Allogeneic Lymph Node Cells Research Articles

Application of the Popliteal Lymph Node Assay to Evaluate an Immunosuppression Protocol

There exists a variety of experimental techniques which are used to evaluate host immunity. None of these in vitro assay systems are optimal, since they are performed ex vivo. Here is described an in vivo model, using four rat strain combinations and a modification of the popliteal lymph node assay, which quantifies the immune response to blood transfusion, in a sensitive and reproducible fashion. The subcutaneous injection of a 5 × 10 5 or 10 × 10 5 (depending on the rat strain) X-irradiated allogeneic lymph node cells into the hind footpad of a rat results in an increase in the weight of the draining popliteal lymph node harvested 7 days later. Injection of 100 × 10 5 X-irradiated cells results in the maximum nodal enlargement (fourfold). A dose of 50 × 10 5 cells results in lymph node enlargement just shy of maximum and so was selected for use in all experiments. When 50 × 10 5 nonirradiated allogeneic lymph node cells are injected, there is up to an eightfold increase in nodal size. To eliminate the effects of nonspecific inflammation, as well as variation in node weights, a stimulation index (SI) was established. Transfusion of a rodent with blood from the same strain as the lymph node donor, 1 week prior to subcutaneous injection, resulted in a significantly lower SI, compared to the SI observed in untreated animals challenged with the same inoculum or third-party controls ( P < 0.05). The significance of all comparisons was assessed by the Students' t test. This simple method of in vivo quantification of the immune response has obvious application to the evaluation of any experimental immunosuppression regimen.

Manipulation of graft-versus-host disease for a graft-versus-leukemia effect after allogeneic bone marrow transplantation in AKR mice with spontaneous leukemia/lymphoma.

Graft-versus-host (GVH) disease can result in a beneficial graft-versus leukemia (GVL) effect after bone marrow transplantation in patients with malignant disease. In this report, we used bacteria-free AKR (H-2k) mice bearing advanced spontaneous T cell leukemia/lymphoma as a moel to evaluate the GVH and GVL effects of bone marrow transplantation using fully incompatible SJL (H-2s) donors. A therapeutic GVL effect, accompanied by increased leukemia-free survival, was obtained only when 0.5 X 10(6) allogeneic lymphocytes (lymph node cells) were added to the marrow inoculum. Transplantation of allogeneic bone marrow without added lymph node cells (or use of syngeneic cells) resulted in a significant increase in leukemia relapse; increasing the dose of allogeneic lymph node cells to 2.0 X 10(6) resulted in significantly higher GVH-associated mortality. Survival and therapeutic benefits were obtained only when the intensity of the GVH reaction was carefully controlled by manipulation of alloreactive lymphocytes present in the marrow. These results suggest, indirectly, that T cell depletion may abolish any GVL effect of marrow transplantation, even if the donor is mismatched with the host at the major histocompatibility complex. The frequency in the spleen of cytotoxic T lymphocytes (CTL) reactive against host alloantigens was estimated using limiting-dilution microcytotoxicity assays at various times after transplantation of allogeneic bone marrow with and without added lymph node cells. The average frequency of CTL was highest in mice that were given marrow plus lymph node cells and tested within the first four weeks after transplantation. The level of CTL activity measured in vitro was dependent on the dose of lymphocytes injected and correlated with both the GVL and GVH effects in vivo. Down-regulation of CTL activity against host, but not third-party, alloantigens in vitro was observed under limiting dilution assay conditions, leading to the suggestion that host-specific regulatory cells may be present in these allogeneic bone marrow chimeras.

A platelet-derived immunoregulatory serum factor with T cell affinity.

A factor in mouse and human serum which enhances immune responses to SRBC and TNP-Ficoll in mice has been described. This factor completely reverses immunosuppression induced by syngeneic lymphoma (RCS) or allogeneic lymph node cells. T lymphocytes bind this factor, since the activity can be removed from serum by absorption with T cell containing spleen cells or cloned cytotoxic T lymphocytes. Furthermore, the factor is active only in the presence of mature T cells. Platelet alpha-granules seem to be the source of this serum factor because 1) the activity is lacking in plasma, in serum prepared in the absence of platelets, and in serum prepared from a patient lacking platelet alpha-granules and 2) isolated mouse or human platelets release an immunostimulatory factor with very similar properties upon incubation with thrombin.

Manipulation of brain DNA synthesis is achieved by using a systemic immunological disease.

