Regulation of cytotoxic T-cell reactivity to syngeneic tumors by the thymus.

Adult thymectomy has been shown to result in the enhanced capacity of splenic T cells to respond to and lyse syngeneic tumor cells in vitro. In addition, T cells from thymectomized mice which kill syngeneic tumor cells do not lyse either normal lymphoid or mitogen-stimulated syngeneic lymphoblast target cells. These findings indicate that the thymus exports a subpopulation of T cells sensitive to adult thymectomy which regulates the generation of cytolytic T cells directed against syngeneic tumor cells.

T and B cell in hapten-specific carrier-determined tolerance.

BDF1 mice were made tolerant by a single i.v. injection of 1 mg of DNAP-gamma1 or by weekly i.v. injections of 0.2 mg of DNP-gamma1 given for a month. In both instances, spleen cells of tolerant animals were fractionated to obtain pure populations of T cells (nonimmunoglobulin-bearing cells), referred to as tolerant T cells, and B cells (immunoglobulin-bearing cells) referred to as tolerant B cells (immunoglobulin-bearing cells) referred to as tolerant B cells. The control cells were similarly fractionated to obtain normal T and B cells. Mixtures of tolerant T cells and normal B cells, or conversely, normal T cells and tolerant B cells were used to repopulate lethally irradiated recipients. These recipients were then immunized with dinitrophenyl-keyhole limpet haemocyanin and in certain instances with other antigen horse red blood cells. The immune response to both antigens was measured using the direct hemolytic plaque assay. It was found that both T and B cells were tolerant and that tolerance was hapten specific at both T- and B-cell levels. While B-cell tolerance was demonstrated at a 1/1 T/B ratio, a 4/1 T/B ratio was necessary to show T-cell tolerance. Thus, the hapten-specific carrier-determined tolerance involves not only B cells but also T cells. The implication of this finding for the cellular mechanism of tolerance in an experimental model closely related to self tolerance is discussed.

Dichotomy between the induction of suppressor cells and immunologic tolerance by adult thymectomy.

Adult thymectomy prevents the development of suppressor T cells without impairing the induction of immunologic tolerance to the same antigenic determinant. This finding demonstrates that the cellular mechanisms underlying immune suppression and immune tolerance are different.

T-cell migration into allografts.

The ability of T and B lymphocytes to migrate into skin allografts undergoing rejection was studied in mice. Spleen cells from CBA/J mice sensitized to transplantation antigens of A/J or C57BL/6 mice were separated on immunabsorbent columns into purified populations of T and B cells, labeled in vitro with 3H-uridine and injected intravenously into CBA/J mice with 7-day old skin iso and allografts (A/J or C57BL/6). The mice were sacrificed 24 h later and studied by autoradiography. After transfer of either unfractionated spleen cells or T cells, large numbers of labeled cells were found in the cellular infiltrate of allografts, whereas extremely few were seen in isografts. In contrast, after transfer of B cells, almost no labeled cells were detected either in the allografts or the isografts, although they, like T cells, homed normally to lymphoid tissue.

Regulation of B cell lymphomagenesis by a malignant Qal+ inducer T cell clone.

A series of Thy-1.2+ Ly-1+ Qa-1+ malignant T cell clones have been isolated from murine sarcoma virus-murine leukemia-Moloney (MSV-MuLV-M)-induced B cell lymphomas or from MSV-MuLV-M-infected B6 mice. These T cell clones enhance both antigen-independent and -dependent lymphocyte differentiation and function. They also induce the differentiation of granulocytes and erythrocytes in the stem cell compartment, a function that parallels the immunopathology of the disease in vivo. The malignant T cell appears to sustain B lymphoma growth in vivo by releasing a factor (BCGF) that promotes B cell proliferation.

Potentiation of cytotoxic T-cell function by virus.

Inoculation of C57BL/6J mice with allogeneic P815 mastocytoma cells in the presence of simian virus 40 (SV40), a DNA tumor virus, led to an enhanced cytolytic T-cell response to P815 in vivo. Cytotoxic function was also augmented if SV40 was given subsequent to a primary immunization, even when mice were given a suboptimal dose of immunizing cells. Although SV40 increased the cell-mediated immune response to allogeneic cells, it did not enhance the antibody response to the soluble antigen dinitrophenyl bovine gamma-globulin, a helper T-cell-dependent response. Thus it appeared that SV40 had a selective adjuvant effect on lymphocyte subpopulations, since it increased cytotoxicity but not helper T-cell function.

Expression of homologues for p53 and p73 in the softshell clam (Mya arenaria), a naturally-occurring model for human cancer.

