incorporation. It was confirmed that the lymphocyte response of patients was depressed. Furthermore, the lymphocytes of 15 out of 27 cancer patients, selected because of their low responses, inhibited the reactivity of normal lymphocytes in co-cultures. The lymphocytes from one patient with Hodgkin's disease were also inhibitory. In contrast, lymphocytes from healthy subjects, patients with chronic lymphocytic leukaemia, lymphosarcoma or multiple myeloma caused no suppression. Experiments with purified cell populations from patients with carcinoma indicated that purified T cells responded to mitogens while unseparated lymphocytes failed to respond and that the inhibitory activity was due to adherent cells, presumably monocytes. There was no evidence for B-cell-mediated suppression. However, in two cases inhibition was caused by isolated T cells of the patients and not by adherent cells. These experiments suggested that one mechanism for the depression of cell-mediated immunity seen in patients with advanced cancer may be the nonspecific suppresssion of certain T-cell functions by circulating monocytes.

Adult↗
PubMed1980

Effector and enhancing lymphoid cells in plasmacytoma-bearing mice. I. Methodological studies on the Winn assay.

Some parameters of the Winn assay for the detection of tumor-suppressing ("effector") and tumor-enhancing lymphoid cells were studied in BALB/c mice. Spleen cells of mice that were preimmunized with mitomycin-C-treated MOPC-104E plasmacytoma cells were inhibitory in this test system for both the MOPC-104E and the HOPC-I plasmacytomas, thus indicating cross-reactivity. Spleen cells taken from mice 6 days after the surgical removal of 15-day-old MOPC-104E tumors inhibited the growth of lethal doses of MOPC-104E cells in normal recipients, but no inhibition was observed 2 days after the removal of 18-day-old tumors. Spleen cells from mice bearing MOPC-104E for 13 days enhanced tumor growth. This enhancement was not influenced significantly by the wide dose range (from 10(5) to 3 x 10(7)) of MOPC-104E cells used to initiate tumors in the lymphoid cell donors, although tendency for stronger enhancing potential occurred after low tumor doses. When spleen cells from donors bearing MOPC-104E for 10 days were injected at the constant tumor-lymphocyte ratio of 1:30 with increasing numbers of tumor cells (from 5 x 10(5) to 2 x 10(6)), tumor inhibition occurred at the lowest dose only, while no significant effect was observed at higher tumor cell doses. When a constant dose (5 x 10(5)) of tumor cells was injected with spleen cells from 10-day tumor-bearers at tumor/lymphocyte ratios of 1:10, u:40 and 1:160, a significant tumor inhibition occurred only at the ratio of 1:40. The relevance of the Winn test to the study of immune mechanisms in tumor-bearing hosts is discussed.

Animals↗
PubMed1980

Effector and enhancing lymphoid cells in plasmacytoma-bearing mice. II. Dynamic changes during tumor progression.

The Winn assay was used for the study of effector (tumor-inhibiting) and enhancing (tumor-promoting) lymphoid cells in BALB/c mice bearing MOPC-104E plasmacytomas. Kinetic studies with thymus, lymphnode, spleen and bone-marrow cells revealed that spleen, lymph node and to a lesser extent bone-marrow cells from 7- and 10-day tumor-bearers inhibited MOPC-104E growth, while at day 13 all three cell populations enhanced tumor growth. However, at day 16 strong tumor inhibition was observed again by spleen cells and to a lesser extent by lymph-node cells and thymocytes. Peritoneal cells from normal and tumor-bearing (7 and 10 days) animals enhanced tumor growth significantly. Separation of spleen cells on nylon wool columns showed that at 10 days the effector cells were T lymphocytes, but at a later stage (35 days) a different effector mechanism was present in the spleen. Treatment of MOPC-104E recipients with carrageenan or silica had little influence on tumor growth, but marked tumor inhibition occurred after lethal irradiation and bone-marrow reconstitution. This latter observation, together with the finding that bone-marrow, lymph node and peritoneal cells from normal donors enhanced tumor growth in several experiments, suggests that this plasmacytoma, like hormone-dependent tumors, requires lymphocyte-derived growth factors.

Animals↗
PubMed1982

Syngeneic and allogeneic cell-mediated cytotoxicity against Marek's disease lymphoblastoid tumor cell lines.

Cell-mediated cytotoxicity (CMC) of lymphocytes obtained from chickens infected with Marek's disease (MD) virus against allogeneic MD lymphoblastoid cell lines has been reported by several research groups. Recently, we established a number of cell lines from MD tumors obtained from highly inbred chickens and characterized for major and minor histocompatibility antigens. Allogeneic versus syngeneic CMC was studied using those cell lines and lymphocytes obtained from chickens 6-8 days post infection with SB-1, a non-oncogenic MD virus. Allogeneic cytotoxicity could be easily demonstrated, while syngeneic cytotoxicity was a rare event. However, increase of the CMC assay period from 4 to 8 h did enhance syngeneic cytotoxicity. Cold inhibition assays demonstrated that the allogeneic cytotoxicity was directed against alloantigens present on spleen lymphocytes sharing the same major histocompatibility antigens as the target cells. Cytotoxicity was not influenced by the sex of either target or effector cells or by the level of virus infectivity of the effector cells.

Animals↗
PubMed1988

Distribution and survival of passively transferred lymphoblasts in normal and tumor-bearing mice.