Previously, we showed that as early as postnatal day 11 an immunological disease, graft-versus-host disease, induced by grafting allogeneic lymph node cells into an immunoincompetent neonatal rat significantly decreases cerebellar histogenesis--i.e., DNA synthesis and the number of newly formed neurons. Here, we report that, subsequent to successful immunotherapy, there was a reversal of the deleterious graft-versus-host disease-induced alterations in DNA synthesis in individual cerebellar germinal cells. Immunotherapy involved treating the diseased rats on postnatal days 11, 12, and 13 with alloantiserum specifically directed against the grafted lymph node cells injected on the day of birth. On postnatal day 14, diseased, serum-treated, and control littermates were injected with [3H]thymidine and, 15 min later, the cerebella were excised and autoradiographed. A 0.72-mm segment of the external granular layer in the cerebellar fissure between lobules VIB and C was searched for labeled cells. The control group had the greatest number of labeled cells, defined by the presence of six or more autoradiographic grains. (43 +/- 4, mean +/- SEM) and the greatest number of grains per cell (9.5 +/- 0.2). Rats with the disease had few labeled cells (4 +/- 2) and the number of grains per cell was low (6.6 +/- 0.6); however, serum treatment increased both the number of labeled cells (26 +/- 8) and the number of grains per cell (7.4 +/- 0.2). These results show that without mononuclear infiltrates or inflammation in the cerebellum, a systemic immunological disease can dramatically decrease DNA synthesis per germinal cell and, moreover, that halting the disease by alloantiserum therapy can reverse this effect. These findings emphasize the sensitive plastic nature of neuronal cell acquisition in the normally developing brain.

Immune Responses to Mouse Neuroblastoma C1300

A 51Cr microcytotoxicity assay was used to study the kinetics of lymph-node- and spleen-cell-mediated cytotoxicity of A/JAX hosts following a single inoculum of the in vivo grown syngeneic neuroblastoma C1300. Lymph node cells (LNCs) demonstrated cytotoxicity which peaked at day 9, decreased as the tumor approached the 0.5-gram weight and increased again as the tumors grew. The increase in cytotoxicity following the depressed state was significantly greater than the initial peak, particularly in those mice bearing large tumors. Three groups of A/JAX mice were evaluated: mice receiving tumor cells alone; mice that were either thymectomized or splenectomized prior to inoculation, and mice that rejected the tumor. The C57Bl/6 served as an allogeneic comparison. Significant cytotoxicity was detected earlier with allogeneic LNCs than with spleen cells. Thymectomy and splenectomy elicited syngeneic LNC immune responses below or equal to the level found in nonimmune A/JAX hosts.

Generation of cell-mediated cytotoxicity to Theileria parva (East Coast fever) after inoculation of cattle with parasitized lymphoblasts.

The participation of genetically restricted cell-mediated lympholysis (CML) in protective immunity against Theileria parva was examined in cattle inoculated with autologous and allogeneic parasitized lymphoblasts. The majority of cattle given 10(7) infected allogeneic cells were protected against infection with the homologous parasite. Animals inoculated with drug-cured lymphoblasts, normal allogeneic lymph node cells (LNC), or a combination of LNC and parasitized allogeneic cells were fully susceptible. The magnitude of the primary CML responses against autologous infected targets (as opposed to that against the allogeneic lymphoblasts inoculated or 3rd-party targets) more accurately correlated with a decreased severity of infection after challenge. Autologous CML responses appeared earlier as cattle were given increasing numbers of parasitized autologous lymphoblasts, and were higher and more persistent with increasing parasitosis. Between 10(2) and 10(5) autologous infected cells induced immunity to T. parva, whereas at least 10(7) allogeneic lymphoblasts were required for comparable protection. The findings provide additional evidence for a protective role of restricted CML responses against T. parva in vivo. When allogeneic infected cells are used for immunization, viable T. parva need to establish infection in host cells to induce adequate protective immunity.

Autoreactive Antibody-Forming Cells Directed against Thymocytes and Thymus-Derived Lymphocytes

Antibody-forming cells with specificities against syngeneic and allogeneic thymocytes are detected in the spleens of normal mice after activation in vitro or in vivo with lipopolysaccharide (LPS). The activity of such cells was measured in a complement-dependent plaque assay employing trypan blue dye to assess zones of lysis. Plaques were rarely seen in the absence of LPS treatment. Anti-immunoglobulin added to the plaque assay abrogated the appearance of plaques, but the addition of LPS had no effect. Furthermore, plaque formation was 2-mercaptoethanol sensitive indicating that the antibody responsible was of the IgM class. Plaque forming cells (PFC) were also detected against syngeneic and allogeneic lymph node cells and to a much lesser extent against splenocytes. The numbers of PFC found against syngeneic, allogeneic, or a mixture of thymocytes was similar and ranged from 1000 to 3000 PFC/10(8) viable spleen cells tested. All murine strains tested, including congenitally athymic nude mice, exhibited anti-thymocyte PFC after LPS activation. C3H/HeJ mice, genetically unresponsive to LPS, did not respond mitogenically to LPS and no anti-thymocyte plaques were observed. These findings suggest that clones of autoreactive B cells are present in normal mice and can be activated by LPS.

Activation of cytotoxic T cells by nonstimulating tumor cells and spleen cell factor(s).

The ability of three cultured mouse tumor lines to stimulate a cytotoxic response in 5-day cultures of allogeneic lymph node cells was studied with a 51Cr release assay. Two lines of mesenchymal origin, P815 and EL-4, were found to be highly stimulatory, whereas the third cell line, CaD2, a mammary gland epithelial tumor, did not stimulate over a wide range of cell concentration. CaD2 cells were shown to contain major antigens similar to those of P815 cells by the specific lysis of both cells by lymphocytes activated to H-2d-bearing peritoneal cells.UV-irradiated P815-cells, like gamma-irradiated CaD2 cells, did not stimulate a cytotoxic response, but both cell lines were found to stimulate a full and specific response to allogeneic lymph node cells if these mixed cultures were supplemented with a supernatant harvested from concanavalin A-stimulated spleen cells.