Homologues for human p53 (Hsp53) and p73 (Hsp73) genes were cloned and expression patterns for their corresponding proteins analysed in tissues from normal and leukemic softshell clams (Mya arenaria). These are the first structural and functional data for p53 and p73 cDNAs and gene products in a naturally occurring, non-mammalian disease model. Core sequence of the predicted clam p53 (Map53) and p73 (Map73) proteins is virtually identical and includes the following highly conserved regions: the transcriptional activation domain (TAD), MDM2 binding site, ATM phosphorylation site, proline rich domain, DNA binding domains (DBDs) II-V, nuclear import and export signals and the tetramerization domain. The core sequence is a structural mosaic of the corresponding human proteins, with the TAD and DBDs resembling Hsp53 and Hsp73, respectively. This suggests that Map53 and Map73 proteins may function similarly to human proteins. Clam proteins have either a short (Map53) or long (Map73) C-terminal extension. These features suggest that Map53 and Map73 may be alternate splice variants of a p63/p73-like ancestral gene. Map73 is significantly upregulated in hemocytes and adductor muscle from leukemic clams. In leukemic hemocytes, both proteins are absent from the nucleus and sequestered in the cytoplasm. This observation suggests that a non-mutational p53/p73-dependent mechanism may be involved in the clam disease. Further studies of these gene products in clams may reveal p53/p73-related molecular mechanisms that are held in common with Burkitt's lymphoma or other human cancers.

T-cell regulation of erythropoiesis during acute lymphoblastic leukemia.

How and where erythropoiesis is maintained during advanced leukemic disease is an important and, as yet, unresolved question in hematology. To address the potential role of T-lymphocytes as cells that regulate CFU-E differentiation during leukemogenesis, an experimental model of disease has been developed in inbred Balb/c mice. Specifically, three-week-old Balb/c By mice were injected with murine sarcoma virus-murine leukemia virus-Moloney (MSV-MuLV-M), which resulted 6-8 months later in the development of immunoblastic T-cell sarcomas with a leukemic phase. Splenic T cells from either normal or tumor-bearing mice were assessed for their relative ability to modulate erythroid differentiation. Quantitatively, T cells, Ly1 or Ly 2,3 T-cell subsets isolated from tumor-bearing animals significantly enhanced erythropoiesis when compared with comparable normal T-cell subsets. These data suggest that the compensatory shift of erythropoiesis from the bone marrow to the spleen observed during leukemogenesis was facilitated by splenic T cells. In this circumstance, the enhanced erythropoietic function may be mediated by splenic T cells, which are selectively activated by virus.

A novel adhesion protein expressed by ciliated epithelium, hemocytes, and leukemia cells in soft-shell clams.

The ontogeny of circulating hemocytes and tumor cells in mollusks has been approached using monoclonal antibodies to normal cells. A monoclonal antibody, previously shown to identify an adhesion related protein (p130), has been used to define the reactivity of cells in tissues from normal soft-shell clams (Mya arenaria) and soft-shell clams with leukemia. Using immunoperoxidase technology, we have determined that hemocytes, connective tissue cells, and a subset of leukemia cells that are adherent share a cross-reactive epitope with cilia.

Unique antigens on neoplastic cells of the soft shell clam Mya arenaria.

Soft shell clams (Mya arenaria) are commonly found in coastal waters of both the eastern and western United States. These invertebrates, which have an open circulatory system, may develop neoplasms of the haemolymph which ultimately kill the host. In this study we have 1) recorded the prevalence of hematopoietic neoplasms (HN) in Mya arenaria within a 50 mile radius of Woods Hole, Massachusetts and 2) utilized cells from one HN bearing clam to generate a series of monoclonal antibodies. Our data show that determinants are expressed on HN cells which are not detected on normal clam haemocytes, suggesting separate ontogenetic pathways of cell differentiation.

Autoimmune vasculitis in MRL/Mp-lpr/lpr mice: orthochromatic basophils participate in the development of delayed-type hypersensitivity angiitis.

The pathogenesis of autoimmune vasculitis is poorly understood. Understanding the immunologic mechanisms governing this disease requires precise identification of the cells which comprise the lesion. In this report, we have evaluated tissue sections from MRL/lpr mice from 16 to 45 weeks of age, representing all stages of clinical vasculitis. We demonstrate that basophil myelocytes participate in the evolution of the delayed-type hypersensitivity (DTH) response which initiates and perpetuates autoimmune vasculitis in these mice. These findings raise questions regarding the immunologic mechanisms by which basophils develop in this lesion and the interaction of basophils. VSMCs and lymphocytes in vasculitic angiodestruction.