Although most circulating lymphocytes are long-lived small lymphocytes, it is the recently-divided medium and large-sized lymphoblasts that most clearly migrate to sites of inflammation, and they do so without regard to immunologic commitment. The tumor-bearing state frequently suppresses macrophage accumulation at inflammatory sites but it is not known whether lymphocyte traffic is similarly affected. We addressed this issue using passively transferred radiolabelled lymphoblasts. Lymphoblasts readily entered s.c. fibrosarcomas transplanted to C57BL/6 mice. Large and small tumors acquired the same number of lymphoblasts per gram of tumor, indicating the absence of an anti-inflammatory reaction. However, the intra-tumoral survival of lymphoblasts was shorter in large than in small tumors. Shortened survival was noted also in spleen and whole body of animals bearing tumors. Shortened survival rather than impaired emigration may contribute to the increase in tumor:lymphocyte ratio observed with large tumors. The adverse effect of tumor bearing on lymphoblast survival may be limited to tumors which acquire a high percentage of passively transferred lymphocytes since intraperitoneal P-815 mastocytomas in DBA/2 mice acquired few labelled lymphoblasts and did not alter lymphoblast survival. These results are important for host resistance to tumor growth and therapeutically for the use of lymphocyte cytotoxic cells induced to proliferate in vitro.

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PubMed1989

Delayed-type hypersensitivity to tumor antigens co-expressed with immunogenic determinants induced by xenogenization.

Previous work has shown that murine lymphoma cells antigenically altered ("xenogenized") by drug treatment elicit strong in vivo resistance to the original cells. Moreover, splenocytes immune to a drug-treated variant (L5178Y/DTIC) of a murine lymphoma exert anti-parental tumor activity in an adoptive transfer system, an effect mediated by L3T4+ lymphocytes and associated with the detection of an anti-L5178Y delayed-type hypersensitivity (DTH) response. We now report that the in vivo activity of the tumor-immune L3T4+ lymphocytes is a radio-sensitive (2,500 rad in vitro) phenomenon that requires collaboration with radio-resistant, silica-sensitive syngeneic cells in the host, and is inhibited by treatment of recipient mice with monoclonal antibodies (MAbs) to the L3T4 antigen or murine interferon-gamma (IFN-gamma). In vitro, the tumor-immune L3T4+ lymphocytes produce interleukin 2 (IL-2), lymphotoxin (LT) and IFN-gamma activities on incubation with L5178Y cells and spleen-adherent cells. These results suggest that the mechanisms of anti-parental tumor protection by xenogenized cells involve specific induction of a DTH response mediated by the "inflammatory" (THI) subset of L3T4+ T lymphocytes and IFN-gamma activated macrophages.

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PubMed1989

Tumorigenic conversion of xeroderma pigmentosum lymphoblastoid cells without karyotypic alteration.

In order to examine the process of malignant transformation of human somatic cells, we studied the tumorigenic conversion of an Epstein-Barr-virus-immortalized lymphoblastoid cell line (LCL) derived from a patient with xeroderma pigmentosum (XP) complementation group A. Repeated irradiation of the XP cells, XP7NI, with UV-light and subsequent treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the acquisition of tumorigenicity in athymic nude mice. The tumorigenicity of XP7NI cells was also induced by TPA treatment alone. The tumors formed in athymic mice were of B-cell lymphoma with characteristic histology, cell surface immunoglobulins and an antigen as detected by a B-cell-specific monoclonal antibody (MAb), CD20. The surface immunoglobulins and the HLA type of these tumor cells were identical with those of the parental cells. These malignantly transformed cells retained the same UV sensitivity, serum requirement, colony-forming ability in soft agar, and normal human karyotype as the parental cells. Unlike other tumorigenic lymphoblastoid cell lines, this XP lymphoblastoid cell line provides a unique case in that process(es) leading to tumorigenicity may be induced by UV and TPA without apparent karyotypic changes.

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PubMed2002

Characteristics of photopheresis treatments for the management of rejection in heart and lung transplant recipients.

Photopheresis has been used in the management of rejection of heart and/or lung transplants. Although its mechanism of action remains unknown, irradiated T-helper cell-induced immunosuppression is the main theory. Since transplant recipients are often lymphopenic and lymphocytes are the target cells in phototherapy, we performed this study to determine which factors affect the cellular yield to undergo irradiation. We reviewed the records of all photophereses performed in our institution between July 1998 and April 2000 using the UVAR (first generation) or XTS (second generation) instruments (Therakos, Exton, PA). Our data included patient's blood volume, absolute lymphocyte count and hematocrit, catheter type, flow rate of collection cycles and centrifuge bowl size, as well as volume, hematocrit, and lymphocyte count of the cell suspension. With a mixed model multivariate analysis we sought to determine which variables predicted the lymphocyte yield. A total of 406 procedures in 25 adult patients was analyzed. There was no significant difference between the lymphocyte yield among the procedures performed with the first- and the second-generation instruments. The patient's absolute lymphocyte count was the only parameter, which positively correlated with the total number of lymphocytes collected for irradiation (P < 0.0001). Indeed, based on the mixed model, the total number of lymphocytes for irradiation can be predicted from the pre-procedure lymphocyte count. Additional studies are necessary to correlate the number of treated cells with patient outcome.

Adult↗