Stimulation of syngeneic and allogeneic lymphoid cells by tumour cells in vitro.

August and Wistar rat lymph node cells were found to respond well to PHA stimulation and in mixed lymphocyte culture, as determined by an increased incorporation of 3H-thymidine. August rat lymph node cells were also stimulated by incubation with irradiated syngeneic tumour cells. Allogeneic Wistar rat lymph node cells produced a larger response to the August tumour cells. The response of syngeneic and allogeneic lymph node cells was reduced by pretreating the tumour cells with a Wistar anti-tumour serum. Pretreating the tumour cells with sera from normal or tumour bearing rats also reduced the response of syngeneic lymph node cells but did not reduce the response of allogeneic lymph node cells.

Relation of the butanol-solubilized rabbit histocompatibility antigens to the suppression of antibody-producing allogeneic lymph node cells.

Soluble antigen prepared by Triton and butanol from cell membrane fragments of rabbit lymph node and spleen has been shown to be associated with the suppression or rejection of allogeneic lymph node cells by its abilities (1) to inhibit or absorb the suppressive antibody in rabbit antileucocyte serum, (2) to induce actively in normal rabbits the rejection of transferred lymph node cells, and (3) to stimulate production of antibodies suppressive for allogeneic lymph node cells. This can be demonstrated in cell transfer or, in vitro, in the hemolytic antibody plaque test. Analysis by chromatography and sucrose density gradient ultracentrifugation shows that the rejection-associated antigen can be found within approximately half the total protein of the butanol extract, and suggests nonidentity of the antigen detectable by inhibition of suppressive antibody and that detectable serologically. Further evidence of nonidentity of the antigens and antibodies detected is given by substantial disproportionality of titers determined in suppressive and serological tests in primary and secondary antisera to whole allogeneic cells, respectively, and in secondary and tertiary antisera to the butanol-solubilized antigen.

Aplastic anaemia in sublethally irradiated mice given allogeneic lymph node cells.

Aplastic anaemia in sublethally irradiated mice given allogeneic lymph node cells.

Suppression of Rabbit Lymph Node Cells by Rabbit Anti-Leucocyte Serum Demonstrated in Vitro by the Antibody Plaque Test

Summary The production of hemolytic antibody plaques by lymph node cells of rabbits injected with sheep erythrocytes can be inhibited by prior incubation of the cells with rabbit anti-rabbit-leucocyte serum and complement. The 50% plaque reduction endpoint of such sera is, over a wide range, inversely proportional to the concentration of the lymph node cell suspension used. This reduction of plaque number can be inhibited by prior incubation of the rabbit anti-rabbit-leucocyte serum with solubilized preparations of the cell antigen associated with the rejection of transferred allogeneic cells. The identity of the antibody causing reduction in number of plaques by allogeneic lymph node cells with the antibody causing reduction of antibody production by such cells when transferred to recipient rabbits has been indicated in two ways. First, in a number of rabbit anti-rabbit-leucocyte serum pools, endpoints obtained by the two methods showed a high degree of correlation, with a 45° line of regression between them. Second, when rabbits were injected with the soluble rejection-associated antigen, and the resulting antisera, individually tested by plaque reduction, were selected on that basis for pools of relatively high and low suppressive activity, the two pools, on subsequent testing in cell-transfer experiments, showed sharp differences in suppressive effect on transferred lymph node cells, in conformity with the activity of the sera in plaque-reduction. In rabbits actively immunized with the rejection-associated antigen and then used as recipients in cell transfer, it was possible to predict roughly the degree of suppression of transferred lymph node cells in these recipients by the plaquereduction titer of the sera of the individual rabbits obtained shortly before cell transfer.

SOLUBILIZATION BY BUTANOL OF RABBIT CELL-ANTIGENS ASSOCIATED WITH THE REJECTION OF TRANSFERRED LYMPH NODE CELLS.

SummaryStudies have been carried out on the solubilization of the rabbit cell-antigen associated with rejection of transferred allogeneic lymph node cells. Using as a preliminary criterion serologic reaction vs cell-suppressive rabbit anti-rabbit-leucocyte sera, soluble antigen has appeared in low concentration in suspensions of liver cell-fragments from normal rabbits kept for some days at 4°C. The antigen was solubilized by the use of butanol at 20% by volume from suspensions of such liver cell-fragments, if the suspensions were previously maintained at 4°C for some time. Butanol extraction could also be applied to Triton-extracted lipoprotein of cell-fragments obtained from normal rabbit lymph node and spleen with serologically active material obtained in solution in about 10 times the yield obtained from liver, relative to mass of tissue. When the butanol-extracted soluble antigen from rabbit lymph node was injected twice into normal rabbits, the sera of the latter showed the property of suppressing a...