Polychlorinated biphenyls (PCBs) selectively disrupt serotonergic cell growth in the developing Spisula embryo.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that exert neurotoxic effects during embryonic development. The present study demonstrates that early embryonic exposure to a mixture of PCBs (Aroclor 1254) results in a decrease in serotonergic cell growth. Using a novel, marine invertebrate embryo model, Spisula solidissima, immunocytochemistry, and confocal microscopy techniques, a dose-dependent decrease in serotonergic cell number was quantified within 24 h of exposure. This effect was seen with doses as low as 1 ppm Aroclor 1254. These findings demonstrate that environmentally relevant doses of Aroclor 1254 impair development of the serotonergic nervous system.

Polychlorinated biphenyls are selectively neurotoxic in the developing Spisula solidissima embryo.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that accumulate to toxic levels in the food chain. Using Spisula solidissima (surf clam) embryos as a developmental model, it was shown that Aroclor 1254 specifically targets two neuronal structures during embryonic development. Embryos were exposed to 1, 10), or 100 ppm Aroclor 1254 or an acetone vehicle control posthatching for 24, 48, and 72 h. Embryos labeled with a serotonin antibody or a neural antigen antibody and a rhodamine-conjugated secondary antibody were viewed by confocal microscopy. The cerebropleural ganglion showed a decrease both in serotonin production and in the size of the serotonin-synthesizing region upon exposure to 10 and 100 ppm Aroclor 1254. These decreases were detectable as early as 48 h postfertilization. When exposed to 100 ppm Aroclor 1254, the primitive neural plexus, which coordinates the movements of the mouth and velum, showed a delay in onset and cessation of expression of a molluscan-specific neural antigen. Exposure to Aroclor 1254 did not affect the overall growth and morphology of the embryos. In addition, analyses of total protein profiles and heat-shock protein 70 levels showed that exposure to Aroclor 1254 did not trigger protein degradation or cause a stress or shock response. These results show that exposure of Spisula embryos to Aroclor 1254 specifically targets neurogenesis while having no effect on the overall growth of the embryo.

Induction of immunoregulatory T cells by adjuvant.

The effect of adjuvant on the generation of suppressor cells in the thymus was investigated. C57Bl/6 mice were injected i.p. with complete Freund's adjuvant (CFA), and the capacity of thymocytes and splenic T cells to suppress the in vitro generation of cytolytic T cells was studied. Suppressor thymocytes were detected within two days after CFA inoculation and preceded the appearance of suppressor T cells in the spleen by six days. Characterization of the suppressor thymocyte populations showed that they inhibited primary but not memory T cell responses in vitro. We suggest that the initial step in the regulation of the primary immune response by adjuvant is the generation of suppressor cells in the thymus. Thymic suppressors subsequently migrate to peripheral lymphoid tissue where they inhibit cytotoxic, but no helper, T cell function.

Vasculitis in MRL/1 pr mice: model of cell-mediated autoimmunity.

The destruction of vascular smooth muscle cells (VSMC) in autoimmune arteritis is a poorly understood phenomenon. To approach this problem, VSMC cultures were established. The interaction of these cells (from normal or autoimmune mice) with lymphocytes was then evaluated. Specifically, splenocytes from MRL/1pr or C3H mice were co-cultivated with MRL/1pr or C3H VSMCs. Massive mononuclear inflammatory cell clusters enveloped MRL/1pr VSMCs which culminated in the detachment of MRL/1pr VSMCs from the culture plate. In contrast, the interaction of splenocytes from normal or autoimmune mice did not destroy normal VSMCs. Further investigation indicated that MRL/1pr VSMCs spontaneously expressed both Ia-k and Ia-d, as assessed by fluorescence microscopy and flow cytometry, and released interleukin-1-like factors--characteristics of accessory cells to T-lymphocyte function. Evaluation of VSMCs accessory function in antigen presentation suggests that these cells may present antigen under specific experimental conditions. As a result of these studies, a novel mechanism of autoimmune vasculitis is proposed. Our hypothesis is that defective biological function of VSMCs from autoimmune mice stimulates a mononuclear inflammatory cell response which culminates in VSMC autodestruction.

The role of vascular smooth muscle cells in experimental autoimmune vasculitis. I. The initiation of delayed type hypersensitivity angiitis.

The destruction of vascular smooth muscle cells (VSMCs) in autoimmune arteritis is a poorly understood phenomenon. For evaluation of the cellular interactions that may contribute to vasculitis, the immunobiology of VSMCs and lymphocytes was explored in vitro. Primary VSMC cultures were established, and the interaction of these cells (from normal or autoimmune mice) with lymphocytes was then assessed. Specifically, splenocytes from MRL/lpr or C3H mice were cocultivated with MRL/lpr or C3H VSMCs. Massive mononuclear cell clusters from normal and autoimmune mice enveloped MRL/lpr VSMCs, which culminated in the detachment of MRL/lpr VSMCs from the culture plate. In contrast, the interaction of SPs from either normal or autoimmune mice did not encompass or destroy normal VSMCs. Further investigation indicated that MRL/lpr, but not C3H, VSMCs spontaneously expressed Ia and released Il-1 like factor(s), which may be at least two mechanisms by which MRL/lpr VSMCs stimulate the in vitro mononuclear cell influx. As a result of these studies, a novel mechanism for the induction of mononuclear cell autoimmune vasculitis is proposed. VSMCs derived from autoimmune mice may stimulate a mononuclear inflammatory cell phlogistic response which culminates in VSMC autodestruction.

Induction of T-cell proliferation by an Ia+ monocytic tumor clone.

Ia+ immature malignant monocyte clones were isolated from retrovirus-induced lymphomas. These lymphomas contained a predominant population of Ly-1+ T cells. In order to explain this anomaly, the functional capacity of one of these clones, ORA I-a, was assessed. The results indicated that ORA I-a could present protein antigen to syngeneic and semisyngeneic antigen-primed lymph node T cells. Allogeneic stimulation, however, could not be induced. Furthermore, ORA I-a-conditioned medium could augment mitogen-dependent and -independent thymocyte proliferation. These studies suggest that the unique populations of malignant antigen-presenting cells can interact with distinct T-cell subpopulations. Functional analyses of these tumor cell lines may thus be useful in elucidating some of the cellular interactions which occur during the course of immunity.

Evolutionary immunobiology.

Identifying the evolutionary origin of inducible, specific immune recognition represents a major objective in developmental immunology. In order to address this issue from an overall phylogenetic perspective, major studies of cellular and humoral immune function are being undertaken using lower vertebrate and invertebrate models. Here, C. Reinisch and G. Litman discuss the application of new technologies, particularly molecular genetic approaches, that is providing important new insights into the genetic mechanisms that have influenced the evolutionary diversification of immunological function.

Specific reactivity of leukemia cells to polyclonal anti-PCB antibodies.

Bivalve molluscs such as the soft shell clam (Mya arenaria) develop leukemias in the hemolymph which are fatal. The prevalence of leukemia in Mya was evaluated using a murine monoclonal antibody which recognizes a leukemia-specific protein expressed by tumor cells. The reactivity with a polyclonal antibody to polychlorinated biphenyls (PCBs) of both normal circulating cells and tumor cells was also determined. Both leukemia prevalence and PCB reactivity were ascertained by flow cytometry. Analytical chemistry was used to quantitate the amount of Aroclor per tumor cell population and compared directly to flow cytometric results. Our results show that the prevalence of leukemia consistently exceeds 60% when clams are retrieved from New Bedford Harbor, a site heavily contaminated with PCBs. Both normal circulating cells and tumor cells are extremely reactive with the PCB antibody. When clams from two other sites were compared with clams from New Bedford Harbor, both disease prevalence and cell reactivity to the PCB antibody were reduced. Our experiments are the first which use the flow cytometer to demonstrate PCBs in cell populations of marine invertebrates. Our results further demonstrate that the presence of polychlorinated biphenyls in vivo is directly correlated with environmentally linked leukemia.

Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice. A histologic and immunocytochemical analysis.

The cellular mechanisms governing the expression of mononuclear cell vasculitis are poorly understood. For determination of the precise sequence of events in the development of vasculitis in autoimmune MRL/lpr mice, histologic sections from 4-20-week-old mice were evaluated with a panel of cytochemical and immunohistochemical stains. The results show that vascular disease in MRL/lpr mice develops as follows: Thy 1+, Ly 1+, L3T4- T cells assemble around predominantly small-to-medium muscular arteries at approximately 8 weeks of age. At 12 weeks of age, an adventitial inflammatory focus forms, composed of large "reactive" mononuclear inflammatory cells adjacent to hypertrophied vascular smooth muscle cells (VSMCs). Blastic Thy 1+, Ly 1+, L3T4- T cells subsequently infiltrate the tunica media, and selective VSMC karyolysis results. Occasional cytotoxic/suppressor T cells, macrophages, and possibly NK cells are noted primarily distal to the infiltration site. The outer zone of the inflammatory infiltrate is composed of mature B cells and occasional B-cell precursors. These findings suggest that cellular constituents of the immune response mediate mononuclear cell vasculitis in MRL/lpr mice.